TW201200591A

TW201200591A - Extraction of lipid from cells and products therefrom

Info

Publication number: TW201200591A
Application number: TW100119199A
Authority: TW
Inventors: Stephen Robert Cherinko; Robert Cody Kertis; Kirk E Apt; Paul Warren Behrens; Jon Milton Hansen; Joseph W Pfeifer Iii; Tracey Lynn Stahl; Ross Zirkle; Krishna Raman; Nasrin Tabayeh Burke; Neil Francis Leininger
Original assignee: Martek Biosciences Corp
Priority date: 2010-06-01
Filing date: 2011-06-01
Publication date: 2012-01-01

Abstract

The present invention relates to processes for obtaining a lipid from a cell by lysing the cell, contacting the cell with a base and/or salt, and separating the lipid. The present invention is also directed to a lipid prepared by the processes of the present invention. The present invention is also directed to microbial lipids having a particular anisidine value, peroxide value, and/or phosphorus content.

Description

201200591 六、發明說明： L發明所雇技術麵域]j 相關專利申請案之交叉引述本申請案主張2010年6月1曰提申之美國申請案號 61/350,363、2010年8月31曰提申之美國申請案號 61/378,923，以及2011年3月15日提申之美國申請案號 61/452,721之利益，其等之各個係以其之整體併入本文中以作為參考資料。發明領域本發明係有關於用於自一細胞獲得一脂質的方法，其等係藉由溶解該細胞、使該細胞之ρ Η提升及/或使該細胞接觸一鹽，以及分離該脂質。本發明亦針對藉由本發明的方法所製備之脂質。本發明亦針對具有特定的曱氧苯胺值、過氧化物值，及/或磷含量之微生物脂質。 C先前技術3 發明背景一種用於自一微生物細胞獲得脂質之典型的方法，例如多不飽和脂肪酸’涉及使能夠產生所欲的脂質之微生物於—發酵器、小池或生物反應器中生長，分離包含一微生物細胞生質之發酵肉湯，乾燥該微生物細胞生質，以及藉由溶劑萃取來分離該脂質。分離的步驟可以包括用水來稀釋一發酵肉湯，離心該經稀釋的肉湯，溶解該等微生物細胞，以及藉由添加一水不互溶溶劑（該脂質為可溶解於其内的’例如’己幻至該混合物而從該等溶解的細胞來萃取胞 201200591 内脂質。自一微生物細胞移除—ρ _ 械力（例如，均質化）、酵素處萃取方法係使用機破裂而溶解-細胞於1酵心學處理來使細胞壁溶劑(例如，異丙醇)而自包含:：。月曰質可以使用-有機之所生成的組錄料;質，及水機械地分離以及醇必須從該心成物予以予以移除。參見，例如’ _ ^ f廢棄物流二者〇1/細。 u開案_⑽/76385和赐然而，使用以上方法之任—者之脂質之工業規模的生產需要大㈣揮發性且可燃的有機溶劑，藉此產的操作條件1萃取的方法中之有機溶劑之使用一防爆脂質回收系統成為必 ^藉此增加了脂質回收的成本。，小於從—微生物細胞萃取脂質的過程中使用有機溶劑會產生—有機溶劑廢棄物流，其需要-完整的溶劑回收系統或-適當的處置方法，其進—步增加脂質萃取之全面的生產成本。舉例而言，對於揮發性有機化合㈣聞的排放之嚴格的限制需要更A的人力以增加^皿和其他的設備之成本。對於不使用—錢溶劑而自—細胞獲得脂質的一方法有1求。已經㈣數個枝用於自—細胞分離一脂質而不使用有機溶劑。舉例㈣，美國專利案號6,75〇，謹揭示一含水的清洗方法，藉此—乳狀液係用含水的清洗溶液予以清洗直到獲得—實質非乳化的脂質為止。缺而，於 201200591 一些具體例中，該方法需要多重的清洗步驟，其等需要很多的成本和時間。美國專利案號7 431 952揭示一方法，藉此將溶解的細胞離心以移除細胞壁碎屑以及接而萃取且純化油。然而，該方法提供了需要廣大的進一步純化之一粗製油。因而，所需要的是一種不使用一揮發性溶劑來自一細胞萃取一脂質的方法，以及其可以使用容易可得到的設備和最小數目的步驟予以執行來提供了高度純的脂質。【發明内容3 發明概要本發明針對-種供用於自一微生物細胞組成物獲得一脂質的方法’該方法包含使該細胞組成物的pH提升至8或以上，以及自該細胞組成物分離一脂質，其中該脂質選擇性地含有少於5%重量計或體積計之一有機溶劑。於-些具體例中，該pH提升溶解該細胞級成物。於一些具體例中，該pH提升去乳化該細胞組成物。於-些具體例中’該方法包含添加鹽至該細胞組成物以去乳化該細胞組成物。於-些具體例中，該添加鹽係在該pH提升之後予以執行於-些具體例中，該方法進一步包含加熱該溶解的細胞組成物以乳化該細胞組成物。於_些具體例中，該加熱係在該pH提升之後予以執行。於一些具體例中，該方法進-步包含再度提升該細胞级成物的pH以去乳化該細驗祕。於_些純例中，該再度提升pH係在該添加鹽或該加熱之後予以執行。 201200591 亦針對一種供用於自一細胞獲得一脂質的方法’ °玄方法包含溶解—細胞以形成-溶解的細胞組成物，使解的細胞1且成物之PH提升至8或以上以去乳化該細匕成物添加鹽至該溶解的細胞組成物以去乳化該細胞、.且成物’以及自該去乳化的細胞組成物分離—脂質，其中該脂質選擇性地含有少於5%重量計或體積計之—有機溶劑。本發月亦針對一種供用於自一細胞組成物獲得—脂質的方法，该方法包含使該細胞組成物的p Η提升至8或以上來溶解該細胞組成物且去乳化該細胞組成物，添加鹽至該細胞組成物，以及自該去乳化的細胞組成物分離一脂質，其中該脂質選擇性地含有少於5%重量計或體積計之一有機溶劑。本發明亦針對一種供用於自一細胞獲得一脂質的方法，s亥方法包含溶解一細胞以形成一溶解的細胞組成物，搜拌该細胞組成物以去乳化該細胞組成物，以及自該去乳化的細胞組成物分離一脂質’其中該脂質選擇性地含有少於5%重量計或體積計之一有機溶劑。於一些具體例中，該方法進一步包含加熱該溶解的細胞組成物以去乳化該細胞纟且成物。於一些具體例中，該加熱係在該添加鹽之後予以執行。於一些臭體例中’該方法進一步包含攪拌該溶解的細胞組成物以去乳化该細胞纟旦成物。於一些具體例中，該授拌係歷時5分鐘至96小時。 201200591 於—些具體例中，該攪拌包含用具有350公分每秒至 9〇〇公分每秒之葉尖速率的一葉輪來攪拌該細胞組成物。於一些具體例中，該方法進一步包含使該溶解的細胞組成物之pH提升以去乳化該細胞組成物。於一些具體例中，使该溶解的細胞組成物之pH提升以去乳化該細胞組成物包含添加鹼。於一些具體例中，第二個鹼係在該添加鹽或該加熱之後添加。於一些具體例中，該加熱係歷時1〇分鐘至96小時。於一些具體例中，該細胞組成物係被加熱至6〇它至1〇〇 C的溫度。於一些具體例中，該細胞組成物係被加熱至9〇 °C至100°(：的溫度。於些具體例中，s亥pH提升包含添加驗。於一些具體例中’該鹼具有1至12的pKb。於一些具體例中，該分離一脂質發生於1〇。〇至1〇〇。〇的溫度。於一些具體例中，該方法包含藉由攪動、混合、摻合、振盪、振動，或其等之一組合來攪拌該溶解的細胞組成物。於一些具體例中，該方法包含以〇 J hp/1〇〇〇 gal至1〇 hp/1000 gal之溶解的細胞組成物來攪拌該溶解的細胞組成物。於一些具體例中，該方法包含用具有2卯似⑺岀至ιοοο ft/min之葉輪葉尖速率的一攪拌器來攪拌該溶解的細胞組成物。於些具體例中，s亥溶解包含機械處理、物理處理、化學處理、酵素處理’或其等之—組合。於一些具體例中， 201200591 該機械處理為均質化。於些具體例中，該鹽係以0.1。/。至20。/。重量計之該溶解的細胞組成物的—量來添加。於—些具體例中該鹽係以0 · 5 /。至15 %重量計之該溶解的細胞組成物的一量來添加至該冷解的細胞組成物。於一些具體例中，該鹽係以2%至 10/。重量计之該溶解的細胞組成物的一量來添加至該溶解的細胞組成物。於一些具體例中，該鹽係選自於以下所構成的群組：鹼金屬鹽、鹼土金屬鹽、硫酸鹽，以及其等之組合。於一些具體例中，δ亥鹽為氣化納。於一些具體例中，該鹽為硫酸鈉。於一些具體例中，該分離包含離心。於一些具體例中，該分離包含離心於30°C至90°C的溫度。於一些具體例中，該方法提供一脂質，該脂質包含至少50%重量計之三酸甘油酯。於一些具體例中，該方法提供一脂質，該脂質具有26 或更少，25或更少’ 20或更少，15或更少，1〇或更少，5或更少，2或更少’或是1或更少之甲氧苯胺值。於一些具體例中，該方法提供一脂質，該脂質具有5或更少，4.5或更少’ 4或更少，3.5或更少，3或更少，2.5或更少，2或更少，丨.5或更少’ 1或更少，0.5或更少，〇.2或更少，或是〇·1或更少的過氧化物值。於一些具體例中，該方法提供一脂質，該脂質具有1〇〇 ppm或更少，95 ppm或更少，90 ppm或更少，85 ρρηι或更 201200591 少，80 ppm或更少，75 ppm或更少，70 ppm或更少，65 ppm 或更少，60 ppm或更少，55 ppm或更少，50 ppm或更少， 45 ppm或更少，40 ppm或更少，35 ppm或更少，30 ppm或更少，25 ppm或更少，20 ppm或更少’ 15 ppm或更少’ 10 ppm 或更少，5 ppm或更少，4 ppm或更少，3 ppm或更少，2 ppm 或更少，或是1 ppm或更少的麟含量。於一些具體例中，該方法提供一脂質，該脂質具有至少10%，至少15%，至少20%，至少25%，至少30%，至少 35%，至少40%，至少45%，或至少50%重量計之所欲的多不飽和脂肪酸(PUFA)。於一些具體例中，該方法提供一脂質，該脂質具有至少10%，至少15%，至少20%，至少25%，至少30%，至少35%，至少40%，至少45%，或至少50%重量計的二十二碳六烯酸（”DHAn)，及/或至少10%，至少 15%，或至少20%以重量計的二十二碳五烯酸("DPAn-6")，及/或至少10%，至少15%，或至少20%以重量計的二十碳五烯酸("EPA”），及/或至少10%，至少15%，至少20%，至少 25%，至少30%，至少35%，至少40°/。，至少45%，或至少 50%重量計之花生油酸("ARA")。於一些具體例中，該細胞為一微生物細胞。於一些具體例中，該方法包含濃縮含有該微生物細胞的一發酵肉湯。於一些具體例中，該細胞為一油籽。於一些具體例中，該油籽係選自於以下所構成的群組：葵花籽、油菜(canola) 籽、油菜子(rapeseed)、亞麻仁（linseed)、蓖麻油（castor oil) 籽、胡荽（coriander)籽、金盞菊（calendula)籽，以及其等之 201200591 基因修飾變異物。於一些具體例中，該方法包含清洗該細胞或細胞組成物。於一些具體例中，該方法包含低溫殺菌該細胞或細胞組成物。於一些具體例中，該方法包含濃縮該溶解的細胞組成物。於一些具體例中，該方法包含精製該脂質。於一些具體例中，該精製係選自於以下所構成的群組：苛性精製法、去膠、酸處理、驗處理、冷卻、加熱、漂白、去臭、脫酸，以及其等之組合。於一些具體例中，該方法包含收穫該脂質，其中該收穫包含泵抽該脂質而不攪拌。本發明亦針對一種脂質，其係藉由本發明的方法之任一者而獲得的。於一些具體例中，該脂質包含一或更多種多不飽和脂肪酸。於一些具體例中，該脂質具有至少10°/。，至少15%，至少20%，至少25%，至少30%，至少35%，至少40%，至少45%，或至少50%重量計之所欲的PUFA。於一些具體例中，該脂質具有至少10%，至少15°/。，至少20%，至少25%，至少30%，至少35%，至少40%，至少45%，或至少50%重量計之DHA，及/或至少10%，至少15%，或至少20%重量計之DPAn-6，及/或至少10%，至少15%，或至少20%重量計之EPA，及/或至少10%，至少15°/。，至少20%，至少25%， 10 201200591 至少30% ’至少35%，至少4〇%，至少桃，或至少篇重量計之ARA。於一些具體例中，該脂質具有3或更少的總香氣強度。於一些具體例中，該脂質具有2或更少的總芳香強度。於一些具體例中’該脂質包含至少1〇%重量計的三醯甘油醋顧份’其中於該三醯甘油酯餾份内之至少12〇/。重量計之脂肪酸為二十碳五烯酸，其中於該三醯甘油酯餾份内之至少25%重量計之脂肪酸為二十二碳六烯酸，以及其中於該三醯甘油酯餾份内之少於5%重量計之脂肪酸為花生油酸。於一些具體例中，該脂質包含至少20%以重量計的二十碳五烯酸以及各少於5%重量計的花生油酸、二十二碳五烯酸n-6、油酸、亞麻油酸、次亞麻油酸、二十稀酸、芬子酸，以及硬脂四烯酸。於一些具體例中，該脂質具有26或更少，25或更少’ 20或更少，15或更少，10或更少，5或更少’ 2或更少’或是1或更少之甲氧苯胺值，及/或5或更少’ 4.5或更少’ 4或更少，3.5或更少，3或更少，2.5或更少，2或更少，1.5或更少，1或更少’ 〇·5或更少，〇·2或更少’或是0.1或更少的過氧化物值，及/或1〇〇 PPm或更少，95 ppm或更少’ 90 ppm 或更少，85 ppm或更少，80 ppm或更少，75 ppm或更少’ 70 ppm或更少，65 ppm或更少，60 ppm或更少，55 ΡΡ111或更少，50 ppm或更少，45 ppm或更少’ 40 ppm或更少，％ ppm 或更少，30 ppm或更少，25 ppm或更少，20 ppm或更少’ 11 201200591 15 Ppm或更少’ 10 PPm或更少，5 ppm或更少，4 ppm或更少’ 3 ppm或更少，2 ppm或更少，或是1 ppm或更少的磷含量。於一些具體例中’該脂質為一粗製脂質。於一些具體例中’該粗製脂質選擇性地具有少於5%重量計或體積計之一有機溶劑。本發明亦針對一種粗製的微生物脂質，其具有26或更少之曱乳本胺值，5或更少的過氧化物值，1〇〇 ppm或更少的磷含量’以及選擇性地少於5%重量計或體積計之一有機溶劑。於一些具體例中，該粗製的微生物脂質具有26或更少，25或更少，20或更少，15或更少，10或更少，5或更少， 2或更少，或是1或更少之甲氧苯胺值，及/或5或更少，4.5 或更少，4或更少，3.5或更少，3或更少，2.5或更少，2或更少，1.5或更少，1或更少，0.5或更少’ 0.2或更少’或是 0.1或更少的過氧化物值，及/或1〇〇 PPm或更少，95 ppm或更少，90 ppm或更少，85 ppm或更少，80 ppm或更少’ 75 ppm 或更少，70 ppm或更少，65 ppm或更少’ 60 ppm或更少’ 55 ppm或更少，50 ppm或更少，45 ppm或更少，40 ppm或更少，35 ppm或更少’ 30 ppm或更少，25 ppm或更少’ 20 ppm 或更少，15 ppm或更少，1〇 ppm或更少’5 ppm或更少’4 ppm 或更少，3 ppm或更少，2 ppm或更少，或是1 PPm或更少的磷含量。於一些具體例中’該粗製的微生物脂質具有至少 12 201200591 10%，至少15%，至少20%，至少25%，至少30%，至少35%，至少40%，至少45%，或至少50%重量計之所欲的PUFA。於一些具體例中，該粗製的微生物脂質具有至少10%，至少15%，至少20%，至少25%，至少30%，至少35%，至少 40%，至少45%，或至少50%重量計之DHA，及/或至少10%，至少15%，或至少20%重量計之DPA n-6，及/或至少10%，至少15%，或至少20%重量計之EPA，及/或至少10%，至少 15%，至少20%，至少25%，至少30%，至少35%，至少40%，至少45%，或至少50%重量計之ARA。本發明亦針對一種經萃取的微生物脂質，其包含至少 70%重量計之三酸甘油醋德份，其中該三酸甘油S旨顧份的二十二碳六烯酸含量為至少50%重量計，其中該三酸甘油酯餾份的二十二碳五烯酸n-6含量為由至少0.5%重量計至 6%重量計，以及其中該油具有26或更少之甲氧苯胺值。本發明亦針對一種經萃取的微生物脂質，其包含至少 70%重量計之三酸甘油酯餾份，其中該三酸甘油酯餾份的二十二碳六稀酸含量為至少40%重量計，其中該三酸甘油酯餾份的二十二碳五烯酸n-6含量為由至少0.5%重量計至 6%重量計，其中該二十二碳六烯酸對該二十二碳五烯酸n-6 之比率係大於6 ·· 1，以及其中該油具有26或更少之曱氧苯胺值。本發明亦針對一種經萃取的微生物脂質，其包含至少大約70%重量計之三酸甘油酯餾份，其中該三酸甘油酯餾份的二十二碳六烯酸含量為至少60%重量計以及其中該油 13 201200591 具有26或更少之曱氧苯胺值。於一些具體例中，該經萃取的脂質具有26或更少’ 25 或更少，20或更少，15或更少，1〇或更少，5或更少，2或更少，或是1或更少之甲氧苯胺值’及/或5或更少’ 4.5或更少，4或更少，3·5或更少，3或更少，2.5或更少’ 2或更少’ 1.5或更少，1或更少，0.5或更少’ 〇·2或更少’或是0.1或更少的過氧化物值，及/或100 ppm或更少，95 ppm或更少’ 90 ppm或更少，85 ppm或更少，80 ppm或更少’ 75 ppm或更少，70 ppm或更少，65 ppm或更少’ 60 ppm或更少’ 55 ppm 或更少，50 ppm或更少，45 ppm或更少，40 ppm或更少’ 35 ppm或更少，30 ppm或更少，25 ppm或更少，20 ppm或更少，15 ppm或更少，10 ppm或更少，5 ppm或更少，4 ppm 或更少，3 ppm或更少，2 ppm或更少，或是1 ppm或更少的磷含量。於一些具體例中，該經萃取的微生物脂質為一粗製脂質或一粗製油。於一些具體例中，該粗製脂質選擇性地具有少於5°/。重量計或體積計之一有機溶劑。本發明亦針對一種供用於獲得一脂質的方法，該方法包含精製本發明的粗製脂質。於一些具體例中，該精製係選自於以下所構成的群組：苛性精製法、去膠、酸處理' 鹼處理、冷卻、加熱、漂白、去臭、脫酸，以及其等之組合。圖式簡單說明併入本文中以及形成本說明書的一部份之附圖闊明了 14 201200591 本發明以及’和詳細說明明明步供使用來解釋本發原貝ρ及K予熟f相關技藝者能力來作出並使用本發第1 4圖提供了說明本發明的方法之概要的流程圖；第5圖為圖，其提供了於各種的pH’溶解的細胞組成物隨時間之電子順磁共振(EpR)。見在將就附圖來說明。於圖式中，類似的參考數目指^完全相_或是功能上相似的元件。此外，— >考數子之最左邊的數字可以視為與首先出現參考數字的圖式為同一的。 C ^ 較佳實施例之詳細說明 “本發明針對—種供用於自一微生物細胞組成物獲得— 吳的方i· »亥方法包含使該細胞組成物的pH提升至8或以人、及自》亥、’田胞組成物分離一脂質，其中該脂質選擇性地 3有）於5%重量計或體積計之—有機溶劑。於—些具體例中，該方法進一步包含添加鹽至該細胞組成物以去乳化該、、、匕.’、成物加熱邊細胞以去乳化該細胞組成物，檀拌該細胞組成物以去乳化該細胞組成物，以及再度提升該細胞、、且成物的ρΗιχ去乳化該細胞組成物之—或更多者。本發月亦針對一種供用於自-細胞獲得-脂質的方、方法包含溶解一細胞以形成一溶解的細胞組成物，使該溶解的細胞組成物之ΡΗ提升至8或以上以去乳化該細触㈣’ &力°該簡的細胞組成物以去乳化該細胞 15 201200591 組成物，以及自該去乳化的細胞組成物分離一脂質，其中該脂質選擇性地含有少於5。/。重量計或體積計之—有機溶劑。該細胞可以為一微生物細胞或一油籽細胞。於一呰具體例中，該方法進一步包含以下的一或更多者：加熱該溶解的細胞組成物以去乳化該細胞組成物，攪拌該溶解的細胞組成物以去乳化該細胞組成物，以及再度提升該溶解的細胞組成物之pH以去乳化該細胞組成物。本發明亦針對一種供用於自一細胞組成物獲得一脂質的方法’ β玄方法包含使該細胞組成物的pH提升至§或以上來溶解該細胞級成物且去乳化該細胞組成物，添加鹽至該細胞組成物，以及自該去乳化的細胞組成物分離一脂質，其中該脂質選擇性地含有少於5%重量計或體積計之一有機溶劑。於一些具體例中，該方法進一步包含加熱該細胞組成物以去乳化該細胞組成物，攪拌該細胞組成物以去乳化該細胞組成物，以及再度提升該細胞組成物的ρΗα去乳化該細胞組成物之一或更多者。本發明針對一種供用於自一微生物細胞獲得一脂質的方法，該方法包含溶解一微生物細胞以形成一溶解的細胞組成物，添加驗至該溶解的細胞組成物以去乳化該細胞組成物’以及自該去乳化的細胞組成物分離一脂質，其中該脂質選擇性地含有少於5%重量計或體積計之一有機溶劑。於一些具體例中，該方法進一步包含以下的一或更多者：添加鹽至該溶解的細胞組成物以去乳化該細胞組成物’加熱該溶解的細胞組成物以去乳化該細胞組成物，攪 201200591 拌該溶解的細胞組成物以去乳化該細胞組成物，以及添加第二個鹼至該溶解的細胞組成物以去乳化該細胞組成物。本發明亦針對一種供用於自一細胞獲得一脂質的方法，該方法包含溶解一細胞以形成一溶解的細胞組成物，添加鹼至該溶解的細胞組成物以去乳化該細胞組成物，添加鹽至該溶解的細胞組成物以去乳化該細胞組成物，以及自該去乳化的細胞組成物分離一脂質，其中該脂質選擇性地含有少於5%重量計或體積計之一有機溶劑。該細胞可以為一微生物細胞或一油籽細胞。於一些具體例中，該方法進一步包含以下的一或更多者：加熱該溶解的細胞組成物以去乳化該細胞組成物，攪拌該溶解的細胞組成物以去乳化該細胞組成物，以及添加第二個鹼至該溶解的細胞組成物以去乳化該細胞組成物。本發明亦針對一種供用於自一細胞獲得一脂質的方法，該方法包含溶解一細胞以形成一溶解的細胞組成物，攪拌該細胞組成物以去乳化該細胞組成物，以及自該去乳化的細胞組成物分離一脂質，其中該脂質選擇性地含有少於5%重量計或體積計之一有機溶劑。本發明亦針對一種脂質，其係藉由本發明的方法之任一者而獲得的。本發明亦針對一種供用於自一細胞獲得一脂質的萃取方法，該方法包含溶解該細胞以形成一溶解的細胞組成物，使該溶解的細胞組成物接觸一第一個鹼，使該溶解的細胞組成物接觸一鹽，加熱該溶解的細胞組成物歷時5分鐘 17 201200591 至96小時使11亥容解的細胞組成物接觸第二個鹼，以及自該溶解的細胞技成物分離-脂質於1〇。(：至_。(：的溫度。本發明亦斜對—種供用於自一細胞獲得一脂質的萃取方法’該方法包含溶解該細胞以形成一溶解的細胞組成物’使該溶解的細胞組成物接觸一鹽，以及攪拌該溶解的細胞組成物歷時5分鐘至％小時來提供一經處理之溶解的細胞組成物，以及自該經處理之溶解的細胞組成物分離一脂質於l〇°C至10(TC的溫度。本發明亦針對一種供用於自一細胞獲得一脂質的萃取方法’該方法包含溶解該細胞以形成一溶解的細胞組成物，使該溶解的細胞組成物接觸一鹽，以及自該溶解的細胞組成物分離一脂質於urc至loot：的溫度。於一些具體例中，該鹼或第二個鹼具有1至丨2的pKb。於一些具體例中’該鹼或第二個鹼具有3至5的pKb。於一些具體例中’ 一種方法包含攪拌該溶解的細胞組成物歷時5分鐘至96小時，1〇分鐘至96小時，1〇分鐘至4小時，I2小時至84小時，或24小時至72小時。於一些具體例中，該方法包含藉由攪動、混合、摻合、振盪、振動，或其等之一組合來攪拌該溶解的細胞組成物。於一些具體例中，該方法包含以〇3 hp/1〇〇〇 gal至1〇 hp/1000 gal的溶解的細胞組成物來搅拌該溶解的細胞組成物。於-些具體例中’該方法包含用具有·〜―至圆 ft/min之f輪葉尖速㈣-磐器來㈣該溶解的細胞組成物。 18 201200591 於一些具體例中，溶解包含一種選自於以下的方法：機械處理、物理處理、化學處理、酵素處理，或其等之一組合。於一些具體例中，該溶解的細胞組成物係以0.1%至 20%重量計，0.5%至15%重量計，或2°/。至10%重量計之該溶解的細胞組成物的一量與一鹽接觸。於一些具體例中，該鹽係選自於以下所構成的群組：鹼金屬鹽、鹼土金屬鹽、硫酸鹽以及其等之組合。於一些具體例中，該鹽為氯化鈉。於一些具體例中，該鹽為硫酸鈉。於一些具體例中，該方法包含加熱該溶解的細胞組成物歷時5分鐘至96小時，10分鐘至4小時，12小時至84小時，或24小時至72小時。於一些具體例中，該分離包含離心。於一些具體例中，該分離包含離心於l〇°C至l〇〇°C的溫度。於一些具體例中，該方法包含在該溶解之前：清洗、離心、蒸發，或其等之一組合，包括該細胞的一肉湯。於一些具體例中，該方法提供一脂質，其具有15或更少之甲氧苯胺值。於一些具體例中，該方法提供一脂質，其包含至少50%重量計之三酸甘油Ϊ旨。於一些具體例中，該方法不添加一有機溶劑至該溶解的細胞組成物。有機溶劑包括極性溶劑、非極性溶劑、水互溶溶劑、水不互溶溶劑，以及其等之組合。於一些具體例中，該方法包含濃縮包含一細胞的一肉 19 201200591 該方法包含濃縮該溶解的細胞組成湯。於一些具體例中物。 μ。^亦針對藉由本文+所朗的—種方法來製備的曰。；—些具體例中’該脂質包含-或更多個多不飽和脂肪酸。於—些具體例中，職質具有至少脳，至少15%，至少 20%，至少 2S。/ 2- , ΟΛΛ/ 芏夕25/。，至少3〇%，至少35%，至少4〇%，至 ^45% ’或至少5()%重量計之所欲的puFA。於—些具體例中’該脂質具有至少·，至少15%，至少20〇/〇，至少25% ’ 至>、30/。，至少35%，至少4〇%，至少45%，或至少5〇%重里计之DHA，及/或至少1〇%，至少15%，或至少2〇%重量 s十之DPA n-6，及/或至少1〇%，至少15%，或至少2〇%重量計之EPA ’及/或至少1〇%，至少15%，至少2〇%，至少25%，至少30% ’至少35%，至少40%，至少45%，或至少50%重量計之ARA。於一些具體例中，該脂質具有26或更少，25 或更少，20或更少，15或更少，10或更少，5或更少，2或更少，或是1或更少之甲氧苯胺值，及/或5或更少，4.5或更少，4或更少，3.5或更少，3或更少，2.5或更少’ 2或更少’ 1.5或更少，1或更少，0.5或更少’ 0.2或更少’或是0.1或更少的過氧化物值，及/或1〇〇 PPm或更少’ 95 PPm或更少’ 90 ppm或更少，85 ppm或更少，80 ppm或更少，75 ppm或更少，70 ppm或更少，65 ppm或更少’ 60 ppm或更少’ 55 ppm 或更少，50 ppm或更少，45 ppm或更少，40 ppm或更少， 35 ppm或更少，30 ppm或更少，25 ppm或更少，20 PPm或更少，15 PPm或更少，或更少’ 5 ppm或更少，4 PPm 20 201200591 或更少，3 ppm或更少，2 ppm或更少，或是1 Ppm或更少的構含量。於一些具體例中，該脂質包含至少10%重量計的三醯甘油酯餾份，其中於該三醯甘油酯餾份内之至少12%重量計之脂肪酸為二十碳五烯酸，其中於該三醢甘油醋德份内之至少25%重量計之脂肪酸為二十二碳六烯酸，以及其中於該三醯甘油酯顧份内之少於5%重量計之脂肪酸為花生油酸。於一些具體例中，該脂質具有26或更少，25或更少’ 20或更少，15或更少，10或更少，5或更少，2或更少，或是1或更少之曱氧苯胺值，及/或5或更少，4.5或更少，4或更少，3.5或更少，3或更少，2.5或更少，2或更少，1.5或更少，1或更少，0.5或更少，0.2或更少，或是0.1或更少的過氧化物值，及/或100 ppm或更少，95 ppm或更少，90 ppm 或更少，85 ppm或更少，80 ppm或更少，75 ppm或更少， 70 ppm或更少，65 ppm或更少，60 ppm或更少，55 ppm或更少，50 ppm或更少，45 ppm或更少，40 ppm或更少’ 35 ppm 或更少，30 ppm或更少，25 ppm或更少，20 ppm或更少， 15 ppm或更少，10 ppm或更少，5 ppm或更少，4 ppm或更少，3 ppm或更少，2 ppm或更少，或是1 ppm或更少的填含量。於一些具體例中，該脂質為一粗製脂質。於一些具體例中，該脂質包含至少20%以重量計的二十碳五烯酸以及各少於5%重量計的花生油酸、二十二碳五浠酸n-6、油酸、亞麻油酸、次亞麻油酸、二十烯酸、芬子酸，以及硬脂四烯酸。於一些具體例中，該脂質具有26或 21 201200591 更少，25或更少，20或更少，15或更少，10或更少’ 5或更少，2或更少，或是1或更少之曱氧苯胺值’及/或5或更少’ 4.5或更少，4或更少，3.5或更少，3或更少，2.5或更少’ 2 或更少，1.5或更少，1或更少，0.5或更少，〇.2或更少’或是0.1或更少的過氧化物值，及/或1〇〇 ppm或更少，95 ppm 或更少，90 ppm或更少，85 ppm或更少，80 ppm或更少’ 75 ppm或更少，70 ppm或更少，65 ppm或更少’ 60 ppm或更少，55 ppm或更少，50 ppm或更少，45 ppm或更少’ 40 ppm 或更少，35 ppm或更少，30 ppm或更少，25 ppm或更少’ 20 ppm或更少，15 ppm或更少，10 ppm或更少’ 5 ppm或更少，4 ppm或更少，3 ppm或更少，2 ppm或更少’或是1 PPm 或更少的磷含量。於一些具體例中，該脂質為粗製油。本發明亦針對一種粗製的微生物脂質，其具有26或更少之甲氧苯胺值，5或更少的過氧化物值，100 PPm或更少的磷含量，以及選擇性地少於5%重量計或體積計之一有機溶劑。於一些具體例中，該粗製的微生物脂質具有26或更少，25或更少，20或更少，15或更少，10或更少’ 5或更少’ 2或更少或是1或更少之甲氧苯胺值，及/或5或更少’ 4.5或更少，4或更少，3.5或更少，3或更少，2.5或更少’ 2或更少，1.5或更少，1或更少，0.5或更少，0.2或更少，或是〇.1 或更少的過氧化物值，及/或100 ppm或更少，95 ppm或更少，90 ppm或更少，85 ppm或更少，80 ppm或更少，75 ppm 或更少，70 ppm或更少，65 ppm或更少，60 ppm或更少’ 55 ppm或更少，50 ppm或更少，45 ppm或更少，PPm或 22 201200591 更少，35 ppm或更少，30 ppm或更少，25 ppm或更少，20 ppm 或更少，15 ppm或更少，10 ppm或更少，5 ppm或更少，4 pprn 或更少’ 3 ppm或更少，2 ppm或更少，或是1 ppm或更少的磷含量。於一些具體例中，該粗製的微生物脂質具有至少 10% ’至少15% ’至少20%，至少25〇/〇，至少30%，至少35%，至少40%，至少45%，或至少50%重量計之所欲的PUFA。於一些具體例中，該粗製的微生物脂質具有至少1〇〇/0，至少15%，至少20%，至少25%，至少30°/。，至少35%，至少 40%，至少45°/。，或至少50%重量計之DHA，及/或至少10%，至少15°/。，或至少20%重量計之DPA n-6，及/或至少10%，至少15°/。，或至少20%重量計之EPA，及/或至少10%，至少 15%，至少20%，至少25%，至少30%，至少35%，至少40%，至少45%，或至少50%重量計之ARA。本發明亦針對一種經萃取的微生物脂質，其包含至少 70%重量計之三酸甘油酯餾份，其中該三酸甘油酯餾份的二十二碳六烯酸含量為至少50%重量計，其中該三酸甘油酯餾份的二十二碳五烯酸n-6含量為由至少0.5%重量計至 6%重量計，以及其中該油具有26或更少之曱氧苯胺值。於一些具體例中，該經萃取的脂質具有26或更少，25或更少， 20或更少，15或更少，10或更少，5或更少，2或更少’或是1或更少之甲氧苯胺值，及/或5或更少’ 4.5或更少’ 4或更少，3.5或更少，3或更少，2.5或更少’ 2或更少’ 1.5或更少，1或更少，0.5或更少，0.2或更少’或是0.1或更少的過氧化物值，及/或100 ppm或更少，95 ppm或更少，90 ppm 23 201200591 或更少，85 ppm或更少，80 ppm或更少，75 ppm或更少， 70 ppm或更少，65 ppm或更少，60 ppm或更少，55 ppm或更少，50 ppm或更少，45 ppm或更少，40 ppm或更少，35 ppm 或更少，30 ppm或更少，25 ppm或更少，20 ppm或更少， 15 ppm或更少，10 ppm或更少，5 ppm或更少，4 ppm或更少，3 ppm或更少，2 ppm或更少，或是1 ppm或更少的填含量。於一些具體例中，該經萃取的脂質為一粗製脂質。本發明亦針對一種經萃取的微生物脂質，其包含至少 70%重量計之三酸甘油酯餾份，其中該三酸甘油酯餾份的二十二碳六烯酸含量為至少40%重量計，其中該三酸甘油酯餾份的二十二碳五烯酸n-6含量為由至少0.5°/。重量計至 6%重量計，其中該二十二碳六烯酸對該二十二碳五烯酸n-6 之比率係大於6 : 1，以及其中該油具有26或更少之曱氧苯胺值。於一些具體例中，該經萃取的脂質具有26或更少， 25或更少，20或更少，15或更少’ 10或更少，5或更少，2 或更少，或是1或更少之曱氧苯胺值，及/或5或更少’ 4.5 或更少，4或更少，3.5或更少，3或更少’ 2.5或更少’ 2或更少，1.5或更少，1或更少，0.5或更少，0.2或更少’或是 0.1或更少的過氧化物值，及/或100 ppm或更少’ 95 PPm4 更少，90 ppm或更少，85 ppm或更少，80 ppm或更少，75 ppm 或更少，70 ppm或更少，65 ppm或更少，60 ppm或更少’ 55 ppm或更少，50 ppm或更少’ 45 ppm或更少，40 ppm或更少，35 ppm或更少’ 30 ppm或更少’ 25 ppm或更少’ 20 ppm 或更少，15 ppm或更少，10 ppm或更少’ 5 ppm或更少’ 4 ppm 24 201200591 或更少，3 ppm或更少，2 ppm或更少，或是1 ppm或更少的麟含量。於一些具體例中，該經萃取的脂質為一粗製脂質。本發明亦針對一種經萃取的微生物脂質，其包含至少大約70%重量計之三酸甘油酯餾份，其中該三酸甘油酯餾份的二十二碳六烯酸含量為至少6 〇 %重量計以及其中該油具有26或更少之甲氧苯胺值。於一些具體例中，該經萃取的脂質具有26或更少，25或更少，20或更少，15或更少， 10或更少，5或更少，2或更少，或是1或更少之甲氧苯胺值，及/或5或更少，4.5或更少，4或更少，3.5或更少，3或更少， 2.5或更少，2或更少，1.5或更少，1或更少，0.5或更少， 0.2或更少，或是0.1或更少的過氧化物值，及/或1〇〇 ppm或更少，95 ppm或更少’ 90 ppm或更少，85 ppm或更少，80 ppm 或更少，75 ppm或更少，70 ppm或更少，65 ppm或更少， 60 ppm或更少，55 ppm或更少’ 50 ppm或更少’ 45 ppm或更少，40 ppm或更少’ 35 ppm或更少，30 ppm或更少，25 ppm 或更少，20 ppm或更少，15 ppm或更少，10 ppm或更少，5 ppm或更少，4 ppm或更少，3 ppm或更少，2 ppm或更少，或是1 ppm或更少的磷含量。於一些具體例中’該經萃取的脂質為一粗製脂質。本發明亦針對一種自物種寇氏隱曱藻 (Crypthecodinium cohnii)的·一微生物所卒取的粗製脂質’其具有100 ppm或更少的磷含量。於一些具體例中，該粗製的脂質具有26或更少，25或更少，20或更少’ 15或更少，10 或更少，5或更少，2或更少’或是1或更少之甲氧苯胺值’ 25 201200591 及/或5或更少，4.5或更少，4或更少’ 3.5或更少，3或更少， 2.5或更少，2或更少，1.5或更少，1或更少，〇.5或更少’ 0.2或更少，或是〇.1或更少的過氧化物值，及/或100 ppm或更少，95 ppm或更少’ 90 ppm或更少，85 ppm或更少’ 80 ppm 或更少，75 ppm或更少，70 ppm或更少’ 65 ppm或更少’ 60 ppm或更少，55 ppm或更少，50 ppm或更少，45 ppm或更少，40 ppm或更少，35 ppm或更少，30 ppm或更少’ 25 ppm 或更少，20 ppm或更少，15 ppm或更少，10 ppm或更少，5 ppm或更少，4 ppm或更少，3 ppm或更少，2 ppm或更少，或是1 ppm或更少的填含量。本發明亦針對一種供用於獲得一脂質的方法，該方法包含精製本發明的粗製脂質。於一些具體例中，該精製係選自於以下所構成的群組：苛性精製法、去膠、酸處理、鹼處理、冷卻、加熱、漂白、去臭、脫酸，以及其等之組合0 綜述一般而言，本發明的方法不使用一有機溶劑俾以萃取或疋用別的方法分離一脂質。因而，於一些具體例中，足以萃取-脂質的-量或濃度之—有機溶劑在本發明的一方法之整個期間不被添加至—包含植物材料之細胞肉湯或含有-微生物細胞的發酵肉湯，隸添加至__細胞組成物，不被添加至—轉的細職錢，或是不被添加至-脂質°於―些具體例中’―有機溶劑可以被添加至-細胞組成物★解的細胞組成物或—去乳化的細胞組成物。 26 201200591 於此等具體例中，該有機溶劑係以少於5%，少於4%，少於 3 /〇 ’少於2% ’少於1%，少於〇 5%，少於〇. 1%，或少於〇〇5% 體積计的濃度來添加。如本文中所使用的，一"有機溶劑” 提及—包括至少一個碳原子的溶劑^如本文中所使用的，溶劑"提及疏水性的或親脂性的一劑，以及不是一脂質。如本文中所使用的，"疏水性的"提及由於大量的水而被排斥的一劑。如本文中所使用的，，，親脂性的"提及溶解於脂處的一劑。。不使用於本發明的一方法中之有機溶劑包括，但不限於，極性溶劑、非極性溶劑、水互溶溶劑、水不互 /谷/容劑，以及其等之組合。有機溶劑之非限制性實例包括經取代的和未經取代的CVC8烷基(例如，己烷與類似物）、 Cs-C!2環烷基、CrCn稀、（^-(：8醇(例如，異丙醇與類似物）、 C1-C8|^、c4-C8醚、Crc8g|、C6-Ci2芳基、Ci_C8醯胺、C5-C12 雜^'基，以及其專之組合。如本文中所定義的一有機溶劑可以選擇性地添加至一溶解的細胞組成物，舉例而言，為 —鹼及/或一鹽的一組份供用於接觸該溶解的細胞組成物。然而，於此等具體例中，該有機溶劑係以如此的濃度存在以致該脂質不會實質被該溶劑（亦即，以少於5%，少於 4〇/〇，少於3〇/〇，少於2%，少於1%，少於〇 5%，少於〇 1%，或少於0观體積計或重量計的濃度）而自該細胞組成物、該溶解的細胞組成物，或該去朝彳卜认Α 扎化的細胞組成物萃取出。於一些具體例中，本發明的—方 + A t Λ 去不包括（例如，用水） >月洗’或是該方法降低一溶解的細胎 ,, ,,, 匕級成物或一去乳化的細胞組成物之清洗的數量。"清浊” ^及用，例如，水或緩 27 201200591 衝液稀釋-組成物以及，例如，藉由離^來移除水或缓衝液的-種方法。清洗-細胞组成物會減少自一細胞獲得的月日質之王面的產量。於本發明中，清洗可以減少i次、2 次、3次或更多次。定義如本文中所使用的’，，脂質"或"油"提及一或更多個脂肪酸（含括游離脂肪酸和脂肪酸的酯）、磷脂質、三醯甘油酯 (triacylglycer〇l)(亦即三酸甘油酯（trigiycerides))、二醯甘油醋、單酿甘油醋、溶血碟月旨質(lys〇ph〇sph〇Hpids)、皂類、 -脂' it ' ID醇與固醇酿、類胡蘿葡素、葉黃素、碳水化合物’以及其他熟悉此藝者所知道的脂質。脂質包括極性脂質及中性脂質。如本文中所使用的，，•極性脂質"提及含有一極性基團且更容易地溶解於極性溶劑内之脂質。極性脂質包括磷脂質。如本文中所使用的，"磷脂質”提及具有一磷酸基團之脂質。如本文中所使用的，"中性脂質"提及不含有極性區域且更容易溶解於非極性溶劑内之脂質。中性脂質包括三醯甘油酯(TAG)。脂肪酸係根據碳鏈的長度及飽和特徵來分類。脂肪酸係根據鏈中的碳長度被稱為短鏈、中鏈或長鏈脂肪酸。當碳原子之間沒有雙鍵存在時，脂肪酸被稱為飽和脂肪酸，以及當雙鍵存在時脂肪酸被稱為不飽和脂肪酸。當只有一個雙鍵存在時，不飽和長鏈脂肪酸為單不飽和的’以及當多於一個雙鍵#在時，不飽和長键脂肪酸為多不飽和的。 28 201200591 存在於脂質中之脂肪酸可以具有4個至28個碳原子。於一些具體例中，一脂質包含一或更多個多不飽和脂肪酸。多不飽和脂肪酸(PUFAs)係根據離脂肪酸的甲基端之第一個雙鍵的位置之來分類：ω-3(η-3)脂肪酸含有第一個雙鍵在第三個碳上，而ω-6(η-6)脂肪酸含有第一個雙鍵在第六個碳上。舉例而言，二十二碳六烯酸("DHA")為具有22個碳的鏈長及6個雙鍵的ω-3長鏈多不飽和脂肪酸(LC PUFA)，通常稱為"22:6 η-3"。就此申請案的目的而言，長鏈多不飽和脂肪酸(LC-PUFAs)係定義為18個及更多個碳鏈長度之脂肪酸，以及較佳為20個或更多個碳鏈長度，含有3個或更多個雙鍵。ω-6系列之LC-PUFAs包括，但不限於，二·升· r亞麻油酸(C20:3n-6)、花生油酸(C20:4n-6) ("ARA")、二十二碳四烯酸(docosatetraenoic acid)或二十二碳四稀酸(adrenic acid) (C22:4n-6)，以及二十二碳五烯酸(C22:5n-6) ("DPAn-6")。 ω-3系列之LC-PUFAs包括，但不限於，二十碳三烯酸 (C20:3n-3)、花生油酸(C20:4n-3)、二十碳五烯酸(C20:5n-3) ("EPA")、二十二碳五烯酸(C22:5n-3)，以及二十二碳六烯酸(C22:6n-3)。LC-PUFA亦包括帶有大於22個碳以及4或更多個雙鍵之脂肪酸，包括，但不限於，C24:6(n-3)與 C28:8(n-3)。術語"脂肪酸"、"多不飽和脂肪酸"，以及"PUFA"不只包括游離脂肪酸形式’還有其他的形式，例如三酿甘油酉旨 (TAG)形式、磷脂質(PL)形式以及其他酯化的形式。如本文中所使用的’術語"酯"及"酯化的"係提及PUFA分子之叛酸 29 201200591 基團中的氫係經另一個取代基置換。典型的酯類係熟悉必匕藝者所知道的，其之論述係提供於Higuchi，T等人， Pro-drugs as Novel Delivery Systems, Vol. 14, A.C.S. Symposium Series, Bioreversible Carriers in Drug Design, Edward B. Roche ed., Amer. Pharma. Assoc., Pergamon Press [\9们），以反 Protective Groups in Organic Chemistry,201200591 VI. INSTRUCTIONS: L technical inventions employed by the invention] j Cross-reference to the related patent application. This application claims the United States application No. 61/350,363, dated August 31, 2010 The benefit of U.S. Application Serial No. 61/378, 923, and U.S. Application Serial No. 61/452, 721, filed on March 15, 2011, is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION The present invention relates to a method for obtaining a lipid from a cell by isolating the cell, raising the ρ Η of the cell and/or contacting the cell with a salt, and isolating the lipid. The invention also targets lipids prepared by the methods of the invention. The invention is also directed to microbial lipids having specific oxime values, peroxide values, and/or phosphorus levels. C Prior Art 3 Background of the Invention A typical method for obtaining lipids from a microbial cell, such as a polyunsaturated fatty acid, involves the growth of a microorganism capable of producing a desired lipid in a fermenter, a cuvette or a bioreactor, A fermentation broth comprising a microbial cell biomass, the microbial cell biomass is dried, and the lipid is separated by solvent extraction. The step of separating may include diluting a fermentation broth with water, centrifuging the diluted broth, dissolving the microbial cells, and adding a water-immiscible solvent (the lipid is soluble in the 'for example' From the dissolved cells, the lipids in the cells 201200591 are extracted from the dissolved cells. The removal from a microbial cell - ρ _ mechanical force (for example, homogenization), the extraction method at the enzyme is dissolved by using a machine rupture - cells in 1 Fermentation treatment to make the cell wall solvent (for example, isopropanol) self-contained:: The enamel can be used - the group of materials produced by organic; the mass, and the mechanical separation of water and the alcohol must be formed from the heart The material is removed. See, for example, ' _ ^ f waste stream 〇 1 / fine. u open case _ (10) / 76385 and the gift, however, using the above method - the production of the industrial scale of the lipid needs to be large (four) Volatile and flammable organic solvent, the use of the organic solvent in the method of extraction 1 to produce an explosion-proof lipid recovery system becomes necessary to increase the cost of lipid recovery. The use of organic solvents in the process of extracting lipids from cells produces an organic solvent waste stream that requires a complete solvent recovery system or a suitable disposal method that further increases the overall production cost of lipid extraction. For example, Strict restrictions on the emission of volatile organic compounds (4) require more manpower to increase the cost of equipment and other equipment. There is a way to obtain lipids from cells without using - money solvents. Several branches are used to separate a lipid from a cell without using an organic solvent. For example, (4), U.S. Patent No. 6,75, discloses an aqueous cleaning method whereby the emulsion is treated with an aqueous cleaning solution. Washing until a substantially non-emulsifying lipid is obtained. In no case, in some specific examples, 201200591, the method requires multiple cleaning steps, which require a lot of cost and time. U.S. Patent No. 7,431,952 discloses a method, This centrifuges the lysed cells to remove cell wall debris and extracts and purifies the oil. However, this method provides the need for extensive One step is to purify one of the crude oils. Thus, what is needed is a method for extracting a lipid from a cell without the use of a volatile solvent, and it can be performed using readily available equipment and a minimum number of steps to provide a high degree of purity. SUMMARY OF THE INVENTION The present invention is directed to a method for obtaining a lipid from a microbial cell composition, the method comprising raising the pH of the cell composition to 8 or more, and from the cell composition Separating a lipid, wherein the lipid selectively contains less than 5% by weight or one volume of one organic solvent. In some embodiments, the pH increase dissolves the cell grade. In some embodiments, the pH Elevating the emulsified cell composition. In some specific examples, the method comprises adding a salt to the cell composition to de-emulsifie the cell composition. In some embodiments, the added salt is carried out after the pH increase. In some embodiments, the method further comprises heating the dissolved cell composition to emulsify the cell composition. In some specific examples, the heating system is performed after the pH is raised. In some embodiments, the method further comprises again increasing the pH of the cell fraction to de-emulsifie the detail. In some of the pure cases, the re-raised pH is performed after the addition of the salt or the heating. 201200591 Also directed to a method for obtaining a lipid from a cell, the method comprises: dissolving - cells to form a dissolved cell composition, raising the pH of the decomposed cells 1 and the composition to 8 or more to de-emulsifie the The fine mash adds salt to the dissolved cell composition to de-emulsifie the cell. And the product' and the cell composition isolated from the emulsified lipid-lipid, wherein the lipid selectively contains less than 5% by weight or volume of the organic solvent. The present invention also relates to a method for obtaining a lipid from a cell composition, the method comprising: raising a p Η of the cell composition to 8 or more to dissolve the cell composition and de-emulsification the cell composition, adding Salt is added to the cell composition, and a lipid is isolated from the emulsified cell composition, wherein the lipid selectively contains less than 5% by weight or one volume of one organic solvent. The present invention is also directed to a method for obtaining a lipid from a cell, the method comprising dissolving a cell to form a dissolved cell composition, mixing the cell composition to de-emulsifie the cell composition, and from there The emulsified cell composition separates a lipid 'where the lipid selectively contains less than 5% by weight or one volume of one organic solvent. In some embodiments, the method further comprises heating the dissolved cell composition to de-emulsifie the cell. In some embodiments, the heating is performed after the addition of the salt. In some odors, the method further comprises agitating the dissolved cell composition to de-emulsifie the cell. In some embodiments, the infusion system lasts from 5 minutes to 96 hours. 201200591 In some embodiments, the agitating comprises agitating the cell composition with an impeller having a tip rate of from 350 centimeters per second to 9 centimeters per second. In some embodiments, the method further comprises increasing the pH of the solubilized cell composition to de-emulsifie the cell composition. In some embodiments, the pH of the solubilized cell composition is raised to de-emulsifie the cell composition comprising the addition of a base. In some embodiments, the second base is added after the addition of the salt or the heating. In some embodiments, the heating system lasts from 1 minute to 96 hours. In some embodiments, the cellular composition is heated to a temperature of 6 Torr to 1 〇〇C. In some embodiments, the cell composition is heated to a temperature of from 9 ° C to 100 ° (in some embodiments, the pH rise of the shai includes an add-on test. In some specific examples, the base has 1 pKb to 12. In some embodiments, the separation of a lipid occurs at a temperature of from 1 Torr to 1 Torr. In some embodiments, the method comprises agitation, mixing, blending, shaking, Vibration, or a combination thereof, to agitate the dissolved cellular composition. In some embodiments, the method comprises dissolving a cellular composition of 〇J hp/1 〇〇〇 gal to 1 〇 hp/1000 gal. The dissolved cellular composition is agitated. In some embodiments, the method comprises agitating the dissolved cellular composition with a stirrer having an impeller tip rate of 2卯(7)岀 to ιοοο ft/min. In the example, the dissolution of shai includes mechanical treatment, physical treatment, chemical treatment, enzyme treatment, or the like. In some specific examples, the mechanical treatment is homogenization in 201200591. In some specific examples, the salt is 0. 1. /. To 20. /. The amount of the dissolved cellular composition is added by weight. In some specific examples, the salt is 0 · 5 /. An amount of the dissolved cell composition to 15% by weight is added to the cold-decomposed cell composition. In some embodiments, the salt is between 2% and 10%. An amount of the dissolved cell composition is added to the dissolved cell composition by weight. In some embodiments, the salt is selected from the group consisting of alkali metal salts, alkaline earth metal salts, sulfates, and combinations thereof. In some specific examples, the delta salt is a gasification nano. In some embodiments, the salt is sodium sulphate. In some embodiments, the separation comprises centrifugation. In some embodiments, the separation comprises centrifuging at a temperature of from 30 °C to 90 °C. In some embodiments, the method provides a lipid comprising at least 50% by weight of triglyceride. In some embodiments, the method provides a lipid having 26 or less, 25 or less '20 or less, 15 or less, 1 or less, 5 or less, 2 or less 'Or 1 or less methoxyaniline value. In some embodiments, the method provides a lipid having 5 or less, 4. 5 or less ' 4 or less, 3. 5 or less, 3 or less, 2. 5 or less, 2 or less, 丨. 5 or less' 1 or less, 0. 5 or less, hehe. 2 or less, or a peroxide value of 1 or less. In some embodiments, the method provides a lipid having 1 〇〇 ppm or less, 95 ppm or less, 90 ppm or less, 85 ρρηι or less 201200591, 80 ppm or less, 75 ppm Or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less Less, 30 ppm or less, 25 ppm or less, 20 ppm or less '15 ppm or less' 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less. In some embodiments, the method provides a lipid having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50 % by weight of the desired polyunsaturated fatty acid (PUFA). In some embodiments, the method provides a lipid having at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50 % by weight of docosahexaenoic acid ("DHAn", and / or at least 10%, at least 15%, or at least 20% by weight of docosapentaenoic acid ("DPAn-6") And/or at least 10%, at least 15%, or at least 20% by weight of eicosapentaenoic acid ("EPA"), and/or at least 10%, at least 15%, at least 20%, at least 25 %, at least 30%, at least 35%, at least 40°/. , at least 45%, or at least 50% by weight of peanut oleic acid ("ARA"). In some embodiments, the cell is a microbial cell. In some embodiments, the method comprises concentrating a fermentation broth containing the microbial cells. In some embodiments, the cell is an oilseed. In some embodiments, the oilseed is selected from the group consisting of: sunflower seeds, canola seeds, rapeseed, linseed, castor oil seeds, and hu Coriander seeds, calendula seeds, and their 201200591 genetic modification variants. In some embodiments, the method comprises washing the cell or cell composition. In some embodiments, the method comprises sterilizing the cell or cell composition at a low temperature. In some embodiments, the method comprises concentrating the dissolved cellular composition. In some embodiments, the method comprises refining the lipid. In some embodiments, the refining is selected from the group consisting of caustic refining, degumming, acid treatment, treatment, cooling, heating, bleaching, deodorization, deacidification, and combinations thereof. In some embodiments, the method comprises harvesting the lipid, wherein the harvesting comprises pumping the lipid without agitation. The invention is also directed to a lipid obtained by any of the methods of the invention. In some embodiments, the lipid comprises one or more polyunsaturated fatty acids. In some embodiments, the lipid has at least 10°/. At least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of the desired PUFA. In some embodiments, the lipid has at least 10%, at least 15°/. At least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of DHA, and/or at least 10%, at least 15%, or at least 20% by weight Preferably, DPAn-6, and/or at least 10%, at least 15%, or at least 20% by weight of EPA, and/or at least 10%, at least 15°/. , at least 20%, at least 25%, 10 201200591 at least 30% 'at least 35%, at least 4%, at least peach, or at least ARA of weight. In some embodiments, the lipid has a total aroma intensity of 3 or less. In some embodiments, the lipid has a total aromatic intensity of 2 or less. In some embodiments, the lipid comprises at least 1% by weight of triterpene glycerol aliquots of at least 12 Å in the triterpene glyceride fraction. The fatty acid by weight is eicosapentaenoic acid, wherein at least 25% by weight of the fatty acid in the triterpene glyceride fraction is docosahexaenoic acid, and wherein the triterpene glyceride fraction is contained therein Less than 5% by weight of the fatty acid is peanut oleic acid. In some embodiments, the lipid comprises at least 20% by weight of eicosapentaenoic acid and less than 5% by weight of peanut oleic acid, docosapentaenoic acid n-6, oleic acid, linseed oil Acid, linoleic acid, benzoic acid, fenic acid, and stearic acid. In some embodiments, the lipid has 26 or less, 25 or less '20 or less, 15 or less, 10 or less, 5 or less '2 or less' or 1 or less The methoxyaniline value, and / or 5 or less ' 4. 5 or less ' 4 or less, 3. 5 or less, 3 or less, 2. 5 or less, 2 or less, 1. 5 or less, 1 or less '〇·5 or less, 〇·2 or less' or 0. 1 or less peroxide value, and / or 1 〇〇 PPm or less, 95 ppm or less ' 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less '70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ΡΡ 111 or less, 50 ppm or less, 45 ppm or less ' 40 ppm or less, % ppm or less, 30 Ppm or less, 25 ppm or less, 20 ppm or less' 11 201200591 15 Ppm or less ' 10 PPm or less, 5 ppm or less, 4 ppm or less ' 3 ppm or less, 2 Ppm or less, or a phosphorus content of 1 ppm or less. In some embodiments, the lipid is a crude lipid. In some embodiments, the crude lipid selectively has less than 5% by weight or one organic solvent. The present invention is also directed to a crude microbial lipid having a lactide value of 26 or less, a peroxide value of 5 or less, a phosphorus content of 1 〇〇ppm or less, and optionally less than 5% by weight or one by volume of an organic solvent. In some embodiments, the crude microbial lipid has 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 Or less methoxyaniline value, and / or 5 or less, 4. 5 or less, 4 or less, 3. 5 or less, 3 or less, 2. 5 or less, 2 or less, 1. 5 or less, 1 or less, 0. 5 or less’ 0. 2 or less' or 0. 1 or less peroxide value, and / or 1 〇〇 PPm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less ' 75 ppm or less , 70 ppm or less, 65 ppm or less '60 ppm or less' 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less' 30 Ppm or less, 25 ppm or less ' 20 ppm or less, 15 ppm or less, 1 〇 ppm or less '5 ppm or less '4 ppm or less, 3 ppm or less, 2 ppm Or less, or a phosphorus content of 1 PPm or less. In some embodiments, the crude microbial lipid has at least 12 201200591 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% The weight of the desired PUFA. In some embodiments, the crude microbial lipid has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight. DHA, and/or at least 10%, at least 15%, or at least 20% by weight of DPA n-6, and/or at least 10%, at least 15%, or at least 20% by weight of EPA, and/or at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight of ARA. The present invention is also directed to an extracted microbial lipid comprising at least 70% by weight of triacetin, wherein the triglyceride S has a docosahexaenoic acid content of at least 50% by weight. , wherein the triglyceride fraction of docosapentaenoic acid n-6 is at least 0. 5% by weight to 6% by weight, and wherein the oil has a methoxyaniline value of 26 or less. The present invention is also directed to an extracted microbial lipid comprising at least 70% by weight of a triglyceride fraction, wherein the triglyceride fraction has a docosahexaenoic acid content of at least 40% by weight, Wherein the triglyceride fraction of docosapentaenoic acid n-6 is at least 0. 5% by weight to 6% by weight, wherein the ratio of the docosahexaenoic acid to the docosapentaenoic acid n-6 is greater than 6 ··1, and wherein the oil has 26 or less Anoxaniline value. The present invention is also directed to an extracted microbial lipid comprising at least about 70% by weight of a triglyceride fraction, wherein the triglyceride fraction has a docosahexaenoic acid content of at least 60% by weight. And wherein the oil 13 201200591 has a oxoaniline value of 26 or less. In some embodiments, the extracted lipid has 26 or less '25 or less, 20 or less, 15 or less, 1 or less, 5 or less, 2 or less, or 1 or less methoxyaniline value 'and / or 5 or less' 4. 5 or less, 4 or less, 3.5 or less, 3 or less, 2. 5 or less ' 2 or less' 1. 5 or less, 1 or less, 0. 5 or less '〇·2 or less' or 0. 1 or less peroxide value, and / or 100 ppm or less, 95 ppm or less '90 ppm or less, 85 ppm or less, 80 ppm or less '75 ppm or less, 70 Ppm or less, 65 ppm or less '60 ppm or less' 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less '35 ppm or less, 30 ppm or Less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less , or a phosphorus content of 1 ppm or less. In some embodiments, the extracted microbial lipid is a crude fat or a crude oil. In some embodiments, the crude lipid optionally has less than 5°/. One of an organic solvent by weight or volume. The invention is also directed to a method for obtaining a lipid comprising refining the crude lipid of the invention. In some embodiments, the refining is selected from the group consisting of caustic refining, degumming, acid treatment, alkali treatment, cooling, heating, bleaching, deodorization, deacidification, and combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and constitute a part of this specification, are in the form of the description of the present invention, and the description of the present invention and the detailed description of the present invention are used to explain the skill of the present invention. A flow chart illustrating the outline of the method of the present invention is provided and used in the context of Figure 14; Figure 5 is a diagram providing electron paramagnetic resonance of various cellular compositions dissolved in pH at time ( EpR). See the description of the drawings. In the drawings, a similar reference number refers to a complete phase or a functionally similar component. In addition, the leftmost digit of the -> test can be considered to be the same as the pattern in which the reference number first appears. C ^ DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT "The present invention is directed to a method for obtaining from a microbial cell composition - Wu's method of raising the pH of the cell composition to 8 or by human, and "Hai, the field cell composition separates a lipid, wherein the lipid is selectively 3) in an amount of 5% by weight or volume - an organic solvent. In some embodiments, the method further comprises adding a salt to the cell The composition is used to de-emulsifie the 、, 匕. ', the object heats the cells to de-emulsifie the cell composition, mixes the cell composition to de-emulsifie the cell composition, and re-emphasizes the cell, and the ρΗιχ of the product to emulsify the cell composition—or More. The present invention also relates to a method for obtaining a lipid from a self-cell, which comprises dissolving a cell to form a dissolved cell composition, raising the enthalpy of the dissolved cell composition to 8 or more to de-emulsifie the fine Touching (4) ' & force the cell composition to de-emulsifie the cell 15 201200591 composition, and separating a lipid from the de-emulsified cell composition, wherein the lipid selectively contains less than 5. /. Weight meter or volume meter - organic solvent. The cell can be a microbial cell or an oil seed cell. In a specific embodiment, the method further comprises one or more of the following: heating the dissolved cell composition to de-emulsifie the cell composition, agitating the dissolved cell composition to de-emulsifie the cell composition, and The pH of the dissolved cell composition is again increased to de-emulsifie the cell composition. The present invention is also directed to a method for obtaining a lipid from a cell composition. The β-synthesis method comprises raising the pH of the cell composition to § or above to dissolve the cell-level product and de-emulsification the cell composition, adding Salt is added to the cell composition, and a lipid is isolated from the emulsified cell composition, wherein the lipid selectively contains less than 5% by weight or one volume of one organic solvent. In some embodiments, the method further comprises heating the cell composition to de-emulsifie the cell composition, agitating the cell composition to de-emulsifie the cell composition, and again increasing the cell composition to de-emulsifie the cell composition. One or more of them. The present invention is directed to a method for obtaining a lipid from a microbial cell, the method comprising dissolving a microbial cell to form a dissolved cell composition, adding the dissolved cell composition to de-emulsification the cell composition' and A lipid is isolated from the de-emulsified cell composition, wherein the lipid selectively contains less than 5% by weight or one volume of one organic solvent. In some embodiments, the method further comprises one or more of the following: adding a salt to the dissolved cell composition to de-emulsifie the cell composition to 'heat the dissolved cell composition to de-emulsifie the cell composition, The dissolved cell composition is stirred to de-emulsifie the cell composition, and a second base is added to the dissolved cell composition to de-emulsifie the cell composition. The present invention is also directed to a method for obtaining a lipid from a cell, the method comprising dissolving a cell to form a dissolved cell composition, adding a base to the dissolved cell composition to deemulsifie the cell composition, and adding a salt The dissolved cellular composition is used to de-emulsifie the cellular composition, and a lipid is isolated from the emulsified cellular composition, wherein the lipid selectively contains less than 5% by weight or one volume of one organic solvent. The cell can be a microbial cell or an oil seed cell. In some embodiments, the method further comprises one or more of: heating the dissolved cell composition to de-emulsifie the cell composition, agitating the dissolved cell composition to de-emulsifie the cell composition, and adding A second base is added to the dissolved cell composition to deemulsifie the cell composition. The invention is also directed to a method for obtaining a lipid from a cell, the method comprising dissolving a cell to form a dissolved cell composition, agitating the cell composition to de-emulsifie the cell composition, and de-emulsification therefrom The cell composition separates a lipid wherein the lipid selectively contains less than 5% by weight or one volume of one organic solvent. The invention is also directed to a lipid obtained by any of the methods of the invention. The present invention is also directed to an extraction method for obtaining a lipid from a cell, the method comprising dissolving the cell to form a dissolved cell composition, contacting the dissolved cell composition with a first base to cause the dissolution The cell composition is contacted with a salt, and the dissolved cell composition is heated for 5 minutes 17 201200591 to 96 hours to allow the 11H solution to contact the second base, and to separate from the dissolved cell technology - lipid 1〇. (: to _. (the temperature of the invention. The present invention is also obliquely used for the extraction method for obtaining a lipid from a cell'. The method comprises dissolving the cell to form a dissolved cell composition' to make the dissolved cell Contacting a salt, and stirring the dissolved cell composition for 5 minutes to % hours to provide a treated dissolved cell composition, and separating a lipid from the treated dissolved cell composition at 10 ° C to 10 (temperature of TC. The present invention is also directed to an extraction method for obtaining a lipid from a cell' which comprises dissolving the cell to form a dissolved cell composition, contacting the dissolved cell composition with a salt, and A temperature at which the lipid is separated from urc to loot: from the dissolved cell composition. In some embodiments, the base or the second base has a pKb of from 1 to 。 2. In some embodiments, the base or the second The base has a pKb of 3 to 5. In some embodiments, one method comprises agitating the solubilized cell composition for 5 minutes to 96 hours, 1 minute to 96 hours, 1 minute to 4 hours, I2 The time is up to 84 hours, or 24 hours to 72 hours. In some embodiments, the method comprises agitating the dissolved cell composition by agitation, mixing, blending, shaking, shaking, or a combination thereof. In some embodiments, the method comprises agitating the dissolved cell composition with a dissolved cell composition of 〇3 hp/1 〇〇〇 gal to 1 〇 hp/1000 gal. In some specific examples, the method comprises (4) The dissolved cell composition is obtained by using a f-tip tip (four)-rhodium with a ~f to a circle ft/min. 18 201200591 In some specific examples, the dissolution comprises a method selected from the group consisting of: mechanical treatment, Physical treatment, chemical treatment, enzyme treatment, or a combination thereof. In some specific examples, the dissolved cell composition is 0. 1% to 20% by weight, 0. 5% to 15% by weight, or 2°/. An amount of the dissolved cellular composition to 10% by weight is contacted with a salt. In some embodiments, the salt is selected from the group consisting of alkali metal salts, alkaline earth metal salts, sulfates, and combinations thereof. In some embodiments, the salt is sodium chloride. In some embodiments, the salt is sodium sulphate. In some embodiments, the method comprises heating the solubilized cell composition for from 5 minutes to 96 hours, from 10 minutes to 4 hours, from 12 hours to 84 hours, or from 24 hours to 72 hours. In some embodiments, the separation comprises centrifugation. In some embodiments, the separation comprises centrifuging at a temperature between 10 ° C and 10 ° C. In some embodiments, the method comprises prior to the dissolving: washing, centrifuging, evaporating, or a combination thereof, including a broth of the cells. In some embodiments, the method provides a lipid having a methoxyaniline value of 15 or less. In some embodiments, the method provides a lipid comprising at least 50% by weight of triglyceride. In some embodiments, the method does not add an organic solvent to the dissolved cellular composition. The organic solvent includes a polar solvent, a non-polar solvent, a water-miscible solvent, a water-immiscible solvent, and combinations thereof. In some embodiments, the method comprises concentrating a meat comprising a cell. 19 201200591 The method comprises concentrating the dissolved cell composition soup. In some specific examples. μ. ^ Also for the 制备 prepared by the method of this article + lang. - In some specific examples, the lipid contains - or more polyunsaturated fatty acids. In some specific examples, the occupationality has at least 脳, at least 15%, at least 20%, and at least 2S. / 2- , ΟΛΛ / 芏 25 25/. , at least 3 %, at least 35%, at least 4 %, to ^45% ' or at least 5 ()% by weight of the desired puFA. In some embodiments, the lipid has at least, at least 15%, at least 20 Å/〇, at least 25% Å to > 30 。. , at least 35%, at least 4%, at least 45%, or at least 5% by weight of DHA, and/or at least 1%, at least 15%, or at least 2% by weight s ten DPA n-6, And/or at least 1%, at least 15%, or at least 2% by weight of EPA 'and/or at least 1%, at least 15%, at least 2%, at least 25%, at least 30% 'at least 35% At least 40%, at least 45%, or at least 50% by weight of ARA. In some embodiments, the lipid has 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less. The methoxyaniline value, and / or 5 or less, 4. 5 or less, 4 or less, 3. 5 or less, 3 or less, 2. 5 or less ' 2 or less' 1. 5 or less, 1 or less, 0. 5 or less’ 0. 2 or less' or 0. 1 or less peroxide value, and / or 1 〇〇 PPm or less '95 PPm or less' 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less , 70 ppm or less, 65 ppm or less '60 ppm or less' 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 Ppm or less, 25 ppm or less, 20 PPm or less, 15 PPm or less, or less '5 ppm or less, 4 PPm 20 201200591 or less, 3 ppm or less, 2 ppm or Less, or 1 Ppm or less. In some embodiments, the lipid comprises at least 10% by weight of a triterpene glyceride fraction, wherein at least 12% by weight of the fatty acid in the triterpene glyceride fraction is eicosapentaenoic acid, wherein At least 25% by weight of the fatty acid in the triterpene glycerol ketone portion is docosahexaenoic acid, and wherein less than 5% by weight of the fatty acid in the triterpene glyceride moiety is peanut oleic acid. In some embodiments, the lipid has 26 or less, 25 or less '20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less. The aniline value, and / or 5 or less, 4. 5 or less, 4 or less, 3. 5 or less, 3 or less, 2. 5 or less, 2 or less, 1. 5 or less, 1 or less, 0. 5 or less, 0. 2 or less, or 0. 1 or less peroxide value, and / or 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 Ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less '35 ppm or less, 30 ppm or Less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less , or a fill content of 1 ppm or less. In some embodiments, the lipid is a crude lipid. In some embodiments, the lipid comprises at least 20% by weight of eicosapentaenoic acid and less than 5% by weight of peanut oleic acid, docosapentaenoic acid n-6, oleic acid, linseed oil Acid, linoleic acid, eicosenoic acid, fenic acid, and stearic acid. In some embodiments, the lipid has 26 or 21 201200591 less, 25 or less, 20 or less, 15 or less, 10 or less '5 or less, 2 or less, or 1 or Less oxoaniline value 'and / or 5 or less' 4. 5 or less, 4 or less, 3. 5 or less, 3 or less, 2. 5 or less' 2 or less, 1. 5 or less, 1 or less, 0. 5 or less, hehe. 2 or less' or 0. 1 or less peroxide value, and / or 1 〇〇 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less '75 ppm or less , 70 ppm or less, 65 ppm or less '60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less '40 ppm or less, 35 ppm or less, 30 Ppm or less, 25 ppm or less '20 ppm or less, 15 ppm or less, 10 ppm or less' 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or Less 'or 1 ppm or less of phosphorus. In some embodiments, the lipid is a crude oil. The invention is also directed to a crude microbial lipid having a methoxyaniline value of 26 or less, a peroxide value of 5 or less, a phosphorus content of 100 PPm or less, and optionally less than 5% by weight. One of the organic solvents. In some embodiments, the crude microbial lipid has 26 or less, 25 or less, 20 or less, 15 or less, 10 or less '5 or less' 2 or less or 1 or Fewer methoxyaniline values, and / or 5 or less' 4. 5 or less, 4 or less, 3. 5 or less, 3 or less, 2. 5 or less ' 2 or less, 1. 5 or less, 1 or less, 0. 5 or less, 0. 2 or less, or 〇. 1 or less peroxide value, and / or 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 Ppm or less, 65 ppm or less, 60 ppm or less '55 ppm or less, 50 ppm or less, 45 ppm or less, PPm or 22 201200591 less, 35 ppm or less, 30 ppm Or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 pprn or less '3 ppm or less, 2 ppm or less Less, or a phosphorus content of 1 ppm or less. In some embodiments, the crude microbial lipid has at least 10% 'at least 15%' at least 20%, at least 25 〇/〇, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% The weight of the desired PUFA. In some embodiments, the crude microbial lipid has at least 1 〇〇/0, at least 15%, at least 20%, at least 25%, at least 30°/. , at least 35%, at least 40%, at least 45°/. , or at least 50% by weight of DHA, and / or at least 10%, at least 15 ° /. , or at least 20% by weight of DPA n-6, and / or at least 10%, at least 15 ° /. , or at least 20% by weight of EPA, and/or at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight ARA. The present invention is also directed to an extracted microbial lipid comprising at least 70% by weight of a triglyceride fraction, wherein the triglyceride fraction has a docosahexaenoic acid content of at least 50% by weight, Wherein the triglyceride fraction of docosapentaenoic acid n-6 is at least 0. 5% by weight to 6% by weight, and wherein the oil has a oxoaniline value of 26 or less. In some embodiments, the extracted lipid has 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less 'or 1 Or less methoxyaniline value, and / or 5 or less ' 4. 5 or less ' 4 or less, 3. 5 or less, 3 or less, 2. 5 or less ' 2 or less' 1. 5 or less, 1 or less, 0. 5 or less, 0. 2 or less' or 0. 1 or less peroxide value, and / or 100 ppm or less, 95 ppm or less, 90 ppm 23 201200591 or less, 85 ppm or less, 80 ppm or less, 75 ppm or less , 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 Ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or Less, or a fill level of 1 ppm or less. In some embodiments, the extracted lipid is a crude lipid. The present invention is also directed to an extracted microbial lipid comprising at least 70% by weight of a triglyceride fraction, wherein the triglyceride fraction has a docosahexaenoic acid content of at least 40% by weight, Wherein the triglyceride fraction of docosapentaenoic acid n-6 is at least 0. 5°/. To a weight of 6% by weight, wherein the ratio of the docosahexaenoic acid to the docosapentaenoic acid n-6 is greater than 6:1, and wherein the oil has 26 or less of anthranilamide value. In some embodiments, the extracted lipid has 26 or less, 25 or less, 20 or less, 15 or less '10 or less, 5 or less, 2 or less, or 1 Or less of the aniline value, and / or 5 or less ' 4. 5 or less, 4 or less, 3. 5 or less, 3 or less' 2. 5 or less ' 2 or less, 1. 5 or less, 1 or less, 0. 5 or less, 0. 2 or less' or 0. 1 or less peroxide value, and / or 100 ppm or less '95 PPm4 less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm Or less, 65 ppm or less, 60 ppm or less '55 ppm or less, 50 ppm or less' 45 ppm or less, 40 ppm or less, 35 ppm or less '30 ppm or less Less '25 ppm or less' 20 ppm or less, 15 ppm or less, 10 ppm or less '5 ppm or less' 4 ppm 24 201200591 or less, 3 ppm or less, 2 ppm or less Less, or a 1 ppm or less lin content. In some embodiments, the extracted lipid is a crude lipid. The present invention is also directed to an extracted microbial lipid comprising at least about 70% by weight of a triglyceride fraction, wherein the triglyceride fraction has a docosahexaenoic acid content of at least 6% by weight. And wherein the oil has a methoxyaniline value of 26 or less. In some embodiments, the extracted lipid has 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 Or less methoxyaniline value, and / or 5 or less, 4. 5 or less, 4 or less, 3. 5 or less, 3 or less, 2. 5 or less, 2 or less, 1. 5 or less, 1 or less, 0. 5 or less, 0. 2 or less, or 0. 1 or less peroxide value, and / or 1 〇〇 ppm or less, 95 ppm or less ' 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less , 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less '50 ppm or less' 45 ppm or less, 40 ppm or less '35 ppm or less, 30 Ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or Less, or a phosphorus content of 1 ppm or less. In some embodiments, the extracted lipid is a crude lipid. The present invention is also directed to a crude lipid which has been drawn from a microorganism of the species Crypthecodinium cohnii, which has a phosphorus content of 100 ppm or less. In some embodiments, the crude lipid has 26 or less, 25 or less, 20 or less '15 or less, 10 or less, 5 or less, 2 or less' or 1 or Less methoxyaniline value ' 25 201200591 and / or 5 or less, 4. 5 or less, 4 or less’ 3. 5 or less, 3 or less, 2. 5 or less, 2 or less, 1. 5 or less, 1 or less, 〇. 5 or less’ 0. 2 or less, or 〇. 1 or less peroxide value, and / or 100 ppm or less, 95 ppm or less '90 ppm or less, 85 ppm or less' 80 ppm or less, 75 ppm or less, 70 Ppm or less '65 ppm or less' 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or Less '25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less , or a fill content of 1 ppm or less. The invention is also directed to a method for obtaining a lipid comprising refining the crude lipid of the invention. In some embodiments, the refining is selected from the group consisting of caustic refining, degumming, acid treatment, alkali treatment, cooling, heating, bleaching, deodorization, deacidification, and the like. Summary In general, the method of the present invention does not use an organic solvent to extract or separate a lipid by other methods. Thus, in some embodiments, the amount or concentration of the organic solvent sufficient to extract - lipids is not added to the cell broth comprising the plant material or the fermented meat containing the microbial cells throughout the process of the invention. Soup, added to the __ cell composition, not added to - the fine money, or not added to - lipid ° in some specific examples - organic solvents can be added to - cell composition ★ Decomposed cell composition or de-emulsified cell composition. 26 201200591 In these specific examples, the organic solvent is less than 5%, less than 4%, less than 3 /〇' less than 2% 'less than 1%, less than 5% 5%, less than 〇. Add 1%, or less than 〇 5% by volume. As used herein, an "organic solvent" refers to a solvent comprising at least one carbon atom, as used herein, a solvent "refers to a hydrophobic or lipophilic agent, and not a lipid As used herein, "hydrophobic" refers to a dose that is rejected due to the large amount of water. As used herein, lipophilic " refers to a solution dissolved in the lipid The organic solvent not used in the method of the present invention includes, but is not limited to, a polar solvent, a non-polar solvent, a water-miscible solvent, a water non-inter-/valley/capacitor, and combinations thereof. Non-limiting examples include substituted and unsubstituted CVC8 alkyl groups (eg, hexanes and analogs), Cs-C! 2 cycloalkyl groups, CrCn dilute, (^-(:8 alcohols (eg, isopropyl) Alcohols and analogs), C1-C8|^, c4-C8 ethers, Crc8g|, C6-Ci2 aryl, Ci_C8 decylamine, C5-C12 heteroatomyl, and combinations thereof. As defined herein An organic solvent may be selectively added to a dissolved cellular composition, for example, an alkali and/or a salt A portion of the composition for contacting the dissolved cell composition. However, in such specific examples, the organic solvent is present in such a concentration that the lipid is not substantially in the solvent (i.e., less than 5%, Less than 4〇/〇, less than 3〇/〇, less than 2%, less than 1%, less than 〇5%, less than 〇1%, or less than 0% by volume or by weight Extracted from the cell composition, the solubilized cell composition, or the cell composition that has been immersed in the sputum. In some embodiments, the present invention - + + A Λ 不 is not included (eg , water) > month wash' or this method to reduce the amount of a dissolved fine tire,,,, 匕 grade or a de-emulsified cell composition cleaning. " turbidity ^ and use, for example , water or slow 27 201200591 Drip dilution - composition and, for example, a method of removing water or buffer by removing ^. The cleaning - cell composition will reduce the moon surface of the moon obtained from a cell Yield. In the present invention, the cleaning can be reduced i times, 2 times, 3 times or more. Definitions as used herein , lipid " or "oil" refers to one or more fatty acids (including esters of free fatty acids and fatty acids), phospholipids, triacylglycerols (ie, triglycerides) Trigiycerides)), diterpene glycerin, glycerin, lys phphings (lys〇ph〇sph〇Hpids), soaps, -fat it' ID alcohols and sterols, carotenoids, Lutein, carbohydrates, and other lipids known to those skilled in the art. Lipids include polar lipids and neutral lipids. As used herein, • polar lipid " refers to a lipid containing a polar group and more readily soluble in a polar solvent. Polar lipids include phospholipids. As used herein, "phospholipid" refers to a lipid having a monophosphate group. As used herein, "neutral lipid" refers to a region that does not contain polar regions and is more soluble in non-polar solvents. Lipids. Neutral lipids include triglyceride (TAG). Fatty acids are classified according to the length and saturation characteristics of the carbon chain. Fatty acids are called short-chain, medium-chain or long-chain fatty acids depending on the length of the carbon in the chain. When there is no double bond between carbon atoms, fatty acids are called saturated fatty acids, and when double bonds are present, fatty acids are called unsaturated fatty acids. When only one double bond is present, unsaturated long-chain fatty acids are monounsaturated. 'And when more than one double bond #, the unsaturated long bond fatty acid is polyunsaturated. 28 201200591 The fatty acid present in the lipid may have from 4 to 28 carbon atoms. In some embodiments, a lipid comprises One or more polyunsaturated fatty acids. Polyunsaturated fatty acids (PUFAs) are classified according to the position of the first double bond from the methyl end of the fatty acid: omega-3 (η-3) fatty acid contains the first The double bond is on the third carbon, while the omega-6 (η-6) fatty acid contains the first double bond on the sixth carbon. For example, docosahexaenoic acid ("DHA") An omega-3 long chain polyunsaturated fatty acid (LC PUFA) having a chain length of 22 carbons and 6 double bonds, commonly referred to as "22:6 η-3". For the purposes of this application, Chain polyunsaturated fatty acids (LC-PUFAs) are defined as fatty acids of 18 or more carbon chain lengths, and preferably 20 or more carbon chain lengths, containing 3 or more double bonds. -6 series of LC-PUFAs include, but are not limited to, 2.8 liters of linoleic acid (C20: 3n-6), peanut oleic acid (C20: 4n-6) ("ARA"), twenty-two carbon four Docosatetraenoic acid or adrenic acid (C22:4n-6), and docosapentaenoic acid (C22:5n-6) ("DPAn-6"). The omega-3 series of LC-PUFAs include, but are not limited to, eicosatrienoic acid (C20: 3n-3), peanut oleic acid (C20: 4n-3), eicosapentaenoic acid (C20: 5n-3) ) ("EPA"), docosapentaenoic acid (C22:5n-3), and docosahexaenoic acid (C22:6n-3). LC-PUFA also Includes fatty acids with greater than 22 carbons and 4 or more double bonds, including, but not limited to, C24:6(n-3) and C28:8(n-3). The term "fatty acids", &quot Polyunsaturated fatty acids ", and "PUFA" include not only free fatty acid forms, but also other forms such as the three-flavored glycerol (TAG) form, the phospholipid (PL) form, and other esterified forms. As used herein, the terms "ester" and "esterified" refer to the resole of the PUFA molecule. 29 201200591 The hydrogen in the group is replaced by another substituent. Typical esters are known to those skilled in the art and are discussed in Higuchi, T et al., Pro-drugs as Novel Delivery Systems, Vol. 14, A. C. S. Symposium Series, Bioreversible Carriers in Drug Design, Edward B. Roche ed. , Amer. Pharma. Assoc. , Pergamon Press [\9), to Anti-Protection Groups in Organic Chemistry,

