TWI675621B - 蛋白質組成物及其製造方法 - Google Patents
蛋白質組成物及其製造方法 Download PDFInfo
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- TWI675621B TWI675621B TW103111562A TW103111562A TWI675621B TW I675621 B TWI675621 B TW I675621B TW 103111562 A TW103111562 A TW 103111562A TW 103111562 A TW103111562 A TW 103111562A TW I675621 B TWI675621 B TW I675621B
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- milk
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- gum
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Abstract
一種蛋白質組成物,其含有:乳鹼性蛋白質級分,及選自於由大豆多醣類、三仙膠、果膠、阿拉伯膠、甘地膠、卡拉膠、刺槐豆膠、酪蛋白鈉、卵磷脂、羧甲基纖維素構成之群組中的至少1種安定劑。根據所述蛋白質組成物,可格外提升乳鹼性蛋白質級分之耐熱性。可在不使乳鹼性蛋白質級分失活下,對蛋白質組成物及包含該組成物的飲食品或飼料、醫藥於90℃以上進行加熱處理。
Description
[0001] 本發明係有關一種有高耐熱性的蛋白質組成物及其製造方法。更詳言之,係有關一種含有乳鹼性蛋白質級分及安定劑的高耐熱性蛋白質組成物及其製造方法。
[0002] 經報導,源自乳汁的鹼性蛋白質級分係具有骨強化作用、牙周病預防作用、脂質代謝改善作用、降血壓作用、皮膚膠原蛋白產生促進作用、免疫系統之控制作用等各種生理機能。而且,為了有效利用此等生理機能,已開發含有乳鹼性蛋白質級分的各類飲食品或飼料、醫藥。惟,周知乳鹼性蛋白質級分在中性區域對加熱呈不穩定,經80℃、10分鐘加熱會生成沉澱(例如參照專利文獻1)。緣此,一般係進行對乳鹼性蛋白質級分在酸性區域進行加熱處理的方法。經報導,若採用此方法,乳鹼性蛋白質級分即使受加熱處理,其骨強化作用亦幾無變化(例如參照非專利文獻1)。 [0003] 然而,依此等方法,對於超過90℃之殺菌,尤為殺菌釜滅菌處理(retort sterilization treatment),乳鹼性蛋白質級分之熱穩定性極低,存有(1)因耐熱性不充分,在90℃以上之加熱殺菌中乳鹼性蛋白質級分易發生凝聚‧沉澱;(2)特別是,在中性區域(pH7左右)以上之環境下進行加熱殺菌時,乳鹼性蛋白質級分容易失活等問題。因此,對含乳鹼性蛋白質級分之液進行加熱處理時(尤為90℃以上),有乳鹼性蛋白質級分失活之可能性,現實在於無法採用殺菌釜滅菌處理等的超高溫加熱處理。如欲在不使乳鹼性蛋白質級分失活下予以摻混於飲食品或飼料、醫藥時有其限制。 [先前技術文獻] [專利文獻] [0004] [專利文獻1]:日本特開平09-191858號公報 [非專利文獻] [0005] [非專利文獻1]:青江誠一郎等人, Bioscience, Biotechnology and Biochemistry, 65卷, 4號, 913-918頁, 2001年
[發明所欲解決之課題] [0006] 本發明係以提供一種可在不使乳鹼性蛋白質級分失活下,對蛋白質組成物於90℃以上進行加熱的蛋白質組成物及其製造方法為課題。 [解決課題之手段] [0007] 亦即,本發明係包含以下構成: (1)一種有加熱穩定性之蛋白質組成物,包括:乳鹼性蛋白質級分,及選自於由大豆多醣類、三仙膠、果膠、阿拉伯膠、甘地膠、卡拉膠、刺槐豆膠、酪蛋白鈉、卵磷脂、羧甲基纖維素構成之群組中的至少1種安定劑。 (2)如(1)之蛋白質組成物,其中,乳鹼性蛋白質級分在其胺基酸組成中係含有15重量%以上之鹼性胺基酸。 (3)如(1)之蛋白質組成物,其中,乳鹼性蛋白質級分係具有以下性質: 1)根據十二烷基硫酸鈉-聚丙烯醯胺膠體電泳(SDS-PAGE),係由分子量3,000~80,000之範圍的數種蛋白質構成; 2)95重量%以上為蛋白質,並包含其他少量之脂肪、灰分; 3)蛋白質係主要包括乳鐵蛋白及乳過氧化酶; 4)蛋白質之胺基酸組成係含有15重量%以上之鹼性胺基酸。 (4)一種飲食品、飼料及醫藥,其係摻混有如(1)至(3)中任一項之蛋白質組成物。 (5)一種乳鹼性蛋白質級分之加熱處理方法,其係使乳鹼性蛋白質級分含有選自於由大豆多醣類、三仙膠、果膠、阿拉伯膠、甘地膠、卡拉膠、刺槐豆膠、酪蛋白鈉、卵磷脂、羧甲基纖維素構成之群組中的至少1種安定劑,並於90℃以上之溫度進行加熱。 (6)如(5)之乳鹼性蛋白質級分之加熱處理方法,其中,乳鹼性蛋白質級分在其胺基酸組成中係含有15重量%以上之鹼性胺基酸。 (7)如(5)之乳鹼性蛋白質級分之加熱處理方法,其中,乳鹼性蛋白質級分係具有以下性質: 1)根據十二烷基硫酸鈉-聚丙烯醯胺膠體電泳(SDS-PAGE),係由分子量3,000~80,000之範圍的數種蛋白質構成; 2)95重量%以上為蛋白質,並包含其他少量之脂肪、灰分; 3)蛋白質係主要包括乳鐵蛋白及乳過氧化酶; 4)蛋白質之胺基酸組成係含有15重量%以上之鹼性胺基酸。 [發明之效果] [0008] 根據本發明,可在不使乳鹼性蛋白質級分失活下,對蛋白質組成物於90℃以上進行加熱。
