CN105189733B - 从凝结芽孢杆菌中产生部分纯化的细胞外代谢产物的方法及其生物应用 - Google Patents
从凝结芽孢杆菌中产生部分纯化的细胞外代谢产物的方法及其生物应用 Download PDFInfo
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- CN105189733B CN105189733B CN201480010615.0A CN201480010615A CN105189733B CN 105189733 B CN105189733 B CN 105189733B CN 201480010615 A CN201480010615 A CN 201480010615A CN 105189733 B CN105189733 B CN 105189733B
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- bacillus coagulans
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Classifications
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- C12R2001/07—Bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C—CHEMISTRY; METALLURGY
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- C—CHEMISTRY; METALLURGY
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Engineering & Computer Science (AREA)
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- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Pest Control & Pesticides (AREA)
- Mycology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
公开了从益生菌菌株凝结芽孢杆菌SBC‑37(以菌株号MTCC 5856保藏于微生物菌种保藏与基因库(Microbial Type Culture Collection and Gene Bank))中产生部分纯化的细胞外代谢物制剂的方法，所述菌株表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌ATCC 7050具有99％遗传同源性。还公开了所述细胞外代谢物制剂针对一组微生物病原的抗菌谱，包括当与协同防腐剂共混物组合时的制剂的协同抗菌作用，所述协同防腐剂共混物包括约61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。单独的细胞外代谢物制剂或所述细胞外代谢物制剂和防腐剂共混物的组合也以协同的方式抑制了微生物生物膜形成。
Description
相关专利申请的交叉引用
本专利申请是于2013年12月24日提交的标题为“METHOD OF PRODUCINGPARTIALLY PURIFIED EXTRACELLULAR METABOLITE PRODUCTS FROM BACILLUS COAGULANSAND BIOLOGICAL APPLICATIONS THEREOF.”的临时专利申请US61920567的非临时申请。
发明背景
发明领域
本发明一般涉及益生菌制剂的抗菌效应。更具体地，本发明涉及(1)用于从益生菌菌株凝结芽孢杆菌SBC-37-01(保藏于微生物菌种保藏与基因库 (Microbial TypeCulture Collection and Gene Bank)，并分配菌株号MTCC 5856)中产生部分纯化的细胞外代谢物制剂的方法，所述菌株表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌 ATCC 7050具有99％遗传同源性，和(2)所述细胞外代谢物制剂针对一组微生物病原的抗菌谱，包括当与协同防腐剂共混物组合时的制剂的协同抗菌作用，所述协同防腐剂共混物包括约61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰(Magnolia officinalis)的超临界流浸膏的约1％w/w的木兰醇。单独的细胞外代谢物制剂或所述细胞外代谢物制剂和防腐剂共混物的组合也显示以协同的方式抑制了微生物生物膜形成。
现有技术的描述
本领域已知，凝结芽孢杆菌的细胞外产物包括培养物凝结芽孢杆菌菌株的上清液或滤液，其适用于哺乳动物的皮肤或粘膜的局部应用，且从而能够用于抑制细菌、酵母、真菌、病毒及其组合的生长(US6905692，“Topical compositions containing probioticBacillus bacteria,spores,and extracellular products and uses thereof”)。本发明关于进一步纯化益生菌凝结芽孢杆菌 SBC37-01(保藏于微生物菌种保藏与基因库，并分配菌株号MTCC 5856) 的培养物的细胞外组分以获得浓缩的细胞外代谢物制剂，所述浓缩的细胞外代谢物制剂当与其自身单独的上清液相比较时、和当与协同的协同防腐剂共混物相组合时表现出增强的抗菌作用，所述协同防腐剂共混物包括约61％ w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约 1％w/w的木兰醇。
本发明的主要目的是公开