McOmie ed.，Plenum Press, New York (1973)之中，其等之各個係以其之整體併入本文中以作為參考資料。常見的酯之實例包括甲基、乙基、三氣乙基、丙基、丁基、戊基、三級丁基、苯甲基、硝苯甲基、曱氧基苯甲基以及二苯甲基。於一些具體例中，一脂質包含至少10%，至少20%，至少30%，至少35%，至少40%，至少50%，至少60%，至少 70%或至少80%重量計之PUFA。於一些具體例中，一脂質包含至少10%，至少20°/。，至少30%，至少35%，至少40%，至少50%，至少60%，至少70%或至少80%重量計之DHA。於一些具體例中，一脂質包含少於50%，少於40%，少於 30%，少於20%，少於15%，少於10%，或少於5%重量計之 EPA。於一些具體例中，一脂質包含少於ι〇〇/0，少於5%，少於2%，少於1%，或少於〇·5°/。以重量計的固醇。於一些具體例中’一或更多個PUFAs係以一或更多個形式之一脂質存在的，例如固醇酯餾份、三酸甘油酯、二酸甘油酯、單酸甘油酯、游離脂肪酸、酯化的脂肪酸、鹼金屬鹽脂肪酸、鹼土金屬鹽脂肪酸，以及其等之組合。於一些具體例中，於本發明的一方法中在離心之後所 30 201200591 分離的一脂質包含至少50%，至少60% ’至少70%，至少 80%，至少90%，至少 95%，或是50%至95%，50%至90%， 50%至85%，50%至80%，50%至75%，60%至95%，60%至 90%，60%至 85%，70%至95%，70%至90%，70%至 85%， 75%至95%，75%至90%，或是75%至85%，重量計之三酸甘油酯。於一些具體例中，該等三酸甘油酯包含至少10%，至少20%，至少30%，至少35%，至少40%，至少50%，至少 60%，至少70%或至少80%重量計之DHA。於一些具體例中，該等三酸甘油酯包含至少50%，至少40%，至少30%，至少20%，至少15%，至少10%，或至少5%重量計之EPA。如本文中所討論的，一脂質在該離心之後之額外的精製可以提供一脂質，其包含至少80%，至少85%，至少90%，至少95°/。’至少99°/。’ 或是80%至99.5%，80%至99%，80% 至97% ’ 80%至95%，80%至90%，85%至99.5%，85%至990/〇， 85%至 97%，85%至 95%，85%至 90%，90%至99.5%，90% 至99% ’ 90%至97%，90%至 95%，95%至99.5%，95%至99%， 95%至97%，97%至99.5°/。，或是98。/。至99.5°/。三酸甘油酯重量計。如本文中所使用的，一"細胞，，提及一含脂質生物材料，例如係衍生自植物或微生物的生物材料。於一些具體例中，適當的植物材料包括，但不限於，植物部分和油籽。油籽包括，但不限於，葵花籽、油菜籽、油菜子、亞麻仁、蓖麻油籽、胡荽籽、金盞菊籽或類似物，以及其等之基因 31 201200591 修飾變異物。依據本文中所說明的方法而由之植物材料及/ 或微生物，例如含油的微生物，所生產的油亦被稱為植物油。由海藻及/或真菌所生產的油亦分別地被稱為藻類油及 /或真菌油。如本文中所使用的，一"微生物細胞"或"微生物"提及生物，例如海藻、細菌、真菌、原生生物，以及其等之組合，例如，單細胞生物。於一些具體例中，一微生物細胞為一真核細胞。適合供本發明使用的一微生物細胞包括，但不限於，金黃蕩（golden algae)(例如，原生藻菌界的微生物]、綠藻（green algae)、石夕藻(diatoms)、二鞭毛藻 (dinoflagellates)(例如，甲薄少cetze)目的微生物，其含括隱曱藻屬的成員例如，舉例而言，寇氏隱甲讓co/zm·/)或寇氏隱曱藻（C1. co/m"))、酵母菌（子囊菌或擔子菌），以及白黴和被孢黴（Mori如e//a)屬的真菌，包括但不限於高山被孢黴 (Mortierella alpina)认反Mortierella sect, schmuckeri。通合供本發明使用之一微生物細胞可以進一步包括，但不限於，存在於下列群組的生物之屬：原生藻菌、//awaiorej、原生單胞菌（Proteromonads)、乳白菌（Opalines)、發生胚菌 {Develpayella) ' 雙抱子菌（Diplophrys)、拉斑絲菌、破囊壺菌、雙克斯菌（所仍％油）、卵菌類 {Oomycetes) > ^ ^ g 11¾ (Hypochytridiomycetes) ' nl·% (Co/ww如·〇«)、網花球菌、毛單胞菌、波球菌（Pe/agococcMj：)、歐力克菌 32 201200591 ((9///co/a)、金黃球菌（yiwreococcw·?)、小殼藻目（Parma/e·?)、石夕藻類Φ⑹〇似）、黃藻類、褐藻類 (Phaeophytes)、黃綠綠類（Eustigmatophytes)、綠鞭薄類 (Raphidophytes)、新转嵐ISynurids)、Axodines [含括色板瘤菌（/?/2/7〇£^"〇»?«//«〇<3/烈）、片内爾菌、隔盤菌 (•Dz'cijoc/za/es))、金錄頂菌（C/zrj^sOmerWes)、束隔金菌 (iS^rez'woc/zrj^i/a/e·?)、水樹藻目（/fyt/rwna/ei·)、智·居金藻目 (Hibberdiales)以反色金暮诌（Chromulinales)。於一些具體例中，供本發明使用的一微生物細胞為網黏菌門（phylum Labyrinthulomycota)的一微生物。於一些具體例中，網黏菌門的一微生物細胞為一破囊壺菌，例如一裂殖壺菌屬（Schizochytrium)氣破嚢壺菌餍 (TTznawWoc/^iWwm)。依據本發明，術語"破囊壺菌"提及破囊壺菌目（Thraustochytriales)的任一成員，其包括破囊壺菌科家族，以及術語"網黏菌（labyrinthulid)"係指網黏菌目之任一成員，其包括網黏菌科家族。網黏菌科家族成員先前被認為是破囊壺菌目成員，但是於最近有關此等生物的分類學分類之修正中，網黏菌科家族現在被認為是網黏菌目之一員。網黏菌目與破囊壺菌目二者皆被認為是網黏菌門（Labyrinthulomycota)之一員。分類學者現在一般將此等群組的微生物與原生藻菌 (Stramenopile)譜系之海藻或海藻-類似原生生物在一起。目前對於破囊壺菌（thraustochytrid)與網黏菌（labyrinthulid)之分類配置可以概述如下： 33 201200591 界：原生藻菌（雜色藻界(C7zromo/?/^a)) 門：網黏菌門（不等鞭毛門（Heterokonta)) 綱·網黏菌綱（網黏菌（Labyrinthulae)) 目：網黏菌目科：網黏菌科目：破囊壺菌屬科：破囊壺菌科為了本發明的目的，說明為破囊壺菌之微生物細胞的菌株包括下列生物：目：破囊壺菌目；科：破囊壺菌科；屬：破囊壺菌屬加(物種：sp.，阿魯迪曼達雷（arudimentale)、金黃色(aureum)、班思寇拉（benthicola) ' 格洛巴森(g/oftosww)、肯内伊、摩提夫（moiivMW)、馬支疋直帝曼違雪（multirudimentale)、厚皮{pachydermum)、普洛利芬盧(pro/z/erww)、紅破囊壺菌，以及紋狀破囊壺菌（WWai·))、巫肯尼亞菌(物種：sp.，阿米巴戴亞、開古蓮西斯（灸、微小 (mbwia)、普羅弗達(pro/_ia)、拉迪亞它如）、沙伊廉斯〇m7e似）、沙勒乃利阿納、赛索凯特羅皮思 (sc/z/zoc/z^yirops)、維蘇爾傑西斯（v/jwr尽ews/s)、約肯西斯 (y or kens is) » 以及 sp. BP-5601)、裂殖壺菌屬謂)(物種：sp.，聚裂殖壺菌、利納赛恩（/iwmaceww)、紅樹林、迷努頓 (wz>zwiw/n) ’以及歐克多斯波偷(ocioiporw/w))、.曰本壺菌屬〇/a/7〇«oc/^Wi/w)(物種：sp.，海洋(Warz«wm))、不遊走壺菌 34 201200591 (物種：sp.，哈利歐堤蒂斯(/2α//ο"Λ·ϊ)、開古蓮西斯(A:ergMe/e?m\s)、普羅弗達(pro/M«也），以及史多其諾伊〇iocc/^m?〇)、歐索爾尼亞菌（4/汾物種：sp.，可勞齊(mjwc/ιή·))，或艾利納菌（五"舶)(物種：sp.，馬利撒爾巴(warba/k)，以及辛諾李菲卡(W⑽。就此發明目的而言’在巫肯尼亞菌（[//Aremb)範圍内所說明之物種被視為疋破囊亞癌屬之成員。乂urantiacochytrium和Oblogospora 為包含於本發明中的網黏菌門之兩種額外的屬。於一些具體例中，一微生物細胞係屬於破囊壺菌屬 (Thraustochystrium)、裂殖壺菌屬，以及其等之混合物。適合供本發明使用之微生物細胞包括，但不限於，選自於以下之網黏菌（Labyrinthulids):目：網黏菌目，科：網黏菌科’屬：網黏菌屬（物種：sp.，阿勒傑利恩西斯 (Ω/ger如也）、可安諾赛斯提斯卜〇伙〇£^怡）、恰特圖尼 (c/zaiiom7)、麥可洛塞斯提斯、麥可洛塞斯提斯阿特蘭提卡、麥可洛塞斯提斯麥可洛塞斯提斯卜㈤啊抬⑽⑽啊叫、海洋(·η>2β)、微小 (winwia)、羅斯可芬西斯（咖⑶及⑼池）' 芳考諾維 (vaM：a«6>Wz·)、維特里納（v加//⑻）、維特里納帕西菲卡 (vitellina pacifica)、維特里納維特 Em(vitemna viteUina), 及索琵菲(z〇Pyh·))、網黏菌屬（Ια6>；η•咐⑹4 (物種：sp，哈利歐堤蒂斯(，及約肯西斯加咖油)）、迷黏菌㈦知咖―m卿）（物種：sp·，海洋）、雙孔蟲(物種： sP_，阿爾切利(⑽〜η·))、派爾侯蘇勒斯菌⑽⑽卿叫(物 35 201200591 種：sp.，海洋（warzTzw·?))、蘇洛迪普洛菲力斯菌 (物種：sp.，史特爾寇來亞從)），以及克廉米多米克沙菌（C7z/aw>^om少χα)(物種：sp.，網黏菌屬 (/a6;/W«i/zM/ozWei)、蒙大拿(wo«iima))(雖然並無與派爾侯蘇勒斯菌（/Vr/ztworMi)、蘇洛迪普洛菲力斯菌 (Sorod/p/op/iOAs)，及克廉米多米克沙菌（CTz/amj/i/om;^)碟切一致之分類配置）。網黏菌門之宿主細胞包括，但不限於，寄存菌株 PTA-10212，PTA-10213，PTA-10214，PTA-10215， PTA-9695，PTA 9696，PTA-9697，PTA-9698，PTA-10208， PTA-10209，PTA-10210，PTA-10211，寄存為 SAM2179 之微生物（由寄存者命名"巫肯尼亞菌（t//kma)SAM2179")，任何破囊壺菌物種(含括在前的巫肯尼亞菌物種，例如維蘇爾傑西斯巫肯尼亞菌（[/· 、阿米巴戴亞巫肯尼亞菌 (t/. 、沙勒乃利阿納巫肯尼亞菌（t/· sarhWawfl)、普羅弗達巫肯尼亞菌（[/· 、拉迪亞它巫肯尼亞菌（C/· ΓίΐΛ如α)、微小巫肯尼亞菌（¢/. 及巫肯Each of McOmie ed., Plenum Press, New York (1973) is incorporated herein by reference in its entirety. Examples of common esters include methyl, ethyl, tri-g-ethyl, propyl, butyl, pentyl, tert-butyl, benzyl, nitrobenzyl, decyloxybenzyl and diphenyl base. In some embodiments, a lipid comprises at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% by weight of PUFA. In some embodiments, a lipid comprises at least 10%, at least 20°/. At least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% by weight of DHA. In some embodiments, a lipid comprises less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% by weight of EPA. In some embodiments, a lipid comprises less than ι〇〇/0, less than 5%, less than 2%, less than 1%, or less than 〇5°/. Sterols by weight. In some embodiments, one or more PUFAs are present in one or more forms of a lipid, such as a sterol ester fraction, a triglyceride, a diglyceride, a monoglyceride, a free fatty acid. An esterified fatty acid, an alkali metal salt fatty acid, an alkaline earth metal salt fatty acid, and combinations thereof. In some embodiments, a lipid isolated by 30 201200591 after centrifugation in a method of the invention comprises at least 50%, at least 60% 'at least 70%, at least 80%, at least 90%, at least 95%, or 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 60% to 95%, 60% to 90%, 60% to 85%, 70% To 95%, 70% to 90%, 70% to 85%, 75% to 95%, 75% to 90%, or 75% to 85% by weight of triglyceride. In some embodiments, the triglycerides comprise at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% by weight. DHA. In some embodiments, the triglycerides comprise at least 50%, at least 40%, at least 30%, at least 20%, at least 15%, at least 10%, or at least 5% by weight of EPA. As discussed herein, additional purification of a lipid after the centrifugation can provide a lipid comprising at least 80%, at least 85%, at least 90%, at least 95°/. 'At least 99°/. ' Or 80% to 99.5%, 80% to 99%, 80% to 97% '80% to 95%, 80% to 90%, 85% to 99.5%, 85% to 990/〇, 85% to 97% %, 85% to 95%, 85% to 90%, 90% to 99.5%, 90% to 99% '90% to 97%, 90% to 95%, 95% to 99.5%, 95% to 99%, 95% to 97%, 97% to 99.5°/. Or 98. /. To 99.5°/. Triglyceride weight meter. As used herein, a "cell, refers to a lipid-containing biomaterial, such as a biological material derived from a plant or microorganism. In some embodiments, suitable plant materials include, but are not limited to, plant parts and oilseeds. Oilseeds include, but are not limited to, sunflower seeds, rapeseed, rapeseed, linseed, castor oil seeds, coriander seeds, calendula seeds or the like, and their genes 31 201200591 modified variants. Oils produced from plant materials and/or microorganisms, such as oil-containing microorganisms, according to the methods described herein are also referred to as vegetable oils. Oils produced from seaweed and/or fungi are also referred to as algae oils and/or fungal oils, respectively. As used herein, a "microbial cell" or "microorganism" refers to a organism, such as a seaweed, a bacterium, a fungus, a protist, and combinations thereof, for example, a single cell organism. In some embodiments, a microbial cell is a eukaryotic cell. A microbial cell suitable for use in the present invention includes, but is not limited to, golden algae (e.g., microorganisms of the genus Algae), green algae, diatoms, dinoflagellates ( Dinoflagellates) (eg, a thin cetze) microorganism of interest, including members of the genus Cryptophyta, for example, 隐隐让 co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co co m")), yeast (ascomycetes or basidiomycetes), and fungi of the genus White mildew and Mortierella (Mori such as e//a), including but not limited to Mortierella alpina, which recognizes Mortierella sect , schmuckeri. One of the microbial cells for use in the present invention may further include, but is not limited to, the genus of organisms present in the following groups: stramenopile, //awaiorej, Proteromonads, Opalines, Embryo {Develpayella) 'Diplophrys, Lactobacillus, Thraustochytrium, Dictyostelium (still % oil), Oomycetes {Oomycetes) > ^ ^ g 113⁄4 (Hypochytridiomycetes) ' nl·% (Co/ww如·〇«), cyanobacteria, Phytophthora, bacillus (Pe/agococcMj:), eutropha 32 201200591 ((9///co/a), golden cocci (yiwreococcw·?), C. algae (Parma/e·?), Shixia algae Φ(6) )like), yellow algae, Phaeophytes, Eustigmatophytes, Raphidophytes, Newly converted to ISynurids), Axodines [including S. sphaeroides (/?/2/7〇£^"〇»?«//«〇<3/lie), typhus, and bacillus • Dz'cijoc/za/es)), C. serrata (C/zrj^sOmerWes), G. glabrata (iS^rez'woc/zrj^i/a/e·?), and water algae (/ Fyt/rwna/ei·), Zhi·Jinjinmu (H Ibberdiales) with Chromulinales. In some embodiments, a microbial cell for use in the present invention is a microorganism of the phylum Labyrinthulomycota. In some embodiments, a microbial cell of the genus Myxobacteria is a Thraustochytrium, such as Schizochytrium, TTznawWoc/^iWwm. According to the invention, the term "Schizochytrium" refers to any member of the Thraustochytriales family, which includes the family Thraustochytrid, and the term "labyrinthulid" Any member of the genus Myxobacteria, which includes the family of the genus Myxobacteria. Members of the family of the genus Myxobacteria were previously considered to be members of the Thraustochytrid family, but in the recent revision of the taxonomic classification of these organisms, the family of the genus Myxobacteria is now considered to be a member of the genus Myxobacteria. Both M. oxysporum and Thraustochytrium are considered to be members of the Labyrinthulomycota. Taxonomists now generally combine these microbes with algae or algae-like protozoa of the Stramenopile lineage. The current classification of thracus tochytrid and labyrinthulid can be summarized as follows: 33 201200591 Boundary: Protozoa (C7zromo/?/^a) Gate: M. (Heterokonta) · · 黏网 Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab Lab For the purpose of the invention, the strains which are described as microbial cells of Thraustochytrium include the following organisms: Mesh: Thraustochytrid; Family: Thraustochytrium; Genus: Thraustochytrium Plus (Species: sp., A Arudimentale, aureum, benthicola 'g/oftosww, Kenney, MotivMW, Ma Zhizhen Zhidi Man Multirudimentale, {pachydermum), proliferate (pro/z/erww), Thraustochytrium, and Thraustochytrium (WWai), W. Kenyan (species: Sp., Amiba Deya, Kaigu Liansis (moxibustion, micro (mbwia), profda (pro/_ia), radia ruthenium), Shaylians 〇 M7e like), Charlene Anna, Saskatchewan Roths (sc/z/zoc/z^yirops), Vesul Jess (v/jwr ews/s), Jokens (y or kens Is) » and sp. BP-5601), Schizochytrium () Species: sp., Trichoderma, Linathon (/iwmaceww), Mangrove, and Mudun (wz>zwiw/ n) 'and 'Ocioiporw/w'), 曰壶〇〇 / a / 7 〇 « oc / ^ Wi / w) (species: sp., ocean (Warz «wm)), Do not swim the chytrid 34 201200591 (Species: sp., Harry Oticis (/2α// ο" Λ·ϊ), Cauclus (A: ergMe/e?m\s), Provoda (pro/M« also), as well as Stochinoyi iocc/^m?〇), Ossuria (4/汾 species: sp., lausch (mjwc/ιή·)), or Elinia (five "Important) (species: sp., warba/k), and chinino Lifica (W (10). For the purposes of this invention 'in Kenyan genus ([// The species described within the scope of Aremb) are considered to be members of the genus Aspergillus subsp., 乂urantiacochytrium and Oblogospora are two additional genera of the genus Myxobacteria contained in the present invention. In some embodiments, a microbial cell line belongs to the genus Thraustochystrium, Schizochytrium, and mixtures thereof. Microbial cells suitable for use in the present invention include, but are not limited to, Labyrinthulids selected from the group consisting of: M. grisea, family: M. genus: Genus: Species: sp ., Alejeri Ensis (Omega/ger Ruyi), Annosisi Sisib 〇 ^ ^ ^ 恰恰、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、、 , McCormite Seth Atlantika, McCormise Seth McCorce Sethb (five) ah (10) (10) ah, ocean (·η > 2β), tiny (winwia), Ross Fencis (Caf (3) and (9) Pool)' Fang Kao Nuowei (vaM: a«6>Wz·), Witrina (v plus // (8)), Viterina pacifica, Vitryna vite Uina, and Sophie (z〇Pyh·), M. genus (Ια6>;η•咐(6)4 (species: sp, Harry Otis ( (and Jorken) Sisca café oil)), viscous bacteria (7) Zhicai-mqing) (species: sp·, ocean), double-hole worm (species: sP_, 阿尔切利((10)~η·)), Pelhousu L. sinensis (10) (10) Qing called (object 35 201200591 species: sp., ocean (war zTzw·?)), F. fuligis (species: sp., Sterling), and C. meliloti (C7z/aw > ^om less χα) Species: sp., genus Mycobacterium (/a6;/W«i/zM/ozWei), Montana (wo«iima)) (although not with S. serrata (/Vr/ztworMi) , Sorod/P/op/iOAs, and C. militaris (CTz/amj/i/om;^) disc-consistent classification configuration). The host cells of Myxomycetes include, but are not limited to, deposited strains PTA-10212, PTA-10213, PTA-10214, PTA-10215, PTA-9695, PTA 9696, PTA-9697, PTA-9698, PTA-10208, PTA-10209, PTA-10210, PTA-10211, microbes registered as SAM2179 (named by the depositer "Wooden Kenyan (t//kma) SAM2179"), any Thraustochytrium species (including the former witch) Kenyan species, such as Vesul Jess, Kenyan bacterium ([/·, Amoebadian, Kenyan bacterium (t/., Charlene, Kenyan genus (t/· sarhWawfl), Provda Witch, Kenya Bacteria ([/·, Radian, Witch Kenyan (C/· ΓίΐΛ such as α), tiny witch Kenyan (¢/. and Wakken)

尼亞菌物種BP-5601，以及含括：紋狀破囊壺菌 (77znmsioc/^rzwm Wriaii/w)、金黄色破囊壺菌、羅斯破囊壺菌（TTirai^oc/^WMW msewm);以及任何日本壺菌屬物種。破囊壺菌目的菌株包括，但不限於破囊壺菌種(23B)(ATCC 20891);紋狀破囊壺菌（Schneider) (ATCC 24473);金黄色破囊壺菌（Goldstein) (ATCC 34304);羅斯破囊壺菌 (Goldstein) (ATCC 28210);曰本壺菌屬物種（L1)(ATCC 36 201200591 28207) ; ATCC 20890 ; ATCC 20892 ;由前述的微生物之任一者所衍生的一突變菌株，以及其等之混合物。裂殖壺菌屬包括，但不限於聚裂殖壺菌少/η·· aggregatum)、裂殖壺菌屬（Schizochytrium limacinum)、裂殖_ 壺菌屬物種（S31) (ATCC 20888)、裂殖壺菌屬物種（S8) (ATCC 20889)、裂殖壺菌屬物種(LC-RM) (ATCC 18915)、裂殖32菌屬物種(SR 21) ’寄存菌株ATCC 28209，寄存的裂殖壺菌屬"則咖_)菌株IF〇 32693，由前述的微生物之任-者所衍生的—突變㈣，以及其等之混口物。於-些具體例中，宿主細胞為—裂殖壺菌屬或一破囊㈣1殖壺_可以藉由連續的二分以及藉由形成抱子囊(其等錢釋放《孢子)二者顿製。“，破囊壺菌只能糟由形成鮮囊（其等繼㈣放遊走孢子）來複製。於一些具體例中，本發明的宿主細胞為—重組型宿主細胞。財發明使㈣-微线細胞之有效㈣養條件包 ;:Γ於，允許脂質生產之有效的培養基、生物反應二以及氧條件。—有致的培養基提及任何的培養基，其中一微生物細胞勺糸 ’破囊壺菌目微生物細其，立目+ 料；。養基典魏包含-水性培養㈣r 之碳、氮，與縣源，以及適當之鹽類、 :物質、金屬，與其他營養物，例如維生素。供本發明使 =生物細胞可以被培養於慣用的發酵生物反應器、震盈燒瓶、試管、微較盤，叹培養㈣。卜此中’培養·適當用於-重組型細胞的-溫度、Pr，、以i £ 37 201200591 氧含量下進行。於一些具體例中，一微生物細胞能夠於12克/升或更少，5克/升或更少，或是3克/升或更少的氣化鈉之鹽度位準下生長。於一些具體例中，一微生物細胞產生一脂質，其包含 ω-3及/或ω-6 PUFA。於一些具體例中’一微生物細胞產生一脂質，其包含DHA、DPA(n-3)、DPA(n-6)、ΕΡΑ、花生油酸(ARA)，或類似物，以及其等之組合。生產包含一PUFA 之脂質的微生物之非限制性實例係揭示於以上以及亦於美國專利案號5,340,594、5,340,742及5,583,019中找到，其等之各個係以其之整體併入本文中以作為參考資料。於一些具體例中，一微生物細胞包含至少30%重量計脂質，至少35%重量計脂質，至少40%重量計脂質，至少50% 重量計脂質，至少60%重量計脂質，至少70%重量計脂質，或至少80%重量計脂質。於一些具體例中，供本發明使用之一微生物細胞能夠產生至少0.1克每公升每小時(g/L/h)的 DHA，至少 0.2 g/L/h 的 DHA，至少 0.3 g/L/h 的 DHA，或至少〇·4 g/L/h的 DHA。方法本發明的方法包含溶解一細胞或細胞生質以形成一溶解的細胞組成物。如本文中所使用的，術語"細胞生質"提及一族群的植物或微生物細胞。如本文中所使用的，術語洛解(lyse)"及"溶解(iysing)"提及一種使一細胞的細胞壁及 /或細胞臈破裂的方法。於一些具體例中，溶解包含一種方 38 201200591 法，例如：機械處理、化學處理、酵素處理、物理處理，或其等之組合。如本文中所使用的’機械處理包括，但不限於，均質化一細胞、施加超音波至一細胞、冷壓—細胞、碾磨一細胞或類似物，以及其等之組合。於一些具體例中，一種方法包含藉由均質化來溶解該細胞。於一些具體例中，一種方法包含用一均質機來溶解該細胞。均質化一細胞可以包括，但不限於，一種方法，其使用一法式壓力細胞破碎機(French pressure cell press)、一音波發生器、一均質機、一球磨機、一棒磨機、一卵石磨機、一玻珠研磨機、一高壓輥磨機、一立軸式衝擊機、一工業摻和機、一高剪切混合機、一混合機、一槳式混合機、一 polytron均質機或類似物，以及其等之組合。於一些具體例中，一細胞係流動通過選擇性地加熱之—均質機。於一些具體例中，適當的岣質化可以包括1至3次通過於高壓及/或低壓之一均質機。於一些具體例中，在均質化的期間之壓力可以為150巴至1，4〇〇巴，150巴至1，2〇〇巴，15〇巴至9〇〇巴’ 150巴至300巴，300巴至1,400巴，300巴至12〇〇巴，300 巴至900巴，400巴至800巴’ 500巴至700巴，或是6〇〇巴。如本文中所使用的’物理處理可以包括，但不限於，加熱一細胞、乾燥一細胞，或類似物，以及其等之組合。加熱一細胞可以包括，但不限於，電阻加熱、對流加熱、蒸汽加熱、於一流體浴中加熱、用太陽能加熱、用聚焦式太陽能加熱，與類似物，其等之任一者可以執行於一 39 201200591 槽、池、管子、導管、燒瓶，或是其他的圍阻裝置之内。於一些具體例中，一細胞係於一槽之内加熱，該槽包括電阻線圈於其之壁之内/於其之壁之上。於一些具體例中，一細胞係於一液體浴中予以加熱，該液體浴包括一管子通經該處。乾燥一細胞可以包括，但不限於，暴露於氣流、暴露於熱（例如，對流熱、一加熱的表面，與類似物）、暴露於太陽能、冷凍乾燥法（凍乾）、喷霧乾燥，以及其等之組合。於一些具體例中，乾燥包含將一細胞塗敷至一選擇性地加熱之旋轉式鼓。如本文中所使用的，化學處理包括，但不限於，使一細胞之pH提升、使一細胞接觸一化學品或類似物。使一細胞之pH提升可以包括，但不限於，添加鹼至一細胞組成物。於一些具體例中，適合供本發明使用的鹼包括，但不限於，氫氧化驗（例如，LiOH、NaOH、KOH、 Ca(0H)2，與類似物，以及其等之組合）、碳酸鹼（例如， Na2C03、K2C03、MgC03，與類似物，以及其等之組合）、碳酸氫鹼(例如，LiHC03、NaHC03、KHC03，與類似物，以及其等之組合），以及其等之組合。一鹼可以以固體（例如，結晶、顆粒、小丸，與類似物）的形式或是液體（例如，一水溶液、一酒精溶液，例如配於甲醇、乙醇、丙醇，與類似物内之氫氧化驗）的形式，以及其等之組合。於一些具體例中，該細胞組成物的pH係提升至8或以上，9或以上， 10或以上，11或以上，12或以上，或是7至13，7至12，7至 40 201200591 1卜 7至 1G ’ 7至9 ’ 8至 13 ’ 8至 12，8至 Π，8至 10，8至9， 9至 12，9至 11，9至 10，1〇至 12，或是 10^^ρΗ。於一些具體例中，使一細胞之ρΗ提升可以包括，但不限於，執行氣鹼方法。於一些具體例中，含氣化鈉與一細胞組成物的一發酵肉湯係接受電解，其會導致氫氧化鈉的形成。氫氧化鈉的形成使該細胞之pH提升》於一些具體例中，一發酵肉湯可以包括氣化鈣或氛化鉀代替氣化鈉或是除了氣化鈉外尚有氣化與或氣化鉀。使此一發酵肉湯接受電解分別地導致氫氧化鈣或氫氧化鉀的形成，藉此使該細胞的pH提升。酵素溶解提及一細胞的細胞壁或細胞膜的溶解，其係藉由使該細胞接觸一或更多個酵素。適合供本發明使用之酵素包括，但不限於，蛋白酶、纖維素酶、半纖維素酶、殼質酶、果膠酶，以及其等之組合。蛋白酶之非限制性實例包括絲胺酸蛋白酶、酥胺酸(theronine)蛋白酶、半胱胺酸蛋白酶、天冬胺酸蛋白酶、金屬蛋白酶、麵胺酸蛋白酶、驗性蛋白酶(alacase)，以及其等之組合。纖維素酶之非限制性實例包括蔗糖酶、麥芽糖酶、乳糖酶、α_葡萄糖苷酶、β-葡萄糖苷酶、澱粉酶、溶菌酶 '神經胺糖酸苷酶、半乳糖苷酶、α-甘露糖苷酶、葡萄醣醛酸酶、玻尿酸酶、聚三葡萄糖酶、葡萄糖腦苷脂酶、半乳糖基神經醯胺酶、乙醢半乳糖胺酶、岩藻糖苷酶、己醣胺酶、艾杜糖酿酸酶、麥芽糖酶-葡萄糖澱粉酶，以及其等之组合。殼質酶之一非限制性貫例包括殼三糖苷酶(chitotriosidase)。果膠酶之非限制性 41 201200591 實例包括果膠酶(pect〇lyase)、果膠酶(pect〇zyme)、聚半乳膽酿酸酶’以及其等之組合。於-些具體例中，-些酵素係由加熱來活化。如本文中所使用的，一"溶解的細胞組成物，，提及一組成物，其包含一或更多個溶解的細胞，含括細胞碎屑和該細胞之其他的内含物’組合以-脂質（來自該等溶解的細 L及選擇性地，含有微生物細胞或植物材料的肉湯。於二具體例中，植物材料係包含於含有該植物材料和水的一肉湯或培養基之内。於—些具體例中，—微生物細胞係包含於含有該微生物細胞和水的一發酵肉湯或培養基之内。於一些具體例中，一溶解的細胞組成物提及一組成物，其包含一或更多個溶解的細胞、細胞碎屑、一脂質、該細胞之天然的内含物，以及來自一肉湯之含水組份。於一些具體例中，一溶解的細胞組成物係以水中油乳狀液的形式，其包含連續的水相和分散的脂質相之一混合物。於一些具體例中’一分散的脂質相係存在以1〇/〇至6〇%，1%至 50〇/〇 ’ 1%至40%，1%至30%，1%至20%，5%至60%，5%至 50〇/〇，5%至40%，5%至30%，5%至20%，10%至60%，10% 至50%，10%至40%，20%至60%，20%至50%，20%至40%， 30%至60% ’ 30°/。至50% ’或是40%至60%的經乳化之溶解的細胞組成物重量計之濃度。縱然不被任何的特定的理論所束缚，據信本發明的方法碎裂或去乳化一經乳化之溶解的細胞組成物，允許一脂質要自該溶解的細胞組成物分離。如本文中所使用的，術 42 201200591 语'•乳狀液”和"經乳化的"提及二或更多不互溶相或層的一混合物，其中一相或層係分散於另一相或層之内。如本文中所使用的，術語”碎裂(break)，，、”碎裂（breakup)"、"去乳化（demulsify)"、"去乳化作用（demulsification)"、，，去乳化 (demulsifying)”，以及"碎裂"提及一種分離一乳狀液的不互溶相或層之方法。舉例而言，去乳化或碎裂一經乳化之溶解的細胞組成物提及一種方法，一經乳化之溶解的細胞組成物係藉由該方法而由具有一或更多個相或層的一乳狀液改變成具有二或更多相或層的一組成物。舉例而言，於一些具體例中，本發明的一方法使一經乳化之溶解的細胞組成物由單相碎裂成二或更多相。於一些具體例中，該二或更多相包括一脂質相及一水相。於一些具體例中，本發明的一方法使一經乳化之溶解的細胞組成物由一或更多個相碎裂成至少三相。於一些具體例中，該三相包括一脂質相、一水相，以及一固相。於一些具體例中，該三相包括一脂質相、一狀液相，以及一水相。於一些具體例中’本發明的方法去乳化一溶解的細胞組成物以形成一去乳化的細胞組成物，其係藉由移除或碎裂至少75%的該乳狀液’至少80%的該乳狀液，至少85%的該乳狀液，至少90%的該乳狀液，至少95%的該乳狀液，至少99%的該乳狀液。於一些具體例中，本發明的方法去乳化一溶解的細胞組成物’其係藉由移除或碎裂75%的該乳狀液至99%的該乳狀液，75%的該乳狀液至95%的該乳狀液’ 75%的§玄乳狀液至90%的該乳狀液，75%的該乳狀液至 43 201200591 85%的該乳狀液，75%的該乳狀液至80%的該乳狀液，80% 的該乳狀液至99%的該乳狀液，80%的該乳狀液至95%的該乳狀液，80%的該乳狀液至90%的該乳狀液，80%的該乳狀液至85%的該乳狀液，85%的該乳狀液至99%的該乳狀液， 85%的該乳狀液至95%的該乳狀液，85%的該乳狀液至9〇〇/0 的該乳狀液，90%的該乳狀液至99%的該乳狀液，9〇%的該乳狀液至95%的邊乳狀液，或是95%的該乳狀液至99%的該乳狀液重量計或體積計。於些具體例中，在溶解該細胞之前，該細胞可以被清洗及/或低溫殺菌。於一些具體例中，清洗該細胞包括使用-水溶液’例如水’來移除任何細胞外的水溶性或水分散性化合物。於-些具體例巾，該細胞可以被清洗一次、 --人、二次，或更多次。於__些具體例中，低溫殺菌該細純括加熱該細胞以使任何不受歡迎的酵素去活性，舉例 =可能降解脂質或降低pUFAs的產量之任何的酵素。於 ”體财’該細胞可以最先被清洗以及接而經低溫殺菌的。生㈣/具體例中，該細胞為植物生物材料以及該植物 2料係在溶解之前形成些具體财，該植物生由自-_移除歧萃取油籽而形成。於—些例中油籽的内部係藉由研磨、礙磨、擠壓、吸出、例中等之組合而從-油籽的外殼移除。於—些具體質化使用本技藝中所知道的方法予以均 s出，例如藉由使油籽通過一壓機來研磨去殼的油 44 201200591 籽成為-塊狀物。於一些具體例中，可以添加水至該物來形成—經乳化之溶解的細胞組成物。於—些具體例中°亥經礼化之溶解的細胞組成物可以使用本技藝中所知道的方法予以過遽以自該溶解的細胞組成物移除的殼碎片。里於些具體例中，用第一個驗處理—溶解的細胞組成物使一經乳化之溶解的細胞組成物碎裂（亦即，去乳化）。於 :些具體例中，用第二個驗處理—溶解的細胞組成物使」經礼化之溶解的細驗成物碎裂（亦即，去乳⑹。於一些具體例中’用-鹽處理一溶解的細胞組成物使一經乳化之容解的細胞組成物碎裂（亦即’去乳化）。於—些具體例中，加熱-溶解的細胞組成物使一經乳化之溶解的細胞組成物碎裂（亦即，去乳化）。於-些具體例中，攪拌一溶解的細胞组成物使—經乳化之溶解的細胞組成物碎裂（料，去乳化）。於一些具體例中，同時的加熱與攪拌一溶解的細胞組成物使-經乳化之溶解的細胞組成物碎裂（亦即，去乳化）。於一些具體例中，前面的處理之一或更多者使-經乳化之溶解的細胞組成物碎裂（亦即，去乳化）。於-些具體例中，本發明的方法包含使一細胞組成物的PH提升以溶解及/或去乳化該細胞組成物。於一些具體例中’本發明时法包纽—轉的細驗絲的pH提升以去乳化該溶解的細胞組成物。於—些具體例中，該pH提升包含使一細胞組成物或溶解的細胞組成物接觸-驗。於-些具體例中’本發明的方法包含使_溶解的細胞組成物接 45 201200591 觸一鹼以去乳化該溶解的細胞組成物。如本文中所使用的，”接觸"提及組合一細胞組成物或一溶解的細胞組成物與一第二組成物（例如，藉由添加一組成物至一細胞組成物或一溶解的細胞組成物，藉由添加一細胞組成物或一溶解的細胞組成物至一組成物，與類似物）^如本文中所使用的，一”組成物"可以包含純的原料或是包括二或更多原料、物質、賦形劑、部分，與類似物的一組合。使一溶解的細胞組成物接觸一第一個鹼使該溶解的細胞組成物之ρ Η 提升。於一些具體例中，一溶解的細胞組成物係與第二個鹼接觸。於一些具體例中，一溶解的細胞組成物或一去乳化的細胞組成物之pH係被再度提升。於一些具體例中，使 pH再度提升包含使一溶解的細胞組成物或去乳化的細胞組成物接觸第二個鹼。於一些具體例中，一溶解的細胞組成物係與一第一個鹼接觸，接而予以加熱，攪拌，或其等之一組合，以及隨後與第二個鹼接觸以提供一經處理之溶解的細胞乳狀液。於一些具體例中，該第一個鹼及/或第二個鹼具有1至 12 ， 1至10 ， 1至8 ， 1至6 ， 1至5 ， 2至12 ， 2至10 ， 2至8 ， 2 至6，2至5，3至 10，3至6，3至5，4至 10，4至8，4至6，5 至10，或是5至8的pKb。如本文中所使用的，術語"pKb"提及鹼之鹼締合常數，Kb，的負對數。Kb提及於水中鹼的游離之平衡常數，其中： B + H2O ^ HB+ + OH ;以及鹼的Kb，B，係定義如同： 46 201200591 κ [ΗΒ+][ΟΗ-] b— [Β] · 適合供本發明使用的鹼包括，但不限於，氫氧化鹼(例如，LiOH、NaOH、ΚΟΗ、Ca(OH)2，與類似物，以及其等之組合）、碳酸鹼(例如，Na2C03、K2C03、MgC03，與類似物，以及其等之組合）、碳酸氫鹼(例如，LiHC03、NaHC03、 KHCO3，與類似物，以及其等之组合），以及其等之組合。一鹼可以以固體(例如，結晶、顆粒、小丸，與類似物）的形式或是液體(例如，一水溶液、一酒精溶液，例如配於曱醇、乙醇、丙醇，與類似物内之氫氧化鹼）的形式，以及其等之組合。因而，一溶劑可以選擇性地存在於一驗之中供本發明使用。如本文中所使用的，"溶劑"提及疏水性的或親脂性的一劑。如本文中所使用的，”疏水性的"提及由於大量的水而被排斥的一劑。如本文中所使用的，”親脂性的"提及溶解於脂質的一劑。於一些具體例中，使一細胞組成物或一溶解的細胞组成物與鹼接觸提升了該溶解的細胞組成物之pH。於一些具體例中，使一溶解的細胞組成物與鹼接觸提升了該溶解的細胞組成物之pH至8或以上，9或以上，10或以上，u或以上’丨2或以上，或7至13 ’ 7至12，7至11，7至10，7至9，8 至 13，8至 12，8至 11，8至 1〇，8至 9，9至 12，9至 11，9至 10，10至12，或10至Π之pH。於一些具體例中，使—溶解的細胞組成物與鹼接觸提供了 8或以下，7或以下，6或以下，或5或以下的pH給該組成物。 47 201200591 於一些具體例中，用驗的添加使該細胞組成物或該溶解的細胞組成物的pH提升抑制了脂質氧化，藉此最小化該溶解的細胞組成物中的自由基的量以至於從本發明的方法獲得的該粗製脂質具有低的過氧化物值（例如，5或更少， 4.5或更少，4或更少，3.5或更少，3或更少，2 5或更少，2 或更少，1.5或更少，1或更少，〇·5或更少，〇 2或更少，或疋0.1或更少）及/或低的甲氧苯胺值(例如，26或更少，25或更少，20或更少’ 15或更少，1〇或更少，5或更少，2或更少，或是1或更少）。如本文中所使用的，術語”過氧化物值 ”或是"PV'|提及在該脂質的氧化的整個期間出現的初級反應產物，例如過氧化物及氫過氧化物，之測量，如本文中所使用的，過氧化物值係以meq/kg來測量。如本文中所使用的，術語"曱氧苯胺值"或是"AV"提及在該脂質氧化的整個期間出現的次級反應產物，例如齡和嗣，之測量。於一些具體例中，於該溶解的細胞組成物内的自由基在用一鹼調整pH之後係使用一電子順磁共振光譜儀，例如，Bruker BioSpin e-scan EPR(系統號碼SC0274) (BrukerNitrogen species BP-5601, and includes: Thraustochytrium (77znmsioc/^rzwm Wriaii/w), Thraustochytrium aureus, Thraustochytrium (TTirai^oc/^WMW msewm); And any Japanese genus Chinensis species. The strains of Thraustochytrium include, but are not limited to, Thraustochytrium (23B) (ATCC 20891); Schneider (ATCC 24473); Golden Stem (Goldstein) (ATCC 34304) ); Goldstein (ATCC 28210); Thripococcus species (L1) (ATCC 36 201200591 28207); ATCC 20890; ATCC 20892; a mutation derived from any of the aforementioned microorganisms A strain, and a mixture thereof. Schizochytrium includes, but is not limited to, Schizochytrium less than η·· aggregatum, Schizochytrium limacinum, Schizochytrium species (S31) (ATCC 20888), and fission Species of the genus Thripococcus (S8) (ATCC 20889), Schizochytrium species (LC-RM) (ATCC 18915), 32 species of fission (SR 21) 'registered strain ATCC 28209, deposited Schizochytrium It belongs to the quotient IF〇32693, which is derived from any of the aforementioned microorganisms, the mutation (4), and the like. In some embodiments, the host cell is a Schizochytrium or a ruptured sac (4) 1 colony _ can be produced by successive dichotomies and by the formation of a sac (which releases the spores). "The Thraustochytrium can only be reproduced by the formation of fresh sacs, which are followed by (4) vaccination. In some specific examples, the host cell of the present invention is a recombinant host cell. The invention makes (4) - microwire Effective cells (4) Conditional package;: 培养基, an effective medium for allowing lipid production, biological reaction 2, and oxygen conditions. - The medium is referred to any medium, and one microbial cell is scooped by 'the bacterium. Finely, stand + material; 养基典魏 contains - water culture (four) r carbon, nitrogen, and county source, and appropriate salts, : substances, metals, and other nutrients, such as vitamins. = Biological cells can be cultured in a conventional fermentation bioreactor, a shake flask, a test tube, a microplate, and a sigh culture (4). In this case, 'culture · appropriate for - recombinant cells - temperature, Pr, to i £ 37 201200591 Oxygen content. In some specific examples, a microbial cell can be at 12 g/l or less, 5 g/l or less, or 3 g/l or less of sodium salt of vaporized sodium. Growth at a certain level. In a specific example, a microbial cell produces a lipid comprising omega-3 and/or omega-6 PUFA. In some embodiments, a microbial cell produces a lipid comprising DHA, DPA(n-3), DPA ( N-6), anthraquinone, arachidonic acid (ARA), or the like, and combinations thereof, etc. Non-limiting examples of the production of microorganisms comprising a lipid of a PUFA are disclosed above and also in U.S. Patent Nos. 5,340,594, 5,340,742. And 5, 583, 019, each of which is incorporated herein by reference in its entirety herein in its entirety in its entirety in its entirety in its entirety, in its entirety Weight, at least 50% by weight lipid, at least 60% by weight lipid, at least 70% by weight lipid, or at least 80% by weight lipid. In some embodiments, one of the microbial cells used in the present invention is capable of producing at least 0.1 g per liter per hour (g/L/h) of DHA, at least 0.2 g/L/h of DHA, at least 0.3 g/L/h of DHA, or at least 〇·4 g/L/h of DHA. The method of the invention comprises dissolving a cell or cell biomass Into a dissolved cell composition. As used herein, the term "cytoplasm" refers to a group of plants or microbial cells. As used herein, the terms lyse "&" Iysing " refers to a method of disrupting the cell wall and/or cell raft of a cell. In some embodiments, the dissolution comprises a method of a method of processing, chemical treatment, enzyme treatment, physical treatment, for example, mechanical treatment, chemical treatment, enzyme treatment, physical treatment , or a combination of these. "Mechanical treatment as used herein includes, but is not limited to, homogenizing a cell, applying ultrasound to a cell, cold-pressing cells, milling a cell or the like, and combinations thereof. In some embodiments, a method comprises dissolving the cell by homogenization. In some embodiments, a method comprises dissolving the cell with a homogenizer. Homogenizing a cell can include, but is not limited to, a method using a French pressure cell press, a sonic generator, a homogenizer, a ball mill, a rod mill, a pebble mill , a bead mill, a high pressure roller mill, a vertical shaft impact machine, an industrial blender, a high shear mixer, a mixer, a paddle mixer, a polytron homogenizer or the like, And a combination of them. In some embodiments, a cell line flows through a homogenizer that is selectively heated. In some embodiments, proper deuteration may include one to three passes through a homogenizer of high pressure and/or low pressure. In some embodiments, the pressure during the homogenization period may be 150 bar to 1,4 bar, 150 bar to 1,2 bar, 15 bar to 9 bar '150 bar to 300 bar, 300 bar to 1,400 bar, 300 bar to 12 bar, 300 bar to 900 bar, 400 bar to 800 bar '500 bar to 700 bar, or 6 bar. 'Physical treatment as used herein may include, but is not limited to, heating a cell, drying a cell, or the like, and combinations thereof. Heating a cell can include, but is not limited to, resistance heating, convection heating, steam heating, heating in a fluid bath, heating with solar energy, heating with focused solar energy, and the like, any of which can be performed on a 39 201200591 Slots, tanks, tubes, conduits, flasks, or other containment devices. In some embodiments, a cell line is heated within a trough comprising a resistive coil within/on the wall thereof. In some embodiments, a cell line is heated in a liquid bath that includes a tube therethrough. Drying a cell can include, but is not limited to, exposure to a gas stream, exposure to heat (eg, convective heat, a heated surface, and the like), exposure to solar energy, freeze drying (lyophilization), spray drying, and The combination of them. In some embodiments, drying comprises applying a cell to a selectively heated rotary drum. As used herein, chemical treatment includes, but is not limited to, raising the pH of a cell, contacting a cell with a chemical or the like. Increasing the pH of a cell can include, but is not limited to, adding a base to a cellular composition. In some embodiments, bases suitable for use in the present invention include, but are not limited to, hydrogen hydroxides (e.g., LiOH, NaOH, KOH, Ca(OH)2, and the like, and combinations thereof, and carbonates). (e.g., Na2C03, K2C03, MgCO3, in combination with analogs, and the like), bicarbonate bases (e.g., LiHC03, NaHC03, KHC03, and the like, combinations thereof, and the like), and combinations thereof. The base may be in the form of a solid (for example, crystals, granules, pellets, and the like) or a liquid (for example, an aqueous solution, an alcohol solution, for example, methanol, ethanol, propanol, and the like. The form of the test, and the combination of it. In some embodiments, the pH of the cell composition is increased to 8 or above, 9 or more, 10 or more, 11 or more, 12 or more, or 7 to 13, 7 to 12, 7 to 40 201200591 1 Bu 7 to 1G '7 to 9 ' 8 to 13 ' 8 to 12, 8 to Π, 8 to 10, 8 to 9, 9 to 12, 9 to 11, 9 to 10, 1 to 12, or 10^ ^ρΗ. In some embodiments, increasing the pH of a cell can include, but is not limited to, performing a gas-base process. In some embodiments, a fermentation broth containing vaporized sodium and a cell composition is subjected to electrolysis, which results in the formation of sodium hydroxide. The formation of sodium hydroxide increases the pH of the cell. In some specific examples, a fermentation broth may include calcium carbonate or potassium sulphate instead of sodium sulphate or gasification and gasification in addition to sodium sulphate. Potassium. The electrolysis of the fermented broth separately results in the formation of calcium hydroxide or potassium hydroxide, thereby increasing the pH of the cells. Enzyme lysis refers to the dissolution of the cell wall or cell membrane of a cell by contacting the cell with one or more enzymes. Enzymes suitable for use in the present invention include, but are not limited to, proteases, cellulases, hemicellulases, chitinases, pectinases, and combinations thereof. Non-limiting examples of proteases include serine proteases, theronine proteases, cysteine proteases, aspartic acid proteases, metalloproteases, facial acid proteases, alacases, and the like. The combination. Non-limiting examples of cellulases include sucrase, maltase, lactase, alpha-glucosidase, beta-glucosidase, amylase, lysozyme 'neuraminase, galactosidase, alpha- Mannosidase, glucuronidase, hyaluronanase, polytriglucosidase, glucocerebrosidase, galactosyl neurodecylase, acetaminogalactosidase, fucosidase, hexosaminidase, Aidu A glyco-enzyme, a maltase-glucoamylase, and combinations thereof. A non-limiting example of a chitinase includes chitotriosidase. Non-limiting pectinase 41 201200591 Examples include pect〇lyase, pect〇zyme, polygalactosidase', and combinations thereof. In some specific examples, some of the enzymes are activated by heating. As used herein, a "dissolved cell composition, refers to a composition comprising one or more lysed cells comprising a combination of cell debris and other inclusions of the cell' a lipid (from the dissolved fine L and, optionally, a broth containing a microbial cell or a plant material. In the second specific example, the plant material is contained in a broth or medium containing the plant material and water. In some specific examples, the microbial cell line is contained within a fermentation broth or medium containing the microbial cells and water. In some embodiments, a dissolved cell composition refers to a composition, Containing one or more dissolved cells, cell debris, a lipid, the natural contents of the cell, and an aqueous component from a broth. In some embodiments, a dissolved cell composition is A form of an oil emulsion in water comprising a mixture of a continuous aqueous phase and a dispersed lipid phase. In some embodiments, a dispersed lipid phase is present at 1〇/〇 to 6〇%, 1% to 50. 〇/〇' 1% to 40% , 1% to 30%, 1% to 20%, 5% to 60%, 5% to 50%/〇, 5% to 40%, 5% to 30%, 5% to 20%, 10% to 60% , 10% to 50%, 10% to 40%, 20% to 60%, 20% to 50%, 20% to 40%, 30% to 60% '30°/. to 50%' or 40% to 60% of the concentration of the emulsified dissolved cell composition. Without being bound by any particular theory, it is believed that the method of the invention fragment or de-emulsifie an emulsified dissolved cell composition, allowing a lipid To be separated from the dissolved cellular composition. As used herein, the term 'an emulsion' and "emulsified" refers to a mixture of two or more immiscible phases or layers, One of the phases or layers is dispersed within another phase or layer. As used herein, the terms "break,", "breakup", "demulsify";,"demulsification",, demulsifying, and "fragment" refer to a method of separating an immiscible phase or layer of an emulsion. For example, De-emulsification or fragmentation The dissolved cell composition refers to a method in which an emulsified dissolved cell composition is changed by the method from one emulsion having one or more phases or layers to having two or more phases or A composition of a layer. For example, in some embodiments, a method of the invention causes an emulsified dissolved cell composition to be cleaved from a single phase into two or more phases. In some embodiments, the two The or more phases comprise a lipid phase and an aqueous phase. In some embodiments, a method of the invention causes an emulsified dissolved cellular composition to be fragmented into at least three phases from one or more phases. In some embodiments, the three phases comprise a lipid phase, an aqueous phase, and a solid phase. In some embodiments, the three phases comprise a lipid phase, a monolithic phase, and an aqueous phase. In some embodiments, the method of the invention de-emulsifies a dissolved cell composition to form a de-emulsified cell composition that is at least 80% by removing or fragmenting at least 75% of the emulsion. The emulsion, at least 85% of the emulsion, at least 90% of the emulsion, at least 95% of the emulsion, at least 99% of the emulsion. In some embodiments, the method of the invention de-emulsifies a dissolved cell composition by removing or fragmenting 75% of the emulsion to 99% of the emulsion, 75% of the emulsion Liquid to 95% of the emulsion '75% § 玄 emulsion to 90% of the emulsion, 75% of the emulsion to 43 201200591 85% of the emulsion, 75% of the milk From 80% of the emulsion, 80% of the emulsion to 99% of the emulsion, 80% of the emulsion to 95% of the emulsion, 80% of the emulsion Up to 90% of the emulsion, 80% of the emulsion to 85% of the emulsion, 85% of the emulsion to 99% of the emulsion, 85% of the emulsion to 95% % of the emulsion, 85% of the emulsion to 9 〇〇 / 0 of the emulsion, 90% of the emulsion to 99% of the emulsion, 9 % of the emulsion Up to 95% of the emulsion, or 95% of the emulsion to 99% of the emulsion weight or volume. In some embodiments, the cells can be washed and/or sterilized at a low temperature prior to dissolving the cells. In some embodiments, washing the cells comprises using an aqueous solution such as water to remove any extracellular water soluble or hydrophobic compounds. In some specific cases, the cells can be washed once, - human, twice, or more. In some specific examples, the low temperature sterilization heats the cells to deactivate any undesired enzymes, for example, any enzyme that may degrade lipids or reduce the yield of pUFAs. In the "body wealth", the cell can be first cleaned and then sterilized by low temperature. In the raw (four) / specific example, the cell is a plant biomaterial and the plant 2 system forms a specific wealth before dissolution, the plant It is formed by extracting oilseeds from -_. In some cases, the interior of the oilseeds is removed from the shell of the oilseed by a combination of grinding, obstructing, squeezing, sucking, and the like. These specific characterizations are carried out using methods known in the art, for example by grinding the oilseeds through a press to grind the dehulled oil 44 201200591 seeds into a mass. In some specific examples, it may be added Water is added to the material to form an emulsified dissolved cell composition. In some specific examples, the dissolved cell composition can be subjected to dissolution by a method known in the art. The shell fragments removed by the cell composition. In some specific examples, the first assay-dissolved cell composition is used to fragment (i.e., de-emulsifie) an emulsified dissolved cell composition. In the specific example, the second one is used. Treatment—dissolved cell composition causes fragmentation of the lysed complex (ie, de-milk (6). In some specific cases, 'solving a dissolved cell composition with a salt to make it emulsified The decomposed cell composition is fragmented (i.e., 'de-emulsified.) In some specific examples, the heated-dissolved cell composition fragments (i.e., deemulsifies) the emulsified dissolved cell composition. In some embodiments, stirring a dissolved cell composition causes the emulsified dissolved cell composition to be fragmented (de- emulsified). In some embodiments, simultaneous heating and stirring of a dissolved cell composition allows - The emulsified dissolved cell composition is fragmented (ie, deemulsified). In some embodiments, one or more of the preceding treatments fragment the emulsified dissolved cellular composition (ie, go Emulsification. In some embodiments, the method of the invention comprises raising the pH of a cell composition to dissolve and/or de-emulsifie the cell composition. In some embodiments, the invention is a method of Finely check the pH of the silk to remove milk The dissolved cell composition. In some embodiments, the pH increase comprises contacting a cell composition or a dissolved cell composition. In some embodiments, the method of the invention comprises dissolving the cell. The composition is contacted with a base to remove the dissolved cellular composition. As used herein, "contact" refers to a combination of a cellular composition or a dissolved cellular composition and a second composition ( For example, by adding a composition to a cell composition or a dissolved cell composition, by adding a cell composition or a dissolved cell composition to a composition, and the like) , a "composition" may comprise a pure starting material or a combination comprising two or more starting materials, materials, excipients, moieties, and the like. contacting a dissolved cell composition with a first base Raise the ρ Η of the dissolved cell composition. In some embodiments, a dissolved cell composition is contacted with a second base. In some embodiments, the pH of a dissolved cell composition or a de-emulsiond cell composition is again increased. In some embodiments, re-upgrading the pH comprises contacting a dissolved cell composition or a deemulsified cell composition with a second base. In some embodiments, a dissolved cell composition is contacted with a first base, followed by heating, agitation, or a combination thereof, and subsequently contacted with a second base to provide a treated solution. Cellular emulsion. In some embodiments, the first base and/or the second base has from 1 to 12, from 1 to 10, from 1 to 8, from 1 to 6, from 1 to 5, from 2 to 12, from 2 to 10, from 2 to 8. , 2 to 6, 2 to 5, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 8, 4 to 6, 5 to 10, or 5 to 8 pKb. As used herein, the term "pKb" refers to the alkali base association constant, Kb, the negative logarithm. Kb refers to the equilibrium constant of the free base in water, where: B + H2O ^ HB + + OH ; and the Kb, B of the base are defined as: 46 201200591 κ [ΗΒ+][ΟΗ-] b— [Β] Bases suitable for use in the present invention include, but are not limited to, alkali hydroxides (e.g., LiOH, NaOH, hydrazine, Ca(OH)2, and the like, and combinations thereof, etc.), carbonate bases (e.g., Na2C03, K2C03) , MgC03, in combination with analogs, and the like, a bicarbonate base (eg, LiHC03, NaHC03, KHCO3, in combination with analogs, and the like), and combinations thereof. A base may be in the form of a solid (eg, crystals, granules, pellets, and the like) or a liquid (eg, an aqueous solution, an alcohol solution, such as hydrogen in a decyl alcohol, ethanol, propanol, and the like). The form of the oxidation base), and combinations thereof. Thus, a solvent can optionally be present in a test for use in the present invention. As used herein, "solvent" refers to a hydrophobic or lipophilic agent. As used herein, "hydrophobic" refers to a dose that is rejected due to the large amount of water. As used herein, "lipophilic" refers to a dose that is dissolved in a lipid. In some embodiments, contacting a cell composition or a dissolved cell composition with a base increases the pH of the solubilized cell composition. In some embodiments, contacting a dissolved cell composition with a base increases the pH of the solubilized cell composition to 8 or above, 9 or more, 10 or more, u or more '丨2 or more, or 7 To 13 '7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 13, 8 to 12, 8 to 11, 8 to 1, 8 to 9, 9 to 12, 9 to 11, 9 to 10, 10 to 12, or 10 to pH of Π. In some embodiments, contacting the dissolved cell composition with a base provides a pH of 8 or less, 7 or less, 6 or below, or 5 or less to the composition. 47 201200591 In some embodiments, the increase in pH of the cell composition or the dissolved cell composition is inhibited by the addition of the test, thereby minimizing the amount of free radicals in the dissolved cell composition. The crude lipid obtained from the process of the invention has a low peroxide value (eg, 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2 5 or less) , 2 or less, 1.5 or less, 1 or less, 〇·5 or less, 〇2 or less, or 疋0.1 or less) and/or low methoxyaniline values (for example, 26 or Less, 25 or less, 20 or less '15 or less, 1 or less, 5 or less, 2 or less, or 1 or less). As used herein, the term "peroxide value" or "PV'| refers to the measurement of primary reaction products, such as peroxides and hydroperoxides, occurring throughout the oxidation of the lipid, As used herein, peroxide values are measured in meq/kg. As used herein, the term "nonoxyaniline value" or "AV" refers to the measurement of secondary reaction products, such as age and sputum, that occur throughout the lipid oxidation. In some embodiments, the free radicals in the dissolved cell composition are subjected to an electron paramagnetic resonance spectrometer after adjusting the pH with a base, for example, Bruker BioSpin e-scan EPR (system number SC0274) (Bruker)

