TWI676682B - 枯草芽孢桿菌分離株及其用途 - Google Patents
枯草芽孢桿菌分離株及其用途 Download PDFInfo
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- TWI676682B TWI676682B TW107130675A TW107130675A TWI676682B TW I676682 B TWI676682 B TW I676682B TW 107130675 A TW107130675 A TW 107130675A TW 107130675 A TW107130675 A TW 107130675A TW I676682 B TWI676682 B TW I676682B
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- bacillus subtilis
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- fermentation
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- C—CHEMISTRY; METALLURGY
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Abstract
本發明提供枯草芽孢桿菌(
Bacillus subtilis)分離株、製備其發酵萃取物之方法、其發酵萃取物、包含其發酵萃取物之組成物,及其發酵萃取物用於促進軟骨細胞再生、增加軟骨細胞外間質分泌及/或治療關節炎之用途。
Description
本發明係關於枯草芽孢桿菌(
Bacillus subtilis)分離株、利用該等分離株製備發酵萃取物、包含該發酵萃取物之組成物及該發酵萃取物於促進軟骨細胞再生、增加軟骨細胞外間質分泌及/或治療關節炎之用途。
退化性關節炎(Osteoarthritis)是全世界最常見的關節疾病且與年齡有直接的關係,是老年人行動不便的頭號原因。有越來越多的證據顯示(Loeser, R.F. 2008, J. Musculoskelet Neuronal Interact ; 8(4):303-306),細胞激素、趨化素和其他炎症介質的產生是造成退化性關節炎中的軟骨降解之因素。這些炎症因子通常透過影響免疫細胞和滑膜細胞而引發關節炎,退化性關節炎會由軟骨細胞產生炎症介質,包括細胞激素和趨化素,例如介白素-1(interleukin-1,簡稱IL-1)、IL-6、IL-7、IL-8、IL-17、IL-18、單核細胞趨化蛋白(monocyte chemotactic protein-1,簡稱MCP-1)、白血病抑制因子(leukemia inhibitory factor,簡稱LIF)、生長調節致癌基因 (growth-regulated oncogene,簡稱GRO)和製瘤素M及活性氧物質(reactive oxygen species,簡稱ROS),例如:一氧化氮(NO),過氧化氫(H2O2)和過氧亞硝酸鹽,以及脂質衍生的炎症介質(例如前列腺素(prostaglandins)和白三烯(leukotrienes)等物質),會促使軟骨細胞產生蛋白水解酶(proteolytic enzymes; proteinases),此包含蛋白聚醣酶(aggrecanase)和基質金屬蛋白酶(matrix metalloproteases,簡稱MMPs)等。
Masahiko Kobayashi等人(2005, Arthritis Rhemu ,52:128-35)的研究顯示,IL-1β會促使軟骨細胞之MMPs(如MMP-1及MMP-13)表現增加,而MMP-1及MMP-13會降解細胞外間質中的第2型膠原蛋白(collagen II)及聚蛋白聚醣(aggrecan)。IL-1b及TNF-α皆可誘發人類軟骨肉瘤細胞(SW1353)產生退化性關節炎相關發炎性物質(例如細胞激素(cytokine)、前列腺素及一氧化氮),及活化蛋白水解酶,亦會抑制軟骨細胞的增生(proliferation),導致第2型膠原蛋白以及聚蛋白聚醣降解。 退化性關節炎患者的軟骨細胞再生速率較慢,且其分泌第2型膠原蛋白以及聚蛋白聚醣等細胞外間質的能力下降,因此關節易產生發炎的現象。世界衛生組織(WHO)之調查顯示全球罹患退化性關節炎的患者逐年增加,而台灣衛生福利部的統計資料亦指出國人膝關節退化的盛行率約為15%,推估有350萬人飽受膝關節疼痛之苦,且隨著運動風氣盛行,很多人的膝蓋常因不當使用,使患者年齡層由60、70歲逐漸下降,出現年輕化趨勢。
由於具有關節退化問題的人口增多,關節保健產品之需求亦隨著增加。然而現行相關產品的原料主要以葡萄醣胺(glucosamine)或硫酸軟骨素(chondroitin sulfate)為主。這些原料主要係萃取自動物組織,然而由於動物疾病(例如:禽流感、狂牛症及口蹄疫等)及魚類重金屬汙染等問題的存在,因此無動物成分的保健產品漸漸受到重視,且使用微生物發酵法生產相關保健產品漸成為各界的研究重點(DeAngelis, P. L. ,2012. Appl. Microbiol. Biotechnol. 94:295-305)。本發明透過篩選具有促進軟骨再生及抗關節發炎效果之微生物發酵物,以開發有潛力應用於骨關節保健的產品。
本發明主要的目的在於提供可產生促進軟骨細胞再生、增加軟骨細胞外間質分泌及/或治療關節炎效果之發酵萃取物的枯草芽孢桿菌分離株因此,本發明提供一種枯草芽孢桿菌分離株,其係寄存於財團法人食品工業發展研究所且寄存編號為BCRC910842或BCRC910843之菌株,或為與寄存於財團法人食品工業發展研究所且寄存編號為BCRC910842或BCRC910843之菌株具有實質上完全相同特徵之變異株。