[0010] 本發明人等為解決上述課題,而進一步對乳鹼性蛋白質級分之熱穩定化致力重複多次研究的結果發現,藉由調製成含有乳鹼性蛋白質級分及大豆多醣類或三仙膠等安定劑的蛋白質組成物,可格外提升乳鹼性蛋白質級分之耐熱性,終至完成本發明。 即,本發明實施形態之蛋白質組成物係包括:乳鹼性蛋白質級分,及選自於由大豆多醣類、三仙膠、果膠、阿拉伯膠、甘地膠、卡拉膠、刺槐豆膠、酪蛋白鈉、卵磷脂、羧甲基纖維素構成之群組中的至少1種安定劑。該蛋白質組成物在酸性至中性、鹼性的廣泛pH範圍,經過90℃以上之高溫加熱處理及120℃以上之殺菌釜滅菌處理仍具有高耐熱性。 較佳為乳鹼性蛋白質級分在其胺基酸組成中含有15重量%以上之鹼性胺基酸。作為乳鹼性蛋白質級分,源自任意來源者皆可使用,惟,對於例如周知具有骨強化作用,且具有以下性質的乳鹼性蛋白質,可在不失去活性下於90℃以上加熱: 1)根據十二烷基硫酸鈉-聚丙烯醯胺膠體電泳(SDS-PAGE),包括分子量3,000~80,000之範圍的數種蛋白質; 2)95重量%以上為蛋白質,並包含其他少量之脂肪、灰分; 3)蛋白質係主要包括乳鐵蛋白及乳過氧化酶; 4)蛋白質之胺基酸組成係含有15重量%以上之鹼性胺基酸。 此外,作為乳鹼性胺基酸,可列舉例如賴胺酸、組胺酸、精胺酸。 [0011] 前述鹼性蛋白質級分可例如藉由使脫脂乳或乳清等乳原料與陽離子交換樹脂接觸而使鹼性蛋白質吸附,並將吸附於該樹脂的鹼性蛋白質級分在0.1M~1M之鹽濃度的溶出液中溶出,回收該溶出級分,再利用逆滲透(RO)膜或電透析(ED)法等進行脫鹽及濃縮,並視需求加以乾燥而得。作為供給源,可列舉乳牛、水牛、人類、豬、綿羊、山羊、馬等的乳汁。 [0012] 再者,作為獲得源自乳汁的乳鹼性蛋白質級分之方法,已知有:使乳汁或源自乳汁的原料與陽離子交換體接觸而使乳鹼性蛋白質吸附後,將吸附於該陽離子交換體的乳鹼性蛋白質級分,在超過pH5、超過離子強度0.5的溶出液中溶出而得之方法(日本特開平5-202098號公報);使用海藻酸膠體而得之方法(日本特開昭61-246198號公報);使用無機多孔性粒子,自乳清獲得之方法(日本特開平1-86839號公報);使用硫酸酯化合物,自乳汁獲得之方法(日本特開昭63-255300號公報)等,在本發明中,可使用以此類方法所得到的乳鹼性蛋白質級分。 [0013] 作為安定劑,更佳為以具有如下性質之成分為主成分的安定劑: (1)如大豆多醣類等分子量大、具被膜性的安定劑。 (2)如三仙膠等在葡萄糖主鏈鍵結有甘露糖與葡萄糖醛酸之類的側鏈相對於直鏈之比例大的安定劑。 (3)在果膠之半乳糖醛酸主鏈具有包含半乳糖、阿拉伯糖、木糖等的多種側鏈的安定劑。 (4)如阿拉伯膠等在半乳糖主鏈鍵結有阿拉伯糖與葡萄糖醛酸之類的側鏈相對於直鏈之比例大的安定劑。 具有此等性質的安定劑可防止加熱時乳鹼性蛋白質級分的凝聚‧沉澱所致之失活,可提高乳鹼性蛋白質級分之耐熱性。 [0014] 另一方面,作為對於乳鹼性蛋白質組成物屬不佳之安定劑,可舉出具有如下性質者: (1)如蔗糖之羥基與脂肪酸反應所形成之蔗糖脂肪酸酯等具有疏水基與親水基,且具有多分子層吸附性的安定劑。 (2)如結冷膠等形成直鏈狀結構的安定劑或如古亞膠、羅望子膠等側鏈相對於直鏈之比例大的安定劑。 如使用此等安定劑時,加熱時乳鹼性蛋白質級分之耐熱性未能提升至高程度。惟,僅含一部分此等安定劑者則不在此限。 [0015] 從而,作為與乳鹼性蛋白質級分混合而構成乳鹼性蛋白質組成物的安定劑,較佳為大豆多醣類、三仙膠、果膠、阿拉伯膠、甘地膠、卡拉膠、刺槐豆膠、酪蛋白鈉、卵磷脂、羧甲基纖維素,茲將此等安定劑的至少1種與乳鹼性蛋白質級分混合而構成乳鹼性蛋白質組成物。此等安定劑當中,有時亦具有作為乳化劑之機能等其他各種機能,可無問題地使用。 [0016] 又,以蛋白質組成物中之乳鹼性蛋白質級分與安定劑的含量比而言,不特別限制,惟以相對於乳鹼性蛋白質級分,含有0.5~100(重量/重量)、較佳為1~40(重量/重量)之安定劑為佳。 [0017] 調製蛋白質組成物之方法不特別限制,例如,為了在溶液中調製,係將乳鹼性蛋白質級分與安定劑分別懸浮或溶解於去離子水並攪拌混合後,調製成飲食品或飼料、醫藥形態而使用。就攪拌混合的條件而言,只要使乳鹼性蛋白質級分與安定劑充分混合即可,亦可視需求一面加熱至40~80℃左右,一面使用ULTRA DISPERSER等予以攪拌混合。另外,該蛋白質組成物之溶液還可視需求,利用UF膜等進行濃縮、或進行冷凍乾燥等,以便容易使用於飲食品、飼料及醫藥。 [0018] 蛋白質組成物在酸性區域至中性、鹼性區域的廣範圍內加熱穩定性高,可進行飲食品、飼料及醫藥之製造通常使用的高溫加熱處理或殺菌釜滅菌處理,亦能以粉末狀進行乾熱殺菌。因此,得以使用蛋白質組成物來調製液狀、膠體狀、粉末狀、顆粒狀等各種形態的飲食品或飼料、醫藥。 [0019] 在蛋白質組成物中,可使用鹽酸或磷酸等無機酸及檸檬酸或乙酸等有機酸、或者氫氧化鈉或碳酸氫鈉等鹼劑來調整pH。