BioSpin，Billerica，ΜΑ)而檢測出。於一些具體例中，該溶解的細胞組成物之一樣本係在測量EPR之前用去離子水以大約1 : 1比率予以稀釋。於一些具體例中，為了測量EpR , 將一自旋捕集化學品添加至該溶解的細胞組成物之—樣本。於一些具體例中，該自旋捕集化學品為本技藝中所知道的任何自旋捕集化學品，包括，但不限於，Ρ0ΒΝ(α_(4_ °比啶基1-氧化物)-Ν-三級丁基硝基酮）或DMPO(5,5'-二甲基 48 201200591 _ 比咯啉-N-氧化物）。於一些具體例中，該自旋捕集化學為大約1.25 Μ以及大約50 pL被添加至該溶解的細胞組成物之大約0.5克樣本。於一些具體例中，含有該自旋捕集化學品的一樣品被孵育於室溫(例如，大約20。〇。於一些具體例中，使用下列光譜儀參數：大約86Hz的調變頻率，大約2高斯的調變振輻，大約5 mW的微波功率，大約2〇秒的時間常數，大約10秒的掃掠時間，大約1〇〇高斯的掃掠寬度，以及大約8的掃描數目。EPR係隨時間予以測量來決定存在於該脂質之中的自由基濃度。於一些具體例中，EpR 係在4小時的過程期間每小時地予以測量。於一些具體例中，該溶解的細胞組成物於以上列舉的參數具有少於〇 i5 X 1〇6 ’ 少於0.14 X 1〇6，少於0.13 X 1〇6，少於〇 12 χ 1〇6，少於〇_11 X 106，少於0.1 X 1〇6，少於〇〇9 χ 1〇6，少於〇〇8 X 10，少於0.07 χ 1〇6，少於〇.〇6 χ 1〇6，或少於〇〇5 χ i〇6的一EPR信號強度（強度或振幅)在4小時之後。於一些具體例中，該溶解的細胞組成物具有〇.05 X 1〇6至〇 15 χ 1〇6, 〇〇5 χ 1〇6至0.14 χ 106，0.05 χ 1〇6至0.13 χ 1〇6，0.05 χ 1〇6至〇 12 χ 106,0.05 χ 106至O.ii χ ι〇6,〇 05 χ 1〇6至〇」χ 1〇6,〇〇5 χ ι〇6 至0.09 χ 106，〇.〇7 χ 1〇6至0.15 χ 1〇6，〇·07 χ 1〇6至〇 13 χ 106，0.07 χ 106至o.ii χ ι〇6，0 〇8 χ 1〇6至〇 14 χ 1〇6，〇〇8 χ 106至0_12 χ 106，〇.〇8 χ 1〇6至〇」χ ι〇6，〇〇9 χ 1〇6至〇 13 χ 1〇6，或是0.09 χ 1〇6至o.ii χ 1〇6的EPR。於一些具體例中，導致以上詳細指明的EPR之該溶解的細胞組成物之？1^為8 至12 ， 8至11 ， 8至1〇， 8至9 ， 9至12 ， 9至11 ， 9至1〇， 1〇至 49 201200591 12，或10至11。於一些具體例中，具有以上詳細指明的一 EPR信號強度之一溶解的細胞組成物會導致一粗製脂質，其具有26或更少，25或更少，20或更少，15或更少，10或更少，5或更少，2或更少，或是1或更少的AV。於一些具體例中，具有以上詳細指明的EPR之一溶解的細胞組成物會導致一粗製脂質，其具有5或更少，4.5或更少，4或更少， 3.5或更少，3或更少，2.5或更少，2或更少，1.5或更少，1 或更少，0.5或更少，0.2或更少，或是0.1或更少的PV。於一些具體例中，一種方法包含使一細胞組成物或溶解的細胞組成物接觸一鹽以去乳化該溶解的細胞組成物。如本文中所使用的，一''鹽”提及一離子的化合物，其係藉由從一酸以一金屬（例如，驗金屬、驗土金屬、過渡金屬，及類似物）或一帶正電荷的化合物(例如，NH4+與類似物）來取代一氫離子而形成。適合供本發明使用的鹽包括，但不限於，鹼金屬鹽、鹼土金屬鹽，或類似物，以及其等之組合。存在於供本發明使用的一鹽之中的帶負電荷的離子的物種包括，但不限於，鹵化物、硫酸鹽、硫酸氫鹽、亞硫酸鹽、磷酸鹽、磷酸氫鹽、磷酸二氫鹽、碳酸鹽、碳酸氫鹽，或類似物，以及其等之組合。於一些具體例中，供本發明使用的一鹽係選自於：氣化鈉、硫酸鈉、碳酸鈉、氣化鈣、硫酸鉀、硫酸鎂、麩胺酸鈉、硫酸銨、氣化鉀、氯化鐵、硫酸鐵、硫酸鋁，以及其等之組合。於一些具體例中，一鹽不包括NaOH。一鹽可以添加為一固體（例如，以結晶質、非晶形的、造粒化的，及/或顆粒的形式），及/或 50 201200591 為含有，舉例而言，水、醇，與類似物之一溶液(例如，一稀液溶液、一飽和的溶液，或一過飽和的溶液），以及其等之組合。於一些具體例中，該鹽係以5 g/l至25 gA，5 g/l至10 gA，10 g/1 至 15 g/卜 15 g/1 至20 g/卜 20 g/1 至25 g/卜或是 10 g/1至20 g/1的一量來添加。於一些具體例中，一細胞組成物或一溶解的細胞組成物之溫度係低於或等於60°C，低於或等於55°C，低於或等於45°C，低於或等於40°C，低於或等於35°C，低於或等於 30°C，或是低於或等於25°C，當添加鹽以去乳化該細胞組成物或該溶解的細胞組成物時。於一些具體例中，一溶解的細胞組成物之溫度為〇°C至60°C，〇°C至55°C，〇°C至50 °C，〇°C 至45°C，〇°C 至40°C，〇°C 至35°C，〇°C 至30°C，〇°C 至25°C，20°C 至60°C，20°C 至55°C，20°C 至50°C，20°C 至 45°C，20°C 至40°C，20°C 至35°C，20°C 至30°C，30°C 至60 °C，30°C 至55°C，30°C 至50°C，30°C 至45°C，30°C 至40°C， 30°C 至40°C，40°C 至60°C，40°C 至55°C，40°C 至50°C，或是50°C至60°C，當添加鹽以去乳化該細胞組成物或該溶解的細胞組成物時。於一些具體例中，該方法包含使一細胞組成物或一溶解的細胞組成物接觸該溶解的細胞組成物或該細胞組成物之20%或更少，15%或更少，10%或更少，7.5%或更少，5% 或更少，或是2%或更少的鹽以重量計。於一些具體例中，一方法包含使一細胞組成物或一溶解的細胞組成物接觸該 51 201200591 細胞組成物或溶解的細胞組成物（例如，總肉湯重）之0.! % 至20〇/〇，0.1〇/〇至15〇/〇，0.1%至10%，0.5%至20〇/〇，0.5〇/〇至 15%，0.5%至 10%，〇_5%至 5%，0.5%至4%，0.5%至3%， 0.5%至2.5%，0.5〇/〇至20/〇，0.5%至 1.5%，0.5%至 1¾，1〇/〇至 20%，1%至 15%，1%至 1〇%，1%至5%，1%至4%，1%至3〇/〇， 1〇/〇至2.5% ’ 1%至2% ’ 1%至 1.5%，1.5%至5%，1.5%至4〇/〇， 1.5%至3%’1_5%至2.5%，1.5%至2%，2%至20%，2%至15〇/〇, 2%至 10%，2%至 5%，2%至4%，2%至3%，2%至2.5%，2.5% 至 5%，2.5%至4%，2.5%至3%，3%至5%，3%至4%，4%至 5% ’ 5%至20%，5%至 15%，5%至 10%，10%至20%，10% 至I5%，或是15%至20。/。的鹽，以重量計。舉例而言，當一溶解的細胞組成物稱重1,000 kg時，接觸0.5%至20%的鹽，以重量計’會需要組合5 kg至200 kg的鹽與該溶解的細胞組成物。於些具體例中’該方法包含加熱一細胞組成物或一溶解的細胞組成物以去乳化該溶解的細胞組成物。於一些具體例中’該細胞組成物或該溶解的細胞組成物係加熱歷時足夠的時間期間用於一鹼及/或一鹽以去乳化一細胞組成物或一溶解的細胞組成物。於一些具體例中，該方法包 3加熱一細胞組成物或一溶解的細胞組成物歷時至少5分鐘，至少10分鐘，至少20分鐘，至少30分鐘，至少1小時，至少2小時’至少4小時，至少8小時，至少12小時，至少18 小時，至少24小時，至少3〇小時，至少36小時，至少42小時，至少48小時，至少54小時，至少6〇小時，至少66小時， 52 201200591 至少72小時，至少78小時，至少84小時，至少90小時或至少96小時。於一些具體例中，該方法包含加熱一溶解的細胞組成物歷時5分鐘至96小時，5分鐘至4小時，5分鐘至2小時，5分鐘至1小時，10分鐘至4小時，1〇分鐘至2小時，ι〇分鐘至1小時，1小時至卯小時，i小時至84小時，i小時至 72小時’ 1小時至60小時，i小時至48小時，丨小時至36小時， 1小時至24小時，1小時至4小時，4小時至96小時，4小時至 84小時，4小時至72小時’ 4小時至60小時，4小時至48小時， 4小時至36小時，4小時至24小時，8小時至96小時，8小時至84小時，8小時至72小時，8小時至60小時，8小時至48小時’ 8小時至36小時’ 8小時至24小時，8小時至12小時，12 小時至96小時，12小時至84小時，12小時至72小時，12小時至60小時’ 12小時至48小時，12小時至36小時，12小時至24小時，24小時至96小時，24小時至84小時，24小時至 72小時，24小時至60小時’ 24小時至48小時，或24小時至 36小時。於一些具體例中，一細胞組成物或一溶解的細胞組成物可以於至少10°C，至少20。(：，至少25°c，至少30°c，至少35°C，至少40°C ’至少45°C，至少50°C，至少55°C，至少60°C ’至少65°C，至少70°C，至少75。(：，至少80°C，至少85°C ’至少90°C ’至少95°C，或至少1〇〇。(：的溫度來加熱。於一些具體例中，一方法包含於UTC至l〇(TC , lot至90 °C，l〇°C 至80t：，10°C 至70°c ’20。(：至 loot：，20°c 至901， 20°C 至80°C，2(TC 至70t，30°C 至 100。〇，3(TC 至90。(：，30 53 201200591 。(：至80°C，3〇°C 至70°C，40°C 至 lOOt，40。(：至90°C，40°C 至80°C，50°C 至 l〇〇eC，50°C 至90°C，50°C 至 80°C，50°C 至 7〇°C，60°C 至 l〇0°C，6〇°C 至90°C，60°C 至80°C，7〇°C 至 100 °C，70°C 至9〇°C，80°C 至 l〇〇°C，80°C 至90。(：，或90°C 至 100 °c的溫度來加熱一細胞組成物或一溶解的細胞組成物。於一些具體例中，一鹽可以在該加熱期間添加至該細胞組成物或該溶解的細胞組成物。於一些具體例中，一細胞組成物或一溶解的細胞組成物可以於一密閉系統中或於具有一蒸發器的一系統中予以加熱。於一些具體例中，一細胞組成物或溶解的細胞組成物可以於具有一蒸發器的一系統中予以加熱以致於存在於該細胞組成物或該溶解的細胞組成物之内的一部分的水係藉由蒸發予以移除。於一些具體例中，一方法包含加熱一細胞組成物或一溶解的細胞組成物於具有一蒸發器的一系統中來移除高至 1%，5%，10%，15%，20%，25%，30%， 35% ’ 40%，45%或50%重量計之存在於該細胞組成物或溶解的細胞組成物之中的水。於一些具體例中，一方法包含加熱一細胞組成物或一溶解的細胞組成物於具有一蒸發器的一系統中來移除1%至50%，1%至45%，1%至40%，1%至 35%，1%至30%，1%至25%，1%至20%，1%至 15%，1%至 10〇/〇，1%至 5%，5%至 50%，5%至45%，5%至40%，5%至 35% ’ 5%至30%，5%至25%，5%至20%，5%至 15%，5%至 10% ’ 10%至50%，10%至45%，10%至40%，10%至35%， 10%至30%，10%至25%，10。/。至20%，10%至 15% , 15%至 54 201200591 50%，15%至45%，15%至40%，15%至35% ’ 15%至30% ’ 15%至25%，15°/。至20%，20%至50%，20%至45%，20%至 40%，20%至35%，20%至30%，20%至25%，25%至50%， 25%至45%，25%至40%，25%至35%，25%至30%，30%至 50% ’ 30%至45% ’ 30%至40%，30%至35%，35%至50%， 35%至45%，35%至40%，40%至50%，40%至45%，或是45% 至 50%。具體例中，該方法包含將—細胞組成物或一溶解的細胞組成物保持於—器皿中料_預定的時間以去乳化該溶解的細胞組成物。於—些具體例中，該方法包含將一細f組成物或-溶解的細胞組成物保持於-器皿中歷時至=分鐘’至㈣分鐘，至少2Q分鐘，至少 1 二’至少2小時’至少4小時，至-小時，至少12小時，至少18小時，至少24小時，至知小時，至少36小時，至少^時’至少48小時’至少54小時，至少叫時，至少 =至少72小時’至少78小時，至少84小時，至少90 =且何。於—些具體财，財純含將一細月組成物或-溶解的細胞組成物保持歷時5分鐘至％小二Γ:至4小時，5分鐘至2小時，5分鐘至1小時，10分分鐘至2小時，分鐘-小時，丨小時至96 =1小輕84小時，丨㈣㈣㈣，丨小時錢小時，小時，1小時至36小時，1小時至24小時，i小時了’ ：小時至96小時’ 4小時至84小時，4小時至72小時，叫時至叫時，4小時至48小時，4小時至％小時，4 55 201200591 小時至24小時，8小時至96小時，8小時至84小時，8小時至 72小時’ 8小時至60小時，8小時至48小時，8小時至36小時， 8小時至24小時，8小時至12小時，12小時至％小時，^小時至84小時，12小時至72小時，12小時至6〇小時，12小時至48小時，12小時至36小時，12小時至24小時，24小時至 96小時，％小時至84小時，24小時至72小時，24小時至6〇小時，24小時至48小時，或24小時至36小時。於一些具體例中，該方法包含使一抗氧化劑接觸一溶解的細胞乳狀液。適合供本發明使用的抗氧化劑包括，作不限於，一生育酚(tocopherol)、一參雙鍵生殖酚、一多酚、白藜蘆醇、一類黃酮類、一類胡蘿動素、番茄紅素、一胡蘿蔔素、葉黃素、抗壞血酸、抗壞血酸棕櫚酸酯，或類似物，以及其等之組合。於一些具體例中，該方法包含允許一經乳化之溶解的細胞組成物靜置，其中該脂質係使用重力而自該經乳化之溶解的細胞組成物予以分離。如本文中所使用的，術語”攪拌（agitating)，，和，，攪拌 (agitation)"提及一種經由施加一力來影響於一溶解的細胞組成物中之運動的方法。於一些具體例中，本發明的方法包含藉由攪動、混合、摻合、振盪、振動，或其等之一組合而來揽拌一細胞組成物或一溶解的細胞組成物。於一些具體例中，攪拌一細胞組成物或一溶解的細胞組成物之方法會去乳化該細胞組成物或該溶解的細胞組成物。於一些具體例中，本發明的方法包含攪拌一溶解的細 56 201200591 胞組成物以0.1 hp/l，000 gal至 l〇 hp/l，000 ga卜 0.5 hp/l，000 gal至8 hp/l，000 gal，1 hp/l，〇〇〇 gai至6 hp/l，000 gal，或是2 hp/l，000 gal至5 hp/l，000 gal之溶解的細胞組成物。於一些具體例中，本發明的方法包含使用一攪拌器來授拌一細胞組成物或一溶解的細胞組成物。於一些具體例中’該攪拌器為一分散型攪拌器，其分散一鹼及/或鹽於該細胞組成物或該溶解的細胞組成物之内。於一些具體例中，一搜拌器具有一個或更多個葉輪。如本文中所使用的， "葉輪"提及一元件，當經配置來在旋轉時授與運動一細胞組成物或一溶解的細胞組成物。適合供本發明使用的葉輪包括直葉片葉輪、拉什頓葉片葉輪（Rusht〇n biade impeller)、軸流葉輪、徑向流葉輪、凹面的葉片盤葉輪、高效能葉輪、螺旋槳、漿' 渦輪機，或類似物，以及其等之組合。於一些具體例中，一方法包括使用一攪拌器來攪拌一細胞組成物或一溶解的細胞組成物，該搜拌器具有9〇 ft/min至 1，200 ft/min、200 ft/min至 1,000 ft/min、300 ft/min 至 800 ft/min、400 ft/min至700 ft/min，或是500 ft/min至600 ft/min的葉輪葉尖速率。於一些具體例中，一方法包括使用一攪拌器來攪拌一細胞組成物或一溶解的細胞組成物，該攪拌器具有350公分/秒至900公分每秒，350公分/秒至850 公分每秒，350公分/秒至800公分/秒，350公分/秒至750公刀/秒，350公分/秒至7〇〇公分/秒，350公分/秒至650公分/ 秒’ 350公分/秒至600公分/秒’ 350公分/秒至550公分/秒， 350公分/秒至5〇〇公分/秒，35〇公分/秒至45〇公分/秒，35〇 57 201200591 公分/秒至400公分/秒，400公分/秒至900公分每秒，400公分/秒至850公分每秒，400公分/秒至800公分/秒，400公分/ 秒至750公分/秒，400公分/秒至700公分/秒，400公分/秒至 650公分/秒，400公分/秒至600公分/秒，400公分/秒至550 公分/秒，400公分/秒至500公分/秒，400公分/秒至450公分/ 秒，450公分/秒至900公分每秒，450公分/秒至850公分每秒，450公分/秒至800公分/秒，450公分/秒至750公分/秒， 450公分/秒至700公分/秒，450公分/秒至650公分/秒，450 公分/秒至600公分/秒，450公分/秒至550公分/秒，450公分/ 秒至500公分/秒，500公分/秒至900公分每秒，500公分/秒至850公分每秒，500公分/秒至800公分/秒，500公分/秒至 750公分/秒，500公分/秒至700公分/秒，500公分/秒至650 公分/秒，500公分/秒至600公分/秒，500公分/秒至550公分/ 秒，550公分/秒至900公分每秒，550公分/秒至850公分每秒，550公分/秒至800公分/秒，550公分/秒至750公分/秒， 550公分/秒至700公分/秒，550公分/秒至650公分/秒，550 公分/秒至600公分/秒，600公分/秒至900公分每秒，600公分/秒至850公分每秒，600公分/秒至800公分/秒，600公分/ 秒至750公分/秒，600公分/秒至700公分/秒，600公分/秒至 650公分/秒，650公分/秒至900公分每秒，650公分/秒至850 公分每秒，650公分/秒至800公分/秒，650公分/秒至750公分/秒，650公分/秒至700公分/秒，700公分/秒至900公分每秒，700公分/秒至850公分每秒，700公分/秒至800公分/秒， 700公分/秒至750公分/秒，750公分/秒至900公分每秒，750 58 201200591 公分/秒至叫分每秒，公分/秒麵公分/秒，800 公至9〇〇公分每秒，800公分/秒至叫分每秒，或是850 ”广至9〇〇公分/秒的葉輪葉尖速帛。如本文中所使用葉輪葉*速率”提及葉輪的最外部分之速率，當其環繞其之中央軸旋轉時。於些具體例中，該授拌（以及選擇性地如本文中所說月之額外的步驟)係於含有—葉輪之容器中執行，其中該葉輪直徑對該容器體積之比率為〇.1至0.5，0.1至〇.4，0.2至〇·5 ’ 〇·2至〇.4 ’ 〇 3至〇 5，或是〇 3至〇 4。於—些具體财，該搜拌（以及選擇性地如本文中所說明之額外的步驟)係於含有-葉輪之容器中執行，其中該葉輪直徑對該容器内徑之比率為至少〇25，至少〇34，至少 0.65 0.25至〇_65 ’ 0.25至0.33 ’ 0.3至〇 6，0.3至〇·5，0.3至 0.4 0.34至0.65，0.34至0.6 ’ 0.34至〇·55，0.37至0.55，0.4 至0.65，0.4至0.6，0.4至0.5，或是〇.42至〇 55。於些具體例中，祝拌包含混合一細胞組成物或一溶解的細胞組成物以致該細胞組成物或該溶解的細胞組成物被放置於由以下所說明的流動條件下：1〇至1〇,〇〇〇, ^000 至 10,000，1，500至 10,000，或是2,000至 10,000的雷諾數。於一些具體例中，一溶解的細胞乳狀液在該搜拌的期間具有2,000或更多，3,000或更多’或是5,0〇〇或更多，或是2 〇〇〇至 10,000 ’ 3,000至 10,000，或是5,000至 1〇,000的雷諾數。於一些具體例中’ 一方法包含攪拌一細胞組成物或一溶解的細胞組成物歷時至少5分鐘，至少1〇分鐘，至少2〇分 59 201200591 鐘，至少30分鐘，至少！小時，至少2小時至少4小時，至少8小時’至少12小時，至少18小時，至少24小時，至少3〇小時’至少36小時’至少42小時，至少仙小時至少54小時，至少60小時，至少66小時，至少72小時至少78小時，至少84小時，至少9〇小時或至少96小時。於一些具體例中， -方法包含搜拌-細胞組成物或一溶解的細胞組成物歷時 5分鐘至96小時，5分鐘至4小時’ 5分鐘至2小時，5分鐘至i 小時，10分鐘至4小時，10分鐘至2小時，1〇分鐘至丨小時， 1小時至96小時，1小時至84小時，1小時至72小時，】小時至60小時’ 1小時至48小時，1小時至36小時，1小時至24小時，1小時至4小時，4小時至96小時，4小時至84小時，4小時至72小時’ 4小時至6〇小時，4小時至48小時，4小時至36 J、時4小時至24小時，8小時至96小時，8小時至84小時， 8小時至72小時，8小時至60小時，8小時至48小時，8小時至36小時，8小時至24小時，8小時至12小時，12小時至96 小時’ 12小時至84小時，12小時至72小時，12小時至60小時’ 12小時至48小時，12小時至36小時，12小時至24小時， 20小時至40小時，24小時至96小時，24小時至84小時，24 小時至72小時’ 24小時至6〇小時，24小時至48小時，或24 小時至36小時。於些具體例中，一方法包含同時地搜拌與加熱一細胞組成物或一溶解的細胞組成物以去乳化該細胞組成物或該溶解的細胞組成物。於一些具體例中，一方法包含攪拌一細胞組成物或一溶解的細胞組成物於至少10°C，至少20 60 201200591 °C，至少25°C，至少30°C，至少35°C，至少40°C，至少45 °C，至少50°C，至少55°C，至少60°C，至少65°C，至少70 °C，至少75°C，至少80°C，至少85°C，至少90°C，至少95 °C，或至少l〇〇°C的溫度。於一些具體例中，一方法包含攪拌一細胞組成物或一溶解的細胞組成物於10°C至l〇〇°C，10 它至90°(：，l〇°C 至80°C，l〇°C 至70°C，20°C 至 l〇〇°C，20°C 至90°C，20°C 至80°C，20°C 至70°C，30°C 至 l〇〇°C，30°C 至 90°C，30°C 至 80°C，30°C 至 70°C，40°C 至 l〇〇°C，40°C 至90 °C，40°C 至 80°C，50°C 至 l〇〇°C，50°C 至90°C，50°C 至 80°C， 50°C 至70°C，60°C 至 l〇〇°C，60°C 至90°C，60°C 至80°C，70 。(：至 l〇〇°C，70°C 至90°C，80°C 至 l〇〇〇°C，80°C 至90°C，或 90°C至l〇〇°C的溫度。於一些具體例中，形成一溶解的細胞組成物，使一溶解的細胞組成物接觸驗或使一溶解的細胞組成物的pH提升，使一溶解的細胞組成物接觸鹽，加熱該溶解的細胞組成物，以及攪拌一溶解的細胞組成物之各種組合可以發生於一單一器皿内。於一些具體例中，形成一細胞組成物，使一細胞組成物接觸驗或使一細胞組成物的pH提升，使一細胞組成物接觸鹽，加熱該細胞組成物，以及授拌一溶解的細胞組成物之各種組合可以發生於一單一器皿内。於一些具體例中，該單一器皿包括一發酵器皿。於一些具體例中，該發酵器皿可以具有至少20,000公升，至少50,000公升，至少100,000公升，至少120,000公升，至少150,000公升，至少200,000公升，或至少220,000公升的體積。於一些 61 201200591 具體例中，該發酵器皿可以具有20,000公升至220,000公升，20,000公升至100,000公升，20,000公升至50,000公升， 50,000公升至220,000公升，50,000公升至150,000公升， 50,000公升至1〇〇,〇〇〇公升，1〇〇,〇〇〇公升至220,000公升， 100,000公升至150,000公升，100,000公升至120,000公升， 150,000公升至220,000公升，150,00〇公升至200,000公升，或是200,000公升至220,000公升的體積。於一些具體例中’形成於一器皿中之一數量的細胞組成物或溶解的細胞組成物可以被轉移至一或更多個搜拌器皿内。於一些具體例中’該等攪拌器盟可具有至少2〇,〇〇〇公升，至少30,000公升，至少40,〇〇〇公升或至少5〇,〇〇〇公升的體積。於一些具體例中，該等攪拌器皿可具有2〇,〇〇〇公升至50,000公升，20,000公升至4〇〇〇〇公升，2〇〇〇〇公升至 3〇,〇〇〇公升，30,000公升至50,000公升，3〇〇〇〇公升至4〇〇〇〇公升或是40,〇〇〇公升至50,〇〇〇公升的體積。於一些具體例中，該等_器皿可具有下列性質的任何組合。於一些具體例中’該等搜拌器皿可具有的2個葉輪。於-些具體例中’該等葉輪為执什頓葉片葉輪。於一些具體例中，該等葉輪係以少等於最小的葉輪的-直徑之距離彼此分離。於-些具體例中，該等葉輪為3〇英忖至 4〇央吋，33英吋至37英吋，33英崎，υ — “ 、寸34央吋，35英吋，36 央吋或37英吋從尖端至尖端。。。目士上具體例中，該等攪拌窃迎具有至少1〇〇〇〇公升，至少〇,000公升，至少30,〇〇〇公升，至夕40,〇〇〇公升或至少5〇〇〇 △升的體積。背'—些具體 62 201200591 =騎授拌益皿具有9〇英时至⑽英时，％英叶至二’ 98外"射，，⑻料，或是1〇2英忖些具體例中，第-個葉輪係座落於灣 15料至2G料，丨6英W糾，或是央忖至18英相及第二個葉輪係座落於第-個葉輪之上的6〇英叶獅Η，65英料75射’ 68英叶，仍英忖， 7〇英忖，71糾’ 72糾，73英十7則，或是75英时。於-些具體例中’一溶解的細胞組成物係以至少5〇啊，至少60啊，或至少70rpm予以攪拌。於—此具體例中，一溶解的細胞組成物係以50啊至70 rpm、50 rpm至6〇啊，或是60rpm至7〇rpm予以授拌。於1具體例中，该細胞組成物、該溶解的細胞組成物，或該脂質係從一器皿予以收穫，其係藉由從該器皿來菜抽該細胁絲、贿解的細触祕，或該脂質。於 —些具體例中，該細胞組成物、該溶解的細胞組成物，或该脂質係從-器皿^以收穫而不祕該器皿。於一些具體例中，該細胞組成物、該溶解的細胞組成物，或該脂質係從-器皿予以收穫，其係從該器皿來果抽，而不，該細胞組成物、該溶解的細胞組成物，或該脂質。於一些具體例中’該細齡絲、贿解的細胞組錢，或該脂質係從-器皿予以收穫而不吹氣。於—些具體例中，藉由以上所說_技術來收穫該細胞組成物、該溶解的細胞组成物’或該脂質導致—粗製脂質，其具有低的甲氧苯胺值(例如，26或更少，25或更少，2〇或更少，1S或更少，ι〇或更 63 201200591 少，5或更少’ 2或更少，或是1或更少）及/或低的磷含量(例如，100 ppm或更少，95 ppm或更少’90 ppm或更少，85 ppm 或更少，80 ppm或更少，75 ppm或更少’ 70 ppm或更少， 65 ppm或更少’ 60 ppm或更少’ 55 ppm或更少，50 ppm或更少’45 ppm或更少，40 ppm或更少，35 ppm或更少，30 ppm 或更少，25 ppm或更少，20 ppm或更少，15 ppm或更少， 10 ppm或更少，5 ppm或更少，4 ppm或更少，3 ppm或更少， 2 ppm或更少’或是1 ppm或更少）。如本文中所說明的，本發明使用使一溶解的細胞組成物接觸一第一個驗或使一溶解的細胞組成物的pH提升，使一溶解的細胞組成物接觸鹽，加熱一溶解的細胞組成物，以及授拌一溶解的細胞組成物之各種組合來提供一經處理之溶解的細胞組成物。如本文中所說明的，本發明使用使一細胞組成物接觸一第一個鹼或使一細胞組成物的pH提升，使一細胞組成物接觸鹽，加熱一細胞組成物，以及撥拌一細胞組成物之各種組合來提供一經處理之細胞組成物。該經處理之細胞組成物或經處理之溶解的細胞組成物與一未經處理之細胞組成物或經處理之溶解的細胞組成物比較為至少部分去乳化的。因而，一經處理之細胞組成物或經處理之溶解的細胞組成物可以被放置於一離心機内以及可以由其分離出一脂質。於一些具體例中，在使一細胞組成物或溶解的細胞組成物的pH提升之後’例如，藉由接觸一第—個鹼，該加熱該細胞組成物或溶解的細胞組成物及/或該攪拌該細胞組 64 201200591 成物或溶解的細胞組成物可以降低該經處理之細胞組成物或經處理之溶解的細胞組成物之pH。為了使一脂質更有效地藉由離心而自一經處理之細胞組成物或經處理之溶解的細胞組成物分離，該經處理之細胞組成物或經處理之溶解的細胞組成物之pH係被再度提升，例如，藉由使該經處理之細胞組成物或該經處理之溶解的細胞組成物與第二個鹼接觸。於一些具體例中，使一經處理之溶解的細胞組成物接觸第二個鹼會使該經處理之細胞組成物或該經處理之溶解的細胞組成物之pH提升。於一些具體例中，一經處理之細胞組成物或一經處理之溶解的細胞組成物係與第二個鹼接觸以使該經處理之細胞組成物或該經處理之溶解的細胞組成物之pH提升至7或以上，7.5或以上，8或以上，8.5或以上，9或以上，9.5或以上，1〇或以上，10.5或以上，11或以上’ 11.5或以上，或是12或以上。於一些具體例中，一經處理之細胞組成物或一經處理之溶解的細胞組成物係與第二個鹼接觸以使該經處理之溶解的細胞組成物之pH提升至7 至 13，7至 12 ’ 7至 11，7至 10，7至9，7至 8，7至 7.5，7.5 至 8，8至 13，8至 12，8至 11，8至 10，8至9，8至 8_5，8.5 至9，9至 12，9至 11，9至 10，9至9.5，9.5至 10，1〇至 12，或10至11 。於一些具體例中，一經處理之細胞組成物或一經處理之溶解的細胞乳狀液之pH為7或更少，6或更少，5或更少， 4或更少，或是3或更少。本發明的方法包含自一經處理之細胞組成物或一經處 65 201200591 理之溶解的細胞組成物分離一脂質。於一些具體例中，一脂質係自一溶解的細胞乳狀液而分離，在一溶解的細胞乳狀液與第二個鹼接觸之後，在該攪拌一溶解的細胞乳狀液之後，或是在使一溶解的細胞乳狀液接觸一鹽之後，其係藉由，舉例而言，允許該經處理之溶解的細胞乳狀液靜止歷時足夠一脂質自該經處理之溶解的細胞乳狀液分離的一時間期間（例如，為一分離的層）。該脂質可以隨後被移除，舉例而言，藉由傾析、撇除、真空吸除、泵抽、吸除、抽除、虹吸，或是用別的方法自該經處理之溶解的細胞乳狀液的表面移除該脂質。於一些具體例中，該分離包含離心一經處理之細胞組成物或一經處理之溶解的細胞組成物（例如，於30°C至1〇〇 °C的溫度），藉此該離心自該經處理之細胞組成物或該經處理之溶解的細胞組成物分離一脂質。於一些具體例中，一種方法包含離心一經處理之細胞組成物或一經處理之溶解的細胞組成物於至少10°c，至少 20°C，至少25°C，至少30°C，至少35°C，至少40°C，至少 45°C，至少50°C，至少55°C，至少60°C，至少65°C，至少 70°C，至少75°C，至少80°C，至少85°C，至少90°C，至少 95°C，或至少l〇〇°C的溫度。於一些具體例中，一種方法包含離心一經處理之細胞組成物或一經處理之溶解的細胞組成物於 10°C 至 100°c，10°c 至90°c，10°c 至80°c，20°c 至 1〇〇 °C，20°C 至90°C，20°C 至80°C，25°C 至 l〇〇°C，25°C 至90°C， 25°C 至80°C，25°C 至75°C，30°C 至 l〇〇°C，30°C 至90°C，30 66 201200591 °〇至80°〇，40°C 至 l〇〇°C，40°C 至90°C，40°C 至80°C，50°C 至 l〇〇°C，50。(：至90°C，50°C 至8(TC，50°C 至70°C，60°C 至 100°C，60〇C 至 90°C，60°C 至 80°C，60°C 至 70°C，70°C 至 100 °C，或是70°C至90°C的溫度。於一些具體例中，離心係以1公斤每分鐘(kg/min)至500 kg/min，1 kg/min至400 kg/min，1 kg/min至300 kg/min，1 kg/min至 200 kg/min，1 kg/min至 100 kg/min，1 kg/min至 75 kg/min ’ 1 kg/min至 50 kg/min，1 kg/min至 40 kg/min，1 kg/min至30 kg/min，1 kg/min至25 kg/min，1 kg/min至 10 kg/min，10 kg/min至500 kg/min，10 kg/min至400 kg/min， 10 kg/min至300 kg/min，10 kg/min至200 kg/min，10 kg/min. 至 100 kg/min，10 kg/min 至 75 kg/min，10 kg/min 至 50 kg/min，10 kg/min至40 kg/min，10 kg/min至30 kg/min，20 kg/min至500 kg/min，20 kg/min至400 kg/min，20 kg/min至 300 kg/min，20 kg/min 至 200 kg/min，20 kg/min 至 100 kg/min，20 kg/min至75 kg/min，20 kg/min至50 kg/min，20 kg/min至40 kg/min，25 kg/min至500 kg/min，25 kg/min至 400 kg/min，25 kg/min 至 300 kg/min，25 kg/min 至 200 kg/min，25 kg/min至 100 kg/min，25 kg/min至 75 kg/min， 25 kg/min至50 kg/min，30 kg/min至60 kg/min，30 kg/min 至 50 kg/min，30 kg/min 至 40 kg/min，50 kg/min 至 500 kg/min，100 kg/min至 500 kg/min，或是200 kg/min至 500 kg/min的（一經處理之細胞組成物或一經處理之溶解的細胞組成物至一微量離心機内之）一進料速率予以實施。 67 201200591 用於S亥分離所需要的總時間可以取決於該經處理之細胞組成物或經處理之溶解的細胞組成物的體積而變化。分離(例如，離心時間）之典型的總時間為至少〇.丨小時，至少 0.2小時，至少0.5小時，至少1小時，至少2小時，至少4小時，至少6小時，至少8小時，至少10小時，至少丨2小時，或0.1小時至24小時，0.5小時至24小時，1小時至12小時，2 小時至10小時，或4小時至8小時。於一些具體例中’本發明的一種方法包含wl，〇〇〇 g至 25.000 g，1，〇〇〇 g至2〇,〇〇〇 g，l，〇〇〇 g至 lo o。。g，2,〇〇〇 g 5.25.000 g > 2,000 g^.20,000 g - 2,000 gJL 15,000 g - 3,000 g 至25,000 g ’ 3,000 g至20,000 g，5,000 g至25,000 g，5,000 g 至20,000 g ’ 5,000 g至 15,000 g，5,000 g至 l〇,〇〇〇 g，5,000 g 至8,000 g ’ l〇,〇〇〇 g至25,000 g, 15,000 g to 25,000 g，或至少 1，000 g，至少2,000, g，至少4,000 g，至少5,000 g，至少 ?，000 g，至少 8,000 g，至少 10,000 g，至少 15,000 g，至少 20.000 g，或至少25,000 g的離心力來離心一經處理之細胞組成物或一經處理之溶解的細胞組成物。如本文中所使用的’ "g"提及標準重力或大概9.8m/s2。於一些具體例中，本發明的一種方法包含以4,000 rpm至14,000 rpm，4,000 rpm 至 10,000 rpm，6,000 rpm至 14,000 rpm，6,000 rpm至 12,000 rpm，8,000至 14,000 rpm，8,000 rpm至 12,000 rpm，或是8,000 rpm至1 〇,〇〇〇 rpm來離心一經處理之細胞組成物或一經處理之溶解的細胞組成物。於一些具體例中，本發明的一種方法包含在自一經處 68 201200591 理之細胞組成物或一經處理之溶解的細胞組成物分離該脂質之後乾燥一脂質俾以從該脂質移除水。於一些具體例中，乾燥該脂質可以包括，但不限於，加熱該脂質以蒸發水。於一些具體例中，在乾燥之後，該脂質具有少於3〇/〇，少於2.5%，少於2%，少於1.5%，少於1%，少於0.5%，少於0.1%，或是〇%以脂質之重量百分比計之水含量。於一些具體例中，在乾燥之後’該脂質具有〇%至3%，0%至2.5%， 0%至2%，〇%至 1.5%，0%至 1%，〇%至0.5%，〇_1%至3%， 0.1%至2.5%，〇.1%至2%，〇.1%至1.5%，〇.1%至1%，〇.1〇/0 至0.5°/〇,0.5%至3%，0.5%至2.5%，0·5%至2%，0.5%至 1.5%， 0.5%至 10/〇，1〇/〇至3%，1〇/0至2.5%，1%至2%，1%至 1.5%， 1.5%至3%，1.5%至2.5%，1.5%至2%，2%至3%，2%至2.5%，或是2.5%至3%以脂質之重量百分比計的水含量。於一些具體例中，一方法進一步包含精製一脂質，其係藉由選自於苛性精製法、去膠、鹼精製法、漂白、去臭、脫酸，或類似物’以及其等之組合的一或更多個方法，以移除一或更多個磷脂質、游離脂肪酸、磷脂、彩色物體、固醇、氣味，以及其他的雜質。如本文中所使用的，一，，精製油"為已經經精製的一粗製脂質或粗製油。如本文中所使用的’ ”一粗製脂質"或”一粗製油"為還沒有經精製的一脂質或油。於一些具體例中’自一去乳化的細胞組成物所分離的該脂質為一粗製脂質。本發明之各種的示範的方法係綱要性地說明於第1-4 圖中。參見第1圖，於一些具體例中，本發明針對一種供用 69 201200591 於自一細胞（101)獲得一脂質（11〇)的方法（1〇〇)，其包含溶解 (102)忒細胞（1〇1)以形成—溶解的細胞組成物。使該溶解的細胞組成物與-第—個驗接觸（1G4)以去乳化溶解的細胞組成物(1〇3)，使與一鹽接觸(1〇5)以去乳化溶解的細胞組成物(103) ’以及予以加熱(1()6)，例如，歷時1()分鐘至叫時，以提供一經處理之溶解的細胞組成物（1〇7)。使該經處理之溶解的細胞組成物（1〇7)與一第二個鹼接觸（108)以及例如，於10C至i〇〇°c的溫度，予以分離(1〇9)以提供— 脂質（110)。參見第2圖，於一些具體例中，本發明針對一種供用於自—細胞獲得一脂質(210)的方法(2⑽），該方法包含溶解 (02) ’細胞(101)以形成一溶解的細胞組成物⑽3)。該溶解的細胞組成物接而與—驗接觸(2G4)以去乳化溶解的細胞組成物（103)以及來提供—經處理之溶解的細胞組成物 (2〇7)。該經處理之溶解的細胞組成物(2〇7)，例如，於1〇它至的溫度，予以分離(209)以提供一脂質（21〇卜參見第3圓，於一些具體例中，本發明針對自一細胞獲得-脂質⑽)的方法該方法包含溶解 ⑽)-細胞⑽)以形成—溶解的細胞組成物⑽)。該溶解的細胞組成物接而與__鹽接觸(狗以絲化溶解的細胞組成物（103)以及來提供—經處理之溶解的細胞組成物 _)，其係’例如’於⑽至赋的溫度，予以分離⑽) 以提供一脂質（310)。參見第4圖，於一些具體例中，本發明針對一種供用於 201200591 自一細胞獲得一脂質（410)的方法（400)，該方法包含溶解 (102)—細胞(ιοί)以形成一溶解的細胞組成物（1〇3)。該溶解的細胞組成物接而與一鹽接觸（405)以去乳化溶解的細胞組成物（103)以及予以攪拌（401)，例如，歷時5分鐘至96小時，以及選擇性地予以加熱（402)，以提供一經處理之溶解的細胞組成物(407)。該經處理之溶解的細胞組成物接而，例如，於至100°C的溫度’予以分離(409)以提供一脂質（410)。於一些具體例中，本發明的一方法包含濃縮含有一微生物細胞的一肉湯，含有植物材料的一肉湯及/或濃縮一溶解的細胞組成物。如本文中所使用的，"濃縮"提及自一組成物移除水。濃縮可以包括，但不限於，蒸發、化學乾燥、離心，與類似物，以及其等之組合。於一些具體例中，含有一微生物細胞的一肉湯或是含有植物材料的一肉湯係予以濃縮以提供至少4%，至少5〇/0，至少10°/。，至少15% ’至少20%，至少25%，或至少30%重量計之肉湯的脂質濃度。於一些具體例中，含有一微生物細胞的一肉湯或是含有植物材料的一肉湯係予以濃縮以提供4%至40°/。’ 4°/。至30%，4%至20%，4%至 15%，5%至40%， 5〇/〇至30%，5%至20%，10%至40%，10%至30%，10%至20%， 15%至40%，15%至30%，20%至40%，20%至30%，25%至 40%，或是30%至40%重量計之肉湯的脂質濃度。於一些具體例中’ 一細胞組成物或一溶解的細胞組成物係予以濃縮以提供至少4%，至少5%，至少10%，至少 15%，至少20%，至少25%，或至少30%重量計之該溶解的 71 201200591 細胞組成物的脂質濃度。於一些具體例中，一細胞組成物或一溶解的細胞組成物係予以濃縮以提供4%至40%，4%至 30〇/〇 ’ 4°/。至20%，4%至 15%，5%至40%，5%至30%，5%至 20%，1〇%至4〇%，1〇%至3〇%，1〇。/0至2〇〇/〇，15%至40%， 15%至30%，20%至40%，20%至30%，25%至40%，或是30% 至40%重量計之該溶解的細胞組成物的脂質濃度。於一些具體例中，藉由本發明的方法所製備之一脂質具有2或更少的總香氣強度。如本文中所使用的，術語"總香氣強度"提及經由一組感覺分析物所給予該脂質的嗅覺感覺等級。如本文中所使用的，術語•，感覺分析物，，提及一經訓練的個體’其提供了對於一物質之感覺特徵的回饋及/ 或評價一物質之感覺特徵。於一些具體例中’藉由本發明的方法所製備之一脂質具有3或更少的總芳香強度。如本文中所使用的，術語"總芳香強度"提及經由—組感覺分析物所給予該脂質的味覺 (gustatory)，或是味覺(taste) ’感覺等級。於一些具體例中，通用型光譜描述性的分析方法係使用來估定樣品之香氣和芳香特徵。此方法使用〇- 15的強度規模，該處〇=無檢測出以及15 =非常高的強度，來測量油之香氣和芳香的屬性。於一些具體例中’藉由本發明的方法所製備之一脂質不會具有特徵如同魚的一餘味。如本文中所使用的，術語 ”餘味”提及該脂質的味道的感覺持續，如同藉由一組感覺分析物予以特徵化的。於一些具體例中’本發明的方法提供了一粗製脂質， 72 201200591 其具有5或更少，4·5或更少，4或更少，3.5或更少，3或更少，2.5或更少’ 2或更少，1.5或更少，1或更少，〇.5或更少，0.2或更少’或是〇.1或更少的過氧化物值(Ρν)β如本文中所使用的’術語"過氧化物值，，或是”PV"提及在該脂質氧化的整個期間出現的初級反應產物，例如過氧化物及氫過氧化物，之測量。於一些具體例中，PV為該脂質的品質以及已發生於該脂質的氧化程度之一指標，具有低的PV(亦即，5或更少）展現出比具有大於5的PV之脂質增高的穩定性與感覺剖繪。於一些具體例中’添加鹼至一溶解的細胞組成物，如以上所討論的，使該溶解的細胞組成物之ρΗ提升以及抑制脂質的氧化，藉此最小化於該溶解的細胞組成物内之自由基的量以至於從本發明的方法獲得的該粗製脂質具有低的PV(亦即，5或更少）。於一些具體例中，本發明的方法提供了一粗製脂質，其具有26或更少，25或更少，20或更少，15或更少，1〇或更少’5或更少，2或更少，或是1或更少之曱氧苯胺值(Av)。如本文中所使用的，術語"甲氧苯胺值，，或是"AV"提及在該脂質氧化的整個期間出現的次級反應產物，例如醛和酮，之測量。於一些具體例中，AV為該脂質的品質以及已發生於邊脂質的氧化程度之一指標。具有低的AV(亦即，％或更少）之—脂質展現iB比具有大於26的AV之脂質增高的穩定吐與感覺n於-些具體射’添加驗至—溶解的細胞组成物’如以上所討論的，使該溶解的細胞組成物之pH提升、及抑物gf·的氧化，藉此最小化於該溶解的細胞組成 73 201200591 物内之自由基的量以至於從本發明的方法獲得的該粗製脂質具有低的AV(亦即，26或更少）。於一些具體例中，本發明的方法提供了一粗製脂質’ 其具有100 ppm或更少，95 ppm或更少，90 ppm或更少，85 ppm或更少，80 ppm或更少，75 ppm或更少，7〇 ppm或更少，65 ppm或更少，60 ppm或更少，55 ppm或更少，50 ppm 或更少，45 ppm或更少，40 ppm或更少，35 ppm或更少， ' 30 ppm或更少，25 ppm或更少，20 ppm或更少’ 15 ppm或更少’ 10 ppm或更少，5 ppm或更少，4 ppm或更少，3 ppm 或更少’ 2 ppm或更少，或是1 ρριη或更少的磷含量。於一些具體例中，本發明的方法提供了一粗製脂質，其具有比設若使用一溶劑（例如，非典型的（atypical)己烧萃取或是FRIOLEX® 方法（Westfalia Separator AG，Germany)) 來執行的萃取有更低的甲氧苯胺值、更低的過氧化物值、更低的磷含量及/或更高的萃取產量》FRI0LEX®方法，其為一種用一水溶性有機溶劑來萃取脂質的方法，如同美國專利案唬5,928,696與國際公開案號w〇 oi/76385與W0 01/76715之中所說明的，其等之各個係以其之整體併入本文中以作為參考資料。於一坚吳體例中刀口热该溶解的細胞組成物引發：反應產物（例如，路和酮）參與相似於梅納反應（璧麗㈣之-反應與存在於該_的細胞組成物之中< 白質-起。該反應據信產生了擁有抗氧化劑活性的產《其降低該脂質的氧化。於一些具體例中，可以添加氣 74 201200591 蛋白質，例如’大豆蛋白質，至該溶解的細胞組成物加抗氧化劑活性。ticm in於該脂質的氧化之降低滅少二增質的AV、減少了該脂質的任何餘味及/或増加該㈣:該贿性。於-些具體例中’穩定性增加至少5%，至少、穩定少15%或至少20%。。’至於-些具體射’-種藉由本發明的一方法脂質、在該脂質的萃取之後剩餘的生質，或其等之*取的以直接地使用作為一食品或食品的成分，例如合可品、嬰兒配方奶粉、飲料、醬汁、牛奶為主的食品（你兒食奶、酸路乳、乾酷與冰淇淋）、油（例如，烹調油或如牛以及烘焙食物中的一成分；營養補給品（例如，以膠橐酱）’ 劑形式）；供用於任何非人類動物之飼料或飼料補>充如，其等之產物(例如，肉、牛奶，或是雞蛋)係供人:(例之該等）；食物補充品：以及藥物（以直接或附加物療4耗用）0術語，，動物"提及任一種屬於動物界之生物，以、法應任何人類動物，及任何非人類動物，其等衍生出^包括如，牛奶、雞蛋、家禽肉、牛肉、豬肉或美羊肉）。具體例中，該脂質及/或生質可以使用於海產食品中。些食品係衍生自，沒有限制，魚、瑕與貝。術語,,產物產衍生自此等動物之任何產物，含括，沒有限括』雖蛋"、牛奶或其他的產物。當將該脂質及/或生質餵此等動物日、多不飽和脂質可被併入該肉、牛奶、雞蛋或此等動物夺，它產物中以增加此等脂質之含量。之其微生物脂質 75 201200591 於一些具體例中，本發明係針對一種依據本發明的方法所萃取之微生物脂質。於一些具體例中’ 一種粗製的微生物脂質具有26或更少’ 25或更少’ 20或更少’ 15或更少， 10或更少，5或更少’ 2或更少’或是1或更少之甲氧苯胺值’ 及/或5或更少，4.5或更少，4或更少，3.5或更少，3或更少， 2.5或更少，2或更少’ 1.5或更少’ 1或更少’ 〇·5或更少， 0.2或更少，或是0.1或更少的過氧化物值’及/或100 PPm4 更少，95 ppm或更少’ 90 ppm或更少’ 85 ppm或更少’ 80 ppm 或更少，75 ppm或更少，70 ppm或更少，65 ppm或更少， 60 ppm或更少，55 ppm或更少，50 ppm或更少，45 ppm或更少，40 ppm或更少’ 35 ppm或更少，30 ppm或更少，25 ppm 或更少，20 ppm或更少，15 ppm或更少，10 ppm或更少，5 ppm或更少，4 ppm或更少，3 ppm或更少，2 ppm或更少，或是1 ppm或更少的填含量。於一些具體例中，該粗製的微生物脂質具有少於5%，少於4%，少於3%，少於2%，或少於1%重量計或體積計的有機溶劑。於一些具體例中，該粗製的微生物脂質具有至少10%，至少15%，至少20%，至少 25%，至少30%，至少35%，至少40%，至少45%，或至少 50%重量計之所欲的PUFA。於一些具體例中，該粗製的微生物脂質具有至少10%，至少15%，至少20%，至少25°/〇，至少30%，至少35°/。，至少40%，至少45%，或至少50。/〇重量计之DHA ’及/或至少1 〇%，至少15%，或至少20%重量計之DPA η-6 ’及/或至少10%，至少15%，或至少20%重量計之EPA，及/或至少10%，至少15°/。，至少20%，至少25%， 76 201200591 至少30%，至少35%，至少卿。，至少娜，或至少5〇%重量計之舰。於-些具體例中，—種依據本發明的方法所萃取之粗製的微生㈣質會導致比設若❹—溶劑（例如，一典型的己院萃取或是fri〇lex⑧方法（勤她BioSpin, Billerica, ΜΑ) was detected. In some embodiments, a sample of the dissolved cellular composition is diluted with deionized water at a ratio of about 1:1 prior to measuring the EPR. In some embodiments, to measure EpR, a spin trapping chemical is added to the sample of the dissolved cellular composition. In some embodiments, the spin trapping chemical is any spin trapping chemical known in the art, including, but not limited to, Ρ0ΒΝ(α_(4_ ° pyridine-1-oxide)-Ν - Tert-butyl butyl ketone) or DMPO (5,5'-dimethyl 48 201200591 _ specific porphyrin-N-oxide). In some embodiments, the spin trap chemistry is about 1. 25 Μ and about 50 pL were added to the dissolved cell composition of about 0. 5 grams of sample. In some embodiments, a sample containing the spin trapping chemistry is incubated at room temperature (eg, about 20. 〇. In some embodiments, the following spectrometer parameters are used: a modulation frequency of about 86 Hz, about 2 Gaussian modulated vibration, microwave power of approximately 5 mW, time constant of approximately 2 〇 seconds, sweep time of approximately 10 seconds, sweep width of approximately 1 〇〇 Gauss, and number of scans of approximately 8. EPR is accompanied by The time is measured to determine the concentration of free radicals present in the lipid. In some embodiments, the EpR is measured hourly during the course of 4 hours. In some embodiments, the dissolved cellular composition is above The listed parameters have less than 〇i5 X 1〇6 ' less than 0. 14 X 1〇6, less than 0. 13 X 1〇6, less than 〇 12 χ 1〇6, less than 〇_11 X 106, less than 0. 1 X 1〇6, less than 〇 χ9 χ 1〇6, less than 〇〇 8 X 10, less than 0. 07 χ 1〇6, less than 〇. EP6 χ 1〇6, or less than 〇 χ5 χ i〇6 An EPR signal strength (intensity or amplitude) after 4 hours. In some embodiments, the dissolved cellular composition has a sputum. 05 X 1〇6 to 〇 15 χ 1〇6, 〇 χ5 χ 1〇6 to 0. 14 χ 106,0. 05 χ 1〇6 to 0. 13 χ 1〇6,0. 05 χ 1〇6 to 〇 12 χ 106,0. 05 χ 106 to O. Ii χ ι〇6, 〇 05 χ 1〇6 to 〇"χ 1〇6, 〇 χ5 χ ι〇6 to 0. 09 χ 106, 〇. 〇7 χ 1〇6 to 0. 15 χ 1〇6, 〇·07 χ 1〇6 to 〇 13 χ 106,0. 07 χ 106 to o. Ii 〇 ι〇6,0 〇8 χ 1〇6 to 〇 14 χ 1〇6, 〇〇8 χ 106 to 0_12 χ 106, 〇. 〇8 χ 1〇6至〇”χ ι〇6,〇〇9 χ 1〇6 to 〇 13 χ 1〇6, or 0. 09 χ 1〇6 to o. Ii χ 1〇6 EPR. In some embodiments, the resulting cellular composition of the EPR as specified above is derived. 1^ is 8 to 12, 8 to 11, 8 to 1 , 8 to 9, 9 to 12, 9 to 11, 9 to 1 , 1 to 49 201200591 12, or 10 to 11. In some embodiments, a cell composition that has one of the EPR signal intensities specified above in detail results in a crude lipid having 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less AV. In some embodiments, a cell composition having one of the EPRs specified above specifically results in a crude lipid having 5 or less, 4. 5 or less, 4 or less, 3. 5 or less, 3 or less, 2. 5 or less, 2 or less, 1. 5 or less, 1 or less, 0. 5 or less, 0. 2 or less, or 0. 1 or less PV. In some embodiments, a method comprises contacting a cell composition or a dissolved cell composition with a salt to deemulsifie the dissolved cell composition. As used herein, a ''salt' refers to an ionic compound by a metal (eg, metal, geotechnical, transition metal, and the like) or a positive charge from an acid. The compound (for example, NH4+ and the like) is formed by substituting a hydrogen ion. Salts suitable for use in the present invention include, but are not limited to, alkali metal salts, alkaline earth metal salts, or the like, and combinations thereof. Species of negatively charged ions in a salt for use in the present invention include, but are not limited to, halides, sulfates, hydrogen sulfates, sulfites, phosphates, hydrogen phosphates, dihydrogen phosphates, a carbonate, a hydrogencarbonate, or the like, and combinations thereof, etc. In some embodiments, the one salt used in the present invention is selected from the group consisting of sodium carbonate, sodium sulfate, sodium carbonate, calcium carbonate, sulfuric acid. Potassium, magnesium sulfate, sodium glutamate, ammonium sulfate, potassium carbonate, ferric chloride, iron sulfate, aluminum sulfate, and combinations thereof, etc. In some embodiments, a salt does not include NaOH. a solid (for example, to crystallize , amorphous, granulated, and/or particulate form), and/or 50 201200591 is a solution containing, for example, water, alcohol, and one of the analogs (eg, a dilute solution, a saturated a solution, or a supersaturated solution), and combinations thereof, etc. In some embodiments, the salt is from 5 g/l to 25 gA, from 5 g/l to 10 gA, from 10 g/1 to 15 g/ Add 15 g/1 to 20 g/b 20 g/1 to 25 g/b or 10 g/1 to 20 g/1. In some specific examples, a cell composition or a dissolved The temperature of the cell composition is lower than or equal to 60 ° C, lower than or equal to 55 ° C, lower than or equal to 45 ° C, lower than or equal to 40 ° C, lower than or equal to 35 ° C, lower than or equal to At 30 ° C, or lower than or equal to 25 ° C, when a salt is added to de-emulsifie the cell composition or the dissolved cell composition. In some embodiments, the temperature of a dissolved cell composition is 〇° C to 60 ° C, 〇 ° C to 55 ° C, 〇 ° C to 50 ° C, 〇 ° C to 45 ° C, 〇 ° C to 40 ° C, 〇 ° C to 35 ° C, 〇 ° C to 30 ° C, 〇 ° C to 25 ° C, 20 ° C to 60 ° C, 20 ° C to 55 ° C, 20 ° C To 50 ° C, 20 ° C to 45 ° C, 20 ° C to 40 ° C, 20 ° C to 35 ° C, 20 ° C to 30 ° C, 30 ° C to 60 ° C, 30 ° C to 55 °C, 30°C to 50°C, 30°C to 45°C, 30°C to 40°C, 30°C to 40°C, 40°C to 60°C, 40°C to 55°C 40 ° C to 50 ° C, or 50 ° C to 60 ° C, when a salt is added to deemulsifie the cell composition or the dissolved cell composition. In some embodiments, the method comprises contacting a cell composition or a dissolved cell composition with the dissolved cell composition or 20% or less, 15% or less, 10% or less of the cell composition. Less, 7. 5% or less, 5% or less, or 2% or less by weight of the salt. In some embodiments, a method comprises contacting a cell composition or a dissolved cell composition with the cell composition of the 51 201200591 or the dissolved cell composition (eg, total broth weight). ! % to 20〇/〇,0. 1〇/〇 to 15〇/〇,0. 1% to 10%, 0. 5% to 20 〇 / 〇, 0. 5〇/〇 to 15%, 0. 5% to 10%, 〇_5% to 5%, 0. 5% to 4%, 0. 5% to 3%, 0. 5% to 2. 5%, 0. 5〇/〇 to 20/〇, 0. 5% to 1. 5%, 0. 5% to 13⁄4, 1〇/〇 to 20%, 1% to 15%, 1% to 1%, 1% to 5%, 1% to 4%, 1% to 3〇/〇, 1〇/〇 To 2. 5% '1% to 2%' 1% to 1. 5%, 1. 5% to 5%, 1. 5% to 4〇/〇, 1. 5% to 3%'1_5% to 2. 5%, 1. 5% to 2%, 2% to 20%, 2% to 15〇/〇, 2% to 10%, 2% to 5%, 2% to 4%, 2% to 3%, 2% to 2. 5%, 2. 5% to 5%, 2. 5% to 4%, 2. 5% to 3%, 3% to 5%, 3% to 4%, 4% to 5% '5% to 20%, 5% to 15%, 5% to 10%, 10% to 20%, 10% To I5%, or 15% to 20. /. Salt, by weight. For example, when a dissolved cell composition weighs 1,000 kg, contact 0. 5% to 20% of the salt, by weight, may require a combination of 5 kg to 200 kg of salt with the dissolved cell composition. In some embodiments, the method comprises heating a cell composition or a dissolved cell composition to de-emulsifie the solubilized cell composition. In some embodiments, the cell composition or the solubilized cell composition is heated for a sufficient period of time for a base and/or a salt to de-emulsifie a cell composition or a dissolved cell composition. In some embodiments, the method package 3 heats a cell composition or a dissolved cell composition for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours. , at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 3 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 6 hours, at least 66 hours, 52 201200591 at least 72 hours, at least 78 hours, at least 84 hours, at least 90 hours or at least 96 hours. In some embodiments, the method comprises heating a dissolved cell composition for 5 minutes to 96 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, 5 minutes to 1 hour, 10 minutes to 4 hours, 1 minute. Up to 2 hours, ι〇 minutes to 1 hour, 1 hour to 卯 hours, i hours to 84 hours, i hours to 72 hours '1 hour to 60 hours, i hours to 48 hours, 丨 hours to 36 hours, 1 hour to 24 hours, 1 hour to 4 hours, 4 hours to 96 hours, 4 hours to 84 hours, 4 hours to 72 hours '4 hours to 60 hours, 4 hours to 48 hours, 4 hours to 36 hours, 4 hours to 24 hours , 8 hours to 96 hours, 8 hours to 84 hours, 8 hours to 72 hours, 8 hours to 60 hours, 8 hours to 48 hours '8 hours to 36 hours' 8 hours to 24 hours, 8 hours to 12 hours, 12 Hours to 96 hours, 12 hours to 84 hours, 12 hours to 72 hours, 12 hours to 60 hours '12 hours to 48 hours, 12 hours to 36 hours, 12 hours to 24 hours, 24 hours to 96 hours, 24 hours to 84 hours, 24 hours to 72 hours, 24 hours to 60 hours '24 hours to 48 hours, or 24 Hours to 36 hours. In some embodiments, a cell composition or a dissolved cell composition can be at least 20 ° C, at least 20. (:, at least 25 ° C, at least 30 ° C, at least 35 ° C, at least 40 ° C 'at least 45 ° C, at least 50 ° C, at least 55 ° C, at least 60 ° C ' at least 65 ° C, at least 70 °C, at least 75. (:, at least 80 ° C, at least 85 ° C 'at least 90 ° C ' at least 95 ° C, or at least 1 〇〇. (: temperature to heat. In some specific examples, a method Contained in UTC to l〇 (TC, lot to 90 °C, l〇°C to 80t:, 10°C to 70°c '20. (: to loot:, 20°c to 901, 20°C to 80 °C, 2 (TC to 70t, 30°C to 100. 〇, 3 (TC to 90. (:, 30 53 201200591. (: to 80 ° C, 3 ° ° C to 70 ° C, 40 ° C to lOOt, 40. (: to 90 ° C, 40 ° C to 80 ° C, 50 ° C to l 〇〇 eC, 50 ° C to 90 ° C, 50 ° C to 80 ° C, 50 ° C to 7 〇 °C, 60°C to l〇0°C, 6〇°C to 90°C, 60°C to 80°C, 7〇°C to 100°C, 70°C to 9〇°C, 80° C to l ° ° C, 80 ° C to 90. (:, or 90 ° C to 100 ° C temperature to heat a cell composition or a dissolved cell composition. In some specific examples, a salt can Add to the cell composition or the during the heating The dissolved cell composition. In some embodiments, a cell composition or a dissolved cell composition can be heated in a closed system or in a system having an evaporator. In some embodiments, a cell The composition or dissolved cell composition can be heated in a system having an evaporator such that the water system present in the cell composition or a portion of the dissolved cell composition is removed by evaporation. In some embodiments, a method comprises heating a cell composition or a dissolved cell composition in a system having an evaporator to remove up to 1%, 5%, 10%, 15%, 20%, 25 %, 30%, 35% '40%, 45% or 50% by weight of water present in the cell composition or dissolved cell composition. In some embodiments, a method comprises heating a cell composition Or a dissolved cell composition is removed in a system with an evaporator from 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1 % to 25%, 1% to 20%, 1% to 15%, 1% to 10〇/〇, 1% to 5%, 5% to 50%, 5% to 45 %, 5% to 40%, 5% to 35% '5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, 5% to 10% '10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10. /. To 20%, 10% to 15%, 15% to 54 201200591 50%, 15% to 45%, 15% to 40%, 15% to 35% '15% to 30%' 15% to 25%, 15° /. Up to 20%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25%, 25% to 50%, 25% to 45 %, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 50% '30% to 45%' 30% to 40%, 30% to 35%, 35% to 50%, 35% to 45%, 35% to 40%, 40% to 50%, 40% to 45%, or 45% to 50%. In a specific example, the method comprises maintaining a cell composition or a dissolved cell composition in a vessel for a predetermined period of time to de-emerge the dissolved cell composition. In some embodiments, the method comprises maintaining a fine f composition or a dissolved cell composition in a vessel for a period of time == minutes to (four) minutes, at least 2Q minutes, at least 1 second 'at least 2 hours' at least 4 hours, to - hour, at least 12 hours, at least 18 hours, at least 24 hours, to know hours, at least 36 hours, at least ^ at least 48 hours 'at least 54 hours, at least called, at least = at least 72 hours' at least 78 hours, at least 84 hours, at least 90 = and what. For some specific wealth, the pure product contains a fine monthly composition or - dissolved cell composition for 5 minutes to % small two: to 4 hours, 5 minutes to 2 hours, 5 minutes to 1 hour, 10 minutes Minutes to 2 hours, minute-hours, 丨 hours to 96 =1 light and light 84 hours, 丨 (4) (four) (four), 丨 hourly hours, hours, 1 hour to 36 hours, 1 hour to 24 hours, i hours ': hours to 96 Hours '4 hours to 84 hours, 4 hours to 72 hours, when calling to call, 4 hours to 48 hours, 4 hours to % hours, 4 55 201200591 hours to 24 hours, 8 hours to 96 hours, 8 hours to 84 hours Hours, 8 hours to 72 hours '8 hours to 60 hours, 8 hours to 48 hours, 8 hours to 36 hours, 8 hours to 24 hours, 8 hours to 12 hours, 12 hours to % hours, ^ hours to 84 hours, 12 hours to 72 hours, 12 hours to 6 hours, 12 hours to 48 hours, 12 hours to 36 hours, 12 hours to 24 hours, 24 hours to 96 hours, % hours to 84 hours, 24 hours to 72 hours, 24 Hours to 6 hours, 24 hours to 48 hours, or 24 hours to 36 hours. In some embodiments, the method comprises contacting an antioxidant with a dissolved cell emulsion. Antioxidants suitable for use in the present invention include, without limitation, tocopherol, one-parallel retinoic phenol, polyphenol, resveratrol, a class of flavonoids, a class of carotenoids, lycopene , a carotenoid, lutein, ascorbic acid, ascorbyl palmitate, or the like, and combinations thereof. In some embodiments, the method comprises allowing the emulsified dissolved cellular composition to stand, wherein the lipid is separated from the emulsified dissolved cellular composition using gravity. As used herein, the terms "agitating," and "agitation" refer to a method of affecting the motion in a dissolved cellular composition by applying a force. The method of the present invention comprises mixing a cell composition or a dissolved cell composition by agitation, mixing, blending, shaking, shaking, or a combination thereof, etc. In some embodiments, stirring one The cell composition or a method of dissolving the cell composition demulsifies the cell composition or the solubilized cell composition. In some embodiments, the method of the invention comprises agitation of a dissolved fine 56 201200591 cell composition to 0. . 1 hp/l,000 gal to l〇 hp/l,000 ga b 0. 5 hp/l,000 gal to 8 hp/l,000 gal, 1 hp/l, 〇〇〇gai to 6 hp/l,000 gal, or 2 hp/l,000 gal to 5 hp/l,000 Cell composition of gal dissolved. In some embodiments, the method of the invention comprises using a blender to mix a cell composition or a dissolved cell composition. In some embodiments, the agitator is a dispersing agitator that disperses a base and/or salt within the cell composition or the dissolved cell composition. In some embodiments, a seeker has one or more impellers. As used herein, "impeller" refers to a component that, when configured to impart motion to a cellular composition or a dissolved cellular composition upon rotation. Impellers suitable for use in the present invention include straight blade impellers, Rushtn biade impellers, axial flow impellers, radial flow impellers, concave vane disc impellers, high efficiency impellers, propellers, slurry 'turbines, Or an analog, and combinations thereof. In some embodiments, a method includes agitating a cell composition or a dissolved cell composition using a stirrer having a range of 9 ft/min to 1,200 ft/min, 200 ft/min to 1,000 ft/min, 300 ft/min to 800 ft/min, 400 ft/min to 700 ft/min, or impeller tip rate from 500 ft/min to 600 ft/min. In some embodiments, a method includes agitating a cell composition or a dissolved cell composition using a stirrer having a temperature of from 350 cm/sec to 900 cm per second, from 350 cm/sec to 850 cm per second. , 350 cm / sec to 800 cm / sec, 350 cm / s to 750 knives / sec, 350 cm / sec to 7 cm / sec, 350 cm / sec to 650 cm / sec '350 cm / sec to 600 Metric/second '350 cm/sec to 550 cm/sec, 350 cm/sec to 5 cm/s, 35 cm/s to 45 cm/s, 35〇57 201200591 cm/sec to 400 cm/sec , 400 cm/sec to 900 cm per second, 400 cm/sec to 850 cm/sec, 400 cm/sec to 800 cm/sec, 400 cm/sec to 750 cm/sec, 400 cm/sec to 700 cm/sec , 400 cm/sec to 650 cm/sec, 400 cm/sec to 600 cm/sec, 400 cm/sec to 550 cm/sec, 400 cm/sec to 500 cm/sec, 400 cm/sec to 450 cm/sec , 450 cm / sec to 900 cm per second, 450 cm / sec to 850 cm per second, 450 cm / sec to 800 cm / sec, 450 cm / sec to 750 cm / sec, 450 cm / Seconds to 700 cm/sec, 450 cm/sec to 650 cm/sec, 450 cm/sec to 600 cm/sec, 450 cm/sec to 550 cm/sec, 450 cm/sec to 500 cm/sec, 500 cm/ Seconds to 900 cm per second, 500 cm/sec to 850 cm per second, 500 cm/sec to 800 cm/sec, 500 cm/sec to 750 cm/sec, 500 cm/sec to 700 cm/sec, 500 cm/ Seconds to 650 cm/sec, 500 cm/sec to 600 cm/sec, 500 cm/sec to 550 cm/sec, 550 cm/sec to 900 cm/sec, 550 cm/sec to 850 cm/sec, 550 cm/ Seconds to 800 cm/sec, 550 cm/sec to 750 cm/sec, 550 cm/sec to 700 cm/sec, 550 cm/sec to 650 cm/sec, 550 cm/sec to 600 cm/sec, 600 cm/ Seconds to 900 cm per second, 600 cm/sec to 850 cm per second, 600 cm/sec to 800 cm/sec, 600 cm/sec to 750 cm/sec, 600 cm/sec to 700 cm/sec, 600 cm/ Seconds to 650 cm/sec, 650 cm/sec to 900 cm/sec, 650 cm/sec to 850 cm/sec, 650 cm/sec to 800 cm/sec, 650 cm/sec to 750 cm/sec, 650 cm/ Second to 700 Minutes/second, 700 cm/sec to 900 cm/sec, 700 cm/sec to 850 cm/sec, 700 cm/sec to 800 cm/sec, 700 cm/sec to 750 cm/sec, 750 cm/sec to 900 Centimeters per second, 750 58 201200591 cm / sec to minutes per minute, centimeters / second centimeters / second, 800 to 9 centimeters per second, 800 centimeters / second to call points per second, or 850" wide 9〇〇 cm/sec impeller tip speed. As used herein, the impeller vane* rate refers to the rate of the outermost portion of the impeller as it rotates about its central axis. In some embodiments, the blending (and optionally the month as described herein) The additional step) is performed in a vessel containing an impeller, wherein the ratio of the diameter of the impeller to the volume of the vessel is 〇. 1 to 0. 5,0. 1 to 〇. 4,0. 2 to 〇·5 〇〇·2 to 〇. 4 〇 3 to 〇 5, or 〇 3 to 〇 4. In some specific terms, the scooping (and optionally the additional steps as described herein) is performed in a vessel containing an impeller having a ratio of impeller diameter to the inner diameter of the vessel of at least ,25, At least 〇34, at least 0. 65 0. 25 to 〇_65 ’ 0. 25 to 0. 33 ’ 0. 3 to 〇 6,0. 3 to 〇·5,0. 3 to 0. 4 0. 34 to 0. 65,0. 34 to 0. 6 ’ 0. 34 to 〇·55,0. 37 to 0. 55. 4 to 0. 65,0. 4 to 0. 6,0. 4 to 0. 5, or 〇. 42 to 〇 55. In some embodiments, the mixed cell composition or a dissolved cell composition is included such that the cell composition or the dissolved cell composition is placed under the flow conditions as described below: 1〇 to 1〇 , 〇〇〇, ^000 to 10,000, 1,500 to 10,000, or 2,000 to 10,000 Reynolds number. In some embodiments, a dissolved cell emulsion has 2,000 or more, 3,000 or more 'or 5,0 or more, or 2 to 10,000' during the soaking period. 3,000 to 10,000, or a Reynolds number of 5,000 to 10,000. In some embodiments, a method comprises agitating a cell composition or a dissolved cell composition for at least 5 minutes, at least 1 minute, at least 2 minutes 59 201200591 minutes, at least 30 minutes, at least! Hours, at least 2 hours for at least 4 hours, at least 8 hours 'at least 12 hours, at least 18 hours, at least 24 hours, at least 3 hours 'at least 36 hours' for at least 42 hours, at least for at least 54 hours, at least 60 hours, at least 60 hours, at least 66 hours, at least 72 hours, at least 78 hours, at least 84 hours, at least 9 hours or at least 96 hours. In some embodiments, the method comprises mixing the cell composition or a dissolved cell composition for 5 minutes to 96 hours, 5 minutes to 4 hours '5 minutes to 2 hours, 5 minutes to 1 hour, 10 minutes to 4 hours, 10 minutes to 2 hours, 1 minute to hour, 1 hour to 96 hours, 1 hour to 84 hours, 1 hour to 72 hours, 】 hour to 60 hours '1 hour to 48 hours, 1 hour to 36 hours Hours, 1 hour to 24 hours, 1 hour to 4 hours, 4 hours to 96 hours, 4 hours to 84 hours, 4 hours to 72 hours '4 hours to 6 hours, 4 hours to 48 hours, 4 hours to 36 J 4 hours to 24 hours, 8 hours to 96 hours, 8 hours to 84 hours, 8 hours to 72 hours, 8 hours to 60 hours, 8 hours to 48 hours, 8 hours to 36 hours, 8 hours to 24 hours, 8 hours to 12 hours, 12 hours to 96 hours '12 hours to 84 hours, 12 hours to 72 hours, 12 hours to 60 hours' 12 hours to 48 hours, 12 hours to 36 hours, 12 hours to 24 hours, 20 hours Up to 40 hours, 24 hours to 96 hours, 24 hours to 84 hours, 24 hours to 72 hours '24 hours to 6 hours, 24 hours to 48 hours, or 24 hours to 36 hours. In some embodiments, a method comprises simultaneously mixing and heating a cell composition or a dissolved cell composition to deemulsifie the cell composition or the solubilized cell composition. In some embodiments, a method comprises agitating a cell composition or a dissolved cell composition at least 10 ° C, at least 20 60 201200591 ° C, at least 25 ° C, at least 30 ° C, at least 35 ° C, at least 40 ° C, at least 45 ° C, at least 50 ° C, at least 55 ° C, at least 60 ° C, at least 65 ° C, at least 70 ° C, at least 75 ° C, at least 80 ° C, at least 85 ° C, at least 90 ° C, at least 95 ° C, or at least 10 ° C temperature. In some embodiments, a method comprises agitating a cell composition or a dissolved cell composition at 10 ° C to 10 ° C, 10 to 90 ° (:, l ° ° C to 80 ° C, l 〇°C to 70°C, 20°C to l〇〇°C, 20°C to 90°C, 20°C to 80°C, 20°C to 70°C, 30°C to l〇〇° C, 30 ° C to 90 ° C, 30 ° C to 80 ° C, 30 ° C to 70 ° C, 40 ° C to l ° ° C, 40 ° C to 90 ° C, 40 ° C to 80 ° C, 50 ° C to l ° ° C, 50 ° C to 90 ° C, 50 ° C to 80 ° C, 50 ° C to 70 ° C, 60 ° C to l ° ° C, 60 ° C to 90 ° C, 60 ° C to 80 ° C, 70. (: to l ° ° C, 70 ° C to 90 ° C, 80 ° C to l ° ° C, 80 ° C to 90 ° C, Or a temperature of from 90 ° C to 10 ° C. In some embodiments, a dissolved cell composition is formed to contact a dissolved cell composition or to raise the pH of a dissolved cell composition. The dissolved cell composition is contacted with a salt, the dissolved cell composition is heated, and various combinations of stirred and dissolved cell components can occur in a single vessel. In some embodiments, a cell is formed. To cause a cell composition to be contacted or to raise the pH of a cell composition, to contact a cell composition with a salt, to heat the cell composition, and to mix a dissolved cell composition in various combinations. In a single vessel, in a particular embodiment, the single vessel comprises a ferment vessel. In some embodiments, the ferment vessel can have at least 20,000 liters, at least 50,000 liters, at least 100,000 liters, at least 120,000 liters, at least 150,000 liters, at least 200,000 liters, or at least 220,000 liters. In some 61 201200591 specific examples, the fermentation vessel can have 20,000 liters to 220,000 liters, 20,000 liters to 100,000 liters, 20,000 liters to 50,000 liters, 50,000 liters to 220,000 liters, 50,000 liters to 150,000 liters, 50,000 liters to 1 liter, liters, 1 liter, liters to 220,000 liters, 100,000 liters to 150,000 liters, 100,000 liters to 120,000 liters, 150,000 liters to 220,000 liters, 150,000 liters Up to 200,000 liters, or 200,000 liters to 220,000 liters. Some specific examples' are formed in the number of cells was one of a group of cells into a vessel or dissolution may be transferred into one or more search stirrer dish. In some embodiments, the agitator may have a volume of at least 2 Torr, 〇〇〇 liters, at least 30,000 liters, at least 40, 〇〇〇 liters, or at least 5 〇, 〇〇〇 liters. In some specific examples, the agitator can have 2 〇, 〇〇〇 liter to 50,000 liters, 20,000 liters to 4 liters, 2 liters to 3 liters, liters liters, 30,000 liters To 50,000 liters, 3 liters to 4 liters or 40, liters to 50, liters of liters. In some embodiments, the _ware can have any combination of the following properties. In some embodiments, the two buckets that the such mixing vessel can have. In some specific examples, the impellers are the Kirchner blade impellers. In some embodiments, the impellers are separated from one another by a distance equal to the smallest impeller diameter. In some specific examples, the impellers range from 3 inches to 4 inches, 33 inches to 37 inches, 33 yos, υ — ”, 34 inches, 35 miles, 36 吋 or 37 miles from the tip to the tip. In the specific example of the guru, the stirring thief has at least 1 liter, at least 〇, 000 liters, at least 30, 〇〇〇 liters, 至40, 〇〇〇 liters or at least 5 〇〇〇 △ liter volume. Back '- some specific 62 201200591 = riding a savory dish with 9 〇 to (10) 英, % Ying Ye to two '98 outside " shoot, (8) Material, or 1 〇 2 忖 In some specific examples, the first impeller is located in the Bay 15 to 2G material, 丨 6 英 W correction, or the 忖 to 18 英 phase and the second impeller system 6 〇叶 Η 座 Η 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 65 75 lb. In some specific examples, 'a dissolved cell composition is stirred at least 5 ,, at least 60 ah, or at least 70 rpm. In this particular example, a dissolved cell composition is 50. Ah to 70 rpm, 50 rpm to 6 〇 , or 60 rpm to 7 rpm, in a specific example, the cell composition, the dissolved cell composition, or the lipid system is harvested from a vessel, which is obtained by drawing from the vessel. The fine silk, the fine touch of the bribe, or the lipid. In some specific examples, the cell composition, the dissolved cell composition, or the lipid system is harvested from the vessel to the organ. In some embodiments, the cell composition, the dissolved cell composition, or the lipid system is harvested from the vessel, and is extracted from the vessel, instead of the cell composition, the dissolved cell. a composition, or the lipid. In some specific examples, the cell of the fine silk, bribe, or the lipid is harvested from the vessel without blowing. In some specific examples, by the above Saying _technology to harvest the cell composition, the dissolved cell composition' or the lipid results in a crude lipid having a low methoxyaniline value (eg, 26 or less, 25 or less, 2 or more) Less, 1S or less, ι〇 or 63 201200591 less, 5 or more '2 or less, or 1 or less) and/or low phosphorus content (eg, 100 ppm or less, 95 ppm or less '90 ppm or less, 85 ppm or less, 80 ppm or Less, 75 ppm or less '70 ppm or less, 65 ppm or less '60 ppm or less' 55 ppm or less, 50 ppm or less '45 ppm or less, 40 ppm or less , 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 Ppm or less, 2 ppm or less 'or 1 ppm or less). As described herein, the present invention employs contacting a dissolved cell composition with a first test or increasing the pH of a dissolved cell composition, contacting a dissolved cell composition with a salt, and heating a dissolved cell. The composition, as well as various combinations of the mixed cell compositions, are provided to provide a treated dissolved cellular composition. As described herein, the present invention employs contacting a cell composition with a first base or increasing the pH of a cell composition, contacting a cell composition with a salt, heating a cell composition, and mixing a cell. Various combinations of compositions provide a treated cell composition. The treated cell composition or treated dissolved cell composition is at least partially deemulsified as compared to an untreated cell composition or treated dissolved cell composition. Thus, the treated cell composition or the treated dissolved cell composition can be placed in a centrifuge and a lipid can be separated therefrom. In some embodiments, after raising the pH of a cell composition or dissolved cell composition, for example, by contacting a first base, the cell composition or dissolved cell composition is heated and/or Stirring the cell group 64 201200591 The composition or dissolved cell composition can reduce the pH of the treated cell composition or the treated dissolved cell composition. In order to allow a lipid to be separated from the treated cell composition or the treated dissolved cell composition by centrifugation more efficiently, the pH of the treated cell composition or the treated dissolved cell composition is re-applied. Lifting, for example, by contacting the treated cell composition or the treated dissolved cell composition with a second base. In some embodiments, contacting a treated dissolved cell composition with a second base increases the pH of the treated cell composition or the treated dissolved cell composition. In some embodiments, the treated cell composition or the treated dissolved cell composition is contacted with a second base to increase the pH of the treated cell composition or the treated dissolved cell composition. To 7 or above, 7. 5 or more, 8 or more, 8. 5 or more, 9 or more, 9. 5 or more, 1 or more, 10. 5 or more, 11 or above' 11. 5 or more, or 12 or more. In some embodiments, the treated cell composition or the treated dissolved cell composition is contacted with a second base to raise the pH of the treated dissolved cell composition to 7 to 13, 7 to 12. '7 to 11, 7 to 10, 7 to 9, 7 to 8, 7 to 7. 5,7. 5 to 8, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 8 to 8_5, 8. 5 to 9, 9 to 12, 9 to 11, 9 to 10, 9 to 9. 5,9. 5 to 10, 1 to 12, or 10 to 11. In some embodiments, the pH of the treated cell composition or the treated dissolved cell emulsion is 7 or less, 6 or less, 5 or less, 4 or less, or 3 or more. less. The method of the present invention comprises isolating a lipid from a treated cell composition or a cell composition dissolved in a solution. In some embodiments, a lipid system is isolated from a dissolved cell emulsion, after contacting a dissolved cell emulsion with a second base, after stirring the dissolved cell emulsion, or After contacting a dissolved cell emulsion with a salt, by allowing, for example, the treated dissolved cell emulsion to remain stationary for a period of time sufficient from the treated dissolved cell emulsion A period of time of separation (eg, a separate layer). The lipid can then be removed, for example, by decantation, decanting, vacuuming, pumping, aspirating, pumping, siphoning, or otherwise treating the dissolved cell milk from the treatment. The surface of the liquid removes the lipid. In some embodiments, the separating comprises centrifuging the treated cell composition or the treated dissolved cell composition (eg, at a temperature of 30 ° C to 1 ° C), whereby the centrifugation is processed from the centrifugation The cell composition or the treated dissolved cell composition separates a lipid. In some embodiments, a method comprises centrifuging a treated cell composition or a treated dissolved cell composition at at least 10 ° C, at least 20 ° C, at least 25 ° C, at least 30 ° C, at least 35 ° C , at least 40 ° C, at least 45 ° C, at least 50 ° C, at least 55 ° C, at least 60 ° C, at least 65 ° C, at least 70 ° C, at least 75 ° C, at least 80 ° C, at least 85 ° C , at least 90 ° C, at least 95 ° C, or at least 10 ° C temperature. In some embodiments, a method comprises centrifuging a treated cell composition or a treated dissolved cell composition at 10 ° C to 100 ° C, 10 ° c to 90 ° C, 10 ° C to 80 ° c, 20°c to 1〇〇°C, 20°C to 90°C, 20°C to 80°C, 25°C to l〇〇°C, 25°C to 90°C, 25°C to 80° C, 25°C to 75°C, 30°C to l〇〇°C, 30°C to 90°C, 30 66 201200591 °〇 to 80°〇, 40°C to l〇〇°C, 40° C to 90 ° C, 40 ° C to 80 ° C, 50 ° C to l ° ° C, 50. (: to 90 ° C, 50 ° C to 8 (TC, 50 ° C to 70 ° C, 60 ° C to 100 ° C, 60 ° C to 90 ° C, 60 ° C to 80 ° C, 60 ° C To 70 ° C, 70 ° C to 100 ° C, or 70 ° C to 90 ° C. In some specific examples, the centrifuge system is 1 kg per minute (kg / min) to 500 kg / min, 1 Kg/min to 400 kg/min, 1 kg/min to 300 kg/min, 1 kg/min to 200 kg/min, 1 kg/min to 100 kg/min, 1 kg/min to 75 kg/min ' 1 Kg/min to 50 kg/min, 1 kg/min to 40 kg/min, 1 kg/min to 30 kg/min, 1 kg/min to 25 kg/min, 1 kg/min to 10 kg/min, 10 Kg/min to 500 kg/min, 10 kg/min to 400 kg/min, 10 kg/min to 300 kg/min, 10 kg/min to 200 kg/min, 10 kg/min. Up to 100 kg/min, 10 kg/min to 75 kg/min, 10 kg/min to 50 kg/min, 10 kg/min to 40 kg/min, 10 kg/min to 30 kg/min, 20 kg/min Up to 500 kg/min, 20 kg/min to 400 kg/min, 20 kg/min to 300 kg/min, 20 kg/min to 200 kg/min, 20 kg/min to 100 kg/min, 20 kg/min Up to 75 kg/min, 20 kg/min to 50 kg/min, 20 kg/min to 40 kg/min, 25 kg/min to 500 kg/min, 25 kg/min to 400 kg/min, 25 kg/min Up to 300 kg/min, 25 kg/min to 200 kg/min, 25 kg/min to 100 kg/min, 25 kg/min to 75 kg/min, 25 kg/min to 50 kg/min, 30 kg/min Up to 60 kg/min, 30 kg/min to 50 kg/min, 30 kg/min to 40 kg/min, 50 kg/min to 500 kg/min, 100 kg/min to 500 kg/min, or 200 kg A feed rate of /min to 500 kg/min (either the treated cell composition or the treated dissolved cell composition into a microcentrifuge) is carried out at a feed rate. 67 201200591 The total time required for S Hai separation may vary depending on the volume of the treated cell composition or the treated dissolved cell composition. The typical total time for separation (eg, centrifugation time) is at least 〇. 丨 hours, at least 0. 2 hours, at least 0. 5 hours, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 丨 2 hours, or 0. 1 hour to 24 hours, 0. 5 hours to 24 hours, 1 hour to 12 hours, 2 hours to 10 hours, or 4 hours to 8 hours. In some embodiments, a method of the invention comprises wl, 〇〇〇 g to 25. 000 g,1,〇〇〇 g to 2〇,〇〇〇 g,l,〇〇〇 g to lo o. . g,2,〇〇〇 g 5. 25. 000 g > 2,000 g^. 20,000 g - 2,000 gJL 15,000 g - 3,000 g to 25,000 g '3,000 g to 20,000 g, 5,000 g to 25,000 g, 5,000 g to 20,000 g ' 5,000 g to 15,000 g, 5,000 g to l〇, 〇〇〇g, 5,000 g to 8,000 g 'l〇, 〇〇〇g to 25,000 g, 15,000 g to 25,000 g, or at least 1,000 g, at least 2,000, g, at least 4,000 g, at least 5,000 g, at least ?, 000 g, at least 8,000 g, at least 10,000 g, at least 15,000 g, at least 20. A centrifuged force of 000 g, or at least 25,000 g, is used to centrifuge a treated cell composition or a treated dissolved cell composition. As used in this article, '"g" refers to standard gravity or approximately 9. 8m/s2. In some embodiments, a method of the present invention comprises from 4,000 rpm to 14,000 rpm, 4,000 rpm to 10,000 rpm, 6,000 rpm to 14,000 rpm, 6,000 rpm to 12,000 rpm, 8,000 to 14,000 rpm, 8,000 rpm to 12,000 rpm, or Between 8,000 rpm and 1 Torr, rpm is used to centrifuge a treated cell composition or a treated dissolved cell composition. In some embodiments, a method of the invention comprises drying a lipid raft to remove water from the lipid after isolating the lipid from a cell composition or a treated cell composition that has been treated. In some embodiments, drying the lipid can include, but is not limited to, heating the lipid to evaporate water. In some embodiments, after drying, the lipid has less than 3 〇 / 〇, less than 2. 5%, less than 2%, less than 1. 5%, less than 1%, less than 0. 5%, less than 0. 1%, or 〇%, of the water content as a percentage by weight of the lipid. In some embodiments, the lipid has a 〇% to 3%, 0% to 2. after drying. 5%, 0% to 2%, 〇% to 1. 5%, 0% to 1%, 〇% to 0. 5%, 〇_1% to 3%, 0. 1% to 2. 5%, hehe. 1% to 2%, 〇. 1% to 1. 5%, hehe. 1% to 1%, 〇. 1〇/0 to 0. 5°/〇,0. 5% to 3%, 0. 5% to 2. 5%, 0.5% to 2%, 0. 5% to 1. 5%, 0. 5% to 10/〇, 1〇/〇 to 3%, 1〇/0 to 2. 5%, 1% to 2%, 1% to 1. 5%, 1. 5% to 3%, 1. 5% to 2. 5%, 1. 5% to 2%, 2% to 3%, 2% to 2. 5%, or 2. 5% to 3% by weight of water by weight of lipid. In some embodiments, a method further comprises refining a lipid by a combination of caustic refining, degumming, alkali refining, bleaching, deodorizing, deacidifying, or the like, and combinations thereof One or more methods to remove one or more phospholipids, free fatty acids, phospholipids, colored objects, sterols, odors, and other impurities. As used herein, a, refined oil " is a crude lipid or crude oil that has been refined. As used herein, "a crude lipid" or "a crude oil" is a lipid or oil that has not been refined. In some embodiments, the lipid isolated from the emulsified cell composition is a crude lipid. Various exemplary methods of the present invention are outlined in Figures 1-4. Referring to Fig. 1, in some embodiments, the present invention is directed to a method (1〇〇) for obtaining a lipid (11〇) from a cell (101) for use in the treatment of 69 201200591, which comprises dissolving (102) sputum cells (1) 〇 1) to form a dissolved cell composition. The dissolved cell composition is contacted with a first test (1G4) to de-emulsifie the dissolved cell composition (1〇3), and contacted with a salt (1〇5) to de-emulsifie the dissolved cell composition ( 103) 'and heat (1 () 6), for example, for 1 minute (minutes) to provide a treated dissolved cell composition (1〇7). The treated dissolved cell composition (1〇7) is contacted (108) with a second base and, for example, at a temperature of 10C to i°°C, is separated (1〇9) to provide - lipid (110). Referring to Figure 2, in some embodiments, the present invention is directed to a method (2(10)) for obtaining a lipid (210) from a cell, the method comprising dissolving (02) 'cell (101) to form a lysed cell. Composition (10) 3). The solubilized cell composition is then contacted (2G4) to de-emulsifie the dissolved cell composition (103) and to provide a treated dissolved cell composition (2〇7). The treated dissolved cell composition (2〇7), for example, is separated (209) at a temperature of 1 Torr to provide a lipid (21 〇 see the 3rd circle, in some specific examples, The invention relates to a method for obtaining a lipid (10) from a cell. The method comprises dissolving (10))-cell (10)) to form a dissolved cell composition (10). The dissolved cell composition is then contacted with __salt (the dog is lysed by the dissolved cell composition (103) and provided by the treated dissolved cell composition _), which is 'for example' at (10) to The temperature is separated (10) to provide a lipid (310). Referring to Figure 4, in some embodiments, the present invention is directed to a method (400) for obtaining a lipid (410) from a cell, the method comprising: dissolving (102)-cell (ιοί) to form a dissolved Cell composition (1〇3). The solubilized cell composition is then contacted with a salt (405) to de-emulsifie the dissolved cell composition (103) and stirred (401), for example, for 5 minutes to 96 hours, and optionally heated (402) ) to provide a treated dissolved cell composition (407). The treated dissolved cell composition is then separated (409), for example, at a temperature of up to 100 ° C to provide a lipid (410). In some embodiments, a method of the invention comprises concentrating a broth containing a microbial cell, a broth containing plant material, and/or concentrating a dissolved cell composition. As used herein, "concentration" refers to the removal of water from a group. Concentration can include, but is not limited to, evaporation, chemical drying, centrifugation, and the like, as well as combinations thereof. In some embodiments, a broth containing a microbial cell or a broth containing plant material is concentrated to provide at least 4%, at least 5 Å/0, at least 10 °/. , at least 15% 'at least 20%, at least 25%, or at least 30% by weight of the lipid concentration of the broth. In some embodiments, a broth containing a microbial cell or a broth containing plant material is concentrated to provide 4% to 40°/. ' 4°/. Up to 30%, 4% to 20%, 4% to 15%, 5% to 40%, 5〇/〇 to 30%, 5% to 20%, 10% to 40%, 10% to 30%, 10% To 20%, 15% to 40%, 15% to 30%, 20% to 40%, 20% to 30%, 25% to 40%, or 30% to 40% by weight of the broth lipid concentration. In some embodiments, a cell composition or a dissolved cell composition is concentrated to provide at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%. The lipid concentration of the dissolved 71 201200591 cell composition was quantified. In some embodiments, a cell composition or a dissolved cell composition is concentrated to provide 4% to 40%, 4% to 30 〇 / ’ ' 4 ° /. To 20%, 4% to 15%, 5% to 40%, 5% to 30%, 5% to 20%, 1% to 4%, 1% to 3%, 1%. /0 to 2〇〇/〇, 15% to 40%, 15% to 30%, 20% to 40%, 20% to 30%, 25% to 40%, or 30% to 40% by weight The lipid concentration of the dissolved cell composition. In some embodiments, one of the lipids prepared by the method of the invention has a total aroma intensity of 2 or less. As used herein, the term "total aroma intensity" refers to the level of olfactory perception of the lipid administered via a panel of sensory analytes. As used herein, the term • sensory analyte, refers to a trained individual' which provides feedback to a sensory characteristic of a substance and/or evaluates a sensory characteristic of a substance. In some embodiments, one of the lipids prepared by the method of the present invention has a total aromatic intensity of 3 or less. As used herein, the term "total aroma intensity" refers to a gustatory, or taste, level of perception imparted to a lipid via a set of sensory analytes. In some embodiments, a general-purpose spectral descriptive analytical method is used to assess the aroma and aroma characteristics of the sample. This method uses the intensity scale of 〇-15, where 〇 = no detect and 15 = very high intensity to measure the aroma and aroma properties of the oil. In some embodiments, one of the lipids prepared by the method of the present invention does not have an aftertaste like a fish. As used herein, the term "aftertaste" refers to the persistence of the taste of the lipid as it is characterized by a set of sensory analytes. In some embodiments, the method of the invention provides a crude lipid, 72 201200591 which has 5 or less, 4.5 or less, 4 or less, 3. 5 or less, 3 or less, 2. 5 or less' 2 or less, 1. 5 or less, 1 or less, 〇. 5 or less, 0. 2 or less' or 〇. 1 or less peroxide value (Ρν) β as used herein, the term 'peroxide value', or 'PV' refers to a primary reaction product that occurs throughout the oxidation of the lipid, for example Measurement of peroxides and hydroperoxides. In some specific examples, PV is an indicator of the quality of the lipid and the degree of oxidation that has occurred in the lipid, with a low PV (ie, 5 or less). Showing a higher stability and sensation profile than a lipid having a PV greater than 5. In some specific examples, 'adding a base to a dissolved cell composition, as discussed above, causes the dissolved cell composition to ρΗ The oxidation of the lipid is enhanced and inhibited, thereby minimizing the amount of free radicals within the dissolved cellular composition such that the crude lipid obtained from the method of the invention has a low PV (i.e., 5 or less). In some embodiments, the methods of the present invention provide a crude lipid having 26 or less, 25 or less, 20 or less, 15 or less, 1 or less '5 or less, 2 Or less, or 1 or less of the aniline value (Av) As used herein, the term "methoxyaniline value, or "AV" refers to the measurement of secondary reaction products, such as aldehydes and ketones, which occur throughout the oxidation of the lipid, in some specific examples. Among them, AV is an indicator of the quality of the lipid and the degree of oxidation that has occurred in the edge lipid. The low AV (i.e., % or less)-lipid exhibits an increase in iB stability over lipids having an AV greater than 26 Spit and sensation n - some specific shots - added to - dissolved cell composition 'as discussed above, the pH of the dissolved cell composition is increased, and the oxidation of the inhibitor gf · is thereby minimized The amount of free radicals in the dissolved cell composition 73 201200591 is such that the crude lipid obtained from the method of the invention has a low AV (ie, 26 or less). In some embodiments, the method of the invention Provides a crude lipid 'which has 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 7 〇 ppm or less , 65 ppm or less, 60 ppm or less, 55 ppm or more Less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, '30 ppm or less, 25 ppm or less, 20 ppm or less' 15 ppm or less '10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less '2 ppm or less, or 1 ρριη or less phosphorus content. In some specific examples, the invention The method provides a crude lipid having a lower methoxygen than extraction using a solvent (eg, atypical burnt extraction or FRIOLEX® method (Westfalia Separator AG, Germany)) Aniline value, lower peroxide value, lower phosphorus content and/or higher extraction yield. FRI0LEX® method, which is a method for extracting lipids with a water-soluble organic solvent, as in U.S. Patent No. 5,928,696. As described in the International Publication Nos. W〇oi/76385 and WO 01/76715, each of which is incorporated herein by reference in its entirety. In the case of Yu Yijian, the cell composition of the cleavage heat is triggered: the reaction product (for example, ketone and ketone) participates in a similar reaction to the Mena reaction (the reaction of 璧丽(四)- and the cell composition present in the _ < White matter - up. This reaction is believed to produce a production that possesses antioxidant activity "which reduces the oxidation of the lipid. In some embodiments, a gas, 201200591 protein, such as 'soy protein, can be added to the dissolved cellular composition plus antioxidant activity. The reduction in oxidation of the lipid by the ticm in the second, the increased quality of the AV, reduces any aftertaste of the lipid and/or adds to the (4): the bribe. In some specific examples, the stability is increased by at least 5%, at least, by 15% or at least 20%. . 'As for some specific shots' - a method by which a lipid, a biomass remaining after extraction of the lipid, or the like is taken directly to use as a food or food ingredient, for example, Products, infant formula, beverages, sauces, milk-based foods (your milk, sour milk, dry and ice cream), oil (for example, cooking oil or a component such as beef and baked goods; nutrition A supplement (for example, in the form of a soy sauce); a feed or feed supplement for any non-human animal; and its products (eg, meat, milk, or eggs) are for humans: (Examples of such); food supplements: and drugs (in the case of direct or additional treatment 4 consumption) 0 term, animal " mention any animal belonging to the animal kingdom, the law should be any human animal, and any Non-human animals, such as milk, eggs, poultry, beef, pork, or mutton. In a specific example, the lipid and/or the raw material can be used in seafood. These foods are derived from, without restrictions, fish, clams and shellfish. The term, product, is derived from any product of these animals, including, without limitation, "album", milk or other products. When the lipid and/or feedstock is fed to such animals, the polyunsaturated lipids can be incorporated into the meat, milk, eggs or such animals to increase the amount of such lipids. Its microbial lipids 75 201200591 In some embodiments, the invention is directed to a microbial lipid extracted by a method according to the invention. In some embodiments, a crude microbial lipid has 26 or fewer '25 or less' 20 or less '15 or less, 10 or less, 5 or less '2 or less' or 1 Or less methoxyaniline value ' and / or 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less '1.5 or more Less '1 or less' 〇·5 or less, 0.2 or less, or 0.1 or less peroxide value' and / or 100 PPm4 less, 95 ppm or less '90 ppm or less '85 ppm or less' 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 Ppm or less, 40 ppm or less '35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or Less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less. In some embodiments, the crude microbial lipid has less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% by weight or volume of organic solvent. In some embodiments, the crude microbial lipid has at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by weight. The desired PUFA. In some embodiments, the crude microbial lipid has at least 10%, at least 15%, at least 20%, at least 25°/〇, at least 30%, at least 35°/. , at least 40%, at least 45%, or at least 50. /〇 by weight of DHA 'and / or at least 1%, at least 15%, or at least 20% by weight of DPA η-6 ' and / or at least 10%, at least 15%, or at least 20% by weight of EPA , and / or at least 10%, at least 15 ° /. , at least 20%, at least 25%, 76 201200591 at least 30%, at least 35%, at least Qing. , at least Na, or at least 5〇% of the weight of the ship. In some embodiments, the crude microbial (4) extracted by the method of the present invention results in a higher ratio than the solvent (e.g., a typical aortic extraction or fri〇lex8 method).

Sep她r AG，Germany))來執行的萃取有更低的曱氧苯胺值、更低的過氧化物值、更低的磷含量及/或更高的萃取產量。從第一組經單離之破囊壺菌微生物所萃取的脂質於-些具體财，本發明進—步針對—種微生物脂質’、係從如同美國公開案號2010/0239533與國際公開案號WO 2010/107415之中說明的—破囊壺菌所萃取的，其等之各個係以其之整體併人本文中以作為參考資料。於一些八體例中4方法包含使―破囊壺菌生長於—培養物中以產生生質以及自該生質萃取—種包含ω-3脂肪酸的脂貝°亥知質可以自一新鮮收穫的生質來萃取或可以自已經計存於預防糟㈣條件下之―先前收穫的生f來萃取。可、使用已夫的方法來培養本發明的破囊壺菌，來自該培養物單離纟質’以及來分析自該生質所萃取的油之脂肪酸 °J、曰參見，例如，美國專利案號5，130,242，以其之整體并本文中以作為參考資料。該脂質可以依據本發明的方法來萃取。 t本發明的微生物脂質可以為衍生自-微生物之任何的月曰質包括’舉例而言：一種粗製油’其係自該微生物的生質所萃取而無進—步的加上；—種精製油，其係藉由用 77 201200591 進一步的加工步驟（例如精製、漂白，及/或去臭）來處理一粗製微生物油所獲得的；一種經稀釋的微生物油，其係藉由稀釋一粗製或精製的微生物油所獲得的；或是一種濃化的油，其係，舉例而言，藉由用進一步的純化方法來處理一粗製或精製的微生物油所獲得以增加該油内的脂肪酸 (例如DHA)之濃度。於一些具體例中，該微生物脂質包含〇%，至少0.1%，至少0.2%，至少0.5%，至少大約1°/。，至少1.5%，至少2%，或至少5%重量計的固醇酯餾份。於一些具體例中，該微生物脂質包含從0°/。至1.5%，0%至2%，0%至5%，1%至1.5%， 0.2%至1.5%，0.2%至2°/。，或是0.2%至5°/〇重量計的固醇酯餾份。於一些具體例中，該微生物脂質包含少於5%，少於 4%，少於3%，或少於2%重量計的固醇酯餾份。於一些具體例中，該微生物脂質包含至少65%，至少 70%，至少75%，至少80%，至少85%，或至少90%重量計的三酸甘油酯馏份。於一些具體例中，該微生物脂質包含 65%至95%，75%至95%，或是80%至95%重量計，或97°/〇重量計，或98%重量計的三酸甘油S旨顧份。於一些具體例中，該微生物脂質包含至少0.5%，至少 1%，至少1.5%，至少2%，至少2.5%，或至少5%重量計的游離脂肪酸餾份。於一些具體例中，該微生物脂質包含由 0.5%至5%，0.5%至2.5%，0.5%至2%，0.5%至 1.5%，0.5% 至 1%，1%至2.5%，1%至5%，1.5%至2.5%，2%至2.5%，或是2%至5%重量計的游離脂肪酸餾份。於一些具體例中， 78 201200591 該微生物脂質包含少於5%，少於4%，少於3%，少於2%，或少於1%重量計的游離脂肪酸餾份。於一些具體例中，該微生物脂質包含至少0.5%，至少 1%，至少1.5%，至少2%，或至少5%重量計的固醇餾份。於一些具體例中，該微生物脂質包含由0.5%至1.5%，1%至 1.5%，0.5%至2°/。，0.5°/。至5%，1%至2%，或是 1%至5%重量計的固醇餾份。於一些具體例中，該微生物脂質包含少於5%，少於4%，少於3%，少於2%，或少於1%重量計的固醇德份。於一些具體例中，該微生物脂質包含至少1.5%，至少 2%，至少2.5%，至少3%，至少3.5%，或至少5%重量計的二酸甘油酯餾份。於一些具體例中，該微生物脂質包含由 1.5%至3%，2%至3%，1.5%至3.5%，1.5%至5%，2.5%至3%， 2.5%至3.5%，或是2.5%至5%重量計的二酸甘油酯德份。於一些具體例中，該微生物脂質包含的不皂化物係少於2%，少於1.5%，少於1%，或少於0.5%重量計之油。存在於該微生物油之中的脂質種類，例如該三酸甘油酯餾份，可以藉由急驟層析法予以分離以及藉由薄膜層析法(TLC)予以分析，或是藉由本技藝中所知道的其他方法予以分離及分析。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三酸甘油酯餾份、游離脂肪酸德份、固醇镏份、二酸甘油酯餾份，及其等之組合，包含至少40%，至少45%，至少50%，至少55%，至少60%， 79 201200591 至少65%，至少70%，至少75%，或至少80%重量計DHA。於一些具體例申，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份，及其等之組合，包含由 40%至45%，40%至50%，40%至60%，50%至60%，55% 至60%，40%至65%，50%至65%，55%至65%，40%至70%， 40%至80%，50%至80%，55%至80%，60%至 80%，或是70% 至80%重量計DHA。於一些具體例中，該微生物脂質包含一固醇酯餾份，其包含45%或更少，40%或更少，35%或更少，30%或更少，25%或更少，20%或更少，15%或更少，或是13%或更少重量計DHA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份，及其等之組合，包含10%或更少、9%或更少、8% 或更少、7%或更少、6%或更少、5%或更少、4%或更少、 3%或更少、2%或更少、或1%或更少以重量計EPA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份，以及其等之組合，包含由2% 至3%，2%至3.5%，2.5%至3.5%，2%至6%，2.5%至6%， 3.0%至6%，3.5%至6%，5%至6%，或是2%至10%重量計 EPA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份 '二酸甘油酯餾份、 80 201200591 極性德份（包括碌脂質餾份），及其等之組合，係實質無EPA 的。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯館份、極性餾份（包括磷脂質餾份），及其等之組合，包含至少5 : !，至少7 :卜至少9 :卜至少1 〇 :卜至少15 : 1，至少20 : 1，至少25 :卜至少30 :卜或至少50 : 1的DHA對EPA重量比，其中該微生物脂質及/或其等之一或更多餾份包含10。/〇或更少以重量計的EPA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份、極性餾份（包括磷脂質餾份），及其等之組合，包含至少5 : 1，但少於20 : 1之DHA對EPA的重量比。於一些具體例中，DHA對EPA的重量比係由5 : 1至18 : 1，由7 : 1至16 : 1，或是由1〇 : 1至15 : 1。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份、極性餾份（包括磷脂質餾份），及其等之組合，包含由0.1%至0.25%，0.2%至0.25%，0.1%至 0.5%，或是〇.1〇/0至1.5%重量計ARA 〇於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份、極性餾份（包括磷脂質餾份），以及其等之組合，包含1.5%或更少，1%或更少，0.5%或更 81 201200591 少，0.2%或更少，或是0.1%或更少以重量計ARA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份、極性餾份（包括填脂質館份），以及其等之組合，係實質無ARA的。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、二酸甘油酯餾份、極性餾份（包括磷脂質餾份），以及其等之組合，包含至少20 : 1，至少30 : 1，至少 35 : 1，至少 40 : 1，至少 60 : 1，至少 80 : 1，至少 100 : 1，至少150 : 1，至少200 : 1，至少250 : 1，或至少300 : 1之 DHA對ARA的重量比。於一些具體例中，該微生物脂質及/ 或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份、極性餾份（包括磷脂質餾份），以及其等之組合，包含由 0.5%至 1%，0.5%至2%，0.5%至2.5%，0.5%至 3%，0_5%至3.5%，0.5%至5%，0.5%至6%，1%至2%，2% 至3%，2%至3.5%，1%至2.5%，1%至3%，1%至3.5%，1% 至5%，或是1〇/〇至6%重量計DPAn-6。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份、極性餾份（包括磷脂質餾份），以及其等之組合，包含6%或更少，5%或更少，3%或更少， 2.5%或更少，2%或更少，1%或更少，或是0.5%或更少以重 82 201200591 量計的DPAn-6。於一些具體例中’該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油醋德份、游離脂肪酸德份、固醇德份、二酸甘油酷顧份、極性德份（包括麟脂質德份），以及其等之組合，係實質無DPAn-6的。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份、極性餾份（包括磷脂質餾份）’以及其等之組合，包含大於6 : 1，of至少8 :卜至少10 : 1 ’至少15 :卜至少20 : 1，至少25 : 1，至少50 : 1 ’或至少100 : 1之DHA 對DPA n-6的重量比。於一些具體例中’該微生物脂質及/ 或其等之一或更多餾份’該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份、極性餾份（包括磷脂質餾份），以及其等之組合，包含各5%或更少，4%或更少’ 3%或更少，2%或更少， 1.5%或更少，1%或更少’或是〇·5%或更少以重量計的亞麻油酸（18:2 n-6)、次亞麻油酸（18:3 n-3)、二十烯酸（2〇:1 n-9)，以及芥子酸(22:1 n-9)。於一些具體例中’該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於固醇酯餾份、三酸甘油酯餾份、游離脂肪酸餾份、固醇餾份、二酸甘油酯餾份、極性餾份（包括磷脂質餾份），以及其等之組合，包含5%或更少，4°/。或更少，3%或更少，2%或更少，1.5%或更少，或是1°/。或更少以重量計的十七酸 (17:0)。於一些具體例中，該微生物脂質及/或其等之一或 83 201200591 更多餾份包含0.01%至5%重量計，0.05%至3%重量計，或 0.1%至1°/。重量計之十七酸。於一些具體例中，一種經萃取的微生物脂質包含至少 70%重量計之二酸甘油自旨餾份’其中該三酸甘油醋傲份的二十二叾反六稀酸含量為至少50%重量計，其中該三酸甘油酉旨镏份的二十二碳五烯酸n-6含量為由至少0.5%重量計至 6%重量計’以及其中該油具有26或更少之甲氧苯胺值。於一些具體例中’ 一種經萃取的微生物脂質包含至少7〇%重量計之三酸甘油酯餾份，其中該三酸甘油酯餾份的二十二石厌六稀酸含®為至少40%重置計’其中該三酸甘油醋傲份的二十二碳五稀酸η-6含量為由至少0.5%重量計至6%重量計，其中該二十二碳六烯酸對該二十二碳五烯酸η_6之比率係大於6: 1，以及其中該脂質具有26或更少之甲氧苯胺值。於一些具體例中，一種經萃取的微生物脂質包含至少70% 重量計之三酸甘油酯餾份，其中該三酸甘油酯餾份的二十二碳六烯酸含量為至少60%重量計以及其中該脂質具有26 或更少之甲氧苯胺值。於一些具體例中，一種具有以上的脂肪酸剖繪之任一者的經萃取的微生物脂質具有26或更少，25或更少，20或更少，15或更少，10或更少，5或更少， 2或更少，或是1或更少之曱氧苯胺值及/或5或更少，4.5或更少，4或更少，3.5或更少，3或更少，2.5或更少，2或更少，1.5或更少，1或更少，〇.5或更少，0.2或更少，或是0.1 或更少的過氧化物值，及/或100 ppm或更少’ 95 ppm或更少’ 90 ppm或更少，85 ppm或更少，80 ppm或更少’ 75 ppm 84 201200591Sep Her AG, Germany)) performs extractions with lower anthracene values, lower peroxide values, lower phosphorus levels and/or higher extraction yields. From the first group of lipids extracted by the microorganisms of the genus Thraustochytrium, the present invention proceeds to a microbial lipid, which is similar to the US Publication No. 2010/0239533 and the International Publication No. Extracted from the genus Thraustochytrium as described in WO 2010/107415, the entire disclosure of which is incorporated herein by reference. In some of the eight methods, the method comprises: growing the Thraustochytrium in the culture to produce the biomass and extracting from the biomass - the lipid containing the omega-3 fatty acid can be freshly harvested. The biomass is extracted or extracted from the previously harvested raw f that has been counted under the precautionary condition (4). The method for cultivating the Thraustochytrium of the present invention can be carried out by using the method of the husband, the isolated fatty acid from the culture and the analysis of the fatty acid extracted from the oil of the biomass. See, for example, the US patent case. No. 5,130,242, the entire disclosure of which is incorporated herein by reference. The lipid can be extracted according to the method of the present invention. t The microbial lipid of the present invention may be any of the protamine derived from the microorganism, including 'for example: a crude oil' which is extracted from the biomass of the microorganism without further stepping; An oil obtained by treating a crude microbial oil with a further processing step (eg, refining, bleaching, and/or deodorization) of 77 201200591; a diluted microbial oil diluted by a crude or Obtained by a refined microbial oil; or a concentrated oil obtained by, for example, treating a crude or refined microbial oil by further purification to increase fatty acids in the oil (eg, The concentration of DHA). In some embodiments, the microbial lipid comprises 〇%, at least 0.1%, at least 0.2%, at least 0.5%, at least about 1°/. At least 1.5%, at least 2%, or at least 5% by weight of the sterol ester fraction. In some embodiments, the microbial lipid comprises from 0°/. To 1.5%, 0% to 2%, 0% to 5%, 1% to 1.5%, 0.2% to 1.5%, 0.2% to 2°/. Or a sterol ester fraction of 0.2% to 5°/〇 by weight. In some embodiments, the microbial lipid comprises less than 5%, less than 4%, less than 3%, or less than 2% by weight of the sterol ester fraction. In some embodiments, the microbial lipid comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% by weight of the triglyceride fraction. In some embodiments, the microbial lipid comprises 65% to 95%, 75% to 95%, or 80% to 95% by weight, or 97°/〇 by weight, or 98% by weight of triglyceride S. The purpose is to share. In some embodiments, the microbial lipid comprises at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, or at least 5% by weight of the free fatty acid fraction. In some embodiments, the microbial lipid comprises from 0.5% to 5%, 0.5% to 2.5%, 0.5% to 2%, 0.5% to 1.5%, 0.5% to 1%, 1% to 2.5%, and 1% to 5%, 1.5% to 2.5%, 2% to 2.5%, or 2% to 5% by weight of the free fatty acid fraction. In some embodiments, 78 201200591 the microbial lipid comprises less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% by weight of the free fatty acid fraction. In some embodiments, the microbial lipid comprises at least 0.5%, at least 1%, at least 1.5%, at least 2%, or at least 5% by weight of the sterol fraction. In some embodiments, the microbial lipid comprises from 0.5% to 1.5%, from 1% to 1.5%, from 0.5% to 2%. , 0.5°/. To 5%, 1% to 2%, or 1% to 5% by weight of the sterol fraction. In some embodiments, the microbial lipid comprises less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% by weight of sterol. In some embodiments, the microbial lipid comprises at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, or at least 5% by weight of the diglyceride fraction. In some embodiments, the microbial lipid comprises from 1.5% to 3%, 2% to 3%, 1.5% to 3.5%, 1.5% to 5%, 2.5% to 3%, 2.5% to 3.5%, or 2.5. % to 5% by weight of diglyceride. In some embodiments, the microbial lipid comprises less than 2%, less than 1.5%, less than 1%, or less than 0.5% by weight of the unsaponifiable matter. The lipid species present in the microbial oil, such as the triglyceride fraction, can be separated by flash chromatography and analyzed by thin film chromatography (TLC), or as known in the art. Other methods are separated and analyzed. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triglyceride fraction, a free fatty acid component, a sterol moiety, a diglyceride fraction, and combinations thereof, comprising at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, 79 201200591, at least 65%, at least 70%, at least 75%, or at least 80% by weight DHA. In some specific embodiments, the microbial lipid and/or one or more fractions thereof are selected from the group consisting of a triglyceride fraction, a free fatty acid fraction, a sterol fraction, a diglyceride fraction, and combinations thereof, comprising from 40% to 45%, 40% to 50%, 40% to 60%, 50% to 60%, 55% to 60%, 40% to 65% 50% to 65%, 55% to 65%, 40% to 70%, 40% to 80%, 50% to 80%, 55% to 80%, 60% to 80%, or 70% to 80% Weight DHA. In some embodiments, the microbial lipid comprises a sterol ester fraction comprising 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20 % or less, 15% or less, or 13% or less by weight of DHA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triglyceride fraction, a free fatty acid fraction, a sterol fraction, a diglyceride fraction, and combinations thereof, etc., comprising 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less by weight of EPA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triglyceride fraction, a free fatty acid fraction, a sterol fraction, A diglyceride fraction, and combinations thereof, comprising from 2% to 3%, 2% to 3.5%, 2.5% to 3.5%, 2% to 6%, 2.5% to 6%, 3.0% to 6% , 3.5% to 6%, 5% to 6%, or 2% to 10% by weight of EPA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. The sterol fraction 'diglyceride fraction, 80 201200591 polarity (including the lipid fraction), and combinations thereof, are substantially EPA-free. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. , a sterol fraction, a diglyceride moiety, a polar fraction (including a phospholipid fraction), and combinations thereof, etc., comprising at least 5: !, at least 7: at least 9: at least 1 〇: at least 15 : 1, at least 20: 1, at least 25: at least 30: or at least 50: 1 by weight of DHA to EPA, wherein the microbial lipid and/or one or more of its fractions comprises 10. /〇 or less by weight of EPA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. A sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof, comprising a weight ratio of DHA to EPA of at least 5:1 but less than 20:1. In some embodiments, the weight ratio of DHA to EPA is from 5:1 to 18:1, from 7:1 to 16:1, or from 1〇:1 to 15:1. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. a sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof, comprising from 0.1% to 0.25%, from 0.2% to 0.25%, from 0.1% to 0.5%, Or 〇.1〇/0 to 1.5% by weight of ARA, in some specific examples, the microbial lipid and/or one or more fractions thereof, the one or more fractions being selected from the group consisting of sterols Ester fraction, triglyceride fraction, free fatty acid fraction, sterol fraction, diglyceride fraction, polar fraction (including phospholipid fraction), and combinations thereof, including 1.5% or more Less, 1% or less, 0.5% or 81 201200591 less, 0.2% or less, or 0.1% or less by weight ARA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. The sterol fraction, the diglyceride fraction, the polar fraction (including the lipid-filled portion), and combinations thereof are substantially free of ARA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof, comprising at least 20: 1, at least 30: 1, at least 35: 1, at least 40: 1, at least 60: 1 , at least 80: 1, at least 100: 1, at least 150: 1, at least 200: 1, at least 250: 1, or at least 300: 1 by weight ratio of DHA to ARA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. a sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof, comprising from 0.5% to 1%, from 0.5% to 2%, from 0.5% to 2.5%, 0.5% to 3%, 0_5% to 3.5%, 0.5% to 5%, 0.5% to 6%, 1% to 2%, 2% to 3%, 2% to 3.5%, 1% to 2.5%, 1% To 3%, 1% to 3.5%, 1% to 5%, or 1%/〇 to 6% by weight of DPAn-6. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. , sterol fraction, diglyceride fraction, polar fraction (including phospholipid fraction), and combinations thereof, including 6% or less, 5% or less, 3% or less, 2.5 % or less, 2% or less, 1% or less, or 0.5% or less of DPAn-6 weighing 82 201200591. In some embodiments, the microbial lipid and/or one or more fractions thereof are selected from the group consisting of a sterol ester fraction, a triglyceride, a free fatty acid component. , sterol, diacid glycerin, polar (including linseed), and combinations thereof, are substantially free of DPAn-6. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. , a sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction) and combinations thereof, etc., comprising greater than 6 : 1, of at least 8 : at least 10 : 1 'at least 15 : At least 20: 1, at least 25: 1, at least 50: 1 ' or at least 100: 1 by weight of DHA to DPA n-6. In some embodiments, the one or more fractions of the microbial lipid and/or one or more fractions thereof are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. a sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof, each containing 5% or less, 4% or less '3% or less, 2% or less, 1.5% or less, 1% or less 'or 〇·5% or less by weight of linoleic acid (18:2 n-6), linoleic acid (18: 3 n-3), eicosenoic acid (2〇: 1 n-9), and sinapic acid (22:1 n-9). In some embodiments, the microbial lipid and/or one or more fractions thereof are selected from the group consisting of a sterol ester fraction, a triglyceride fraction, and a free fatty acid fraction. , a sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof, etc., comprising 5% or less, 4°/. Or less, 3% or less, 2% or less, 1.5% or less, or 1°/. Or less of heptadecanoic acid (17:0) by weight. In some embodiments, the microbial lipid and/or one of the same or 83 201200591 more fractions comprise from 0.01% to 5% by weight, from 0.05% to 3% by weight, or from 0.1% to 1%. Heptatypic acid by weight. In some embodiments, an extracted microbial lipid comprises at least 70% by weight of diglycerin from the fraction "wherein the triglyceride is at least 50% by weight of the hexadecane hexahydrate The docosapentaenoic acid n-6 content of the triglyceride is from at least 0.5% by weight to 6% by weight 'and wherein the oil has a methoxyaniline value of 26 or less . In some embodiments, an extracted microbial lipid comprises at least 7% by weight of a triglyceride fraction, wherein the triglyceride fraction has at least 40% The reset meter' wherein the trisuccinic acid spectroscopy has an eta-6 content of at least 0.5% by weight to 6% by weight, wherein the docosahexaenoic acid is 20 The ratio of eicosapentaenoic acid η_6 is greater than 6: 1, and wherein the lipid has a methoxyaniline value of 26 or less. In some embodiments, an extracted microbial lipid comprises at least 70% by weight of a triglyceride fraction, wherein the triglyceride fraction has a docosahexaenoic acid content of at least 60% by weight and Wherein the lipid has a methoxyaniline value of 26 or less. In some embodiments, an extracted microbial lipid having any of the above fatty acid profiles has 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 Or less, 2 or less, or 1 or less of the aniline value and / or 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or Less, 2 or less, 1.5 or less, 1 or less, 〇.5 or less, 0.2 or less, or a peroxide value of 0.1 or less, and/or 100 ppm or less '95 ppm or less' 90 ppm or less, 85 ppm or less, 80 ppm or less ' 75 ppm 84 201200591