本發明另提供一種製備枯草芽孢桿菌發酵萃取物之方法,其包含:(a) 將前述之枯草芽孢桿菌分離株接種於培養基中;(b) 在適當環境下培養該經接種之培養基以獲得發酵物;(c) 自該發酵物中去除該枯草芽孢桿菌菌株以獲得上清液;(d) 將該上清液以體積比為約1:1至約1:9之比例與100%乙醇混合以產生沉澱物;及(e) 收集該沉澱物。
本發明另提供一種枯草芽孢桿菌發酵萃取物,其可自如前述之方法獲得。
本發明另提供一種組成物,其包含治療有效量之前述枯草芽孢桿菌發酵萃取物。
本發明另提供一種前述之枯草芽孢桿菌發酵萃取物的用途,其係用於製備促進軟骨細胞再生、增加軟骨細胞外間質分泌及/或治療關節炎之藥物。
參考以下對本發明之各態樣、實例、及伴隨相關描述之化學圖式及表格的詳細描述,可更容易地瞭解本發明。在揭示及描述本發明之菌株、發酵萃取物、組成物及/或方法之前,應瞭解,除非由申請專利範圍另外特別地指出,否則本發明不受限於特定製備方法、載劑或調配物、或將本發明發酵萃取物調配成用於局部、經口或非經腸投予之產物或組合物的特定模式,此係由於熟習相關技術之通常知識者非常清楚此等事情是可以加以變化的。亦應瞭解,本文所用之術語僅用於描述特定態樣之目的而不意欲用於限制性本發明之範疇。
必須指出,除非上下文另外清楚規定,否則如本說明書及隨附申請專利範圍中所用之單數形式「一」及「該」包括複數個所指標的物。因此,除非上下文另外需要,否則單數術語應包括複數且複數術語應包括單數。
範圍在本文中通常表述為「約」一個特定值及/或至「約」另一個特定值。當表述此類範圍時,一態樣為包括一個特定值及/或至另一個特定值之範圍。類似地,當值藉由使用字「約」表述為近似值時,應瞭解特定值可形成另一態樣。另外應瞭解,每一範圍之各端點皆有顯著性,一端點與另一端點既有相關性,亦彼此獨立。
本發明提供一種枯草芽孢桿菌(
Bacillus subtilis)分離株,其係寄存於財團法人食品工業發展研究所且寄存編號分別為BCRC910842及BCRC910843之菌株(上述菌株另寄存於萊布尼茨研究所DSMZ-德國微生物及細胞培養保藏中心,寄存編號分別為DSM32867和DSM32870),或為與寄存於財團法人食品工業發展研究所且寄存編號分別為BCRC910842及BCRC910843之菌株具有實質上完全相同特徵之變異株。
枯草芽孢桿菌,或稱枯草桿菌,為芽孢桿菌屬的一種細菌,存在於土壤及植物體表,或可在人體腸道內共生。枯草芽孢桿菌是公認安全(generally recognized as safe,簡稱GRAS)的微生物,故可直接用於食品和飼料添加劑中。
本發明所述寄存編號為BCRC910842(或DSM32867)之枯草芽孢桿菌菌株(下稱P32-7B菌株)及寄存編號為BCRC910843(或DSM32870)之枯草芽孢桿菌菌株(下稱P12-3C)菌株可為枯草芽孢桿菌199菌株(下稱199菌株,購自食品工業發展研究所,貨品編號為BCRC 14199)之突變株。其中,P12-3C菌株具有如SEQ ID NO. 1所示之16S DNA序列以及如SEQ ID NO. 2所示之gyrB序列;P32-7B菌株具有如SEQ ID NO. 3所示之16S DNA序列以及如SEQ ID NO. 4所示之gyrB序列。
依據財團法人食品工業發展研究所所出具之鑑定報告,上述P12-3C菌株根據16S rDNA之鑑定結果,與
Bacillus subtilis群(
Bacillus subtilis subsp. inaquosorum、
Bacillus subtilis subsp. subtilis、
Bacillus subtilis subsp. spizizenii、
Bacillus tequilensis、
Bacillus velezensis、
Bacillus vallismortis、
Bacillus mojavensis、
Bacillus amyloliquefaciens等)之相似性高達99%以上;而根據gyr B基因序列分析,其最接近
Bacillus subtilis subsp. subtilis,相似性為98.9%。由此可知,上述P12-3C菌株為
Bacillus subtilis subsp. subtilis,且為新菌株。
依據財團法人食品工業發展研究所所出具之鑑定報告,上述P32-7B菌株根據16S rDNA之鑑定結果,與
Bacillus subtilis群(
Bacillus subtilis subsp. inaquosorum、
Bacillus subtilis subsp. subtilis、
Bacillus subtilis subsp. spizizenii、
Bacillus tequilensis、
Bacillus velezensis等)之相似性高達99%以上;而根據gyr B基因序列分析,其最接近
Bacillus subtilis subsp. subtilis,相似性為99.0%。由此可知,上述P32-7B菌株為
Bacillus subtilis subsp. subtilis,且為新菌株。
本發明另提供一種製備枯草芽孢桿菌發酵萃取物之方法,其包含: (a) 將前述之枯草芽孢桿菌分離株接種於培養基中; (b) 在適當環境下培養該經接種之培養基以獲得發酵物; (c) 自該發酵物中去除該枯草芽孢桿菌菌株以獲得上清液; (d) 將該上清液以體積比為約1:1至約1:9之比例與100%乙醇混合以產生沉澱物;及 (e) 收集該沉澱物。
上述步驟(a)中之培養基可為任何習知用於培養枯草芽孢桿菌之培養基,例如可為RM培養基(含2% 酵母萃取物(yeast extract)、2%大豆蛋白腖(soy peptone)、4% 葡萄糖、0.2% K
2HPO
4與0.07% MgSO
4)、營養培養液(Nutrient Broth,簡稱NB)、胰蛋白酶大豆培養液 (Tryptic Soy Broth,簡稱TSB)、馬鈴薯液體培養基(Potato dextrose Broth,簡稱PDB)及蛋白腖培養基(含2% 大豆蛋白腖與4% 葡萄糖)。