又,包圍蛋白質組成物的環境只要為可維持該蛋白質組成物之加熱穩定性的pH,則可不特別調整pH地進行高溫加熱處理或殺菌釜滅菌處理,惟可依據含蛋白質組成物之飲食品或飼料、醫藥中所要求的品質,來適當選擇加熱殺菌條件及pH。 [0020] 就蛋白質組成物而言,可僅使用該蛋白質組成物製成飲食品或飼料、醫藥,亦可適當與醣類或脂質、香料等其他的飲食品、飼料及醫藥通常所含有的原材料等混合而製成飲食品、飼料或醫藥。 [0021] 以下,茲示出參考例、實施例及試驗例對本發明詳細加以說明,為此等僅單純列示本發明之實施形態,本發明非受此等任何限定。 [0022] (參考例1:乳鹼性蛋白質級分的調製1) 將填充有400g陽離子交換樹脂(磺化CHITO PEARL;富士紡織股份有限公司製)的管柱(直徑5cm×高30cm)以去離子水充分清洗後,對該管柱以流速25ml/min流通未殺菌脫脂乳40公升(pH6.7)。通液後,將該管柱以去離子水充分清洗,使用含0.98M氯化鈉的0.02M碳酸緩衝液(pH7.0)將吸附於樹脂的鹼性蛋白質級分溶出。其後,對該溶出液利用逆滲透(RO)膜進行脫鹽、濃縮後,予以冷凍乾燥而得到21g之粉末狀鹼性蛋白質級分(參考例品A)。針對所得乳鹼性蛋白質級分,利用月桂基硫酸鈉-聚丙烯醯胺膠體電泳(SDS-PAGE)進行測定的結果,分子量係分布於3,000~80,000之範圍,成分組成係如表1所示。又,使用6N鹽酸以110℃、24小時予以水解後,利用胺基酸分析裝置(L-8500型、日立製作所製)分析其胺基酸組成,將其結果示於表2。進而,利用ELISA法分析其蛋白質組成,結果如表3所示,含有40%以上之乳鐵蛋白及乳過氧化酶。 [0023] [表1]
[0024]
[0025]
[0026] (參考例2:乳鹼性蛋白質級分的調製2) 將填充有30kg陽離子交換樹脂(SP TOYOPEARL;TOSOH股份有限公司製)的管柱(直徑100cm×高10cm)以去離子水充分清洗後,對該管柱以流速10公升/min流通經121℃加熱殺菌30秒的乾酪乳清3t(pH6.2)。通液後,將該管柱以去離子水充分清洗,使用含0.9M氯化鈉的0.1M檸檬酸緩衝液(pH5.7)將吸附於樹脂的鹼性蛋白質級分溶出。其後,對該溶出液利用電透析(ED)法進行脫鹽、濃縮後,予以冷凍乾燥而得到183g之粉末狀鹼性蛋白質級分(參考例品B)。 [0027] 試驗例1 將參考例品A之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將0.2重量%之作為安定劑的大豆多醣類溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以50℃、8000rpm予以攪拌混合3分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。 另一方面,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係依以下方法實施。 SDS-PAGE:將15μl之各試料以15μl之樣本緩衝液(sample buffer)(0.5M Tris-HCl (pH6.8)1.25ml、丙三醇1.0ml、10%SDS 2.0ml、2-巰基乙醇0.5ml、0.1%BPB 0.25ml)稀釋,並於100℃加熱5分鐘。其後將各試料各取15μl在14%聚丙烯醯胺膠體(TEFCO SDS-PAGE mini)中進行電泳。作為分子量標記係使用Kaleidoscope Prestained Standards(BioRad)。將其結果示於表4。 [0028] [0029] 據表4之結果,含有乳鹼性蛋白質及大豆多醣類之蛋白質組成物的溶液在pH2~9未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,該蛋白質組成物非僅在酸性側呈穩定,在中性及鹼性側加熱穩定性亦極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH2~9,縱使於120℃加熱10分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。由此試驗結果而言,可明瞭該蛋白質組成物即使經過殺菌釜滅菌處理,仍可不使乳鹼性蛋白質級分失活而保持活性。另一方面,僅含乳鹼性蛋白質級分的溶液在pH2~9完全無法確認出乳鹼性蛋白質級分之譜帶。此表示因加熱發生變性、分解,而導致乳鹼性蛋白質級分喪失其機能。 [0030] 試驗例2 將參考例品B之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將作為安定劑的三仙膠以0.04%(重量%)溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以50℃、9500rpm予以攪拌混合3分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。又,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係以與試驗例1同樣的方法實施。將結果示於表5。 [0031] [表5][0032] 據表5之結果,含有包括乳鹼性蛋白質級分及三仙膠之蛋白質組成物的溶液在pH2~9未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,該蛋白質組成物非僅在酸性側呈穩定,在中性及鹼性側加熱穩定性亦極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH2~9,縱使於130℃加熱10分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。依此,可明瞭該蛋白質組成物即使經過殺菌釜滅菌處理,仍可充分保持乳鹼性蛋白質級分之活性。 [0033] 試驗例3 將參考例品A之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將0.2重量%之作為安定劑的果膠溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以40℃、8000rpm予以攪拌混合3分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。又,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係以與試驗例1同樣的方法實施。將結果示於表6。 [0034] [表6][0035] 據表6之結果,含有乳鹼性蛋白質級分及果膠之蛋白質組成物的溶液在pH2~9未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,該蛋白質組成物非僅在酸性側呈穩定,在中性及鹼性側加熱穩定性亦極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH2~9,縱使於120℃加熱8分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。由此結果,可明瞭該蛋白質組成物即使在殺菌釜滅菌處理後,仍可充分保持乳鹼性蛋白質級分之活性。 [0036] 試驗例4 將參考例品B之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將0.1重量%之作為安定劑的阿拉伯膠溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以40℃、8000rpm予以攪拌混合3分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。又,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係以與試驗例1同樣的方法實施。 [0037] 依試驗例4之結果,含有乳鹼性蛋白質級分及阿拉伯膠之蛋白質組成物的溶液在pH3~5未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,該蛋白質組成物在酸性側加熱穩定性極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH3~5,縱使於120℃加熱7分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。由此試驗結果而言,可明瞭該蛋白質組成物即使在殺菌釜滅菌處理後,仍可充分保持乳鹼性蛋白質級分之活性。 [0038] 試驗例5 將參考例品A之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將0.1重量%之作為安定劑的甘地膠溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以40℃、8000rpm予以攪拌混合4分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。又,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係以與試驗例1同樣的方法實施。 [0039] 依試驗例5之結果,含有乳鹼性蛋白質級分及甘地膠之蛋白質組成物的溶液在pH3~8未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,該蛋白質組成物非僅在酸性側呈穩定,在中性及鹼性側加熱穩定性亦極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH3~8,縱使於120℃加熱7分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。由此試驗結果而言,可明瞭該蛋白質組成物即使在殺菌釜滅菌處理後,仍可充分保持乳鹼性蛋白質級分之活性。 [0040] 試驗例6 將參考例品B之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將0.2重量%之作為安定劑的卡拉膠溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以40℃、8000rpm予以攪拌混合3分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。