或更：70 ppm或更少’ 65 ppm或更少，60 ppm或更少， 55 Ppm或更少，50 ppm或更少，ppm或更少，40 ppm或更少，35 ppm或更少’ 3〇 ??01或更少，25 ppm或更少，20 ppm 或更少，15 ppm或更少，1〇 ppm4更少，5 ??〇1或更少，4 ppm 或更少，3 ppm或更少，2 ppm或更少，或是} ppm或更少的磷含量。於一些具體例中，一種具有以上的脂肪酸剖繪之任—者的經萃取的微生物脂質係自一經單離之破囊壺菌微生物所萃取的，該經單離之破囊壺菌微生物具有根據ATCC 存取號碼PTA-9695、PTA-9696、PTA-9697，或是PTA-9698 所寄存之破囊壺菌物種的特徵。於一些具體例中，一種具有以上的脂肪酸剖繪之任一者的經萃取的微生物脂質為一粗製脂質。於一些具體例中，該粗製脂質具有少於5%重量什或體積計的有機溶劑。於一些具體例中，該依據本發明的方法所萃取之微生物脂質導致之更低的曱氧苯胺值、更低的過氧化物值、更低的磷含量及/或更高的萃取產量，設若萃取係使用一溶劑(例如，非典型的（atypical)己烷萃取或疋FRIOLEX 方法（Westfalia Separator AG，Germany))來執行的。從第二組經單離之破囊壺菌微生物所萃取的脂質於一些具體例中，本發明進一步針對一種微生物脂質，其係從如同美國申請案號12/729,013與 PCT/US2010/028175之中說明的一破囊壺菌所萃取的，其等之各個係以其之整體併入本文中以作為參考資料。於—些具體例中，該方法包含使一破囊壺菌生長於一培養物中以 85 201200591 產生一生質以及自該生質萃取一種包含ω-3脂肪酸的脂質。該脂質可以自一新鮮收穫的生質來萃取或可以自已經貯存於預防糟蹋的條件下之一先前收穫的生質來萃取。可以使用已知的方法來培養本發明的破囊壺菌，來自該培養物單離一生質，以及來分析自該生質所萃取的油之脂肪酸剖繪。參見，例如，美國專利案號5，130,242，以其之整體併入本文中以作為參考資料。該脂質可以依據本發明的方法來萃取。本發明的微生物脂質可以為衍生自一微生物之任何的脂質，包括，舉例而言：一種粗製油，其係自該微生物的生質所萃取而無進一步的加工；一種精製油，其係藉由用進一步的加工步驟(例如精製、漂白，及/或去臭）來處理一粗製微生物油所獲得的；一種經稀釋的微生物油，其係藉由稀釋一粗製或精製的微生物油所獲得的；或是一種濃化的油，其係，舉例而言，藉由用進一步的純化方法來處理一粗製或精製的微生物油所獲得以增加該油内的脂肪酸 (例如DHA)之濃度。於一些具體例中，該微生物脂質包含〇%，至少0.1%，至少0.2%，至少0.5%，至少1°/。，至少1.5%，至少2%，或至少5%重量計的固醇酯餾份。於一些具體例中，該微生物脂質包含0°/。至 1_5%，0%至2%，0%至5%，1%至 1.5%，0.2% 至1.5%，0.2%至2%，或是0.2%至5%重量計的固醇酯餾份。於一些具體例中，該微生物脂質包含5%或更少，4%或更少，3%或更少，2%或更少，1%或更少，0.5%或更少，0.3% 86 201200591 或更少，0.2%或更少，0.5%或更少，0.4%或更少，0.3%或更少，或是0.2%或更少以重量計的固醇酯餾份。於一些具體例中，該微生物脂質包含至少35%，至少 40%，至少45%，至少50%，至少55%，至少60%，至少65%，至少70%，至少75%，至少80%，至少85%，或至少90%重量計的三醯甘油酯餾份。於一些具體例中，該微生物脂質包含35%至98%，35%至90%，35%至80%，35°/。至70%，35% 至70%，35%至65%，40%至70%，40%至65%，40%至55%， 40%至 50%，65%至95%，75%至95%，75%至98%，80%至 95%，80%至98%，90%至96%，90%至97%，90%至98%， 90%，95%，97% ’或是98%重量計的三酿甘油醋德份。於一些具體例中，該微生物脂質包含至少10%，至少 11%，至少12%，至少13%，至少14%，至少15%，至少16%，至少17% ’至少18°/。，至少19%，或至少20%重量計的二醯甘油酯餾份。於一些具體例中，該微生物脂質包含10%至 45%，10%至40% ’ 10%至35%，10%至30%，15。/。至40%， 15%至35%，或是15%至30%重量計的二醯甘油酯餾份。於一些具體例中，該微生物脂質包含至少0.2%，至少0.3%，至少0.4% ’至少0.5。/。，至少1%，至少5%，至少10%，至少 11% ’至少12%，至少13°/。，至少14%，至少15%，至少160/〇，至少17%，至少18% ’至少19%，或至少2〇%重量計的丨，2_ 二醯甘油酯餾份。於一些具體例中，該微生物脂質包含0.2% 至45%，0.2%至30%，0.2%至20%，0.2%至 10%，0.2%至5%， 0.2%至 1%，0.2%至0.8%，〇.4〇/0至45%，0.4%至30%，0.4% 87 201200591 至20%，0.4%至 1〇%，〇.4〇/。至5〇/0, 〇.4〇/〇至 1〇/〇, 0.4%至0.8%， 0.5%至 1°/。，0.5%至0.8%，10%至45%，10%至40%，10%至 350/。，10°/〇至30%，15%至40%，15%至35%，15%至30%，或是15%至25%重量計的二醯甘油酯餾份。於一些具體例中’該微生物脂質包含至少0.1%，至少〇_2%，至少〇 5%，至少1%，至少2% ’至少2.5 %，或至少3%重量計的1，3-二醯甘油酯餾份。於一些具體例中’該微生物脂質包含至少0.3%，至少 0.4% ’至少0.5% ’至少1%，至少1.5%，至少2%，或至少 5°/〇重量計的固醇餾份。於一些具體例中，該微生物脂質包含0.3%至5%，0.3%至2%，0.3%至 1.5%，0.5%至 1.5%，1% 至 1.5%，0.5%至2%，0.5%至5%，1%至2%，或是 1%至5% 重量計的固醇餾份。於一些具體例中，該微生物脂質包含 5°/。或更少，4%或更少，3%或更少，2%或更少，1.5%或更少，或是1%或更少以重量計的固醇餾份。於一些具體例中，該微生物脂質包含至少2%，至少 5%，或至少8%重量計的磷脂質餾份。於一些具體例中，該微生物脂質包含2°/。至25%，2%至20%，2%至15%，2%至 10%，5%至25%，5%至20%，5%至20%，5%至 1〇%，或是 7%至9%重量計的磷脂質餾份。於一些具體例中，該微生物脂質包含少於20%，少於15%，少於10%，少於9%，或少於 8%重量計的鱗脂質餾份。於一些具體例中，該微生物脂質係實質無磷脂質的。於一些具體例中，該微生物脂質包含的不皂化物係少 88 201200591 於2%，少於1.5%，少於1% ,或少於〇 5%重量計之油。存在於該微生物脂質内的之脂質種類，例如三醯甘油醋德份，可以藉由急驟層析法予以分離以及係藉由薄膜層析法(TLC)予以分析，或藉由本技藝中所知道的其他方法予以分離及予以分析。於一些具體例中，該微生物脂質及/或其等之一或更多餾份’該一或更多餾份係選自於三醯甘油酯餾份、游離脂肪酸餾份、固醇餾份、二醯甘油酯餾份，及其等之組合，包含至少5%，至少10%，多於1〇%，至少12%，至少13%，至少14%，至少15% ’至少16%，至少π%，至少18%，至少19% ’至少20%，至少25%，至少30%，最少（least)35%，至少40% ’或至少45°/❶重量計EPA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三醯甘油酯餾份、游離脂肪酸餾份、固醇餾份、二醢甘油酯餾份，以及其等之組合，該方法包含5%至55%，50/〇至50%，5%至45%，5%至40%，5%至35%，5%至30%，10% 至55%，10%至50%，10%至45%，10%至40%，10%至35%， 10%至30%，至少12%至55%，至少12%至50%，至少12%至 45%，至少12%至40%，至少12%至35%，或至少12%至30%， 15%至55%，15%至50%，15%至45%，15%至40%，15%至 35%，15%至30%，15%至25%，15%至20%，20%至55〇/〇， 20%至50%，20%至45%，20%至40%，或是20%至30%重量計EPA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三醯甘油酯餾份、二 89 201200591 醯甘油酯餾份、固醇餾份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，包含至少5%，至少10%，至少15%，至少20%，至少25%，至少30%，至少35%，至少40%，至少50%，或至少60%重量計DHA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三醯甘油酯餾份、二醯甘油酯餾份、固醇餾份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，包含5°/。至60%，5%至55%，5%至50%，5°/〇至 40%，10%至60%，10%至 50%，10%至40%，20%至60%， 25%至60%，25%至50%，25%至45%，30%至50%，35%至 50%，或是30°/。至40%重量計DHA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三醯甘油酯餾份、二醯甘油酯餾份、固醇餾份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，包含10%或更少，9%或更少，8%或更少，7%或更少，6% 或更少，5%或更少，4%或更少，3°/❶或更少，2%或更少，或是1%或更少以重量計DHA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三醯甘油酯餾份、二醯甘油酯餾份、固醇餾份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，包含 1°/。至 10%，1%至5%，2%至5%，3%至5%，或是3%至 10% 重量計之脂肪酸為DHA。於一些具體例中，該微生物脂質及/或其等之一或更多館份，該一或更多館份係選自於三醯甘油酯餾份、二醯甘油酯餾份、固醇餾份、固醇酯餾份、 90 201200591 游離脂肪酸餾份、磷脂質餾份，以及其等之組合，係實質無DHA的。於一些具體例中，該微生物脂質及/或其等之一或更多德份，該一或更多德份係選自於三酷甘油酯德份、二醢甘油酯餾份、固醇餾份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，包含0.1%至5%，0.1% 至少於5%，0.1%至4%，0.1%至3%，0.1%至2%，0.2%至5%， 0.2%至少於5%，0.2%至4%，0.2%至3°/。，0.2°/。至2%，0.3% 至2%，0.1%至 0.5%，0.2%至 0.5%，0.1%至 0.4%，0.2%至 0.4%，0.5%至2%，1%至2%，0.5%至 1.5%，或是 1%至 1.5% 重量計ARA。於一些具體例中，該微生物脂質及/或其等之一或更多德份，該一或更多餾份係選自於三醯甘油S旨鶴份、二醯甘油酯餾份、固醇餾份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，包含5%或更少，少於5%，4%或更少，3°/。或更少，2%或更少，1.5%或更少， 1%或更少，0.5%或更少，0.4%或更少，0.3%或更少，0.2% 或更少，或是0.1%或更少以重量計ARA。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三醯甘油醋德份、二醯甘油醋顧份、固醇館份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，係實質無ARA的。於一些具體例中，該微生物脂質及/或其等之一或更多德份*該一或更多顧份係選自於三酿甘油醋餾份、二醯甘油S旨德份、固醇餾份、固醇S旨德份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，包含0.4% 至2%，0.4%至3%，0.4%至4%，0.4%至5%，0.4%至少於5%， 91 201200591 0.5%至 1%，0.5%至2%，0.5%至3%，0.5%至4%，0.5%至5%， 0.5%至少於5%，1%至2%，1%至3%，1%至4%，1%至5%，或是1%至少於5%重量計DPA n-6。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三醯甘油酯餾份、二醢甘油酯餾份、固醇餾份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，包含5%，少於5%，4%或更少，3%或更少，2%或更少，1% 或更少，0.75%或更少，0.6%或更少，或是0.5%或更少以重量計DPAn-6。於一些具體例中，該微生物脂質及/或其等之一或更多餾份’該一或更多餾份係選自於三醯甘油酯餾份、二醯甘油酯餾份、固醇餾份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，係實質無DPAn-6 的。於一些具體例中，該微生物脂質及/或其等之一或更多餾份，該一或更多餾份係選自於三醯甘油酯餾份、二醯甘油酯餾份、固醇餾份、固醇酯餾份、游離脂肪酸餾份、磷脂質餾份，以及其等之組合，包含脂肪酸，有5%或更少，少於5% ’ 4%或更少，3°/❶或更少，或是2%或更少重量計的油酸（18:1 n-9)、亞麻油酸（18:2 n-6)、次亞麻油酸（18:3 n_3)、二十烯酸(20 : 1 n-9)、芥子酸(22:1 n-9)，硬脂四烯酸 (18:4 n-3)，或其等之組合。於一些具體例中，一種經萃取的微生物脂質包含至少 20%重量計的二十碳五烯酸以及各少於5%重量計的花生油酸、二十二碳五稀酸n-6、油酸、亞麻油酸、次亞麻油酸' 二十烯酸、芥子酸，以及硬脂四烯酸。於一些具體例中， 92 201200591 一種經萃取的微生物脂質包含至少10%重量計的三醯甘油醋餾份，其中於該三醯甘油酯餾份内之至少12%重量計之該等脂肪酸為二十碳五烯酸，其中於該三醯甘油酯餾份内之至少25%重量計之該等脂肪酸為二十二碳六烯酸，以及其中於該三醯甘油酯餾份内之少於5%重量計之該等脂肪酸為花生油酸。於一些具體例中，一種具有以上的脂肪酸剖繪之任一者之經萃取的微生物脂質具有26或更少，25或更少，20或更少，15或更少，10或更少，5或更少，2或更少，或是1或更少之曱氧苯胺值，及/或5或更少，4.5或更少， 4或更少，3.5或更少，3或更少，2.5或更少，2或更少，1.5 或更少，1或更少，0.5或更少，0.2或更少，或是0.1或更少的過氧化物值，及/或100 ppm或更少，95 ppm或更少，90 ppm或更少，85 ppm或更少，80 ppm或更少，75 ppm或更少，70 ppm或更少，65 ppm或更少，60 ppm或更少，55 ppm 或更少，50 ppm或更少，45 ppm或更少，40 ppm或更少， 35 ppm或更少，30 ppm或更少，25 ppm或更少，20 ppm或更少，15 ppm或更少，10 ppm或更少，5 ppm或更少，4 ppm 或更少，3 ppm或更少，2 ppm或更少，或是1 ppm或更少的磷含量。於一些具體例中，一種具有以上的脂肪酸剖繪之任一者的經萃取的微生物脂質係自一經單離之破囊壺菌微生物所萃取的，該經單離之破囊壺菌微生物具有根據ATCC 存取號碼PTA-10208、PTA-10209、PTA-10210、PTA-1021 卜 PTA-10212、PTA-10213、PTA-10214，或是PTA-10215所寄存之破囊壺菌物種的特徵。於一些具體例中，一種具有以 93 201200591 上的脂肪酸剖繪之任一者之經萃取的微生物脂質為一粗製脂質。於一些具體例中，該粗製脂質具有少於5%重量計或體積計的有機溶劑。於一些具體例中，一種依據本發明的方法所萃取之微生物脂質導致比設若使用一典型的己烷萃取或是一 FRIOLEX®方法（Westfa丨ia Separat〇r AG，Germany) 來執行之萃取為更低的甲氧苯胺值，及/或更低的過氧化物值，及/或更低的磷含量。於一些具體例中，一種藉由本發明的方法之任一者所獲得之脂質包含至少20%重量計的二十碳五烯酸以及各少於5%重量計的花生油酸、二十二碳五稀酸n_6、油酸、㈣ ’由西文-人亞麻油酸、二十稀酸、芥子g楚，以及硬脂四歸酸" 於一些具體例中，一種藉由本發明的方法之任—者所獲〜之脂質包含至少1〇%重量計的三酿甘油_份其中於， =酿甘油賴份内之至少12%重量計之該等脂肪酸為= 碳五烯醆，其中於該三醯甘油酯餾份内之至少25%重' 之°亥等脂肪酸為二十二碳六烯酸，以及其中於該= 十 :::份内之少於5%重量計之該等脂肪酸為花生些具體例中’―種具有以上的脂肪酸剖♦之任—者、本發明的方法之任-者所獲得之脂f具有26或更少之藉由更少’ 20或更少，15或更少，1()或更少，裱 5或或更少’或是1或更少之曱氧苯職及/或5或更少3尺2 二T更少，3·5或更少，3或更少，2.5或更少， S更少’ 1或更少，0.5或更少，〇_2或更少，乂的過氣化物值，及/或1〇。ppm或更少，95 _或更=更 94 201200591 90 PPm或更少’ 85 ppm或更少，8〇 ppm或更少，75靜或更少，70 ppm或更少’ 65 ppm或更少，6()解或更少，55 ppm 或更少’50Ppm或更少，45ppm或更少，心㈣或更少， 35 ppm或更少，3〇 ppm或更少，25 ppm或更少，2()卯滅更少，15 PPm或更少’ 1G ppm或更少，5觸或更少，4 ppm 或更少’ 3 ppm或更少，2ppm或更少，或是丨啊或更少的麟含量。於-些具體例中，—種具有以上的脂肪酸剖繪之任-者之藉由本發明的方法之任—者所萃取之脂質係自一經單離之破囊錢微生物解取的，該經單離之破囊壺菌微生物具有根據ATCC存取號碼PTA_1〇2〇8、pTA_1〇2〇9、 PTA-10210 ^ PTA-10211 . PTA-l〇2l2 . pTA_1〇213 . PTA-1()214，或是PTA_1G215所寄存之破囊㈣物種的特徵。於一些具體例中，一種具有以上的脂肪酸剖繪之任一者之藉由本發明的方法之任-者所獲得之脂f為一粗製脂質。於一些具體例中，該粗製脂質具有少於5%重量計或體積計的有機溶劑。於一些具體例中’―種依據本發明的方法所萃取之脂質導致更低的甲氧苯胺值、更低的過氧化物值、更低的磷含量及/或更高的萃取產量，設若萃取係使用一溶劑（例如’非典型的（atypical)己烷萃取或是FRI〇LEX® 方法（Westfalia Separator AG，Germany))來執行的。從一經單離的微生物物種寇氏隱甲藻所萃取的脂質於一些具體财，+發明進一步針對_種自物種寇氏隱曱藻的一微生物所萃取的粗製脂質。於—些具體例中，該方法包含使物種寇氏隱甲藻的一微生物生長於一培養物 95 201200591 中以產生一生質以及自該生質的萃取一種包含ω_3脂肪酸的脂質。該脂質可以自一新鮮收穫的生質來萃取或可以自已經貯存於預防糟蹋的條件下之一先前收穫的生質來萃取。可以使用已知的方法來培養物種寇氏隱甲藻的一微生物，以及來自該培養物單離一生質。參見，例如，美國專利案號7，163,811，以其之整體併入本文中以作為參考資料。該脂質可以依據本發明的方法來萃取。於一些具體例中，依據本發明的萃取方法自物種寇氏隱曱藻的一微生物所萃取之該粗製脂質相比於使用一典型的己烷萃取法可以具有更低的磷含量。於一些具體例中，自物種寇氏隱甲藻的一微生物所萃取之該粗製脂質包含 100 ppm或更少，95 ppm或更少，90 ppm或更少，85 ppm或更少，80 ppm或更少’ 75 ppm或更少，70 ppm或更少’ 65 ppm 或更少，60 ppm或更少，55 ppm或更少，50 ppm或更少， 45 ppm或更少，40 ppm或更少，35 ppm或更少，30 ppm或更少，25 ppm或更少’ 20 ppm或更少’ 15 ppm或更少，10 ppm 或更少，5 ppm或更少，4 ppm或更少，3 ppm或更少，2 ppm 或更少，或是1 PPm或更少的磷含量。於一些具體例中’該粗製油具有26或更少，25或更少’ 20或更少，15或更少’ 10或更少，5或更少’或是2或更少，或是1或更少之甲氧苯胺值及/或5或更少’ 4.5或更少’ 4或更少，3.5或更少’ 3或更少，2.5或更少，2或更少，1.5或更少，1或更少’ 〇.5或更少，0.2或更少’或是〇.1或更少的過氧化物值。於一些具體例中，一種依據本發明的方法所萃取之粗製的微生物脂 96 201200591 質導致更低的甲氧苯胺值、更低的過氧化物值、更低的磷含量及/或更高的萃取產量，設若萃取係使用一溶劑(例如，非典型的（atypical)己規萃取或是FRI〇LEX⑯方法 Separator AG，Germany))來執行的。已經廣泛地說明本發明，參考本文中所提供的實施例而獲知進-步的了解。此等實施例僅供為了闡明的目的以及不打算成為限制。下列實施例為一種方法和藉由本發明的方法所製備之-脂質之例證，但是非限制。多樣的條件和參數之其他適當的修飾和適應於從一細胞萃取一脂質所通常遭遇到的，以及其等對熟悉此藝者會變得明顯的，以及係落在本發明的精神與範缚之内。實施例實施例1 將一含有微生物細胞（裂殖壺菌屬）的細胞肉湯(2〇,〇〇〇 kg)加熱至60 C。將酵素（亦即，驗性蛋白酶L FG 0.5%)添加至該細胞生質以溶解細胞且形成一經乳化之溶解的細胞組成物。該經乳化之溶解的細胞組成物係首先用一第一個驗(NaOH ’ 250 kg的50% w/w溶液）予以處理直到該溶解的細胞組成物之pH為10.4至10.6為止。接著，將一鹽（固體NaCl，以2°/〇，以重量計，之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。該溶解的細胞組成物接而加熱至85t至102Ό的溫度以及保持在該溫度位準歷時 24小時至70小時。繼而將第二個鹼(Na〇H，50% w/w溶液， 4〇 kg)添加至該溶解的細胞組成物直到pH為在8之上為 97 201200591 止。繼而離心該溶解的細胞組成物以將該溶解的細胞組成物分離成三相：含有—脂質層的—頂相、含有-乳狀液層的-中間相’以及含有—固體層的—底相。該溶解的細胞組成物接而予以離心於4〇t：至8(rC使用WestfaUa RSE1 1〇Or more: 70 ppm or less '65 ppm or less, 60 ppm or less, 55 Ppm or less, 50 ppm or less, ppm or less, 40 ppm or less, 35 ppm or less' 3〇??01 or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 1〇ppm4 less, 5 ??〇1 or less, 4 ppm or less, 3 ppm Or less, 2 ppm or less, or a phosphorus content of < ppm or less. In some embodiments, an extracted microbial lipid system having any of the above fatty acid profiles is extracted from an individual isolated from the thraustochytrid microorganism, and the isolated Thraustochytrium microorganism has a basis The ATCC access number PTA-9695, PTA-9696, PTA-9697, or the characteristics of the Thraustochytrium species deposited by PTA-9698. In some embodiments, an extracted microbial lipid having any of the above fatty acid profiles is a crude lipid. In some embodiments, the crude lipid has less than 5% by weight or volume of organic solvent. In some embodiments, the microbial lipid extracted by the method of the present invention results in a lower oxime value, a lower peroxide value, a lower phosphorus content, and/or a higher extraction yield. The extraction is carried out using a solvent (for example, atypical hexane extraction or 疋FRIOLEX method (Westfalia Separator AG, Germany)). Lipids extracted from a second group of isolated Thraustochytrium microorganisms. In some embodiments, the present invention is further directed to a microbial lipid, as in U.S. Application Serial No. 12/729,013 and PCT/US2010/028175. The various extracts of the described Thraustochytrium are described herein in their entirety as a reference. In some embodiments, the method comprises growing a Thraustochytrium in a culture to produce a biomass at 85 201200591 and extracting a lipid comprising an omega-3 fatty acid from the biomass. The lipid may be extracted from a freshly harvested biomass or may be extracted from previously harvested biomass that has been stored under conditions that prevent dross. The known method can be used to culture the Thraustochytrium of the present invention, from which the culture is isolated, and to analyze the fatty acid profile of the oil extracted from the biomass. See, for example, U.S. Patent No. 5,130,242, the disclosure of which is incorporated herein by reference. The lipid can be extracted according to the method of the present invention. The microbial lipid of the present invention may be any lipid derived from a microorganism, including, for example, a crude oil extracted from the biomass of the microorganism without further processing; a refined oil by Obtained by processing a crude microbial oil with further processing steps (eg, refining, bleaching, and/or deodorization); a diluted microbial oil obtained by diluting a crude or refined microbial oil; Or a concentrated oil obtained, for example, by treatment of a crude or refined microbial oil by further purification to increase the concentration of fatty acids (e.g., DHA) within the oil. In some embodiments, the microbial lipid comprises 〇%, at least 0.1%, at least 0.2%, at least 0.5%, at least 1°/. At least 1.5%, at least 2%, or at least 5% by weight of the sterol ester fraction. In some embodiments, the microbial lipid comprises 0°/. To 1_5%, 0% to 2%, 0% to 5%, 1% to 1.5%, 0.2% to 1.5%, 0.2% to 2%, or 0.2% to 5% by weight of the sterol ester fraction. In some embodiments, the microbial lipid comprises 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, 0.3% 86 201200591 Or less, 0.2% or less, 0.5% or less, 0.4% or less, 0.3% or less, or 0.2% or less by weight of the sterol ester fraction. In some embodiments, the microbial lipid comprises at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, At least 85%, or at least 90% by weight of the triterpene glyceride fraction. In some embodiments, the microbial lipid comprises from 35% to 98%, from 35% to 90%, from 35% to 80%, and 35°/. Up to 70%, 35% to 70%, 35% to 65%, 40% to 70%, 40% to 65%, 40% to 55%, 40% to 50%, 65% to 95%, 75% to 95% %, 75% to 98%, 80% to 95%, 80% to 98%, 90% to 96%, 90% to 97%, 90% to 98%, 90%, 95%, 97% 'or 98 % by weight of the three-branched glycerin vinegar. In some embodiments, the microbial lipid comprises at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17% 'at least 18°/. At least 19%, or at least 20% by weight of the diterpene glyceride fraction. In some embodiments, the microbial lipid comprises from 10% to 45%, from 10% to 40% '10% to 35%, from 10% to 30%, 15. /. Up to 40%, 15% to 35%, or 15% to 30% by weight of the diterpene glyceride fraction. In some embodiments, the microbial lipid comprises at least 0.2%, at least 0.3%, at least 0.4% 'at least 0.5. /. , at least 1%, at least 5%, at least 10%, at least 11% 'at least 12%, at least 13°/. , at least 14%, at least 15%, at least 160 / 〇, at least 17%, at least 18% 'at least 19%, or at least 2% by weight of hydrazine, 2 - diglyceride fraction. In some embodiments, the microbial lipid comprises 0.2% to 45%, 0.2% to 30%, 0.2% to 20%, 0.2% to 10%, 0.2% to 5%, 0.2% to 1%, 0.2% to 0.8. %, 〇.4〇/0 to 45%, 0.4% to 30%, 0.4% 87 201200591 to 20%, 0.4% to 1%, 〇.4〇/. To 5〇/0, 〇.4〇/〇 to 1〇/〇, 0.4% to 0.8%, 0.5% to 1°/. , 0.5% to 0.8%, 10% to 45%, 10% to 40%, 10% to 350/. 10 ° / 〇 to 30%, 15% to 40%, 15% to 35%, 15% to 30%, or 15% to 25% by weight of the diterpene glyceride fraction. In some embodiments, the microbial lipid comprises at least 0.1%, at least 〇2%, at least 〇5%, at least 1%, at least 2% 'at least 2.5%, or at least 3% by weight of 1,3-dioxene. A glyceride fraction. In some embodiments, the microbial lipid comprises at least 0.3%, at least 0.4% ' at least 0.5% ' at least 1%, at least 1.5%, at least 2%, or at least 5°/〇 by weight of the sterol fraction. In some embodiments, the microbial lipid comprises 0.3% to 5%, 0.3% to 2%, 0.3% to 1.5%, 0.5% to 1.5%, 1% to 1.5%, 0.5% to 2%, 0.5% to 5%. %, 1% to 2%, or 1% to 5% by weight of the sterol fraction. In some embodiments, the microbial lipid comprises 5°/. Or less, 4% or less, 3% or less, 2% or less, 1.5% or less, or 1% or less by weight of the sterol fraction. In some embodiments, the microbial lipid comprises at least 2%, at least 5%, or at least 8% by weight of the phospholipid fraction. In some embodiments, the microbial lipid comprises 2°/. Up to 25%, 2% to 20%, 2% to 15%, 2% to 10%, 5% to 25%, 5% to 20%, 5% to 20%, 5% to 1%, or 7 From 1 to 9% by weight of the phospholipid fraction. In some embodiments, the microbial lipid comprises less than 20%, less than 15%, less than 10%, less than 9%, or less than 8% by weight of the squamous lipid fraction. In some embodiments, the microbial lipid is substantially phospholipid free. In some embodiments, the microbial lipid comprises less than 5% by weight of the unsaponifiable compound at 2%, less than 1.5%, less than 1%, or less than 5% by weight of the oil. The lipid species present in the microbial lipids, such as triterpene glycerol, can be separated by flash chromatography and analyzed by thin film chromatography (TLC), or as known in the art. Other methods are separated and analyzed. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triterpene glyceride fraction, a free fatty acid fraction, a sterol fraction, A diglyceride fraction, and combinations thereof, comprising at least 5%, at least 10%, more than 1%, at least 12%, at least 13%, at least 14%, at least 15% 'at least 16%, at least π %, at least 18%, at least 19% 'at least 20%, at least 25%, at least 30%, least 35%, at least 40%' or at least 45°/❶ by weight EPA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triterpene glyceride fraction, a free fatty acid fraction, a sterol fraction, Diterpene glyceride fraction, and combinations thereof, the method comprises 5% to 55%, 50/〇 to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, at least 12% to 55%, at least 12% to 50%, at least 12% to 45%, at least 12% to 40%, at least 12% to 35%, or at least 12% to 30%, 15% to 55%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 55〇/〇, 20% to 50%, 20% to 45%, 20% to 40%, or 20% to 30% by weight of EPA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of triterpene glyceride fractions, two 89 201200591 醯 glyceride fractions, solids The alcohol fraction, the sterol ester fraction, the free fatty acid fraction, the phospholipid fraction, and combinations thereof, etc., comprise at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% At least 35%, at least 40%, at least 50%, or at least 60% by weight of DHA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triterpene glyceride fraction, a diterpene glyceride fraction, a sterol distillate The fraction, the sterol ester fraction, the free fatty acid fraction, the phospholipid fraction, and combinations thereof, etc., comprise 5°/. Up to 60%, 5% to 55%, 5% to 50%, 5°/〇 to 40%, 10% to 60%, 10% to 50%, 10% to 40%, 20% to 60%, 25% Up to 60%, 25% to 50%, 25% to 45%, 30% to 50%, 35% to 50%, or 30°/. Up to 40% by weight of DHA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triterpene glyceride fraction, a diterpene glyceride fraction, a sterol distillate a fraction, a sterol ester fraction, a free fatty acid fraction, a phospholipid fraction, and the like, including 10% or less, 9% or less, 8% or less, 7% or less, 6 % or less, 5% or less, 4% or less, 3°/❶ or less, 2% or less, or 1% or less by weight of DHA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triterpene glyceride fraction, a diterpene glyceride fraction, a sterol distillate Parts, sterol ester fractions, free fatty acid fractions, phospholipid fractions, and combinations thereof, including 1°/. To 10%, 1% to 5%, 2% to 5%, 3% to 5%, or 3% to 10% by weight of the fatty acid is DHA. In some embodiments, the microbial lipid and/or one or more thereof, the one or more pavilions are selected from the group consisting of a triterpene glyceride fraction, a diterpene glyceride fraction, a sterol distillate Part, sterol ester fraction, 90 201200591 free fatty acid fraction, phospholipid fraction, and combinations thereof, are substantially free of DHA. In some embodiments, the microbial lipid and/or one or more of the components thereof are selected from the group consisting of trisodium glyceride, diterpene glyceride fraction, and sterol distillation. a portion, a sterol ester fraction, a free fatty acid fraction, a phospholipid fraction, and combinations thereof, comprising 0.1% to 5%, 0.1% at least 5%, 0.1% to 4%, 0.1% to 3%, 0.1% to 2%, 0.2% to 5%, 0.2% at least 5%, 0.2% to 4%, 0.2% to 3°/. , 0.2°/. Up to 2%, 0.3% to 2%, 0.1% to 0.5%, 0.2% to 0.5%, 0.1% to 0.4%, 0.2% to 0.4%, 0.5% to 2%, 1% to 2%, 0.5% to 1.5 %, or 1% to 1.5% by weight ARA. In some embodiments, the microbial lipid and/or one or more of the fractions thereof, the one or more fractions are selected from the group consisting of triterpenoid glycerin S, diterpene glyceride fraction, sterol The fraction, the sterol ester fraction, the free fatty acid fraction, the phospholipid fraction, and combinations thereof, etc., comprise 5% or less, less than 5%, 4% or less, 3°/. Or less, 2% or less, 1.5% or less, 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% Or less by weight ARA. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of triterpene glycerol vinegar, diterpene glycerol vinegar, sterol The sterol ester fraction, the free fatty acid fraction, the phospholipid fraction, and combinations thereof are substantially ARA-free. In some embodiments, the microbial lipid and/or one or more of the components thereof are selected from the group consisting of triglyceride fraction, diterpene glycerol S, and sterol. Distillate, sterol S, free fatty acid fraction, phospholipid fraction, and combinations thereof, including 0.4% to 2%, 0.4% to 3%, 0.4% to 4%, 0.4% to 5% , 0.4% at least 5%, 91 201200591 0.5% to 1%, 0.5% to 2%, 0.5% to 3%, 0.5% to 4%, 0.5% to 5%, 0.5% at least 5%, 1% to 2%, 1% to 3%, 1% to 4%, 1% to 5%, or 1% by weight of DPA n-6. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triterpene glyceride fraction, a diterpene glyceride fraction, a sterol distillate a combination of a sterol ester fraction, a free fatty acid fraction, a phospholipid fraction, and the like, comprising 5%, less than 5%, 4% or less, 3% or less, 2% or less , 1% or less, 0.75% or less, 0.6% or less, or 0.5% or less by weight of DPAn-6. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triterpene glyceride fraction, a diterpene glyceride fraction, a sterol distillate The fraction, the sterol ester fraction, the free fatty acid fraction, the phospholipid fraction, and the like, are substantially free of DPAn-6. In some embodiments, the microbial lipid and/or one or more fractions thereof, the one or more fractions are selected from the group consisting of a triterpene glyceride fraction, a diterpene glyceride fraction, a sterol distillate a portion, a sterol ester fraction, a free fatty acid fraction, a phospholipid fraction, and combinations thereof, comprising a fatty acid, 5% or less, less than 5% '4% or less, 3°/❶ or Less, or 2% or less by weight of oleic acid (18:1 n-9), linoleic acid (18:2 n-6), linoleic acid (18:3 n_3), eicosene Acid (20: 1 n-9), sinapic acid (22:1 n-9), stearic acid (18:4 n-3), or combinations thereof. In some embodiments, an extracted microbial lipid comprises at least 20% by weight of eicosapentaenoic acid and less than 5% by weight of peanut oleic acid, docosapentaenoic acid n-6, oleic acid , linoleic acid, linoleic acid 'eicosenoic acid, sinapic acid, and stearic acid. In some embodiments, 92 201200591 an extracted microbial lipid comprising at least 10% by weight of a triterpene glycerin fraction, wherein at least 12% by weight of the fatty acid in the triterpene glyceride fraction is two Decapentaenoic acid, wherein at least 25% by weight of the fatty acid in the triterpene glyceride fraction is docosahexaenoic acid, and wherein less than 5 of the triterpene glyceride fraction The % by weight of the fatty acid is peanut oleic acid. In some embodiments, an extracted microbial lipid having any of the above fatty acid profiles has 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 Or less, 2 or less, or 1 or less of the aniline value, and / or 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 Or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or a peroxide value of 0.1 or less, and/or 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm Or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less Less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less of phosphorus. In some embodiments, an extracted microbial lipid system having any one of the above fatty acid profiles is extracted from an isolated S. cerevisiae microorganism, and the isolated Thraustochytrium microorganism has a basis ATCC Access Number PTA-10208, PTA-10209, PTA-10210, PTA-1021, PTA-10212, PTA-10213, PTA-10214, or the characteristics of the Thraustochytrium species deposited by PTA-10215. In some embodiments, an extracted microbial lipid having a fatty acid profile on 93 201200591 is a crude lipid. In some embodiments, the crude lipid has less than 5% by weight or volume of organic solvent. In some embodiments, the microbial lipid extracted by the method of the present invention results in an extraction performed by using a typical hexane extraction or a FRIOLEX® method (Westfa丨ia Separat〇 AG, Germany). Low methoxyaniline values, and/or lower peroxide values, and/or lower phosphorus levels. In some embodiments, a lipid obtained by any of the methods of the present invention comprises at least 20% by weight of eicosapentaenoic acid and less than 5% by weight of peanut oleic acid, twenty-two carbon five Dilute acid n_6, oleic acid, (iv) 'from Western-human linoleic acid, twenty dilute acid, mustard g Chu, and stearic acid " In some specific examples, one of the methods of the present invention - The lipid obtained by the solution comprises at least 1% by weight of the tri-branched glycerin, wherein at least 12% by weight of the glycerol-based glycerol is at least 12% by weight of the pentadiene quinone, wherein the triterpenoid At least 25% by weight of the fatty acid in the glyceride fraction is docosahexaenoic acid, and wherein less than 5% by weight of the fatty acid is peanuts in the = ten::: In a specific example, the fat f obtained by the method of the above-described fatty acid profile, or the method of the present invention has 26 or less by less than 20 or less, 15 or less. , 1 () or less, 裱 5 or less 'or 1 or less of oxybenzoquinone and / or 5 or less 3 ft 2 2 T less, 3 · 5 or less 3 or less, 2.5 or less, S less' 1 or less, 0.5 or less, or less 〇_2, qe vapor over value, and / or 1〇. Ppm or less, 95 _ or more 94 201200591 90 PPm or less '85 ppm or less, 8 〇 ppm or less, 75 pm or less, 70 ppm or less ' 65 ppm or less, 6 () solution or less, 55 ppm or less '50 Ppm or less, 45 ppm or less, heart (four) or less, 35 ppm or less, 3 〇 ppm or less, 25 ppm or less, 2 () Less annihilation, 15 PPm or less '1G ppm or less, 5 touches or less, 4 ppm or less' 3 ppm or less, 2 ppm or less, or 丨 or less Lin content. In some specific examples, the lipids extracted by any of the above fatty acid profiles are obtained by dissociating the lipids from the isolated sac. The microorganisms of Thraustochytrium are isolated according to ATCC access numbers PTA_1〇2〇8, pTA_1〇2〇9, PTA-10210^PTA-10211. PTA-l〇2l2. pTA_1〇213. PTA-1()214, Or the characteristics of the sac (4) species registered by PTA_1G215. In some embodiments, a fat f obtained by any of the above methods of fatty acid profile is a crude lipid. In some embodiments, the crude lipid has less than 5% by weight or volume of organic solvent. In some embodiments, the lipid extracted by the method according to the invention results in a lower methoxyaniline value, a lower peroxide value, a lower phosphorus content and/or a higher extraction yield. This is carried out using a solvent such as 'atypical hexane extraction or FRI(R) LEX® method (Westfalia Separator AG, Germany). The lipid extracted from the isolated microbial species Cryptococcus faecalis is further specific to the crude lipid extracted from a microorganism of the species C. elegans. In some embodiments, the method comprises growing a microorganism of the species Cryptococcus faecalis in a culture 95 201200591 to produce a biomass and extracting a lipid comprising the omega-3 fatty acid from the biomass. The lipid may be extracted from a freshly harvested biomass or may be extracted from previously harvested biomass that has been stored under conditions that prevent dross. A known method can be used to culture a microorganism of the species C. elegans, and from the culture. See, for example, U.S. Patent No. 7,163,811, incorporated herein by reference in its entirety herein. The lipid can be extracted according to the method of the invention. In some embodiments, the crude lipid extracted from a microorganism of the species C. elegans according to the present invention may have a lower phosphorus content than a typical hexane extraction process. In some embodiments, the crude lipid extracted from a microorganism of the species C. faecalis comprises 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or Less '75 ppm or less, 70 ppm or less' 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less , 35 ppm or less, 30 ppm or less, 25 ppm or less '20 ppm or less' 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 Ppm or less, 2 ppm or less, or phosphorus content of 1 PPm or less. In some embodiments, the crude oil has 26 or less, 25 or less '20 or less, 15 or less '10 or less, 5 or less' or 2 or less, or 1 Or less methoxyaniline value and / or 5 or less '4.5 or less' 4 or less, 3.5 or less '3 or less, 2.5 or less, 2 or less, 1.5 or less , 1 or less '〇.5 or less, 0.2 or less' or a peroxide value of 〇.1 or less. In some embodiments, a crude microbial lipid 96 201200591 extracted according to the method of the present invention results in a lower methoxyaniline value, a lower peroxide value, a lower phosphorus content, and/or higher. The extraction yield is carried out if the extraction is carried out using a solvent (for example, atypical self-extraction or FRI(R) LEX16 method Separator AG, Germany). The present invention has been broadly described, and further understanding is obtained with reference to the embodiments provided herein. These examples are for illustrative purposes only and are not intended to be limiting. The following examples are illustrative of one method and the lipids prepared by the method of the present invention, but are not limiting. Other suitable modifications and adaptations to a variety of conditions and parameters are commonly encountered in the extraction of a lipid from a cell, and as will become apparent to those skilled in the art, as well as in the spirit and scope of the present invention. within. EXAMPLES Example 1 A cell broth (2 〇, 〇〇〇 kg) containing microbial cells (Schizochytrium) was heated to 60 C. An enzyme (i.e., an experimental protease L FG 0.5%) is added to the cell biomass to dissolve the cells and form an emulsified dissolved cell composition. The emulsified dissolved cell composition was first treated with a first test (NaOH '250 kg of 50% w/w solution) until the pH of the dissolved cell composition was 10.4 to 10.6. Next, a salt (solid NaCl, at a concentration of 2 ° / Torr, the amount of the dissolved cell composition) was added to the dissolved cell composition. The solubilized cell composition is then heated to a temperature of from 85 t to 102 Torr and maintained at this temperature level for a period of from 24 hours to 70 hours. A second base (Na〇H, 50% w/w solution, 4 〇 kg) was then added to the dissolved cell composition until the pH was above 97 at 2012 201200591. The dissolved cellular composition is then centrifuged to separate the dissolved cellular composition into three phases: a top phase containing a lipid layer, an intermediate phase containing an emulsion layer, and a bottom phase containing a solid layer . The solubilized cell composition is then centrifuged at 4 〇t: to 8 (rC using WestfaUa RSE1 1〇)

Centrifuge (Westfalia Separator Industry GmbH，Germany)，操作於6,000 rpms以30 kg/min的一進料速率以自該溶解的細胞組成物分離一脂質。該離心提供了三相：含有一脂質的一上相、含有一乳狀液的一中間相，以及含有一固態/液態乳狀液的一底相《該溶解的細胞組成物之ρΗ·維持於7 $ 至8.5於該離心期間。整個2〇,〇〇〇吆的批量之離心的總時間為大概10至11小時。將該脂質層分離以及具有大概1%重量計的溼氣含量。實施例2 將含有微生物細胞（寇氏隱甲藻）的一細胞肉湯(5 〇〇 g) 從大概7〇/〇生質濃縮至13·5%生質，重量計之肉湯。該肉湯係以10,000 psi的壓力予以均質化(2次通過）以形成一溶解的細胞組成物。該溶解的細胞組成物係用一鹼（亦即， Na〇H，10 g的50% w/w溶液）予以處理直到該溶解的細胞組成物之pH為10.4至10.6為止。將一鹽（固體Naa，以2%重量計之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。接而將該溶解的細胞組成物加熱至以^至％。的溫度以及保持在該溫度範圍歷時15分鐘至2小時。接而將該溶解的細胞組成物予以離心於7〇〇c至9(rc的溫度使用BenchCentrifuge (Westfalia Separator Industry GmbH, Germany), operating at 6,000 rpms at a feed rate of 30 kg/min to separate a lipid from the dissolved cell composition. The centrifugation provides three phases: an upper phase containing a lipid, an intermediate phase containing an emulsion, and a bottom phase containing a solid/liquid emulsion. 7 $ to 8.5 during this centrifugation. The total time for centrifugation of the whole batch is about 10 to 11 hours. The lipid layer was separated and had a moisture content of about 1% by weight. Example 2 A cell broth (5 〇〇 g) containing microbial cells (C. faecalis) was concentrated from approximately 7 〇/〇 biomass to 13.5% biomass, and the broth was weighed. The broth was homogenized (2 passes) at 10,000 psi to form a dissolved cell composition. The solubilized cell composition was treated with a base (i.e., Na〇H, 10 g of a 50% w/w solution) until the pH of the solubilized cell composition was 10.4 to 10.6. A salt (solid Naa, an amount of the dissolved cell composition in an amount of 2% by weight) was added to the dissolved cell composition. The dissolved cell composition is then heated to between 5% and 5%. The temperature is maintained at this temperature range for 15 minutes to 2 hours. The dissolved cell composition is then centrifuged at 7〇〇c to 9 (the temperature of the rc is used in the Bench)

Top Sigma 6K15 Centrifuge (SIGMA Laborzentrifugen 98 201200591Top Sigma 6K15 Centrifuge (SIGMA Laborzentrifugen 98 201200591

GmbH，Germany)，操作於5，400 rpm以將該溶解的細胞組成物分離成二相·含有一脂質的一上相、含有一乳狀液的一中間相，以及含有一固態/液態乳狀液的一底相。該溶解的細胞組成物之pH於該離心期間係維持於6.5至8 5。離心的總時間為5分鐘。將該脂質層分離以及具有大概1%重量計的渔氣含量。實施例3 將含有微生物細胞（裂殖壺菌屬）的一細胞肉湯(2〇,〇〇〇 kg)加熱至60°C。將酵素（亦即，鹼性蛋白酶(3&/奶6)2.4 L FG 0.5%)添加至該細胞生質以溶解細胞且形成一溶解的細胞組成物。接著，將一鹽（固體Na2S04，2,000 kg，或是1 〇%，重量計’之該溶解的細胞組成物）添加至該溶解的細胞組成物。繼而將該溶解的細胞組成物授拌歷時24小時至48小時於室溫。繼而將該溶解的細胞組成物予以離心於4〇°C至75 °C 使用 Westfalia RSE110 Centrifuge (Westfalia Separator Industry GmbH，Germany)，操作於6,000 rpm以40 kg/min 的一進料速率以自該溶解的細胞組成物分離一脂質《該離心提供了三相：含有一脂質的一上相 '含有一乳狀液的一中間相，以及含有一固態/液態乳狀液的一底相。整個20,000 kg的批量之離心的總時間為大概8至9 hrs。將該脂質層從經離心溶解的細胞組成物分離。實施例4 提供含有微生物細胞(ATCC存取號碼PTA-10208)之一經低溫殺菌的細胞肉湯(500 g)。將酵素（亦即，鹼性蛋白酶 99 201200591 (β/αί〇2·4 L FG 0.5%)添加至該細胞生質以溶解細胞且形成經彳Ub之溶解的細胞㈣物。該經乳化之溶解的細胞組成物_ -帛-錄予讀理（料，洲溶液) 來調整該溶解的細胞組成物之PH至1G.5。接著，將一鹽（固體NaCI，以2% ’重量計，之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。該溶解的細胞組成物接而加熱至9 5 °C的溫度以及保持在該溫度位準歷時2小時同時揽拌該溶解的細胞組成物。繼而將第二個鹼（亦即，NaOH之 25%溶液)添加至該溶解的細胞組成物直到阳為8 3為止。接而將該溶解的細胞組成物以5，剛r p m予以離心5分鐘以分離該溶解的細胞組成物以及產生一脂質層及少量的乳狀液層0 實施例5 提供含有微生物細胞(ATCC存取號碼pta-9695)且經濃縮及經低溫殺菌的一細胞肉湯(5〇〇g)。將酵素（亦即，鹼性蛋白酶L FG 0.5%)添加至該細胞生質以溶解細胞且形成一經乳化之溶解的細胞組成物。該經乳化之溶解的細胞組成物係用—鹼（亦即，Na〇H之25%溶液）予以處理來調整該溶解的細胞組成物之]3^1至1〇 5 ^接著，將一鹽（固體Naa，以2%，重量計，之該溶解的細胞組成物的— 量）添加至該溶解的細胞組成物。接而將該溶解的細胞組成物加熱至95°C的溫度以及保持在該溫度位準歷時丨小時同時攪拌該溶解的細胞組成物以及pH降低至8·5。在1小時之後於具有總計10毫升之發酵肉湯内，有大約1毫升層的油 100 201200591 (脂質）以及大約6宅升的乳狀液層。將該溶解的細胞組成物加熱歷時總計220分鐘以及該乳狀液層開始消失。接而將該溶解的細胞組成物以5，100 rpm予以離心歷時5分鐘以分離該溶解的細胞組成物。該脂質的萃取產量為58·8重量％。粗製油之甲乳本胺值(AV)為11.3。細胞斷裂產量（ce丨丨breakage yield)係於93%至95%重量計的範圍内。實施例6 提供一含有根據ATCC存取號碼PTA-9695所寄存之經單離之破囊壺菌的微生物細胞之經低溫殺菌的細胞肉湯 (473 g)。將酵素（亦即’驗性蛋白酶(j/c<3^aiSe)2.4 l FG 0.5%) 添加至該細胞生質以溶解細胞且形成一經乳化之溶解的細胞組成物。該經乳化之溶解的細胞組成物係用一第一個驗 (亦即，NaOH之25%溶液）予以處理來調整該溶解的細胞組成物之pH至10.62。接著，將一鹽（固體Naa，以2%，重量计’之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。該溶解的細胞組成物接而被加熱至95。(：的溫度以及保持在該溫度位準歷時3小時同時攪拌該溶解的細胞組成物。繼而將第二個驗（亦即，NaOH之25%苛性驗溶液)添加至該溶解的細胞組成物直到pH為8.13為止。接而將該溶解的細胞組成物以5，100 rpm予以離心歷時5分鐘以分離該溶解的細胞組成物以及產生等量的一脂質層及一乳狀液層。為了決定是否使該pH提升會增加該脂質層的產量，所以添加額外的第二個鹼（亦即，NaOH之25%溶液）至該經分離之溶解的細胞組成物直到pH為9.02為止以及將該溶解的細胞 101 201200591 組成物以5，1〇〇啊再次予以離心歷時5分鐘。此導致相似產量的脂質層。將額外的第二個驗再次添加至該經分離之溶解的細胞組成物直到PH_.12為止以及將該溶解的細胞組成物以5，1〇〇rpm再次予以離心歷時5分鐘。再者，此導致相似產量的脂質層。實施例7 提供-含有微生物細邮取存取號碼ρτΑ 9695)之經低溫«的細胞肉湯(47Gg)。該細胞生質雜械地予以均質化以溶解細胞以及形成一經乳化之溶解的細胞組成物。該經乳化之溶解的細胞組成物係用—第—個驗（亦即， NaOH之25%雜）予叫理來調㈣轉的細胞組成物之 pH至10.5。接著，將一鹽（固體Naa，以2%，重量計之該溶解的細胞組成物的―量）添加域轉的細胞組成物。該溶解的細触成物—σ^95。⑶溫度以及保持在該溫度位準歷時3小時同時㈣該溶解的細胞組成物。繼而將第二個鹼（亦即，Na0H之25%溶液）添加至該溶解的細胞組成物直到pH為8.07為止。接而將該溶解的細胞組成物以 5，10 0 rp m予以離心歷時5分鐘以分離該溶解的細胞組成物以及產生一脂質層及一乳狀液層，其中該乳狀液層係比該脂質層更大。為了決定是否該pH提升會增加該脂質層的產量，所以將額外的第二個鹼添加至該經分離之溶解的細胞組成物直到pH為9.11為止以及將該溶解的細胞組成物以 5，100 rpm再次予以離心歷時5分鐘。此導致相似產量的脂質層。將額外的第二個鹼再次添加至該經分離之溶解的細胞 102 201200591 組成物直到ρ Η為1 ο. ο 9為止以及將該溶解的細胞組成物以 5，100卬!11再次予以離心歷時5分鐘。再者，該(仇匀導致相似產量的脂質層。實施例8 含有微生物細胞（寇氏隱甲藻）的一細胞肉湯係使用於一減少的生物素試驗發酵器中。2〇,0〇〇 kg之經清洗、濃縮，及經低溫殺菌的肉湯係予以收穫。於低溫殺菌的起動將此拔出。使其維持歷時大概1天，在予以轉移且予以均質化之前。原料係以813巴/1次通過予以均質化以及收集回至一處理槽之内。通過顯微鏡檢查，估計溶解了大概8〇%的細胞。該肉湯係在處理開始之前予以加熱至約4〇。〇。將pH調整至10.5且添加2%NaCl以及加熱至66°c。於此時已經形成一顯著的油層以及pH已經降低至9.5在1-2小時之後。該肉湯保持在66 C過夜。第二天’將該肉湯離心於WestfaHaRSE_110、用所安裝之155 mm環閘。黏性為大約18〇⑪於仙乞。離心機係提供以48kg/mm，且5-l〇psi反壓關於輕相以及3〇psi反壓關於重相。進料溫度係、維持於賊。於廢棄物相之中沒有油存在以及僅可看見幾滴在添加異丙醇之後。表1顯示出執行於由此程序所獲得的粗製油上之分析的結果。 103 201200591 表1.使用實施例8的方法所獲得的粗製油之規格· %油 87.79 DHA (毫克/克） 531.02 % DHA 60.49 PV 1.95 (0.6*) AV 15 %FFA 0.18 填（ppm) 8.65 銅（ppm) 0.22 鐵（ppm) 0.7 船（ppm) 0.63 *離心的油之PV. 於所提供的20,000 1^的肉湯之中，1〇.5%重量計（2，100 kg)為生質。於該生質之中，50%重量計為油（1，〇50 kg)。油之中’58.9%重量計為DHA(618kg)。在進行以上所說明的方法之後，於該脂質層中有592.5 kg的原料，其中大約87.8% 重量計(520.2 kg)為油。因而，自該生質之油的萃取產量為 49.5%。油之中，60.5〇/〇重量計(314.6 kg)為DHA，藉此導致從該生質之51%重量計DHA的萃取產量。實施例9 提供含有微生物細胞（裂殖壺菌屬），經清洗、濃縮，及低溫殺菌的一細胞肉湯（大約500g)。該肉湯係用一鹼（亦即’ NaOH之25%溶液）予以化學處理而無先前的細胞溶解的步驟。鹼之添加使該肉湯之pH由5.8提升至U.2。鹼的添加與pH的提升溶解了細胞以形成一溶解的細胞組成物。接著，將一鹽（固體NajO4，以5❶/。，重量計，之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。該溶解的細胞 104 201200591 組成物接而被加熱至90°C至95°C的範圍内之溫度且保持在該溫度位準歷時90分鐘以及該溶解的細胞組成物之pH降低至9.7。在90分鐘之後，每45毫升的發酵肉湯有大約2.5毫升的油層以及沒有任何溼氣損失。在3小時之後，pH已經降低至9.2。接而將該溶液以大約5,1 〇〇 rpm離心歷時5分鐘以分離該溶解的細胞組成物以及出產一脂質層。該脂質的萃取產量為81°/。重量計。粗製油之甲氧苯胺值(AV)為2〇」。細胞斷裂產量係於92%至98%重量計的範圍内。實施例10 提供含有微生物細胞（裂殖壺菌屬），經清洗、濃縮，及低溫殺菌的一細胞肉湯（大約5〇〇g)。該肉湯係用一鹼（亦即’ NaOH之25%溶液）予以化學處理而無先前的細胞溶解的步驟。驗之添加使該肉湯之pH由4.8提升至11。鹼的添加與 pH的提升溶解了細胞以形成一溶解的細胞組成物。接著，將一鹽（固體NaCn，以2% ’重量計，之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。該溶解的細胞組成物接而被加熱至90。(：至95°C的範圍内之溫度且保持在該溫度位準歷時3.5小時以及該溶解的細胞組成物之pH降低至8.7 且沒有任何渔氣損失。接而將該溶液以大約5，100 rpm予以離心歷時5分鐘來分離該溶解的細胞組成物以及出產一脂質層。該脂質的萃取產量在3.5小時之後為92°/。重量計。使該溶解的細胞組成物之一部份維持歷時6小時以及該溶解的細胞組成物之pH降低至8 6。接而將該溶液以大約 5，100 rpm予以離心歷時5分鐘來分離該溶解的細胞組成物 105 201200591 以及出產一脂質層。該脂質的萃取產量在6小時之後為89% 重量計。粗製油之曱氧苯胺值(AV)為14.4。細胞斷裂產量為95%重量計。實施例11 提供含有微生物細胞(裂殖壺菌屬），經清洗、濃縮，及低溫殺菌的一細胞肉湯（大約500g)。該肉湯係用一驗（亦即，N a Ο Η之5 0 %溶液）予以化學處理而無先前的細胞溶解的步驟。鹼之添加使該肉湯之pH由5.8提升至11.2。鹼的添加與pH的提升溶解了該等細胞以形成一溶解的細胞組成物。該溶解的細胞組成物接而於真空下加熱至70°C以將水含量從88.7%降低至85.5%。在蒸發的期間，pH降低至10.36。將該溶液以大約5,100 rpm予以離心歷時5分鐘來分離該溶解的細胞組成物以及出產一脂質層。該脂質的萃取產量為 83.9%重量計。粗製油之甲氧苯胺值(AV)為10.5。細胞斷裂產量為93.17%重量計。該方法係予以重覆’除了該溶解的細胞組成物係於真空下被加熱至70°C以將水含量從88.7%降低至79.2%之外。該脂質的萃取產量為87.5%重量計當水含量降低至79 2%時以及細胞斷裂產量為92.3%重量計。該方法亦予以重覆以將水含量從88.7%降低至80.8%。該脂質的萃取產量為9〇%重量計當水含量降低至80.8%時以及細胞斷裂產量為95 9%重量計。實施例12 提供含有微生物細胞（裂殖壺菌屬）’經清洗、濃縮，及 106 201200591 低溫殺菌的一細胞肉湯（大約500g)。該肉湯係用一鹼（亦即，NaOH之5〇%溶液）予以化學處理而無先前的細胞溶解的步驟。驗之添加使該肉湯之pH由5.6提升至lu。驗的添加與pH的提升溶解了該等細胞以形成—溶解的細胞組成物。該溶解的細胞組成物接而被加熱至9 〇 °C於—密閉系統中斤時40分鐘。在40分鐘之後，每40毫升的發酵肉湯有大約1毫升層的油（脂質）。接而將該溶液以大約5，1〇〇 rpm予以離心歷時5分鐘來分離該溶解的細胞組成物以及出產—脂質層。该脂質的萃取產量為85.1 %重量計。粗製油之甲氧苯胺值(AV)為16.3。細胞斷裂產量為97.6%重量計。實施例13 提供含有微生物細胞（裂殖壺菌屬），經清洗、濃縮，及低溫殺菌的一細胞肉湯（大約500g)。該肉湯係用—鹼（亦即，NaOH之50%溶液）予以化學處理而無先前的細胞溶解的步驟。鹼之添加使該肉湯之pH由4.9提升至11.2。鹼的添加與pH的提升溶解了該等細胞以形成一溶解的細胞組成物。該溶解的細胞組成物接而予以混合於室溫歷時4小時。接而將該浴液以大約5，1 〇〇 rpm予以離心歷時5分鐘來分離該溶解的細胞組成物以及出產少量的脂質層。該溶解的細胞組成物之一部份係予以混合於室溫歷時大約96小時。將該溶液以大約5，1〇〇 rpm予以離心歷時5分鐘來分離該溶解的細胞組成物以及出產更多量的脂質層。脂質的萃取產量為61.4%重量計。粗製油之甲氧苯胺值(av) 為 22.6。 107 201200591 實施例14 提供含有微生物細胞(ATCC存取號碼PTA-9695)，經清洗、濃縮，及低溫殺菌的一細胞肉湯（大約5〇〇g)。該肉湯係用一鹼（亦即’ MaOH之50%溶液）予以化學處理而無先前的細胞溶解的步驟。鹼之添加使該肉湯之pH由5 6提升至 11.1。鹼的添加與pH的提升溶解了該等細胞以形成一溶解的細胞組成物。該溶解的細胞組成物接而予以加熱於7〇。〇至75 C的範圍内歷時3小時。接而將該溶液以大約5，1〇〇 rpm 予以離心歷時5分鐘來分離該溶解的細胞組成物以及出產一脂質層。該脂質的萃取產量為84 4%重量計。該溶解的細胞組成物之一部份係被加熱歷時總計5小時。接而將该溶液以大約5，i〇〇 rpm予以離心歷時5分鐘來分離該溶解的細胞組成物以及出產相似的脂質層。脂質的萃取產量為87.3%重量計。細胞斷裂產量為891%重量計。實施例15 提供含有微生物細胞（裂殖壺菌屬），經清洗、濃縮，及低溫殺菌的一細胞肉湯（大約5〇〇g)。該肉湯係用一鹼（亦即，NaOH之50%溶液）予以化學處理而無先前的細胞溶解的步驟。驗之添加使該肉湯之阳由73提升至u。鹼的添加與 pH的提升溶解了料細胞以形成—溶解的細胞組成物。接著，將-鹽（固體Na2S〇4 ’以5%，重量計’之該溶解的細胞組成物的-4)添加至該溶解的細齡成物。該溶解的細胞組成物接而被加熱至901的溫度以及保持在該溫度位準歷時2小時。同時維持9〇t的溫度歷時額外的2至4小時，將含 108 201200591 有該溶解的細胞組成物的器孤打開以允許水的蒸發。接而將該溶液以大約5，丨〇〇 rp m予以離心歷時5分鐘來分離該溶解的細胞組成物以及出產一脂質層。該脂質的萃取產量為大於70%重量計。粗製油之曱氧笨胺值(AV)為11.6。實施例16 提供含有微生物細胞（ATCC存取號碼PTA-9695)的一細胞肉湯(9,925 kg)。該細胞肉湯係用水以丄：i比率重量計予以稀釋來形成20,000 kg之經稀釋的肉湯。該肉湯的固體含篁在稀釋之前為16.13%重量計以及在稀釋之後為8 25% 重量計。將經稀釋的肉湯混合以及用一去泥離心機以6,4〇〇 rpm予以離心來移除細胞外的水溶性或水分散性化合物。濃縮物（1〇,250 kg)係從該離心機來收集以及具有1〇 5%重量計的固體含量《該所收集的濃縮物係被加熱至62〇(：至64。(：來低溫殺菌該濃縮物。將酵素（亦即，鹼性蛋白酶〇4/ca/ose)2.4 L FG 0.5%)添加至該經低溫殺菌的濃縮物以溶解該等細胞以及形成一經乳化之溶解的細胞組成物。該經乳化之溶解的細胞組成物係用一驗（亦即，Na〇H之25% 溶液）予以處理來調整該溶解的細胞組成物之13^1至丨丨。接著’將一鹽（固體NajO4’以5%，以重量計，之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。該溶解的細胞組成物接而被加熱至95。(：的溫度以及保持在該溫度位準歷時10小時至12小時同時攪拌該溶解的細胞組成物。在攪拌之後，該溶解的細胞組成物之pH為8.6以及有非常少的乳狀液層。允許該攪拌槽冷卻至60。(：以及該溶解的細胞組成 109 201200591 物之pH增加至9.6同時冷卻。該溶解的細胞組成物之pH係藉由添加構酸而降低至8_2。蛾酸之添加不會損害該脂質層及該非常少的乳狀液層之分離。該溶解的細胞組成物接而以 5，100 rpm、以48 kg/min的一進料速率予以離心歷時5分鐘於60 °C至63 °C來分離該溶解的細胞組成物以及出產具有 1.7%至2_3°/。重量計的澄氣含量之一脂質層。實施例17 π货嘗另做玍物細脃（冠氏隱甲藻），經清洗、濃縮，低溫殺菌的-細胞肉湯(50〇g)。該肉湯係以8 _至12 〇( ㈣的壓力（2次通過）予以均質化卿成—溶解的細胞心物。該溶解的細胞組祕_ _驗（騎，聽^請〉液)。予以處理直龍轉的細驗絲物.8至8.2的 pHi 止。將一鹽（固體Na2S04，以5。/。重量_ + _ 抓沾1 笪叶之該溶解的細胞組万物的-量)添加至該溶解的細胞組物接而被㈣㈣。⑽溫度叹彳贿解的細胞… 将在該溫度。該溶解白，、田胞組成物之pH係藉由添㈣( 而祜給即，NaOH之12.5%溶液而破維持於7.8至8·2的位準歷時中伴隨很少古/ / > \ 小時於一密閉系矣吸恨夕或沒有任何水分損失。成物U + & 接而將該溶解的細胞I. 风物U大約5，1G0 rpm予以離心歷細胞缸忐札此f 5分鐘來分離該溶解6 匕、、且成物以及出產一油層。此 40毫井ίΛ , 软大約2毫升的一油層另樣品内。油的萃取產B氐„ 之曱氧贫^ 座量為73%重量計。粗製胺值(AV)為13_5。細胞斷釗計。產量為82%至86%重i 實施例18 110 201200591 提供含有微生物細胞（裂殖壺菌屬）的一細胞肉湯 (1，000 g)。將酵素（亦即’驗性蛋白酶(伽2·4 L FG 〇·5%)添加至該細胞生質以溶解細胞且形成'經乳化之溶解的細胞組成物。該經乳化之溶解的細胞組成物係用一驗 (亦印，Na〇H之12.5%溶液）予以處理來調整該溶解的細胞組成物之pH由7.21至10.52。接著，將一鹽（固體Naa，以 2%，重量計，之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。接而將該肉湯分隔成4部分，且各部分經保持在4個不同的溫度和時間：1)將試驗#1保持在9〇。匸歷時22 小時；2)將試驗#2保持在90°C歷時2小時以及接而保持在25 C歷時20小時；3)將試驗#3保持在6〇。(：歷時22小時；以及 4)將試驗#4保持在25°C歷時22小時。個別的試驗接而予以離心而無進一步pH調整。關於試驗#1、#2，和#3，該肉湯係以大概6,600卬m(4,800之g力）予以離心歷時5分鐘來分離該溶解的細胞組成物。因為試驗#4之分離於4,800之g力不佳（<20%) ’ g力增加至15,000以及該肉湯係於15,000之g力旋轉歷時5分鐘。該脂質的萃取產量依照重量百分比與甲氧苯胺值(AV)係列舉於以下的表中。表2.當變化該溶解的細胞組成物之溫度和加熱的時間時之條件及結果· 試驗# 處理時間和溫度離心條件 AV 油產量(％) 1 90°C歷時22小時 pH = 6.22 g力=4,800 58.7 51.4 2 90°C歷時2小時，25°C 歷時20小時 pH = 8.19 g力=4,800 109.2 82.2 3 6〇°C歷時22小時 pH = 8.38 g力=4,800 91.2 27.2 4 25°C歷時22小時 pH = 10.03 g力=15,000 105.2 55.7 111 201200591 於表2中的甲氧苯胺值係比預期更高的。介於先前的實施例與此實施例之間的一個差異為允許該溶解的細胞組成物靜置歷時一段長的時間期間在萃取脂質之前。假定在萃取之前之長的時間期間導致存在於該溶解的細胞組成物之中的溶解的氧之氧化。增加的氧化接而導致曱氧笨胺值的增加。於最高的溫度加熱歷時最長的時間（試驗#1)之試驗具有最低的甲氧笨胺值之事實支持此假說’因為一溶解的細胞組成物之溶解的氧含量一般而言隨著溫度增加而減少。增加的甲氧苯胺值因而據信為—反常現象，其為延遲自該溶解的細胞組成物萃取脂質的結果。於生產中，不會有從該溶解的細胞組成物萃取脂質的延遲時間以及曱氧笨胺值會與26或更少的甲氧苯胺值之先前的結果一致。實施例19 k供含有彳政生物細胞（裂殖壺菌屬）的一細胞肉湯 (1,000 g)。該肉湯接而分成3部分以及稀釋如下：丨）試驗#1 完全未被稀釋且供使用作為對照部分；2)試驗#2係用水稀釋25%;以及3)試驗#3係用水稀釋5〇。/(^將酵素（亦即，鹼性蛋白酶L FG 〇.5%)添加至該細胞生質以溶解細胞且形成一經乳化之溶解的細胞組成物。該經乳化之溶解的細胞組成物係用一鹼（亦即，Na〇Hi12 5%溶液）予以處理來調整該溶解的細胞組成物之pH由6.8至10.6 ^接著，將一鹽（固體NaCl，以2%，重量計，之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。該肉湯接而被加熱至9(TC以及維持歷時20小時。在維持的時間之後，將各 112 201200591 試驗之肉湯劃分成2 ’且將一半離心asis以及另一半使其之pH調整至大概8.5在離心前。兩部分接而都以大概8,545 rpm (8,000之g力）予以離心歷時5分鐘。依照重量百分比與甲氧苯胺值(AV)之脂質的萃取產量係列舉於以下的表中。表3.當變化稀釋經低溫殺菌的肉湯之稀釋度時之條件及結果. 試驗# 經稀釋的？離心條件 AV 油產量(％) 1 沒有稀釋 pH = 6.0 g力=8,000 51.8 81.2 la 沒有稀釋 pH = 8.4 g力=8,000 44.3 78.1 2 用水25%稀釋 pH = 5.5 g力=8,000 76.1 88.9 2a 用水25%稀釋 pH = 8.4 g力=8,000 85.3 82.1 3 用水50%稀釋 Γ pH = 5.7 g力=8,000 68.5 85.0 3a 用水50%稀釋 pH = 8.5 g力=8,000 79.6 84.0 於表3中之甲氧笨胺值係比預期更高。介於先前的實施例（除實施例18之外）與此實施例之間的一個差異為允許該溶解的細齡成物#置歷時—段長_關間在萃取脂質之則。假定在萃取之前之長的時間期間導致存在於該溶解的、”田胞組成物之中的溶解的氧之氧化。增加的氧化接而導致甲氧笨胺值的增加。增加的甲氧笨胺值因而據信為-反常見象其為延遲從該溶解的細胞組成物萃取脂質的結果於生產中’不會有從該溶解的細胞組成物萃取脂質的延遲夺間以及甲氧笨胺值會與26或更少的曱氧苯胺值之先前的結果一致。實施例20 從各種的發酵批次獲得的細胞肉湯係制於實施例2 113 201200591 中所說明的方法予以處理，除了添加鹽之時間的選擇（例如，在均質化前及在均質化後）以及不同的鹽的量之外。所生成之經分離的脂質係予以分析以及分析係提供於表4中。表4_不同的鹽添加的時機和所添加的鹽量之使用本發明的方法所獲得的脂質之規格. 發酵批次 P2137 P2137 P4167 P4167 P2137 P2137 NaCl的添加· 之前之前之後之後之後之後 %NaCl重量計 2 5 2 5 5 5 %脂質· 27 37 51 72 59 15 %起始固體重量計 13 12.9 19.2 19.2 13.3 7.5+ %固體重量計在離心前 19.7 21.7 19.7 20.1 20.2 8.6 均質化前或在均質化後；％脂質提及以三酸甘油酷形式之脂質的百分比此操作係經轉以具有低百分比的起始固體。表4中提供的資料展現出在均質化後添加該鹽導致比在均質化前添加該鹽有更高的％脂質值。表4中提供的資料亦展現出稀釋該樣品會導致較低的％脂質值。實施例21 使用自微生物細胞（裂殖壺菌屬）所獲得的經驗性蛋白㈣心㈣酵素處理之溶解的細胞組成物的—樣品。該樣时具有大概5.5的pH。该樣品劃分成4份更小的樣品以及該等樣品的3份之pH係藉由添加氫氧化鈉而分別地調整至大概7.4、大概1G.5,以及大概12。該等樣品係用去離子水以】： !比率予以稀釋。將Ρ〇ΒΝ(α_(44定基n氧化物)况三級丁基石肖基酮’⑶心㈣作為—自旋捕集化學品添加抓 g的經稀釋的樣品之各個。該等樣品係用一Βγ-γ印学 114 201200591 e-scan EPR (電子順磁共振）光譜儀（系統號碼SC〇274) (Bruker BioSpin，Billerica，ΜΑ)予以測量來測量由脂質的氧化而存在之自由基的量◦該等樣品係孵育於室溫(2〇。〇以及含有樣品之50 μί的ΡΟΒΝ之各個係使用下列光譜儀參數在調整pHs之後以每小時的間隔予以測試歷時4小時：86 Hz的調變頻率，2.U高斯的調變振輻，519 mW的微波功率，20.48秒的時間常數，10.49秒的掃掠時間，1〇〇高斯的掃掠寬度，以及8的掃描數目。EPR光譜儀讀數的結果係提供於第5圖中。於第5圖中的資料展現出最初於pH 5.5之樣品之自由基的位準為最高的以及於pH 10.5和12之樣品之自由基的位準為最低的。該資料亦展現出在4小時期間自由基形成的速率於PH 10.5之樣品為最慢的以及於pH 5.5之樣品為最高的。該資料亦展現出添加鹼至該溶解的細胞組成物抑制了脂質氧化，以及因而導致於該粗製脂質與精製油内之低的AV。實施例22 油籽係自一油菜子植物予以萃取以及接而通過一研磨磨碾機以使油籽的外殼破裂且碎裂。該等油籽接而經由已知的構件予以去殼，例如經由吸入，以由油籽的殼來移除種子的食用部分（内部）。去殼的油籽可以藉由使其等通過一壓機予以均質化或予以排出而來研磨去殼的油籽成為一塊狀物俾以溶解油籽的細胞。可以添加水來形成一經乳化之溶解的細胞組成物。該經乳化之溶解的細胞組成物係予以過濾以自該溶解的細胞組成物移除任何過量的殼碎片。該 115 201200591 經乳化之溶解的細胞組成物係用一鹼（亦即，Na0H之25% 溶液）予以處理來調整該溶解的細胞組成物之PH至11。接著將一鹽（固體NaCU，以2%重量計之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。該溶解的細胞組成物接而被加熱至90 C的溫度以及保持在該位準歷時6小時至48小時同時攪拌該溶解的細胞組成物。該溶解的細胞組成物接而以5，100 rpm予以離心歷時5分鐘來分離該溶解的細胞組成物以及產生一脂質層及一乳狀液層。實施例23 藉由己烷萃取所獲得的粗製脂質之比較分析自一批使用實施例2中所說明的方法所獲得的粗製脂質係予以分析來決定各種的規格。額外的粗製脂質係使用一典型的己烷方法對於實施例2中使用之同樣的微生物細胞而獲得。己烷萃取方法包括喷霧乾燥一發酵肉湯，添加己烷至該經噴霧乾燥的生質以獲得15%至20°/。固體重量叶的一溶液。該溶液接而予以均質化來溶解該等細胞以形成一溶解的細胞組成物。將該溶解的細胞組成物予以離心以及移除含有脂質與己烷的一層。接而將己烷自該脂質移除。分析的結果係提供於表5中。 116 201200591 表5.使用本發明的方法或是己烷萃取方法所獲得的脂質之規格. 發酵批次 A B C D E F G 萃取方法 Ex. 2 己烷己烧己烧己炫· 己坑己烷 AV· 5.9 ND ND ND 14.7 17.18 6.7 PV* 1.21 0.65 1.56 0.46 ND 0.85 0.3 %脂質· 89.61 86.94 84.31 86.75 85.53 86.05^ 86.54 DHA ί毫克/克） 537.47 508.15 459.32 465.31 510.49 495.82 506.33 % DHA* 59.98 58.39 54.49 53.65 59.71 57.68 58.51 AV =甲氧苯胺值；PV =過氧化物值；〇/。脂質提及以三酸甘油酯形式之脂質的百分比；％DHA提及該脂質内的DHA之百分比表5中提供的資料展現出藉由本發明的方法所獲得之粗製脂質與係藉由典型的己烷萃取方法所製備的脂質相比顯出較優秀的曱氧笨胺值、脂質的百分比、DHA的量以及 DHA的百分比。經由FRIOLEX®方法所獲得的粗製脂質之比較分析實施例24 從使用於實施例1和3中所說明的方法之各種的發酵批次所獲得的粗製脂質係予以分析來決定各種的規格，額外的粗製月曰貝係使用FRIOLEX®方法（Westfalia Separator AG，Germany)所獲得的，其係一種依照美國專利案號 5,928,696以及國際公開案號w〇 01/76385和 WO 01/76715 之中所說明的用一水溶性有機溶劑萃取脂質的方法。分析的結果係提供於表6中。 117 201200591 表6.使用本發明的方法或是FRIOLEX®方法所獲得的脂質之規格. 發酵批次 A B B B B C C C 萃取方法 Ex. 1 Ex. 1 Ex. 3 Ex. la Ex. lb Ex. 1 Ex. 1 FRIOLEX® AV· 3.1 1.6 3.9 300 7.1 3.5 4 36 PV* 1.8 0.17 0.14 6.16 0.34 0 0 0.35 %脂質* 96.27 93.67 92.55 87.20 94.42 95.14 94.31 93.92 DHA (毫克） 452.4 458.16 455.17 414.66 471.55 416.41 416.05 415.38 萃取產量 (%) 93.5 87 ND 96 ND 94 94 94 * AV =甲氧苯胺值；pv =過氧化物值；％脂質提及以三酸甘油酯形式之脂質的百分比 a:該溶解的細胞組成物不予以加熱。 b:在萃取之前允許該溶解的細胞組成物靜置歷時3週。表6中提供的資料展現出藉由本發明的方法所獲得之粗製脂質與藉由一 FRIOLEX®方法予以製備的脂質相比顯現出較優秀的曱氧笨胺值（伴隨該溶解的細胞組成物不予以加熱時所獲得的脂質之例外）。藉由本發明的方法所製備之脂質展現出為使用FRIOLEX®方法所製備的一脂質之甲氧苯胺值之4.4%至19.7%之甲氧苯胺值。據#藉由本發明的方法所製備之一脂質已經增加了穩定性。舉例而言，如表6中所顯示的，本發明的一方法係使用來由-溶解的細胞組成物萃取一脂質，其中在萃取方法之前允許該溶解的細胞組成物靜置歷時3週。據信於一溶解的’.田H成物中之-脂質之甲氧苯胺值隨著時間而增加，以及因而’會預期到從3週長的溶解的細胞組成物所萃取的月曰質已、盈增加了曱氧苯胺值。然而，如表6中所顯示的，使用本發明的-方法而從3週長的溶解的細胞組成物獲得 118 201200591 的脂質具有一曱氧苯胺值，其為藉由FRIOLEX®方法戶的一脂質之甲氧苯胺值的19.7%。實施例25 使用於實施例16中所說明的方法而自微生物細& (ATCC存取號碼PTA-9695)的一肉湯所獲得的粗製脂質係予以分析來決定各種的規格。額外的粗製脂質係使用 FRIOLEX®方法(Westfalia Separator AG，Germany)而自微生物細胞（ATCC存取號碼PTA-9695)的一肉湯所獲得，該 FRIOLEX®方法係一種依照美國專利案號5,928,696與國際公開案號W0 01/76385和W0 01/76715之中說明之用一水溶性有機溶劑（例如，異丙醇）萃取脂質的方法。分析的結果係提供於表7中。表7.粗製脂質比較實施例16 Friolex® %油 9.19 93.73 DHA (毫克/克） 570.68 574.33 % DHA 62.1 61.27 % FFA 1.13 0.22 PV 0 0.74 AV 10 73.9 鐵(ppm) 0.11 0 銅（ppm) 0.67 0.3 船(ppm) 0.21 0 _ 磷(ppm) 5.22 7.20 萃取產量（％) 61.4 45.3 表7中提供的資料展現出藉由本發明的方法所獲得之粗製脂質與藉由FRIOLEX®方法所製備的脂質相比顯現出 119 201200591 較優秀的甲氧苯胺值(AV)以及過氧化物值(pv)。該資料亦展現出藉由本發明的方法所獲得之脂質的萃取產量與經由 ™〇LEX%法賴得的脂f料取產量相比為較優秀的。實施例26 粗製脂質的精製粗製脂質係使用於實施例1中所概述的方法以及 FRI0LEX®方法所獲得。該等”脂質係相繼地藉由：υ 去膠和苛性精製法；2)漂白；3)冷卻過濾;以及·抗氧化劑去臭予以進-步加工。關於粗製脂f、苛性精製脂質、漂白的脂質，以及去臭的脂質之資料係呈現於表如㈣中。精製油之比較係呈現於表9中。 ^自_LEX⑯方法獲得_ f (實施例24)GmbH, Germany), operating at 5,400 rpm to separate the dissolved cell composition into two phases, an upper phase containing a lipid, an intermediate phase containing an emulsion, and a solid/liquid emulsion a bottom phase of the liquid. The pH of the dissolved cell composition was maintained at 6.5 to 85 during the centrifugation. The total time for centrifugation is 5 minutes. The lipid layer was separated and had a fish gas content of about 1% by weight. Example 3 One cell broth (2 〇, 〇〇〇 kg) containing microbial cells (Schizochytrium) was heated to 60 °C. An enzyme (i.e., alkaline protease (3 & / milk 6) 2.4 L FG 0.5%) was added to the cell biomass to dissolve the cells and form a dissolved cell composition. Next, a salt (solid Na2SO4, 2,000 kg, or 1% by weight, by weight of the dissolved cell composition) is added to the dissolved cell composition. The solubilized cell composition is then allowed to mix for 24 hours to 48 hours at room temperature. The dissolved cell composition was then centrifuged at 4 ° C to 75 ° C using Westfalia RSE 110 Centrifuge (Westfalia Separator Industry GmbH, Germany), operated at 6,000 rpm at a feed rate of 40 kg/min to dissolve therefrom. The cell composition separates a lipid "This centrifugation provides three phases: an upper phase containing a lipid" an intermediate phase containing an emulsion, and a bottom phase containing a solid/liquid emulsion. The total time for centrifugation of the entire 20,000 kg batch is approximately 8 to 9 hrs. The lipid layer was separated from the cell composition dissolved by centrifugation. Example 4 A cell broth (500 g) containing one of microbial cells (ATCC accession number PTA-10208) was sterilized by low temperature. An enzyme (i.e., alkaline protease 99 201200591 (β/αί〇2·4 L FG 0.5%) is added to the cell biomass to dissolve the cells and form a cell (tetra) dissolved by the 彳Ub. The emulsified dissolution The cell composition _ - 帛 - recorded into the reading (material, continent solution) to adjust the pH of the dissolved cell composition to 1G.5. Next, a salt (solid NaCI, based on 2% 'weight, An amount of the dissolved cell composition is added to the dissolved cell composition. The dissolved cell composition is then heated to a temperature of 95 ° C and maintained at the temperature level for 2 hours while mixing the solution. a cell composition. A second base (i.e., a 25% solution of NaOH) is then added to the dissolved cell composition until the yang is 8 3. The dissolved cell composition is then 5, just rpm Centrifuge for 5 minutes to separate the dissolved cell composition and produce a lipid layer and a small amount of emulsion layer. Example 5 provides a microbial cell (ATCC accession number pta-9695) and concentrated and sterilized. Cell broth (5〇〇g). Enzyme (ie, alkaline egg) White enzyme L FG 0.5%) is added to the cell biomass to dissolve the cells and form an emulsified dissolved cell composition. The emulsified dissolved cell composition is made with a base (ie, 25% of Na〇H) The solution is treated to adjust the dissolved cell composition] 3^1 to 1〇5 ^ Next, a salt (solid Naa, based on 2% by weight, of the dissolved cell composition) is added To the dissolved cell composition, the dissolved cell composition was then heated to a temperature of 95 ° C and maintained at this temperature level for a while while stirring the dissolved cell composition and the pH was lowered to 8.5. After 1 hour in a fermentation broth with a total of 10 ml, there was approximately 1 ml of layer of oil 100 201200591 (lipid) and approximately 6 liters of emulsion layer. The dissolved cell composition was heated for a total of 220 minutes. And the emulsion layer began to disappear. The dissolved cell composition was then centrifuged at 5,100 rpm for 5 minutes to separate the dissolved cell composition. The extracted yield of the lipid was 58.8% by weight. Oil-to-milk lactam value (AV) 11.3. Cell breakage yield is in the range of 93% to 95% by weight. Example 6 provides a bacterium that contains the isolated bacterium that is deposited according to the ATCC accession number PTA-9695. Low temperature bactericidal cell broth (473 g) of microbial cells. Enzyme (also known as 'probe protease (j/c) <3^aiSe) 2.4 l FG 0.5%) is added to the cell biomass to dissolve the cells and form an emulsified dissolved cell composition. The emulsified dissolved cell composition was treated with a first test (i.e., a 25% solution of NaOH) to adjust the pH of the solubilized cell composition to 10.62. Next, a salt (solid Naa, an amount of the dissolved cell composition of 2% by weight) was added to the dissolved cell composition. The solubilized cell composition was then heated to 95. The temperature of (: and the temperature maintained at the temperature level for 3 hours while stirring the dissolved cell composition. A second test (ie, a 25% caustic solution of NaOH) is then added to the dissolved cell composition until The pH was 8.13. The dissolved cell composition was then centrifuged at 5,100 rpm for 5 minutes to separate the dissolved cell composition and produce an equal amount of a lipid layer and an emulsion layer. Increasing this pH will increase the yield of the lipid layer, so an additional second base (i.e., a 25% solution of NaOH) is added to the separated dissolved cell composition until the pH is 9.02 and the solution is dissolved. Cell 101 201200591 The composition was again centrifuged at 5, 1 Torr for 5 minutes. This resulted in a similar yield of lipid layer. An additional second test was added again to the isolated dissolved cell composition until PH_.12 The dissolved cell composition was again centrifuged at 5,1 rpm for 5 minutes. Again, this resulted in a similar yield of lipid layer. Example 7 Provided - Containing Microbial Fine Mail Number ρτΑ 9695) of the low temperature << cell broth (47Gg). The cell biomass is homogenized to dissolve the cells and form an emulsified dissolved cellular composition. The emulsified solubilized cell composition is conditioned to adjust the pH of the (4) transformed cell composition to 10.5 by a first test (i.e., 25% of NaOH). Next, a salt (solid Naa, the amount of the dissolved cell composition in an amount of 2% by weight) was added to the domain-transformed cell composition. The dissolved fine contact is - σ^95. (3) The temperature and the temperature at which the temperature was maintained for 3 hours while (iv) the dissolved cell composition. A second base (i.e., a 25% solution of NaOH) was then added to the dissolved cell composition until the pH was 8.07. The dissolved cell composition is then centrifuged at 5,10 rpm for 5 minutes to separate the dissolved cell composition and produce a lipid layer and an emulsion layer, wherein the emulsion layer is The lipid layer is larger. In order to determine whether this pH increase would increase the yield of the lipid layer, an additional second base was added to the separated solubilized cell composition until the pH was 9.11 and the dissolved cell composition was 5,100. The rpm was again centrifuged for 5 minutes. This results in a lipid layer of similar yield. An additional second base is again added to the isolated solubilized cell 102 201200591 composition until ρ Η is 1 ο. ο 9 and the dissolved cell composition is again centrifuged at 5,100 卬! 5 minutes. Furthermore, this (cause uniform results in a lipid layer of similar yield. Example 8 A cell broth containing microbial cells (C. faecalis) is used in a reduced biotin test fermenter. 2〇, 0〇〇kg is washed, concentrated, and sterilized by low temperature sterilization. It is pulled out at the start of low temperature sterilization. It is maintained for about 1 day, before being transferred and homogenized. Bar/1 was homogenized and collected back into a treatment tank. It was estimated by microscopy that approximately 8% of the cells were dissolved. The broth was heated to about 4 之前 before the start of the treatment. The pH was adjusted to 10.5 and 2% NaCl was added and heated to 66 ° C. At this point a significant oil layer had formed and the pH had been reduced to 9.5 after 1-2 hours. The broth was kept at 66 C overnight. 'The broth was centrifuged on WestfaHaRSE_110, with the installed 155 mm ring brake. The viscosity was about 18〇11 in 乞. The centrifuge was supplied at 48kg/mm and the 5-l〇psi back pressure was related to the light phase and 3〇psi back pressure on heavy phase. Feed temperature system, Hold the thief. No oil is present in the waste phase and only a few drops are visible after the addition of isopropanol. Table 1 shows the results of the analysis performed on the crude oil obtained from this procedure. 103 201200591 Table 1 The specification of the crude oil obtained by the method of Example 8 · % oil 87.79 DHA (mg/g) 531.02 % DHA 60.49 PV 1.95 (0.6*) AV 15 % FFA 0.18 Fill (ppm) 8.65 Copper (ppm) 0.22 iron (ppm) 0.7 ship (ppm) 0.63 * PV of centrifuged oil. Among the 20,000 1^ broth provided, 1〇.5% by weight (2,100 kg) is the raw material. Among them, 50% by weight is oil (1, 〇 50 kg). Among the oils, '58.9% by weight is DHA (618 kg). After carrying out the method described above, there are 592.5 kg of raw materials in the lipid layer. , about 87.8% by weight (520.2 kg) is oil. Therefore, the extraction yield of the oil from the biomass is 49.5%. Among the oils, 60.5 〇 / 〇 weight (314.6 kg) is DHA, thereby causing The extraction yield of DHA of 51% by weight of the biomass. Example 9 Providing a microbial cell containing a microbial cell (Schizochytrium), washed and concentrated And a low-temperature sterilized one-cell broth (about 500 g). The broth is chemically treated with a base (ie, a 25% solution of NaOH) without prior cell lysis. The addition of alkali makes the broth The pH is increased from 5.8 to U.2. The addition of base and the increase in pH dissolve the cells to form a dissolved cellular composition. Next, a salt (solid NajO4, an amount of 5 Å/., by weight of the dissolved cell composition) was added to the dissolved cell composition. The solubilized cells 104 201200591 composition was then heated to a temperature in the range of 90 ° C to 95 ° C and maintained at this temperature level for 90 minutes and the pH of the dissolved cell composition was lowered to 9.7. After 90 minutes, each 45 ml of fermentation broth had approximately 2.5 ml of oil layer and no moisture loss. After 3 hours, the pH has been lowered to 9.2. The solution was then centrifuged at approximately 5,1 rpm for 5 minutes to separate the dissolved cell composition and produce a lipid layer. The extracted yield of the lipid was 81 ° /. Weight meter. The crude oil has a methoxyaniline value (AV) of 2 〇. Cell disruption yield is in the range of 92% to 98% by weight. Example 10 A cell broth (about 5 〇〇g) containing microbial cells (Schizochytrium), washed, concentrated, and sterilized at low temperature was provided. The broth was chemically treated with a base (i.e., a 25% solution of NaOH) without the previous step of cell lysis. The addition was increased to raise the pH of the broth from 4.8 to 11. The addition of a base and an increase in pH dissolve the cells to form a dissolved cellular composition. Next, a salt (solid NaCn, an amount of the dissolved cell composition of 2% by weight) was added to the dissolved cell composition. The dissolved cell composition is then heated to 90. (: a temperature in the range of 95 ° C and maintained at this temperature level for 3.5 hours and the pH of the dissolved cell composition was lowered to 8.7 without any loss of fish gas. The solution was then approximately 5,100 The lysed cell composition was separated by centrifugation for 5 minutes at rpm, and a lipid layer was produced. The extraction yield of the lipid was 92° after 3.5 hours. The fraction of the dissolved cell composition was maintained for a duration of time. The pH of the dissolved cell composition was lowered to 8 6 hours and the solution was centrifuged at about 5,100 rpm for 5 minutes to separate the dissolved cell composition 105 201200591 and produce a lipid layer. The extraction yield was 89% by weight after 6 hours. The crude oil had an oxyaniline value (AV) of 14.4. The cell rupture yield was 95% by weight. Example 11 Provided a microbial cell (Schizochytrium), A cell broth (approximately 500 g) that has been washed, concentrated, and sterilized at low temperature. The broth is chemically treated with a test (ie, 50% solution of Na Ο 而) without prior cell lysis steps. Alkali The addition increased the pH of the broth from 5.8 to 11.2. The addition of the base and the increase in pH dissolved the cells to form a dissolved cellular composition. The dissolved cell composition was then heated to 70° under vacuum. C reduced the water content from 88.7% to 85.5%. During the evaporation period, the pH was lowered to 10.36. The solution was centrifuged at about 5,100 rpm for 5 minutes to separate the dissolved cell composition and produce a lipid layer. The extraction yield of lipid was 83.9% by weight. The crude oil had a methoxyaniline value (AV) of 10.5. The cell rupture yield was 93.17% by weight. The method was repeated 'except that the dissolved cell composition was under vacuum. It was heated to 70 ° C to reduce the water content from 88.7% to 79.2%. The extraction yield of the lipid was 87.5% by weight when the water content was reduced to 79 2% and the cell break yield was 92.3% by weight. The method was also repeated to reduce the water content from 88.7% to 80.8%. The extraction yield of the lipid was 9% by weight when the water content was reduced to 80.8% and the cell break yield was 959% by weight. Providing microbial cells Schizochytrium) 'washed, concentrated, and 106 201200591 low-temperature sterilized one-cell broth (approximately 500g). The broth is chemically treated with a base (ie, 5% NaOH solution) without The previous step of cell lysis. The addition of the broth increased the pH from 5.6 to lu. The addition of the test and the increase in pH dissolved the cells to form a dissolved cell composition. The dissolved cell composition was It is heated to 9 〇 ° C for 40 minutes in a closed system. After 40 minutes, there were approximately 1 ml of oil (lipid) per 40 ml of fermentation broth. The solution was then centrifuged at about 5,1 rpm for 5 minutes to separate the dissolved cell composition and the production-lipid layer. The extracted yield of the lipid was 85.1% by weight. The crude oil had a methoxyaniline value (AV) of 16.3. The cell breakage yield was 97.6% by weight. Example 13 A cell broth (about 500 g) containing a microbial cell (Schizochytrium), which was washed, concentrated, and sterilized at a low temperature was provided. The broth is chemically treated with a base (i.e., a 50% solution of NaOH) without the previous step of cell lysis. The addition of a base increased the pH of the broth from 4.9 to 11.2. The addition of a base and the increase in pH dissolve the cells to form a dissolved cellular composition. The solubilized cell composition was then mixed at room temperature for 4 hours. The bath was then centrifuged at about 5,1 rpm for 5 minutes to separate the dissolved cell composition and produce a small amount of lipid layer. A portion of the dissolved cell composition is mixed at room temperature for about 96 hours. The solution was centrifuged at approximately 5,1 rpm for 5 minutes to separate the dissolved cell composition and produce a greater amount of lipid layer. The extraction yield of the lipid was 61.4% by weight. The crude oil had a methoxyaniline value (av) of 22.6. 107 201200591 Example 14 A cell broth (about 5 〇〇g) containing microbial cells (ATCC accession number PTA-9695), which was washed, concentrated, and sterilized at low temperature was provided. The broth is chemically treated with a base (i.e., a 50% solution of 'MaOH) without the previous step of cell lysis. The addition of a base increased the pH of the broth from 5 6 to 11.1. The addition of a base and an increase in pH dissolve the cells to form a dissolved cellular composition. The dissolved cell composition was then heated to 7 Torr. 〇 It takes 3 hours to reach the range of 75 C. The solution was then centrifuged at about 5,1 rpm for 5 minutes to separate the dissolved cell composition and produce a lipid layer. The extracted yield of the lipid was 84 4% by weight. A portion of the dissolved cellular composition is heated for a total of 5 hours. The solution was then centrifuged at approximately 5, i rpm for 5 minutes to separate the dissolved cell composition and produce a similar lipid layer. The extraction yield of the lipid was 87.3% by weight. Cell disruption yield was 891% by weight. Example 15 A cell broth (about 5 gram) containing microbial cells (Schizochytrium), washed, concentrated, and sterilized at low temperature was provided. The broth was chemically treated with a base (i.e., a 50% solution of NaOH) without the previous step of cell lysis. Addition of the test increases the yang of the broth from 73 to u. The addition of a base and an increase in pH dissolve the cells to form a dissolved cell composition. Next, a salt (solid Na2S〇4 ' at 5% by weight of the dissolved cell composition of -4) was added to the dissolved fine-aged product. The dissolved cell composition was then heated to a temperature of 901 and maintained at this temperature level for 2 hours. While maintaining a temperature of 9 Torr for an additional 2 to 4 hours, a device containing 108 201200591 having the dissolved cell composition was allowed to open to allow evaporation of water. The solution was then centrifuged at about 5, 丨〇〇 rp m for 5 minutes to separate the dissolved cell composition and produce a lipid layer. The lipid has an extraction yield of greater than 70% by weight. The crude oil had an oxo-amine value (AV) of 11.6. Example 16 A cell broth (9,925 kg) containing microbial cells (ATCC accession number PTA-9695) was provided. The cell broth was diluted with water to a weight ratio of 丄:i to form a 20,000 kg diluted broth. The solids of the broth were 16.13% by weight prior to dilution and 8255% by weight after dilution. The diluted broth was mixed and centrifuged at 6,4 rpm to remove extracellular water-soluble or water-dispersible compounds. The concentrate (1 〇, 250 kg) was collected from the centrifuge and had a solids content of 〇 5% by weight. The collected concentrate was heated to 62 〇 (: to 64. (: to low temperature sterilization) The concentrate. The enzyme (ie, alkaline protease 〇4/ca/ose) 2.4 L FG 0.5%) is added to the sterilized concentrate to dissolve the cells and form an emulsified dissolved cell composition. The emulsified dissolved cell composition is treated with a test (i.e., a 25% solution of Na〇H) to adjust the dissolved cell composition from 13^1 to 丨丨. Then 'one salt ( Solid NajO4' is added to the dissolved cell composition at 5% by weight of the dissolved cell composition. The dissolved cell composition is then heated to 95. (: temperature and retention) The dissolved cell composition is stirred simultaneously at this temperature level for 10 hours to 12 hours. After stirring, the dissolved cell composition has a pH of 8.6 and a very small emulsion layer. The agitation tank is allowed to cool to 60. (: and the dissolved cell composition 109 201200591 The pH is increased to 9.6 while cooling. The pH of the dissolved cell composition is reduced to 8-2 by the addition of phytic acid. The addition of molybdic acid does not impair the separation of the lipid layer and the very small emulsion layer. The dissolved cell composition was then centrifuged at 5,100 rpm at a feed rate of 48 kg/min for 5 minutes at 60 ° C to 63 ° C to separate the dissolved cell composition and yield 1.7% to 2_3 ° /. The weight of one of the gas content of the lipid layer. Example 17 π goods taste another 玍 fine 脃 (C. sinensis), washed, concentrated, low temperature sterilization - cell broth (50 〇 g). The broth is homogenized into a dissolved cell heart with a pressure of 8 _ to 12 〇 ((4). The dissolved cell group secret _ _ test (rid, listen ^ please) Liquid). Treat the straight wire of the straight dragon. The pH of the mixture is from 8 to 8.2. A salt (solid Na2S04, 5% by weight _ + _ 沾 1 1 之之溶解溶解溶解溶解溶解- Amount) added to the dissolved cell group is then (4) (4). (10) Temperature sighs the bribes of the cells ... will be at this temperature. The dissolve white, the field The pH of the cell composition is maintained by a 12.5% solution of NaOH, which is maintained at a level of 7.8 to 8.2, with a small amount of ancient / / > \ hours in a closed system. Absorbs the hate or does not have any loss of water. The substance U + & then the dissolved cells I. The wind U is about 5,1G0 rpm to centrifuge the cell cylinder for 5 minutes to separate the dissolved 6 匕, And the production and production of a layer of oil. This 40 millimeters Λ, soft about 2 ml of an oil layer in another sample. The extraction of oil is based on the amount of 73% by weight of the oxygen. The crude amine value (AV) is 13_5. The cell is broken. The yield is 82% to 86% by weight. Example 18 110 201200591 Provided a cell broth (1,000 g) of a microbial cell (Schizochytrium). Add the enzyme (ie, 'German 2·4 L FG 〇·5%) to the cell biomass to dissolve The cells form an 'emulsified dissolved cell composition. The emulsified dissolved cell composition is treated with a test (also printed as a 12.5% solution of Na〇H) to adjust the pH of the dissolved cell composition. From 7.21 to 10.52. Next, a salt (solid Naa, at 2% by weight, an amount of the dissolved cell composition) is added to the dissolved cell composition. The broth is then divided into 4 Part, and each part is maintained at 4 different temperatures and times: 1) keep test #1 at 9 〇. 匸 last for 22 hours; 2) keep test #2 at 90 °C for 2 hours and then keep At 25 C for 20 hours; 3) Hold test #3 at 6 〇. (: 22 hours; and 4) Hold test #4 at 25 ° C for 22 hours Individual tests were then centrifuged without further pH adjustment. For trials #1, #2, and #3, the broth was centrifuged for approximately 5,600 卬m (4,800 g force) for 5 minutes to separate the dissolution. Cell composition. Because the separation of test #4 is not good at 4,800 g ( <20%) The 'g force was increased to 15,000 and the broth was rotated at 15,000 g for 5 minutes. The extraction yield of this lipid is shown in the following table in terms of weight percent and methoxyaniline value (AV) series. Table 2. Conditions and results when changing the temperature of the dissolved cell composition and the time of heating · Test # Treatment time and temperature Centrifugation conditions AV oil yield (%) 1 90 ° C for 22 hours pH = 6.22 g force = 4,800 58.7 51.4 2 90°C for 2 hours, 25°C for 20 hours pH = 8.19 g force = 4,800 109.2 82.2 3 6〇°C for 22 hours pH = 8.38 g force = 4,800 91.2 27.2 4 25°C for 22 hours pH = 10.03 g force = 15,000 105.2 55.7 111 201200591 The methoxyaniline values in Table 2 were higher than expected. One difference between the previous embodiment and this example is that the dissolved cellular composition is allowed to stand for a long period of time prior to lipid extraction. It is assumed that the oxidation of dissolved oxygen present in the dissolved cellular composition is caused during a long period of time prior to extraction. The increased oxidation linkage results in an increase in the value of the oxime oxamine. The fact that the test at the highest temperature for the longest period of time (test #1) has the lowest methoxylamine value supports this hypothesis 'because the dissolved oxygen content of a dissolved cell composition generally increases with temperature cut back. The increased methoxyaniline value is therefore believed to be an anomaly as a result of delaying the extraction of lipids from the solubilized cellular composition. In production, there is no delay in extracting lipids from the solubilized cell composition and the value of the oxime oxime will be consistent with previous results for methoxybenzamide values of 26 or less. Example 19 k is a cell broth (1,000 g) containing a scorpion biological cell (Schizochytrium). The broth was then divided into 3 parts and diluted as follows: 丨) Test #1 was completely undiluted and used as a control part; 2) Test #2 was diluted 25% with water; and 3) Test #3 was diluted with water 5 〇 . / (^ The enzyme (i.e., alkaline protease L FG 〇. 5%) is added to the cell biomass to dissolve the cells and form an emulsified dissolved cell composition. The emulsified dissolved cell composition is used A base (i.e., Na〇Hi12 5% solution) is treated to adjust the pH of the dissolved cell composition from 6.8 to 10.6. Next, a salt (solid NaCl, 2% by weight, of the dissolved An amount of the cell composition was added to the dissolved cell composition. The broth was then heated to 9 (TC and maintained for 20 hours. After the maintenance time, the broth of each 112 201200591 test was divided into 2 'And centrifuge half asis and the other half to adjust the pH to approximately 8.5 before centrifugation. Both parts were centrifuged at approximately 8,545 rpm (8,000 g force) for 5 minutes. Percentage by weight and methoxyaniline value The extraction yields of (AV) lipids are listed in the following table. Table 3. Conditions and results when varying dilutions of diluted sterilized broth. Test # Diluted? Centrifugal conditions AV oil yield (% 1 does not dilute pH = 6.0 g force = 8, 000 51.8 81.2 la No dilution pH = 8.4 g Force = 8,000 44.3 78.1 2 Dilute with water 25% pH = 5.5 g Force = 8,000 76.1 88.9 2a Dilute with water 25% pH = 8.4 g force = 8,000 85.3 82.1 3 Dilute with water 50% Γ pH = 5.7 g force = 8,000 68.5 85.0 3a 50% dilution with water pH = 8.5 g force = 8,000 79.6 84.0 The methoxylamine values in Table 3 are higher than expected. Between the previous examples (except for Example 18) A difference between this and the embodiment is that the dissolved ageing product is allowed to be aged - the length of the segment is between the lipids, and it is assumed that the dissolution is caused during a long period of time before the extraction. The oxidation of dissolved oxygen in the composition of the field cell. The increased oxidation leads to an increase in the value of the methoxyantamine. The increased methoxylamine value is therefore believed to be - counteracting as it is delayed from the dissolution The result of extracting the lipid from the cell composition in production 'does not delay the extraction of lipids from the dissolved cell composition and the methoxylamine value will be consistent with the previous results of 26 or less of the oxoaniline value. Example 20 Cell meat obtained from various fermentation batches The soup was processed in the method described in Example 2 113 201200591, except for the choice of time to add the salt (for example, before homogenization and after homogenization) and the amount of different salts. The isolated lipid lines were analyzed and analyzed and are provided in Table 4. Table 4 - Timing of different salt additions and amounts of added salts The specifications of the lipids obtained using the method of the invention. Fermentation batches P2137 P2137 P4167 P4167 Addition of P2137 P2137 NaCl · Before and after before and after and after % NaCl weight 2 5 2 5 5 5 % lipid · 27 37 51 72 59 15 % starting solid weight 13 13.9 19.2 19.2 13.3 7.5+ % solid weight in centrifugation The first 19.7 21.7 19.7 20.1 20.2 8.6 before homogenization or after homogenization; % lipid refers to the percentage of lipids in the form of triglycerides. This operation is converted to have a low percentage of starting solids. The data presented in Table 4 shows that the addition of the salt after homogenization resulted in a higher % lipid value than the addition of the salt prior to homogenization. The data presented in Table 4 also shows that dilution of the sample results in a lower % lipid value. Example 21 Using an empirical protein obtained from a microbial cell (Schizochytrium) (IV) A sample of a dissolved cell composition treated with a cardiac (tetra) enzyme. This sample has a pH of approximately 5.5. The sample was divided into 4 smaller samples and the pH of 3 of the samples was adjusted to approximately 7.4, approximately 1 G.5, and approximately 12, respectively, by the addition of sodium hydroxide. These samples were diluted with deionized water at a ratio of :: !. The Ρ〇ΒΝ(α_(44-defined n-oxide) condition of the third-grade butyl-stone succinyl ketone '(3) heart (four) is used as a spin-trapping chemical to add each of the diluted samples of g. These samples are used one. Βγ-γ印学114 201200591 e-scan EPR (electron paramagnetic resonance) spectrometer (system number SC〇274) (Bruker BioSpin, Billerica, ΜΑ) is measured to measure the amount of free radicals present by oxidation of lipids. The samples were incubated at room temperature (2 〇. 〇 and each of the 50 μίΡΟΒΝ containing samples with the following spectrometer parameters were tested at hourly intervals after adjusting pHs for 4 hours: 86 Hz modulation frequency, 2 .U Gaussian modulated vibration, microwave power of 519 mW, time constant of 20.48 seconds, sweep time of 10.49 seconds, sweep width of 1 〇〇 Gauss, and number of scans of 8. The results of EPR spectrometer readings are provided In Figure 5, the data in Figure 5 shows that the level of free radicals in the sample initially at pH 5.5 is the highest and the level of free radicals in the samples at pH 10.5 and 12 is the lowest. Also exhibits free radicals during 4 hours The rate of the sample at pH 10.5 is the slowest and the sample at pH 5.5 is the highest. The data also shows that the addition of base to the dissolved cell composition inhibits lipid oxidation and thus results in the crude lipid and refining Low AV in oil. Example 22 Oilseeds were extracted from a rapeseed plant and passed through a grinder to break and break the outer shell of the oilseed. The member is dehulled, for example by inhalation, to remove the edible portion (internal) of the seed from the shell of the oil seed. The dehulled oil seed can be ground by homogenizing or discharging it through a press. The dehulled oilseeds become a mass of cells that dissolve the oilseeds. Water can be added to form an emulsified dissolved cell composition. The emulsified dissolved cell composition is filtered from the dissolved cells. The composition removes any excess shell debris. The 115 201200591 emulsified dissolved cell composition is treated with a base (ie, a 25% solution of NaOH) to adjust the dissolved cell composition. PH to 11. A salt (solid NaCU, an amount of the dissolved cell composition of 2% by weight) is then added to the dissolved cell composition. The dissolved cell composition is then heated to 90 C. And dissolving the dissolved cell composition while maintaining the level of the standard for 6 hours to 48 hours. The dissolved cell composition is then centrifuged at 5,100 rpm for 5 minutes to separate the dissolved cell composition and A lipid layer and an emulsion layer were produced.Example 23 Comparative Analysis of Crude Lipids Obtained by Hexane Extraction A batch of crude lipids obtained using the method described in Example 2 was analyzed to determine various specification. Additional crude lipids were obtained using the same hexane method for the same microbial cells as used in Example 2. The hexane extraction process comprises spray drying a fermentation broth and adding hexane to the spray dried biomass to obtain 15% to 20°/. A solution of solid weight leaves. The solution is then homogenized to dissolve the cells to form a dissolved cellular composition. The dissolved cell composition was centrifuged and a layer containing lipid and hexane was removed. Hexane was then removed from the lipid. The results of the analysis are provided in Table 5. 116 201200591 Table 5. Specification of lipids obtained by the method of the present invention or hexane extraction method. Fermentation batch ABCDEFG Extraction method Ex. 2 Hexane hexane burned hexazone · Hexane hexane AV· 5.9 ND ND ND 14.7 17.18 6.7 PV* 1.21 0.65 1.56 0.46 ND 0.85 0.3% lipid · 89.61 86.94 84.31 86.75 85.53 86.05^ 86.54 DHA ί mg / g) 537.47 508.15 459.32 465.31 510.49 495.82 506.33 % DHA* 59.98 58.39 54.49 53.65 59.71 57.68 58.51 AV = A Oxyaniline value; PV = peroxide value; 〇 /. The lipid refers to the percentage of lipid in the form of triglyceride; % DHA refers to the percentage of DHA in the lipid. The information provided in Table 5 shows that the crude lipid obtained by the method of the present invention is typical of The lipids prepared by the alkane extraction method showed superior oxime oxime values, percentage of lipids, amount of DHA, and percentage of DHA. Comparative analysis of crude lipids obtained by the FRIOLEX® method Example 24 The crude lipids obtained from the various fermentation batches used in the methods described in Examples 1 and 3 were analyzed to determine various specifications, additional The crude laurel is obtained using the FRIOLEX® method (Westfalia Separator AG, Germany), which is used in accordance with the teachings of U.S. Patent No. 5,928,696 and International Publication No. WO 01/76385 and WO 01/76715. A method of extracting lipids by a water-soluble organic solvent. The results of the analysis are provided in Table 6. 117 201200591 Table 6. Specifications of lipids obtained using the method of the invention or the FRIOLEX® method. Fermentation batch ABBBBCCC Extraction method Ex. 1 Ex. 1 Ex. 3 Ex. la Ex. lb Ex. 1 Ex. 1 FRIOLEX ® AV· 3.1 1.6 3.9 300 7.1 3.5 4 36 PV* 1.8 0.17 0.14 6.16 0.34 0 0 0.35 % Lipid* 96.27 93.67 92.55 87.20 94.42 95.14 94.31 93.92 DHA (mg) 452.4 458.16 455.17 414.66 471.55 416.41 416.05 415.38 Extraction yield (%) 93.5 87 ND 96 ND 94 94 94 * AV = methoxyaniline value; pv = peroxide value; % lipid refers to the percentage of lipid in the form of triglyceride a: the dissolved cell composition is not heated. b: The solubilized cell composition was allowed to stand for 3 weeks prior to extraction. The data provided in Table 6 shows that the crude lipid obtained by the method of the present invention exhibits superior oxime oxime value compared to the lipid prepared by a FRIOLEX® method (the cellular composition accompanying the lysis does not An exception to the lipid obtained when heated). The lipid prepared by the method of the present invention exhibited a methoxyaniline value of 4.4% to 19.7% of a lipid methoxyaniline value prepared by the FRIOLEX® method. According to #, one of the lipids prepared by the method of the present invention has increased stability. For example, as shown in Table 6, a method of the present invention is used to extract a lipid from a cell composition that is dissolved, wherein the dissolved cell composition is allowed to stand for 3 weeks prior to the extraction process. It is believed that the methoxyaniline value of the lipid in the dissolved 'H-forms' increases with time, and thus 'the urinary tract extracted from the 3 weeks of dissolved cell composition is expected to have been , surplus increased the value of aniline. However, as shown in Table 6, the lipid obtained from the 3-week-dissolved cell composition was obtained using the method of the present invention. The lipid of 118 201200591 has a decyloxyaniline value which is a lipid of the FRIOLEX® method. 19.7% of the methoxyaniline value. Example 25 The crude lipids obtained from a broth of Microbial Fines & (ATCC Accession No. PTA-9695) were analyzed using the method described in Example 16 to determine various specifications. Additional crude lipids were obtained from a broth of microbial cells (ATCC Accession No. PTA-9695) using the FRIOLEX® method (Westfalia Separator AG, Germany), which is in accordance with U.S. Patent No. 5,928,696 and International A method of extracting a lipid with a water-soluble organic solvent (for example, isopropyl alcohol) as described in the publications WO 01/76385 and WO 01/76715. The results of the analysis are provided in Table 7. Table 7. Crude lipids Comparative Example 16 Friolex® % oil 9.19 93.73 DHA (mg/g) 570.68 574.33 % DHA 62.1 61.27 % FFA 1.13 0.22 PV 0 0.74 AV 10 73.9 Iron (ppm) 0.11 0 Copper (ppm) 0.67 0.3 Ship (ppm) 0.21 0 _ phosphorus (ppm) 5.22 7.20 Extraction yield (%) 61.4 45.3 The data provided in Table 7 shows that the crude lipid obtained by the method of the present invention appears as compared with the lipid prepared by the FRIOLEX® method. 119 201200591 Excellent methoxyaniline value (AV) and peroxide value (pv). This data also shows that the extraction yield of the lipid obtained by the method of the present invention is superior to that obtained by the TM〇LEX% method. Example 26 Purification of Crude Lipids Crude lipids were obtained using the method outlined in Example 1 and the FRI0LEX® method. These "lipids" are successively processed by: υ 胶 and caustic refining; 2) bleaching; 3) cooling filtration; and · antioxidant deodorization for further processing. About crude fat f, caustic refined lipid, bleached The lipids, as well as the deodorized lipids, are presented in Tables (4). The comparison of refined oils is presented in Table 9. ^ Obtained from the _LEX16 method _ f (Example 24)