步驟(a)中之接種量可為每克培養基接種約10
7CFU至約10
10CFU之該分離株,較佳可為每克培養基接種約10
8CFU至約10
9CFU之該分離株。於接種前,另可先將該枯草芽孢桿菌分離株活化,例如可於營養瓊脂皿上活化培養後,以無菌水刮洗下菌體作為菌體懸浮液,再用於接種。
步驟(b)中,該經接種之培養基可在含氧下於約20°C至約40°C之溫度中進行培養。舉例而言,可於30°C之環境下進行搖瓶培養,以使含氧量充足。培養時間可視其接種量及培養溫度進行調整,例如可為2至10天,較佳可為約5天。
步驟(c)中,自該發酵物中去除該枯草芽孢桿菌菌株之方法可為一般之分離方法,例如可為離心或過濾等,本發明不加以限制。
步驟(d)中,係將該上清液以體積比為約1:1至約1:9之比例與100%乙醇混合來產生沉澱物。該上清液與100%乙醇之混合體積比可視情況做調整,較佳可為約1:4。如本技術領域中具通常知識者可理解,亦可使用其他比例之乙醇替換前述100%乙醇,僅需使混合後乙醇之濃度達到與前述之最終乙醇濃度相同即可。
步驟(e)中,可藉由任何習知諸如離心或過濾等方法使沉澱物沉降聚集,並於去除上清液後收集該沉澱物。
較佳地,於步驟(e)之後,更包含:(f) 乾燥該沉澱物。本發明不限制該沉澱物之乾燥方法,例如可為加熱乾燥、真空乾燥、冷凍乾燥等。藉此,可使該沉澱物形成乾燥顆粒或粉末等,以利其應用時與其他成分混合。
較佳地,於步驟(f)之後,更包含:(g) 將該沉澱物溶入適當的溶劑中。舉例而言,步驟(g)中之溶劑可為磷酸鹽緩衝劑(phosphate buffered saline,簡稱PBS)、醋酸緩衝劑、鹽酸緩衝劑(HCl buffer solution)或純水等,然不以此為限。
本發明另提供一種枯草芽孢桿菌發酵萃取物,其可自前述之方法獲得。
根據本發明之萃取物,當利用高效液相層析(high performance liquid chromatography)在以下條件下測定時,該枯草芽孢桿菌發酵萃取物在滯留時間為約2.73min、約2.97min、約3.23min及約5.86min處分別具有4個主要峰: HPLC機型:Water 2695 separation module 偵測器:Water 2996 photodiode array detector 管柱::RP-18 (LiChroCART®100,5 μm,Merck) 流速:0.5 mL/min 移動相:0.005M戊磺酸鈉(sodium pentanesulfonate)溶液/乙腈(acetonitrile),95∶5(v/v) 測定波長:258nm 注射體積:10 μL 分析時間:10 min。
於某些實施例中,該枯草芽孢桿菌發酵萃取物於上述條件之效液相層析的測定結果可如圖1A(P12-3C菌株)或圖1B(P32-7B菌株)所示。
本發明又提供一種組成物,其包含治療有效量之前述枯草芽孢桿菌發酵萃取物。
術語「治療有效量」係指萃取物或化合物經投與後可一定程度上減輕所治療疾病或病症之一或多種症狀的足夠量。結果可為減輕及/或緩解疾病之病徵、症狀或病因或生物系統之任何其他所需的改變。如下文所指出,確實的需要量將在個體之間有變化,此視個體之疾病病況、身體狀況、年齡、性別、物種及體重、組合物之特性及配方等而定。給藥方案可經調整以誘導最佳治療反應。舉例而言,可每日投予若干分次劑量,或可依治療情形之緊急程度按比例減少劑量。因此,很難指定確實的「治療有效量」。然而,本發明領域中具有通常知識者使用常規實驗即可確定適當的治療有效量。
如本文所用之術語「個體」表示任何動物,較佳為哺乳動物,且更佳為人類。個體之實例包括人類、非人類靈長類動物、齧齒動物、兔、羊、豬、牛、馬、狗及貓。
舉例而言,該組成物可為食品組成物或醫藥組成物。
於本發明之實施態樣中,該發酵萃取物可在食品製造過程中,添加於習用之食品中(亦即可食用之食品或飲品或其前驅物)來獲得本發明之食品組成物。幾乎所有之食品皆可添加根據本發明之該萃取物。可添加根據本發明之該萃取物之食品包含,但不限於糖果、烘焙食品、冰淇淋、乳製品、甜品及風味小點、小吃、肉類替代產品、快餐食品、湯類、麵食、麵條、罐頭食品、冷凍食品、乾製食品、冷藏食品、油脂、嬰兒食品、軟食物、或麵包塗醬或其混合物。
根據本發明之醫藥組成物可以習知製劑形式向患者經口或非經腸投予組成物,習知製劑形式諸如膠囊、微囊、錠劑、粒劑、散劑、糖衣錠、丸劑、栓劑、注射劑、懸浮液及糖漿。適合組成物可利用常用方法使用諸如以下之習知、有機或無機載劑來製備:賦形劑(例如蔗糖、澱粉、甘露醇、山梨醇、乳糖、葡萄糖、纖維素、滑石、磷酸鈣或碳酸鈣)、黏合劑(例如纖維素、甲基纖維素、羥甲基纖維素、聚丙基吡咯啶酮、聚乙烯吡咯啶酮、明膠、阿拉伯膠、聚乙二醇、蔗糖或澱粉)、崩解劑(例如澱粉、羧甲基纖維素、羥丙基澱粉、經低取代之羥丙基纖維素、碳酸氫鈉、磷酸鈣或檸檬酸鈣)、潤滑劑(例如硬脂酸鎂、輕質無水矽酸、滑石或月桂基硫酸鈉)、調味劑(例如檸檬酸、薄荷腦、甘胺酸或橘子粉)、防腐劑(例如苯甲酸鈉、亞硫酸氫鈉、對羥基苯甲酸甲酯或對羥基苯甲酸丙酯)、穩定劑(例如檸檬酸、檸檬酸鈉或乙酸)、懸浮劑(例如甲基纖維素、聚乙烯吡咯啶酮或硬脂酸鋁)、分散劑(例如羥丙基甲基纖維素)、稀釋劑(例如水)及底蠟(例如可可脂、白凡士林或聚乙二醇)。
本發明之醫藥組成物可藉由本發明領域中已知之任何方法局部或全身投予,包括但不限於藉由肌肉內、皮內、靜脈內、皮下、腹膜內、鼻內、經口、黏膜或外部途徑投予。適當的投藥途徑、調配方法及投藥時程可由本發明領域中具有通常知識者來決定。在本發明中,醫藥組合物可根據相應投藥途徑以多種方式調配,諸如液體溶液、懸浮液、乳液、糖漿、錠劑、丸劑、膠囊、持續釋放調配物、散劑、顆粒、安瓿、注射液、輸注液、套組、軟膏、洗劑、擦劑、乳膏或其組合。視情況,其可經滅菌或與任何醫藥學上可接受之載劑或賦形劑混合,其中有許多醫藥學上可接受之載劑或賦形劑已為一般技術者所知。