又,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係以與試驗例1同樣的方法實施。 [0041] 依試驗例6之結果,含有乳鹼性蛋白質及卡拉膠之蛋白質組成物的溶液在pH4~8未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,該蛋白質組成物非僅在酸性側呈穩定,在中性及鹼性側加熱穩定性亦極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH4~8,縱使於120℃加熱6分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。由此試驗結果而言,可明瞭該蛋白質組成物即使在殺菌釜滅菌處理後,仍可充分保持乳鹼性蛋白質級分之活性。 [0042] 試驗例7 將參考例品A之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將0.15重量%之作為安定劑的刺槐豆膠溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以40℃、8000rpm予以攪拌混合3分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。又,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係以與試驗例1同樣的方法實施。 [0043] 依試驗例7之結果,含有乳鹼性蛋白質及刺槐豆膠之蛋白質組成物的溶液在pH4~7未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,蛋白質組成物非僅在酸性側呈穩定,在中性及鹼性側加熱穩定性亦極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH4~7,縱使於120℃加熱5分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。由此試驗結果而言,可明瞭該蛋白質組成物即使在殺菌釜滅菌處理後,仍可充分保持乳鹼性蛋白質級分之活性。 [0044] 試驗例8 將參考例品B之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將0.15重量%之作為安定劑的酪蛋白鈉溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以40℃、9500rpm予以攪拌混合3分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。又,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係以與試驗例1同樣的方法實施。 [0045] 依試驗例8之結果,含有乳鹼性蛋白質及刺槐豆膠之蛋白質組成物的溶液在pH5~9未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,該蛋白質組成物非僅在酸性側呈穩定,在中性及鹼性側加熱穩定性亦極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH5~9,縱使於120℃加熱5分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。由此試驗結果而言,可明瞭該蛋白質組成物即使在殺菌釜滅菌處理後,仍可充分保持乳鹼性蛋白質級分之活性。 [0046] 試驗例9 將參考例品A之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將0.25重量%之作為安定劑的卵磷脂溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以40℃、8000rpm予以攪拌混合3分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。又,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係以與試驗例1同樣的方法實施。 [0047] 依試驗例9之結果,含有乳鹼性蛋白質及卵磷脂之蛋白質組成物的溶液在pH3~8未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,該蛋白質組成物非僅在酸性側呈穩定,在中性及鹼性側加熱穩定性亦極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH3~8,縱使於120℃加熱6分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。由此試驗結果而言,可明瞭該蛋白質組成物即使在殺菌釜滅菌處理後,仍可充分保持乳鹼性蛋白質級分之活性。 [0048] 試驗例10 將參考例品B之乳鹼性蛋白質級分以10mg%濃度溶解於去離子水(A液)。將0.15重量%之作為安定劑的羧甲基纖維素溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以40℃、8000rpm予以攪拌混合3分鐘而調製成蛋白質組成物。