AOX提及抗氧化劑色8b.使用NaCl而自-(實施例1)獲得的脂質加工步驟 FFA % PV AV DHA _ (毫克/克） 406.7 產量％ (脂質） Ν/Α Ν/Α 的粗製脂質苛性精製脂質 <0.1 0.27 2.3 410.8 85.9 86^7^ 經漂白的脂質 <0.1 0.16 0.8 404.3 97.0 過濾的脂質 <0.1 0.37 1.0 414.1 59.3 60^7^ 經去臭的脂質 w/AOX 0.06 <0.1 1.8 379.9* 94.9 87.1 *註釋：用高油酸的葵花油(H〇SO)之增為臟(毫克/克)減少^ 120 201200591 表9.精製油比較AOX mentions antioxidant color 8b. Lipid processing step obtained from - (Example 1) using NaCl FFA % PV AV DHA _ (mg/g) 406.7 Yield % (lipid) Ν/Α Ν/Α crude lipid caustic Refined lipid <0.1 0.27 2.3 410.8 85.9 86^7^ Bleached lipid <0.1 0.16 0.8 404.3 97.0 Filtered lipid <0.1 0.37 1.0 414.1 59.3 60^7^ Deodorized lipid w/AOX 0.06 <0.1 1.8 379.9* 94.9 87.1 *Note: Increased by high oleic sunflower oil (H〇SO) to dirty (mg/g)^ 120 201200591 Table 9. Comparison of refined oils

1主釋：用HOSO之增加的稀釋為DHA(毫克/克)減少的原因表8a、表8b，以及表9中提供的資料展現出藉由的方法所製備之精製油與藉由FRIOLEX®方法所製^發明精製油相比展現出較低的曱氧苯胺值。一實施例27 感覺剖繪比較於實施例26中所獲得的精製油係藉由_組8個至感覺分析物予以分析。感覺分析物係根據香氣、芳香固及餘味來評價各種的脂質規格以提供各脂質之,，總香氣= 度"。通用型光譜描述性的分析方法係使用來估定樣。；；氣和芳香特徵。此方法使用〇 _ 15的強度規模，該處〇 =無 /則出以及15 m的強度，來測量油之香氣和芳香^ 性°感覺資料的結果係提供於表10中。 121 201200591 ----- ----— 規格 ___香氣整體的強度 —1,1 _ 参海洋複合％綠色複合物草本的堅果烘烤的顏料的其他 Friolex® 3 1 1 1 0 0 0 Ex. 1 2 — — 0 1 1 0 0 0 芳香S 整體的強度参海洋複合也綠色才复合物草本的堅果烘烤的顏料的其他 Friolex® 4叫 1 1 2 0 0 0 Ex. 1 3 0 1 2 0 0 0 餘味 Friolex® -草本的/稍微魚的 Ex. 1 . 箪本的表ίο中&供的資料展現出藉由本發明的方法所製備之精製油與藉由FRIOLEX®方法所製備之一精製油相比展現出較優秀的感覺資料。如以上所顯示出的，藉由本發明所提供的脂質分別地具有3和2之總香氣強度，而，FRIOLEX® 脂質分別地提供了 4和3的總香氣強度。實施例28 比較實施例一種用於自微生物獲得脂質而不使用有機溶劑之萃取方法係揭示於美國專利案號6,750,048之中。一種自藉由本發明的方法所製備之粗製脂質而獲得的精製油以及一種自藉由美國專利案號6,750,048中所揭示的萃取方法來製備的粗製脂質而獲得的精製油之比較係提供於表丨丨中。 122 201200591 表11 ·關於藉由本發明的方法（實施例1)所製備之一脂質以 f由於美國專利案號6,750,048之中的-種方法所製備之一月曰質之比較資料. 實施例1 U S*P.N. 6,750,048 DHA (毫克/克） 379.9 346 % DHA 40.18 37.3 FFA% 0.06 ND PV <0.1 0.46 AV 1.8 ND 鐵(ppm) <0.02 0.26 銅（ppm) <0.02 <0.05 船（ppm) <0.1 <0.20 砷(ppm) <0.1 <0.20 ~~ 汞(ppm) <0.01 <0.20 %溼氣及揮發物 <0.01 0.02 不皂化物（％) 1.33 ND 〜經由IR之反式脂肪酸(％) <1 ND ~~ 表中提供的資料11展現出自藉由本發明的方法所製備之粗製脂質而獲得的精製油與自藉由美國專利案號 6,750,048所揭示的萃取方法來製備的粗製脂質而獲得的精製油相比展現出較優秀的性質。實施例29 該經單離之破囊壺菌（ATCC存取號碼pTa_9695)係予以特徵化以進行分類學的分類。自在低潮(low tide)期間之潮間帶棲息地收集樣品。將水、沈積物、活植物材料及腐化的植物/動物碎屑放入無菌 50毫升試管内。將各樣品的部分連同水塗佈在單離培養式之固體瓊月曰平板上。單離培養基係由：5〇〇毫升的人、生海水、500毫升的蒸餾水、1克葡萄糖、！克甘油、13克瓊脂、 t 123 201200591 1克麩胺酸鹽、0.5克酵母萃取物、0.5克酪蛋白水解產物、j 毫升維生素溶液（100毫克/升硫胺素、0.5毫克/升生物素、0.5 毫克Bi2)、1毫升微量礦物溶液(PH金屬，每公升含：6.0克1 Main release: Reasons for reduced DHA (mg/g) reduction with HOSO Table 8a, Table 8b, and the information provided in Table 9 show the refined oil prepared by the method and by the FRIOLEX® method The refined oil produced by the invention exhibits a lower value of the oxime aniline. An Example 27 Comparison of Sensory Profiles The refined oil obtained in Example 26 was analyzed by a group of 8 to sensory analytes. The sensory analytes were evaluated for various lipid specifications based on aroma, aroma, and aftertaste to provide a total aroma = degree ". A general-purpose spectral descriptive analytical method is used to estimate the sample. ;; gas and aromatic characteristics. This method uses the intensity scale of 〇 -15, where 〇 = no / then and 15 m of strength, to measure the aroma and aroma of the oil. The results of the sensory data are provided in Table 10. 121 201200591 ----- ----- Specifications ___ Aroma overall strength - 1,1 _ ginseng compound% green compound herbaceous nuts baking pigments other Friolex® 3 1 1 1 0 0 0 Ex. 1 2 — — 0 1 1 0 0 0 Aromatic S The overall strength of the marine compound is also green and the composite of the herbaceous nut of the baking pigment of the other Friolex® 4 is called 1 1 2 0 0 0 Ex. 1 3 0 1 2 0 0 0 Afternoon Friolex® - Herbal/Slightly Fish Ex. 1 . The table of 箪 & && The data provided demonstrates the refined oil prepared by the method of the present invention and prepared by the FRIOLEX® method. A refined oil exhibits superior sensory data. As indicated above, the lipids provided by the present invention have a total aroma intensity of 3 and 2, respectively, while FRIOLEX® lipids provide a total aroma intensity of 4 and 3, respectively. EXAMPLE 28 Comparative Example An extraction method for obtaining lipids from microorganisms without the use of organic solvents is disclosed in U.S. Patent No. 6,750,048. A comparison of a refined oil obtained by the crude lipid prepared by the method of the present invention and a refined oil obtained by the crude lipid prepared by the extraction method disclosed in U.S. Patent No. 6,750,048 is provided in the specification. In the middle. 122 201200591 Table 11 - Comparison of one of the lipids prepared by the method of the present invention (Example 1), which is prepared by the method of U.S. Patent No. 6,750,048. Example 1 US *PN 6,750,048 DHA (mg/g) 379.9 346 % DHA 40.18 37.3 FFA% 0.06 ND PV <0.1 0.46 AV 1.8 ND Iron (ppm) <0.02 0.26 Copper (ppm) <0.02 <0.05 Ship (ppm) &lt ;0.1 <0.20 Arsenic (ppm) <0.1 <0.20 ~~ Mercury (ppm) <0.01 <0.20% Moisture and Volatile <0.01 0.02 Unsaponifiable (%) 1.33 ND ~ Via IR Fatty Acid (%) <1 ND ~~ The data provided in the Table 11 shows the refined oil obtained from the crude lipid prepared by the method of the present invention and the extraction method disclosed in U.S. Patent No. 6,750,048. The refined oil obtained by the crude lipid exhibits superior properties compared to the refined oil obtained. Example 29 The isolated Thraustochytrium (ATCC accession number pTa_9695) was characterized for taxonomic classification. Samples were collected from intertidal habitats during low tide. Water, sediment, live plant material and decayed plant/animal debris were placed in sterile 50 ml tubes. Portions of each sample were coated with water on a single culture medium solid Qiongyue plate. The isolated medium consists of: 5 ml of human, raw sea water, 500 ml of distilled water, 1 g of glucose,! Glycerol, 13 g agar, t 123 201200591 1 g glutamate, 0.5 g yeast extract, 0.5 g casein hydrolysate, j ml vitamin solution (100 mg/L thiamine, 0.5 mg/L biotin, 0.5 mg Bi2), 1 ml trace mineral solution (PH metal, per liter: 6.0 g

FeCl36H2〇、6.84 克 H3B〇3、0.86 克 MnCl24H2〇、0.06 克 ZnCh、0.026 克 CoC126H20、0.052 克 NiS04H20、0.002 克FeCl36H2〇, 6.84 g H3B〇3, 0.86 g MnCl24H2〇, 0.06 g ZnCh, 0.026 g CoC126H20, 0.052 g NiS04H20, 0.002 g

CuS〇45H2〇及 0.0〇5克Na2Mo〇42H2〇)，以及5〇〇毫克各青黴素G及硫酸鏈黴素所組成。於20-25°C下在黑暗中辉育該等瓊J旨平板。2至4天後’在放大倍數下檢查該等瓊脂平板，並以無菌牙籤剔取細胞之菌落並在新鮮的培養基平板上重複劃線。將細胞重複地在新鮮培養基上劃線，直到移除已污染的微生物為止。將來自瓊脂平板之菌落轉移至含有半強度海水及〇毫升）經高壓滅菌之新孵化的豐年蝦幼苗懸浮液之培養皿 (petri dish)。在2-3天之後，該等豐年蝦幼蝦變得過度生長且具有孢子囊聚簇。於排放時釋放的遊走孢子為二鞭毛類 (biflagellate)’其可活躍地自成熟的孢子囊游離，其壁殘跡在釋出孢子之後仍清楚可見(在位相差）。孢子囊之直徑經測定為12.5微米至25微米，且遊走孢子之大小為2 5微米至2 8 微米x4.5微米至4.8微米。每一各別孢子囊有8至24個孢子。穩定後的遊走孢子放大並快速進行均體分裂 division)以得到四分體、八分體，且最後得到孢子囊聚簇。四分體形成作用係在孢子囊成熟前之非常早期的階段開始。此等特性係與裂殖壺菌屬阶/^0咖的細)相符。該經單離之破囊壺菌（ATCC存取號碼ΡΤΑ_9695)係進 124 201200591 一步根據其之18s rRNA基因與已知物種的18s rRNA之相似性而予以特徵化。來自該破囊壺菌（ATCC存取號碼 PTA-9695)的總基因體DNA係藉標準程序(Sambrook J. and Russell D. 2001· Molecular cloning: A laboratory manual，第 3版。Cold Spring Harbor Laboratory Press, Cold Spring Harbor，New York)而製備並使用於18s RNA基因之PCR擴增。18s rRNA之PCR擴增係使用先前所說明的引子予以進行(Honda等人，*/.五46(6) 1999)。使用染色體DNA模板之PCR條件如下：0.2 μΜ dNTPs、0.1 μΜ各引子、8% DMSO、200毫微克（ng)染色體DNA、2.5 U PfuUltra® 融合 HS DNA 聚合酶（Stratagene)，以及 IX PfuUltra®緩衝劑（Stratagene)配於50微升總體積。PCR操作程序包括下列步驟：（1) 95°C歷時2分鐘；（2)95°C歷時45秒； (3)55°C歷時30秒；（4)72°C歷時2分鐘；（5)重複步驟2-4計40 個循環；（6)72°C歷時5分鐘；以及(7)保持於6°C。使用以上所說明的染色體模板，PCR擴增出產具有預期的大小之有區別的DNA產物。PCR產物係依據製造者之指示而被選殖至載體pJET1.2/鈍的（Fermentas)之内，以及使用所提供的標準引子來決定插入序列。表12顯示來自該破囊壺菌（ATCC存取號碼PTA-9695) 的 18s rRNA序列與在 National Center for Biotechnology Information (NCBI)電子資料庫内之DNA序列的比較。簡單地說，”％同一性”係藉由使用DNA對齊的標準，VectorNTI 程式（Invitrogen)的"AlignX"程式中之計分矩陣 125 201200591 "swgapdnamt"予以決定。"％涵蓋••係取自從NCBI電子資料庫進行Basic Local Alignment Search Tool (BLAST)計算的結果且為含括在該對齊的節段内之質詢長度（query length) 的百分率。表12: 18srRNA序列之比較破囊壺菌 %同一性計算#1 %涵蓋計算#2 聚破囊壺菌(Thraustochytrium aggregatum) (p) 98 90 破1壺菌餍(Thraustochutriidae) sp.HUl 84 86 破1查菌爆(Thraustochutriidae) spU 84 91 馬堤盧蒂曼達雷破囊壺菌 81 88 破囊壺菌屣(Thraus(ochutriidae) sp, FW19 81 85 裂殖壺菌屬切.ATCC 20888 81 95 (P):表示部分序列如表12中所顯示的，已發現就％同一性而言，來自該破囊壺菌（ATCC存取號碼PTA-9695)的18s rRNA與Honda, D.等人在J· Euk. Micro. 46(6): 637-647 (1999)中所提供之聚破囊壺菌的18s rRNA基因序列緊密相關，雖然並非相同。聚破囊壺菌（Thraustochytrium aggregatum)所公開之 18s rRNA序列為一部分序列，其在該序列之中間具有大概71 DNA核苷酸間隔。就百分比涵蓋而言，本發明之分離株之 18s rRNA基因序列與裂殖壺菌屬sp.ATCC 20888之關係比與聚破囊壺菌之關係更密切。高守恆性蛋白質，例如肌動蛋白及β-微管蛋白 (0-扣1)111〖11)，連同183『1^八基因，已經廣泛使用來作為用於評估生物間之種系發生關係的標記(Baldauf, S. M. Am. Nat. 154, S178 (1999))。來自破囊壺菌（ATCC存取號碼PTA-9695) 126 201200591 的總基因體DNA亦可使用作為一用於肌動蛋白及β-微管蛋白基因之PCR擴增的模板。以用於來自聚破囊壺菌之肌動蛋白及β-微管蛋白DNA序列的守恆區所設計之引子來進行 PCR擴增。 PCR條件及染色體DNA模板係如下：〇.2 μΜ dNTPs、CuS〇45H2〇 and 0.0〇5g Na2Mo〇42H2〇), and 5〇〇mg of each penicillin G and streptomycin sulfate. These qi plates were bred in the dark at 20-25 °C. After 2 to 4 days, the agar plates were examined at magnification and the colonies of the cells were picked up with a sterile toothpick and repeatedly streaked on fresh medium plates. The cells were repeatedly streaked on fresh medium until the contaminated microorganisms were removed. The colonies from the agar plates were transferred to a petri dish containing a semi-strength seawater and 〇 milliliters of autoclaved freshly hatched brine shrimp seedling suspension. After 2-3 days, the brine shrimp pups become overgrown and have sporangia clusters. The migratory spores released upon discharge are biflagellates, which are actively freed from the mature sporangia, and their wall remnants are clearly visible after the spores are released (in phase difference). The diameter of the sporangia was determined to be 12.5 micrometers to 25 micrometers, and the size of the migratory spores was 25 micrometers to 28 micrometers x 4.5 micrometers to 4.8 micrometers. Each individual sporangia has 8 to 24 spores. The stabilized tourospores are amplified and rapidly divided into divisions to obtain tetrads, octants, and finally sporangia clusters. The tetrad formation begins at a very early stage before the sporangia matures. These characteristics are consistent with the fines of Schizochytrium genus/^0 coffee. The isolated Thraustochytrium (ATCC accession number 9699695) is affixed to 124 201200591 in a step based on the similarity of its 18s rRNA gene to the 18s rRNA of known species. The total genomic DNA from the Thraustochytrium (ATCC accession number PTA-9695) was borrowed from the standard procedure (Sambrook J. and Russell D. 2001. Molecular cloning: A laboratory manual, 3rd edition. Cold Spring Harbor Laboratory Press , Cold Spring Harbor, New York) was prepared and used for PCR amplification of the 18s RNA gene. PCR amplification of 18s rRNA was performed using the primers previously described (Honda et al., */. Wu 46(6) 1999). The PCR conditions using the chromosomal DNA template were as follows: 0.2 μΜ dNTPs, 0.1 μΜ each primer, 8% DMSO, 200 ng (ng) chromosomal DNA, 2.5 U PfuUltra® fusion HS DNA polymerase (Stratagene), and IX PfuUltra® buffer (Stratagene) is formulated in a total volume of 50 μl. The PCR procedure consists of the following steps: (1) 95 ° C for 2 minutes; (2) 95 ° C for 45 seconds; (3) 55 ° C for 30 seconds; (4) 72 ° C for 2 minutes; (5) Repeat steps 2-4 for 40 cycles; (6) 72 °C for 5 minutes; and (7) for 6 °C. Using the chromosomal template described above, PCR amplified a differentiated DNA product of the expected size. The PCR product was cloned into the vector pJET1.2/Fermentas according to the manufacturer's instructions, and the inserted primers were used to determine the insertion sequence. Table 12 shows a comparison of 18s rRNA sequences from the Thraustochytrium (ATCC accession number PTA-9695) with DNA sequences in the National Center for Biotechnology Information (NCBI) electronic library. Simply put, "% identity" is determined by using the DNA alignment standard, the VectorNTI program (Invitrogen)'s "AlignX" program scoring matrix 125 201200591 "swgapdnamt". "% Coverage•• is taken from the results of the Basic Local Alignment Search Tool (BLAST) calculations from the NCBI Electronic Library and is the percentage of the query length enclosed in the aligned segments. Table 12: Comparison of 18srRNA sequences for Thraustochytrid % identity calculation #1 % Covered calculation #2 Thraustochytrium aggregatum (p) 98 90 Thraustochutriidae sp.HUl 84 86 Broken 1 查菌爆(Thraustochutriidae) spU 84 91 玛卢蒂蒂 81 81 81 81 81 81 81 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 88 P): indicates that the partial sequence is as shown in Table 12, and it has been found that in terms of % identity, 18s rRNA from the Thraustochytrium (ATCC accession number PTA-9695) and Honda, D. et al. · Euk. Micro. 46(6): The 18s rRNA gene sequence of the genus Thraustochytrium provided in 637-647 (1999) is closely related, albeit not identical. The 18s disclosed by Thraustochytrium aggregatum The rRNA sequence is a partial sequence having a ratio of approximately 71 DNA nucleotides in the middle of the sequence. In terms of percentage, the relationship between the 18s rRNA gene sequence of the isolate of the present invention and the Schizochytrium sp. ATCC 20888 is More closely related to the polycystin. Highly conserved proteins, such as muscle eggs White and β-tubulin (0-keel 1) 111 〖11), together with the 183 『1 八 gene, have been widely used as markers for assessing the phylogenetic relationships between organisms (Baldauf, SM Am. Nat) 154, S178 (1999)). Total genomic DNA from Thraustochytrium (ATCC accession number PTA-9695) 126 201200591 can also be used as a template for PCR amplification of actin and β-tubulin genes. PCR amplification was carried out using primers designed for use in the conserved regions of the actin and the β-tubulin DNA sequences of Thraustochytrium. PCR conditions and chromosomal DNA template are as follows: Μ.2 μΜ dNTPs,

0.1 uM各引子、8% DMSO、200毫微克染色體DNA，2.5 U0.1 uM primer, 8% DMSO, 200 ng chromosomal DNA, 2.5 U

Herculase® II 融合 DNA 聚合酶（Stratagene)，以及 ιχ Herculase®緩衝液(Stratagene)配於50 μΐ^總體積中。pcr操作程序包括下列步驟：（1)95°C歷時2分鐘；（2)95°C歷時30 秒；（3) 55°C歷時30秒；（4)72°C歷時2分鐘；（5)重複步驟2_4 計40個循環；（6)72°C歷時5分鐘；以及(7)保持於6°C。使用以上所說明的染色體模板，PCR擴增出產具有預期的大小之有區別的DNA產物。分別的PCR產物係依據製造者之指示而被選殖至載體pJETl.2/鈍的（Fermentas)之内，以及使用所提供的標準引子來決定各個插入序列。表13顯示出與可得自公開資料庫之肌動蛋白序列比較，來自破囊壺菌（ATCC存取號碼PTA-9695)的肌動蛋白胺基酸序列之同一性。同一性係藉由使用蛋白質對齊的標準，VectorNTI程式的"AlignX”程式中之計分矩陣 "blosum62mt2"予以決定。 !； 127 201200591 表13 :肌動蛋白蛋白序列％同一性之比較破囊壺菌 %同一性破囊壺菌(Thraustochytriidae) sp. RT49 98 裂殖壺菌屬·ϊ/λ ATCC 20888 96 紋狀破囊壺菌 96 聚破囊壺菌 96 海洋曰本壺菌 95 金黄色破囊壺菌 95 表14顯示出與可得自公開資料庫之β-微管蛋白序列比較，來自破囊壺菌（ATCC存取號碼ΡΤΑ-9695)的β-微管蛋白胺基酸序列之同一性。同一性係藉由使用蛋白質對齊的標準，VectorNTI程式的"AlignX”程式中之計分矩陣 ”blosum62mt2"予以決定。表14 : β-微管蛋白蛋白序列％同一性之比較破囊壺菌 %同一性開古蓮西斯不遊走壺菌 100 史多其諾伊不遊走壺菌幻occ/?/«o/) 100 海洋曰本壺菌 100 網黏菌属(Ζα办r/n/Zm/a)平N8 100 破囊壺菌·Φ. RT49 100 聚破囊壺菌 100 破囊壺菌讲HU1 100 金黄色破囊壺菌 100 肯内伊破囊壺菌 100 破囊壺菌sp. #32 100 破囊壺菌切.PW]9 100 聚裂殖壺菌 100 裂殖壺菌屬苹· ATCC 20888 100 128 201200591 根據上述特徵，該經單離的破囊壺菌（ATCC存取號碼 PTA-9695)據信代表一新的裂殖壺菌屬物種以及因而亦稱為裂殖壺菌屬sp. ATCC PTA-9695。實施例30 在如以下所說明之不同的培養條件下，該經單離的破囊壺菌（ATCC存取號碼PTA-9695)生產高位準的細胞生長。典型的培養基與培養條件係顯示於表15中。而且，觀察到高位準的脂肪酸和DHA(亦即，大於50%重量計的乾燥細胞重為脂肪酸且大於50%重量計之脂肪酸甲酯為DHA)。 129 201200591 表15 :器皿培養基成分 NaCl KC1 MgS04-7H20 (NH4)2S04 CaCl2T 154(酵母萃取物) KH2P〇4 度濃 L L L L L L L /, / .// // 2··0·0·6·2·0·2 範圍 0-25、5-20，或 10-15 0-5、0.25-3，或 0.5-2 0-10、2-8，或 3-6 0-10、0.25-5，或 0.5-3 0.1-5、0.15-3，或 0.2-1 0-20、1-15，或 5-10 0.1-10 、 0.5-5 ，或 1-3 高壓蒸氣滅菌後(金屬）檸檬酸 mg/L 3.5 0.1-100、1-50，或 2-25 FeS04_7H20 mg/L 10.30 0.1-100、1-50，或 5-25 MnCl2-4H20 mg/L 3.10 0.1-100、1-50，或 2-25 ZnS04-7H20 mg/L 3.10 0.1-100、1-50，或 2-25 CoC12.6H20 mg/L 0.04 0.001-1、0.005-0.5，或 0.01-0.1 Na2Mo04-2H20 mg/L 0.04 0.001-1、0.005-0.5，或 0.01-0.1 CuS04-5H20 mg/L 2.07 0.1-100、0.5-50，或 1-25 NiS04_6H20 mg/L 2.07 0.1-100、0.5-50，或 1-25 高壓蒸氣滅菌後(維生素）硫胺素 mg/L 9.75 0.1-100、1-50，或 5-25 維生素B12 mg/L 0.16 0.1-100、0.1-10，或 0.1-1 CaVi-泛酸 mg/L 3.33 0.1-100、0.1-50，或 1-10 高壓蒸氣滅菌後(碳）葡萄糖 g/L 30.0 5-150、10-100，或 20-50 氮進料：成分濃度 nh4oh mL/L 21.6 0-150、10-100，或 15-50 典型的培養條件會包括下列： pH 約 6_5- -約8.5，約6.5-約8.0，或是約7.0- 約7.5 溫度：攝氏約17-約30度、攝氏約20-約25度，或是攝氏約22至約23度溶解的氧：約5-約100%飽和、約10-約80%飽和，或約20- •約50%飽和葡萄糖控制在：約 5-約 50 g/L、約 10-約 40 g/L，或約 20— 約 35 g/L。 130 201200591Herculase® II fusion DNA polymerase (Stratagene), and ιχ Herculase® buffer (Stratagene) were combined in a total volume of 50 μM. The pcr operating procedure includes the following steps: (1) 95 ° C for 2 minutes; (2) 95 ° C for 30 seconds; (3) 55 ° C for 30 seconds; (4) 72 ° C for 2 minutes; (5) Repeat steps 2_4 for 40 cycles; (6) 72 °C for 5 minutes; and (7) for 6 °C. Using the chromosomal template described above, PCR amplified a differentiated DNA product of the expected size. The respective PCR products were selected to the vector pJETl.2/Fermentas according to the manufacturer's instructions, and the individual primers were used to determine the individual insertion sequences. Table 13 shows the identity of the actin amino acid sequences from Thraustochytrium (ATCC accession number PTA-9695) compared to the actin sequences available from the public database. Identity is determined by using the standard for protein alignment, the scoring matrix in the "AlignX" program of the VectorNTI program "blosum62mt2".; 127 201200591 Table 13: Comparison of the % identity of actin protein sequences Threustochytriidae sp. RT49 98 Schizochytrium ϊ/λ ATCC 20888 96 Thraustochytrium 96 Polycystis sinensis 96 Marine sputum bacillus 95 Golden yellow broken Chlamydia serovar 95 Table 14 shows the identity of the β-tubulin amino acid sequence from Thraustochytrium (ATCC accession number 695-9695) compared to the β-tubulin sequence available from the public database. The identity is determined by using the standard of protein alignment, the scoring matrix "blosum62mt2" in the "AlignX" program of the VectorNTI program. Table 14: Comparison of the % identity of β-tubulin protein sequences %% 同一开莲莲莲莲莲 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Zm/a) Flat N8 100 Thraustochytrium·Φ. RT49 100 Polycystis sinensis 100 Thraustochytrium sinensis HU1 100 Golden Thraustochytrium 100 Kennedy Thraustochytrium 100 Thraustochytrium sp. #32 100 Thraustochytrium cut. PW]9 100 Fission Phytophthora 100 Schizochytrium genus ATCC 20888 100 128 201200591 According to the above features, the isolated Thraustochytrium (ATCC accession number PTA-9695) is believed to represent a new Schizochytrium species and Thus also known as Schizochytrium sp. ATCC PTA-9695. Example 30 Production of the isolated Thraustochytrium (ATCC accession number PTA-9695) under different culture conditions as described below High level of cell growth. Typical media and culture conditions are shown in Table 15. Also, high levels of fatty acids and DHA were observed (i.e., greater than 50% by weight of dry cells weighed as fatty acids and greater than 50% by weight The fatty acid methyl ester is DHA. 129 201200591 Table 15: Vessel medium composition NaCl KC1 MgS04-7H20 (NH4)2S04 CaCl2T 154 (yeast extract) KH2P〇4 degree thick LLLLLLL /, / .// // 2··0 ·0·6·2·0·2 range 0-25, 5-20, or 10-15 0-5, 0.25-3, or 0.5-2 0-10, 2-8, or 3-6 0-10, 0.25-5, or 0.5-3 0.1-5, 0.15-3, or 0.2-1 0-20, 1-15, or 5-10 0.1-10, 0.5-5, or 1- 3 After autoclaving (metal) citric acid mg/L 3.5 0.1-100, 1-50, or 2-25 FeS04_7H20 mg/L 10.30 0.1-100, 1-50, or 5-25 MnCl2-4H20 mg/L 3.10 0.1-100, 1-50, or 2-25 ZnS04-7H20 mg/L 3.10 0.1-100, 1-50, or 2-25 CoC12.6H20 mg/L 0.04 0.001-1, 0.005-0.5, or 0.01-0.1 Na2Mo04-2H20 mg/L 0.04 0.001-1, 0.005-0.5, or 0.01-0.1 CuS04-5H20 mg/L 2.07 0.1-100, 0.5-50, or 1-25 NiS04_6H20 mg/L 2.07 0.1-100, 0.5-50 , or 1-25 after autoclaving (vitamins) thiamine mg/L 9.75 0.1-100, 1-50, or 5-25 vitamin B12 mg/L 0.16 0.1-100, 0.1-10, or 0.1-1 CaVi - Pantothenic acid mg/L 3.33 0.1-100, 0.1-50, or 1-10 After autoclaving (carbon) Glucose g/L 30.0 5-150, 10-100, or 20-50 Nitrogen feed: Ingredient concentration nh4oh mL /L 21.6 0-150, 10-100, or 15-50 Typical culture conditions will include the following: pH about 6_5--about 8.5, about 6.5-about 8.0, or about 7.0-about 7.5 Temperature: from about 17 to about 30 degrees Celsius, from about 20 to about 25 degrees Celsius, or from about 22 to about 23 degrees Celsius dissolved oxygen: from about 5 to about 100% saturated, from about 10 to about 80% saturated, or about 20- • About 50% saturated glucose is controlled at: about 5 to about 50 g/L, from about 10 to about 40 g/L, or from about 20 to about 35 g/L. 130 201200591

於給料碳與氮之培養物中、伴隨8200 pPmCrM22.5°C 及20%溶解的氧於pH 7 〇，在7天的培養之後，該分離株生產140克/升之乾燥細胞重，且7〇%重量計之脂肪酸含量。使用閉環(Closed loop)氨進料並維持pH於7_〇下。於此等條件下’ ω-3生產力為8 92克/(升*天），伴隨4 7克/升epa(5〇/〇重量計的脂肪酸）以及56.3克/升DHA(57%重量計的脂肪酸)於 7天内。於給料碳與氮之培養物中、伴隨3640 ppm C1·於22.5°C 及20%溶解的氧於pH 7 〇，在7天的培養之後，該分離株生產82克/升之乾燥細胞重，且58%重量計的脂肪酸含量。於此等條件下，ω-3生產力為4_5 g/(L*天），伴隨2丨克/升 EPA(4.3%重量計的脂肪酸）以及2 8 5克/升DHA(5 8 7%重量計的脂肪酸)於7天内。於給料碳與氮之培養物中、伴隨98〇 ppm cn^22.5t 及20%溶解的氧於pH 7.0，在7天的培養之後，該分離株生產60克/升之乾燥細胞重，且53。/(>重量計的脂肪酸含量。於此等條件下，ω-3生產力為2.8 g/(L*天），伴隨1.1克/升 EPA(3·4%重量計的脂肪酸）以及18 4克/升1)11八(56 8%重量計的脂肪酸)於7天内。實施例31 油係自该經單離的破囊壺菌微生物（八1(：：(：：存取號碼 PTA-9695)之-生質樣品(樣品A)予以萃取。該生質樣品係生產於給料碳與氮之培養物中、伴隨98〇 ppm cn^22.5〇c 及20%溶解的氧於pH 7.0。油係藉由己烷萃取方法自生質樣 131 201200591In a feed carbon and nitrogen culture, with 8200 pPmCrM 22.5 ° C and 20% dissolved oxygen at pH 7 该, after 7 days of culture, the isolate produced 140 g / liter of dry cell weight, and 7脂肪酸% by weight of the fatty acid content. A closed loop ammonia feed was used and the pH was maintained at 7 Torr. Under these conditions, 'ω-3 productivity is 8 92 g / (l * day), with 47 g / l epa (5 〇 / 〇 weight of fatty acids) and 56.3 g / liter of DHA (57% by weight Fatty acid) within 7 days. In a feed carbon and nitrogen culture, with 3640 ppm C1 at 22.5 ° C and 20% dissolved oxygen at pH 7 该, after 7 days of culture, the isolate produced 82 g/L dry cell weight. And 58% by weight of the fatty acid content. Under these conditions, the omega-3 productivity is 4_5 g/(L*day) with 2 g/L EPA (4.3% by weight fatty acid) and 285 g/L DHA (5 8 7% by weight). Fatty acids) within 7 days. In a feed carbon and nitrogen culture, with 98 〇ppm cn^22.5t and 20% dissolved oxygen at pH 7.0, after 7 days of culture, the isolate produced 60 g/L dry cell weight, and 53 . /(>The fatty acid content of the weight. Under these conditions, the omega-3 productivity is 2.8 g/(L*day), with 1.1 g/L EPA (3.4% by weight fatty acid) and 18 4 g / l 1) 11 (56 8% by weight of fatty acids) within 7 days. Example 31 An oil system was extracted from the isolated strain of Thraustochytrium microorganism (eight: 1 (::: access number PTA-9695) - a biomass sample (sample A). The biomass sample was produced. In feedstock carbon and nitrogen cultures, with 98〇ppm cn^22.5〇c and 20% dissolved oxygen at pH 7.0. Oils are self-generated by hexane extraction method.

品A予以萃取以出產微生物油樣品A1。簡單地說，乾燥的生質係利用不鎮鋼管及不錄鋼球軸承而用己院予以磨碎歷時大概2小時。真空過濾該漿體並收集濾液。使用一旋轉蒸發器來移除己烷。油亦使用FRIOLEX®方法(GEA Westfalia Separator UK Ltd.，Milton Keynes ’ England)自生質樣 'A 予以萃取以出產微生物油樣品A2。個別的脂質種類係使用低壓急驟層析法而由微生物油樣品A1和A2予以單離，以及決定各類的重量百分比。各類的脂肪酸剖繪係使用附有火焰離子化偵檢器之氣相層析儀(GC-FID)予以決定為脂肪酸曱酯(FAME)。急驟層析法-急驟層析法係使用來分離存在於該等粗製油内的脂質種類，以及決定存在於該油内之各類的重量百分比。該層析系統係利用具有3毫升/分鐘之由石油醚及乙酸乙酯所組成的流動相之矽凝膠（Silica Gel) 60 (EMD Chemicd ’ Gibbstown ’ NJ)。使用一階段梯度以選擇性洗提得自管柱之各脂質種類。該流動相梯度係自1 〇〇%石油醚開始並以50%乙酸乙酯結束（繼而進行100%曱醇清洗）。德份係使用Gilson FC 204大床溶餾份收集器（Gilson，Inc， Middleton，WI)收集於10毫升試管内。各管係藉由薄膜層析法(TLC)予以分析且含各脂質種類（如同藉由在具有預期的滯留係數(Rf)之TLC平板上之單一斑點而判斷）之管子係予以儲集’濃縮至乾無，以及予以稱重。總德份含量接而以重量方式決定。 TLC分析-薄膜層析法係於矽凝膠板上予以實施。使用 132 201200591 由石油醚：乙醚：乙酸(80 : 20 : 1)所組成之溶劑系統洗提該等平板以及使用蛾蒸汽進行視覺化測定。各斑點之Rf值接而與各脂質種類之報告文獻值比較。脂肪酸分析-生質和經單離的脂質種類之樣品係予以分析可作為FAME之脂肪酸組成物。將樣品直接稱重至螺旋蓋試管之内，以及將配於甲苯内之1毫升的C19:0内標準 (NuCheck ’ Elysian，MN)和配於曱醇内之2毫升的1.5 N HC1 添加至各管。將該等管子予以短暫地渦漩以及放置於一加熱組内於100°C歷時2小時。從該加熱組移開該等管子，允許冷卻，以及添加配於水内之1毫升飽和的NaC卜將該等管子再次予以渦旋，離心，以及將頂（有機)層的一部分放置於一 GC小玻璃瓶内並藉由gC_FID予以分析。使用利用 Nu-Chek-Prep GLC 參考標準物（Nu_Chek prep，Inc，Product A was extracted to produce microbial oil sample A1. To put it simply, the dry biomass is ground in a hospital with a non-town steel pipe and a non-recorded steel ball bearing for about 2 hours. The slurry was vacuum filtered and the filtrate was collected. A rotary evaporator was used to remove the hexane. The oil was also extracted from the plastid sample 'A using the FRIOLEX® method (GEA Westfalia Separator UK Ltd., Milton Keynes 'England) to produce the microbial oil sample A2. Individual lipid species were isolated by microbial oil samples A1 and A2 using low pressure flash chromatography and the weight percentages of the various types were determined. Various types of fatty acid profiles were determined to be fatty acid oxime esters (FAME) using a gas chromatograph (GC-FID) with a flame ionization detector. The flash chromatography-flash chromatography method is used to separate the lipid species present in the crude oil and to determine the weight percentage of the various types present in the oil. The chromatographic system utilizes a Silica Gel 60 (EMD Chemicd '' Gibbstown' NJ) having a mobile phase consisting of petroleum ether and ethyl acetate at 3 ml/min. A one-stage gradient was used to selectively elute the individual lipid species from the column. The mobile phase gradient was started with 1% petroleum ether and ended with 50% ethyl acetate (then 100% sterol cleaning). Desserts were collected in 10 ml tubes using a Gilson FC 204 large bed fraction collector (Gilson, Inc, Middleton, WI). Each tube was analyzed by thin film chromatography (TLC) and stored in a tube system containing each lipid species (as judged by a single spot on a TLC plate with the expected retention coefficient (Rf)). Nothing to do, and to weigh. The total German content is determined by weight. TLC analysis - thin film chromatography was carried out on a gel plate. The plates were eluted using a solvent system consisting of petroleum ether: diethyl ether: acetic acid (80:20:1) using 132 201200591 and visualized using moth steam. The Rf value of each spot is then compared to the reported literature values for each lipid species. Fatty Acid Analysis - Samples of the biomass and isolated lipid species were analyzed as fatty acid compositions of FAME. The sample was weighed directly into a screw-capped test tube, and 1 ml of C19:0 internal standard (NuCheck 'Elysian, MN) in toluene and 2 ml of 1.5 N HCl in sterol were added to each. tube. The tubes were briefly vortexed and placed in a heating group at 100 ° C for 2 hours. Remove the tubes from the heating set, allow for cooling, and add 1 ml of saturated NaC in water to vortex the tubes again, centrifuge, and place a portion of the top (organic) layer in a GC. The vial was analyzed by gC_FID. Use the Nu-Chek-Prep GLC reference standard (Nu_Chek prep, Inc,

ElySian ’ MN)所產生之3點内標準校準曲線來定量FAME並根據滯留時間暫時地辨識。存在之脂肪酸係以總FAME之毫克/克及％來表達。樣ooAl係藉由將該粗製油溶解於己炫内以及施加至該管柱之頂部而製備。在使用急驟層析法之樣品的分餾之後，固醇酯餾份佔丨2%重量計之粗製油，三醯甘油酯（TAG) 餾份佔82.7¾重量計之粗製油，游離脂肪酸(FFA)餾份佔〇.9〇/°重里計之粗製油，以及二醯甘油酯（DAG)餾份佔2.90/〇重置汁之粗製油。樣品A1粗製油及經單離的餾份之總脂肪酸剖繪係分別計算為毫克/克及％ FAME而顯示於以下的表 16和表17中。 133 201200591 表16 :依照毫克每克FAME來計算的樣品A1之脂肪酸剖繪生質粗製油固醇酯 TAG FFA DAG Wt. % ΝΑ 38% 1.2% 82.7% 0.9 2.9% 脂肪酸 FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） C12:0* 0.6 0.0 1.9 3.2 1.7 0.0 C14:0# 5.7 13.6 12.8 20.2 13.0 17.6 C14:l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 1.3 3.4 3.1 3.1 2.1 2.6 C16:0* 105.5 239.5 222.2 274.3 183.3 225.1 C16:l* 0.0 0.0 0.8 0,0 0.8 0.0 C18:0* 6.4 16.4 43.1 16.8 9.8 14.0 C18:1N9* 0.0 3.8 1.9 3.3 1.0 3.5 C18:1M7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0,0 0.0 0.0 0.0 0.0 C20:0* 1.8 5.5 13.0 4.7 2.0 2.9 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20 ： 1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 0.6 0.0 C20:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.8 7.3 0.8 0.0 1.2 C20:4 N7 0.0 0.0 0.8 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.0 3.4 0.0 2.6 2.0 1.9 C22:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 1.5 4.1 1.5 3.5 2.1 2.1 C20:5 N3* 18.2 39.5 3.5 38.4 30.6 42.8 C24:0* 0.0 0.0 6.3 0.0 0.0 0.0 C22:4N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 11.9 29.5 8.9 26,9 14.8 18.7 C22:5 N3* 1.1 4.7 0.9 3.6 3.4 2.7 C22:6N3* 253.5 569.7 107.3 556.5 352.8 451.4 全部FAMES之總和 408.6 934.0 435.4 958.0 620.1 786.4 134 201200591 表17 :樣品A1的脂肪酸剖繪之依照總FAME之百分比生質粗製油固醇酯 TAG FFA DAG 脂肪酸 %FAME % FAME % FAME % FAME % FAME % FAME CI2:0* 0.1 0.0 0.4 0.3 0.3 0.0 C14:0* 1.4 1.5 2.9 2.1 2.1 2.2 C14:l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.3 0.4 0.7 0.3 0.3 0.3 C16:0* 25.8 25.6 51.0 28.6 29.6 28.6 C16:1* 0.0 0.0 0.2 0.0 0.1 0.0 C18:0* 1.6 1.8 9.9 1.8 1.6 1.8 C18:1N9* 0.0 0.4 0.4 0.3 0.2 0.4 C18:1N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:0* 0.4 0.6 3.0 0.5 0.3 0.4 C18:3N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4N3 0.0 0.0 0.0 0.0 0.1 0.0 C20:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.1 1.7 0.1 0.0 0.1 C20:4 N7 0.0 0.0 0.2 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 0.3 0.4 0.0 0.3 0.3 0.2 C22:1N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N3 0.4 0.4 0.4 0.4 0.3 0.3 C20:5N3* 4.5 4.2 0.8 4.0 4.9 5.4 C24:0* 0.0 0.0 1.4 0.0 0.0 0.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5N6* 2.9 3.2 2.1 2.8 2.4 2.4 C22:5N3* 0.3 0.5 0.2 0.4 0.5 0.3 C22:6N3* 62.0 61.0 24.6 58.1 56.9 57.4 FAME %之總和 100.0 100.0 100.0 100.0 100.0 100.0 135 201200591 樣品A 2係藉由將該粗製油溶解於己烷内以及施加至該管柱之頂部而製備。在使用急驟層析法之樣品的分餾之後，該固醇酯餾份佔0.8%重量計之粗製油，三醯甘油酯 (TAG)餾份佔83.4%重量計之粗製油，游離脂肪酸(FFA)餾份佔1.8%重量計之粗製油，以及二醯甘油酯（DAG)餾份佔 5.6%重量計之粗製油。樣品A2粗製油及經單離的餾份之總脂肪酸剖繪係分別計算為毫克/克及％ FAME而顯示於以下的表18和表19中。 136 201200591 表18 :依照毫克每克FAME來計算的樣品A2之脂肪酸剖繪生質粗製油固醇酯 TAG FFA DAG Wt. % ΝΑ ΝΑ 0.8% 83.4% 1.8% 5.6% 脂肪酸 FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） C12:0* 0.6 0.0 0.0 1.5 0.0 1.0 C14:0* 5.7 13.2 8.9 14.1 9.5 5.4 C14:l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 1.3 3.3 2.8 3.4 2.1 2.2 C16:0* 105.5 233.7 183.8 246.1 159.7 137.3 C16:l* 0.0 0.0 0.0 0.8 0.0 0.0 C18:0* 6.4 16.6 23.6 16.9 11.3 5.6 C18:1N9* 0.0 7.6 5.0 4.3 2.4 2.6 C18:l N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2N6* 0.0 2.2 0.7 1.6 0.8 5.1 C20:0* 1.8 5.2 12.1 5.5 2.6 1.1 C18:3N3* 0.0 0.0 0.0 0.0 0·0 0.0 C20:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4N3 0.0 0.0 0.0 0.8 1.0 0.0 C20:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.3 0.0 0.0 C22:0* 0.0 0.7 6.0 1.3 0.8 0.0 C20:4N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.0 3.0 0.0 3.1 2.3 1.2 C22:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 1.5 4.1 1.4 4.3 2.7 1.0 C20:5N3* 18.2 38.6 2.7 38.6 39.5 45.5 C24:0* 0.0 0.0 4.7 0.6 0.0 0.3 C22-.4N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 11.9 28.2 8.6 29.6 18.0 14.7 C22:5N3* 1.1 3.4 0.0 3.5 2.5 2.2 C22:6N3* 253.5 566.7 102.2 575.0 475.3 447.2 全部FAME'S之總和 408.6 926.5 362.3 951.3 730.4 672.5 137 201200591 表19 :依照總FAME之百分比之樣品A2的脂肪酸剖繪The standard calibration curve within 3 points generated by ElySian 'MN) quantifies the FAME and is temporarily identified based on the residence time. The fatty acids present are expressed in milligrams per gram and % of total FAME. The ooAl was prepared by dissolving the crude oil in the sleek and applying it to the top of the column. After fractional distillation of the sample using flash chromatography, the sterol ester fraction accounts for 2% by weight of the crude oil, and the triterpene glyceride (TAG) fraction accounts for 82.73⁄4 by weight of the crude oil, free fatty acid (FFA). The crude oil of the fraction of 〇.9 〇 / °, and the crude oil of the diglyceride (DAG) fraction of 2.90 / 〇 reset juice. The total fatty acid profiles of sample A1 crude oil and isolated fractions were calculated as mg/g and % FAME, respectively, and are shown in Tables 16 and 17 below. 133 201200591 Table 16: Fatty acid crude oil sterols of sample A1 calculated in milligrams per gram of FAME TAG FFA DAG Wt. % ΝΑ 38% 1.2% 82.7% 0.9 2.9% Fatty acid FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) C12:0* 0.6 0.0 1.9 3.2 1.7 0.0 C14:0# 5.7 13.6 12.8 20.2 13.0 17.6 C14 :l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 1.3 3.4 3.1 3.1 2.1 2.6 C16:0* 105.5 239.5 222.2 274.3 183.3 225.1 C16:l* 0.0 0.0 0.8 0,0 0.8 0.0 C18:0* 6.4 16.4 43.1 16.8 9.8 14.0 C18:1N9* 0.0 3.8 1.9 3.3 1.0 3.5 C18:1M7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2 N6* 0.0 0,0 0.0 0.0 0.0 0.0 C20:0* 1.8 5.5 13.0 4.7 2.0 2.9 C18:3 N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20 : 1 N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4 N3 0.0 0.0 0.0 0.0 0.6 0.0 C20:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.8 7.3 0.8 0.0 1.2 C20:4 N7 0.0 0.0 0.8 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.0 3.4 0.0 2.6 2.0 1.9 C22:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 C20:4 N3 1.5 4.1 1.5 3.5 2.1 2.1 C20:5 N3* 18.2 39.5 3.5 38.4 30.6 42.8 C24:0* 0.0 0.0 6.3 0.0 0.0 0.0 C22:4N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 11.9 29.5 8.9 26,9 14.8 18.7 C22:5 N3* 1.1 4.7 0.9 3.6 3.4 2.7 C22:6N3* 253.5 569.7 107.3 556.5 352.8 451.4 Total of all FAMES 408.6 934.0 435.4 958.0 620.1 786.4 134 201200591 Table 17: Fatty acid profile of sample A1 Percentage according to total FAME Raw crude oil sterol ester TAG FFA DAG Fatty acid % FAME % FAME % FAME % FAME % FAME % FAME CI2: 0* 0.1 0.0 0.4 0.3 0.3 0.0 C14:0* 1.4 1.5 2.9 2.1 2.1 2.2 C14:l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.3 0.4 0.7 0.3 0.3 0.3 C16:0* 25.8 25.6 51.0 28.6 29.6 28.6 C16:1* 0.0 0.0 0.2 0.0 0.1 0.0 C18:0* 1.6 1.8 9.9 1.8 1.6 1.8 C18:1N9* 0.0 0.4 0.4 0.3 0.2 0.4 C18:1N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:0* 0.4 0.6 3.0 0.5 0.3 0.4 C18:3N3* 0.0 0.0 0.0 0.0 0.0 0.0 C20:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18 :4N3 0.0 0.0 0.0 0.0 0.1 0.0 C20:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.0 0.1 1.7 0.1 0.0 0.1 C20:4 N7 0.0 0.0 0.2 0.0 0.0 0.0 C20 :3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 0.3 0.4 0.0 0.3 0.3 0.2 C22:1N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N3 0.4 0.4 0.4 0.4 0.3 0.3 C20:5N3 * 4.5 4.2 0.8 4.0 4.9 5.4 C24:0* 0.0 0.0 1.4 0.0 0.0 0.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5N6* 2.9 3.2 2.1 2.8 2.4 2.4 C22: 5N3* 0.3 0.5 0.2 0.4 0.5 0.3 C22: 6N3* 62.0 61.0 24.6 58.1 56.9 57.4 FAME % sum 100.0 100.0 100.0 100.0 100.0 100.0 135 201200591 Sample A 2 is prepared by dissolving the crude oil in hexane and applying it to the tube Prepared at the top of the column. After fractionation of the sample using flash chromatography, the sterol ester fraction accounts for 0.8% by weight of the crude oil, and the triterpene glyceride (TAG) fraction accounts for 83.4% by weight of the crude oil, free fatty acid (FFA). The fraction accounted for 1.8% by weight of the crude oil, and the diterpene glyceride (DAG) fraction accounted for 5.6% by weight of the crude oil. The total fatty acid profile of the sample A2 crude oil and the isolated fraction was calculated as mg/g and % FAME, respectively, and is shown in Tables 18 and 19 below. 136 201200591 Table 18: Fatty acid profile of crude oil in sample A2 calculated in milligrams per gram of FAME TAG FFA DAG Wt. % ΝΑ ΝΑ 0.8% 83.4% 1.8% 5.6% Fatty acid FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) C12:0* 0.6 0.0 0.0 1.5 0.0 1.0 C14:0* 5.7 13.2 8.9 14.1 9.5 5.4 C14 :l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 1.3 3.3 2.8 3.4 2.1 2.2 C16:0* 105.5 233.7 183.8 246.1 159.7 137.3 C16:l* 0.0 0.0 0.0 0.8 0.0 0.0 C18:0* 6.4 16.6 23.6 16.9 11.3 5.6 C18: 1N9* 0.0 7.6 5.0 4.3 2.4 2.6 C18:l N7 0.0 0.0 0.0 0.0 0.0 0.0 C18:2N6* 0.0 2.2 0.7 1.6 0.8 5.1 C20:0* 1.8 5.2 12.1 5.5 2.6 1.1 C18:3N3* 0.0 0.0 0.0 0.0 0·0 0.0 C20:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C18:4N3 0.0 0.0 0.0 0.8 1.0 0.0 C20:2 N6* 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.3 0.0 0.0 C22:0* 0.0 0.7 6.0 1.3 0.8 0.0 C20:4N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.0 3.0 0.0 3.1 2.3 1.2 C22:l N9* 0.0 0.0 0.0 0.0 0. 0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 1.5 4.1 1.4 4.3 2.7 1.0 C20:5N3* 18.2 38.6 2.7 38.6 39.5 45.5 C24:0* 0.0 0.0 4.7 0.6 0.0 0.3 C22-.4N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:1N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 11.9 28.2 8.6 29.6 18.0 14.7 C22:5N3* 1.1 3.4 0.0 3.5 2.5 2.2 C22:6N3* 253.5 566.7 102.2 575.0 475.3 447.2 Total of all FAME's 408.6 926.5 362.3 951.3 730.4 672.5 137 201200591 Table 19: Fatty acid profile of sample A2 according to the percentage of total FAME

注意到樣品A1和A2係分別地使用一典型的己燒萃取以及FRIQLEX%·*予以萃卜設若料樣品❹本發^ 方法來萃取，預期表16-19的脂肪酸剖繪要為實質相同的 138 201200591 實施例32 在使用如實施例31中所述之Fri〇lex方法而自發酵肉湯萃取油後，該粗製油係經由精製、漂白、及脫臭步驟予以進一步加工來獲得最終油。使該最終油經高油酸的葵花油稀釋以獲得具有大概4〇〇毫克/克之DHa含量的最終商業油。個別的脂質種類係予以單離以及各類的脂肪酸剖繪係使用附有火焰離子化偵檢器之氣相層析儀(GC_FID)予以測定為脂肪酸甲酿（fame)。急鄉層析法-急驟層析法係使用來分離存在於該最終油内的脂質種類’以及來決定存在於該油内之各類的重量百分比。該層析系統係利用具有3毫升/分鐘之由石油醚及乙酸乙醋所組成的流動相之矽凝膠6〇(emd chemical， Gibbstown，NJ)。使用階段梯度以選擇性洗提得自管柱之各脂質種類。該流動相梯度係自⑽％石⑽開始並以5〇% 乙酸乙醋結束(繼而進行1〇〇%甲醇清洗)。館份係使用GUs〇n FC 204大床,谷顧份收集器（GUs〇n，心，沾她t〇n，WI)收集於10毫升試㈣。各管係藉由_層析法(TLC)予以分析且3各月日質種類（如同藉由在具有預期的滯留係、數(間之 ΤΚ平板上之單-斑點而判斷)之管子係予以儲集，濃縮至乾燥，以及予以稱重。總齡含量接而以重量方式決定。 TL C分析—薄膜層析法係於石夕凝膠板上予以實施。使用 W㈣：乙醚：乙酸⑽：2G:1)所組成之溶劑系統洗提該專平板以及使_;魏_視覺化敎。各㈣之娜接而與各脂f義之料讀值比較。 f； 139 201200591 脂肪酸分析-最終油樣品和經單離的脂質種類係予以分析可作為FAME之脂肪酸組成物。將樣品直接稱重至螺旋蓋試管之内，以及將配於甲苯内之丨毫升的cl9:〇内標準 (NuCheck ’ Elysian ’ MN)和配於甲醇内之2毫升的1 5 N HC1 添加至各管。將該等管子予以短暫地渦漩以及放置於一加熱組内於100。(：歷時2小時。從該加熱組移開該等管子，允許冷卻’以及添加配於水内之丨毫升飽和的Naa。將該等管子再次予以渦漩，離心，以及將頂（有機)層的一部分放置於一 GC小玻璃瓶内並藉由gc-FID予以分析。使用利用It is noted that the samples A1 and A2 are separately extracted using a typical hexane extraction and FRIQLEX%** extraction method, and the fatty acid profile of Tables 16-19 is expected to be substantially the same 138. 201200591 Example 32 After extracting the oil from the fermentation broth using the Fri〇lex method as described in Example 31, the crude oil was further processed through a refining, bleaching, and deodorizing step to obtain a final oil. The final oil was diluted with high oleic sunflower oil to obtain a final commercial oil having a DHa content of about 4 mg/g. Individual lipid species were isolated and various fatty acid profiles were determined using a gas chromatograph (GC_FID) with a flame ionization detector to determine fatty acid fame. The Rapid Chromatography-Jet chromatographic method is used to separate the lipid species present in the final oil' and to determine the weight percent of the species present in the oil. The chromatographic system utilizes a gel phase 6 (emd chemical, Gibbstown, NJ) having a mobile phase consisting of petroleum ether and ethyl acetate at 3 ml/min. A stage gradient is used to selectively elute the individual lipid species from the column. The mobile phase gradient begins with (10)% stone (10) and ends with 5% acetic acid ethyl acetate (and then 1% methanol wash). The museum uses the GUs〇n FC 204 bed, the Gu Gu part collector (GUs〇n, heart, dip her t〇n, WI) collected in 10 ml test (4). Each tube was analyzed by _ Chromatography (TLC) and 3 months of daily quality (as evidenced by a tube system with the expected retention system, number (single-spot on the plate) The mixture was concentrated to dryness and weighed. The total age content was determined by weight. TL C analysis - thin film chromatography was carried out on a stone gel plate. W (four): diethyl ether: acetic acid (10): 2G : 1) The solvent system consists of eluting the special plate and visualizing the _; Each (4) of the Na is then compared with the readings of the various fats. f; 139 201200591 Fatty Acid Analysis - The final oil sample and the isolated lipid species were analyzed as fatty acid compositions of FAME. The sample was weighed directly into a screw-capped test tube, and the cl9: 〇 ml standard (NuCheck ' Elysian ' MN) in toluene and 2 ml of 1 5 N HCl in methanol were added to each tube. The tubes are briefly vortexed and placed in a heating group at 100. (: 2 hours. Remove the tubes from the heating set, allow for cooling' and add 5% of saturated Naa in water. The tubes are again vortexed, centrifuged, and top (organic) layer Part of it is placed in a small glass vial and analyzed by gc-FID.