適合調配物可利用常用方法使用諸如以下之習知、有機或無機載劑來製備:賦形劑(例如蔗糖、澱粉、甘露醇、山梨醇、乳糖、葡萄糖、纖維素、滑石、磷酸鈣或碳酸鈣)、黏合劑(例如纖維素、甲基纖維素、羥甲基纖維素、聚丙基吡咯啶酮、聚乙烯吡咯啶酮、明膠、阿拉伯膠、聚乙二醇、蔗糖或澱粉)、崩解劑(例如澱粉、羧甲基纖維素、羥丙基澱粉、經低取代之羥丙基纖維素、碳酸氫鈉、磷酸鈣或檸檬酸鈣)、潤滑劑(例如硬脂酸鎂、輕質無水矽酸、滑石或月桂基硫酸鈉)、調味劑(例如檸檬酸、薄荷腦、甘胺酸或橘子粉)、防腐劑(例如苯甲酸鈉、亞硫酸氫鈉、對羥基苯甲酸甲酯或對羥基苯甲酸丙酯)、穩定劑(例如檸檬酸、檸檬酸鈉或乙酸)、懸浮劑(例如甲基纖維素、聚乙烯吡咯啶酮或硬脂酸鋁)、分散劑(例如羥丙基甲基纖維素)、稀釋劑(例如水)及底蠟(例如可可脂、白凡士林或聚乙二醇)。
本發明所言之外部途徑亦可稱為局部投藥,包含但不限於以吹氣或吸入投藥。局部投藥之各類製劑實例包含軟膏、乳液、乳霜、凝膠、發泡體,以經皮貼片輸送之製劑,吸入或吹氣用之粉末、噴霧劑、氣溶膠、膠囊或藥匣,或滴劑(例如眼用或鼻用滴劑),供霧化用溶液/懸浮液、栓劑、陰道藥栓、駐留灌腸劑及咀嚼劑或可吸入之錠劑或藥片或脂質或為膠囊製劑。
軟膏、乳霜及凝膠可例如配合水性或油性基質,且添加適用增稠劑及/或膠凝劑及/或溶劑調配。該基質因此可包含例如水及/或油,如液態鏈烷或植物油,如花生油或蓖麻油,或溶劑如聚乙二醇。可依據基質性質使用之增稠劑及膠凝劑包含軟質鏈烷、硬脂酸鋁、鯨醯硬脂基醇、聚乙二醇、毛脂肪、蜜蠟、羧基聚亞甲基及纖維素衍生物,及/或單硬脂酸甘油酯,及/或非離子性乳化劑。
乳液可配合水性或油性基質調配,且通常亦含有一或多種乳化劑、安定劑、分散劑、懸浮劑或增稠劑。
外塗用粉末可配合任何適用之粉末狀基質形成,例如滑石、乳糖或澱粉。滴劑可配合水性或非水性基質調配,且亦包括一或多種分散劑、溶解劑、懸浮劑或保存劑。
噴霧組合物可例如調配成水溶液或懸浮液,或調配成自預加壓袋輸送之氣溶膠,如劑量吸入器,且配合使用適用之液化推進劑。適用於吸入用之氣溶膠組合物可為懸浮液或溶液,該氣溶膠組合物可視情況含有額外之技藝中習知之調配佐藥,如介面活性劑例如油酸或卵磷脂及共溶劑例如乙醇。
局部用製劑可藉由每天對欲作用之區域施用一或多次投藥;且在皮膚區域上較佳使用覆蓋貼片。可藉由黏著劑儲存系統持續或延長輸送。
本發明另提供一種前述枯草芽孢桿菌發酵萃取物的用途,其係用於製備促進軟骨細胞再生之藥物。亦即,本發明提供一種於一個體中促進其軟骨細胞再生之方法,其包含給予該個體治療有效量之前述萃取枯草芽孢桿菌發酵萃取物及視需要之醫藥上可接受之載劑或賦形劑。
於本發明之一具體實施例中,於豬軟骨細胞為模型之測試中,以枯草芽孢桿菌發酵萃取物共同培養可顯著促進軟骨細胞增生,代表枯草芽孢桿菌發酵萃取物具有促進軟骨細胞增生之療效。
本發明另提供一種前述枯草芽孢桿菌發酵萃取物的用途,其係用於製備增加軟骨細胞外間質分泌之藥物。亦即,本發明提供一種於一個體中增加其增加軟骨細胞外間質分泌之方法,其包含給予該個體治療有效量之前述萃取枯草芽孢桿菌發酵萃取物及視需要之醫藥上可接受之載劑或賦形劑。
於本發明之一具體實施例中,於豬軟骨細胞為模型之測試中,以枯草芽孢桿菌發酵萃取物共同培養可顯著提升軟骨細胞的GAGs含量,代表枯草芽孢桿菌發酵萃取物具有增加軟骨細胞外間質分泌之療效。
本發明另提供一種前述枯草芽孢桿菌發酵萃取物的用途,其係用於製備治療關節炎之藥物。亦即,本發明提供一種於一個體中治療其關節炎之方法,其包含給予該個體治療有效量之前述萃取枯草芽孢桿菌發酵萃取物及視需要之醫藥上可接受之載劑或賦形劑。
於本發明之一具體實施例中,於人類軟骨肉瘤細胞(SW-1353)為模型之測試中,以枯草芽孢桿菌發酵萃取物共同培養可調節IL-1b誘發生成ROS及MMPs表現量,代表枯草芽孢桿菌發酵萃取物具有治療關節炎之療效。
以下之非限制性之實例有助於本發明所屬技術領域中具通常知識者實施本發明。該等實例不應視為過度地限制本發明。本發明所屬技術領域中具有通常知識者可在不背離本發明之精神或範疇的情況下對本文所討論之實施例進行修改及變化,而仍屬於本發明之範圍。
以下實驗結果係出於說明目的而提供,而並不意欲限制本發明範圍。
實例 1 . 枯草芽孢桿菌發酵萃取物製備(1). 枯草芽孢桿菌P12-3C菌株及P32-7B菌株之製備: 將枯草芽孢桿菌199菌株於營養瓊脂皿、30°C培養3天後,以無菌水洗下菌體,調整菌體濃度大於1×10
8cfu/mL。取0.5 mL菌液置於無菌平板中,將平板置於紫外光燈箱,以1% EMS(ethyl methane sulfonate)處理後,再以100 mJ劑量之UV光照射後,以無菌水連續稀釋,將不同稀釋濃度之菌液,取0.1 mL塗抹於營養瓊脂皿或篩選培養基上,並在避光條件下於30°C培養1至2天後,挑選獲得枯草芽孢桿菌P12-3C菌株。 續以前述相同方法突變P12-3C菌株,並挑選獲得枯草芽孢桿菌P32-7B菌株。
(2). 199菌株、P12-3C菌株及P32-7B菌株之發酵萃取物製備 將199菌株、P12-3C菌株及P32-7B菌株之甘油保存管於營養瓊脂皿(Nutrient agar plate)活化,於30°C培養3天,反覆活化二次後,加入5 mL無菌水,以L棒刮洗下菌體成為菌體懸浮液10
9CFU/mL,並以此為種菌進行後續實驗。將前述菌體懸浮液分別以1%(v/v)接種量接種於含50 mL RM培養基之250 mL平底搖瓶中,在30°C及轉速150 rpm下搖瓶培養5天。將發酵液於3000 rpm下離心10分鐘,以去除菌體並獲得上清液。取10 mL上清液與40 mL之100%乙醇混合進行沉澱。經3000 rpm離心15分鐘,取其沉澱物。重複乙醇沉澱步驟二次後,以真空方式乾燥其沉澱物,即為發酵萃取物。其中,前述以199菌株、P12-3C菌株及P32-7B菌株發酵獲得之發酵萃取物含量分別為1.68 g/L、1.20 g/L及1.73 g/L。