其後,使用乳酸或氫氧化鈉溶液將該蛋白質組成物調成pH1~10之10份試料。將其各取2ml分注至安瓿管中,於90℃、100℃、110℃、120℃、130℃加熱4分鐘。對作為對照區之不含安定劑而僅有乳鹼性蛋白質級分的溶液(A液),以與上述同樣的方法調整pH並於110℃加熱4分鐘。依目視判定加熱處理後之各試料及對照區的凝聚‧沉澱狀態。又,為了確認經加熱處理之乳鹼性蛋白質級分的分解程度,而針對加熱處理後之各試料及對照區之試樣,利用聚丙烯醯胺膠體電泳(SDS-PAGE),進行乳鹼性蛋白質級分之譜帶圖形的解析。SDS-PAGE係以與試驗例1同樣的方法實施。 [0049] 依試驗例10之結果,含有乳鹼性蛋白質及羧甲基纖維素之蛋白質組成物的溶液在pH3~7未生成可由目視觀察到的凝聚‧沉澱,而且利用SDS-PAGE可確認乳鹼性蛋白質級分之譜帶。由此可知,該蛋白質組成物非僅在酸性側呈穩定,在中性及鹼性側加熱穩定性亦極高。更且,延長加熱時間並檢測是否可確認乳鹼性蛋白質級分的結果,在pH3~7,縱使於120℃加熱6分鐘亦未發生凝聚‧沉澱,並可確認乳鹼性蛋白質級分之譜帶。由此試驗結果而言,可明瞭該蛋白質組成物即使在殺菌釜滅菌處理後,仍可充分保持乳鹼性蛋白質級分之活性。 [0050] 試驗例11 對試驗例1~試驗例10中所調製之試料,使用針對乳鹼性蛋白質級分之抗體並利用ELISA法測定將pH調整至7並於140℃加熱5分鐘時的抗原性。作為比較對象,係將未與安定劑混合的乳鹼性蛋白質級分於140℃加熱5分鐘,設使其與抗體反應時的反應性為1,並以相對於其之比算出各蛋白質組成物與抗體的反應性,予以示於表7。 [0051]
※以設乳鹼性蛋白質級分之與抗體的反應性為1時的比表示。 ※數值係表示平均值±標準差(n=6)。 ※a表示與對照比較時有顯著差異(p<0.05)。 [0052] 由表7可明瞭,蛋白質組成物之與抗體的反應性相對於將只有乳鹼性蛋白質級分加熱時之與抗體的反應性直到140℃均維持1以上。從而,本發明之蛋白質組成物對於140℃之超高溫加熱處理呈穩定。 [0053] 試驗例12 對試驗例1~試驗例10中所調製之試料,將pH調整至7,並測定於140℃加熱5分鐘時的成骨細胞增殖活性。作為比較對象,係將與安定劑混合的乳鹼性蛋白質級分於140℃加熱5分鐘,設其成骨細胞增殖活性為1,並將蛋白質組成物相對於其之成骨細胞增殖活性的比示於表8。 [0054]
[0055] 由表8之結果可明瞭,蛋白質組成物之增殖活性相對於將只有乳鹼性蛋白質級分加熱時之增殖活性,直到至140℃均維持1以上。從而,本發明之蛋白質組成物對於所謂140℃之超高溫加熱處理呈穩定。 [0056] 試驗例13 將含有以與試驗例2同樣方法調製的蛋白質組成物(50mg%乳鹼性蛋白質級分、0.04重量%三仙膠)的溶液調整至pH2~9,各取150ml填充至蒸煮袋(殺菌袋,retort pouch)並予以密封。將作為對照之僅含LF的溶液調整至pH2~9後各取150ml填充至蒸煮袋並予以密封。對此等溶液利用殺菌釜滅菌機(第1種壓力容器;TYPE:RCS-4CRTGN;日阪製作所製)於120℃加熱4分鐘。於25℃保存加熱後之各試料,依目視觀察隨時間經過是否有凝聚‧沉澱,並且利用聚丙烯醯胺膠體電泳(SDS-PAGE)進行乳鹼性蛋白質級分之譜帶圖形的解析。 其結果,將作為對照之僅含LF的溶液調整至pH2~9者,於第1天可確認發生凝聚‧沉澱,但將含有蛋白質組成物的溶液調整至pH2~9者,保存後經過1個月亦未確認發生凝聚‧沉澱。此外,利用SDS-PAGE對乳鹼性蛋白質級分之譜帶圖形進行解析的結果,將含有蛋白質組成物的溶液調整至pH2~9者在保存開始時起至保存後1個月,仍可確認乳鹼性蛋白質級分之譜帶,且未看出其他變化。據此試驗結果,可明瞭本發明之蛋白質組成物對於殺菌釜滅菌處理之情況亦屬有效。 [0057] 試驗例14 對200g以與試驗例2同樣方法調製的蛋白質組成物(50mg%乳鹼性蛋白質級分、0.04重量%三仙膠)混合800g還原脫脂奶粉溶液(3重量%脫脂奶粉),調製成含有蛋白質組成物的溶液(1)。作為對照,調製200g乳鹼性蛋白質級分溶液(50mg%乳鹼性蛋白質級分之溶液)中混有800g還原脫脂奶粉溶液(脫脂奶粉3重量%)的溶液(2)、及僅有1000g還原脫脂奶粉溶液(脫脂奶粉3重量%)的溶液(3)。將各溶液各取150ml填充至蒸煮袋並予以密封。對此等溶液利用殺菌釜滅菌機(第1種壓力容器、TYPE:RCS-4CRTGN;日阪製作所製)於120℃加熱20分鐘。其結果,在溶液(1)、(3)中均未看出凝聚‧沉澱,且風味亦良好;然而在溶液(2)中可看出凝聚‧沉澱。據此試驗結果,可明瞭本發明之蛋白質組成物對於殺菌釜滅菌處理之情況極為有效。 [0058] 試驗例15 將參考例品A之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液)。將作為安定劑之0.4重量%大豆多醣類、0.04重量%三仙膠、0.05重量%羅望子膠及0.1重量%蔗糖脂肪酸酯分別溶解於去離子水(B液)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以50℃、8000rpm予以攪拌混合3分鐘而調製成蛋白質組成物。使用乳酸或氫氧化鈉溶液將該蛋白質組成物調製成pH6.5。