Nu-Chek-Prep GLC 參考標準物（Nu-Chek Prep，Inc.， Elysian ’ MN)所產生之3點内標準校準曲線來定量FAme並根據滞留時間暫時地辨識。存在之脂肪酸係以總FAME之毫克/克及％來表達。該樣品係藉由將250毫克的最終油溶解於600 pL的己烧内以及施加至該管柱之頂部而製備。在使用急驟層析法之樣品的分餾之後’固醇酯餾份佔丨.2%重量計之最終油，三醯甘油酯（triacylglyceride) (TAG)餾份佔92· 1 %重量計之最終油，游離脂肪酸(FFA)餾份佔2.1%重量計之最終油，固醇餾份佔1.1%之最終油，二醯甘油酯（DAG)餾份佔2 8%重量計之最終油。所儲集的餾份之TLC分析顯示出FFA和固醇餾份係分別地與TAG和DAG混合。分別計算為毫克/克及％ FAME之 FRIOLEX®最終油及經單離的餾份之總脂肪酸剖繪係顯示於以下的表20和表21中。 140 201200591 表20 :依照毫克每克的FAME來計算的脂肪醆剖、徐最終油固醇酯 TAG FFA 固醇 ---___ dag Wt. % ΝΑ 1.2 92.1 2.1 1.1 2.8 脂肪酸 FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） C12:0* 0.0 0.0 1.0 0.0 1.2 0.6 C14:0* 11.5 5.1 11.3 6.0 9.6 5.7 C14:l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 2.3 0.0 2.3 1.2 2.0 1.9 C16:0* 183.3 80.0 180.8 99.9 149.3 132.2 C16:l* 0.0 0.0 0.9 0.0 0.8 0.6 C18:0* 19.6 17.5 19.6 7.5 16.2 6.7 C18:l N9* 243.3 242.8 249.6 62.9 190.5 84.0 C18:l N7 1.9 1.7 2.0 0.8 1.9 0.9 C18:2N6* 13.8 5.6 13.8 6.2 14.3 9.1 C20:0* 4.3 6.6 4.5 1.5 3.6 1.4 C18:3N3* 0.0 0.0 0.3 0.0 0.0 0.0 C20:l N9* 0.0 0.0 0.8 0.0 0.8 0.0 C18:4N3 0.0 0.0 0.7 1.3 0.9 0.4 C20:2 N6* 0.0 0.0 0.6 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.3 0.0 0.0 0.0 C22:0* 3.3 61.0 3.2 1.1 3.0 1.2 C20:4N7 0.0 0.0 0.0 0.0 0.0 0.0 C20-.3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.7 0.0 1 2.3 1.4 1.9 1.3 C22:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 2.4 4.5 3.0 2.2 2.6 1.3 C20:5N3* 28.1 3.0 27.7 38.6 25.6 43.2 C24:0* 1.4 64.3 1.4 0.0 2.0 1.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 20.0 7.6 21.0 10.1 17.2 14.4 C22:5N3* 2.8 0.0 3.1 3.7 .3.4 2.9 C22:6 N3* 407.1 72.5 417.4 443.6 350.5 428.5 全部FAME'S 之總和 936.1 572.1 967.6 688.0 797.3 737.3 141 201200591 表21 :依照總FAME之百分比的脂肪酸剖繪最終油固醇酯 TAG FFA 固醇 DAG 脂肪酸 % FAME % FAME % FAME % FAME % FAME % FAME C12:0* 0.0 0.0 0.1 0.0 0.2 0.1 C14:0* 1.2 0.9 1.2 0.9 1.2 0.8 C14:l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.2 0.0 0.2 0.2 0.2 0.3 C16:0* 19.6 14.0 18.7 14.5 18.7 17.9 C16:l* 0.0 0.0 0.1 0.0 0.1 0.1 C18:0* 2.1 3.1 2.0 1.1 2.0 0.9 C18:l N9* 26.0 42.4 25.8 9.1 23.9 11.4 C18:l N7 0.2 0.3 0.2 0.1 0.2 0.1 C18:2N6* 1.5 1.0 1.4 0.9 1.8 1.2 C20:0* 0.5 1.1 0.5 0.2 0.5 0.2 C18:3N3* 0.0 0.0 0.0 0.0 0,0 0.0 C20:l N9* 0.0 0.0 0.1 0.0 0.1 0.0 C18:4N3 0.0 0.0 0.1 0.2 0.1 0.1 C20:2N6* 0.0 0.0 0.1 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.4 10.7 0.3 0.2 0.4 0.2 C20:4N7 0.0 0.0 0.0 0.0 0.0 0.0 C20:3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 0.2 0.0 0.2 0.2 0.2 0.2 C22:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N3 0.3 0.8 0.3 0.3 0.3 0.2 C20:5N3* 3.0 0.5 2.9 5.6 3.2 5.9 C24:0* 0.2 11.2 0.1 0.0 0.2 0.1 C22:4N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 2.1 1.3 2.2 1.5 2.2 1.9 C22:5N3* 0.3 0.0 0.3 0.5 0.4 0.4 C22:6N3* 43.6 12.7 43.1 64.5 44.0 58.1 FAME % 之總和 100 100 100 100 100 100 142 201200591 注思到表20和21的脂肪酸剖繪係自使用FRI〇LEX⑧方法所萃取的樣品而獲得的。設若該等樣品使財發明的方法來萃取，職表2G和21的麟㈣％要為實質相同的。實施例33 °亥經單離的破囊壺菌微生物(ATCC存取號碼PTA-9695) 之已二天的接種體燒瓶係在給料碳與氮之培養物、伴隨98〇 ppm Cl·(破囊壺菌培養基）中予以製備。依據下列程序進行突變誘發：將一個無菌已丁=2日的燒瓶中之大概5〇毫升，傾倒至一個無菌的40毫升玻璃均質機中。該培養物在均質機中接受 50次猛衝。將該培養物量吸出以及過濾通過置於一個5〇毫升的無菌管中之一個無菌的5〇微米篩網過濾器（該篩網係使用作為留住菌落的較大型群集及同時讓較小的聚簇與單 —細胞通過該50微米篩網之一構件）。在一個無菌的5〇毫升管中收集全部的濃縮浸出物。將浸潰過的培養物進行渦旋並在含破囊壺菌培養基之試管内製成高至i : 1〇〇倍位準的稀釋液。渦漩該等經稀釋浸出物樣品，然後添加2〇〇微升接種體至一個100 X 15毫米、含有4-5粒玻璃珠(3毫米玻璃珠）之破囊壺菌培養基瓊脂培養皿（100x15毫米）。將各平盤溫和地攪拌，以使得玻璃珠將接種體均勻地塗佈在平盤上。自平盤上移除玻璃珠及讓平盤加蓋靜置歷時大概5分鐘至乾燥。當該程序在暗光中進行時，將無菌通風櫥與鄰近區域的燈光關閉。僅存在使該程序得以進行之間接與微弱之極少量的光。 143 "u^〇〇59l 田照射該等樣品時，將五重複的平盤的蓋移開，以及將平盤窨从於XL父聯器（Spectronics Corporation，New York) 的底板j* 其仅準該交聯器所輸送的功率係以微焦耳為單位以及係尋求達到9〇%·95%的殺死率。五重複的對照組平 &係使用4 ^相同的操作程序接種未經突變誘發的細胞。此等細皰數目r 盤取出你使用來計算％殺死率。一旦照射完成後，將平、替換蓋子，以及依序以封口膜(parafilm)與鋁箔來 °该等平盤在第一週務必在黑暗中生長，藉此其等無法修補受損的基因。將平盤置於22.5°C房中大約10天，然後計數菌落。當進彳-具丁終叶數之後，以一個無菌的接種環挑取個別的菌落以及重複劃線於新的破囊壺菌培養基平盤上。將各菌落分植在個別的平盤上。當平盤生長稍密時，使用-接種環採集一樣品，以及接種至含有5〇毫升的破囊壺菌培養基之一個無菌的250毫升震盪燒瓶中。將此燒瓶置於22.5t房中的一個200 rpm振盪器上。在丁=7日’將該震盪燒瓶的培養物收穫至一個50毫升的無菌管中。測定pH值且旋轉樣品以收集生質小丸。將各樣品沖洗及再懸浮於異丙醇與蒸餾水的 50: 50混合物中，然後再旋轉。將所收集的小丸冷凍乾燥、稱重，以及進行FAME分析。表22_28中的資料代表使用上述方法所產生之突變體。 144 201200591 韜粼钬 Ws696-vldf?£^-44:uulv卷涸洄嘟輕镩：(N(N<The standard calibration curve within 3 points generated by the Nu-Chek-Prep GLC reference standard (Nu-Chek Prep, Inc., Elysian 'MN) was used to quantify FAme and was temporarily identified based on the residence time. The fatty acids present are expressed in milligrams per gram and % of total FAME. The sample was prepared by dissolving 250 mg of the final oil in 600 pL of calcined and applied to the top of the column. After the fractionation of the sample using flash chromatography, the 'co-alcohol ester fraction accounted for 2% by weight of the final oil, and the triacylglyceride (TAG) fraction accounted for 92. 1% by weight of the final oil. The free fatty acid (FFA) fraction accounts for 2.1% by weight of the final oil, the sterol fraction accounts for 1.1% of the final oil, and the diterpene glyceride (DAG) fraction accounts for 28.8% by weight of the final oil. TLC analysis of the collected fractions showed that the FFA and sterol fractions were mixed with TAG and DAG, respectively. The total fatty acid profiles of the FRIOLEX® final oil and the isolated fractions, calculated in milligrams per gram and % FAME, respectively, are shown in Tables 20 and 21 below. 140 201200591 Table 20: Fat profile calculated in milligrams per gram of FAME, final oil sterol ester TAG FFA sterol---___ dag Wt. % ΝΑ 1.2 92.1 2.1 1.1 2.8 Fatty acid FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) C12:0* 0.0 0.0 1.0 0.0 1.2 0.6 C14:0* 11.5 5.1 11.3 6.0 9.6 5.7 C14 :l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 2.3 0.0 2.3 1.2 2.0 1.9 C16:0* 183.3 80.0 180.8 99.9 149.3 132.2 C16:l* 0.0 0.0 0.9 0.0 0.8 0.6 C18:0* 19.6 17.5 19.6 7.5 16.2 6.7 C18: l N9* 243.3 242.8 249.6 62.9 190.5 84.0 C18:l N7 1.9 1.7 2.0 0.8 1.9 0.9 C18:2N6* 13.8 5.6 13.8 6.2 14.3 9.1 C20:0* 4.3 6.6 4.5 1.5 3.6 1.4 C18:3N3* 0.0 0.0 0.3 0.0 0.0 0.0 C20 :l N9* 0.0 0.0 0.8 0.0 0.8 0.0 C18:4N3 0.0 0.0 0.7 1.3 0.9 0.4 C20:2 N6* 0.0 0.0 0.6 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.3 0.0 0.0 0.0 C22:0* 3.3 61.0 3.2 1.1 3.0 1.2 C20:4N7 0.0 0.0 0.0 0.0 0.0 0.0 C20-.3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 1.7 0.0 1 2.3 1.4 1.9 1.3 C22:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4 N3 2.4 4.5 3.0 2.2 2.6 1.3 C20:5N3* 28.1 3.0 27.7 38.6 25.6 43.2 C24:0* 1.4 64.3 1.4 0.0 2.0 1.0 C22:4 N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 20.0 7.6 21.0 10.1 17.2 14.4 C22:5N3* 2.8 0.0 3.1 3.7 .3.4 2.9 C22:6 N3* 407.1 72.5 417.4 443.6 350.5 428.5 All FAME'S Total 936.1 572.1 967.6 688.0 797.3 737.3 141 201200591 Table 21: Fatty acid profile according to the percentage of total FAME Final oil sterol ester TAG FFA sterol DAG Fatty acid % FAME % FAME % FAME % FAME % FAME % FAME C12:0* 0.0 0.0 0.1 0.0 0.2 0.1 C14:0* 1.2 0.9 1.2 0.9 1.2 0.8 C14:l* 0.0 0.0 0.0 0.0 0.0 0.0 C15:0 0.2 0.0 0.2 0.2 0.2 0.3 C16:0* 19.6 14.0 18.7 14.5 18.7 17.9 C16:l* 0.0 0.0 0.1 0.0 0.1 0.1 C18:0* 2.1 3.1 2.0 1.1 2.0 0.9 C18:l N9* 26.0 42.4 25.8 9.1 23.9 11.4 C18:l N7 0.2 0.3 0.2 0.1 0.2 0.1 C18:2N6* 1.5 1.0 1.4 0.9 1.8 1.2 C20:0* 0.5 1.1 0.5 0.2 0.5 0.2 C18:3N3* 0.0 0.0 0.0 0.0 0,0 0.0 C20:l N9* 0.0 0.0 0.1 0.0 0.1 0.0 C18:4N3 0.0 0.0 0.1 0.2 0.1 0.1 C20:2N6* 0.0 0.0 0.1 0.0 0.0 0.0 C20:3 N6 0.0 0.0 0.0 0.0 0.0 0.0 C22:0* 0.4 10.7 0.3 0.2 0.4 0.2 C20:4N7 0.0 0.0 0.0 0.0 0.0 0.0 C20: 3 N3 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N6* 0.2 0.0 0.2 0.2 0.2 0.2 C22:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N5 0.0 0.0 0.0 0.0 0.0 0.0 C20:4N3 0.3 0.8 0.3 0.3 0.3 0.2 C20:5N3 * 3.0 0.5 2.9 5.6 3.2 5.9 C24:0* 0.2 11.2 0.1 0.0 0.2 0.1 C22:4N9 0.0 0.0 0.0 0.0 0.0 0.0 C24:l N9* 0.0 0.0 0.0 0.0 0.0 0.0 C22:5 N6* 2.1 1.3 2.2 1.5 2.2 1.9 C22: 5N3* 0.3 0.0 0.3 0.5 0.4 0.4 C22:6N3* 43.6 12.7 43.1 64.5 44.0 58.1 FAME % sum 100 100 100 100 100 100 142 201200591 Note that the fatty acid profiles in Tables 20 and 21 have been extracted using the FRI〇LEX8 method. Obtained from the sample. If these samples are to be extracted by the method of the invention, the syllabus of the 2G and 21 is essentially the same. Example 33 A two-day inoculum flask of the Thraustochytrium microorganism (ATCC accession number PTA-9695) was applied to a feed carbon and nitrogen culture with 98 〇ppm Cl· Prepared in the culture medium). Mutation induction was performed according to the following procedure: Approximately 5 ml of a sterile diced = 2 day flask was poured into a sterile 40 ml glass homogenizer. The culture received 50 bursts in a homogenizer. The culture was aspirated and filtered through a sterile 5 〇 micron mesh filter placed in a 5 liter sterile tube (this screen was used as a larger cluster to retain colonies and at the same time to allow for smaller clusters) Clusters and mono-cells pass through one of the 50 micron screens). Collect all concentrated extracts in a sterile 5 ml tube. The impregnated culture was vortexed and a dilution of up to i: 1 〇〇 level was made in a test tube containing Thraustochytrium medium. Vortex the diluted extract samples, then add 2 μL of the inoculum to a 100 X 15 mm, Thraustochytrium medium agar dish containing 4-5 glass beads (3 mm glass beads) (100x15 Mm). The pans were gently agitated so that the glass beads uniformly spread the inoculum on the pan. Remove the glass beads from the flat plate and let the flat plate cover for about 5 minutes to dry. When the procedure is performed in dim light, the sterile fume hood and the lighting in the adjacent area are turned off. There is only a small amount of light that allows the program to be connected and weak. 143 "u^〇〇59l When illuminating the samples, remove the cover of the five repeating pans and remove the flat pan from the bottom plate j* of the XL parent connector (Spectronics Corporation, New York) The power delivered by the cross-linker is in microjoules and seeks to achieve a kill rate of 9〇%·95%. Five replicates of the control group were vaccinated with cells that were not mutated using 4 ^ identical procedures. The number of these fine blister r disks is taken out to calculate the % kill rate. Once the irradiation is completed, the cover will be replaced, replaced, and sequentially with a parafilm and an aluminum foil. These flat disks must be grown in the dark in the first week, so that the damaged genes cannot be repaired. The plates were placed in a 22.5 °C room for approximately 10 days and colonies were counted. After the number of leaves in the sputum, the individual colonies were picked with a sterile inoculating loop and repeatedly streaked onto a new plate of Thraustochytrium medium. Each colony was planted on individual flat plates. When the plates were slightly densely grown, a sample was collected using the inoculation loop and inoculated into a sterile 250 ml shake flask containing 5 mL of Thraustochytrium medium. The flask was placed on a 200 rpm shaker in a 22.5 t room. The culture of the shake flask was harvested into a 50 ml sterile tube at deg = 7 days. The pH was measured and the sample was rotated to collect the raw pellets. Each sample was rinsed and resuspended in a 50:50 mixture of isopropanol and distilled water and then spun. The collected pellets were freeze dried, weighed, and subjected to FAME analysis. The data in Table 22_28 represents the mutants produced using the above method. 144 201200591 韬粼钬 Ws696-vldf?£^-44: uulv 涸洄涸洄镩: (N(N<

突變體10 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.14 0.00 1.57 0.00 0.00 29.20 0.24 0.00 丨 0.00 0.13 瓦· 0.00 0.00 0.00 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.00 0.36 0.75 突變體9 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.15 0.00 1.72 0.00 0.00 30.11 0.27 0.00 0.00 0.13 1 0.00 0.00 0.00 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.00 0.33 0.73 突變體8 0.00 0.00 0.00 0.00 0.00 *—Μ 〇 0.00 〇 0.00 00 0.00 0.00 30.97 0.16 0.00 0.00 0.16 m (N 0.00 0.00 0.00 0.03 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.41 0.60 突變體5 0.00 0.00 0.00 0.00 0.00 0.07 0.00 0.18 0.00 1.29 0.00 0.00 29.86 0.27 0.00 0.00 0.12 1.57 0.00 0.00 0.00 0.00 0.00 0.00 0.46 0.00 0.00 0.00 0.00 0.32 0.89 突變體4 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.12 0.00 2.36 0.00 0.00 30.33 0.25 0.00 0.00 0.27 (N (N 0.00 0.00 0.00 0.07 0.00 0.00 0.34 0.00 0.00 0.00 0.00 0.44 0.80 突變體3 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.13 0.00 Pi 0.00 0.00 29.97 0.14 0.00 0.00 0.17 1.47 0.00 0.00 0.00 0.00 0.00 0.00 0.45 0.00 0.00 0.00 0.00 0.42 0.57 突變體2 0.00 0.00 0.00 0.00 0.00 0.08 ! 0.00 0.17 0.00 1.49 0.00 0.00 29.96 0.31 0.00 0.00 0.13 00 rn 0.00 0.00 0.00 0.00 0.00 0.00 0.42 0.00 0.00 0.00 0.00 0.32 0.94 突變體1 0.00 0.00 0.00 0.00 0.00 0.10 0.00 〇 0.00 IT) 00 0.00 0.00 28.75 0.20 ! 〇.〇〇 0.00 0.15 1.22 0.00 0.00 0.00 0.03 0.00 0.00 0.36 0.00 0.00 0.00 0.00 0.38 0.55 對照 ATCC PTA-9695 0.00 0.00 0.00 0.00 0.00 0.10 I 0.00 0.11 0.00 ON 0.00 0.00 30.98 0.27 ! 〇.〇〇 0.00 0.12 1.29 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.55 脂肪酸 08:0 09:0 10:0 〇 12:0 12:1 13:0 j 13:1 14:0 14:1 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18:3 n-6 18:3 n-3 18:4 n-3 20:0 20:1 n-9 20:2 20:3 n-9 20:3 n-6 20:3 n-3 20:4 ARA 145 201200591 忘鱗？F<N<N< 突變體10 2.81 0.09 0.00 0.00 0.00 0.00 tn ro 0.18 58.52 0.00 0.00 37.87 0.39 突變體9 2.81 0.09 0.00 0.00 0.00 0.00 3.01 0.17 57.53 0.00 0.09 37.72 0.38 突變體8 2.54 0.07 0.00 0.00 0.00 0.00 3.34 0.20 56.62 0.00 0.00 54.71 0.50 突變體5 2.83 0.10 0.00 0.00 0.00 0.00 2.87 0.18 57.98 0.00 0.00 30.62 | 0.36 | 突變體4 3.64 0.07 0.00 0.00 0.00 0.00 3.35 0.20 54.87 0.00 0.00 49.78 0.51 突變體3 2.40 0.09 0.00 0.00 0.00 0.00 3.43 0.23 57.85 0.00 0.00 47.39 0.51 突變體2 3.01 0.09 0.00 0.00 0.00 0.00 2.94 0.21 57.56 0.00 0.00 31.23 0.35 突變體1 2.94 0.08 0.00 0.00 0.00 0.00 3.19 0.18 58.63 0.00 0.08 46.10 0.46 對照 ATCC PTA-9695 2.62 0.08 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 脂肪酸 20:5 n-3 EPA 22:0 (N CN 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 24:1 脂肪未知 146 201200591 韜翱钬 wln696-vHdt餱路哞UUHV 卷 gs嘟嫵镩：突變體22 0.00 0.00 0.00 0.00 0.00 0.10 0.00 0.13 0.00 1.81 0.00 0.00 30.81 0.33 0.00 0.00 0.14 | 1.33 0.00 0.00 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.00 0.34 0.69 2.87 0.08 突變體21 0.00 0.00 0.00 0.00 0.00 0.09 0.00 i 0.16 0.00 0.00 0.00 30.05 0.23 0.00 0.00 0.16 1.34 0.00 0.00 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.00 0.35 0.73 2.90 0.08 突變體20 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.17 0.00 «·«·· 0.00 0.00 28.84 0.23 0.00 0.00 0.14 1.28 0.00 0.00 0.00 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.33 0.83 3.03 0.08 突變體16 0.00 0.00 0.00 0.00 0.00 〇 0.00 i 0.12 0.00 〇〇 Ο) 0.00 0.00 30.18 0.24 0.00 0.00 0.12 1.21 0.00 0.00 1 0.00 0.03 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.37 0.62 2.66 0.08 突變體15 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.00 1.83 0.00 0.00 31.79 0.21 0.00 0.00 0.15 VO rn 0.00 0.00 1 0.00 i 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.00 0.36 0.64 2.52 0.08 突變體14 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.14 0.00 1.75 0.00 0.00 29.92 0.28 0.00 0.00 0.13 1.31 0.00 1 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.33 0.80 2.97 0.08 突變體13 0.00 0.00 0.00 ! 0.00 0.00 0.08 0.00 0.16 0.00 1.43 0.00 0.00 30.27 0.26 0.00 0.00 0.15 1.44 0.00 ! 0.00 0.00 0.00 0.00 0.00 0.42 0.06 0.00 0.00 0.00 0.36 0.72 2.72 0.09 突變體11 0.00 0.00 0.00 0.00 0.00 0.10 0.00 0.15 0.00 1.89 0.00 0.00 31.08 I 0.32 ! 0.00 0.00 0.24 \〇 0.00 0.00 0.00 0.05 0.00 0.00 0.38 0.00 0.00 0.00 0.00 0.43 0.79 3.17 0.08 對照 ATCC ΡΤΑ-9695 0.00 0.00 0.00 1 0.00 0.00 0.10 0.00 <0 0.00 0.00 0.00 30.98 0.27 0.00 0.00 0.12 Os (N 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.55 2.62 0.08 脂肪酸 08:0 09:0 10:0 11:0 12:0 12:1 13:0 13:1 14:0 14:1 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18:3 n-6 18:3 n-3 18:4 n-3 20:0 20:1 n-9 20:2 20:3 n-9 20:3 n-6 20:3 n-3 20:4 ARA 20:5 n-3 EPA 22:0 % 147 201200591 忘碥？Fe(N^ 突變體22 0.00 0.00 0.00 0.00 3.02 0.18 56.65 0.00 0.08 43.55 0.38 突變體21 0.00 0.00 0.00 0.00 3.01 0.18 | 57.45 | 0.00 0.00 | 40.23 | 0.39 突變體20 0.00 0.00 0.00 0.00 2.98 0.17 58.58 0.00 0.00 | 37.71 | 0.37 突變體16 0.00 0.00 0.00 0.00 3.16 0.16 57.38 0.00 0.07 47.32 0.44 突變體15 0.00 0.00 0.00 0.00 3.07 0.19 56.02 0.00 0.00 48.46 0.47 突變體14 0.00 0.00 0.00 0.00 2.97 0.17 57.63 0.00 0.00 38.41 0.36 突變體13 0.00 0.00 0.00 0.00 3.06 0.19 57.52 0.00 0.00 37.00 | 0.39 | 突變體11 0.00 0.00 0.00 0.00 3.25 0.20 55.17 0.00 0.00 46.19 0.47 對照 ATCC ΡΤA-9695 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 脂肪酸 (N (N 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 脂肪未知 148 201200591 韜激·钬 w g696-vldtti??^磘4φυυ1ν 卷S涸嘟輕寸<N< 突變體35 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.10 0.00 os 0.00 0.39 30.78 0.16 0.00 0.00 0.12 1.34 0.09 0.00 0.00 0.00 0.00 0.00 0.40 0.14 0.00 0.00 0.00 0.00 0.28 2.59 0.08 突變體34 0.00 0.00 0.00 0.00 0.00 〇 0.00 0.12 0.00 2.01 i 0.00 0.54 32.28 0.26 0.00 0.00 0.16 1.37 Ο 0.00 0.00 0.00 0.00 0.00 0.40 0.13 0.00 0.00 0.00 0.00 0.24 2.51 0.08 突變體33 0.00 0.00 0.00 0.00 0.00 0.10 0.00 0.12 1 0.00 Os 0.00 0.70 30.26 0.29 0.00 0.00 0.26 1.32 0.10 0.00 0.00 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.00 0.34 3.28 0.08 突變體30 0.00 0.00 0.00 0.00 0.00 0.08 0.00 1 0.14 0.00 vo 0.00 0.00 29.50 0.26 0.00 0.00 0.13 1.30 1 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.35 0.32 2.91 0.08 突變體29 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.16 i 0.00 i vp 0.00 0.00 29.70 0.26 0.00 0.00 0.13 1.32 0.00 0.00 0.00 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.00 0.35 0.68 2.90 0.09 突變體27 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.14 0.00 ON 0.00 0.00 30.21 0.22 0.00 0.00 0.16 1.31 0.00 0.00 0.00 1 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.00 0.35 0.69 2.85 0.08 突變體26 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.13 0.00 1.71 0.00 0.00 30.32 0.22 0.00 0.00 0.18 1.31 0.00 1 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.59 2.70 0.08 突變體24 0.00 0.00 0.00 0.00 0.00 〇 0.00 0.12 0.00 ί 1.98 0.00 0.00 30.61 0.19 0.00 0.00 0.15 3 0.00 0.00 0.00 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.38 0.61 2.62 0.08 對照 ATCC PTA-9695 0.00 0.00 0.00 0.00 0.00 0.10 0.00 ο 0.00 ON 0.00 0.00 30.98 0.27 0.00 0.00 0.12 <N 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.55 2.62 0.08 脂肪酸 08:0 09:0 10:0 11:0 11:1 12:0 12:1 13:0 1 13:1 14:0 14:1 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 η-9 18:1 η-7 18:2 18:3 η-6 18:3 η-3 18:4 η-3 20:0 20:1 η-9 20:2 20:3 η-9 20:3 η-6 20:3 η-3 20:4 ARA 20:5 η-3 EPA 22:0 % 149 201200591 忘鴉？f173< 突變體35 0.00 0.00 0.00 0.00 3.56 0.24 56.73 0.00 0.07 51.55 1.25 突變體34 0.00 0.00 0.00 0.00 3.26 0.15 54.96 0.00 0.07 46.43 (N 突變體33 0.00 0.00 0.00 0.00 3.43 0.18 55.92 0.00 0.07 46.95 (Ν 突變體30 0.00 0.00 0.00 0.00 3.11 0.18 58.52 0.00 0.00 38.16 0.82 突變體29 0.00 0.00 0.00 0.00 3.10 0.18 57.96 0.00 0.00 38.60 0.37 突變體27 0.00 0.00 0.00 0.00 3.05 0.19 57.46 0.00 0.00 38.86 0.39 突變體26 0.00 0.00 0.00 0.00 3.11 0.18 57.46 0.00 0.00 43.50 0.42 突變體24 0.00 0.00 0.00 0.00 3.10 0.16 57.03 0.00 0.08 47.80 0.45 對照ATCC PTA-9695 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 脂肪酸 CN (N 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 24:1 脂肪未知 201200591 n^^w^^^^s696-vlduuHv#gg^«^:s3< 突變體44 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.15 0.00 1.75 0.00 0.50 30.18 0.22 0.00 0.00 0.13 0.10 0.00 0.00 0.00 0.00 0.00 0.40 0.49 0.00 0.00 0.00 0.00 0.23 2.80 0.08 突變體43 0.00 0.00 0.00 0.00 0.00 0.12 0.00 0.08 0.00 2.17 0.00 0.62 43.37 s 0.00 0.00 0.26 2.21 1 0.09 0.05 0.00 0.00 0.00 0.00 0.61 0.15 0.00 0.00 0.00 0.00 0.35 3.47 0.14 突變體42 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.12 0.00 0.00 0.36 25.77 0.10 0.00 0.00 0.10 (N 0.12 0.00 0.00 0.00 0.00 0.00 0.36 0.15 0.00 0.00 0.00 0.00 0.30 3.21 0.07 突變體40 0.00 0.00 0.00 0.00 0.00 0.09 0.00 〇 0.00 •«Η 0.00 0.55 30.25 0.21 0.00 0.00 0.16 a\ (N 1 0.09 0.00 0.00 0.00 0.00 0.00 0.39 0.14 0.00 0.00 0.00 0.00 0.24 2.80 0.08 突變體39 0.00 0.00 0.00 0.00 0.00 〇 0.00 0.12 0.00 Cs 0.00 0.54 30.84 0.22 0.00 0.00 0.16 1.30 0.09 0.00 0.00 0.00 0.00 0.00 0.38 0.00 0.00 0.00 0.00 0.00 0.27 2.72 0.07 突變體38 0.00 0.00 0.00 0.00 0.00 0.00 1 0.00 0.24 0.00 0.00 0.71 32.28 0.23 0.00 0.00 0.28 Os 0.25 0.00 0.00 0.00 0.00 0.00 0.43 0.43 0.00 0.00 0.00 0.00 0.24 4.00 0.14 突變體37 0.00 0.00 0.00 0.00 0.00 0.00 0.09 0.00 ON Os 0.00 0.48 28.04 0.26 0.00 0.00 0.13 U1 0.08 0.00 0.00 0.06 0.00 0.00 0.31 0.00 0.00 0.00 0.00 0.00 0.31 2.77 0.07 突變體36 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.44 0.00 (N 0.00 2.12 26.95 0.00 0.00 0.00 0.95 00 ! 0.37 0.00 0.00 0.00 0.00 0.00 0.34 0.00 0.00 0.00 0.00 0.00 0.41 5.36 0.00 對照 ATCC ΡΤA-9695 0.00 0.00 0.00 0.00 0.00 0.10 0.00 0.11 0.00 1.79 i 0.00 0.00 30.98 0.27 0.00 0.00 0.12 1.29 ! 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.55 2.62 0.08 脂肪酸 08:0 09:0 10:0 11:0 12:0 12:1 13:0 13:1 14:0 14:1 j 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18:3 n-6 18:3 n-3 18:4 n-3 20:0 20:1 n-9 20:2 20:3 n-9 20:3 n-6 20:3 n-3 20:4 ARA 20:5 n-3 EPA 22:0 % £： 151 201200591 忘碥？fs(n< 突變體44 0.00 0.00 0.00 0.00 3.33 0.17 56.76 0.00 0.09 41.20 1.20 突變體43 0.00 0.00 0.00 0.00 2.37 0.33 41.61 0.00 0.06 56.44 0.90 突變體42 0.00 0.00 0.00 0.00 3.89 0.30 60.99 0.00 0.08 42.48 1.31 突變體40 0.00 0.00 0.00 0.00 3.29 0.17 57.09 0.00 0.09 42.59 1.20 突變體39 0.00 0.00 0.00 0.00 3.48 0.17 56.24 0.00 0.08 45.74 00 (N 突變體38 1 . 0.00 0.00 0.00 0.00 2.57 0.00 54.20 0.00 0.00 18.08 0.73 突變體37 1 0.00 0.00 0.00 0.06 3.94 0.19 58.57 0.00 0.08 54.86 1.36 突變體36 ! ! 0.00 0.00 0.00 0.00 2.40 0.00 57.52 0.00 0.00 12.73 0.29 對照 ATCC PTA-9695 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 脂肪酸 22:1 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 脂肪未知 201200591 韜翱钬 wg696-vHd^^^^331v卷堀涸嘟辆镩：9<n<Mutant 10 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.14 0.00 1.57 0.00 0.00 29.20 0.24 0.00 丨0.00 0.13 瓦· 0.00 0.00 0.00 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.00 0.36 0.75 Mutant 9 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.15 0.00 1.72 0.00 0.00 30.11 0.27 0.00 0.00 0.13 1 0.00 0.00 0.00 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.00 0.33 0.73 Mutant 8 0.00 0.00 0.00 0.00 0.00 *—Μ 〇0.00 〇0.00 00 0.00 0.00 30.97 0.16 0.00 0.00 0.16 m (N 0.00 0.00 0.00 0.03 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.41 0.60 Mutant 5 0.00 0.00 0.00 0.00 0.00 0.07 0.00 0.18 0.00 1.29 0.00 0.00 29.86 0.27 0.00 0.00 0.12 1.57 0.00 0.00 0.00 0.00 0.00 0.00 0.4 0.00 0.00 0.00 0.00 0.002 0.89 Mutant 4 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.12 0.00 2.36 0.00 0.00 30.33 0.25 0.00 0.00 0.27 (N (N 0.00 0.00 0.00 0.07 0.00 0.00 0.34 0.00 0.00 0.00 0.00 0.44 0.80 Mutant 3 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.13 0.00 Pi 0.00 0.00 29.97 0.14 0.00 0.00 0.17 1.47 0.00 0.00 0.00 0.00 0.00 0.00 0.45 0.00 0.00 0.00. 00 0.42 0.57 mutant 2 0.00 0.00 0.00 0.00 0.00 0.08 ! 0.00 0.17 0.00 1.49 0.00 0.00 29.96 0.31 0.00 0.00 0.13 00 rn 0.00 0.00 0.00 0.00 0.00 0.00 0.42 0.00 0.00 0.00 0.00 0.22 0.94 Mutant 1 0.00 0.00 0.00 0.00 0.00 0.000 0.00 〇 0.00 IT) 00 0.00 0.00 28.75 0.20 ! 〇.〇〇0.00 0.15 1.22 0.00 0.00 0.00 0.03 0.00 0.00 0.36 0.00 0.00 0.00 0.00 0.38 0.55 Control ATCC PTA-9695 0.00 0.00 0.00 0.00 0.00 0.10 0.1 0.00 0.00 0.00 0.00 0.00 0.00 0.00.30 0.27 ! 〇.〇〇0.00 0.12 1.29 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.35 0.55 fatty acid 08:0 09:0 10:0 〇12:0 12:1 13:0 j 13:1 14:0 14:1 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18:3 n-6 18:3 n-3 18: 4 n-3 20:0 20:1 n-9 20:2 20:3 n-9 20:3 n-6 20:3 n-3 20:4 ARA 145 201200591 Forget the scales? F<N<N< mutant 10 2.81 0.09 0.00 0.00 0.00 0.00 tn ro 0.18 58.52 0.00 0.00 37.87 0.39 Mutant 9 2.81 0.09 0.00 0.00 0.00 0.00 3.01 0.17 57.53 0.00 0.09 37.72 0.38 Mutant 8 2.54 0.07 0.00 0.00 0.00 0.00 3.34 0.20 56.62 0.00 0.00 54.71 0.50 Mutant 5 2.83 0.10 0.00 0.00 0.00 0.00 2.87 0.18 57.98 0.00 0.00 30.62 | 0.36 | Mutant 4 3.64 0.07 0.00 0.00 0.00 0.00 3.35 0.20 54.87 0.00 0.00 49.78 0.51 Mutant 3 2.40 0.09 0.00 0.00 0.00 0.00 3.43 0.23 57.85 0.00 0.00 47.39 0.51 Mutant 2 3.01 0.09 0.00 0.00 0.00 0.00 2.94 0.21 57.56 0.00 0.00 31.23 0.35 Mutant 1 2.94 0.08 0.00 0.00 0.00 0.00 3.19 0.18 58.63 0.00 0.08 46.10 0.46 Control ATCC PTA-9695 2.62 0.08 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 Fatty acid 20:5 n-3 EPA 22:0 (N CN 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 24:1 Fat unknown 146 201200591 韬翱钬wln696-vHdt 糇路哞UUHV Volume gs mutter: Mutant 22 0.00 0.00 0.00 0.00 0.00 0.000 0.00 0.13 0.00 1.81 0.00 0.00 30.81 0.3 3 0.00 0.00 0.14 | 1.33 0.00 0.00 0.00 0.00 0.00 0.00 0.3 0.00 0.00 0.00 0.00 0.34 0.69 2.87 0.08 Mutant 21 0.00 0.00 0.00 0.00 0.00 0.09 0.00 i 0.16 0.00 0.00 0.0030.05 0.23 0.00 0.00 0.16 1.34 0.00 0.00 0.00 0.00 0.00 0.008 0.00 0.00 0.00 0.00 0.35 0.73 2.90 0.08 Mutant 20 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.17 0.00 «·«·· 0.00 0.00 28.84 0.23 0.00 0.00 0.14 1.28 0.00 0.00 0.00 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.33 0.83 0.08 0.08 Mutant 16 0.00 0.00 0.00 0.00 0.00 〇0.00 i 0.12 0.00 〇〇Ο) 0.00 0.00 30.18 0.24 0.00 0.00 0.12 1.21 0.00 0.00 1 0.00 0.03 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.37 0.62 2.66 0.08 Mutant 15 0.00 0.00 0.00 0.00 0.00 0.00 0.13 0.00 1.83 0.00 0.00 31.79 0.21 0.00 0.00 0.15 VO rn 0.00 0.00 1 0.00 i 0.00 0.00 0.00 0.40 0.00 0.00 0.00 0.00 0.36 0.64 2.52 0.08 Mutant 14 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.14 0.00 1.75 0.00 0.00 29.92 0.28 0.00 0.00 0.13 1.31 0.00 1 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.33 0.80 2.97 0.08 Mutant 13 0.00 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.16 0.00 1.43 0.00 0.00 30.27 0.26 0.00 0.00 0.15 1.44 0.00 0.00 0.00 0.00 0.00 0.00 0.42 0.06 0.00 0.00 0.00 0.36 0.72 2.72 0.09 Mutant 11 0.00 0.00 0.00 0.00 0.00 0.10 0.00 0.15 0.00 1.89 0.00 0.00 31.08 I 0.32 ! 0.00 0.00 0.24 \〇0.00 0.00 0.00 0.05 0.00 0.00 0.38 0.00 0.00 0.00 0.00 0.43 0.79 3.17 0.08 Control ATCC ΡΤΑ-9695 0.00 0.00 0.00 1 0.00 0.00 0.10 0.00 <0 0.00 0.00 0.00 30.98 0.27 0.00 0.00 0.12 Os (N 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.55 2.62 0.08 Fatty Acid 08:0 09:0 10:0 11:0 12:0 12:1 13:0 13:1 14:0 14 :1 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18:3 n-6 18:3 n-3 18:4 n-3 20:0 20:1 n-9 20:2 20:3 n-9 20:3 n-6 20:3 n-3 20:4 ARA 20:5 n-3 EPA 22:0 % 147 201200591 Forgot? Fe(N^ mutant 22 0.00 0.00 0.00 0.00 3.02 0.18 56.65 0.00 0.08 43.55 0.38 Mutant 21 0.00 0.00 0.00 0.00 3.01 0.18 | 57.45 | 0.00 0.00 | 40.23 | 0.39 Mutant 20 0.00 0.00 0.00 0.00 2.98 0.17 58.58 0.00 0.00 | 0.37 mutant 16 0.00 0.00 0.00 0.00 3.16 0.16 57.38 0.00 0.07 47.32 0.44 Mutant 15 0.00 0.00 0.00 0.00 3.07 0.19 56.02 0.00 0.00 48.46 0.47 Mutant 14 0.00 0.00 0.00 0.00 2.97 0.17 57.63 0.00 0.00 38.41 0.36 Mutant 13 0.00 0.00 0.00 0.00 3.06 0.19 57.52 0.00 0.00 37.00 | 0.39 | Mutant 11 0.00 0.00 0.00 0.00 3.25 0.20 55.17 0.00 0.00 46.19 0.47 Control ATCC ΡΤA-9695 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 Fatty Acids (N (N 22:2 22: 3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 Fat unknown 148 201200591 钬钬 g w g696-vldtti??^磘4φυυ1ν 卷 S涸嘟Light inch <N< Mutant 35 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.10 0.00 os 0.00 0.39 30.78 0.16 0.00 0.00 0.12 1.34 0.09 0.00 0.00 0.00 0.00 0.00 0.40 0.14 0.00 0.00 0.00 0.00 0.28 2.5 9 0.08 Mutant 34 0.00 0.00 0.00 0.00 0.00 〇0.00 0.12 0.00 2.01 i 0.00 0.54 32.28 0.26 0.00 0.00 0.16 1.37 Ο 0.00 0.00 0.00 0.00 0.00 0.40 0.13 0.00 0.00 0.00 0.00 0.24 0.28 0.08 Mutant 33 0.00 0.00 0.00 0.00 0.00 0.10 0.00 0.12 1 0.00 Os 0.00 0.70 30.26 0.29 0.00 0.00 0.26 1.32 0.10 0.00 0.00 0.00 0.00 0.00 0.37 0.00 0.00 0.00 0.00 0.00 0.34 3.28 0.08 Mutant 30 0.00 0.00 0.00 0.00 0.00 0.008 0.00 1 0.14 0.00 vo 0.00 0.00 29.50 0.26 0.00 0.00 0.13 1.30 1 0.00 0.00 0.00 0.00 0.00 0.00 0.35 0.00 0.00 0.00 0.00 0.35 0.32 2.91 0.08 Mutant 29 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.16 i 0.00 i vp 0.00 0.00 29.70 0.26 0.00 0.00 0.13 1.32 0.00 0.00 0.00 0.00 0.00 0.00 0.4 0.00 0.00 0.00 0.005 0.68 0.68 2.90 0.09 Mutant 27 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.14 0.00 ON 0.00 0.00 30.21 0.22 0.00 0.00 0.16 1.31 0.00 0.00 0.00 0.00 0.00 0.00 0.000 0.00 0.00 0.00 0.00 0.35 0.69 2.85 0.08 Mutant 26 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.13 0.00 1.71 0.00 0.00 30.32 0.22 0.00 0.00 0 .18 1.31 0.00 1 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.59 2.70 0.08 Mutant 24 0.00 0.00 0.00 0.00 0.00 〇0.00 0.12 0.00 ί 1.98 0.00 0.00 30.61 0.19 0.00 0.00 0.15 3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.38 0.61 2.62 0.08 Control ATCC PTA-9695 0.00 0.00 0.00 0.00 0.00 0.10 0.00 ο 0.00 ON 0.00 0.00 30.98 0.27 0.00 0.00 0.12 <N 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.55 2.62 0.08 Fatty Acid 08:0 09:0 10:0 11:0 11:1 12:0 12:1 13:0 1 13:1 14:0 14:1 15:1 16:0 16:1 16:2 16:3 17:0 18 :0 18:1 η-9 18:1 η-7 18:2 18:3 η-6 18:3 η-3 18:4 η-3 20:0 20:1 η-9 20:2 20:3 Η-9 20:3 η-6 20:3 η-3 20:4 ARA 20:5 η-3 EPA 22:0 % 149 201200591 Forgot? F173 < mutant 35 0.00 0.00 0.00 0.00 3.56 0.24 56.73 0.00 0.07 51.55 1.25 mutant 34 0.00 0.00 0.00 0.00 3.26 0.15 54.96 0.00 0.07 46.43 (N mutant 33 0.00 0.00 0.00 0.00 3.43 0.18 55.92 0.00 0.07 46.95 (Ν mutant 30 0.00 0.00 0.00 0.00 3.11 0.18 58.52 0.00 0.00 38.16 0.82 Mutant 29 0.00 0.00 0.00 0.00 3.10 0.18 57.96 0.00 0.00 38.60 0.37 Mutant 27 0.00 0.00 0.00 0.00 3.05 0.19 57.46 0.00 0.00 38.86 0.39 Mutant 26 0.00 0.00 0.00 0.00 3.11 0.18 57.46 0.00 0.00 43.50 0.42 Mutant 24 0.00 0.00 0.00 0.00 3.10 0.16 57.03 0.00 0.08 47.80 0.45 Control ATCC PTA-9695 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 Fatty Acid CN (N 22:2 22:3 22:4 n-6 22: 5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 24:1 Fat unknown 201200591 n^^w^^^^s696-vlduuHv#gg^«^:s3< Mutant 44 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.15 0.00 1.75 0.00 0.50 30.18 0.22 0.00 0.00 0.13 0.10 0.00 0.00 0.00 0.00 0.00 0.40 0.49 0.00 0.00 0.00 0.00 0.23 2.80 0.08 Mutant 43 0.00 0.00 0.00 0.00 0.00 0.12 0.00 0.08 0.00 2.17 0.00 0.62 43.37 s 0.00 0.00 0.26 2.21 1 0.09 0.05 0.00 0.00 0.00 0.00 0.61 0.15 0.00 0.00 0.00 0.00 0.35 3.47 0.14 Mutant 42 0.00 0.00 0.00 0.00 0.00 0.08 0.00 0.12 0.00 0.00 0.36 25.77 0.10 0.00 0.00 0.10 (N 0.12 0.00 0.00 0.00 0.00 0.00 0.36 0.15 0.00 0.00 0.00 0.00 0.30 3.21 0.07 Mutant 40 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.24 2.80 0.08 Mutant 39 0.00 0.00 0.00 0.00 0.00 〇0.00 0.12 0.00 Cs 0.00 0.54 30.84 0.22 0.00 0.00 0.16 1.30 0.09 0.00 0.00 0.00 0.00 0.00 0.3 0.00 0.00 0.00 0.00 0.00 0.007 7.72 0.07 Mutant 38 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1 0.00 0.24 0.00 0.00 0.71 32.28 0.23 0.00 0.00 0.28 Os 0.25 0.00 0.00 0.00 0.00 0.00 0.43 0.43 0.00 0.00 0.00 0.00 0.24 4.00 0.14 Mutant 37 0.00 0.00 0.00 0.00 0.00 0.00 0.09 0.00 O Os 0.00 0.48 28.04 0.26 0.00 0.00 0.13 U1 0.08 0.00 0.00 0.06 0.00 0.00 0.31 0.00 0.00 0.00 0.00 0.00. 31 2.77 0.07 Mutant 36 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.44 0.00 (N 0.00 2.12 26.95 0.00 0.00 0.00 0.95 00 ! 0.37 0.00 0.00 0.00 0.00 0.00 0.34 0.00 0.00 0.00 0.00 0.001 5.36 0.00 Control 0.00CC ΡΤA-9695 0.00 0.00 0.00 0.00 0.00 0.10 0.00 0.11 0.00 1.79 i 0.00 0.00 30.98 0.27 0.00 0.00 0.12 1.29 ! 0.00 0.00 0.00 0.00 0.00 0.00 0.3 0.00 0.00 0.00 0.00 0.37 0.55 2.62 0.08 Fatty acid 08:0 09:0 10:0 11:0 12:0 12:1 13:0 13:1 14:0 14:1 j 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18 :3 n-6 18:3 n-3 18:4 n-3 20:0 20:1 n-9 20:2 20:3 n-9 20:3 n-6 20:3 n-3 20:4 ARA 20:5 n-3 EPA 22:0 % £: 151 201200591 Forgot? Fs(n<mutant 44 0.00 0.00 0.00 0.00 3.33 0.17 56.76 0.00 0.09 41.20 1.20 Mutant 43 0.00 0.00 0.00 0.00 2.37 0.33 41.61 0.00 0.06 56.44 0.90 Mutant 42 0.00 0.00 0.00 0.00 3.89 0.30 60.99 0.00 0.08 42.48 1.31 Mutant 40 0.00 0.00 0.00 0.00 3.29 0.17 57.09 0.00 0.09 42.59 1.20 Mutant 39 0.00 0.00 0.00 0.00 3.48 0.17 56.24 0.00 0.08 45.74 00 (N mutant 38 1 . 0.00 0.00 0.00 0.00 2.57 0.00 54.20 0.00 0.00 18.08 0.73 Mutant 37 1 0.00 0.00 0.00 0.06 3.94 0.19 58.57 0.00 0.08 54.86 1.36 Mutant 36 ! ! 0.00 0.00 0.00 0.00 2.40 0.00 57.52 0.00 0.00 12.73 0.29 Control ATCC PTA-9695 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 Fatty Acid 22:1 22:2 22:3 22 :4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 Fat Unknown 201200591 韬翱钬wg696-vHd^^^^331v 堀涸堀涸镩镩:9<n<

突變體52 0.00 0.00 0.00 0.00 0.00 〇 0.00 0.09 0.00 1.85 0.00 0.53 32.53 0.22 0.00 0.00 0.15 1.35 0.06 0.00 0.00 0.00 0.00 0.00 0.41 0.19 0.00 0.00 0.00 0.00 0.24 2.44 突變體51 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.10 0.00 〇〇 0.00 0.53 30.79 0.21 0.00 | 〇.〇〇 0.18 1.48 0.09 0.00 0.00 0.00 0.00 0.00 0.40 0.16 0.00 0.00 0.00 0.00 0.24 2.94 突變體50 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.13 0.00 00 0.00 0.55 30.46 0.18 0.00 0.00 0.17 VO rn 1 0.10 0.00 0.00 0.00 0.00 0.00 0.39 0.19 0.00 0.00 0.00 0.00 0.22 2.71 突變體49 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.09 0.00 (Ν 'sO 0.00 0.48 31.21 0.17 0.00 0.00 ! 0.14 0.08 0.00 0.00 0.00 0.00 0.00 0.42 0.13 0.00 0.00 0.00 0.00 0.21 2.46 突變體48 0.00 0.00 0.00 0.00 0.00 0.07 0.00 〇 0.00 1.52 0.00 0.46 29.07 0.17 0.00 0.00 0.12 0.09 0.00 0.00 0.00 0.00 0.00 0.42 0.24 0.00 0.00 0.00 0.00 0.18 2.93 突變體47 0.00 0.00 0.00 0.00 0.00 0.11 0.00 0.09 0.00 so 00 0.00 0.57 30.07 0.18 0.00 0.00 0.16 00 (N 0.09 0.00 0.00 0.05 0.00 0.00 0.37 〇 0.00 0.00 0.00 0.00 0.26 2.66 突變體46 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.10 0.00 2.07 0.00 0.76 24.90 0.24 0.00 0.00 0.24 0.07 0.00 0.00 0.12 0.00 0.00 0.29 0.13 0.00 0.00 0.05 0.12 0.65 4.28 突變體45 0.00 0.00 0.00 0.00 0.00 0.10 0.00 〇 0.00 0.00 0.41 28.79 0.19 0.00 0.00 〇 0.08 0.00 0.00 0.00 0.00 0.00 0.36 0.15 0.00 0.00 0.00 0.00 0.29 3.05 對照 ATCC ΡΤA-9695 0.00 0.00 0.00 0.00 0.00 0.10 I 0.00 〇 0.00 On 0.00 0.00 30.98 0.27 0.00 0.00 0.12 1.29 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.55 2.62 脂肪酸 08:0 09:0 10:0 〇 11:1 12:0 12:1 13:0 13:1 14:0 14:1 ! 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18:3 n-6 18:3 n-3 18:4 n-3 20:0 20:1 n-9 20:2 20:3 n-9 20:3 n-6 20:3 n-3 20:4 ARA 20:5 n-3 EPA 153 201200591 客碥？F9(N< 突變體52 0.08 0.00 0.00 0.00 0.00 3.24 0.17 55.06 0.00 0.07 51.93 1.19 突變體51 0.08 0.00 0.00 0.00 0.00 3.18 0.17 56.19 0.00 0.10 43.01 00 (Ν 突變體50 0.08 0.00 0.00 0.00 0.00 3.17 0.16 56.83 0.00 0.10 44.93 1.31 突變體49 0.09 0.00 0.00 0.00 0.00 3.32 0.17 56.45 0.00 〇 48.60 1.35 突變體48 0.09 0.00 0.00 0.00 0.00 3.07 0.17 58.65 0.00 0.10 35.41 g 突變體47 1 ___ ! 0.07 0.00 0.00 0.00 0.05 3.46 0.18 56.70 0.00 0.10 54.80 卜突變體46 0.06 0.00 0.00 0.00 0.07 4.28 0.27 58.32 0.00 0.15 58.95 突變體45 0.07 0.00 0.00 0.00 0.06 3.59 0.25 57.74 0.00 0.07 48.91 對照 ATCC PTA-9695 0.08 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 脂肪酸 22:0 (N 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 脂肪未知 201200591 突變體65 I 0*00 | 丨 0.00 | | 0.00 | I 0.00 | I 0.00 | | 0.08 | | 0.00 | I ο-m | I 0.00 | 00 I 0.00 | 1 0.48 1 1 30.03 1 1 0.25 1 I 0.00 | 1 0.00 I 1_0J31 1L35_I | 0.07 | 0.00 | 0.00 | 0.00 I 0.00 | 0.00 | 0.41 | 0.21 | 0.00 | 0.00 0.00 | 0.00 | 0.19 | 2.82 0.09 | 突變體61 I 0.00 I 0.00 | 0.00 | 0.00 I 0.00 | 0.10 | 0.00 I 0.09 I 0.00 00 I 0.00 | 1 0.51 1 1 31.15 1 1 0.26 1 I 0.00 | 1 0.00 I 1^〇J4_1 | 0.07 | 0.00 | I 0.00 | 0.00 I 0.00 | 0.00 | 0.39 | 0.13 | 0.00 | 0.00 0.00 | 0.00 | 0.24 | 2.59 0.08 | 突變體60 I 0.00 | 0.00 | 0.00 丨 0.00 I 0.00 | 0.08 | 0.00 | 0.14 I 0.00 〇\ I 0.00 ! 1 0.50 1 1 29.39 1 1 0.22 1 I 0.00 | 1 0.00 I 1_0J31 ! 0.10 | 0.00 | 0.00 | 0.00 1 0.00 | 0.00 | 0.39 | 0.14 | 0.00 | 0.00 0.00 | 0.00 | 0.20 | 2.72 0.08 突變體58 I 0.00 | 0.00 | 0.00 | 0.00 I 0.00 | 0.08 | 0.00 | 0.16 I 0.00 Os to I 0.00 | 1_M5_1 1 30.65 ] 1 0.25 1 I 0.00 | 1 0.00 I !_0J2_1 | 0.09 | I 0.00 | I 0.00 | 1 0.00 1 ! o.oo | 0.00 | 0.43 | 0.17 | 0.00 | 0.00 0.00 | 0.00 | 0.51 | 5.76 0.09 突變體57 I 0.00 1 0.00 I 0.00 | 0.00 I 0.00 | 0.08 | 0.00 | 0.13 I 0.00 On I 0.00 1 0.52 1 1 29.37 1 10^4I I 0.00 | 1 0.00 I 1 0.16 1 1 L37_1 o.ll 1 0.00 | 0.00 | 0.00 I 0.00 | 0.00 | 0.38 | 0.20 | 0.00 | 0.00 0.00 | 0.00 | 0.25 | 2.78 0.08 突變體56 I 0.00 1 0.00 | 0.00 | 0.00 I 0.00 | 0.08 | 0.00 I 0.09 I 0.00 ί v〇 r—^ | 0.00 | 1 0.51 1 1 31.08 1 I 0.26 | I 0.00 | 1 0.00 I 0^4_1 00 rn I 0.06 | I 0.00 | ! 0.00 | 0.00 1 0.00 | 0.00 | 0.42 | 0.19 | 0.00 | 0.00 0.00 | 0.00 | 0.22 | 2.67 0.09 突變體55 I 0.00 I 0.00 I 0.00 I 0.00 I 0.00 | 0.12 | 0.00 | 0.08 | 0.00 1_2J3_I I 0.00 | 1 0.48 1 1 33.01 1 | 0.26 | I 0.00 | 1 0.00 I 0.14 | ! 1.37 I 0.00 | 0.00 | 0.00 | 0.00 I 0.00 | 0.00 | 0.40 | 0.13 | 0.00 | 0.00 0.00 | 0.00 | 0.26 | 2.81 0.08 | 突變體54 | 0.00 1 0.00 I 0.00 I 0.00 I 0.00 ! | 0.08 | I 0.00 I 1_0^2_I I 0.00 1 Γ^ί VO I 0.00 | I 0.52 J 1 29.54 1 | 0.23 | I 0.00 | 1 0.00 I 1 0.14 I 0.08 I 0.00 1 0.00 1 0.00 I 0.00 | 0.00 1 0.39 I 0.16 J 0.00 1 0.00 0.00 1 0.00 1 0.21 | 2.78 0.08 突變體53 | 0.00 I 0.00 1 0.00 | 0.00 I 0.00 I 0.09 I 0.00 j d I 0.00 1 1 1-14_1 I 0.00 | I 0.53 I 1 30.13 1 0.21 | ί 0.00 | 0.00 I 0.15 1 0.08 I 0.00 1 0.00 1 0.00 1 0.00 I 0.00 1 0.38 1 0.19 I 0.00 1 0.00 0.00 1 0.00 1 0.25 I 2.75 0.08 對照 ATCC PTA-9695 | 0.00 | 0.00 I 0.00 1 0.00 丨 0.00 I 0.10 , | 0.00 1 〇 I 0.00 1 | 1.79 _ I I 0.00 | | 0.00 1 1 30.98 1 I 0.27 I I 0.00 1 I 0.00 I 0.12 I as (N 0.00 I 0.00 1 0.00 1 0.00 1 0.00 1 0.00 1 0.39 I 0.00 1 0.00 1 0.00 0.00 1 0.37 1 0.55 I 2.62 0.08 脂肪酸 08:0 09:0 10:0 〇 12:0 12:1 13:0 ， 13:1 1 14:0 I 14:1 I 15:1 1 16:0 1 16:1 I 16:2 I 16:3 I 17:0 I 18:0 I 18:1 n-9 I 18:1 n-7 I 18:2 1 18:3 n-6 1 18:3 n-3 I 18:4 n-3 I 20:0 I 20:1 n-9 I 20:2 1 20:3 n-9 20:3 n-6 I 20:3 n-3 I 20:4 ARA I 20:5 n-3 EPA 22:0 155 201200591 ;«?FI>3^ 突變體65 0.00 0.00 | 0.00 | 0.00 3.18 0.17 57.56 0.00 0.08 36.21 1.17 突變體61 0.00 0.00 0.00 0.06 3.42 1_Oil_1 55.91 1 0.00 I 0.07 50.40 1.48 突變體60 0.00 0.00 0.00 0.00 3.30 1_m_i 58.21 1 0.00 I 0.10 36.97 突變體58 0.00 0.00 0.00 0.00 2.62 0.59 54.09 0.00 0.11 33.96 0.77 突變體57 0.00 0.00 0.00 0.00 3.43 0.20 57.76 0.00 0.09 43.97 1.35 突變體56 0.00 0.00 0.00 0.00 3.19 0.17 56.38 0.00 0.10 41.92 Os (N 突變體55 0.00 0.00 0.00 0.00 3.25 0.17 1 54.04 1 0.00 0.07 48.81 1.19 突變體54 0.00 0.00 0.00 0.00 3.20 0.18 58.07 0.00 0.09 39.59 1.19 突變體53 0.00 0.00 0.00 0.00 3.47 0.18 56.99 0.00 0.09 I 45.83 I 00 CN 對照 ATCC PTA-9695 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 脂肪酸 CN (N 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 脂肪 %未知 201200591 «瀚钬 ws696-vHdt^^-哞 3U1V 卷涸涸嘟麵镩：8<n< 突變體74 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.05 0.00 0.47 ,23.95 0.00 0.00 0.00 0.00 S 0.00 0.00 0.00 1 0.00 0.22 0.00 0.27 0.00 突變體73 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.97 0.00 0.50 24.50 0.00 0.00 0.00 1 〇.〇〇 1.11 0.00 0.00 o.oo 0.00 0.22 0.00 0.29 0.00 突變體72 ATCC PTA-9698 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.00 0.00 2.29 0.00 0.40 ,33.49 __0^1__ 0.00 0.00 0.13 ! 2.24 0.00 0.00 0.00 1 0.00 0.16 0.00 0.52 0.00 突變體71 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.22 2.27 [0.19 0.46 ,28.89 0.19 0.00 0.00 0.00 1.51 0.00 0.00 0.00 1 0.00 0.22 0.00 ' 0.44 0.00 突變體70 ATCC PTA-9697 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.21 0.00 0.44 28.14 0.00 0.00 0.00 0.00 1.49 0.00 0.00 0.00 1 0.00 0.17 0.00 i 0.37 0.00 突變體69 0.00 0.00 0.00 0.00 | 0.00 0.00 0.00 0.00 0.00 2J1__I 0.00 0.47 t 27.79 | 0.00 0.00 0.00 0.00 1.44 ! o.oo ! 0.00 0.00 I 0.00 0.19 0.00 0.35 0.00 突變體68 ATCC PTA-9696 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.09 0.00 0.47 ,25.84 0.00 0.00 0.00 0.00 CM <N 0.00 0.00 0.00 I 0.00 0.21 0.00 0.31 0.00 突變體67 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.07 0.00 0.48 26.20 0.00 0.00 0.00 0.00 (N (N 0.00 0.00 0.00 I 0.00 0.20 0.00 0.32 0.00 突變體66 , 1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.29 0.00 0.47 ；31.02 0.19 0.00 0.00 0.00 oo vo 0.00 0.00 0.00 I 0.00 0.18 0.00 0.41 0.00 對照 ATCC PTA-9695 0.00 0.00 0.00 0.00 0.00 0.15 0.00 0.00 0.00 2.42 0.00 0.55 39.19 0.43 0.00 0.00 0.16 1.67 0.00 1 o.oo 0.00 I 0.00 0.00 0.00 0.49 0.00 脂肪酸 08:0 09:0 10:0 11:0 1 11:1 12:0 12:1 13:0 13:1 14:0 14:1 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18:3 n-6 18:3 n-3 18:4 n-3 20:0 20:1 n-9 157 201200591 銮稱？F8(N< 突變體74 0.00 0.00 0.00 0.00 0.39 4.53 0.34 0.00 ! 〇.〇〇 0.00 ! 〇.〇〇 ! 3.19 ! 0.57 62.23 0.00 0.00 46.63 0.76 突變體73 0.00 0.00 0.00 0.00 0.38 4.43 0.35 0.00 0.00 0.00 ! 〇.〇〇 I 3.17 | 0.48 61.83 0.00 0.00 ! 48.27 0.78 突變體72 ATCC PTA-9698 0.00 0.00 0.00 0.00 0.27 2.74 0.12 0.00 0.00 0.00 0.00 2.79 0.27 53.06 0.00 0.00 63.40 〇〇突變體71 0.00 0.00 0.00 0.00 0.30 3.09 0.43 0.00 0.00 0.00 0.00 3.46 0.25 55.62 0.00 0.00 40.38 2.46 突變體70 ATCC PTA-9697 0.00 0.00 0.00 0.00 0.37 4.12 0.38 0.00 0.00 0.00 0.00 2.95 0.61 58.03 0.00 0.00 53.65 0.73 突變體69 0.00 0.00 0.15 0.00 0.24 3.32 0.48 0.00 0.00 0.00 0.00 2.72 0.34 59.74 0.00 0.00 48.80 0.67 突變體68 ATCC PTA-9696 0.00 0.00 0.00 0.00 0.27 I 3.97 0.44 0.00 0.00 0.00 0.00 2.66 0.42 61.42 0.00 0.00 49.49 0.66 突變體67 0.00 0.00 0.00 0.00 0.33 3.86 0.35 0.00 0.00 0.00 0.00 3.17 0.46 60.60 0.00 0.00 48.51 0.73 突變體66 0.00 0.00 0.00 0.00 0.16 2.30 0.46 0.00 0.00 0.00 ! 0.00 2.83 0.18 57.01 0.00 0.00 49.32 0.82 對照ATCC PTA-9695 0.00 0.00 0.00 0.00 0.18 0.33 0.00 0.00 0.00 0.00 2.62 0.18 49.52 0.00 0.00 52.70 0.35 脂肪酸 20:2 20:3 n-9 20:3 n-6 20:3 n-3 20:4 ARA 20:5 n-3 EPA 22:0 CN (N 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 24:1 脂肪 %未知 158 201200591 實施例34 微生物之單離在低潮期間，自包括沿著北美洲西海岸（加州、俄勒尚州及華盛頓州）及夏威夷的海灣與潮口之潮間帶棲息地收集樣品。將水、沈積物、活的植物材料及腐化的植物/動物碎屑置於無菌的50毫升管中。將各樣品的部分連同水塗佈在單離培養基之固態瓊脂平板上。單離培養基係由：5〇〇毫升的人造海水、500毫升的蒸餾水、i克葡萄糖、甘油、 13克瓊脂、1克麩胺酸鹽、〇.5克酵母萃取物、〇 5克酪蛋白水解產物、1毫升維生素溶液(1〇0毫克/升硫胺素、〇 5毫克/ 升生物素、0.5毫克B,2)、1毫升微量礦物溶液(ρπ金屬，每公升含：6.0 克 FeCl36H20、6_84 克 Η3Β〇3、0.86 克 MnCl24H20、0.06 克 ZnCl2、0.026 克 CoC126H20、0.052 克 NiS04H20、0.002克CuS045H20及0.005克Na2Mo042H20)，以及500毫克各青黴素G及硫酸鏈黴素所組成。於2〇_25。(：下在黑暗中孵育該等瓊脂平板。2至4天後，在放大倍數下檢查該等瓊脂平板，並以無菌牙籤剔取細胞之菌落並在新鮮的培養基平板上重複劃線。將細胞重複地在新鮮培養基上劃線，直到移除已污染的微生物為止。經單離的微生物中之二種係根據ATCC存取號碼PTA-10212與PTA-10208予以寄存。根據ATCC存取號碼PTA-10212所寄存之經單離的微生物之分類特徵根據ATCC存取號碼PTA-10212 ("PTA-10212”)所寄存 159 201200591 之經單離的微生物之培養物呈現為白色、濕的、塗抹菌落形式，而無可見的分離孢子囊群。 PTA-10212係生長在固態與液態FFM、固態KMV、KMV 泥（1%)、KMV肉湯，以及MH肉湯中以進一步檢視生長特徵。觀察到PTA-10212在KMV與MH上生長快速。參見，例如 ’ Porter D.，1989. Phylum Labyrinthulomycota. In Margulis, L., Corliss, J.O., Melkonian, M., Chapman, D.J. (Eds.) Handbook of Protoctista, Jones and Bartlett, Boston, pp. 388-398 (KMV) ; Honda等人，Myco/· iiej· 102:439-448 (1998)(MH);以及美國專利案號5，130,242(FFM)。 PTA-l〇212在固態FFM培養基生長數曰之後、在KMV 培養基與ΜΗ肉湯中生長72小時之後，進行下列觀察。孢子囊並未群集在任一培養基之中或之上，及孢子囊非常地小 (5至10微米）。ΡΤΑ-1〇212並未展現裂殖壺菌屬分裂型之大量的四分體特徵。在轉移至新鮮的固態培養基約24小時之後，出現變形蟲狀細胞。此等變形蟲狀細胞在數日之後消失且在液態或泥狀培養基中不明顯。不同於Yokoyama，R.等人於Μ少codewce 48(6): 329-341 (2007)所述之當生長於液態培養基時具有"位於燒瓶底部的小型沙粒"外觀之如;-<2”"〇(：/2>^1//72，？丁八-10212並不沉降至燒瓶底部而是懸浮於KMV與MH液態培養基中。抱子囊的稍密度係不如裂殖壺菌屬（Schizochytrium')或謂典型所具有者，其等亦具有pTA_1〇212中所缺乏之強健的外質網絡。當大部分的物種在數小時期間藉由 160 201200591 較大型孢子囊的分裂而進行小孢子囊或同化細胞之營養分裂時’ PTA-10212形成啞鈴形狀的拉長型同化細胞，其然後形成一薄的峽部及當啞鈴的終端分開時被拉斷。所生成的細胞似乎為小型同化細胞。未觀察到變形蟲狀細胞直接轉形成σ亞鈐形狀的同化細胞。觀察到典型的二鞭毛遊走抱子游動，但相當地少》ΡΤΑ_10212並無繁殖力，其藉由營養分裂而分裂。雖然觀察到遊走孢子游動，但未觀察到直接釋出遊走孢子。營養細胞係非常小的（2微米至5微米）。使用流通技術進一步檢視ΡΤΑ_丨〇2丨2，其中藉由將一小刀之生長於瓊脂的菌落懸浮於一滴的半強度海水中而製備顯微鏡載玻片。藉由該技術’觀察到初生孢子囊為球狀及直徑大概10微米。壁非常薄以及當原生質體的均體分裂起始時並未觀察到殘跡。重複的均體分裂產生8至16個較小 (直徑4至5微米）的次生抱子囊。次生孢子囊維持靜息達數小時’然後再度釋出-種無定形的原生質體。無定形原生質體藉由收聚與牽引作用來分裂，最減生典型㈣鈴狀中間階段以及最後產生4-8個直徑2.5-2.8微米的小型球體。後者休止數分鐘至1_2小時’錢改變形狀(拉長)及變成二鞭毛的遊走孢子’ 2.3.2.5 X 3,7_3 9微米。遊走抱子的數量幕多’及當其等休止時可精確測量。遊走抱子接而圓滿完成大且比維蘇爾傑西斯巫肯尼亞菌（C7⑹心 visurgensis)^ 0 PTA-10212係以其之l8s rRNA基因與已知物種的相似 161 201200591 性為基礎予以進一步特徵化。基因體DNA係藉由標準程序而自PTA-10212來製備。見，舉例而言，Sambrook J. andMutant 52 0.00 0.00 0.00 0.00 0.00 〇0.00 0.09 0.00 1.85 0.00 0.53 32.53 0.22 0.00 0.00 0.15 1.35 0.06 0.00 0.00 0.00 0.00 0.001 0.19 0.00 0.00 0.00 0.00 0.24 2.44 Mutant 51 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.10 0.00 〇〇0.00 0.53 30.79 0.21 0.00 | 〇.〇〇0.18 1.48 0.09 0.00 0.00 0.00 0.00 0.00 0.40 0.16 0.00 0.00 0.00 0.00 0.24 2.94 Mutant 50 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.13 0.00 00 0.00 0.55 30.46 0.18 0.00 0.00 0.17 VO rn 1 0.10 0.00 0.00 0.00 0.00 0.00 0.39 0.29 0.00 0.00 0.00 0.00 0.22 2.71 Mutant 49 0.00 0.00 0.00 0.00 0.00 0.09 0.00 0.09 0.00 (Ν 'sO 0.00 0.48 31.21 0.17 0.00 0.00 ! 0.14 0.08 0.00 0.00 0.00 0.00 0.002 0.13 0.00 0.00 0.00 0.00 0.21 2.46 Mutant 48 0.00 0.00 0.00 0.00 0.00 0.07 0.00 〇0.00 1.52 0.00 0.46 29.07 0.17 0.00 0.00 0.12 0.09 0.00 0.00 0.00 0.00 0.002 0.24 0.00 0.00 0.00 0.00 0.18 2.93 Mutant 47 0.00 0.00 0.00 0.00 0.00 0.11 0.00 0.09 0.00 so 00 0.00 0.57 30.07 0.18 0.00 0.00 0.16 00 (N 0.09 0.00 0.00 0.05 0.00 0.00 0.37 〇0.00 0.00 0.00 0.00 0.26 2.66 Mutant 46 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.10 0.00 2.07 0.00 0.76 24.90 0.24 0.00 0.00 0.24 0.07 0.00 0.00 0.12 0.00 0.00 0.29 0.13 0.00 0.00 0.05 0.12 0.65 4.28 Mutant 45 0.00 0.00 0.00 0.00 0.00 0.10 0.00 〇0.00 0.00 0.41 28.79 0.19 0.00 0.00 〇0.08 0.00 0.00 0.00 0.00 0.00 0.36 0.15 0.00 0.00 0.00 0.00 0.29 3.05 Control ATCC ΡΤA-9695 0.00 0.00 0.00 0.00 0.00 0.10 I 0.00 〇0.00 On 0.00 0.00 30.98 0.27 0.00 0.00 0.12 1.29 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.00 0.00 0.00 0.00 0.37 0.55 2.62 Fatty acid 08:0 09:0 10:0 〇11:1 12:0 12:1 13:0 13:1 14:0 14:1 ! 15: 1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18:3 n-6 18:3 n-3 18:4 n -3 20:0 20:1 n-9 20:2 20:3 n-9 20:3 n-6 20:3 n-3 20:4 ARA 20:5 n-3 EPA 153 201200591 Customer? F9 (N< mutant 52 0.08 0.00 0.00 0.00 0.00 3.24 0.17 55.06 0.00 0.07 51.93 1.19 Mutant 51 0.08 0.00 0.00 0.00 0.00 3.18 0.17 56.19 0.00 0.10 43.01 00 (Ν Mutant 50 0.08 0.00 0.00 0.00 0.00 3.17 0.16 56.83 0.00 0.10 44.93 1.31 Mutant 49 0.09 0.00 0.00 0.00 0.00 3.32 0.17 56.45 0.00 〇48.60 1.35 Mutant 48 0.09 0.00 0.00 0.00 0.00 3.07 0.17 58.65 0.00 0.10 35.41 g Mutant 47 1 ___ ! 0.07 0.00 0.00 0.00 0.05 3.46 0.18 56.70 0.00 0.10 54.80 Body 46 0.06 0.00 0.00 0.00 0.07 4.28 0.27 58.32 0.00 0.15 58.95 Mutant 45 0.07 0.00 0.00 0.00 0.06 3.59 0.25 57.74 0.00 0.07 48.91 Control ATCC PTA-9695 0.08 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 Fatty Acid 22:0 (N 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 Fat unknown 201200591 Mutant 65 I 0*00 | 丨0.00 | | 0.00 I 0.00 | I 0.00 | | 0.08 | | 0.00 | I ο-m | I 0.00 | 00 I 0.00 | 1 0.48 1 1 30.03 1 1 0.25 1 I 0.00 | 1 0.00 I 1_0J31 1L35_I | 0.07 | 0.00 | 0.00 | I 0.00 | 0.00 | 0 .41 | 0.21 | 0.00 | 0.00 0.00 | 0.00 | 0.19 | 2.82 0.09 | Mutant 61 I 0.00 I 0.00 | 0.00 | 0.00 I 0.00 | 0.10 | 0.00 I 0.09 I 0.00 00 I 0.00 | 1 0.51 1 1 31.15 1 1 0.26 1 I 0.00 | 1 0.00 I 1^〇J4_1 | 0.07 | 0.00 | I 0.00 | 0.00 I 0.00 | 0.00 | 0.39 | 0.13 | 0.00 | 0.00 0.00 | 0.00 | 0.24 | 2.59 0.08 | Mutant 60 I 0.00 | 0.00 |丨0.00 I 0.00 | 0.08 | 0.00 | 0.14 I 0.00 〇\ I 0.00 ! 1 0.50 1 1 29.39 1 1 0.22 1 I 0.00 | 1 0.00 I 1_0J31 ! 0.10 | 0.00 | 0.00 | 0.00 1 0.00 | 0.00 | 0.39 | 0.00 | 0.00 0.00 | 0.00 | 0.20 | 2.72 0.08 Mutant 58 I 0.00 | 0.00 | 0.00 | 0.00 I 0.00 | 0.08 | 0.00 | 0.16 I 0.00 Os to I 0.00 | 1_M5_1 1 30.65 ] 1 0.25 1 I 0.00 | !_0J2_1 | 0.09 | I 0.00 | I 0.00 | 1 0.00 1 ! o.oo | 0.00 | 0.43 | 0.17 | 0.00 | 0.00 0.00 | 0.00 | 0.51 | 5.76 0.09 Mutant 57 I 0.00 1 0.00 I 0.00 | 0.00 I 0.00 | 0.08 | 0.00 | 0.13 I 0.00 On I 0.00 1 0.52 1 1 29.37 1 10^4I I 0.00 | 1 0.00 I 1 0.16 1 1 L37_1 o.ll 1 0.00 | 0.00 | 0.00 I 0.00 | 0.00 | 0.20 | 0.00 | 0.00 0.00 | 0.00 | 0.25 | 2.78 0.08 Mutant 56 I 0.00 1 0.00 | 0.00 | 0.00 I 0.00 | 0.08 | 0.00 I 0.09 I 0.00 ί v〇r—^ | 0.00 | 1 0.51 1 1 31.08 1 I 0.26 | I 0.00 | 1 0.00 I 0^4_1 00 rn I 0.06 | I 0.00 | ! 0.00 | 0.00 1 0.00 | 0.00 | 0.42 | 0.19 | 0.00 | 0.00 0.00 | 0.00 | 0.22 | 2.67 0.09 Mutant 55 I 0.00 I 0.00 I 0.00 I 0.00 I 0.00 | 0.12 | 0.00 | 0.08 | 0.00 1_2J3_I I 0.00 | 1 0.48 1 1 33.01 1 | 0.26 | I 0.00 | 1 0.00 I 0.14 | ! 1.37 I 0.00 | 0.00 | 0.00 | 0.00 I 0.00 | 0.40 | 0.13 | 0.00 | 0.00 0.00 | 0.00 | 0.26 | 2.81 0.08 | Mutant 54 | 0.00 1 0.00 I 0.00 I 0.00 I 0.00 ! | 0.08 | I 0.00 I 1_0^2_I I 0.00 1 Γ^ί VO I 0.00 | I 0.52 J 1 29.54 1 | 0.23 | I 0.00 | 1 0.00 I 1 0.14 I 0.08 I 0.00 1 0.00 1 0.00 I 0.00 | 0.00 1 0.39 I 0.16 J 0.00 1 0.00 0.00 1 0.00 1 0.21 | 2.78 0.08 Mutant 53 | I 0.00 1 0.00 | 0.00 I 0.00 I 0.09 I 0.00 jd I 0.00 1 1 1-14_1 I 0.00 | I 0.53 I 1 30.13 1 0.21 | ί 0.00 | 0.00 I 0.15 1 0.08 I 0.00 1 0.00 1 0.00 1 0.00 I 0.00 1 0.38 1 0.19 I 0.00 1 0.00 0.00 1 0.00 1 0.25 I 2.75 0.08 Control ATCC PTA-9695 | 0.00 | 0.00 I 0.00 1 0.00 丨0.00 I 0.10 , | 0.00 1 〇I 0.00 1 | 1.79 _ II 0.00 | 0.00 1 1 30.98 1 I 0.27 II 0.00 1 I 0.00 I 0.12 I as (N 0.00 I 0.00 1 0.00 1 0.00 1 0.00 1 0.00 1 0.39 I 0.00 1 0.00 1 0.00 0.00 1 0.37 1 0.55 I 2.62 0.08 Fatty acid 08:0 09:0 10:0 〇12:0 12:1 13:0 , 13:1 1 14:0 I 14:1 I 15:1 1 16:0 1 16:1 I 16:2 I 16:3 I 17 :0 I 18:0 I 18:1 n-9 I 18:1 n-7 I 18:2 1 18:3 n-6 1 18:3 n-3 I 18:4 n-3 I 20:0 I 20:1 n-9 I 20:2 1 20:3 n-9 20:3 n-6 I 20:3 n-3 I 20:4 ARA I 20:5 n-3 EPA 22:0 155 201200591 ;« ?FI>3^ Mutant 65 0.00 0.00 | 0.00 | 0.00 3.18 0.17 57.56 0.00 0.08 36.21 1.17 Mutant 61 0.00 0.00 0.00 0.06 3.42 1_Oil_1 55.91 1 0.00 I 0.07 50.40 1.48 Mutant 60 0.00 0.00 0.00 0.00 3.30 1_m_i 58.21 1 0.00 I 0.10 36.97 Mutant 58 0.00 0.00 0.00 0.00 2.62 0.59 54.09 0.00 0.11 33.96 0.77 Mutant 57 0.00 0.00 0.00 0.00 3.43 0.20 57.76 0.00 0.09 43.97 1.35 Body 56 0.00 0.00 0.00 0.00 3.19 0.17 56.38 0.00 0.10 41.92 Os (N mutant 55 0.00 0.00 0.00 0.00 3.25 0.17 1 54.04 1 0.00 0.07 48.81 1.19 Mutant 54 0.00 0.00 0.00 0.00 3.20 0.18 58.07 0.00 0.09 39.59 1.19 Mutant 53 0.00 0.00 0.00 0.00 3.47 0.18 56.99 0.00 0.09 I 45.83 I 00 CN Control ATCC PTA-9695 0.00 0.00 0.00 0.00 3.19 0.18 56.88 0.00 0.00 46.83 0.85 Fatty Acids CN (N 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24:0 Fat%Unknown 201200591 «瀚钬ws696-vHdt^^-哞3U1V 涸涸涸涸镩:8<n< Mutant 74 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 O.oo 0.00 0.22 0.00 0.29 0.00 Mutant 72 ATCC PTA-9698 0.00 0.00 0.00 0.00 0.00 0.13 0.00 0.00 0.00 2.29 0.00 0.40 ,33.49 __0^1__ 0.00 0.00 0.13 ! 2.24 0.00 0.00 0.00 1 0.00 0.16 0.00 0.52 0.00 Mutant 71 0 .00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.22 2.27 [0.19 0.46 ,28.89 0.19 0.00 0.00 0.00 1.51 0.00 0.00 0.00 1 0.00 0.22 0.00 ' 0.44 0.00 Mutant 70 ATCC PTA-9697 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2. 0.00 0.00 0.44 28.14 0.00 0.00 0.00 0.00 1.49 0.00 0.00 0.00 1 0.00 0.17 0.00 i 0.37 0.00 Mutant 69 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2J1__I 0.00 0.47 t 27.79 | 0.00 0.00 0.00 0.00 1.44 ! o.oo ! 0.00 0.00 I 0.00 0.19 0.00 0.35 0.00 Mutant 68 ATCC PTA-9696 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.009 0.00 0.47 ,25.84 0.00 0.00 0.00 0.00 CM <N 0.00 0.00 0.00 0.00 0.00 0.001 0.00 0.31 0.00 Mutant 67 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.07 0.00 0.48 26.20 0.00 0.00 0.00 0.00 (N (N 0.00 0.00 0.00 I 0.00 0.20 0.00 0.32 0.00 Mutant 66 , 1 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.29 0.00 0.47; 31.02 0.19 0.00 0.00 0.00 oo vo 0.00 0.00 0.00 I 0.00 0.18 0.00 0.41 0.00 Control ATCC PTA-9695 0.00 0.00 0.00 0.00 0.00 0.15 0.00 0.00 0.00 2.42 0.00 0 .55 39.19 0.43 0.00 0.00 0.16 1.67 0.00 1 o.oo 0.00 I 0.00 0.00 0.00 0.49 0.00 Fatty acid 08:0 09:0 10:0 11:0 1 11:1 12:0 12:1 13:0 13:1 14 :0 14:1 15:1 16:0 16:1 16:2 16:3 17:0 18:0 18:1 n-9 18:1 n-7 18:2 18:3 n-6 18:3 N-3 18:4 n-3 20:0 20:1 n-9 157 201200591 Nickname? F8 (N< mutant 74 0.00 0.00 0.00 0.00 0.39 4.53 0.34 0.00 ! 〇.〇〇0.00 ! 〇.〇〇! 3.19 ! 0.57 62.23 0.00 0.00 46.63 0.76 Mutant 73 0.00 0.00 0.00 0.00 0.38 4.43 0.35 0.00 0.00 0.00 ! .〇〇I 3.17 | 0.48 61.83 0.00 0.00 ! 48.27 0.78 Mutant 72 ATCC PTA-9698 0.00 0.00 0.00 0.00 0.27 2.74 0.12 0.00 0.00 0.00 0.00 2.79 0.27 53.06 0.00 0.00 63.40 〇〇 mutant 71 0.00 0.00 0.00 0.00 0.30 3.09 0.43 0.00 0.00 0.00 0.00 3.46 0.25 55.62 0.00 0.00 40.38 2.46 Mutant 70 ATCC PTA-9697 0.00 0.00 0.00 0.00 0.37 4.12 0.38 0.00 0.00 0.00 0.00 2.95 0.61 58.03 0.00 0.00 53.65 0.73 Mutant 69 0.00 0.00 0.15 0.00 0.24 3.32 0.48 0.00 0.00 0.00 0.00 2.72 0.34 59.74 0.00 0.00 48.80 0.67 Mutant 68 ATCC PTA-9696 0.00 0.00 0.00 0.00 0.27 I 3.97 0.44 0.00 0.00 0.00 0.00 2.66 0.42 61.42 0.00 0.00 49.49 0.66 Mutant 67 0.00 0.00 0.00 0.00 0.33 3.86 0.35 0.00 0.00 0.00 0.00 3.17 0.46 60.60 0.00 0.00 48.51 0.73 Mutant 66 0.00 0.00 0.00 0.00 0.16 2.30 0.46 0.00 0.00 0.00 ! 0.00 2. 83 0.18 57.01 0.00 0.00 49.32 0.82 Control ATCC PTA-9695 0.00 0.00 0.00 0.00 0.18 0.33 0.00 0.00 0.00 0.00 2.62 0.18 49.52 0.00 0.00 52.70 0.35 Fatty acid 20:2 20:3 n-9 20:3 n-6 20:3 n- 3 20:4 ARA 20:5 n-3 EPA 22:0 CN (N 22:2 22:3 22:4 n-6 22:5 n-6 22:5 n-3 22:6 n-3 DHA 24 :0 24:1 fat % unknown 158 201200591 Example 34 Microorganisms inhabit during the low tide period, including the intertidal zone between the Gulf and the tidal zone along the west coast of North America (California, Oregon, and Washington) and Hawaii Samples were collected. Water, sediment, live plant material and spoiled plant/animal debris were placed in sterile 50 ml tubes. A portion of each sample was coated with water on a solid agar plate of the isolated medium. The isolation medium consists of: 5 ml of artificial seawater, 500 ml of distilled water, i g of glucose, glycerol, 13 g of agar, 1 g of glutamate, 5 g of yeast extract, and 5 g of casein hydrolyzed. Product, 1 ml of vitamin solution (1 〇 0 mg / liter of thiamine, 〇 5 mg / liter of biotin, 0.5 mg B, 2), 1 ml of trace mineral solution (ρπ metal, per liter: 6.0 g FeCl36H20, 6_84 Kezhen 3Β〇3, 0.86 g MnCl24H20, 0.06 g ZnCl2, 0.026 g CoC126H20, 0.052 g NiS04H20, 0.002 g CuS045H20 and 0.005 g Na2Mo042H20), and 500 mg each of penicillin G and streptomycin sulfate. At 2〇_25. (: Incubate the agar plates in the dark. After 2 to 4 days, inspect the agar plates at magnification, and pick up the colonies of the cells with a sterile toothpick and repeat the streaking on the fresh medium plate. The fresh medium is repeatedly streaked until the contaminated microorganisms are removed. Two of the isolated microorganisms are registered according to the ATCC access numbers PTA-10212 and PTA-10208. According to the ATCC access number PTA- The classified characteristics of the detached microorganisms registered in 10212 are white, wet, smear colonies according to the culture of the detached microorganisms registered 159 201200591 according to ATCC accession number PTA-10212 ("PTA-10212") Form, without visible separation of sporangia. PTA-10212 was grown in solid and liquid FFM, solid KMV, KMV mud (1%), KMV broth, and MH broth to further examine growth characteristics. PTA was observed. -10212 grows rapidly on KMV and MH. See, for example, 'Porter D., 1989. Phylum Labyrinthulomycota. In Margulis, L., Corliss, JO, Melkonian, M., Chapman, DJ (Eds.) Handbook of Protoctista, Jones and Bartlett, Boston, pp. 388-398 (KMV); Honda et al, Myco/. iiej. 102: 439-448 (1998) (MH); and U.S. Patent No. 5,130,242 (FFM). PTA- After the growth of the solid FFM medium for several hours, in the KMV medium and the broth for 72 hours, the following observations were made. The sporangia were not clustered in or on any medium, and the sporangia were very small ( 5 to 10 μm. ΡΤΑ-1〇212 does not exhibit a large number of tetrad characteristics of Schizochytrium genus. After about 24 hours of transfer to fresh solid medium, amoeba-like cells appear. The worm cells disappeared after a few days and were not apparent in the liquid or muddy medium. Unlike Yokoyama, R. et al., Μ少 codewce 48(6): 329-341 (2007), when grown in liquid medium When there is "small sand at the bottom of the flask"appearance;-<2""〇(:/2>^1//72,? D8-10212 does not settle to the bottom of the flask but is suspended In KMV and MH liquid medium. The slightly dense density of the sac is not as good as Schizochytrium or Who has type, which also has other lack of robustness in the missing pTA_1〇212 ectoplasm network. When most species undergo mitochondrial or assimilated cell vegetative division by splitting 160 201200591 larger sporangia during a few hours, 'PTA-10212 forms a dumbbell-shaped elongated assimilating cell, which then forms a thin The isthmus is pulled off when the terminal of the dumbbell is separated. The resulting cells appear to be small assimilated cells. It was not observed that the amoeba cells directly transformed into sigma-like assimilating cells. It was observed that the typical two-flagellate wandering wandering, but quite less, ΡΤΑ_10212 has no fecundity, which is split by nutrient splitting. Although the migration of the spores was observed, no direct release of the spores was observed. The vegetative cell line is very small (2 microns to 5 microns). Further investigation of ΡΤΑ_丨〇2丨2 was carried out using a flow-through technique in which a microscope slide was prepared by suspending a small knife-grown colony of agar in a drop of half-strength seawater. The primary sporangia were observed to be spherical and approximately 10 microns in diameter by this technique. The walls were very thin and no remnants were observed when the homogenous division of the protoplasts began. Repeated homogenous division produces 8 to 16 smaller (4 to 5 micrometers in diameter) secondary sacs. The secondary sporangia remain resting for a few hours and then re-release of the amorphous protoplast. Amorphous protoplasts divide by trapping and traction, minimizing the typical (four) bell-shaped intermediate stage and finally producing 4-8 small spheres of 2.5-2.8 microns in diameter. The latter rested for a few minutes to 1_2 hours. The money changed shape (elongated) and became a two-flagellated migratory spore '2.3.2.5 X 3,7_3 9 microns. The number of wandering cages is large and can be accurately measured when it is stopped. Wandering the sling and succeeding in the completion of the large and more than Visius Jessian witch Kenyan bacteria (C7 (6) heart visurgensis) ^ 0 PTA-10212 is further characterized by its similar l8s rRNA gene and known species 161 201200591. Genomic DNA was prepared from PTA-10212 by standard procedures. See, for example, Sambrook J. and