另將前述P12-3C菌株及P32-7B菌株發酵獲得之發酵萃取物分別利用高效液相層析在以下條件下測定: HPLC機型:Water 2695 separation module 偵測器:Water 2996 photodiode array detector 管柱::RP-18 (LiChroCART®100,5 μm,Merck) 流速:0.5 mL/min 移動相:0.005M戊磺酸鈉溶液/乙腈,95∶5(v/v) 測定波長:258nm 注射體積:10 μL 分析時間:10 min。
P12-3C菌株及P32-7B菌株之發酵萃取物的測定結果分別如圖1A、1B所示,皆於滯留時間約為約2.73min、約2.97min、約3.23min及約5.86min處分別具有4個主要峰。
其中,用於HPLC之發酵萃取物的濃度可為40 mg/ml,且4個主要峰的面積比率可如下表1所示。 表1、發酵萃取物之主要峰面積比率
菌株編號 | 峰面積 (%) | |||
主要峰1 | 主要峰2 | 主要峰3 | 主要峰4 | |
P12-3C | 40.22 | 35.96 | 19.64 | 3.78 |
P32-7B | 43.83 | 37.3 | 13.47 | 4.79 |
實例 2 . 枯草芽孢桿菌發酵萃取物治療 關節炎之活性本實施例是利用介白素(IL-1β)誘發人類軟骨肉瘤細胞(SW1353)產生如IL-8、ROS及MMP-1、MMP-3和MMP-13等退化性關節炎相關細胞激素之表現,並評估上述發酵樣品調節IL-1β對SW1353之誘發活性,以了解上述發酵樣品應用於抗關節炎及開發關節軟骨保健產品之潛力。
(1). 人類軟骨肉瘤細胞(SW1353)培養: 將SW1353細胞(獲自購自食品工業發展研究所,編號為BCRC60548)培養於添加10%胎牛血清(fetal bovine serum,簡稱FBS)與2 mM L-穀胺醯胺的Leibovitz’s L-15培養基(HyClone),於37°C、0% CO
2的細胞培養箱進行培養。待細胞生長至約8-9分滿時,進行繼代培養。培養皿上貼附之細胞先用10 mL PBS(購自Sigma,pH 7.4)清洗,反覆清洗2次後去除上清液。再加入0.5%胰蛋白酶(Trypsin) 1 mL處理細胞2-3分鐘,再加入Leibovitz’s L-15培養基並收集細胞懸浮液,細胞懸浮液分盤培養於37°C、0% CO
2培養箱,進行繼代培養。
(2).枯草芽孢桿菌發酵萃取物調節IL-1b誘發SW1353細胞生成IL-8之效果 將上述199菌株、P12-3C菌株及P32-7B菌株發酵所獲得之發酵萃取物分別以PBS回溶,進行抗關節發炎活性分析。
將步驟(1). 之SW1353細胞接種於24孔盤(1´10
5cells/well),並於37°C培養,隔天更換新培養基,及分別加入前述發酵萃取物(劑量100 mg/mL)作用1小時後,去除上清液,並以PBS清洗3次,加入濃度為10 ng/mL之IL-1b(10 ml/well)並反應24小時後,收集上清液,分析其中之發炎細胞激素IL-8 表現量。IL-8分析係利用三明治型酵素連結免疫分析法(sandwich-ELISA,購自eBioscience公司)進行分析。方法簡述如下:先於每一孔洞加入IL-8的1級抗體100
l,4°C下反應一晚,利用試劑所附之清洗液將孔盤清洗4次並移除清洗液後,每一孔洞加入分析緩衝液(assay buffer) 200 ml,室溫下作用1小時,利用清洗液將孔盤清洗4次,移除清洗液,每一孔洞加入測試樣品或標準品100 ml,室溫下作用2小時,利用清洗液將孔盤清洗4次,移除清洗液,每一孔洞加入IL-8的2級抗體100
l,室溫下作用1小時,利用清洗的溶液將孔盤清洗4次,移除清洗液,加入Avidin-HRP 100
l至孔盤內,室溫下作用30分鐘,利用清洗的溶液將孔盤清洗4次,移除清洗液,加入TMB 100
l至孔盤內,室溫下作用15分鐘,加入2N硫酸50 ml至孔盤內,中止反應,測量波長450 nm之吸光值。
實驗結果如圖2所示,P12-3C菌株之發酵萃取物可以抑制IL-1b所誘發之SW1353的IL-8表現,其抑制率約為14%,顯示其具有調節退化性關節炎、開發關節軟骨保健產品之潛力。P32-7B菌株之發酵萃取物同樣可以抑制IL-1b所誘發之SW1353的IL-8表現,其抑制率約為33%,顯示P32-7B菌株具有與P12-3C菌株相似且更佳之效果,且P32-7B菌株之效果甚至優於對照組葡萄糖胺(抑制率30%)。
實例 3. 枯草芽孢桿菌發酵萃取物促進軟骨細胞增生、增加細胞外間質產生本實驗主要目的是利用分離自豬膝關節的軟骨細胞,評估枯草芽孢桿菌發酵萃取物誘導關節軟骨細胞再生、細胞外間質(extracellular matrix,簡稱ECM)生成(如第2型膠原蛋白及醣胺聚醣)及刺激ECM相關基因表現的能力,以評估發酵樣品誘導關節軟骨再生之活性。
(1). 豬膝關節的軟骨細胞培養: 將取自豬之後腿骨膝關節的初代軟骨細胞加入含10%FBS之DMEM培養基(購自GIBCO公司)中,調整細胞數至約10
5cells/mL(代數P0),取10 mL細胞懸浮液置入於直徑10公分的培養皿,於37°C、5% CO
2培養箱內培養至約八至九分滿後,將培養皿上貼附之軟骨細胞先用PBS10 mL清洗,清洗2次並去除上清液,再加入0.5% Trypsin 1mL處理細胞2-3分鐘,加入含10%FBS之DMEM培養基,製成細胞懸浮液備用(代數P1)。
(2). 枯草芽孢桿菌發酵萃取物對軟骨細胞之細胞增生之影響 於24孔盤種入步驟(1).之豬軟骨細胞(1´10
5cells/well),並於37°C、5% CO