對所得試料,依循試驗例1之方法,於120℃加熱4分鐘的前後,依目視確認各試料的凝聚‧沉澱狀態,並利用SDS-PAGE確認蛋白質組成物的分解程度。將結果示於表9。 [0059] [0060] 據表9之結果,加熱前之蛋白質組成物均未發生凝聚‧沉澱,呈透明,且亦可確認乳鹼性蛋白質級分之譜帶。於加熱後,含有大豆多醣類及三仙膠的蛋白質組成物係呈透明,且亦可確認乳鹼性蛋白質級分之譜帶,惟含有羅望子膠及蔗糖脂肪酸酯的蛋白質組成物均呈半透明、或者發生凝聚‧沉澱,以SDS-PAGE也無法確認乳鹼性蛋白質級分之譜帶。 [實施例1] [0061] 將參考例品B之乳鹼性蛋白質級分以50mg%濃度溶解於去離子水(A液;300g)。將0.4重量%之作為安定劑的大豆多醣類溶解於去離子水(B液;300g)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以50℃、9500rpm予以攪拌混合3分鐘而調製成600g之本發明蛋白質組成物。 [實施例2] [0062] 將參考例品A之乳鹼性蛋白質級分以40mg%濃度溶解於去離子水(A液;10kg)。將作為安定劑的三仙膠以0.08重量%溶解於去離子水(B液;10kg)。混合A液與B液,利用TK HOMO MIXER(MARK II 160型;特殊機化工業製),以3600rpm予以攪拌混合30分鐘,進而以分級分子量10kDa之UF膜予以濃縮,而調製成10kg之本發明蛋白質組成物。 [實施例3] [0063] 將參考例品B之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液;1000kg)。將0.4重量%之作為安定劑的果膠溶解於去離子水(B液;1000kg)。混合A液與B液,利用TK HOMO MIXER(MARK II 2500型;特殊機化工業製),以40℃、3600rpm予以攪拌混合40分鐘,進而予以冷凍乾燥,製備成本發明之蛋白質組成物3.9kg。 [實施例4] [0064] 將參考例品A之乳鹼性蛋白質級分以100mg%濃度溶解於去離子水(A液;500g)。將0.4重量%之作為安定劑的大豆多醣類溶解於去離子水(B液;500g)。混合A液與B液,利用ULTRA DISPERSER(ULTRA-TURRAX T-25;IKA JAPAN公司製),以40℃、9500rpm予以攪拌混合3分鐘。其後,對上述溶液添加80g山梨糖醇、4g酸味料、4g香料、10g果膠、10g乳清蛋白質濃縮物、2g乳酸鈣、890g水,予以攪拌混合,調製成本發明蛋白質組成物。予以填充至200ml之附瓶蓋鋁箔便利包(cheer pack),並以85℃、20分鐘進行殺菌後予以密封,而調製成包含本發明蛋白質組成物的膠體狀食品10袋。調製之膠體狀食品均未看出沉澱等,且未感受到風味異常。 [實施例5] [0065] 將參考例品B之乳鹼性蛋白質級分以500mg%濃度溶解於去離子水 (A液;200g)。將4重量%之作為安定劑的大豆多醣類溶解於去離子水(B液;200g)。混合100g麥芽糖醇、20g還原糖漿、2g酸味料、2g香料、200g前述A液、200g前述B液、及476g水,調製成本發明蛋白質組成物。將該蛋白質組成物填充至50ml之玻璃瓶,以90℃、15分鐘進行殺菌後予以密封,而調製成含有本發明蛋白質組成物的飲料20瓶。調製之飲料均未看出沉澱,且未感受到風味異常。 [實施例6] [0066] 對0.2kg實施例2中所調製之蛋白質組成物(20mg%乳鹼性蛋白質級分、0.04重量%三仙膠)摻混12kg大豆粕、14kg脫脂奶粉、4kg大豆油、2kg玉米油、28kg棕櫚油、15kg玉米澱粉、9kg小麥粉、2kg米糠、9kg維生素混合物、2.8kg纖維素、2kg礦物質混合物,以120℃、4分鐘進行殺菌,調製成100kg犬類飼養用飼料。 [實施例7] [0067] 對3kg實施例3中所調製之蛋白質組成物(50mg%乳鹼性蛋白質級分、0.2重量%果膠)摻混5kg乾酪素、5kg大豆蛋白質、1kg魚油、3kg紫蘇油、19kg糊精、6kg礦物質混合物、1.95kg維生素混合物、2kg乳化劑、4kg安定劑、0.05kg香料,予以填充至200ml之蒸煮袋,並利用殺菌釜滅菌機(第1種壓力容器;TYPE:RCS-4CRTGN;日阪製作所製)以121℃、20分鐘進行殺菌,調製成50kg經腸營養劑。
無。
[0009] 無
Claims (6)
- 一種有加熱穩定性之骨強化用蛋白質組成物,包括:乳鹼性蛋白質級分,及選自於由大豆多醣類、三仙膠、果膠、阿拉伯膠、甘地膠、卡拉膠、刺槐豆膠、酪蛋白鈉、卵磷脂、羧甲基纖維素構成之群組中的至少1種安定劑;其中,該乳鹼性蛋白質級分係具有以下性質:1)根據十二烷基硫酸鈉-聚丙烯醯胺膠體電泳(SDS-PAGE)係由分子量3,000~80,000之範圍的數種蛋白質構成;2)95重量%以上為蛋白質,並包含其他少量之脂肪、灰分;3)蛋白質係主要包括乳鐵蛋白及乳過氧化酶;4)蛋白質之胺基酸組成係含有15重量%以上之鹼性胺基酸。
- 如申請專利範圍第1項之有加熱穩定性之骨強化用蛋白質組成物,其中,該安定劑係選自於由阿拉伯膠、甘地膠構成之群組中的至少1種。
- 一種骨強化用飲食品,其係摻混有如申請專利範圍第1或2項之有加熱穩定性之骨強化用蛋白質組成物。