Russell D. 2001. Molecular cloning: A laboratory manual，第 3版。Cold Spring Harbor Laboratory Press, Cold Spring Harbor，New York。簡述為：（1)將來自對數中期培養物的500 微升細胞予以離心。將細胞再度離心，以及用小孔徑的管頭自細胞小丸移除所有的水；（2)用200微升的溶解緩衝液 (20mMTrispH8.0、125微克/毫升的蛋白酶K、50mM氣化鈉、10mMEDTApH8.0、0.5%SDS)來再懸浮小丸；（3)細胞在50°C溶解歷時1小時；（4)將溶解混合物量吸至2毫升的鎖相凝膠管（PLG-Eppendorf)中；（5)添加等體積的P : C : I 以及允許混合歷時1.5小時；（6)將該等管子以12,000 X g離心歷時5分鐘；（7)自PLG管内將凝膠上方的水相移除且將等體積的氣仿添加至該水相，以及混合歷時3〇分鐘；（8)將該等管子以14,000 X g離心歷時大概5分鐘；（9)自氣仿將頂層 (含水相）吸出，及置於一個新的管子中；（10)添加01體積的 3M NaOAC且加以混合（倒置數次）；（⑴添加2體積的 100%EtOH且加以混合(倒置數次），而基因體〇να沉澱物在本時期形成；（12)該等管子係在一微量離心機中於4〇c及以 14,000 X g離心歷時大概15分鐘；（13)將液體平緩地傾倒掉，及基因體DNA剩餘在該管子的底部；（14)以〇 5毫升的 70%乙醇來清洗小丸；（15)該等管子係在一微量離心機中於 4C及14,000 xg離心歷時大概5分鐘；（⑹將玢⑽平緩地傾倒掉，及乾燥基因體DNA小丸；以及（17)將一適宜體積的 162 201200591 水與RNase直接添加至基因體DNA的小丸。18s rRNA基因之聚合酶鏈反應擴增係以先前所述的引子來進行(Honda等人，·/. A/Vcro. 46(6): 637-647 (1999)。使用染色體DNA 模板之PCR條件如下：0.2 μΜ dNTPs、0.1 μΜ各引子、8% DMS0、200毫微克染色體DNA、2_5 U Herculase® II融合 DNA聚合酶（Stratagene)，以及Herculase®緩衝液（Stratagene) 配於50微升總體積。PCR操作程序包括下列步驟：（1)95°C 歷時2分鐘；（2)95°C歷時35秒；（3)55°C歷時35秒；（4)72°C 歷時1分鐘又30秒；（5)重複步驟2-4計30個循環；（6)72°C歷時5分鐘；以及(7)保持於4°C。使用以上所說明的染色體模板，PCR擴增出產具有預期的大小之有區別的DNA產物。PCR產物係依據製造者之指示而被選殖至載體pJET1.2/純的（Fermentas)之内，以及使用所提供的標準引子來決定插入序列。種系發生分析在中等的證明之下，將PTA-10212置於包括厚皮破囊壺菌（Thraustochytrium pachydefmum)與聚破囊壺菌（Thfaustochytrium aggregatum)之讀系內。厚皮破囊壺菌（7：的孢子囊具有非常厚的細胞壁。聚破囊壺菌（Γ· 形成明顯可見的不透明孢子囊群之群集。PTA-10212並未顯示此等特性中之任一者。在其他分類群’諸如巫肯尼亞菌、71· gaeriner/wm、尤，及紅樹林裂殖壺菌（& 中，曾述及存在眾多的變形蟲狀細胞；然而，與該等分類群相關聯之敘述說明係與該分離株所觀察到的特徵不同。並且， 163 201200591 PTA-10212並未顯現對於此等分類群中任一者之種系發生親和性。表29顯示來自根據ATCC存取號碼ΡΤΑ-10212所寄存的被生物之 18s rRNA序列與National Center for Biotechnology Information(NCBI)電子資料庫内的DNA序列之比較。使用二種不同的計算來測定百分比同一性。"計算#1"將出現在序列中之來自非同源區域或部份序列的任何"間隔”納入考量(AlignX-載體NTI預設設定）。，，計算#2”不包括間隔之計算罰分(AlignX_載體ΝΤΓ同一性，'矩陣設定）。表29: 18srRNA序列之比較破囊壺菌 %同一性計算#1 %同一性計算#2 厚皮破囊壺菌 85% 93% 聚破囊壺菌（P) 83% 92% Thraustochytrium gaertnerium 82% 92% 維蘇爾傑西斯巫肯尼亞菌 82% 92% 裂殖壺菌屬取-9695 80% 92% 红樹林裂殖壺菌 80% 91% 裂殖壺菌 M sp. ATCC 20888 80% 90% Aurantiochytrium limiacinum 79% 90% (P):表示部份序列如表29中所顯示的，已發現就％同一性而言，來自根據ATCC存取號碼PTA-1〇212所寄存的微生物之⑻姻八基因序列係與NCB!資料庫内可取得之l8s刪絲因序列二近’雖目同。-般認為在生物屬於不同的屬或種之際，仍可具有非常相近的18s rRNA基因序列。基於以上的特徵分析，該經單離的微生物（ATCC存取 164 201200591 號碼PTA-10212)據信代表一種新的破囊壺菌屬物種以及因而亦命名為破囊壺菌屬 (TVzraMsioc/z声η·Μ/« )«?/?. ATCCPTA-10212 〇根據ATCC存取號碼PTA-10208所寄存之經單離的微生物之分類特徵根據ATCC存取號碼PTA-10208所寄存的微生物 ("PTA-10208")係經認定為根據ATCC存取號碼PTA-9695所寄存的微生物（"PTA-9695”)之一種亞分離株（自一培養物單離出之一個別細胞及保有一種分離與獨特的培養物）， "PTA-9695"係描述於美國公開案號2010/0239533與國際公開案號W0 2010/107415之中。 PTA-10208具有PTA-9695的分類特徵。發現PTA-9695 於排放時有二鞭毛遊走孢子，其可活躍地自成熟的孢子囊游離，其之壁殘跡在釋出孢子之後仍清楚可見(在位相差）。測得PTA-9695孢子囊的直徑為12.5微米至25微米，以及遊走孢子的尺寸為2.5微米至2.8微米x4.5微米至4.8微米。每個個別的PTA-9695孢子囊具有8至24個孢子。穩定後的 PTA-9695遊走孢子增大及迅速進行均體分裂而形成四分體、八分體’及最後形成孢子囊聚簇。四分體形成作用係在孢子囊成熟前之非常早期的階段開始。此等特性係與裂殖壺菌屬OSc/n’zoc/y/iriw/w)相符。就％同一性而言，發現 PTA-10208所共有之PTA-9695的18s rRNA基因序列係與 Honda等人於，J.五政始46(6): 637-647 (1999)中所提供之聚破囊壺菌（Z aggregaiww)的18s rRNA基因序列非常 165 201200591 相近的，雖然並非一致。所發表之聚破囊壺菌 aggregaiwm)的 18s rRNA序列係一部份序列，在序列中間具有約71個DNA核苷酸之一個間隔。 PTA·9695據信代表一種新的裂殖壺菌屬物種。因此’亞分離株PTA_10208亦命名為裂殖壺菌屬物種 jp.) ATCCPTA-10208。實施例35 根據ATCC存取號碼PTA-10212所寄存之經單離的微生物之生長特徵該經單離的微生物（ATCC存取號碼pTA_ i 〇2丨2)係於以下所說明之個別的發酵操作中檢查生長特徵。典型的培養基與培養條件係顯示於表3 〇中。 166 201200591 表30 : PTA-10212器皿培養基成分濃度 Na2S〇4 g/L 31.0 NaCl g/L 0.625 KC1 g/L 1.0 MgS04-7H20 g/L 5.0 (NH4)2S04 g/L 0.44 MSG1H20 g/L 6.0 CaCl2 g/L 0.29 T 154(酵母萃取物） g/L 6.0 KH2P〇4 g/L 0.8 高壓蒸氣滅菌後(金屬）檸檬酸 mg/L 3.5 FeS04-7H20 mg/L 10.30 MnCl2.4H20 mg/L 3.10 ZnS047H20 mg/L 3.10 CoC12-6H20 mg/L 0.04 Na2Mo04-2H20 mg/L 0.04 CuS045H20 mg/L 2.07 NiS04_6H20 mg/L 2.07 高壓蒸氣滅菌後(維生素）硫胺素 mg/L 9.75 維生素B12 mg/L 0.16 CaA泛酸 mg/L 2.06 生物素 mg/L 3.21 高壓蒸氣滅菌後(碳）甘油 g/L 30.0 氮進料：成分濃度 MSG1H20 g/L 17 10-100，或 15-50 典型的培養條件會包括下列：範圍 0-50、15-45，或 25-35 0-25、0.1-10，或 0.5-5 0-5、0.25-3，或 0.5-2 0-10、2-8，或 3-6 0-10、0.25-5，或 0.05-3 0-10、4-8，或 5-7 0.1-5, 0.15-3，或 0.2-1 0-20、0.1-10，或 1-7 0.1-10、0.5-5，或 0.6-1.8 0.1-5000、10-3000，或 3-2500 0.1-100、1-50，或 5-25 0.1-100、1-50，或 2-25 0.01-100、1-50，或 2-25 0-1、0.001-0.1，或 0.01-0.1 0.001-1、0.005-0.5，或 0.01-0.1 0.1-100、0.5-50，或 1-25 0.1-100、0.5_50，或 1-25 0.1-100、1-50，或 5-25 0.01-100、0.05-5，或 0.1-1 0.1-100、0.1-50，或 1-10 0.1-100、0.1-50，或 1-10 5-150、10-100，或 20-50 0-150 、Russell D. 2001. Molecular cloning: A laboratory manual, 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Briefly stated: (1) Centrifuge 500 microliters of cells from a mid-log phase culture were centrifuged. Centrifuge the cells again and remove all water from the cell pellet with a small pore size tube; (2) use 200 μl of lysis buffer (20 mM Tris pH 8.0, 125 μg/ml proteinase K, 50 mM sodium sulphate, 10 mM EDTA pH 8.0, 0.5% SDS) to resuspend the pellets; (3) the cells were lysed at 50 ° C for 1 hour; (4) the amount of the dissolved mixture was aspirated into 2 ml of phase-locked gel tubes (PLG-Eppendorf); (5) Add an equal volume of P: C: I and allow mixing for 1.5 hours; (6) Centrifuge the tubes at 12,000 X g for 5 minutes; (7) Remove the aqueous phase above the gel from the PLG tube And adding an equal volume of gas to the aqueous phase, and mixing for 3 minutes; (8) centrifuging the tubes at 14,000 X g for about 5 minutes; (9) aspirating the top layer (aqueous phase) from the gas And placed in a new tube; (10) Add 01 volume of 3M NaOAC and mix (invert several times); ((1) add 2 volumes of 100% EtOH and mix (invert several times), while the genome is 〇 Να precipitates formed during this period; (12) the tubes were used in a microcentrifuge at 4 ° C and at 14,000 X g centrifugation for approximately 15 minutes; (13) gently pour the liquid, and the genomic DNA remains at the bottom of the tube; (14) wash the pellet with 5 ml of 70% ethanol; (15) the tube Centrifuge at 4C and 14,000 xg for approximately 5 minutes in a microcentrifuge; ((6) gently pour the sputum (10) and dry the genomic DNA pellets; and (17) add a suitable volume of 162 201200591 water directly to the RNase Pellets to the genomic DNA. Polymerase chain reaction amplification of the 18s rRNA gene was carried out using the primers previously described (Honda et al., A/Vcro. 46(6): 637-647 (1999). The PCR conditions using the chromosomal DNA template were as follows: 0.2 μΜ dNTPs, 0.1 μΜ each primer, 8% DMS0, 200 ng chromosomal DNA, 2_5 U Herculase® II fusion DNA polymerase (Stratagene), and Herculase® buffer (Stratagene) At 50 μl total volume, the PCR procedure consists of the following steps: (1) 95 ° C for 2 minutes; (2) 95 ° C for 35 seconds; (3) 55 ° C for 35 seconds; (4) 72 ° C 1 minute and 30 seconds; (5) repeat steps 2-4 for 30 cycles; (6) 72 °C for 5 minutes And (7) maintained at 4° C. Using the chromosomal template described above, PCR was used to amplify a differentiated DNA product of the expected size. The PCR product was cloned into the vector pJET1.2/Fermentas according to the manufacturer's instructions, and the inserted primers were used to determine the insertion sequence. Phylogenetic analysis Under moderate proof, PTA-10212 was placed in a reading system including Thraustochytrium pachydefmum and Thfaustochytrium aggregatum. Thraustochytrium sclerotia (7: sporangia has a very thick cell wall. Polycystis sinensis (Γ· forms a cluster of clearly visible opaque sporangia. PTA-10212 does not show any of these characteristics) In other taxa such as W. Kenyan, 71. gaeriner/wm, especially, and mangrove schizophyllum (&, there are numerous amoeba-like cells present; however, with these taxa The associated narrative description differs from that observed for this isolate. Moreover, 163 201200591 PTA-10212 does not appear to have affinity for the germline of any of these taxa. Table 29 shows from the ATCC deposit. Take the comparison of the 18s rRNA sequence registered by the number ΡΤΑ-10212 with the DNA sequence in the National Center for Biotechnology Information (NCBI) electronic database. Two different calculations are used to determine the percent identity. "Calculation#1&quot ; any "intervals from non-homologous regions or partial sequences that appear in the sequence are taken into account (AlignX-carrier NTI default setting)., calculation #2" does not include the interval Penalty (AlignX_carrier ΝΤΓ identity, 'matrix setting). Table 29: Comparison of 18srRNA sequences. Thresococcal serotype % identity calculation #1 % identity calculation #2 Thraustochytrium 85% 93% Chlamydia (P) 83% 92% Thraustochytrium gaertnerium 82% 92% Vesul Jessie Kenyan bacteria 82% 92% Schizochytrium -9695 80% 92% Mangrove Schizochytrium 80% 91% cracked Cloacae M sp. ATCC 20888 80% 90% Aurantiochytrium limiacinum 79% 90% (P): indicates that the partial sequence is as shown in Table 29, and has been found to be from the ATCC access number PTA in terms of % identity. -1〇212 The microbial (8) octopus gene sequence deposited with the NCB! The l8s deletion sequence available in the NCB! database is similar. It is considered that when the organism belongs to a different genus or species, Still having a very similar 18s rRNA gene sequence. Based on the above characteristic analysis, the isolated microorganism (ATCC access 164 201200591 number PTA-10212) is believed to represent a new species of Thraustochytrium and thus also named For the genus Thraustochytrium (TVzraMsioc/z sound η·Μ/« )«?/?. ATCCPTA-10212微生物 According to the ATCC access number PTA-10208, the classified characteristics of the detached microorganisms registered according to the ATCC access number PTA-10208 ("PTA-10208") are determined to be based on the ATCC access number PTA -9695 A sub-isolated strain of the microorganism ("PTA-9695") (single individual cells from a culture and maintaining a separate and unique culture), "PTA-9695" It is disclosed in U.S. Publication No. 2010/0239533 and International Publication No. WO 2010/107415. PTA-10208 has a classification feature of PTA-9695. It was found that PTA-9695 had two flagellar migration spores when it was discharged, which was actively released from the mature sporangia, and its wall residue was clearly visible after the spores were released (in phase difference). The PTA-9695 sporangia was measured to have a diameter of 12.5 micrometers to 25 micrometers, and the size of the zoospores was 2.5 micrometers to 2.8 micrometers x 4.5 micrometers to 4.8 micrometers. Each individual PTA-9695 sporangia has 8 to 24 spores. The stabilized PTA-9695 grows spores and rapidly undergoes homogenous division to form tetrads, octants, and finally sporangia clusters. The tetrad formation system begins at a very early stage before the sporangia matures. These properties are consistent with Schizochytrium OSc/n'zoc/y/iriw/w). In terms of % identity, the 18s rRNA gene sequence of PTA-9695 shared by PTA-10208 was found to be aggregated by Honda et al., J. Wu Zhengshi 46(6): 637-647 (1999). The 18s rRNA gene sequence of Zulggregaiww is very similar to 165 201200591, although not identical. The published 18s rRNA sequence of Thraustochytrium aggregaiwm is a partial sequence with an interval of about 71 DNA nucleotides in the middle of the sequence. PTA·9695 is believed to represent a new species of Schizochytrium. Therefore, the sub-isolated strain PTA_10208 was also named as Schizochytrium sp. jp.) ATCCPTA-10208. Example 35 Growth characteristics of detached microorganisms registered according to ATCC accession number PTA-10212 The detached microorganisms (ATCC accession number pTA_i 〇2丨2) are subjected to individual fermentation operations as described below. Check the growth characteristics. Typical cultures and culture conditions are shown in Table 3 〇. 166 201200591 Table 30: PTA-10212 ware medium concentration Na2S〇4 g/L 31.0 NaCl g/L 0.625 KC1 g/L 1.0 MgS04-7H20 g/L 5.0 (NH4)2S04 g/L 0.44 MSG1H20 g/L 6.0 CaCl2 g/L 0.29 T 154 (yeast extract) g/L 6.0 KH2P〇4 g/L 0.8 After autoclaving (metal) citrate mg/L 3.5 FeS04-7H20 mg/L 10.30 MnCl2.4H20 mg/L 3.10 ZnS047H20 Mg/L 3.10 CoC12-6H20 mg/L 0.04 Na2Mo04-2H20 mg/L 0.04 CuS045H20 mg/L 2.07 NiS04_6H20 mg/L 2.07 After autoclaving (vitamin) Thiamine mg/L 9.75 Vitamin B12 mg/L 0.16 CaA pantothenic acid Mg/L 2.06 Biotin mg/L 3.21 After autoclaving (carbon) Glycerol g/L 30.0 Nitrogen feed: Component concentration MSG1H20 g/L 17 10-100, or 15-50 Typical culture conditions will include the following: 0-50, 15-45, or 25-35 0-25, 0.1-10, or 0.5-5 0-5, 0.25-3, or 0.5-2 0-10, 2-8, or 3-6 0- 10, 0.25-5, or 0.05-3 0-10, 4-8, or 5-7 0.1-5, 0.15-3, or 0.2-1 0-20, 0.1-10, or 1-7 0.1-10, 0.5-5, or 0.6-1.8 0.1-5000, 10-3000, or 3-2500 0.1-100, 1-5 0, or 5-25 0.1-100, 1-50, or 2-25 0.01-100, 1-50, or 2-25 0-1, 0.001-0.1, or 0.01-0.1 0.001-1, 0.005-0.5, Or 0.01-0.1 0.1-100, 0.5-50, or 1-25 0.1-100, 0.5_50, or 1-25 0.1-100, 1-50, or 5-25 0.01-100, 0.05-5, or 0.1- 1 0.1-100, 0.1-50, or 1-10 0.1-100, 0.1-50, or 1-10 5-150, 10-100, or 20-50 0-150,

pH 溫度：溶解的氧：甘油控制在：約6.5-約9.5、約6.5—約 8.0，或約6.8-約7_8 ; 攝氏約15-約30度、攝氏約18-約28度，或攝氏約21至約23度；約0.1-約100%飽和、約5-約50%飽和，或約 10-約30%飽和；及/或約5_約50 g/L、約 10-約40 g/L，或約 15—約35 g/L。 167 201200591 於給料碳（甘油）與氮之培養物中、伴隨1〇〇〇 ppm cr於 22.5C及20%溶解的氧於pH 73，ΡΤΑ_1〇21α體積為1〇公升的發酵器中在138小時的培養之後，生產26.2克/升的乾燥細胞重。脂質產量為7.9克/升；ω_3產量為5·3克/升；ΕΡΑ 產量為3.3克/升；以及DHA產量為L8克/升。脂肪酸含量為 3〇·3%重量計；EPA含量為41.4%的脂肪酸甲酯（FAME);以及DHA含量為26.2°/。的FAME。脂質生產力為1.38克/升/天，以及ω-3生產力為〇·92克/升/天於此等條件下，伴隨〇 57克/ 升/天ΕΡΑ生產力以及〇·3ΐ克/升/天dha生產力。於給料碳（甘油）與氮之培養物中、伴隨1〇〇〇 ppm C1·於 22_5°C及20%溶解的氧於pH 7.3，PTA-10212在體積為10公升的發酵器中在189小時的培養之後，生產38.4克/升的乾燥細胞重。脂質產量為18克/升；ω-3產量為12克/升；EPA產量為5克/升；以及DHA產量為6.8克/升。脂肪酸含量為45% 重量計；EPA含量為27.8%的FAME;以及DHA含量為37.9% 的FAME。脂質生產力為2·3克/升/天，以及ω_3生產力為1.5 克/升/天於此等條件下，伴隨0.63克/升/天ΕΡΑ生產力以及 0.86克/升/天DHA生產力。於給料碳（甘油）與氮之培養物中、伴隨1000 ppm C1·於 22_5°C 及20°/〇溶解的氧於pH 6.8-7.7, PTA-10212在體積為 1〇公升的發酵器中在189小時的培養之後，生產13克/升的乾燥細胞重。脂質產量為5_6克/升；ω-3產量為3.5克/升；EPA 產量為1.55克/升；以及DHA產量為1.9克/升。脂肪酸含量為 38%重量計；ΕΡΑ含量為29.5%的FAME ;以及DHA含量為 168 201200591 36%的FAME。脂質生產力為〇·67克/升/天，以及ω_3生產力為0.4克/升/天於此等條件下，伴隨〇2〇克/升/天ερα生產力以及0.24克/升/天DHA生產力。於給料碳(甘油）與氮之培養物中 '伴隨1〇〇〇 pprn CT於 22.5-28.5°C 及20%溶解的氧於pH 6.6-7_2，PTA-10212在體積為10公升的發酵器中在191小時的培養之後，生產36.7克/ 升-48.7克/升的乾燥細胞重。脂質產量為15 2克/升_ 25 3克/ 升；ω-3產量為9_3克/升—13.8克/升；EPA產量為2.5克/ 升-3.3克/升；以及DHA產量為5_8克/升-11克/升。脂肪酸含量為42.4% - 53%重量計；ΕΡΑ含量為9.8% - 22%的 FAME ;以及DHA含量為38.1% - 43.6%的FAME。脂質生產力為1，9克/升/天-3.2克/升/天，以及ω-3生產力為1_2克/升/ 天- 1.7克/升/天於此等條件下，伴隨〇.31克/升/天_ 〇41克/ 升/天ΕΡΑ生產力以及〇‘72克/升/天-1.4克/升/天DHA生產力。根據ATCC存取號碼ΡΤΑ-10208所寄存之經單離的微生物之生長特徵該經單離的微生物（ATCC存取號碼ΡΤΑ-10208)係於以下所說明之個別的發酵操作中檢查生長特徵。典型的培養基與培養條件係顯示於表31中。 169 201200591 表31 : PTA-10208器皿培養基成分濃度範圍 Nfl2S〇4 g/L 31.0 0-50、15—45，或25-35 NaCl g/L 0.625 0-25、0.1-10，或0.5-5 成分濃度範圍 Nfl2S〇4 g/L 8.8 0-25、2-20，或3-10 NaCl g/L 0.625 0-25、0.1-10，或0.5-5 KC1 g/L 1.0 〇-5 ' 0.25-3，或0.5-2 MgS04-7H20 g/L 5.0 〇-1〇、2-8，或3-6 (NH4)2S〇4 g/L 0.42 〇-1〇、0.25-5，或0.05-3 CaCl2 g/L 0.29 0.1-5、0.15-3，或0.2-1 T 154(酵母萃取物） g/L 1.0 〇-20、0.1-10，或0.5-5 kh2po4 g/L 1.765 01-10、0.5-5，或 1-3 高壓蒸氣滅菌後(金屬）檸檬酸 mg/L 46.82 0.1-5000、10-3000，或40-2500 FeS04-7H20 mg/L 10.30 0.1-100 、 1-50 ，或5-25 MnCl2-4H20 mg/L 3.10 0.1-100、1-50，或2-25 ZnS04-7H20 mg/L 9.3 0.01-100、1-50，或2-25 CoC12-6H20 mg/L 0.04 〇-1、0.001-(U，或0.01-0.1 Na2Mo04-2H20 mg/L 0.04 0.001-1、0.005-0.5，或0.01-0-1 CuS04-5H20 mg/L 2.07 0.1-100、0.5-50，或 1-25 NiS04.6H20 mg/L 2.07 0.1-100、0.5-50，或 1-25 高壓蒸氣滅菌後(維生素）硫胺素 mg/L 9.75 0.1-100、1-50，或5-25 Ca!4-泛酸 mg/L 3.33 0.1-100、0.1-50，或 1-10 生物素 mg/L 3.58 0.1-100、0.1-50，或 1-10 高壓蒸氣滅菌後(碳）葡萄糖 g/L 30.0 5-150 、 10-100 ，或20-50 氮進料：成分濃度 NH4〇H mL/L 23.6 0-150、l〇-l〇〇，或 15-50 典型的培養條件會包括下列： pH 溫度· 溶解的氧：葡萄糖控制在：約6.5-約8.5、約6.5-約8.0，或約7.0-約8_0 ; 攝氏約Π-約30度、攝氏約20-約28度，或攝氏約22至大約24度；約2-約100%飽和、約5-約50%飽和，或約7-約20%飽和；及/或約5-約50 g/L、約 10-約40 g/L，或約20-約35 g/L。 170 201200591 於給料碳（葡萄糖)與氮之培養物中、伴隨1000 ppm cl· 於22_5°C及20%溶解的氧於在氮進料的期間以及之後的 10%溶解的氧，PTA-10208在體積為10公升的發酵器中在 200小時的培養之後，生產95克/升的乾燥細胞重。脂質產量為53.7克/升；ω-3產量為37克/升；EPA產量為14.3克/升；以及DHA產量為21克/升。脂肪酸含量為57%重量計；ερα 含量為27.7%的FAME ;以及DHA含量為39.1%的FAME。脂質生產力為6.4克/升/天，以及ω_3生產力為4.4克/升/天於此等條件下，伴隨1.7克/升/天ΕΡΑ生產力以及2.5克/升/天DHA 生產力。於給料碳（葡萄糖)與氮之培養物中、伴隨1〇〇〇 ppm cl-於22_51於pH 7.5及20%溶解的氧在氮進料的期間以及之後的10%溶解的氧，PTA-10208在體積為1〇公升的發酵器中在139小時的培養之後，生產56克/升的乾燥細胞重。脂質產量為53克/升；ω-3產量為34克/升；EPA產量為11.5克/升；以及DHA產量為22克/升。脂肪酸含量為58%重量計；ΕρΑ 含里為21.7%的FAME，以及DHA含量為41.7%的FAME。脂質生產力為9.2克/升/天，以及ω_3生產力為5.9克/升/天於此等條件下，伴隨2克/升/天ΕΡΑ生產力以及3.8克/升/天dha 生產力。於給料碳（葡萄糖)與氮之培養物中、伴隨1〇〇〇ppmC1-於22.5 C於pH 7.0及20%溶解的氧在氮進料的期間以及之後的10%溶解的氧，PTA-10208在體積為2〇00公升的發酵器中在167小時的培養之後，生產93.8克/升的乾燥細胞重。脂 171 201200591 質產量為47.2克/升；ω-3產量為33_1克/升；EPA產量為10_5 克/升，以及DHA產量為20.4克/升。脂肪酸含量為50 6。/〇重量計；EPA含量為23°/。的FAME ;以及DHA含量為42_6%的 FAME »脂質生產力為6.8克/升/天，以及ω_3生產力為4.7 克/升/天於此等條件下，伴隨1.5克/升/天ΕΡΑ生產力以及2 9 克/升/天DHA生產力。於給料碳（葡萄糖)與氮之培養物中 '伴隨1〇〇〇 ppm C1-於22.5°C於pH 7.0及20%溶解的氧在氮進料的期間以及之後的10°/。溶解的氧，PTA-10208在體積為2〇〇〇公升的發酵器中在168小時的培養之後，生產105克/升的乾燥細胞重。脂質產量為46.4g/L ; ω-3產量為33克/升；EPA產量為10.7克/ 升，以及DHA產量為20.3克/升。脂肪酸含量為43.9%重量計；ΕΡΑ含量為24%的FAME ;以及DHA含量為43.7%的 FAME。脂質生產力為6.6克/升/天，以及ω_3生產力為47 克/升/天於此等條件下’伴隨1.5克/升/天ΕΡΑ生產力以及2.9 克/升/天DHA生產力。於給料碳（葡萄糖)與氮之培養物中、伴隨1000 ppmC1_ 於22.5°C於pH 7.0及20%溶解的氧在氮進料的期間以及之後的10%溶解的氧，PTA-〗0208在體積為2000公升的發酵器中在168小時的培養之後’生產64.8克/升的乾燥細胞重。脂質產量為38.7 g/L ; ω-3產量為29.9 g/L ; EPA產量為8 5 g/L，以及DHA產量為16.7 g/L。脂肪酸含量為59.6%重量計；EPA含量為23%的FAME ;以及DHA含量為42.3%的 FAME。脂質生產力為5.53 g/L/天，以及ω_3生產力為3 8 172 201200591 §/17天於此等條件下，伴隨1.2£/17天£?八生產力以及2.3 g/L/天DHA生產力。實施例36 微生物菌株ATCC PTA-10208和PTA-10212的脂肪酸剖綠四個生質樣品（PTA-10208樣品#1 ; PTA-10208樣品#2 ; PTA-10212樣品# 1 ;及PTA-10212樣品#2)係藉由溶劑萃取作用來分析總粗製油含量，藉由高效液相層析法/蒸發光散射檢測作用（HPLC/ELSD)來測定脂質種類，藉由HPLC/質譜法 (HPLC/MS)來分析三醯甘油酯（TAG)，以及藉由附有火焰離子化偵檢作用的氣相層析儀(GC-FID)來測定脂肪酸(FA)剖繪。藉由使用己烷的溶劑研磨作用來測定各冷凍乾燥生質的粗脂質含量且與藉由直接轉酯作用所生成的FAME總量 (毫克/克)相比較，以及藉由GC/F1D分析將所生成的脂肪酸甲基醋類(FAME)量化。所萃取的粗脂質中之脂肪酸亦藉由轉酯化作用予以量化以及使用GC/FID分析將所生成的 FAME量化。使用具有ELSD與大氣壓力化學離子化 -MS(APCI-MS)鑑定之正相HPLC來測定所萃取的粗脂質中之所有中性脂質（NL)與游離脂肪酸(FFA)的重量百分比。該方法分離與量化固醇酯（SE)、TAG、游離脂肪酸(FFA)、1，3-二醯甘油酯（1，3-DAG)、固醇、1,2-二醯甘油酯（1，2-DAG)，以及單醯甘油（MAG)。結果係顯示於以下的表32與33之内。注意到表32與33的脂肪酸剖繪係自使用一溶劑所萃取的樣品而獲得的。設若該等樣品使用本發明的方法來萃取’預期表32與33的脂肪酸剖繪要為實質相同的。 173 201200591 TAG與磷脂質（PL)係自四個生質樣品（pTA]0208樣品 #1 ; PTA-10208樣品#2 ; PTA-10212樣品#1 ;及PTA-10212 樣品#2)所萃取的粗製油中單離出。TAG係使用低壓急驟層析法予以單離以及PL係使用固相萃取(SPE)予以單離。各經單離的餾份之同一性係藉由薄膜層析法(TLC)予以確認。該經單離的TAG和PL餾份之脂肪酸剖繪係依循直接使用 GC-FID的轉酯作用予以決定為fame。結果係顯示於以下的表34和35中。亦在來自微生物菌株ATCC pTA_1〇2i2之二個額外的生質樣品（PTA-丨0212樣品#3和PTA-10212樣品#4)測定經單離的脂質種類之總粗製油含量與脂肪酸剖繪。粗製油係自藉由己烧萃取之各樣品所獲得，以及個別的脂質種類係使用低壓急驟層析法予以單離。該生質、粗製油，及經單離的備份之脂肪酸剖繪係使用GC-FID來決定為FAME。結果係顯示於以下的表36-39之内。注意到表36-39的脂肪酸剖繪係自使用一典型的己烷萃取所萃取的樣品而獲得的。設若該等樣品使用本發明的方法來萃取，預期表36_39的脂肪酸剖繪要為實質相同的。個別的脂質種類係由使用FRI〇lex®方法而事先萃取之來自微生物菌株ATCC PTA_1〇212的粗製油樣品 (PTA-10212樣品#5)予以單離’以及各類的脂肪酸剖繪係使用GC-FID予以決定為FAME。結果係顯示於以下的表4〇和 41中。注意到表40和41的脂肪酸剖繪係自使用FRI〇LEX(g) 方法所萃取的樣品所獲得。設若該等樣品使用本發明的方 174 201200591 法來萃取，預期表40與41的脂肪酸刮繪要為實質相同的。個別的脂質種類係使用具有ELSD和APCI-MS鑑定之正相HPLC而由來自微生物菌株ATCC PTA-10208的粗製油之樣品(PTA-10208樣品#3)所單離。實驗程序粗製油卒取作用-粗製油係使用溶劑研磨作用而自冷凍乾燥的生質樣品予以萃取。舉例而言，將大概3克的生質稱重置入一瑞典式管中。在瑞典式管中添加3個滾珠軸承與 30毫升的己烷’以氣丁二烯橡膠塞密封該管及置於一振盈器中2小時。所生成的淤漿係使用布赫納(Buchner)漏斗與惠特曼(Whatman)濾紙予以過濾。收集過濾後的液體，在真空中移除溶劑，以及以重量方式測定剩餘粗製脂質的量。脂肪酸分析-生質、經萃取之粗製脂質，和經單離的脂質種類之樣品係予以分析作為F A ME之脂肪酸組成物。簡單地說，將冷凍乾燥的生質和經單離的脂質種類直接稱重至一螺旋蓋試管之内’同時將粗製油的樣品溶解於己烷内以提供大概2毫克/毫升的濃度。將含有内標準的甲苯和配於甲醇内之1.5 N HC1添加至各管。將該等管子予以滿璇，接而加蓋以及加熱至100。(：歷時2小時。允許該等管子冷卻，以及添加配於水内之飽和的NaCl。將該等管子再次予以渦旋及離心以允許該等層來分離。繼而將有機層的一部分放置於一GC小玻璃瓶内並藉由GC-FID予以分析。使用利用pH Temperature: Dissolved Oxygen: Glycerol is controlled at: from about 6.5 to about 9.5, from about 6.5 to about 8.0, or from about 6.8 to about 7-8; from about 15 to about 30 degrees Celsius, from about 18 to about 28 degrees Celsius, or about 21 degrees Celsius. Up to about 23 degrees; from about 0.1 to about 100% saturated, from about 5 to about 50% saturated, or from about 10 to about 30% saturated; and/or from about 5 to about 50 g/L, from about 10 to about 40 g/L , or about 15 to about 35 g / L. 167 201200591 In a feedstock of carbon (glycerol) and nitrogen, with 1 〇〇〇 ppm cr dissolved at 22.5 C and 20% oxygen at pH 73, ΡΤΑ_1〇21α in a volume of 1 liter of fermenter at 138 hours After the cultivation, a dry cell weight of 26.2 g/liter was produced. The lipid yield was 7.9 g/l; the ω_3 yield was 5.3 g/l; the ΕΡΑ yield was 3.3 g/l; and the DHA yield was L8 g/l. The fatty acid content was 3 〇·3% by weight; the EPA content was 41.4% fatty acid methyl ester (FAME); and the DHA content was 26.2 °/. FAME. Lipid productivity was 1.38 g/L/day, and omega-3 productivity was 〇·92 g/L/day under these conditions, with 〇57 g/L/天ΕΡΑ productivity and 〇·3 ΐ/L/day dha productive forces. In feedstock carbon (glycerol) and nitrogen cultures, with 1 〇〇〇ppm C1 at 22_5 ° C and 20% dissolved oxygen at pH 7.3, PTA-10212 in a 10 liter fermenter at 189 hours After the cultivation, 38.4 g/L of dry cells were produced. The lipid yield was 18 g/l; the omega-3 yield was 12 g/l; the EPA yield was 5 g/l; and the DHA yield was 6.8 g/l. The fatty acid content is 45% by weight; the EPA content is 27.8% FAME; and the DHA content is 37.9% FAME. The lipid productivity was 2.3 g/L/day, and the ω_3 productivity was 1.5 g/L/day under these conditions, with a productivity of 0.63 g/L/day and a DHA productivity of 0.86 g/L/day. In a feedstock of carbon (glycerol) and nitrogen, dissolved in oxygen at pH 6.8-7.7 with 1000 ppm C1 at 22_5 ° C and 20 ° / ,, PTA-10212 in a fermenter with a volume of 1 liter After 189 hours of cultivation, 13 g/L of dry cells were produced to weigh. The lipid yield was 5-6 g/l; the omega-3 yield was 3.5 g/l; the EPA yield was 1.55 g/l; and the DHA yield was 1.9 g/l. The fatty acid content is 38% by weight; the hydrazine content is 29.5% FAME; and the DHA content is 168 201200591 36% FAME. The lipid productivity was 〇·67 g/L/day, and the ω_3 productivity was 0.4 g/L/day under these conditions, with 〇2〇g/L/day ερα productivity and 0.24 g/L/day DHA productivity. In feedstock carbon (glycerol) and nitrogen cultures 'with 1 〇〇〇 pprn CT at 22.5-28.5 ° C and 20% dissolved oxygen at pH 6.6-7_2, PTA-10212 in a 10 liter fermenter After 191 hours of cultivation, a dry cell weight of 36.7 g / liter - 48.7 g / liter was produced. Lipid yield was 15 2 g / liter - 25 3 g / liter; omega-3 yield was 9_3 g / liter - 13.8 g / liter; EPA production was 2.5 g / liter - 3.3 g / liter; and DHA production was 5 _ 8 g / -11 grams / liter. The fatty acid content is from 42.4% to 53% by weight; the hydrazine content is from 9.8% to 22% of FAME; and the DHA content is from 38.1% to 43.6% of FAME. Lipid productivity is 1,9 g / liter / day - 3.2 g / liter / day, and omega-3 productivity is 1_2 g / liter / day - 1.7 g / liter / day under these conditions, accompanied by 〇. 31 g / l / day _ 〇 41 g / liter / day ΕΡΑ productivity and 〇 '72 g / liter / day - 1.4 g / liter / day DHA productivity. Growth characteristics of detached microorganisms registered according to ATCC access number ΡΤΑ-10208 The detached microorganisms (ATCC access number ΡΤΑ-10208) are examined for growth characteristics in the individual fermentation operations described below. Typical medium and culture conditions are shown in Table 31. 169 201200591 Table 31: PTA-10208 ware medium concentration range Nfl2S〇4 g/L 31.0 0-50, 15-45, or 25-35 NaCl g/L 0.625 0-25, 0.1-10, or 0.5-5 Concentration range Nfl2S〇4 g/L 8.8 0-25, 2-20, or 3-10 NaCl g/L 0.625 0-25, 0.1-10, or 0.5-5 KC1 g/L 1.0 〇-5 ' 0.25-3 , or 0.5-2 MgS04-7H20 g/L 5.0 〇-1〇, 2-8, or 3-6 (NH4)2S〇4 g/L 0.42 〇-1〇, 0.25-5, or 0.05-3 CaCl2 g /L 0.29 0.1-5, 0.15-3, or 0.2-1 T 154 (yeast extract) g/L 1.0 〇-20, 0.1-10, or 0.5-5 kh2po4 g/L 1.765 01-10, 0.5-5 , or 1-3 after autoclaving (metal) citric acid mg / L 46.82 0.1-5000, 10-3000, or 40-2500 FeS04-7H20 mg / L 10.30 0.1-100, 1-50, or 5-25 MnCl2 -4H20 mg/L 3.10 0.1-100, 1-50, or 2-25 ZnS04-7H20 mg/L 9.3 0.01-100, 1-50, or 2-25 CoC12-6H20 mg/L 0.04 〇-1, 0.001- (U, or 0.01-0.1 Na2Mo04-2H20 mg/L 0.04 0.001-1, 0.005-0.5, or 0.01-0-1 CuS04-5H20 mg/L 2.07 0.1-100, 0.5-50, or 1-25 NiS04.6H20 Mg/L 2.07 0.1-100, 0.5-50, or 1-25 high pressure vapor off Post-bacteria (vitamin) Thiamine mg/L 9.75 0.1-100, 1-50, or 5-25 Ca! 4-pantothenic acid mg/L 3.33 0.1-100, 0.1-50, or 1-10 biotin mg/L 3.58 0.1-100, 0.1-50, or 1-10 After autoclaving (carbon) Glucose g/L 30.0 5-150, 10-100, or 20-50 Nitrogen feed: Component concentration NH4〇H mL/L 23.6 0-150, l〇-l〇〇, or 15-50 typical culture conditions will include the following: pH Temperature · Dissolved Oxygen: Glucose is controlled at: from about 6.5 to about 8.5, from about 6.5 to about 8.0, or about 7.0- About 8_0; about 摄- about 30 degrees Celsius, about 20 to about 28 degrees Celsius, or about 22 to about 24 degrees Celsius; about 2 to about 100% saturated, about 5 to about 50% saturated, or about 7 to about 20 % saturated; and/or from about 5 to about 50 g/L, from about 10 to about 40 g/L, or from about 20 to about 35 g/L. 170 201200591 In the feed carbon (glucose) and nitrogen cultures, with 1000 ppm cl· at 22_5 ° C and 20% dissolved oxygen during the nitrogen feed and after 10% dissolved oxygen, PTA-10208 is After 200 hours of cultivation in a 10 liter volume in a fermenter, 95 g/L dry cell weight was produced. The lipid yield was 53.7 g/l; the omega-3 yield was 37 g/l; the EPA yield was 14.3 g/l; and the DHA yield was 21 g/l. The fatty acid content was 57% by weight; the ερα content was 27.7% FAME; and the DHA content was 39.1% FAME. The lipid productivity was 6.4 g/L/day, and the ω_3 productivity was 4.4 g/L/day with 1.7 g/L/day productivity and 2.5 g/L/day DHA productivity. In a feed carbon (glucose) and nitrogen culture, with 1 〇〇〇ppm cl- at 22_51 at pH 7.5 and 20% dissolved oxygen during the nitrogen feed and after 10% dissolved oxygen, PTA-10208 After 139 hours of cultivation in a fermenter having a volume of 1 liter, a dry cell weight of 56 g/liter was produced. The lipid yield was 53 g/l; the omega-3 yield was 34 g/l; the EPA yield was 11.5 g/l; and the DHA yield was 22 g/l. The fatty acid content was 58% by weight; ΕρΑ contained 21.7% of FAME, and DHA with a DHA content of 41.7%. The lipid productivity was 9.2 g/L/day, and the ω_3 productivity was 5.9 g/L/day under conditions such as 2 g/L/day productivity and 3.8 g/L/day dha productivity. In a feed carbon (glucose) and nitrogen culture, with 1 〇〇〇ppmC1 at 22.5 C dissolved at pH 7.0 and 20% oxygen during the nitrogen feed and after 10% dissolved oxygen, PTA-10208 After 167 hours of cultivation in a fermenter having a volume of 2 00 liters, a dry cell weight of 93.8 g/liter was produced. The fat 171 201200591 yield was 47.2 g/l; the omega-3 yield was 33_1 g/l; the EPA yield was 10_5 g/l, and the DHA yield was 20.4 g/l. The fatty acid content is 50 6 . / 〇 weight meter; EPA content is 23 ° /. FAME; and DME content of 42_6% FAME »lipid productivity of 6.8 g / liter / day, and ω_3 productivity of 4.7 g / liter / day under these conditions, with 1.5 g / liter / day ΕΡΑ productivity and 2 9 G/L/D DHA productivity. In the feed carbon (glucose) and nitrogen cultures, '1 〇〇〇 ppm C1 - dissolved at pH 7.0 and 20% at 22.5 ° C during the nitrogen feed and 10 ° / after. Dissolved oxygen, PTA-10208, produced a dry cell weight of 105 g/l after 168 hours of cultivation in a 2 liter liter fermenter. The lipid yield was 46.4 g/L; the omega-3 yield was 33 g/l; the EPA yield was 10.7 g/l, and the DHA yield was 20.3 g/l. The fatty acid content was 43.9% by weight; the FAME having a strontium content of 24%; and the FAME having a DHA content of 43.7%. The lipid productivity was 6.6 g/L/day, and the ω_3 productivity was 47 g/L/day under conditions of '1.5 g/L/day productivity and 2.9 g/L/day DHA productivity. In a feed carbon (glucose) and nitrogen culture, with 1000 ppm C1 at 22.5 ° C at pH 7.0 and 20% dissolved oxygen during the nitrogen feed and after 10% dissolved oxygen, PTA - 0208 in volume After 168 hours of cultivation in a 2000 liter fermenter, '64.8 g/L dry cell weight was produced. The lipid yield was 38.7 g/L; the omega-3 yield was 29.9 g/L; the EPA yield was 85 g/L, and the DHA yield was 16.7 g/L. The fatty acid content was 59.6% by weight; the EPA content was 23% FAME; and the DHA content was 42.3% FAME. Lipid productivity was 5.53 g/L/day, and ω_3 productivity was 3 8 172 201200591 §/17 days under these conditions, with 1.2 £/17 days £8 productivity and 2.3 g/L/day DHA productivity. Example 36 Microbial strains ATCC PTA-10208 and PTA-10212 fatty acid green four samples (PTA-10208 sample #1; PTA-10208 sample #2; PTA-10212 sample #1; and PTA-10212 sample# 2) The total crude oil content is analyzed by solvent extraction, and the lipid species is determined by high performance liquid chromatography/evaporative light scattering detection (HPLC/ELSD) by HPLC/MS (HPLC/MS). The triterpene glyceride (TAG) was analyzed, and the fatty acid (FA) profile was determined by a gas chromatograph (GC-FID) with flame ionization detection. The crude lipid content of each freeze-dried biomass was determined by solvent milling using hexane and compared to the total amount of FAME (mg/g) produced by direct transesterification, and by GC/F1D analysis. The fatty acid methyl vinegar (FAME) produced was quantified. The fatty acids in the extracted crude lipids were also quantified by transesterification and the generated FAME was quantified using GC/FID analysis. Normal phase HPLC with ELSD and atmospheric pressure chemical ionization-MS (APCI-MS) identification was used to determine the weight percent of all neutral lipids (NL) and free fatty acids (FFA) in the extracted crude lipids. The method separates and quantifies sterol ester (SE), TAG, free fatty acid (FFA), 1,3-dioxyl glyceride (1,3-DAG), sterol, 1,2-dioxyl glyceride (1, 2-DAG), and monoterpene glycerol (MAG). The results are shown in Tables 32 and 33 below. It is noted that the fatty acid profiles of Tables 32 and 33 were obtained from samples extracted using a solvent. It is assumed that the samples are extracted using the method of the present invention and that the fatty acid profiles of Tables 32 and 33 are expected to be substantially identical. 173 201200591 TAG and phospholipids (PL) are derived from four crude samples (pTA) 0208 sample #1; PTA-10208 sample #2; PTA-10212 sample #1; and PTA-10212 sample #2) Single out of the oil. The TAG is isolated by a low pressure flash stratification method and the PL system is separated by solid phase extraction (SPE). The identity of each individual fraction was confirmed by thin film chromatography (TLC). The fatty acid profile of the isolated TAG and PL fractions was determined to be fame following the direct transesterification using GC-FID. The results are shown in Tables 34 and 35 below. The total crude oil content and fatty acid profile of the isolated lipid species were also determined in two additional biomass samples (PTA-丨0212 sample #3 and PTA-10212 sample #4) from the microbial strain ATCC pTA_1〇2i2. The crude oil was obtained from each sample extracted by calcination, and the individual lipid species were isolated by low pressure flash chromatography. The crude oil, crude oil, and isolated fatty acid profile are determined to be FAME using GC-FID. The results are shown in Tables 36-39 below. It is noted that the fatty acid profiles of Tables 36-39 were obtained from samples extracted using a typical hexane extraction. If the samples are extracted using the method of the present invention, it is expected that the fatty acid profiles in Table 36-39 are substantially identical. Individual lipid species were isolated from the crude oil sample (PTA-10212 sample #5) from the microbial strain ATCC PTA_1〇212 previously extracted using the FRI〇lex® method and the various fatty acid profiles were GC- The FID is determined to be FAME. The results are shown in Tables 4 and 41 below. It is noted that the fatty acid profiles of Tables 40 and 41 were obtained from samples extracted using the FRI(R) LEX(g) method. If the samples are extracted using the method of the invention 174 201200591, it is expected that the fatty acid scribing of Tables 40 and 41 is substantially the same. Individual lipid species were isolated from crude oil samples (PTA-10208 Sample #3) from the microbial strain ATCC PTA-10208 using normal phase HPLC with ELSD and APCI-MS identification. Experimental Procedure Crude Oil Stroke - The crude oil was extracted from the freeze-dried biomass sample using solvent milling. For example, approximately 3 grams of raw biomass is reset into a Swedish tube. Three ball bearings were added to the Swedish tube and 30 ml of hexane was sealed with a gas butadiene rubber stopper and placed in a vibrator for 2 hours. The resulting slurry was filtered using a Buchner funnel and Whatman filter paper. The filtered liquid was collected, the solvent was removed in vacuo, and the amount of residual crude lipid was determined by weight. Fatty Acid Analysis - Biomass, extracted crude lipids, and samples of isolated lipid species were analyzed as fatty acid compositions of F A ME . Briefly, the lyophilized biomass and the detached lipid species were weighed directly into a screw cap tube' while the crude oil sample was dissolved in hexane to provide a concentration of approximately 2 mg/ml. Toluene containing the internal standard and 1.5 N HCl1 in methanol were added to each tube. The tubes are filled, then capped and heated to 100. (: 2 hours. Allow the tubes to cool, and add saturated NaCl in water. The tubes are again vortexed and centrifuged to allow the layers to separate. Then a portion of the organic layer is placed in one GC small glass bottle and analyzed by GC-FID.

Nu-Chek-Prep GLC參考標準物(NuChek，ElySian，MN)所產生之3點内標準校準曲線來定量FAME。存在於萃取物中的 175 201200591 脂肪酸係以毫克/克的形式及以重量百分比的形式來表達。假設藉由GC-FID分析時之反應係與内標準相當，而估算樣品中的脂肪含量。 HPLC/ELSD/MS 方法- 儀器 Agilent 1100 HPLC，Alltech 3300 ELSD，The standard calibration curve within 3 points generated by the Nu-Chek-Prep GLC reference standard (NuChek, ElySian, MN) was used to quantify FAME. The 175 201200591 fatty acid present in the extract is expressed in mg/gram form and in weight percent. It is assumed that the reaction content in the GC-FID analysis is equivalent to the internal standard, and the fat content in the sample is estimated. HPLC/ELSD/MS Method - Instrumentation Agilent 1100 HPLC, Alltech 3300 ELSD,

Agilent 1100 MSD 管柱 Phenomenex Luna二氧化石夕，250 x 4_6 mm，5 μιη粒徑w/保護管柱移動相 A — 99.5%&^(Omnisolv) 0.4%異丙醇（Omnisolv) 0.1%乙酸 B - 99.9% 乙醇(Omnisolv，95 : 5 的乙醇：IPA) 0.1%乙酸梯度時間，min. % A %B 0 100 0 5 100 0 15 85 10 20 0 100 25 0 100 26 100 0 35 100 0Agilent 1100 MSD column Phoenomenex Luna dioxide, 250 x 4_6 mm, 5 μηη particle size w/protective column mobile phase A — 99.5% & ^ (Omnisolv) 0.4% isopropanol (Omnisolv) 0.1% acetic acid B - 99.9% ethanol (Omnisolv, 95:5 ethanol: IPA) 0.1% acetic acid gradient time, min. % A %B 0 100 0 5 100 0 15 85 10 20 0 100 25 0 100 26 100 0 35 100 0

管柱溫度流速注射體積 ELSD檢測 MSDColumn temperature flow rate injection volume ELSD detection MSD

30°C 1.5 mL/min 5 μί30 ° C 1.5 mL / min 5 μί

溫度35°C，氣流 1.2L/min 質量範圍200 - 1200，碎裂器225 V 乾燥氣溫度350°C 汽化器溫度325°C 毛細管電壓3500 V 電暈電流10 μA 176 201200591 固相萃取作用-PL餾份係使用置於一個Vac Elut裝置 (Varian Inc，Palo Alto，USA)中之2 克的胺丙基匣（Biotage， Uppsala ’ Sweden)，藉由固相萃取(SPE)而自粗製脂質來分離。以15毫升的己烷調節該匣，以及將~6〇毫克的各樣品溶於1毫升的CHC13且施用至該匣。以15毫升之2 : 1的CHC13 : 異丙醇清洗該管柱，以洗提所有的中性脂質，及予以棄置。然後以15毫升之配於乙醚中的2%乙酸(HOAc)來洗提脂肪酸，及予以棄置《以15毫升之ό : 1的曱醇：氣仿來洗提餾份，將其收集、在氮氣下乾燥，以及加以稱重。急驟層析法-使用急驟層析法來分離存在於粗製油中之脂質種類。將溶於己烷中之大概200毫克的粗製油注入至管柱頂部。該層析系統係利用具有5毫升/分鐘(表6_7)或3毫升/分鐘（表8-13)之由石油醚和乙酸乙酯所組成的流動相之矽凝膠60(EMD Chemica卜 Gibbstown，NJ)。使用一階段梯度以選擇性洗提得自管柱之各脂質種類。該流動相梯度係自100%石油醚開始並以50%乙酸乙酯結束（繼而進行丨曱醇清洗）。餾份係使用Gilson FC 204大床溶館份收集器 (Gilson，Inc. ’ Middleton，WI)收集於1〇毫升試管内。各管係藉由薄膜層析法(TLC)予以分析且含個別的脂質種類(如同藉由在具有預期的滯留係數(Rf)之TLC平板上之單— 點而判斷）之管子係予以儲集，濃縮至乾燥，以及予以稱重。總餾份含量接而以重量方式決定。 TLC分析-薄膜層析法係於矽凝膠板上予以實施。使用由石油醚：乙醚：乙酸(80:20 : 1)所組成之溶劑系統來洗Temperature 35 ° C, gas flow 1.2 L / min mass range 200 - 1200, chipper 225 V drying gas temperature 350 ° C vaporizer temperature 325 ° C capillary voltage 3500 V corona current 10 μA 176 201200591 solid phase extraction - PL distillation The fractions were isolated from crude lipids by solid phase extraction (SPE) using 2 g of aminopropyl hydrazine (Biotage, Uppsala 'Swedish) placed in a Vac Elut apparatus (Varian Inc, Palo Alto, USA). The mash was adjusted with 15 ml of hexane, and ~6 〇 of each sample was dissolved in 1 ml of CHC13 and applied to the mash. The column was washed with 15 ml of 2:1 CHC13: isopropanol to elute all neutral lipids and discarded. The fatty acid was then eluted with 15 ml of 2% acetic acid (HOAc) in diethyl ether, and discarded. The fraction was eluted with 15 ml of hydrazine: 1 sterol: gas, collected, and purged with nitrogen. Dry down and weigh. Flash Chromatography - Rapid chromatography is used to separate the lipid species present in the crude oil. Approximately 200 mg of crude oil dissolved in hexane was injected into the top of the column. The chromatographic system utilizes a mobile phase consisting of 5 ml/min (Table 6-7) or 3 ml/min (Tables 8-13) of a mobile phase consisting of petroleum ether and ethyl acetate (EMD Chemica Gibbstown, NJ). A one-step gradient is used to selectively elute each lipid species from the column. The mobile phase gradient begins with 100% petroleum ether and ends with 50% ethyl acetate (followed by decyl alcohol cleaning). Fractions were collected in 1 mL tubes using a Gilson FC 204 Large Bed Fraction Collector (Gilson, Inc.' Middleton, WI). Each tube was analyzed by thin film chromatography (TLC) and contained with individual lipid species (as judged by a single point on a TLC plate with the expected retention coefficient (Rf)). , concentrated to dryness, and weighed. The total fraction content is then determined by weight. TLC analysis - thin film chromatography was carried out on a gel plate. Washed with a solvent system consisting of petroleum ether: diethyl ether: acetic acid (80:20:1)