2培養箱內培養,隔天更換新培養基,及加入前述P12-3C菌株發酵所獲得之發酵萃取物,至如圖3所示之不同濃度(最終體積為1000 ml/well),待細胞與發酵萃取物分別作用4天(4D)、7天(7D)及14天(14D)後,移除上清液,以PBS清洗2次,再加入0.5% Trypsin 0.3 mL處理細胞2至3分鐘,再加入1mL PBS洗下細胞,將細胞收集於1.5 mL離心管內,以PBS反覆清洗2次後,以1500 rpm離心10 min移除上清液後,收集細胞團進行冷凍乾燥。冷凍乾燥後的細胞加入1 ml新配製的木瓜酵素,於60°C反應24小時後,以3000 rpm離心10 min,收集上清液進行DNA含量分析。
DNA含量分析方法簡述如下,於96孔盤內置入上清液10 ml,並加入100 ml DNA染劑(DNA染劑配方:200 µg Hoechst 33258(購自Sigma公司)溶於1L pH7.4的Tris緩衝液(含100 mM NaCl及10 mM Tris-buffer)),均勻混合後,以螢光偵測儀分析螢光值(激發波長350 nm、發射波長455 nm)。統計分析細胞數及螢光反應值之相關性,並建立檢量線,以評估各組實驗之軟骨細胞數。
實驗結果如圖3所示,不論是P12-3C菌株之發酵萃取物或對照組硫酸軟骨素A(簡稱CS4)及硫酸軟骨素C(簡稱CS6),細胞數皆隨著培養時間增加而增加,至7天後細胞增生倍率達最高,持續培養至14天細胞數反而下降,顯示軟骨細胞培養7天後,由於細胞密度過高,故細胞即開始老化死亡。P12-3C菌株之發酵萃取物促進軟骨細胞增生的效果與對照組CS4及CS6相近,顯示其具有促進軟骨細胞增生之效果,而增加樣品濃度並無明顯增加細胞增生的效果。
(3). 枯草芽孢桿菌發酵萃取物對軟骨細胞之細胞外間質分泌量之影響 關節軟骨之細胞外間質的主要成份為膠原蛋白及醣胺聚醣(glycosaminoglycan,簡稱GAGs),故分析軟骨細胞的GAGs含量,以了解樣品是否能刺激細胞外間質產生。將(1).之細胞懸浮液利用台盼藍(trypan blue)計算細胞數,於24孔盤種入細胞,接種細胞量1´10
5cells/well、體積為0.9 mL/well,並於37°C、5% CO
2培養箱內培養,隔天更換新培養基(含10%FBS之DMEM培養基),並加入P12-3C菌株發酵所獲得之發酵萃取物至圖4所示之不同濃度(最終體積為1 mL/well,每二天更新培養基並加入新的樣品)。作用4天(4D)、7天(7D)及14天(14D)後,移除上清液,以PBS清洗細胞2次,再加入0.5% Trypsin 0.3 mL處理細胞2-3分鐘,再加入PBS 1mL,將細胞收集於1.5 mL離心管內,以1500 rpm離心10 min,並反覆以PBS 1 mL進行清洗2次後,移除上清液後,將細胞進行冷凍乾燥。冷凍乾燥後的細胞加入新配製的木瓜酵素1 mL,於60°C反應24小時後, 3000 rpm離心10 min,取上清液或標準品CS6(sigma)10 ml,置於96孔盤內,並加入DMB染劑130 ml 均勻混合後,以分光光度計分析波長525 nm之吸光值。
實驗結果如圖4所示,軟骨細胞培養7天及14天後,與控制組相較,培養基中加入P12-3C菌株之發酵萃取物(250 mg/mL) 有助於GAGs產生,其效果與CS4(500 mg/mL)及CS6(500 mg/mL)類似,顯示P12-3C菌株之發酵萃取物具有促進軟骨細胞之ECM產生的效果。
(4). 枯草芽孢桿菌發酵萃取物對軟骨細胞ECM相關基因表現之影響 由於如圖4所示,P12-3C菌株之發酵萃取物可以增加軟骨細胞GAGs分泌,促進軟骨ECM產生,因此進一步利用即時定量聚合酶連鎖反應(Real-time Quantitative Polymerase Chain Reaction,簡稱qRT-PCR)分析發酵萃取物刺激軟骨細胞之Aggrecan、Col I (collgen I)及Col II (collgen II)等三個與ECM合成相關基因表現量,所有基因表現量均以持家基因(house keeping gene) GAPDH基因的表現量來作校正。
即時定量聚合酶連鎖反應,是利用專一的引子探針(primer probe)會在聚合酶連鎖反應過程中產生螢光,再利用螢光偵測系統來偵測每個循環(cycle)所釋放出的螢光量,進而推算出每個循環所產生的產物含量,達到即時定量分析基因表現量的目的。實驗方法簡述如下,將(1)之細胞懸浮液利用台盼藍計算細胞數,於24孔盤種入細胞,接種細胞量1´10
5cells/well、體積為0.9 ml/well,並於37°C、5% CO
2培養箱內培養,隔天更換新培養基(含10% FBS之DMEM培養基),並加入前述P12-3C菌株發酵所獲得之發酵萃取物至如圖5所示之不同濃度(最終體積為1 mL/well,每二天更新培養基並加入新的樣品)。作用7天後,移除上清液,以PBS清洗細胞2次,再加入0.5% Trypsin 0.3 mL處理細胞2-3分鐘,再加入PBS 1mL,將細胞收集於1.5 mL離心管內,抽取其mRNA,並進行反轉錄反應以製備cDNA,作為qRT-PCR反應的材料。qRT-PCR反應條件如下:於96孔盤,每一孔依序加入混合溶液(mixture solution),每一孔依序加入稀釋後的cDNA樣品50 ml (1ng/well),將96孔盤放至冰上,至-20°C冰箱取出SYBR Green Master Matrix,振盪後每個well依序加入10 ml SYBR Green Master Matrix,取出封膜黏貼於96孔盤上,黏貼完全後即可上機, Real-Time PCR循環溫度如下:Stage 1: 50°C反應2分鐘;Stage 2: 95°C反應10分鐘;Stage 3: 95°C反應15秒、60°C反應1分鐘,共進行40個循環。此實驗共分析Col I (Collagen I)、Col II (Collagen II)及Aggrecan等3個基因的表現量,qRT-PCR所使用的核酸引子序列如表2。 表2、qRT-PCR所引用的核酸引子序列