- 一種骨強化用飼料,其係摻混有如申請專利範圍第1或2項之有加熱穩定性之骨強化用蛋白質組成物。
- 一種骨強化用醫藥,其係摻混有如申請專利範圍第1或2項之有加熱穩定性之骨強化用蛋白質組成物。
- 一種骨強化用乳鹼性蛋白質級分之加熱處理方法,其係使乳鹼性蛋白質級分含有選自於由大豆多醣類、三仙膠、果膠、阿拉伯膠、甘地膠、卡拉膠、刺槐豆膠、酪蛋白鈉、卵磷脂、羧甲基纖維素構成之群組中的至少1種安定劑,並於90℃以上之溫度進行加熱;其中,該乳鹼性蛋白質級分係具有以下性質:1)根據十二烷基硫酸鈉-聚丙烯醯胺膠體電泳(SDS-PAGE)係由分子量3,000~80,000之範圍的數種蛋白質構成;2)95重量%以上為蛋白質,並包含其他少量之脂肪、灰分;3)蛋白質係主要包括乳鐵蛋白及乳過氧化酶;4)蛋白質之胺基酸組成係含有15重量%以上之鹼性胺基酸。
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JP2007246413A (ja) * | 2006-03-14 | 2007-09-27 | Snow Brand Milk Prod Co Ltd | 乳由来塩基性タンパク質含有組成物 |
JP2007259733A (ja) * | 2006-03-28 | 2007-10-11 | Sanei Gen Ffi Inc | 発酵乳食品用安定剤および該安定剤を含有する発酵乳食品 |
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JP4624959B2 (ja) * | 2006-05-19 | 2011-02-02 | 三栄源エフ・エフ・アイ株式会社 | 密封容器入り乳飲料用安定剤 |
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JP2010180219A (ja) * | 2010-03-04 | 2010-08-19 | Snow Brand Milk Prod Co Ltd | ラクトフェリン組成物 |
CA2823505A1 (en) * | 2011-01-26 | 2012-08-02 | Megmilk Snow Brand Co., Ltd. | Agent for improving peripheral sensitivity of skin |
-
2013
- 2013-03-28 JP JP2013070417A patent/JP6309202B2/ja active Active
-
2014
- 2014-03-25 MY MYPI2015703020A patent/MY169153A/en unknown
- 2014-03-25 US US14/779,647 patent/US9861121B2/en not_active Expired - Fee Related
- 2014-03-25 CN CN201480018942.0A patent/CN105072920A/zh active Pending
- 2014-03-25 EP EP14773646.6A patent/EP2984935A4/en not_active Withdrawn
- 2014-03-25 WO PCT/JP2014/058209 patent/WO2014157156A1/ja active Application Filing
- 2014-03-27 TW TW103111562A patent/TWI675621B/zh active
-
2015
- 2015-09-02 PH PH12015501937A patent/PH12015501937A1/en unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004352669A (ja) * | 2003-05-29 | 2004-12-16 | Snow Brand Milk Prod Co Ltd | ラクトフェリン組成物 |
Also Published As
Publication number | Publication date |
---|---|
JP6309202B2 (ja) | 2018-04-11 |
CN105072920A (zh) | 2015-11-18 |
EP2984935A1 (en) | 2016-02-17 |
PH12015501937B1 (en) | 2016-01-11 |
WO2014157156A1 (ja) | 2014-10-02 |
US20160044949A1 (en) | 2016-02-18 |
US9861121B2 (en) | 2018-01-09 |
TW201517807A (zh) | 2015-05-16 |
EP2984935A4 (en) | 2016-11-30 |
PH12015501937A1 (en) | 2016-01-11 |
JP2014193125A (ja) | 2014-10-09 |
MY169153A (en) | 2019-02-18 |
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