S 177 201200591 提該等平板以及使用碘蒸汽進行視覺化測定。各斑點之Rf 值接而與各脂質種類之報告文獻值比較。 TAG和PL傲份之分析-該經單離的TAG和PL餾份係予以分析為脂肪酸曱酯（FAME)之脂肪酸組成物。將該TAG餾份溶解於己烷内以提供大概1-2毫克/毫升的濃度。將1毫升整分部分的溶液在氮氣下予以濃縮至乾燥。將含有内標準的甲苯，及配於甲醇内之1.5 N HC1添加至各管。將該等管子予以渦旋，接而加蓋且加熱至1 〇〇。〇歷時2小時。將内標準和HC1甲醇直接地添加至含有該PL餾份之管子中以及予以加熱。允許該等管子冷卻，以及添加配於水内之飽和的 NaCl。將該等管子再次予以渦漩並離心以允許該等層來分離。繼而將該有機層的一部分放置於一GC小玻璃瓶内以及藉由GC-FID予以分析。使用利用Nu_chek-Prep GLC 502B 參考標準物(NuChek ’ Elysian，MN)所產生之3點内標準校準曲線來定量FAME。存在於萃取物中之脂肪酸係表達為毫克/克以及為FAME之％。結果 PTA 10208 樣品#1 PTA-10208樣品#1之生質與經萃取之粗製脂質的脂肪酸剖繪係使用GC/HD予以決定。生質内的脂肪酸係藉由稱重28.6毫克的生質直接地至一 FAMe管中而予以原位轉酯化’而該經萃取之粗製脂質的一樣品係藉由稱重55.0毫克的粗製脂質至一 50毫升體積的燒瓶之内以及轉移1毫升至一單獨的FAME管來製備。該生質的估計粗製脂質含量使用 178 201200591 具FID檢測之GC經判定為53.2%(以FAME的總和），同時 52.0% (wt/wt)脂質係自該乾的生質予以萃取，提供97.8%總脂質的回收率。該粗製脂質使用GC/HD經判定為91.9%脂肪酸（以FAME的總和）。該粗製脂質内含有的主要脂肪酸為 C16:0(182.5毫克/克）、C20:5 n-3(186.8毫克/克），以及C22:6 n-3 (423.1 毫克/克）。藉由稱重55.0毫克的粗製脂質至一50毫升體積的燒瓶之内以及轉移一整分部分至一 HPLC小玻璃瓶用於 HPLC/ELSD/MS分析來測定該經萃取之粗製脂質的脂質種類剖繪。依據HPLC/ELSD/MS分析，該粗製脂質含有0.2% 固醇酯（SE)、95.1% TAG、0.4%固醇，以及0.5% 1，2-二醯甘油酯（DAG)。TAG餾分的5%含括了在TAG峰後直接洗提出之一峰，但並未產生一個可辨識的質譜。來自此樣品之經單離的TA G當藉由急驟層析法來決定時構成了大概92.4%的粗製油。在SPE單離作用後，藉由重量或TLC並未檢測到PL。TAG内含有之主要的脂肪酸(>50 毫克/克)為C16:0(189毫克/克）、C20:5 n-3(197毫克/克），以及C22:6n-3(441 毫克/克）。 PTA-10208 樣品#2 PTA-10208樣品#2之生質與經萃取之粗製脂質的脂肪酸剖繪係使用GC/FID予以決定。生質内的脂肪酸係藉由稱重32.0毫克的生質直接地至一FAME管中而予以原位轉酯化，而該經萃取之粗製脂質的一樣品係藉由稱重60.1毫克的粗製脂質至一50毫升體積的燒瓶之内以及轉移1毫升至 179 201200591 一單獨的FAME管來製備。該生質的估計粗製脂質含量使用具FID檢測之GC經判定為52.4°/。（以FAME的總和），同時 48.0%(wt/wt)脂質係自該乾的生質予以萃取’提供91.7。/〇總脂質的回收率。該粗製脂質使用GC/FID經判定為95.3%脂肪酸（以F A ME的總和）。該粗製脂質内含有的主要脂肪酸為〇16:0(217.5毫克/克）、〇20:5 11-3(169.3毫克/克），以及〇22:6 n-3(444.1 毫克/克）。藉由稱重60.1毫克的粗製脂質至一 50毫升體積的燒瓶之内以及轉移一整分部分至一 HPLC小玻璃瓶用於 HPLC/ELSD/MS分析來測定該經萃取之粗製脂質的脂質種類剖繪。依據HPLC/ELSD/MS分析，該粗製脂質含有0.2% SE、95.70/〇 TAG、0.3%固醇，以及0.7% 1，2-DAG。TAG餾分的5.1%含括了在TAG峰後直接洗提出之一峰，但並未產生一個可辨識的質譜。來自此樣品之經單離的TAG構成了大概93.9%的粗製油。在SPE單離作用後，藉由重量或TLC並未檢測到PL» TAG 内含有之主要的脂肪酸(>50毫克/克）為C16:0 (218毫克/ 克）、C20:5 n-3(167毫克/克）以及C22:6 n-3 (430毫克/克）。 PTA-10208 樣品 #3 來自根據ATCC存取號碼PTA-10208所寄存之微生物的粗製油之一樣品（樣品PTA-10208 #3)係使用 HPLC/ELSD/MS予以分析。回收了總計98.38%的脂質，帶有佔0.32%之固醇酯（SE)餾份、佔96.13%之TAG餾份、佔 0.22%之1,3-二醯甘油酯（DAG)餾份、佔0.78%之1,2-DAG餾 180 201200591 份，以及佔0.93%之固醇餾份。 PTA-10212 樣品 #1 PTA-10212樣品#1之生質與經萃取之粗製脂質的脂肪酸剖繪係使用GC/FID予以決定。生質内的脂肪酸係藉由稱重27.0毫克的生質直接地至一 FAME管中而予以原位轉酯化，而該經萃取之粗製脂質的一樣品係藉由稱重52.5毫克的粗製脂質至一50毫升體積的燒瓶之内以及轉移1毫升至一單獨的FAME管來製備。該生質的估計粗製脂質含量使用具FID檢測之GC經判定為38.3%(以FAME的總和），同時 36.3%(wt/wt)脂質係自該乾的生質予以萃取，提供94.6%總脂質的回收率。該粗製脂質使用GC/FID經判定為83.2%脂肪酸（以FAME的總和）。該粗製脂質内含有之主要脂肪酸為 C16:0(328.5毫克/克）、C20:5 n-3(90.08毫克/克），以及C224 n_3(289.3毫克/克）。藉由稱重52.5毫克的粗製脂質至一 50毫升體積的燒瓶之内以及轉移整分部分至一 HPLC小玻璃瓶用於 HPLC/ELSD/MS分析來測定該經萃取之粗製脂質的脂質種類剖繪。依據HPLC/ELSD/MS分析，該粗製脂質含有0.2% SE、64.2% TAG、1.9% FFA、2.8% 1，3-DAG、1.4%固醇、 18.8% 1,2-DAG，以及0.5% MAG。TAG餾分的3.4%含括了在TAG峰後直接洗提出之一峰，但並未產生一個可辨識的質譜。來自此樣品之經單離的TAG構成了大概49.8%的粗製油。經單離的PL構成大概8.1%的該粗製油。TAG餾份内含 181 201200591 有之主要的脂肪酸(>50毫克/克）為C16:0(400毫克/克）、 C20:5 n-3(91 毫克/克），以及C22:6 n-3(273毫克/克）°PL餾份内含有之主要的脂肪酸(>50毫克/克）為C16:0 (98毫克/ 克）' C20:5 n-3(33毫克/克），以及C22:6 n-3(227毫克/克）。 PTA-10212 樣品 #2 PTA-10212樣品#2之生質與經萃取之粗製脂質的脂肪酸剖繪係使用GC/FID予以決定。生質内的脂肪酸係藉由稱重29.5毫克的生質直接地至一FAME管中而予以原位轉酯化，而該經萃取之粗製脂質的一樣品係藉由稱重56.5毫克的粗製脂質至一50毫升體積的燒瓶之内以及轉移1毫升至一單獨的FAME管來製備。該生質的估計粗製脂質含量使用具FID檢測之GC經判定為40_0%(以FAME的總和），同時 41.3%(wt/wt)脂質係自該乾的生質予以萃取，提供106.1%的總脂質回收率。該粗製脂質使用GC/FID經判定為82.8%脂肪酸（以FAME的總和）。該粗製脂質内含有之主要的脂肪酸為C16:0(327.3毫克/克）、C20:5 n-3(92.5毫克/克）’以及C22:6 n-3(277.6毫克/克）。藉由稱重56.5毫克的粗製脂質至一50毫升體積的燒瓶之内以及轉移一整分部分至一 HPLC小玻璃瓶用於 HPLC/ELSD/MS #才斤I ^ $ I亥& # t月旨質#月旨質5¾ 類剖繪。依據HPLC/ELSD/MS分析，該粗製脂質含有0.2% SE、58.20/〇 TAG、2.3% FFA、3.4% 1，3-DAG、1.7%固醇、 23.4%1，2-〇八〇，以及0.6%]\4八〇。丁八〇德分的3.3%含括了在TAG峰後直接洗提出之一峰，但並未產生一個可辨識的 182 201200591 質譜。來自此樣品之經單離的TAG構成了大概51.9%的粗製油。經單離的PL構成大概8.8%的該粗製油。TAG餾份内含有之主要的脂肪酸（>50毫克/克）為C16:0(402毫克/克）、 C20:5 n-3(92毫克/克），以及C22:6 n-3(245毫克/克）。PL餾份内含有之主要的脂肪酸(>50毫克/克）為C16:0(121毫克/ 克）、C20:5 n-3(48毫克/克），以及C22:6n-3 (246毫克/克）。 183 201200591 表32 : PTA-10208和PTA-10212生質與經萃取之粗製脂質的脂肪酸剖繪（毫克/克） PTA-10208 樣品#1 生質 PTA-10208 樣品#1 粗製脂質 PTA-10208 樣品#2 生質 PTA-10208 樣品#2 粗製脂質 PTA-10212 樣品#1 生質 PTA- 10212 樣品#1 粗製脂質 PTA-10212 樣品#2 生質 PTA-10212 樣品#2 粗製脂質脂肪酸 FAME (毫克/克） FAME (去克/克） FAME (€克/克） FAME (毫克/克） FAME (4免/克） FAME (毫克/克） FAME (秦克/克） FAME (i免/克） C12:0 1.47 2.43 1.80 3.14 0.99 1.90 0.87 1.91 C14:0 11.62 20.12 16.72 31.03 5.51 12.91 5.97 13.69 C14:l 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 2.43 3.75 3.60 6.22 9.13 20.42 9.39 20.81 C16:0 105.04 182.47 117.72 217.49 145.87 328.45 147.87 327.27 C16:l 0.00 0.00 0.06 0.01 6.26 14.53 7.46 16.89 C18:0 5.37 8.96 4.77 8.37 6.77 15.39 6.77 15.15 C18:l n-9 0.00 3.26 0.00 3.09 0.03 4.04 0.08 5.87 C18:l n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:0 1.48 1.79 1.40 1.85 1.60 3.09 1.67 3.20 C18:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:4 n-3 0.91 1.61 1.10 2.00 2.28 2.56 2.21 2.64 C20:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C22:0 0.10 0.00 0.08 0.00 0.30 0.12 0.35 0.24 C20:4 n-7 0.81 0.45 0.67 0.41 0.00 0.00 0.00 0.00 C20:4 n-6 7.22 12.23 6.84 12.18 1.19 2.26 1.31 2.32 C22:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-5 0.63 0.52 0.00 0.46 0.00 0.00 0.00 0.00 C20:4 n-3 3.45 5.45 3.33 5.58 0.00 2.40 0.00 2.34 C20:3 n-3 0.09 0.00 0.11 0.00 0.00 0.00 0.00 0.00 C20:5 n-3 107.31 186.83 92.99 169.32 40.32 90.08 43.15 92.54 C22:4 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C24:0 0.60 0.00 0.52 0.00 2.81 6.83 2.74 6.53 C24:l n-9 1.55 3.26 0.85 2.04 0.43 1.34 0.42 1.24 C22:5 n-6 9.66 15.84 10.27 17.98 2.42 4.68 2.32 4.21 C22:5 n-3 20.44 35.13 9.92 17.50 2.41 4.94 2.69 5.23 C22:6 n-3 246.98 423.10 245.96 444.08 139.58 289.34 137.35 277.57 FAME 的總和 527.15 907.18 518.71 942.75 367.89 805.29 372.63 799.68 184 201200591 表33 : PTA-10208和PTA-10212生質與經萃取之粗製脂質的脂肪酸剖繪(％) PTA-10208 樣品#1 生質 PTA- 10208 樣品#1 粗製脂質 PTA- 10208 樣品#2 生質 PTA- 10208 樣品#2 粗製脂質 PTA- 10212 樣品#1 生質 PTA- 10212 樣品#1 粗製脂質 PTA- 10212 樣品#2 生質 PTA- 10212 樣品#2 粗製脂質脂肪酸 % FAME % FAME % FAME % FAME % FAME % FAME % FAME % FAME C12:0 0.28 0.27 0.35 0.33 0.27 0.24 0.23 0.24 C14:0 2.20 2.22 3.22 3.29 1.50 1.60 1.60 1.71 C14:l 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 0.46 0.41 0.69 0.66 2.48 2.54 2.52 2.60 C16:0 19.93 20.11 22.70 23.07 39.65 40.79 39.68 40.93 C16:l 0.00 0.00 0.01 0.00 1.70 1.80 2.00 2.11 C18:0 1.02 0.99 0.92 0.89 1.84 1.91 1.82 1.89 C18:l n-9 0.00 0.36 0.00 0.33 0.01 0.50 0.02 0.73 C18:l n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:0 0.28 0.20 0.27 0.20 0.43 0.38 0.45 0.40 C18:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:l n-9 0.00 0.00 0.00 0.00 0.00 . 0.00 0.00 0.00 C18:4 n-3 0.17 0.18 0.21 0.21 0.62 0.32 0.59 0.33 C20:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C22:0 0.02 0.00 0.01 0.00 0.08 0.02 0.09 0.03 C20:4 n-7 0.15 0.05 0.13 0.04 0.00 0.00 0.00 0.00 C20:4 n-6 1.37 1.35 1.32 1.29 0.32 0.28 0.35 0.29 C22:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-5 0.12 0.06 0.00 0.05 0.00 0.00 0.00 0.00 C20:4 n-3 0.65 0.60 0.64 0.59 0.00 0.30 0.00 0.29 C20:3 n-3 0.02 0.00 0.02 0.00 0.00 0.00 0.00 0.00 C20:5 n-3 20.36 20.59 17.93 17.96 10.96 11.19 11.58 11.57 C22:4 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C24:0 0.11 0.00 0.10 0.00 0.76 0.85 0.74 0.82 C24:l n-9 0.29 0.36 0.16 0.22 0.12 0.17 0.11 0.16 C22:5 n-6 1.83 1.75 1.98 1.91 0.66 0.58 0.62 0.53 C22:5 n-3 3.88 3.87 1.91 1.86 0.65 0.61 0.72 0.65 C22:6 n-3 46.85 46.64 47.42 47.10 37.94 35.93 36.86 34.71 FAME %的總和 100 100 100 100 100 100 100 100 185 201200591 表34: PTA-10208和PTA-10212經單離的TAG之脂肪酸剖繪 PTA- 10208 樣品#1 PTA-10208 樣品#1 PTA- 10208 樣品#2 PTA-10208 樣品#2 PTA-10212 樣品#1 PTA-10212 樣品#1 PTA- 10212 樣品#2 PTA- 10212 樣品#2 脂肪酸 FAME (毫克/克） % FAME FAME (毫克/克） % FAME FAME (毫克/克） % FAME FAME (毫克/克） % FAME C12:0 2.57 0.27 3.35 0.36 0.00 0.00 0.00 0.00 C14:0 21.07 2.23 31.37 3.41 14.05 1.61 14.45 1.69 C14:l 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 3.89 0.41 6.17 0.67 23.27 2.66 23.14 2.71 C16:0 189.28 20.07 218.78 23.75 399.51 45.75 402.43 47.07 C16:l 0.00 0.00 0.00 0.00 15.23 1.74 17.62 2.06 C18:0 9.21 0.98 8.07 0.88 22.70 2.60 23.10 2.70 C18:l n-9 3.35 0.36 3.64 0.40 6.12 0.70 7.48 0.87 C18:l n-7 0.00 0.00 0.00 0.00 <0.1 <0.1 <0.1 <0.1 C18:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:0 1.86 0.20 1.55 0.17 4.76 0.55 5.32 0.62 C18:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:4 n-3 1.64 0.17 2.00 0.22 2.25 0.26 2.24 0.26 C20:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C22:0 0.00 0.00 0.00 0.00 0.55 0.06 0.89 0.10 未知 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-7 0.39 0.04 0.05 0.01 0.00 0.00 0.00 0.00 C20:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-6 12.79 1.36 11.82 1.28 2.33 0.27 2.25 0.26 C22:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-5 0.39 0.04 0.07 0.01 0.00 0.00 0.00 0.00 C20:4 n-3 5.52 0.59 5.09 0.55 2.87 0.33 2.98 0.35 C20:5 n-3 197.14 20.90 166.68 18.10 91.17 10.44 91.78 10.74 C24:0 0.00 0.00 0.00 0.00 6.93 0.79 7.36 0.86 C22:4 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C24:l n-9 1.08 0.11 <0.1 <0.1 0.00 0.00 0.00 0.00 C22:5 n-6 15.88 1.68 16.57 1.80 4.01 0.46 3.39 0.40 C22:5 n-3 36.05 3.82 16.00 1.74 4.53 0.52 5.07 0.59 C22:6 n-3 440.99 46.76 429.83 46.67 273.02 31.26 245.38 28.70 FAME 的總和 943.11 - 921.03 - 873.31 - 854.89 - 總％ FAME - 100.00 - 100.00 - 100.00 - 100.00 186 201200591 表35 : PTA-10212經單離的PL之脂肪酸剖繪 PTA-10212 樣品#1 PTA-10212 樣品#1 PTA-10212 樣品#2 PTA-10212 樣品#2 脂肪酸 FAME (毫免/克） % FAME FAME (毫克/克） % FAME C12:0 0.00 0.00 0.00 0.00 C14:0 0.93 0.22 1.89 0.39 C14:l 0.00 0.00 0.00 0.00 C15:0 3.44 0.82 5.07 1.05 C16:0 98.29 23.50 120.98 25.00 C16:l 1.15 0.27 3.07 0.63 C18:0 3.25 0.78 3.72 0.77 C18:l n-9 1.12 0.27 0.95 0.20 C18:l n-7 <0.1 <0.1 0.02 0.003 C18:2n-6 0.00 0.00 0.00 0.00 C20:0 <0.1 <0.1 <0.1 <0.1 C18:3 n-3 0.00 0.00 0.00 0.00 C20:l n-9 0.00 0.00 0.00 0.00 C18:4 n-3 3.71 0.89 3.24 0.67 C20:2 n-6 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 C22:0 0.00 0.00 0.00 0.00 未知 42.33 10.12 44.71 9.24 C20:4 n-7 0.00 0.00 0.00 0.00 C20:3 n-3 0.00 0.00 0.00 0.00 C20:4 n-6 0.84 0.20 1.54 0.32 C22:l n-9 0.00 0.00 0.00 0.00 C20:4 n-5 0.00 0.00 0.00 0.00 C20:4 n-3 <0.1 <0.1 0.27 0.06 C20:5 n-3 33.39 7.98 47.91 9.90 C24:0 <0.1 <0.1 0.01 0.001 C22:4 n-9 0.00 0.00 0.00 0.00 C24:l n-9 0.00 0.00 0.00 0.00 C22:5 n-6 3.08 0.74 3.82 0.79 C22:5 n-3 <0.1 <0.1 0.66 0.14 C22:6 n-3 226.68 54.20 246.09 50.85 FAME的總和 418.21 - 483.94 - 總％ FAME - 100 - 100 187 201200591 PTA-10212 樣品 #3 ΡΤΑ-10212樣品#3之生質中的脂質含量經估算為34% 以FAME之總和，以及在溶劑萃取作用後所得的粗製油量為 37 %重量計，提供生質中所存在的脂肪之1〇9%回收率。在使用急驟層析法之分潑之後，大概46%的該粗製油係單離為TAG，28%係單離為DAG。該粗製油含有3〇9毫克/克的 DHA和毫克/克的EPA。該經單離的TAG含有341毫克/克的DHA和274毫克/克的EPA。該經單離的dag餘份含有262 毫克/克的DHA和237毫克/克的EPA。該生質、經萃取的粗製油，以及經單離的餾份之總脂肪酸剖繪係分別計算為毫克/克及％FAME而顯示於以下的表36和表37中。 188 201200591 表36:ΡΤΑ-10212樣品#3生質與經萃取之粗製脂質的脂肪酸别繪（毫克/克）生質粗製油 TAG DAG Wt. % ΝΑ 37.2% 46.0% 27.9% 脂肪酸 FAME (毫克/克） FAME (臺克/克） FAME (毫免/克） FAME (毫克/克） C12:0 0.0 0.0 0.0 0.0 C14:0 3.6 10.3 11.5 9.4 C14:l 0.0 0.0 0.0 0.0 C15:0 4.1 10.6 9.8 6.6 C16:0 70.5 181.8 231.7 111.3 C16:l 6.7 19.1 18.7 17.1 C18:0 2.4 10.2 14.2 0.0 C18:l n-9 0.0 6.7 0.0 0.0 C18:l n-7 0.0 1.2 0.0 0.0 C18:2 n-6 0.0 1.8 0.0 0.0 C20:0 0.0 2.4 0.0 0.0 C18:3 n-3 0.0 0.0 0.0 0.0 C20:l n-9 0.0 0.3 0.0 1.7 C18:4 n-3 1.9 3.4 3.2 4.4 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 3.3 0.0 0.0 0.0 C20:4 n-7 0.0 2.1 1.5 0.0 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 6.8 17.9 21.4 13.8 C22:l n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 1.3 1.3 0.0 C20:4 n-3 3.0 8.5 10.9 6.4 C20:5 n-3 102.0 263.6 274.2 237.4 C24:0 0.0 1.7 3.9 0.0 C22:4 n-9 0.0 0.0 0.0 0.0 C24:l n-9 0.0 0.0 4.2 0.0 C22:5 n-6 3.2 8.3 10.7 6.1 C22:5 n-3 3.8 10.4 15.1 6.6 C22:6 n-3 131.2 309.4 341.1 261.9 FAME的總和 342.4 871.1 973.2 682.6 189 201200591 表37 : PTA-10212樣品#3生質與經萃取之粗製脂質的脂肪酸剖繪(％) 生質粗製油 TAG DAG Wt. % ΝΑ 37.2% 46.0% 27.9% 脂肪酸 FAME (毫克/克） FAME (毫克/克） FAME (毫龙/克） FAME (毫免/克） C12:0 0.0 0.0 0.0 0.0 C14:0 1.1 1.2 1.2 1.4 C14:l 0.0 0.0 0.0 0.0 C15:0 1.2 1.2 1.0 1.0 C16:0 20.6 20.9 23.8 16.3 C16:l 2.0 2.2 1.9 2.5 C18:0 0.7 1.2 1.5 0.0 C18:l n-9 0.0 0.8 0.0 0.0 C18:l n-7 0.0 0.1 0.0 0.0 C18:2 n-6 0.0 0.2 0.0 0.0 C20:0 0.0 0.3 0.0 0.0 C18:3 n-3 0.0 0.0 0.0 0.0 C20:l n-9 0.0 0.0 0.0 0.2 C18:4 n-3 0.6 0.4 0.3 0.6 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 1.0 0.0 0.0 0.0 C20:4 n-7 0.0 0.2 0.2 0.0 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 2.0 2.1 2.2 2.0 C22:l n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.1 0.1 0.0 C20:4 n-3 0.9 1.0 1.1 0.9 C20:5 n-3 29.8 30.3 28.2 34.8 C24:0 0.0 0.2 0.4 0.0 C22:4 n-9 0.0 0.0 0.0 0.0 C24:l n-9 0.0 0.0 0.4 0.0 C22:5 n-6 0.9 1.0 1.1 0.9 C22:5 n-3 1.1 1.2 1.6 1.0 C22:6 n-3 38.3 35.5 35.1 38.4 總％ FAME 100.0 100.0 100.0 100.0 190 201200591 PTA-10212 樣品 #4 PTA-10212樣品#4含有以FAME之總和來測定之大概 23°/〇的脂質，使用己烷萃取作用回收其中的1〇7%。在使用急驟層析法之分餾之後，大概42%的該粗製油係單離為 TAG，18%係單離為DAG。該粗製油含括275毫克/克的DHA 和209毫克/克的EPA。該經單離的TAG含有296毫克/克的 DHA和205毫克/克的EPA。該經單離的DAG餾份含有245毫克/克的DHA和219毫克/克的EPA。該生質、經萃取的粗製油，以及經單離的餾份之總脂肪酸剖繪係顯示於以下的表 38(毫克/克)和表39(%FAME)中。 191 201200591 表38 : PTA-10212樣品#4生質與經萃取之粗製脂質的脂肪酸剖繪(毫克/克）生質粗製油 TAG DAG wt. % ΝΑ 24.7% 42.2% 18.4% 脂肪酸 FAME (毫克/克） FAME (毫克/克） FAME (i克/克） FAME (i：见/克） C12:0 0.0 0.0 2.1 2.4 C14:0 2.0 8.3 9.8 9.6 C14:l 0.0 0.0 0.0 0.0 C15:0 4.8 16.8 0.4 0.9 C16:0 63.3 210.6 285.7 138.0 C16:l 1.6 6.7 7.4 7.5 C18:0 2.8 12.2 19.9 4.6 C18:l n-9 0.0 3.7 0.7 1.1 C18:l n-7 0.0 0.0 0.3 1.2 C18:2n-6 0.0 0.0 0.0 0.0 C20:0 0.0 3.3 6.0 1.5 C18:3 n-3 0.0 0.0 0.0 0.0 C20:l n-9 0.0 0.0 0.7 1.2 C18:4 n-3 1.4 3.8 3.6 5.0 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.4 0.0 C22:0 1.5 0.0 1.9 0.0 C20:4 n-7 0.0 0.0 0.9 0.6 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 2.5 10.1 13.0 10.3 C22:l n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.0 0.8 0.7 C20:4 n-3 1.4 6.3 8.6 6.0 C20:5 n-3 57.6 209.1 205.4 219.0 C24:0 0.0 2.6 0.8 0.0 C22:4 n-9 0.1 0.0 0.0 0.0 C24:l n-9 0.0 0.0 1.1 0.5 C22:5 n-6 1.4 6.1 7.9 4.5 C22:5 n-3 4.0 15.8 20.8 12.9 C22:6 n-3 87.7 275.0 296.4 244.8 FAME的總和 232.2 790.1 894.8 672.4 192 201200591 表39:ΡΤΑ-10212樣品#4生質與經萃取之粗製脂質的脂肪酸剖繪(％) 生質粗製油 TAG DAG Wt. % ΝΑ 24.7% 42.2% 18.4% 脂肪酸 FAME (i:免/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） C12:0 0.0 0.0 0.2 0.4 C14:0 0.9 1.0 1.1 1.4 C14:l 0.0 0.0 0.0 0.0 C15:0 2.1 2.1 0.0 0.1 C16:0 27.3 26.7 31.9 20.5 C16:l 0.7 0.8 0.8 1.1 C18:0 1.2 1.5 2.2 0.7 C18:l n-9 0.0 0.5 0.1 0.2 C18:l n-7 0.0 0.0 0.0 0.2 C18:2n-6 0.0 0.0 0.0 0.0 C20:0 0.0 0.4 0.7 0.2 C18:3 n-3 0.0 0.0 0.0 0.0 C20:l n-9 0.0 0.0 0.1 0.2 C18:4 n-3 0.6 0.5 0.4 0.7 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 0.6 0.0 .0.2 0.0 C20:4 n-7 0.0 0.0 0.1 0.1 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 1.1 1.3 1.5 1.5 C22:l n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.0 0.1 0.1 C20:4 n-3 0.6 0.8 1.0 0.9 C20:5 n-3 24.8 26.5 23.0 32.6 C24:0 0.0 0.3 0.1 0.0 C22:4 n-9 0.0 0.0 0.0 0.0 C24:l n-9 0.0 0.0 0.1 0.1 C22:5 n-6 0.6 0.8 0.9 0.7 C22:5 n-3 1.7 2.0 2.3 1.9 C22:6 n-3 37.8 34.8 33.1 36.4 總％ FAME 100.0 100.0 100.0 100.0 193 201200591 PTA-10212 樣品 #5 一粗製油的樣品係使用FRIOLEX®方法（GEA Westfalia Separator UK Ltd.，Milton Keynes，England)而自 PTA-10212的一生質予以萃取以出產微生物油PTA-10212 樣品#5。個別的脂質種類係使用低壓急驟層析法而由 PTA-10212樣品#5予以單離，以及測定各類的重量百分比。各類的脂肪酸剖繪係使用GC/HD予以測定。簡單地說，該樣品係藉由將240毫克的粗製油溶解於 600 μι的己烷内以及施加至該管柱之頂部而製備。在使用急驟層析法之樣品的分餾之後，所有餾分的合併重量為240 毫克，而得1〇〇%的回收率。固醇酯餾份佔0.9%，TAG餾份佔42.6%，游離脂肪酸(FFA)餾份佔1.3%，固醇餾份佔2.2%，以及DAG餾份佔41.6%。FRIOLEX®粗製油及經單離的餾份之總脂肪酸剖繪係分別計算為毫克/克及％ FAME而顯示於以下的表40和表41中。 194 201200591 表40 : PTA-10212樣品#5粗製油之脂肪酸剖繪（毫克/克) 粗製油 TAG DAG wt. % ΝΑ 42.6% 41.6% 脂肪酸 FAME (毫克/克） FAME (秦克/克） FAME (毫克/克） C12:0 0 0.7 1.0 C14:0 7.7 7.7 8.5 C14:l 0 0.0 0.0 C15:0 10.3 11.7 9.3 C16:0 179.3 217.7 134.6 C16:l 18.1 16.3 25.9 C18:0 8.1 13.2 2.3 C18:l n-9 4.7 8.4 0.7 C18:l n-7 0 1.8 1.0 C18:2n-6 1.8 3.3 0.7 C20:0 1.9 3.6 0.2 C18:3 n-3 0 0.0 0.0 C20:l n-9 0 0.7 1.0 Cl8:4 n-3 3.1 2.8 3.8 C20:2 n-6 0 0.0 0.0 C20:3 n-6 0 0.6 0.4 C22:0 0 1.5 0.0 C20:4 n-7 0 1.0 0.7 C20:3 n-3 0 0.0 0.0 C20:4 n-6 12.7 16.1 13.6 C22:l n-9 0 0.0 0.0 C20:4 n-5 0 1.5 0.8 C20:4 n-3 6.5 9.3 6.4 C20:5 n-3 213.3 223.7 252.8 C24:0 2.3 4.4 0.6 C22:4 n-9 0 1.9 0.9 C24:l n-9 0 0.0 0.0 C22:5 n-6 7.9 9.5 8.3 C22:5 n-3 13 20.6 9.7 C22:6 n-3 305.6 327.4 353.8 FAME的總和 796.6 905.3 837.4 195 201200591 表41 : PTA-10212樣品#5粗製油之脂肪酸剖繪(％) 粗製油 TAG DAG 脂肪酸 % FAME % FAME % FAME C12:0 0 0.1 0.1 C14:0 1 0.9 1.0 C14:l 0 0.0 0.0 C15:0 1.3 1.3 1.1 C16:0 22.5 24.0 16.1 C16:l 2.3 1.8 3.1 C18:0 1 1.5 0.3 C18:l n-9 0.6 0.9 0.1 C18:l n-7 0 0.2 0.1 C18:2 n-6 0.2 0.4 0.1 C20:0 0.2 0.4 0.0 C18:3 n-3 0 0.0 0.0 C20:l n-9 0 0.1 0.1 C18:4 n-3 0.4 0.3 0.5 C20:2 n-6 0 0.0 0.0 C20:3 n-6 0 0.1 0.0 C22:0 0 0.2 0.0 C20:4 n-7 0 0.1 0.1 C20:3 n-3 0 0.0 0.0 C20:4 n-6 1.6 1.8 1.6 C22:l n-9 0 0.0 0.0 C20:4 n-5 0 0.2 0.1 C20:4 n-3 0.8 1.0 0.8 C20:5 n-3 26.8 24.7 30.2 C24:0 0.3 0.5 0.1 C22:4 n-9 0 0.2 0.1 C24:l n-9 0 0.0 0.0 C22:5 n-6 1 1.1 1.0 C22:5 n-3 1.6 2.3 1.2 C22:6 n-3 38.4 36.2 42.3 總％ FAME 100 100 100 196 201200591 實施例37 粗製油係經由精製、漂白、及脫臭步驟予以進一步加工來獲得精製油。使該精製油經高油酸的葵花油稀釋以獲得具有大概400毫克/克之DHA含量的最終油。個別的脂質種類係予以單離以及各類的脂肪酸剖繪係使用GC-FID予以測定為FAME。 PTA-10208最終油 PTA-10208最終油# 1 -5之脂肪酸剖繪係總結於表42-43 中，含括與該經單離的TAG餾份相關聯之剖繪（表44-45)以及與該經單離的固醇/DAG餾份相關聯之剖繪（表46-47)。最終油内之個別的脂質種類亦使用急驟層析法（表48) 及具有ELSD與APCI-MS驗證的正相HPLC(表49)來測定。 197 201200591 表42 : ΡΤΑ-1〇2〇8最終油的脂肪酸剖繪（毫克/克) 脂肪酸 PTA-10208 最終油#1 PTA-10208 最終油#2 PTA-10208 最終油#3 PTA-10208 最終油#4 PTA-10208 最終油#5 C12:0 FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） 2.5 2.4 2.8 2.7 2.7 C14:0 16.1 14.9 21.0 18.4 17.5 C14:l 0.0 0.0 0.0 0.0 0.0 C15:0 3.8 3.6 4.4 3.9 3.9 C】6:0 192.1 179.1 193.1 184.3 194.6 C16:l 0.4 0.5 0.5 0.5 0.5 C17:0 0.6 0.5 0.9 0.8 2.1 C18:0 12.8 13.9 11.5 12.3 12.9 C18:l n-9 23.5 82.0 25.7 26.0 29.5 C18:l n-7 0.2 0.7 0.1 0.1 0.1 Cl8:2 n-6 3.7 8.1 4.0 4.1 4.3 C20:0 4.3 4.1 3.7 4.0 4.0 Cl8:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:l n-9 <0.1 0.1 <0.1 <0.1 <0.1 Cl8:4 n-3 2.4 2.5 2.8 2.7 2.8 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 0.2 0.1 0.1 0.1 0.1 C22:0 1.2 1.8 1.0 1.1 1.1 C20:4 n-7 1.7 1.6 1.7 1.8 1.6 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 12.9 12.1 13.5 13.5 13.3 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 1.6 1.4 1.5 1.7 1.5 C20:4 n-3 6.0 5.7 6.0 6.0 6.1 C20:5 η·3 173.8 163.3 196.4 209.6 197.9 C24:0 1.4 1.6 1.3 1.3 1.0 C22:4 \n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 3.4 3.2 2.3 2.6 2.3 C22:5 n-6 14.9 14.0 14.4 13.0 12.9 C22:5 n-3 43.9 41.3 32.8 40.3 36.9 C22:6 n-3 394.8 373.7 373.2 374.3 364.2 FAME的總和 918.1 932.2 914.7 925.1 914.1 198 201200591 表43 : PTA-10208最終油的脂肪酸剖繪(％) PTA-10208 最終油#1 PTA-10208 最終油#2 PTA-10208 最終油#3 PTA-10208 最終油#4 PTA-10208 最終油#5 脂肪酸 % FAME % FAME % FAME % FAME % FAME C12:0 0.3 0.3 0.3 0.3 0.3 C14:0 1.8 1.6 2.3 2.0 1.9 C14:l 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.5 0.4 0.4 C16:0 20.9 19.2 21.1 19.9 21.3 C16:l <0.1 <0.1 <0.1 <0.1 0.1 C17:0 0.1 0.1 0.1 0.1 0.2 C18:0 1.4 1.5 1.3 1.3 1.4 C18:l n-9 2.6 8.8 2.8 2.8 3.2 C18:l n-7 <0.1 0.1 <0.1 <0.1 <0.1 C18:2 n-6 0.4 0.9 0.4 0.4 0.5 C20:0 0.5 0.4 0.4 0.4 0.4 C18:3n-3 0.0 0.0 0.0 0.0 0.0 C20:l n-9 <0.1 <0.1 <0.1 <0.1 <0.1 Cl 8:4 n-3 0.3 0.3 0.3 0.3 0.3 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.2 0.2 0.2 0.2 0.2 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.4 1.3 1.5 1.5 1.5 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 \n-5 0.2 0.2 0.2 0.2 0.2 C20:4 \n-3 0.7 0.6 0.7 0.7 0.7 C20:5 n-3 18.9 17.5 21.5 22.7 21.6 C24:0 0.1 0.2 0.1 0.1 0.1 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 0.4 0.3 0.2 0.3 0.2 C22:5 n-6 1.6 1.5 1.6 1.4 1.4 C22:5 n-3 4.8 4.4 3.6 4.4 4.0 C22:6 n-3 43.0 40.1 40.8 40.5 39.9 199 201200591 表—雒的TAG脂肪酸剖繪:PTA-10208最終油(毫克/克) PTA-10208 最終油#1 PTA-10208 最終油#2 PTA-10208 最終油#3 PTA-10208 最終油#4 PTA-10208 最終油#5 脂肪酸 FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） C12:〇 2.5 2.3 2.7 2.5 2.6 C14:〇 16.3 15.1 21.3 18.6 18.1 C14:l 0.0 0.0 0.0 0.0 0.0 C15:〇 3.9 3.6 4.4 4.0 4.0 C16:〇 194.2 181.9 196.1 186.1 199.8 C16:l 0.4 0.4 0.6 0.5 0.7 C17:〇 0.6 0.5 0.9 0.8 0.8 C18:〇 12.9 14.2 11.7 12.5 13.2 Cl 8:1 n-9 24.3 84.0 26.8 26.1 34.0 Cl 8:1 n-7 0.1 0.7 0.1 0.1 0.3 Cl8:2 n-6 3.2 7.7 3.4 3.5 4.0 C20:0 4.4 4.2 3.8 4.0 4.2 Cl8:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:l n-9 <0.1 0.2 <0.1 <0.1 0.1 Cl 8:4 n-3 2.5 2.4 2.8 2.6 2.7 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 0.2 0.2 0.1 0.1 0.1 C22:0 1.2 1.9 1.0 1.1 1.1 C20:4 n-7 1.7 1.6 1.8 1.8 1.7 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 13.2 12.3 13.8 13.7 13.8 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 1.6 1.5 1.6 1.7 1.5 C20:4 n-3 6.1 5.7 6.1 5.9 6.2 C20:5 n-3 176.0 166.1 199.0 211.2 204.2 C24:0 1.2 1.3 1.0 1.1 1.2 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 3.3 3.2 2.2 2.5 2.4 C22:5 n-6 15.0 14.2 14.7 13.2 13.5 C22:5 n-3 44.4 42.0 33.3 40.5 38.3 C22:6 n-3 397.9 378.4 376.4 375.5 375.5 FAME的總和 926.9 945.7 925.5 929.6 944.1 200 201200591 表45 :經單離的TAG脂肪酸剖繪：ΡΤΑ-1〇2〇8最終油（％) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 最終油#1 最終油#2 最終油#3 最終油#4 最終油#5 脂肪酸 % FAME % FAME % FAME % FAME % FAME C12:0 0.3 0.2 0.3 0.3 0.3 C14:0 1.8 1.6 0.3 0.3 0.3 C14:l 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.5 0.4 0.4 C16:0 20.9 19.2 21.2 20.0 21.2 C16:l <0.1 <0.1 0.1 0.1 0.1 C17:0 0.1 0.1 0.1 0.1 0.1 C18:0 1.4 1.5 1.3 1.3 1.4 C18:l n-9 2.6 8.9 2.9 2.8 3.6 C18:l n-7 <0.1 0.1 <0.1 <0.1 <0.1 C18:2 n-6 0.3 0.8 0.4 0.4 0.4 C20:0 0.5 0.4 0.4 0.4 0.4 C18:3 n-3 0.0 0.0 0.0 0.0 0,0 C20:l n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4 n-3 0.3 0.3 0.3 0.3 0.3 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.2 0.2 0.2 0.2 0.2 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.4 1.3 1.5 1.5 1.5 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 0.2 0.2 0.2 0.2 0.2 C20:4 n-3 0.7 0.6 0.7 0.6 0.7 C20:5 n-3 19.0 17.6 21.5 22.7 21.6 C24:0 0.1 0.1 0.1 0.1 0.1 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 0.4 0.3 0.2 0.3 0.3 C22:5 n-6 1.6 1.5 1.6 1.4 1.4 C22:5 n-3 4.8 4.4 3.6 4.4 4.1 C22:6 n-3 42.9 40.0 40.7 40.4 39.8 201 201200591 (表毫單離的师漏脂肪心幢：ΡΤΑ·刪8最終油 PTA-10208 最終油#1 PTA-10208 最終油#2 PTA-10208 最終油#3 PTA-10208 最終油#4 PTA-10208 最終油#5 脂肪酸 FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） FAME (毫克/克） C12:〇 1.9 2.1 2.9 2.1 1.9 C14:0 9.9 9.5 9.7 10.3 8.0 C14:l 0.0 0.0 0.0 0.0 0.0 C15:〇 2.4 2.3 2.2 2.3 2.0 C16:〇 132.6 128.6 110.1 116.8 106.4 C16:l 0.2 0.3 <0.1 0.3 0.4 C17:0 0.3 0.2 0.3 0.3 0.3 C18:0 7.3 8.1 6.4 6.8 6.1 Cl 8:1 n-9 15.0 55.1 47.4 19.0 30.1 C18:l n-7 0.4 0.7 0.1 <0.1 0.2 Cl8:2 n-6 13.1 16.7 21.6 13.5 18.4 C20:0 2.0 2.1 1.2 1.8 1.4 Cl8:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:l n-9 <0.1 <0.1 <0.1 <0.1 <0.1 Cl8:4 n-3 2.3 2.4 2.4 2.4 2.0 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.6 1.0 0.5 0.6 0.5 C20:4 n-7 0.8 0.9 2.1 0.9 0.7 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 5.7 5.8 4.8 6.1 4.5 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 <0.1 <0.1 <0.1 0.6 <0.1 C20:4 n-3 2.7 2.7 2.1 2.7 2.0 C20:5 n-3 92.9 94.5 91.9 111.6 84.8 C24:0 1.2 1.3 1.1 1.1 1.3 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 1.9 2.0 1.2 1.5 1.2 C22:5 n-6 7.8 8.0 6.7 7.0 5.5 C22:5 n-3 22.2 22.9 13.9 20.7 14.2 C22:6 n-3 246.3 252.7 223.5 240.3 196.3 FAME的總和 569.3 619.8 552.1 568.7 488.2 202 201200591 表47:經單離的固醇/DAG脂肪酸剖繪：PTA-10208最終油(％) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 最終油#1 最終油#2 最終油#3 最終油#4 最終油#5 脂肪酸 % FAME % FAME % FAME % FAME % FAME C12:0 0.3 0.3 0.5 0.4 0.4 C14:0 1.7 1.5 1.8 1.8 1.6 C14:l 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.4 0.4 0.4 Cl 6:0 23.3 20.8 19.9 20.5 21.8 C16:l <0.1 <0.1 <0.1 <0.1 0.1 C17:0 0.0 0.0 0.1 0.1 0.1 C18:0 1.3 1.3 1.2 1.2 1.2 Cl 8:1 n-9 2.6 8.9 8.6 3.3 6.2 C18:l n-7 0.1 0.1 <0.1 <0.1 <0.1 Cl8:2 n-6 2.3 2.7 3.9 2.4 3.8 C20:0 0.4 0.3 0.2 0.3 0.3 Cl8:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:l n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4n-3 0.4 0.4 0.4 0.4 0.4 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.1 0.1 0.4 0.2 0.1 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.0 0.9 0.9 1.1 0.9 C22-.1 n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 <0.1 <0.1 <0.1 0.1 <0.1 C20:4 n-3 0.5 0.4 0.4 0.5 0.4 C20:5 n-3 16.3 15.2 16.6 19.6 17.4 C24:0 0.2 0.2 0.2 0.2 0.3 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 0.3 0.3 0.2 0.3 0.2 C22:5 n-6 1.4 1.3 1.2 1.2 1.1 C22:5 n-3 3.9 3.7 2.5 3.6 2.9 C22:6 n-3 43.3 40.8 40.5 42.3 40.2 203 201200591 表48 :藉由急驟層析法之脂質種類的分離(wt%) 脂質種類之分離 PTA-10208 最終油#1 PTA-10208 最終油#2 PTA-10208 最終油#3 PTA-10208 最終油#4 PTA-10208 最終油#5 TAG 93.4 95.4 94.0 95.7 95.1 固醇/DAG 3.1 2.9 2.6 3.0 2.9 回收率(％) 96.5 98.3 96.6 98.7 98.0 表49 :藉由HPLC-ELSD之脂質種類的分離(wt%) 固醇酯 TAG FFA 固醇 1,3- DAG 1，2- DAG MAG 總計 PTA-10208 最終油#1 0.4 90.8 ND 0.8 0.5 0.5 N.D. 93.0 PTA-10208 最終油#2 0.4 88.5 ND 0.6 0.6 0.6 N.D. 90.7 PTA-10208 最終油#3 0.3 89.4 ND 0.8 0.6 0.5 N.D. 91.6 PTA-10208 最終油#4 0.3 88.0 ND 0.8 0.5 0.5 N.D. 90.1 PTA-10208 最終油#5 0.3 86.3 ND 0.7 0.8 0.5 N.D. 88.6 PTA-10208 最終油#6 0.36 100.76 ND 0.84 0.54 0.61 N.D. 103.11 ND=未檢測出 PTA-10212最终油 DHA係以41.63%與366.9毫克/克存在於？丁八-10212最終油中，而EPA係以16.52%存在。測定個別脂肪酸剖繪及總結於表50中。 204 201200591 表50 : PTA-10212最終油的脂肪酸剖繪(％FAME) 脂肪酸 % FAME C6:0 ND C7:0 ND C8:0 ND C9:0 ND C10:0 ND C11:0 ND C12:0 ND C13:0 ND C14:0 0.84 C14:l ND C15:0 1.33 C16:0 27.09 C16:l 1.03 C17:0 0.34 C17:l ND C18:0 1.26 C18:l n-9 2.14 C18:l n-7 0.18 C19:0 ND C18:2 n-6 0.58 C20:0 0.32 C18:3 n-3 ND C20:l n-9 ND C18:3 n-6 ND C20:2 n-6 0.26 C20:3 n-6 ND C22:0 0.14 C20:3 n-3 ND C20:4 n-6 1.34 C22:l n-9 ND C23:0 ND C20:5 n-3 16.53 C24:0 0.53 C24:l n-9 ND C22:5 n-6 1.50 C22:5 n-3 1.30 C22:6 n-3 41.63 未知 0.87 ND =未檢測出 205 201200591 實施例3 8 一個根據ATCC存取號碼PTA_1〇2〇8和1〇212所寄存之經單離的微生物之已二天的接種體燒瓶係於依據表3〇和31 之培養基中予以製備為一給料碳與氮之培養物。依據下列程序進行突變誘發：將一個無菌已T=2日的燒瓶中之大概5〇毫升，傾倒至— 個無菌的40毫升玻璃均質機中。該培養物在均質機中接受 50次猛衝。將該培養物量吸出以及過濾通過置於一個5〇毫升的無菌管中之一個無菌的50微米筛網過濾器（該篩網係使用作為留住菌落的較大型群集及同時讓較小的聚簇與單 —細胞通過該50微米篩網之一構件）。在一個無菌的5〇毫升管十收集全部的ί農縮浸出物。將浸軟的培養物進行渦旋並製成问至1 · 100倍位準的稀釋液。渦旋該等經稀釋浸出物樣品，然後添加200微升接種體至一個1〇〇 χ 15毫米、含有 4-5粒玻璃珠(3毫米玻璃珠)之培養基瓊脂培養瓜（ι〇〇χΐ5毫米）。將各平盤溫和地攪拌，以使得玻璃珠將接種體均勻地塗佈在平盤上。自平盤上移除玻璃珠及讓平盤加蓋靜置歷夺大概5刀鐘至乾燥。當該程序在暗光中進行時，將無菌通風櫥與鄰近(1域的燈光關閉。僅存在使該程序得以進行之間接與微弱之極少量的光。 §照射該等樣品時，將五重複的平盤的蓋移開，以及將平盤置於XL交聯器（Spectr〇nics Corporation，New York) 的底板上。該交聯器所輸送的功率係以微焦耳為單位以及其位準係尋求達到9〇%_95%的殺死率。五重複的對照平盤 206 201200591 係使用相同的操作程序來接種未經突變誘發的細胞。此等，，，田胞數目係使用來計算％殺科。—旦照射完祕，將平盤取出f換蓋子’以及依序以封口膜與銘猪包裹平盤。 4平盤在帛-週務必在黑暗巾生長，藉此其等無法修補受損的基因。將平盤置於22.5°C$中大約10天，然後計數菌落。當進行最終計數之後，以一個無菌的接種環挑取個別的菌落以及重複劃線於新的培養基平盤上。將各菌落分植在個別的平盤上。當平盤生長稠密時，使用一接種環採集一樣品，以及接種至含有50毫升的培養基之一個無菌的250毫升震盈燒瓶中。將此燒瓶置於22.5°C房中的一個200 rpm振盪器上。在T=7日，將該震盪燒瓶的培養物收穫至一個50毫升的無菌管中。測定pH值且離心樣品以收集生質小丸。將各樣品沖洗及再懸浮於異丙醇與蒸餾水的50 : 50混合物中，然後再離心。將所收集的小丸予以冷冰乾燥、稱重，以及進行FAME分析。於表51和52内之資料代表使用上述方法分別自菌株PTA-10208和PTA-10212所產生之突變體。 207 201200591 表51 : PTA-10208突變體脂肪酸對照 PTA-10208 突變體1 PTA-10209 突變體2 PTA-10210 突變體3 PTA-10211 % 08:0 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 % 12:0 0.11 0.10 0.22 0.19 % 12:1 0.00 0.00 0.00 0.00 % 13:0 0.19 0.19 0.15 0.16 % 13:1 0.00 0.00 0.00 0.00 % 14:0 1.94 1.82 2.98 2.59 % 14:1 0.00 0.00 0.00 0.00 % 15:1 2.66 2.22 1.76 1.66 % 16:0 24.87 24.97 23.71 25.01 % 16:1 0.20 0.25 0.07 0.07 % 16:2 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 % 17:0 1.49 1.21 0.62 0.66 % 18:0 1.13 1.14 0.91 1.01 % 18:1 η-9 0.07 0.07 0.06 0.06 % 18:1 η-7 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 % 18:3 η-6 0.00 0.00 0.05 0.04 % 18:3 η-3 0.09 0.08 0.17 0.14 % 18:4 η-3 0.00 0.00 0.00 0.00 % 20:0 0.31 0.33 0.24 0.30 % 20:1 η-9 0.00 0.04 0.00 0.00 % 20:2 0.00 0.00 0.05 0.00 % 20:3 η-9 0.00 0.00 0.00 0.00 % 20:3 η-6 0.12 0.13 0.08 0.04 % 20:3 η-3 0.42 0.42 0.08 0.06 %20:4ARA 0.68 0.67 1.44 1,11 % 20:5 η-3 ΕΡΑ 6.56 6.47 11.99 9.87 % 22:0 0.07 0.07 0.06 0.07 % 22:1 0.00 0.00 0.00 0.00 % 22:2 0.11 0.09 0.10 0.08 % 22:3 0.00 0.00 0.00 0.00 % 22:4 η-6 0.00 0.00 0.00 0.00 % 22:5 η-6 2.32 2.36 2.36 2.36 % 22:5 η-3 0.48 0.66 0.66 0.52 % 22:6 η-3 DHA 51.58 52.27 48.17 49.35 % 24:0 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 %脂肪 47.87 49.41 66.00 63.12 %未知 4.61 4.45 4.07 4.64 208 201200591 表52 : PTA-10212突變體脂肪酸對照 PTA-10212 突變體1 PTA-10213 突變體2 PTA-10214 突變體3 PTA-10215 % 08:0 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 % 12:0 0.00 0.00 0.00 0.00 % 12:1 0.00 0.00 0.00 0.00 % 13:0 0.00 0.00 0.21 0.20 % 13:1 0.00 0.00 0.00 0.00 % 14:0 0.68 0.77 0.62 0.97 % 14:1 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.00 0.00 % 16:0 17.36 19.94 15.27 23.61 % 16:1 1.45 2.33 1.40 2.57 % 16:2 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 % 17:0 0.20 0.21 0.18 0.27 % 18:0 0.78 0.82 0.79 0.81 % 18:1 η-9 0.00 0.00 0.00 0.00 % 18:1 η-7 0.18 0.27 0.20 0.19 % 18:2 0.00 0.00 0.00 0.00 % 18:3 η-6 0.00 0.00 0.00 0.00 % 18:3 η-3 0.00 0.00 0.00 0.00 % 18:4 η-3 0.00 0.00 0.00 0.00 % 20:0 0.00 0.00 0.00 0.00 % 20:1 η-9 0.00 0.00 0.00 0.00 % 20:2 0.00 0.00 0.00 0.00 % 20:3 η-9 0.00 0.00 0.00 0.00 % 20:3 η-6 0.00 0.00 0.00 0.00 % 20:3 η-3 0.90 0.77 0.99 0.66 %20:4ARA 1.43 1.32 1.65 0.72 % 20:5 η-3 ΕΡΑ 13.33 14.93 14.14 8.54 % 22:0 0.00 0.00 0.00 0.00 % 22:1 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 % 22:4 η-6 0.00 0.00 0.00 0.00 % 22:5 η-6 2.39 1.95 2.59 2.18 % 22:5 η-3 0.73 0.79 0.80 0.68 % 22:6 η-3 DHA 59.18 54.31 59.89 56.39 % 24:0 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00 %脂肪 45.69 38.08 42.88 48.48 %未知 1.38 1.58 1.27 2.19 209 201200591 實施例39 將含有微生物細胞（裂殖壺菌屬）之2種細胞肉湯（大概 13.3 kg)加熱至60°C於一20公升發酵器中。該發酵器具有2 個具有15 cm的一直徑之6-葉片拉什頓葉輪。頂葉輪係放在 12公升標記的位置以及底葉輪係放在頂葉輪之下10 cm的位置。第一種細胞肉湯係以307公分/秒予以連續不斷地攪拌。第二種細胞肉湯係以464公分/秒予以連續不斷地攪拌。將酵素（亦即，鹼性蛋白酶(Alcalase)2.4 L FG 0.5%)添加至該細胞生質以溶解細胞且形成一經乳化之溶解的細胞組成物。該經乳化之溶解的細胞組成物係首先用一鹼 (NaOH，250 kg的50% w/w溶液）予以處理直到該溶解的細胞組成物之pH為10.4至10.6為止。接著，將一鹽（固體NaC卜以2%，重量計，之該溶解的細胞組成物的一量）添加至該溶解的細胞組成物。該溶解的細胞組成物接而被加熱至90°C 的溫度以及保持在該溫度位準歷時20小時。拿取各細胞肉湯的一樣品且將pH調整至8.0以及放置於一 50毫升試管中。將測試管子予以離心以及測量油萃取資料。油萃取資料係提供於表53中。 210 201200591 表53.在50毫升管子内於pH 8.0由萃取測試而來的結果. 307公分/秒測試萃取之濕的尚湯(g) 回收的油之#量⑻ °/。產量 (油/肉湯） °/。產量 (油/固體)* 49.990 3.881 7.76 27.81 50.814 2.747 5.41 19.36 50.772 2.418 4.76 17.05 464公分/秒測試萃取之濕的肉湯(g) 回收的油之f量(g) %產量 (油/肉湯） %產量 (油/固體r 51.154 7.067 13.81 49.13 51.092 7.055 13.81 49.11 50.132 6.606 13.18 46.86 *根據未處理之經低溫殺菌的肉湯之固體含量表53中提供的資料展現出較高的攪拌速度導致更大的回收的油之質量、由該肉湯之更大的％油產量，以及由該未處理之經低溫殺菌的肉湯之固體含量之更大的％油產量。結論本文中所說明之各種的具體例或選擇之全體可以組合以任何的變化以及所有的變化。縱然本發明已經就其等之一些具體例予以特定地顯示及說明，熟悉此藝者會了解到其等已經僅經由實例予以呈現，且非限制，以及可以在其中作出形式上和細節上之各種的改變而不背離本發明的精神與範疇。因而，本發明的寬度與範疇不應被以上所說明的例示性具體例之任一者所限制，而只應該根據下列申請專利範圍和其等之均等物來定義。本文中引用的所有的文件，含括期刊文章或摘要、公 211 201200591 開的或對應的美國或外國的專利申請案、核發的或外國的專利，或是任何的其他的文件，係各自整個地併入本文中以作為參考資料，含括於引用的文件中所呈現之所有的資料、表、圖，以及文字。【圖式簡單說明】第1-4圖提供了說明本發明的方法之概要的流程圖；第5圖為一圖，其提供了於各種的pH，溶解的細胞組成物隨時間之電子順磁共振(EPR)。【主要元件符號說明 100、200、300、400...方法 101…細胞 102…溶解 103…溶解的細胞組成物 104…與一第一個鹼接觸 105…與一鹽接觸 106…加熱 1〇7…經處理之溶解的細胞組成物 108…與一第二個鹼接觸 109…分離 110…脂質 204…與一驗接觸 207…經處理之溶解的細胞組成物 209…分離 210…脂質 305…與一鹽接觸 307…經處理之溶解的細胞組成物 309…分離 310…脂質 401…搜拌 402…熱 405···與一鹽接觸 407…經處理之溶解的細胞組成物 409…分離 410…脂質 212S 177 201200591 These plates were evaluated and visualized using iodine vapor. The Rf value of each spot is then compared to the reported literature values for each lipid species. Analysis of TAG and PL Prominence - The isolated TAG and PL fractions were analyzed as fatty acid oxime esters (FAME) fatty acid compositions. The TAG fraction was dissolved in hexane to provide a concentration of approximately 1-2 mg/ml. A 1 ml portion of the solution was concentrated to dryness under nitrogen. Toluene containing the internal standard and 1.5 N HCl1 in methanol were added to each tube. The tubes were vortexed, capped and heated to 1 Torr. It lasted 2 hours. The internal standard and HC1 methanol were added directly to the tube containing the PL fraction and heated. Allow the tubes to cool and add saturated NaCl in water. The tubes were again swirled and centrifuged to allow the layers to separate. A portion of the organic layer was then placed in a GC vial and analyzed by GC-FID. FAME was quantified using a 3-point standard calibration curve generated using the Nu_chek-Prep GLC 502B reference standard (NuChek 'Elysian, MN). The fatty acid present in the extract is expressed in milligrams per gram and is % of FAME. Results Fatty acid profiles of PTA 10208 sample #1 PTA-10208 sample #1 and extracted crude lipids were determined using GC/HD. The fatty acids in the biomass were transesterified in situ by weighing 28.6 mg of the crude directly into a FAMe tube' and a sample of the extracted crude lipid was weighed 55.0 mg of crude lipid by weighing Prepare by placing into a 50 ml volume flask and transferring 1 ml to a separate FAME tube. The estimated crude lipid content of the biomass was determined to be 53.2% (to the sum of FAME) using a GC with FID detection, while 52.0% (wt/wt) of the lipid system was extracted from the dried biomass to provide 97.8%. Total lipid recovery. The crude lipid was judged to be 91.9% fatty acid (to the sum of FAME) using GC/HD. The main fatty acids contained in the crude lipid were C16:0 (182.5 mg/g), C20:5 n-3 (186.8 mg/g), and C22:6 n-3 (423.1 mg/g). The lipid profile of the extracted crude lipid was determined by weighing 55.0 mg of crude lipid into a 50 ml volumetric flask and transferring an aliquot to an HPLC vial for HPLC/ELSD/MS analysis. painted. The crude lipid contained 0.2% sterol ester (SE), 95.1% TAG, 0.4% sterol, and 0.5% 1,2-diethylene glycol ester (DAG) according to HPLC/ELSD/MS analysis. 5% of the TAG fraction included a peak eluted directly after the TAG peak, but did not produce an identifiable mass spectrum. The isolated TA G from this sample, when determined by flash chromatography, constituted approximately 92.4% crude oil. After the SPE was isolated, no PL was detected by weight or TLC. The main fatty acids (> 50 mg/g) contained in the TAG are C16:0 (189 mg/g), C20:5 n-3 (197 mg/g), and C22:6n-3 (441 mg/g). ). PTA-10208 Sample #2 PTA-10208 Sample #2 Biomass and extracted crude lipids are profiled using a GC/FID. The fatty acids in the biomass were transesterified in situ by weighing 32.0 mg of the crude directly into a FAME tube, and a sample of the extracted crude lipid was weighed by 60.1 mg of crude lipid. Prepare into a 50 ml volume flask and transfer 1 ml to 179 201200591 a separate FAME tube. The estimated crude lipid content of this biomass was determined to be 52.4 °/ using a GC with FID detection. (To the sum of FAME), while 48.0% (wt/wt) of the lipid system was extracted from the dried biomass, providing 91.7. /〇 Total lipid recovery. The crude lipid was judged to be 95.3% fatty acid (as a sum of F A ME) using GC/FID. The main fatty acids contained in the crude lipid were 〇16:0 (217.5 mg/g), 〇20:5 11-3 (169.3 mg/g), and 〇22:6 n-3 (444.1 mg/g). The lipid species profile of the extracted crude lipid was determined by weighing 60.1 mg of crude lipid into a 50 ml volumetric flask and transferring an aliquot to an HPLC vial for HPLC/ELSD/MS analysis. painted. The crude lipid contained 0.2% SE, 95.70/〇 TAG, 0.3% sterol, and 0.7% 1,2-DAG according to HPLC/ELSD/MS analysis. 5.1% of the TAG fraction included a peak eluted directly after the TAG peak, but did not produce an identifiable mass spectrum. The isolated TAG from this sample constituted approximately 93.9% crude oil. After the SPE was isolated, the major fatty acids (>50 mg/g) contained in PL»TAG were not detected by weight or TLC as C16:0 (218 mg/g), C20:5 n-3 (167 mg/g) and C22:6 n-3 (430 mg/g). PTA-10208 Sample #3 A sample of crude oil (sample PTA-10208 #3) from a microorganism deposited according to ATCC accession number PTA-10208 was analyzed using HPLC/ELSD/MS. A total of 98.38% of the lipid was recovered, with a 0.32% sterol ester (SE) fraction, a 96.13% TAG fraction, and a 0.22% 1,3-dioxyl glyceride (DAG) fraction. 0.78% of 1,2-DAG distillate 180 201200591 parts, and 0.93% of sterol fraction. PTA-10212 Sample #1 PTA-10212 Sample #1 Biomass and extracted crude lipids are profiled using GC/FID. The fatty acids in the biomass were transesterified in situ by weighing 27.0 mg of the crude directly into a FAME tube, and a sample of the extracted crude lipid was weighed by 52.5 mg of crude lipid. Prepare by placing into a 50 ml volume flask and transferring 1 ml to a separate FAME tube. The estimated crude lipid content of the biomass was determined to be 38.3% (to the sum of FAME) using the FID-detected GC, while 36.3% (wt/wt) of the lipid system was extracted from the dried biomass to provide 94.6% total lipid. Recovery rate. The crude lipid was judged to be 83.2% fatty acid (as a sum of FAME) using GC/FID. The main fatty acids contained in the crude lipid were C16:0 (328.5 mg/g), C20:5 n-3 (90.08 mg/g), and C224 n_3 (289.3 mg/g). The lipid species of the extracted crude lipids were determined by weighing 52.5 mg of crude lipid into a 50 ml volumetric flask and transferring the whole fraction to an HPLC vial for HPLC/ELSD/MS analysis. . The crude lipid contained 0.2% SE, 64.2% TAG, 1.9% FFA, 2.8% 1,3-DAG, 1.4% sterol, 18.8% 1,2-DAG, and 0.5% MAG, according to HPLC/ELSD/MS analysis. A 3.4% fraction of the TAG fraction included a peak eluted directly after the TAG peak, but did not produce an identifiable mass spectrum. The isolated TAG from this sample formed approximately 49.8% crude oil. The isolated PL constitutes approximately 8.1% of the crude oil. The TAG fraction contains 181 201200591. The main fatty acids (> 50 mg/g) are C16:0 (400 mg/g), C20:5 n-3 (91 mg/g), and C22:6 n- The main fatty acid (>50 mg/g) contained in the 3 (273 mg/g) °PL fraction is C16:0 (98 mg/g) 'C20:5 n-3 (33 mg/g), and C22: 6 n-3 (227 mg/g). PTA-10212 Sample #2 PTA-10212 Sample #2 Biomass and extracted crude lipids are profiled using a GC/FID. The fatty acids in the biomass were transesterified in situ by weighing 29.5 mg of the crude directly into a FAME tube, and a sample of the extracted crude lipid was weighed by 56.5 mg of crude lipid. Prepare by placing into a 50 ml volume flask and transferring 1 ml to a separate FAME tube. The estimated crude lipid content of the biomass was determined to be 40_0% (to the sum of FAME) using the FID-detected GC, while 41.3% (wt/wt) of the lipid system was extracted from the dried biomass to provide 106.1% of the total. Lipid recovery. The crude lipid was judged to be 82.8% fatty acid (to the sum of FAME) using GC/FID. The main fatty acids contained in the crude lipid were C16:0 (327.3 mg/g), C20:5 n-3 (92.5 mg/g)' and C22:6 n-3 (277.6 mg/g). By weighing 56.5 mg of crude lipid into a 50 ml volumetric flask and transferring an aliquot to an HPLC vial for HPLC/ELSD/MS #才斤 I ^ $ I海&# t月The purpose of the quality of the month of the 53⁄4 class. The crude lipid contained 0.2% SE, 58.20/〇TAG, 2.3% FFA, 3.4% 1,3-DAG, 1.7% sterol, 23.4% 1,2-indole, and 0.6 according to HPLC/ELSD/MS analysis. %]\4 gossip. A 3.3% Ding Biaode score included a peak directly after the TAG peak, but did not produce an identifiable 182 201200591 mass spectrum. The isolated TAG from this sample constituted approximately 51.9% crude oil. The isolated PL constitutes approximately 8.8% of the crude oil. The major fatty acids (> 50 mg/g) contained in the TAG fraction were C16:0 (402 mg/g), C20:5 n-3 (92 mg/g), and C22:6 n-3 (245). Mg/g). The main fatty acids (> 50 mg/g) contained in the PL fraction are C16:0 (121 mg/g), C20:5 n-3 (48 mg/g), and C22:6n-3 (246 mg). /g). 183 201200591 Table 32: Fatty acid profiles of PTA-10208 and PTA-10212 raw and extracted crude lipids (mg/g) PTA-10208 Sample #1 Biomass PTA-10208 Sample #1 Crude Lips PTA-10208 Sample# 2 Biomass PTA-10208 Sample #2 Crude Lips PTA-10212 Sample #1 Biomass PTA-10212 Sample #1 Crude Lips PTA-10212 Sample #2 Biomass PTA-10212 Sample #2 Crude Fatty Acid FAME (mg/g) FAME (g/g) FAME (kg/g) FAME (mg/g) FAME (4 ex/g) FAME (mg/g) FAME (Qin/g) FAME (i free/g) C12:0 1.47 2.43 1.80 3.14 0.99 1.90 0.87 1.91 C14:0 11.62 20.12 16.72 31.03 5.51 12.91 5.97 13.69 C14:l 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 2.43 3.75 3.60 6.22 9.13 20.42 9.39 20.81 C16:0 105.04 182.47 117.72 217.49 145.87 328.45 147.87 327.27 C16:l 0.00 0.00 0.06 0.01 6.26 14.53 7.46 16.89 C18:0 5.37 8.96 4.77 8.37 6.77 15.39 6.77 15.15 C18:l n-9 0.00 3.26 0.00 3.09 0.03 4.04 0.08 5.87 C18:l n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:0 1.48 1.79 1.40 1.85 1.60 3.09 1.67 3.20 C18:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:4 n-3 0.91 1.61 1.10 2.00 2.28 2.56 2.21 2.64 C20:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.002 C02:0 0.10 0.00 0.08 0.00 0.30 0.12 0.35 0.24 C20:4 N-7 0.81 0.45 0.67 0.41 0.00 0.00 0.00 0.00 C20:4 n-6 7.22 12.23 6.84 12.18 1.19 2.26 1.31 2.32 C22:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-5 0.63 0.52 0.00 0.46 0.00 0.00 0.00 0.00 C20:4 n-3 3.45 5.45 3.33 5.58 0.00 2.40 0.00 2.34 C20:3 n-3 0.09 0.00 0.11 0.00 0.00 0.00 0.00 0.00 C20:5 n-3 107.31 186.83 92.99 169.32 40.32 90.08 43.15 92.54 C22:4 n- 9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C24:0 0.60 0.00 0.52 0.00 2.81 6.83 2.74 6.53 C24:l n-9 1.55 3.26 0.85 2.04 0.43 1.34 0.42 1.24 C22:5 n-6 9.66 15.84 10.27 17.98 2.42 4.68 2.32 4.21 C22: 5 n-3 20.44 35.13 9.92 17.50 2.41 4.94 2.69 5.2 3 C22:6 n-3 246.98 423.10 245.96 444.08 139.58 289.34 137.35 277.57 FAME total 527.15 907.18 518.71 942.75 367.89 805.29 372.63 799.68 184 201200591 Table 33: Fatty acid profiles of PTA-10208 and PTA-10212 biomass and extracted crude lipids (%) PTA-10208 Sample #1 Biomass PTA-10208 Sample #1 Crude Lips PTA-10208 Sample #2 Biomass PTA-10208 Sample #2 Crude Lips PTA-10212 Sample #1 Biomass PTA- 10212 Sample #1 Crude Lipid PTA-10212 Sample #2 Biomass PTA-10212 Sample #2 Crude Lipid Fatty Acid % FAME % FAME % FAME % FAME % FAME % FAME % FAME % FAME C12:0 0.28 0.27 0.35 0.33 0.27 0.24 0.23 0.24 C14:0 2.20 2.22 3.22 3.29 1.50 1.60 1.60 1.71 C14:l 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 0.46 0.41 0.69 0.66 2.48 2.54 2.52 2.60 C16:0 19.93 20.11 22.70 23.07 39.65 40.79 39.68 40.93 C16:l 0.00 0.00 0.01 0.00 1.70 1.80 2.00 2.11 C18:0 1.02 0.99 0.92 0.89 1.84 1.91 1.82 1.89 C18:l n-9 0.00 0.36 0.00 0.33 0.01 0.50 0.02 0.73 C18:l n-7 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:2 n- 6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:0 0.28 0.20 0.27 0.20 0.43 0.38 0.45 0.40 C18:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C 18 :4 n-3 0.17 0.18 0.21 0.21 0.62 0.32 0.59 0.33 C20:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.0022:0 0.02 0.00 0.01 0.00 0.08 0.02 0.09 0.03 C20:4 n-7 0.15 0.05 0.13 0.04 0.00 0.00 0.00 0.00 C20:4 n-6 1.37 1.35 1.32 1.29 0.32 0.28 0.35 0.29 C22:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-5 0.12 0.06 0.00 0.05 0.00 0.00 0.00 0.00 C20:4 n-3 0.65 0.60 0.64 0.59 0.00 0.30 0.00 0.29 C20:3 n-3 0.02 0.00 0.02 0.00 0.00 0.00 0.00 0.00 C20:5 n-3 20.36 20.59 17.93 17.96 10.96 11.19 11.58 11.57 C22:4 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C24:0 0.11 0.00 0.10 0.00 0.76 0.85 0.74 0.82 C24:l n-9 0.29 0.36 0.16 0.22 0.12 0.17 0.11 0.16 C22:5 n-6 1.83 1.75 1.98 1.91 0.66 0.58 0.62 0.53 C22:5 n-3 3.88 3.87 1.91 1.86 0.65 0.61 0 .72 0.65 C22:6 n-3 46.85 46.64 47.42 47.10 37.94 35.93 36.86 34.71 FAME % of total 100 100 100 100 100 100 100 100 185 201200591 Table 34: PTA-10208 and PTA-10212 fatty acid profiles of isolated TAG PTA-10208 Sample #1 PTA-10208 Sample #1 PTA-10208 Sample #2 PTA-10208 Sample #2 PTA-10212 Sample #1 PTA-10212 Sample #1 PTA- 10212 Sample #2 PTA- 10212 Sample #2 Fatty Acid FAME (mg/g) % FAME FAME (mg/g) % FAME FAME (mg/g) % FAME FAME (mg/g) % FAME C12:0 2.57 0.27 3.35 0.36 0.00 0.00 0.00 0.00 C14:0 21.07 2.23 31.37 3.41 14.05 1.61 14.45 1.69 C14:l 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C15:0 3.89 0.41 6.17 0.67 23.27 2.66 23.14 2.71 C16:0 189.28 20.07 218.78 23.75 399.51 45.75 402.43 47.07 C16:l 0.00 0.00 0.00 0.00 15.23 1.74 17.62 2.06 C18:0 9.21 0.98 8.07 0.88 22.70 2.60 23.10 2.70 C18:l n-9 3.35 0.36 3.64 0.40 6.12 0.70 7.48 0.87 C18:l n-7 0.00 0.00 0.00 0.00 <0.1 <0.1 <0.1 <0.1 C18:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:0 1.86 0.20 1.55 0.17 4.76 0.55 5.32 0.62 C18:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C18:4 n-3 1.64 0.17 2.00 0.22 2.25 0.26 2.24 0.26 C20:2 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C22: 0 0.00 0.00 0.00 0.00 0.55 0.06 0.89 0.10 Unknown 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-7 0.39 0.04 0.05 0.01 0.00 0.00 0.00 0.00 C20:3 n-3 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.006 C20:4 n -6 12.79 1.36 11.82 1.28 2.33 0.27 2.25 0.26 C22:l n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C20:4 n-5 0.39 0.04 0.07 0.01 0.00 0.00 0.00 0.00 C20:4 n-3 5.52 0.59 5.09 0.55 2.87 0.33 2.98 0.35 C20:5 n-3 197.14 20.90 166.68 18.10 91.17 10.44 91.78 10.74 C24:0 0.00 0.00 0.00 0.00 6.93 0.79 7.36 0.86 C22:4 n-9 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 C24:l n-9 1.08 0.11 <0.1 <0.1 0.00 0.00 0.00 0.00 C22:5 n-6 15.88 1.68 16.57 1.80 4.01 0.46 3.39 0.40 C22:5 n-3 36.05 3.82 16.00 1.74 4.53 0.52 5.07 0.59 C22:6 n-3 440.99 46.76 429.83 46.67 273.02 31.26 245.38 28.70 FAME Total 943.11 - 921.03 - 873.31 - 854.89 - Total % FAME - 100.00 - 100.00 - 100.00 - 100.00 186 201200591 Table 35: PTA-10212 Alkaline PL fatty acid profile PTA-10212 Sample #1 PTA-10212 Sample #1 PTA-10212 Sample #2 PTA-10212 Sample #2 Fatty Acid FAME (mmol/g) % FAME FAME (mg/g) % FAME C12:0 0.00 0.00 0.00 0.00 C14:0 0.93 0.22 1.89 0.39 C14:l 0.00 0.00 0.00 0.00 C15:0 3.44 0.82 5.07 1.05 C16:0 98.29 23.50 120.98 25.00 C16:l 1.15 0.27 3.07 0.63 C18:0 3.25 0.78 3.72 0.77 C18:l n-9 1.12 0.27 0.95 0.20 C18:l n-7 <0.1 <0.1 0.02 0.003 C18:2n-6 0.00 0.00 0.00 0.00 C20:0 <0.1 <0.1 <0.1 <0.1 C18:3 n-3 0.00 0.00 0.00 0.00 C20:l n-9 0.00 0.00 0.00 0.00 C18:4 n-3 3.71 0.89 3.24 0.67 C20:2 n-6 0.00 0.00 0.00 0.00 C20:3 n-6 0.00 0.00 0.00 0.00 C22:0 0.00 0.00 0.00 0.00 unknown 42.33 10.12 44.71 9.24 C20:4 n-7 0.00 0.00 0.00 0.00 C20:3 n-3 0.00 0.00 0.00 0.00 C20:4 n-6 0.84 0.20 1.54 0.32 C22:l n- 9 0.00 0.00 0.00 0.00 C20:4 n-5 0.00 0.00 0.00 0.00 C20:4 n-3 <0.1 <0.1 0.27 0.06 C20:5 n-3 33.39 7.98 47.91 9.90 C24:0 <0.1 <0.1 0.01 0.001 C22:4 n-9 0.00 0.00 0.00 0.00 C24:l n-9 0.00 0.00 0.00 0.00 C22:5 n-6 3.08 0.74 3.82 0.79 C22:5 n-3 <0.1 <0.1 0.66 0.14 C22:6 n-3 226.68 54.20 246.09 50.85 Total of FAME 418.21 - 483.94 - Total % FAME - 100 - 100 187 201200591 PTA-10212 Sample #3 ΡΤΑ-10212 Sample #3 in the lipid content of the biomass It is estimated that 34% of the total of FAME, and the amount of crude oil obtained after solvent extraction is 37% by weight, providing a recovery of 1% to 9% of the fat present in the biomass. After the fractionation using flash chromatography, approximately 46% of the crude oil system was separated from TAG and 28% was isolated as DAG. The crude oil contains 3〇9 mg/g DHA and mg/g EPA. The isolated TAG contained 341 mg/g DHA and 274 mg/g EPA. The detached dag remainder contained 262 mg/g DHA and 237 mg/g EPA. The crude oil, the extracted crude oil, and the total fatty acid profile of the isolated fractions were calculated in milligrams per gram and % FAME, respectively, and are shown in Tables 36 and 37 below. 188 201200591 Table 36: ΡΤΑ-10212 Sample #3 Raw Acid and Extracted Crude Lipids Fatty Acids (mg/g) Biomass Crude Oil TAG DAG Wt. % ΝΑ 37.2% 46.0% 27.9% Fatty Acids FAME (mg/g FAME (Tax/g) FAME (Milligram/g) FAME (mg/g) C12:0 0.0 0.0 0.0 0.0 C14:0 3.6 10.3 11.5 9.4 C14:l 0.0 0.0 0.0 0.0 C15:0 4.1 10.6 9.8 6.6 C16 :0 70.5 181.8 231.7 111.3 C16:l 6.7 19.1 18.7 17.1 C18:0 2.4 10.2 14.2 0.0 C18:l n-9 0.0 6.7 0.0 0.0 C18:l n-7 0.0 1.2 0.0 0.0 C18:2 n-6 0.0 1.8 0.0 0.0 C20:0 0.0 2.4 0.0 0.0 C18:3 n-3 0.0 0.0 0.0 0.0 C20:l n-9 0.0 0.3 0.0 1.7 C18:4 n-3 1.9 3.4 3.2 4.4 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 N-6 0.0 0.0 0.0 0.0 C22:0 3.3 0.0 0.0 0.0 C20:4 n-7 0.0 2.1 1.5 0.0 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 6.8 17.9 21.4 13.8 C22:l n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 1.3 1.3 0.0 C20:4 n-3 3.0 8.5 10.9 6.4 C20:5 n-3 102.0 263.6 274.2 237.4 C24:0 0.0 1.7 3.9 0.0 C22:4 n-9 0.0 0.0 0.0 0.0 C24:l n-9 0.0 0.0 4.2 0.0 C22:5 n-6 3.2 8.3 10.7 6.1 C22:5 n-3 3.8 10.4 15.1 6.6 C22:6 n-3 131.2 309.4 341.1 261.9 The sum of FAME 342.4 871.1 973.2 682.6 189 201200591 Table 37: PTA-10212 sample #3 Fatty acid and fatty acid profile of extracted crude lipids (%) Raw crude oil TAG DAG Wt. % ΝΑ 37.2% 46.0% 27.9% Fatty acid FAME (mg/g) FAME (mg/g) FAME (Millies per gram) FAME (milli-free/g) C12:0 0.0 0.0 0.0 0.0 C14:0 1.1 1.2 1.2 1.4 C14:l 0.0 0.0 0.0 0.0 C15:0 1.2 1.2 1.0 1.0 C16:0 20.6 20.9 23.8 16.3 C16:l 2.0 2.2 1.9 2.5 C18:0 0.7 1.2 1.5 0.0 C18:l n- 9 0.0 0.8 0.0 0.0 C18:l n-7 0.0 0.1 0.0 0.0 C18:2 n-6 0.0 0.2 0.0 0.0 C20:0 0.0 0.3 0.0 0.0 C18:3 n-3 0.0 0.0 0.0 0.0 C20:l n-9 0.0 0.0 0.0 0.2 C18:4 n-3 0.6 0.4 0.3 0.6 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 1.0 0.0 0.0 0.0 C20:4 n-7 0.0 0.2 0.2 0.0 C20 :3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 2.0 2.1 2.2 2.0 C22:l n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.1 0.1 0.0 C20:4 n-3 0.9 1.0 1.1 0.9 C20 :5 n-3 29.8 30.3 28.2 34.8 C24:0 0.0 0.2 0.4 0.0 C22:4 n-9 0 .0 0.0 0.0 0.0 C24:l n-9 0.0 0.0 0.4 0.0 C22:5 n-6 0.9 1.0 1.1 0.9 C22:5 n-3 1.1 1.2 1.6 C22:6 n-3 38.3 35.5 35.1 38.4 Total % FAME 100.0 100.0 100.0 100.0 190 201200591 PTA-10212 Sample #4 PTA-10212 Sample #4 contained approximately 23°/〇 of lipid measured by the sum of FAME, and 1% 7% of it was recovered by hexane extraction. After fractional distillation using flash chromatography, approximately 42% of the crude oil system was isolated as TAG and 18% was isolated as DAG. The crude oil contains 275 mg/g DHA and 209 mg/g EPA. The isolated TAG contained 296 mg/g DHA and 205 mg/g EPA. The isolated DAG fraction contained 245 mg/g DHA and 219 mg/g EPA. The crude fatty acid, extracted crude oil, and the total fatty acid profile of the isolated fractions are shown in Table 38 (mg/g) and Table 39 (% FAME) below. 191 201200591 Table 38: PTA-10212 sample #4 Fatty acid profile of raw and extracted crude lipids (mg/g) Biomass crude oil TAG DAG wt. % ΝΑ 24.7% 42.2% 18.4% Fatty acid FAME (mg/g FAME (mg/g) FAME (i g/g) FAME (i: see / g) C12:0 0.0 0.0 2.1 2.4 C14:0 2.0 8.3 9.8 9.6 C14:l 0.0 0.0 0.0 0.0 C15:0 4.8 16.8 0.4 0.9 C16:0 63.3 210.6 285.7 138.0 C16:l 1.6 6.7 7.4 7.5 C18:0 2.8 12.2 19.9 4.6 C18:l n-9 0.0 3.7 0.7 1.1 C18:l n-7 0.0 0.0 0.3 1.2 C18:2n-6 0.0 0.0 0.0 0.0 C20:0 0.0 3.3 6.0 1.5 C18:3 n-3 0.0 0.0 0.0 0.0 C20:l n-9 0.0 0.0 0.7 1.2 C18:4 n-3 1.4 3.8 3.6 5.0 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 N-6 0.0 0.0 0.4 0.0 C22:0 1.5 0.0 1.9 0.0 C20:4 n-7 0.0 0.0 0.9 0.6 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 2.5 10.1 13.0 10.3 C22:l n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.0 0.8 0.7 C20:4 n-3 1.4 6.3 8.6 6.0 C20:5 n-3 57.6 209.1 205.4 219.0 C24:0 0.0 2.6 0.8 0.0 C22:4 n-9 0.1 0.0 0.0 0.0 C24: l n-9 0.0 0.0 1.1 0.5 C22: 5 n-6 1.4 6.1 7.9 4.5 C22: 5 n-3 4.0 15.8 20.8 12.9 C22:6 n-3 87.7 275.0 296.4 244.8 The sum of FAME 232.2 790.1 894.8 672.4 192 201200591 Table 39: ΡΤΑ-10212 sample #4 Fatty acid and extracted crude lipid profile (%) Raw crude oil TAG DAG Wt. % ΝΑ 24.7% 42.2% 18.4% Fatty Acid FAME (i: free / gram) FAME (mg / gram) FAME (mg / gram) FAME (mg / gram) C12:0 0.0 0.0 0.2 0.4 C14:0 0.9 1.0 1.1 1.4 C14: l 0.0 0.0 0.0 0.0 C15:0 2.1 2.1 0.0 0.1 C16:0 27.3 26.7 31.9 20.5 C16:l 0.7 0.8 0.8 1.1 C18:0 1.2 1.5 2.2 0.7 C18:l n-9 0.0 0.5 0.1 0.2 C18: l n-7 0.0 0.0 0.0 0.2 C18:2n-6 0.0 0.0 0.0 0.0 C20:0 0.0 0.4 0.7 0.2 C18:3 n-3 0.0 0.0 0.0 0.0 C20:l n-9 0.0 0.0 0.1 0.2 C18:4 n-3 0.6 0.5 0.4 0.7 C20:2 n-6 0.0 0.0 0.0 0.0 C20:3 n-6 0.0 0.0 0.0 0.0 C22:0 0.6 0.0 .0.2 0.0 C20:4 n-7 0.0 0.0 0.1 0.1 C20:3 n-3 0.0 0.0 0.0 0.0 C20:4 n-6 1.1 1.3 1.5 1.5 C22:l n-9 0.0 0.0 0.0 0.0 C20:4 n-5 0.0 0.0 0.1 0.1 C20:4 n-3 0.6 0.8 1.0 0.9 C20:5 n-3 24.8 26.5 23.0 32.6 C24:0 0.0 0.3 0.1 0.0 C22:4 n-9 0.0 0.0 0.0 0. 0 C24: l n-9 0.0 0.0 0.1 0.1 C22: 5 n-6 0.6 0.8 0.9 0.7 C22: 5 n-3 1.7 2.0 2.3 1.9 C22: 6 n-3 37.8 34.8 33.1 36.4 Total % FAME 100.0 100.0 100.0 100.0 193 201200591 PTA-10212 Sample #5 A sample of crude oil was extracted from a biomass of PTA-10212 using the FRIOLEX® method (GEA Westfalia Separator UK Ltd., Milton Keynes, England) to produce microbial oil PTA-10212 sample #5. Individual lipid species were isolated by PTA-10212 sample #5 using low pressure flash chromatography and the weight percentages of the various types were determined. Various types of fatty acid profiles were determined using GC/HD. Briefly, the sample was prepared by dissolving 240 mg of crude oil in 600 μl of hexane and applying it to the top of the column. After fractionation of the sample using flash chromatography, the combined weight of all fractions was 240 mg, resulting in a recovery of 1%. The sterol ester fraction accounted for 0.9%, the TAG fraction accounted for 42.6%, the free fatty acid (FFA) fraction accounted for 1.3%, the sterol fraction accounted for 2.2%, and the DAG fraction accounted for 41.6%. The total fatty acid profiles of the FRIOLEX® crude oil and the isolated fractions were calculated as mg/g and %FAME, respectively, and are shown in Tables 40 and 41 below. 194 201200591 Table 40: PTA-10212 sample #5 Crude oil fatty acid profile (mg/g) Crude oil TAG DAG wt. % ΝΑ 42.6% 41.6% Fatty acid FAME (mg/g) FAME (Qinke/g) FAME ( Mg/g) C12:0 0 0.7 1.0 C14:0 7.7 7.7 8.5 C14:l 0 0.0 0.0 C15:0 10.3 11.7 9.3 C16:0 179.3 217.7 134.6 C16:l 18.1 16.3 25.9 C18:0 8.1 13.2 2.3 C18:l n -9 4.7 8.4 0.7 C18: l n-7 0 1.8 1.0 C18: 2n-6 1.8 3.3 0.7 C20: 0 1.9 3.6 0.2 C18: 3 n-3 0 0.0 0.0 C20: l n-9 0 0.7 1.0 Cl8: 4 n -3 3.1 2.8 3.8 C20:2 n-6 0 0.0 0.0 C20:3 n-6 0 0.6 0.4 C22:0 0 1.5 0.0 C20:4 n-7 0 1.0 0.7 C20:3 n-3 0 0.0 0.0 C20:4 N-6 12.7 16.1 13.6 C22:l n-9 0 0.0 0.0 C20:4 n-5 0 1.5 0.8 C20:4 n-3 6.5 9.3 6.4 C20:5 n-3 213.3 223.7 252.8 C24:0 2.3 4.4 0.6 C22: 4 n-9 0 1.9 0.9 C24:l n-9 0 0.0 0.0 C22:5 n-6 7.9 9.5 8.3 C22:5 n-3 13 20.6 9.7 C22:6 n-3 305.6 327.4 353.8 Total of FAME 796.6 905.3 837.4 195 201200591 Table 41: PTA-10212 Sample #5 Crude Oil Fatty Profile (%) Crude Oil TAG DAG Fatty Acid % FAME % FAME % FAME C12:0 0 0.1 0.1 C14:0 1 0.9 1.0 C14:l 0 0.0 0.0 C15:0 1.3 1.3 1.1 C16:0 22.5 24.0 16.1 C16:l 2.3 1.8 3.1 C18:0 1 1.5 0.3 C18:l n-9 0.6 0.9 0.1 C18: l n-7 0 0.2 0.1 C18: 2 n-6 0.2 0.4 0.1 C20: 0 0.2 0.4 0.0 C18: 3 n-3 0 0.0 0.0 C20: l n-9 0 0.1 0.1 C18: 4 n-3 0.4 0.3 0.5 C20:2 n-6 0 0.0 0.0 C20:3 n-6 0 0.1 0.0 C22:0 0 0.2 0.0 C20:4 n-7 0 0.1 0.1 C20:3 n-3 0 0.0 0.0 C20:4 n-6 1.6 1.8 1.6 C22: l n-9 0 0.0 0.0 C20: 4 n-5 0 0.2 0.1 C20: 4 n-3 0.8 1.0 0.8 C20: 5 n-3 26.8 24.7 30.2 C24:0 0.3 0.5 0.1 C22:4 n- 9 0 0.2 0.1 C24: l n-9 0 0.0 0.0 C22: 5 n-6 1 1.1 1.0 C22: 5 n-3 1.6 2.3 1.2 C22: 6 n-3 38.4 36.2 42.3 Total % FAME 100 100 100 196 201200591 EXAMPLE 37 The crude oil is further processed through a refining, bleaching, and deodorizing step to obtain a refined oil. The refined oil was diluted with high oleic sunflower oil to obtain a final oil having a DHA content of about 400 mg/g. Individual lipid species were isolated and various fatty acid profiles were determined using GC-FID to determine FAME. PTA-10208 final oil PTA-10208 final oil #1 -5 fatty acid profile is summarized in Tables 42-43, including the cross-sections associated with the isolated TAG fraction (Tables 44-45) and A cross-section associated with the isolated sterol/DAG fraction (Tables 46-47). Individual lipid species in the final oil were also determined using flash chromatography (Table 48) and normal phase HPLC (Table 49) with ELSD and APCI-MS validation. 197 201200591 Table 42: Fatty acid profile of ΡΤΑ-1〇2〇8 final oil (mg/g) Fatty acid PTA-10208 Final oil #1 PTA-10208 Final oil #2 PTA-10208 Final oil #3 PTA-10208 Final oil #4 PTA-10208 Final Oil #5 C12:0 FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) 2.5 2.4 2.8 2.7 2.7 C14:0 16.1 14.9 21.0 18.4 17.5 C14:l 0.0 0.0 0.0 0.0 0.0 C15:0 3.8 3.6 4.4 3.9 3.9 C]6:0 192.1 179.1 193.1 184.3 194.6 C16:l 0.4 0.5 0.5 0.5 0.5 C17:0 0.6 0.5 0.9 0.8 2.1 C18:0 12.8 13.9 11.5 12.3 12.9 C18:l n-9 23.5 82.0 25.7 26.0 29.5 C18:l n-7 0.2 0.7 0.1 0.1 0.1 Cl8:2 n-6 3.7 8.1 4.0 4.1 4.3 C20:0 4.3 4.1 3.7 4.0 4.0 Cl8:3 n -3 0.0 0.0 0.0 0.0 0.0 C20:l n-9 <0.1 0.1 <0.1 <0.1 <0.1 Cl8:4 n-3 2.4 2.5 2.8 2.7 2.8 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 0.2 0.1 0.1 0.1 0.1 C22:0 1.2 1.8 1.0 1.1 1.1 C20:4 n-7 1.7 1.6 1.7 1.8 1.6 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 12.9 12.1 13.5 13.5 13.3 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 1.6 1.4 1.5 1.7 1.5 C20 :4 n-3 6.0 5.7 6.0 6.0 6.1 C20:5 η·3 173.8 163.3 196.4 209.6 197.9 C24:0 1.4 1.6 1.3 1.3 1.0 C22:4 \n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 3.4 3.2 2.3 2.6 2.3 C22:5 n-6 14.9 14.0 14.4 13.0 12.9 C22:5 n-3 43.9 41.3 32.8 40.3 36.9 C22:6 n-3 394.8 373.7 373.2 374.3 364.2 The sum of FAME 918.1 932.2 914.7 925.1 914.1 198 201200591 Table 43: PTA- Fatty Acid Profile of 10208 Final Oil (%) PTA-10208 Final Oil #1 PTA-10208 Final Oil #2 PTA-10208 Final Oil #3 PTA-10208 Final Oil #4 PTA-10208 Final Oil #5 Fatty Acid% FAME % FAME % FAME % FAME % FAME C12:0 0.3 0.3 0.3 0.3 0.3 C14:0 1.8 1.6 2.3 2.0 1.9 C14:l 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.5 0.4 0.4 C16:0 20.9 19.2 21.1 19.9 21.3 C16:l <0.1 <0.1 <0.1 <0.1 0.1 C17:0 0.1 0.1 0.1 0.1 0.2 C18:0 1.4 1.5 1.3 1.3 1.4 C18:l n-9 2.6 8.8 2.8 2.8 3.2 C18:l n-7 <0.1 0.1 <0.1 <0.1 <0.1 C18: 2 n-6 0.4 0.9 0.4 0.4 0.5 C20: 0 0.5 0.4 0.4 0.4 0.4 C18: 3n-3 0.0 0.0 0.0 0.0 0.0 C20: l n-9 <0.1 <0.1 <0.1 <0.1 <0.1 Cl 8:4 n-3 0.3 0.3 0.3 0.3 0.3 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.2 0.2 0.2 0.2 0.2 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.4 1.3 1.5 1.5 1.5 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 \n-5 0.2 0.2 0.2 0.2 0.2 C20:4 \n-3 0.7 0.6 0.7 0.7 0.7 C20:5 n-3 18.9 17.5 21.5 22.7 21.6 C24:0 0.1 0.2 0.1 0.1 0.1 C22: 4 n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 0.4 0.3 0.2 0.3 0.2 C22:5 n-6 1.6 1.5 1.6 1.4 1.4 C22:5 n-3 4.8 4.4 3.6 4.4 4.0 C22:6 n-3 43.0 40.1 40.8 40.5 39.9 199 201200591 Table - TAG Fatty Acid Profile: PTA-10208 Final Oil (mg/g) PTA-10208 Final Oil #1 PTA-10208 Final Oil #2 PTA-10208 Final Oil #3 PTA-10208 Final Oil #4 PTA-10208 Final Oil #5 Fatty Acid FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) C12: 〇2.5 2.3 2.7 2.5 2.6 C14: 〇16.3 15.1 21.3 18.6 18.1 C14:l 0.0 0.0 0.0 0.0 0.0 C15: 〇3.9 3.6 4.4 4.0 4.0 C16: 〇194.2 181.9 196.1 186.1 199.8 C16:l 0.4 0.4 0.6 0.5 0.7 C17: 〇0.6 0.5 0.9 0.8 0.8 C18: 〇12.9 14.2 11.7 12.5 13.2 Cl 8:1 n -9 24.3 84.0 26.8 26.1 34.0 Cl 8:1 n-7 0.1 0.7 0.1 0.1 0.3 Cl8:2 n-6 3.2 7.7 3.4 3.5 4.0 C20:0 4.4 4.2 3.8 4.0 4.2 Cl8:3 n-3 0.0 0.0 0.0 0.0 0.0 C20 :l n-9 <0.1 0.2 <0.1 <0.1 0.1 Cl 8:4 n-3 2.5 2.4 2.8 2.6 2.7 C20: 2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 0.2 0.2 0.1 0.1 0.1 C22:0 1.2 1.9 1.0 1.1 1.1 C20:4 n -7 1.7 1.6 1.8 1.8 1.7 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 13.2 12.3 13.8 13.7 13.8 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 1.6 1.5 1.6 1.7 1.5 C20:4 n-3 6.1 5.7 6.1 5.9 6.2 C20:5 n-3 176.0 166.1 199.0 211.2 204.2 C24:0 1.2 1.3 1.0 1.1 1.2 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 3.3 3.2 2.2 2.5 2.4 C22:5 n-6 15.0 14.2 14.7 13.2 13.5 C22:5 n-3 44.4 42.0 33.3 40.5 38.3 C22:6 n-3 397.9 378.4 376.4 375.5 375.5 The sum of FAME 926.9 945.7 925.5 929.6 944.1 200 201200591 Table 45: Individual TAG fatty acid profile: ΡΤΑ-1〇2〇8 final oil (%) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final oil #1 Final oil #2 Final oil #3 Final oil# 4 Final Oil #5 Fatty Acid% FAME % FAME % FAME % FAME % FAME C12:0 0.3 0.2 0.3 0.3 0.3 C14:0 1.8 1.6 0.3 0.3 0.3 C14:1 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.5 0.4 0.4 C16: 0 20.9 19.2 21.2 20.0 21.2 C1 6:l <0.1 <0.1 0.1 0.1 0.1 C17:0 0.1 0.1 0.1 0.1 0.1 C18:0 1.4 1.5 1.3 1.3 1.4 C18:l n-9 2.6 8.9 2.9 2.8 3.6 C18:l n-7 <0.1 0.1 <0.1 <0.1 <0.1 C18:2 n-6 0.3 0.8 0.4 0.4 0.4 C20:0 0.5 0.4 0.4 0.4 0.4 C18:3 n-3 0.0 0.0 0.0 0.0 0,0 C20:l n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18:4 n-3 0.3 0.3 0.3 0.3 0.3 C20:2 n-6 0.0 0.0 0.0 0.0 0.0 C20:3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.2 0.2 0.2 0.2 0.2 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.4 1.3 1.5 1.5 1.5 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 0.2 0.2 0.2 0.2 0.2 C20:4 n-3 0.7 0.6 0.7 0.6 0.7 C20:5 n-3 19.0 17.6 21.5 22.7 21.6 C24:0 0.1 0.1 0.1 0.1 0.1 C22:4 n -9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 0.4 0.3 0.2 0.3 0.3 C22:5 n-6 1.6 1.5 1.6 1.4 1.4 C22:5 n-3 4.8 4.4 3.6 4.4 4.1 C22:6 n-3 42.9 40.0 40.7 40.4 39.8 201 201200591 (The table is separated from the teacher's fat core building: ΡΤΑ·Delete 8 final oil PTA-10208 Final oil #1 PTA-10208 Final oil #2 PTA-10208 Final oil #3 PTA-10208 Final oil #4 PTA -10208 Final Oil #5 Fatty Acid FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) FAME (mg/g) C12: 〇1.9 2.1 2.9 2.1 1.9 C14:0 9.9 9.5 9.7 10.3 8.0 C14:l 0.0 0.0 0.0 0.0 0.0 C15:〇2.4 2.3 2.2 2.3 2.0 C16:〇132.6 128.6 110.1 116.8 106.4 C16:l 0.2 0.3 <0.1 0.3 0.4 C17:0 0.3 0.2 0.3 0.3 0.3 C18:0 7.3 8.1 6.4 6.8 6.1 Cl 8:1 n-9 15.0 55.1 47.4 19.0 30.1 C18:l n-7 0.4 0.7 0.1 <0.1 0.2 Cl8: 2 n-6 13.1 16.7 21.6 13.5 18.4 C20:0 2.0 2.1 1.2 1.8 1.4 Cl8:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:l n-9 <0.1 <0.1 <0.1 <0.1 <0.1 Cl8: 4 n-3 2.3 2.4 2.4 2.4 2.0 C20: 2 n-6 0.0 0.0 0.0 0.0 0.0 C20: 3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.6 1.0 0.5 0.6 0.5 C20:4 n-7 0.8 0.9 2.1 0.9 0.7 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 5.7 5.8 4.8 6.1 4.5 C22:l n-9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 <0.1 <0.1 <0.1 0.6 <0.1 C20:4 n-3 2.7 2.7 2.1 2.7 2.0 C20:5 n-3 92.9 94.5 91.9 111.6 84.8 C24:0 1.2 1.3 1.1 1.1 1.3 C22:4 n-9 0.0 0.0 0.0 0.0 0.0 C24:l n-9 1.9 2.0 1.2 1.5 1.2 C22:5 n-6 7.8 8.0 6.7 7.0 5.5 C22:5 n-3 22.2 22.9 13.9 20.7 14.2 C22:6 n-3 246.3 252.7 223.5 240.3 196.3 The sum of FAME 569.3 619.8 552.1 568.7 488.2 202 201200591 Table 47 : Alkaline sterol/DAG fatty acid profile: PTA-10208 final oil (%) PTA-10208 PTA-10208 PTA-10208 PTA-10208 PTA-10208 Final oil #1 Final oil #2 Final oil #3 Final oil #4 末油#5 Fatty Acid% FAME % FAME % FAME % FAME % FAME C12:0 0.3 0.3 0.5 0.4 0.4 C14:0 1.7 1.5 1.8 1.8 1.6 C14:l 0.0 0.0 0.0 0.0 0.0 C15:0 0.4 0.4 0.4 0.4 0.4 Cl 6:0 23.3 20.8 19.9 20.5 21.8 C16:l <0.1 <0.1 <0.1 <0.1 0.1 C17:0 0.0 0.0 0.1 0.1 0.1 C18:0 1.3 1.3 1.2 1.2 1.2 Cl 8:1 n-9 2.6 8.9 8.6 3.3 6.2 C18:l n-7 0.1 0.1 <0.1 <0.1 <0.1 Cl8: 2 n-6 2.3 2.7 3.9 2.4 3.8 C20: 0 0.4 0.3 0.2 0.3 0.3 Cl8: 3 n-3 0.0 0.0 0.0 0.0 0.0 C20: l n-9 <0.1 <0.1 <0.1 <0.1 <0.1 C18: 4n-3 0.4 0.4 0.4 0.4 0.4 C20: 2 n-6 0.0 0.0 0.0 0.0 0.0 C20: 3 n-6 <0.1 <0.1 <0.1 <0.1 <0.1 C22:0 0.1 0.2 0.1 0.1 0.1 C20:4 n-7 0.1 0.1 0.4 0.2 0.1 C20:3 n-3 0.0 0.0 0.0 0.0 0.0 C20:4 n-6 1.0 0.9 0.9 1.1 0.9 C22-.1 n- 9 0.0 0.0 0.0 0.0 0.0 C20:4 n-5 <0.1 <0.1 <0.1 0.1 <0.1 C20: 4 n-3 0.5 0.4 0.4 0.5 0.4 C20: 5 n-3 16.3 15.2 16.6 19.6 17.4 C24:0 0.2 0.2 0.2 0.2 0.3 C22: 4 n-9 0.0 0.0 0.0 0.0 0.0 C24: l n-9 0.3 0.3 0.2 0.3 0.2 C22: 5 n-6 1.4 1.3 1.2 1.2 1.1 C22: 5 n-3 3.9 3.7 2.5 3.6 2.9 C22: 6 n-3 43.3 40.8 40.5 42.3 40.2 203 201200591 Table 48: by flash chromatography Separation of lipid species (wt%) Separation of lipid species PTA-10208 Final oil #1 PTA-10208 Final oil #2 PTA-10208 Final oil #3 PTA-10208 Final oil #4 PTA-10208 Final oil #5 TAG 93.4 95.4 94.0 95.7 95.1 Sterol/DAG 3.1 2.9 2.6 3.0 2.9 Recovery (%) 96.5 98.3 96.6 98.7 98.0 Table 49: Separation of lipid species by HPLC-ELSD (wt%) Sterol ester TAG FFA Sterol 1,3- DAG 1,2- DAG MAG Total PTA-10208 Final Oil #1 0.4 90.8 ND 0.8 0.5 0.5 ND 93.0 PTA-10208 Final Oil #2 0.4 88.5 ND 0.6 0.6 0.6 ND 90.7 PTA-10208 Final Oil #3 0.3 89.4 ND 0.8 0.6 0.5 ND 91.6 PTA-10208 Final oil #4 0.3 88.0 ND 0.8 0.5 0.5 ND 90.1 PTA-10208 Final oil #5 0.3 86.3 ND 0.7 0.8 0.5 ND 88.6 PTA-10208 Final oil #6 0.36 100.76 ND 0.84 0.54 0.61 N.D. 103.11 ND=Not detected PTA-10212 Final oil DHA is present at 41.63% and 366.9 mg/g? Ding 8-10212 is the final oil, while EPA is present at 16.52%. The individual fatty acids were determined and summarized in Table 50. 204 201200591 Table 50: Fatty acid profile of PTA-10212 final oil (%FAME) Fatty acid % FAME C6:0 ND C7:0 ND C8:0 ND C9:0 ND C10:0 ND C11:0 ND C12:0 ND C13 :0 ND C14:0 0.84 C14:l ND C15:0 1.33 C16:0 27.09 C16:l 1.03 C17:0 0.34 C17:l ND C18:0 1.26 C18:l n-9 2.14 C18:l n-7 0.18 C19 :0 ND C18:2 n-6 0.58 C20:0 0.32 C18:3 n-3 ND C20:l n-9 ND C18:3 n-6 ND C20:2 n-6 0.26 C20:3 n-6 ND C22 :0 0.14 C20:3 n-3 ND C20:4 n-6 1.34 C22:l n-9 ND C23:0 ND C20:5 n-3 16.53 C24:0 0.53 C24:l n-9 ND C22:5 n -6 1.50 C22:5 n-3 1.30 C22:6 n-3 41.63 Unknown 0.87 ND = not detected 205 201200591 Example 3 8 A single order registered under the ATCC access numbers PTA_1〇2〇8 and 1〇212 The two-day inoculum flask of the isolated microorganism was prepared as a feed carbon and nitrogen culture in the medium according to Tables 3 and 31. Mutation induction was performed according to the following procedure: Approximately 5 ml of a sterile T=2 day flask was poured into a sterile 40 ml glass homogenizer. The culture received 50 bursts in a homogenizer. The culture was aspirated and filtered through a sterile 50 micron mesh filter placed in a 5 liter sterile tube (this screen was used as a larger cluster to retain colonies and at the same time to allow for smaller clusters) With single-cells passing through one of the 50 micron screens). Collect all the extracts of the cultivator in a sterile 5 〇 ml tube. The macerated culture was vortexed and made into a dilution of 1 · 100 times. Vortex the diluted leachate samples, then add 200 μl of the inoculum to a 1 〇〇χ 15 mm medium agar cultured melon containing 4-5 glass beads (3 mm glass beads) (ι 5 mm ). The flat pans were gently agitated so that the glass beads uniformly spread the inoculum on the flat pan. Remove the glass beads from the flat plate and let the flat plate cover for about 5 knives to dry. When the procedure is carried out in dim light, the sterile fume hood is adjacent to the adjacent (lights in the 1 field are closed. There is only a small amount of light that allows the program to be connected and weak. § Five repetitions when illuminating the samples The lid of the flat plate is removed and the flat plate is placed on the bottom plate of the XL cross-linker (Spectr〇nics Corporation, New York). The power delivered by the cross-linker is in microjoules and its level Seeking to achieve a kill rate of 9〇%_95%. Five replicates of the control plate 206 201200591 The same procedure was used to inoculate cells that were not mutated. These, the number of field cells was used to calculate % killing - After the irradiation, remove the flat plate and replace the cover with the cover film and the flat film in the order of the sealing film. 4 flat plate in the 帛-week must grow in the dark towel, so that it can not repair the damaged Genes. Place the plate in 22.5 ° C for approximately 10 days, then count the colonies. After the final count, pick individual colonies with a sterile inoculating loop and repeat scribing on the new medium plate. Each colony is planted in individual flat When the plate is densely grown, a sample is taken using an inoculating loop and inoculated into a sterile 250 ml shake flask containing 50 ml of medium. The flask is placed in a 200 rpm room at 22.5 °C. On the shaker, the culture of the shake flask was harvested into a 50 ml sterile tube at T = 7. The pH was measured and the sample was centrifuged to collect the raw pellets. Each sample was rinsed and resuspended in isopropanol. In a 50:50 mixture with distilled water, then centrifuged again. The collected pellets were cold-dried, weighed, and subjected to FAME analysis. The data in Tables 51 and 52 represent the use of the above methods from strains PTA-10208 and Mutant produced by PTA-10212. 207 201200591 Table 51: PTA-10208 mutant fatty acid control PTA-10208 Mutant 1 PTA-10209 Mutant 2 PTA-10210 Mutant 3 PTA-10211 % 08:0 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 % 12:0 0.11 0.10 0.22 0.19 % 12:1 0.00 0.00 0.00 0.00 % 13 :0 0.19 0.19 0.15 0.16 % 13:1 0.00 0.00 0.00 0.00 % 14:0 1.94 1.82 2.98 2.59 % 14:1 0.00 0.00 0.00 0.00 % 15:1 2.66 2.22 1.76 1.66 % 16:0 24.87 24.97 23.71 25.01 % 16:1 0.20 0.25 0.07 0.07 % 16: 2 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 17:0 1.49 1.21 0.62 0.66 % 18:0 1.13 1.14 0.91 1.01 % 18:1 η-9 0.07 0.07 0.06 0.06 % 18:1 η-7 0.00 0.00 0.00 0.00 % 18:2 0.00 0.00 0.00 0.00 % 18:3 η-6 0.00 0.00 0.05 0.04 % 18:3 η-3 0.09 0.08 0.17 0.14 % 18:4 η-3 0.00 0.00 0.00 0.00 % 20:0 0.31 0.33 0.24 0.30 % 20:1 η-9 0.00 0.04 0.00 0.00 % 20:2 0.00 0.00 0.05 0.00 % 20:3 η-9 0.00 0.00 0.00 0.00 % 20:3 η-6 0.12 0.13 0.08 0.04 % 20:3 η-3 0.42 0.42 0.08 0.06 %20:4ARA 0.68 0.67 1.44 1,11 % 20:5 η-3 ΕΡΑ 6.56 6.47 11.99 9.87 % 22:0 0.07 0.07 0.06 0.07 % 22:1 0.00 0.00 0.00 0.00 % 22:2 0.11 0.09 0.10 0.08 % 22 :3 0.00 0.00 0.00 0.00 % 22:4 η-6 0.00 0.00 0.00 0.00 % 22:5 η-6 2.32 2.36 2.36 2.36 % 22:5 η-3 0.48 0.66 0.66 0.52 % 22:6 η-3 DHA 51.58 52.27 48.17 49.35 % 24:0 0.00 0. 00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00% fat 47.87 49.41 66.00 63.12 %Unknown 4.61 4.45 4.07 4.64 208 201200591 Table 52: PTA-10212 mutant fatty acid control PTA-10212 Mutant 1 PTA-10213 Mutant 2 PTA-10214 Mutant 3 PTA-10215 % 08:0 0.00 0.00 0.00 0.00 % 09:0 0.00 0.00 0.00 0.00 % 10:0 0.00 0.00 0.00 0.00 % 11:0 0.00 0.00 0.00 0.00 % 11:1 0.00 0.00 0.00 0.00 % 12:0 0.00 0.00 0.00 0.00 % 12:1 0.00 0.00 0.00 0.00 % 13:0 0.00 0.00 0.21 0.20 % 13:1 0.00 0.00 0.00 0.00 % 14:0 0.68 0.77 0.62 0.97 % 14:1 0.00 0.00 0.00 0.00 % 15:1 0.00 0.00 0.00 0.00 % 16:0 17.36 19.94 15.27 23.61 % 16:1 1.45 2.33 1.40 2.57 % 16:2 0.00 0.00 0.00 0.00 % 16:3 0.00 0.00 0.00 0.00 % 17:0 0.20 0.21 0.18 0.27 % 18:0 0.78 0.82 0.79 0.81 % 18:1 η-9 0.00 0.00 0.00 0.00 % 18:1 η-7 0.18 0.27 0.20 0.19 % 18:2 0.00 0.00 0.00 0.00 % 18:3 η-6 0.00 0.00 0.00 0.00 % 18:3 η-3 0.00 0.00 0.00 0.00 % 18:4 η-3 0.00 0.00 0.00 0.00 % 20:0 0.00 0.00 0.00 0.00 % 20:1 η-9 0.00 0.00 0.00 0.00 % 2 0:2 0.00 0.00 0.00 0.00 % 20:3 η-9 0.00 0.00 0.00 0.00 % 20:3 η-6 0.00 0.00 0.00 0.00 % 20:3 η-3 0.90 0.77 0.99 0.66 %20:4ARA 1.43 1.32 1.65 0.72 % 20 :5 η-3 ΕΡΑ 13.33 14.93 14.14 8.54 % 22:0 0.00 0.00 0.00 0.00 % 22:1 0.00 0.00 0.00 0.00 % 22:2 0.00 0.00 0.00 0.00 % 22:3 0.00 0.00 0.00 0.00 % 22:4 η-6 0.00 0.00 0.00 0.00 % 22:5 η-6 2.39 1.95 2.59 2.18 % 22:5 η-3 0.73 0.79 0.80 0.68 % 22:6 η-3 DHA 59.18 54.31 59.89 56.39 % 24:0 0.00 0.00 0.00 0.00 % 24:1 0.00 0.00 0.00 0.00% fat 45.69 38.08 42.88 48.48 %Unknown 1.38 1.58 1.27 2.19 209 201200591 Example 39 Two kinds of cell broth (about 13.3 kg) containing microbial cells (Schizochytrium) were heated to 60 ° C at a 20 Liter in the fermenter. The fermenter has two 6-blade Rushton impellers with a diameter of 15 cm. The top impeller is placed at the 12 liter mark and the bottom impeller is placed 10 cm below the top impeller. The first cell broth was continuously stirred at 307 cm/sec. The second cell broth was continuously stirred at 464 cm/sec. An enzyme (i.e., Alcalase 2.4 L FG 0.5%) was added to the cell biomass to dissolve the cells and form an emulsified dissolved cell composition. The emulsified dissolved cell composition was first treated with a base (NaOH, 250 kg of a 50% w/w solution) until the pH of the dissolved cell composition was 10.4 to 10.6. Next, a salt (solid NaC Bu, 2% by weight, an amount of the dissolved cell composition) was added to the dissolved cell composition. The solubilized cell composition was then heated to a temperature of 90 ° C and maintained at this temperature level for 20 hours. A sample of each cell broth was taken and the pH was adjusted to 8.0 and placed in a 50 ml tube. Centrifuge the test tube and measure the oil extraction data. Oil extraction data is provided in Table 53. 210 201200591 Table 53. Results from the extraction test at pH 8.0 in a 50 ml tube. 307 cm/sec Test the extracted wet soup (g) The amount of oil recovered (8) °/. Yield (oil/broth) °/. Yield (oil/solids)* 49.990 3.881 7.76 27.81 50.814 2.747 5.41 19.36 50.772 2.418 4.76 17.05 464 cm/sec test extracted wet broth (g) amount of oil recovered (g) % yield (oil/broth) % yield (oil/solids r 51.154 7.067 13.81 49.13 51.092 7.055 13.81 49.11 50.132 6.606 13.18 46.86 *Based on the solid content of untreated sterilized broth, the information provided in Table 53 shows that higher agitation speeds result in greater The quality of the recovered oil, the greater % oil yield from the broth, and the greater % oil yield from the solids content of the untreated, low temperature sterilized broth. Conclusion The various specificities described herein The present invention may be combined with any changes and all changes. The present invention has been specifically shown and described with respect to some specific examples thereof, and those skilled in the art will appreciate that they have been presented by way of example only. The invention is not limited thereto, and various changes in form and details may be made therein without departing from the spirit and scope of the invention. The scope should not be limited by any of the illustrative specific examples described above, but only by the scope of the following claims and their equivalents. All documents cited herein include journal articles or abstracts. , or the corresponding US or foreign patent application, issued or foreign patent, or any other document, which is incorporated herein by reference in its entirety for reference. All the materials, tables, figures and texts presented in the document. [Simplified illustration of the drawings] Figures 1-4 provide a flow chart illustrating an overview of the method of the present invention; Figure 5 is a diagram providing Electron paramagnetic resonance (EPR) of dissolved cell composition over time at various pHs. [Main component symbol description 100, 200, 300, 400... Method 101... Cell 102... Dissolved 103... Dissolved cell composition 104...contacting a first base 105...contacting a salt 106...heating 1〇7...treated dissolved cell composition 108...with a second base contact 109...separation 110...lipid 204...and Test contact 207... treated dissolved cell composition 209...separation 210...lipid 305...in contact with a salt 307...treated dissolved cell composition 309...separation 310...lipid 401...search mix 402...hot 405·· Contact with a salt 407... Treated dissolved cell composition 409...isolation 410...lipid 212

Claims

201200591 VII. Patent Application Range: 1. A method for obtaining a lipid from a microbial cell composition, comprising: raising a pH of the cell composition to 8 or above; and separating a lipid from the cell composition, Wherein the lipid contains less than 5% of an organic solvent of a halo meter. 2. The method of claim 1, wherein the pH increase dissolves the cell composition. 3. The method of claim 1, wherein the pH is raised to emulsifie the cell composition. 4. The method of any one of claims 1-3, further comprising adding a salt to the cellular composition to de-emulsifie the cellular composition. The method of claim 4, wherein the added salt is performed after the positive lift. 6·=Shenqing Patent Range No. 5 of the method of claim 5, further comprising heating the cell composition to de-emulsifie the cell composition. The method of claim 6 wherein the heating is performed after the pH is raised. 8. ^Apply to the method of diverting the scope of the first fiscal term, the further method 2 (4) the pH of the cell composition is increased again to de-emulsifie the cell composition 9' = the item of the claim, wherein the re-increase is in the added salt Or after the heating is performed. A method for obtaining a lipid from a cell, comprising: 213 201200591 dissolving a cell to form a dissolved cell composition; raising the pH of the dissolved cell composition to 8 or more to de-emulsifie the a cell composition; adding a salt to the dissolved cell composition to de-emulsifie the cell composition; and separating a lipid from the de-emulsified cell composition, wherein the lipid contains less than 5% by weight of one organic solvent. 11. A method for obtaining a lipid from a cell composition, comprising: raising a pH of a cell composition to 8 or more to dissolve the cell composition and de-emulsification the cell composition; adding a salt to the cell a composition; and separating a lipid from the emulsified cell composition, wherein the lipid contains less than 5% by weight of one organic solvent. 12. The method of claim 10, wherein the method further comprises heating the dissolved cellular composition to de-emulsifie the cellular composition. 13. The method of claim 12, wherein the heating is performed after the addition of the salt. 14. The method of any of claims 1-13, further comprising agitating the solubilized cell composition to de-emulsifie the cell composition. 15. The method of any one of claims 10-14, further comprising raising the p Η of the dissolved cellular composition to de-emulsifie the cellular composition. 16. The method of claim 15, wherein the pH raising of the dissolved cell composition is performed after the addition of the salt or the heating is performed 214 201200591. 17. The method of any one of the preceding claims, wherein the pH increase comprises the addition of a base. The method of any of the preceding claims, wherein the test has a pKb of from 1 to 12. 19. If you apply for a patent scope! The method of any one of the items 4 to 18, wherein the method comprises the method of mixing, mixing, blending, oscillating, vibrating, or a group thereof to mix the dissolved cell composition. The method of any one of the preceding claims, wherein the dissolution comprises mechanical treatment, physical treatment, chemical treatment, enzyme treatment, or a combination thereof. </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; The amount of the cell composition is added. 23. The method of any one of the preceding claims, wherein the salt is selected from the group consisting of a metal salt, a soil metal salt, a sulphate, and combinations thereof. The method of any one of claims 1 to 23, which comprises centrifugation. The method of any one of claims 1 to 24, wherein the formula further comprises a lipid comprising at least 50% by weight of a triglyceride. The method of any one of the preceding claims, wherein the party to 215 201200591 provides a lipid having a oxoaniline value of 26 or less. 27. The method of any one of claims 10-26, wherein the cell is a microbial cell. 28. The method of any one of claims 1-9, wherein the method further comprises concentrating a fermentation broth containing the microbial cell. 29. The method of any one of claims 10-26, wherein the cell is an oilseed. 30. The method of claim 29, wherein the oilseed is selected from the group consisting of: sunflower seed, canola, rapeseed, linseed, castor oil (castor oil) seeds, coriander seeds, calendula seeds, and genetic modification variants thereof. The method of any one of claims 1 to 30, further comprising washing the cell or the cell composition. The method of any one of claims 1 to 31, further comprising sterilizing the cell or the cell composition at a low temperature. The method of any one of claims 1 to 32, further comprising concentrating the dissolved cellular composition. 34. The method of any one of claims 1 to 3, further comprising refining the lipid. 35. The method of claim 34, wherein the refining is selected from the group consisting of: refining, degumming, acid treatment, alkali treatment, cooling, heating, bleaching, deodorization, deacidification, and The combination of them. The method of any of claims 1 to 35, further comprising 216 201200591 comprising harvesting the lipid, wherein the harvesting comprises pumping the lipid without agitation. 37. A method for obtaining a lipid from a cell, comprising: dissolving a cell to form a dissolved cell composition; agitating the cell composition to de-emulsifie the cell composition; and de-emulsification the cell The composition separates a lipid wherein the lipid contains less than 5% by weight of one organic solvent. 38. The method of claim 37, wherein the agitating comprises agitating the cell composition with a leaf wheel having a tip rate of from 350 centimeters per second to 900 centimeters per second. 39. A lipid obtained by the method of any one of claims 1 to 38. 40. The lipid of claim 39, wherein the lipid comprises one or more polyunsaturated fatty acids. 41. The lipid of claim 39, wherein the lipid comprises at least 35% by weight of docosahexaenoic acid. The lipid of any one of claims 39-41, wherein the lipid has a total aroma intensity of 3 or less. The lipid of any one of claims 39-41, wherein the lipid has a total aromatic intensity of 2 or less. 44. The lipid of any one of claims 39-43, wherein the lipid has a oxoaniline value of 26 or less. The lipid of any one of claims 39-44, wherein the lipid has a photo-content of 100 ppm or less. The lipid of any one of claims 39-45, wherein the lipid 217 201200591 has a peroxide value of 5 or less. 47. The fat f of claim 39, which comprises at least the weight of eicosapentaenoic acid and less than 5% by weight of arachidonic acid, docosapentaenoic acid n-6, oleic acid , linoleic acid, linoleic acid, eicosenoic acid, sinapic acid, and stearic acid. 48. The lipid of claim 39, comprising at least 1% by weight of a diterpene glyceride fraction, wherein at least 12% by weight of the fatty acid in the triterpene glyceride fraction is twenty carbons Pentaenoic acid wherein at least 25% by weight of the fatty acid in the triterpene glyceride fraction is docosahexaenoic acid, and wherein less than 5% by weight of the triterpene glyceride fraction The fatty acid is peanut oleic acid. 49. The lipid of claim 47 or 48, wherein the lipid has a peroxide value of 5 or less. The lipid of any one of claims 39-49, wherein the lipid is a crude oil. 51. A crude microbial lipid having a peroxide value of 26 or less methoxybenzamine value of 5 or less, a dish content of 1 〇〇ppm or less, and less than 5% by weight An organic solvent. 52. An extracted microbial lipid comprising at least 70% by weight of a triglyceride fraction wherein the triglyceride fraction has a docosahexaenoic acid content of at least 50% by weight, wherein The diglycidonic acid n-6 content of the triglyceride fraction is from at least 0.5% by weight to 6% by weight 'and wherein the oil has a oxoaniline value of 26 or less. 53. An extracted microbial lipid comprising at least 7% by weight of three 218 201200591 r glyceryl citrate fraction, wherein the triglyceride fraction has a docosahexaenoic acid content of at least 40 % by weight, wherein the triglyceride fraction has a docosapentaenoic acid n-6 content of from at least 0.5% by weight to 6% by weight, wherein the docosahexaenoic acid is The ratio of eicosapentaenoic acid n_6 is greater than 6:1 ' and wherein the oil has a oxoaniline value of 26 or less. 54. An extracted microbial lipid comprising at least about 7% by weight of a triglyceride fraction, wherein the triglyceride fraction has a docosahexaenoic acid content of at least 60% by weight And wherein the oil has a oxoaniline value of 26 or less. The extracted microbial lipid according to any one of claims 52 to 54, wherein the oil has a peroxide value of 5 or less. 56. The extracted microbial cerambye of any one of claims 52-55, wherein the oil has a phosphorus content of 100 ppm or less. 57. The extracted microbial lipid of any of claims 52-56, wherein the lipid is a crude oil. - In the case of (4)-fat f, the financial method contains crude lipids that are difficult to apply for patents 50, 5 or 57. 59. The method of claim 58, wherein the refining is selected from the group consisting of: caustic refining, degumming, acid treatment, treatment, bleaching, deodorization, deacidification, and the like combination. 219