Gene | Primer | SEQ ID |
Collagen I | F: CGATGGCTGCACGAGTCACAC R:CAGGTTGGGATGGAGGGAGTTTAC | NO. 5 NO. 6 |
Collagen II | F:CCGGGCAGAGGGCAATAGCAGGTT R:CAATGATGGGGAGGCGTGAG | NO. 7 NO. 8 |
Aggrecan | F:CCAGAATCTAGCAGGGAGTCATC R:AGGCAGAGGTGGCTTCAGTC | NO. 9 NO. 10 |
GAPDH | F:CTGCCCCTTCTGCTGATGC R:TCCACGATGCCGAAGTTGTC | NO. 11 NO. 12 |
實驗結果如圖5所示,其中之倍率(ratio)係指軟骨細胞培養7天後,樣品處理組之Aggrecan、Col I 及Col II等三個基因的表現量(以GAPDH為校正依據),與對照組表現量的倍率關係。 如圖5所示,軟骨細胞培養7天後,P12-3C菌株之發酵沉澱物處理組之Aggrecan、Col I及Col II等基因表現量明顯較對照組增加,其中高劑量P12-3C菌株之發酵沉澱物組別(250 mg/mL)之Aggrecan為對照組的4倍、Col II為對照組的2倍,低劑量P12-3C菌株之發酵沉澱物組別(100 mg/mL)亦有略微刺激Aggrecan 及Col II表現的效果,且效果皆較正對照組(CS6)更佳。綜合以上實驗結果顯示,P12-3C菌株之發酵沉澱物確實可以促進軟骨細胞合成更多的細胞外間質,其機制可能與促進軟骨細胞之Aggrecan 及Col II基因表現有關。
實例 4. 枯草芽孢桿菌發酵萃取物調節人類關節軟骨細胞發炎之活性評估利用介白素(IL-1b)誘發人類軟骨肉瘤細胞(SW-1353)產生ROS及IL-8表現量增加,以評估枯草芽孢桿菌發酵萃取物是否具有減緩退化性關節炎之效果。
(1). 枯草芽孢桿菌發酵萃取物調節IL-1b誘發SW1353生成ROS之效果 細胞內活性氧物質(ROS)的產生量與發炎息息相關,故ROS可以做為評估細胞發炎的指標,分析樣品是否具有調節細胞內ROS含量的效果,可以用來初步評估樣品抗關節炎的潛力。將長約八至九分滿之T-75 flask之SW1353細胞進行繼代培養,將貼附之軟骨細胞先用1X PBS 10mL清洗,清洗2次並去除上清液後,再加入0.5% Trypsin 0.5 mL處理細胞2至3分鐘,將細胞收集於50 ml離管內,利用台盼藍計數細胞,於96孔盤種入1´10
4cells/well ,並於37°C培養,隔天更換新培養基,及加入P12-3C菌株之發酵萃取物作用1小時 ,以PBS 清洗3次,加入配製好濃度10 ng/ml的IL-1b於96well孔盤之細胞中(100 ml/well),並反應24小時,加入50 mM DCFH
2100 ml/well(購自sigma)作用30分鐘、避光反應, 以PBS 沖洗細胞 3 次,加入PBS 100 ml,以螢光偵測儀分析螢光值(激發波長480 nm、發射波長530 nm)。
ROS生成量分析結果如圖6所,SW1353培養液中加入IL-1b (10 ng/mL),ROS生成量由約250 RFU(relative fluorescent unit)升高至300 RFU;若先於SW1353培養液加入P12-3C菌株發酵萃取物 (100 mg/mL)作用1小時後,去除上清液,並用PBS清洗3次,再加新鮮的培養基及IL-1b (10 ng/mL)作用24小時,則ROS生成量會減少,且隨P12-3C菌株發酵萃取物添加量增多,ROS生成量隨之減少,呈劑量效應。此外P12-3C的效果與葡萄糖胺(100 mg/mL;購自sigma)或抗氧化劑水溶性維生素E (Trolox,100 mM/mL;購自Aldrich)的效果類似,顯示P12-3C酒精沉澱物具有抑制SW1353發炎的效果,具有開發抗關節炎產品之潛力。
(2). P12-3C菌株發酵萃取物調節IL-1b誘發MMPs表現量之效果 採用發炎細胞激素蛋白質陣列(MMP蛋白質陣列分析;購自RayBiotech) 分析P12-3C菌株發酵萃取物調節IL-1b誘發MMPs表現量之效果。結果如圖7所示,其中a.為控制組;b. 為IL-1b 10 ng/mL之誘導組;c.為IL-1b及P12-3C 100 mg/mL;d.為IL-1b及葡萄糖胺100 mg/mL。
如圖7所示,IL-1b與SW1353作用24小時後,MMP-1、MMP-3、MMP-8及MMP-10等相關金屬蛋白酶的表現量皆明顯增加,證實IL-1b會誘發SW1353產生類似關節炎的反應。
利用影像分析軟體半定量分析基質金屬蛋白酶表現量的變化,結果如圖8所示,P12-3C菌株發酵萃取物可以抑制因IL-1b誘發人類軟骨肉瘤細胞之金屬蛋白酶MMP-1、MMP-3、MMP-8及MMP-10表現量增加的現象,同時亦會抑制MMP-13的表現,而葡萄糖胺只能抑制金屬蛋白酶MMP-1、MMP-3及MMP-8的表現量,顯示P12-3C菌株發酵萃取物的效果優於葡萄糖胺。
(3). 枯草芽孢桿菌發酵萃取物調節MMP-1、MMP-3、MMP-13、TIMP-1及IL-8之效果 為進一步確定枯草芽孢桿菌發酵萃取物調節IL-1b誘發SW1353之MMPs表現的活性,及是否有劑量效應,故評估不同劑量P12-3C菌株發酵萃取物調節MMP-1、MMP-3、MMP-13、TIMP-1(分析試劑購自R&D公司)及IL-8(分析試劑購自eBioscience公司)等金屬蛋白酶及發炎細胞激素的活性。
結果如圖9所示 ,P12-3C菌株發酵萃取物(劑量25、50及100 mg/mL)可以抑制因IL-1b誘發SW1353之金屬蛋白酶MMP-1、MMP-3及MMP-13的表現,活性呈現劑量效應;此外其同時亦會抑制另一個發炎指標IL-8的表現,且促進金屬蛋白酶抑制劑TIMP-1的表現量增加,進而達到抗關節發炎的效果。比較P12-3C菌株發酵萃取物與臨床藥葡萄糖胺抑制IL-1β分泌的效果,結果顯示當兩者的濃度均為100 mg/mL 時,P12-3C菌株發酵萃取物所測到的MMP-1、MMP-3、MMP-13及IL-8表現量均比葡萄糖胺組低,效果優於葡萄糖胺,顯示P12-3C菌株發酵萃取物具有調節退化性關節炎、開發關節軟骨保健產品之潛力。
上述實施例僅為說明本發明之原理及其功效,而非限制本發明。習於此技術之人士對上述實施例所做之修改及變化仍不違背本發明之精神。本發明之權利範圍應如後述之申請專利範圍所列。
圖1A顯示P12-3C菌株之發酵萃取物的HPLC圖譜。 圖1B顯示P32-7B菌株之發酵萃取物的HPLC圖譜。 圖2顯示199菌株、P12-3C菌株及P32-7B菌株之發酵萃取物調節IL-1b誘發SW1353生成IL-8之效果。 圖3顯示P12-3C菌株之發酵萃取物促進軟骨細胞增生的效果。 圖4顯示P12-3C菌株之發酵萃取物對軟骨細胞醣胺聚醣(glycosaminoglycan,簡稱GAGs)分泌量之影響。 圖5顯示P12-3C菌株之發酵萃取物對軟骨細胞細胞外間質(extracellular matrix,簡稱ECM)相關基因表現之影響。 圖6顯示P12-3C菌株之發酵萃取物調節IL-1b誘發SW1353生成ROS之效果。 圖7顯示P12-3C菌株之發酵萃取物物調節IL-1b誘發SW1353之金屬蛋白酶(MMP-1、MMP-3、MMP-8、MMP-10及MMP-13)表現之效果。 圖8顯示P12-3C菌株之發酵萃取物調節金屬蛋白酶(MMP-1、MMP-2、MMP-3、MMP-8、MMP-9、MMP-10、MMP-13、TIMP-1、TIMP-2及TIMP-4)表現量之效果。 圖9顯示P12-3C菌株之發酵萃取物調節IL-1b誘發SW1353生成MMP-1、MMP-3、MMP-13及IL-8及TIMP-1之效果
枯草芽孢桿菌分離株(P32-7B): 財團法人食品工業發展研究所生物資源保存及研究中心;2018年7月13日;BCRC910842;及 萊布尼茨研究所DSMZ-德國微生物及細胞培養保藏中心;2018年7月18日;DSM32867。 枯草芽孢桿菌分離株(P12-3C): 財團法人食品工業發展研究所生物資源保存及研究中心;2018年7月13日;BCRC910843;及 萊布尼茨研究所DSMZ-德國微生物及細胞培養保藏中心;2018年7月18日;DSM32870。
<110> 財團法人食品工業發展研究所
<120> 枯草桿菌分離株及其用途
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Claims (15)
- 一種枯草芽孢桿菌(Bacillus subtilis)分離株,其係寄存於財團法人食品工業發展研究所且寄存編號為BCRC910842或BCRC910843之菌株。
- 一種製備枯草芽孢桿菌發酵萃取物之方法,其包含:(a)將如請求項1之枯草芽孢桿菌分離株接種於培養基中;(b)在適當環境下培養該經接種之培養基以獲得發酵物;(c)自該發酵物中去除該枯草芽孢桿菌菌株以獲得上清液;(d)將該上清液以體積比為約1:1至約1:9之比例與100%乙醇混合以產生沉澱物;及(e)收集該沉澱物。
- 如請求項2之方法,其中步驟(a)中之接種量為每克培養基接種約107CFU至約1010CFU之該分離株。
- 如請求項2或3之方法,於步驟(b)中,該經接種之培養基係在含氧下培養於約20℃至約40℃之溫度中。
- 如請求項2或3之方法,於步驟(d)中,該上清液與100%乙醇之混合體積比為約1:4。
- 如請求項2或3之方法,其中更包含:(f)乾燥該沉澱物。
- 如請求項6之方法,其中更包含:(g)將該沉澱物溶入適當的溶劑中。
- 如請求項7之方法,其中該溶劑為磷酸鹽緩衝劑、醋酸緩衝劑、鹽酸緩衝劑或純水。
- 一種枯草芽孢桿菌發酵萃取物,其可自如請求項2至8中任一項之方法獲得。
- 如請求項9之枯草芽孢桿菌發酵萃取物,當其利用高效液相層析(high performance liquid chromatography)在以下條件下測定時,在滯留時間為約2.73min、約2.97min、約3.23min及約5.86min下分別具有4個主要峰:HPLC機型:Water 2695 separation module偵測器:Water 2996 photodiode array detector管柱::RP-18(LiChroCART®100,5μm,Merck)流速:0.5mL/min移動相:0.005M戊磺酸鈉(sodium pentanesulfonate)溶液/乙腈(acetonitrile),95:5(v/v)測定波長:258nm注射體積:10μL分析時間:10min。
- 一種醫藥組成物,其包含治療有效量之如請求項9或10之枯草芽孢桿菌發酵萃取物。
- 一種食品組成物,其包含如請求項9或10之枯草芽孢桿菌發酵萃取物。
- 一種如請求項9或10之枯草芽孢桿菌發酵萃取物的用途,其係用於製備促進軟骨細胞再生之藥物。
- 一種如請求項9或10之枯草芽孢桿菌發酵萃取物的用途,其係用於製備增加軟骨細胞外間質分泌之藥物。
- 一種如請求項9或10之枯草芽孢桿菌發酵萃取物的用途,其係用於製備治療關節炎之藥物。
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FR3007655B1 (fr) * | 2013-06-28 | 2016-12-23 | Lesaffre & Cie | Souche bacillus subtilis pour le traitement et/ou la prevention de maladies inflammatoires chroniques |
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