1)从益生菌菌株凝结芽孢杆菌SBC37-01(保藏于微生物菌种保藏与基因库，菌株号MTCC 5856)中产生部分纯化的细胞外代谢物制剂的方法，所述菌株表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌 NBRC 3887和凝结芽孢杆菌ATCC 7050具有99％遗传同源性。
2)所述细胞外代谢物制剂针对一组微生物病原的抗菌谱，包括当与协同防腐剂共混物组合时的制剂的协同抗菌作用，所述协同防腐剂共混物包括约 61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。
3)单独的或与协同防腐剂共混物组合的所述细胞外代谢物制剂的微生物生物膜抑制潜能，所述协同防腐剂共混物包括约61％w/w的百里酚，约 38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。
本发明满足了前述的目的并提供了另外的相关优点。
发明概述
本发明描述了
(1)用于从益生菌菌株凝结芽孢杆菌SBC37-01(保藏于微生物菌种保藏与基因库，并分配菌株号MTCC 5856)中产生部分纯化的细胞外代谢物制剂的方法，所述菌株表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌ATCC7050具有99％遗传同源性；
(2)所述细胞外代谢物制剂针对一组微生物病原的抗菌谱，包括当与协同防腐剂共混物组合时的制剂的协同抗菌作用，所述协同防腐剂共混物包括约61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇和
(3)单独的或与协同防腐剂共混物组合的所述细胞外代谢物制剂的微生物生物膜抑制潜能，所述协同防腐剂共混物包括约61％w/w的百里酚，约 38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。
(4)单独的或与协同防腐剂共混物组合的所述细胞外代谢物制剂针对水不溶性葡聚糖中的粘附变形链球菌(S.mutans)的抗菌活性和抗生酸作用，所述协同防腐剂共混物包括约61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。
本发明提供了以下优点。
1)公开了用于从益生菌菌株凝结芽孢杆菌SBC37-01(保藏于微生物菌种保藏与基因库，并分配菌株号MTCC 5856)中产生部分纯化的细胞外代谢物制剂的纯化方法，所述菌株表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌ATCC 7050具有99％遗传同源性；
(2)公开了所述细胞外代谢物制剂针对一组微生物病原的抗菌谱，包括当与协同防腐剂共混物组合时的制剂的协同抗菌作用，所述协同防腐剂共混物包括约61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。
(3)公开了单独的或与协同防腐剂共混物组合的所述细胞外代谢物制剂的微生物生物膜抑制潜能，所述协同防腐剂共混物包括约61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。
(4)公开了单独的或与协同防腐剂共混物组合的所述细胞外代谢物制剂针对水不溶性葡聚糖中的粘附变形链球菌(S.mutans)的抗菌活性和抗生酸作用，所述协同防腐剂共混物包括约61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。
本发明的其他特征和优点将从以下与附图结合的更详细的说明中变得清楚，所述说明以举例的方式示出了本发明的原理。
附图简述
图1显示了用于从益生菌菌株凝结芽孢杆菌SBC37-01(保藏于微生物菌种保藏与基因库，菌株号MTCC 5856)中产生部分纯化的细胞外代谢物制剂的纯化方法的流程图，所述菌株表现出与已知细菌菌株凝结芽孢杆菌 ATCC 31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌ATCC 7050具有 99％遗传同源性；
图2显示了用于协同抗菌研究的棋盘肉汤微稀释法(checkerboard broth micro-dilution method)的示意图。
图3A-3B，3C-3D，3E-3F，3G-3H，3I-3J，3K-3L，3M-3N，3O-3P分别显示了天然防腐剂共混物和单独的凝结芽孢杆菌MTCC 5856的细胞外代谢物以及二者组合对以下的生长和生存力的作用：铜绿假单胞菌 (Pseudomonas aeruginosa)ATCC 9027，大肠杆菌(Escherichia coli)ATCC 25922，阿博尼沙门氏菌(Salmonella abony)NCIM 2257，变形链球菌 (Streptococcus mutans)MTCC 1943，痤疮丙酸杆菌(Propionibacterium acnes)ATCC 11827，金黄色葡萄球菌(Staphylococcus aureus)ATCC 29213，表皮葡萄球菌(Staphlococcus epidermidis)ATCC 14990和蜡样芽孢杆菌(Bacillus cereus)ATCC14579。
图4A，4B，4C，4D和4E分别显示了天然防腐剂共混物和单独的凝结芽孢杆菌MTCC5856的细胞外代谢物以及二者组合对以下的生长和生存力的作用：铜绿假单胞菌(Pseudomonas aeruginosa)ATCC 9027，大肠杆菌 (Escherichia coli)ATCC 25922，变形链球菌(Streptococcus mutans)MTCC 1943，金黄色葡萄球菌(Staphylococcus aureus)ATCC29213和表皮葡萄球菌(Staphlococcus epidermidis)ATCC 14990
图5A和5B分别显示了在2％蔗糖存在下，天然防腐剂共混物和凝结芽孢杆菌MTCC5856的细胞外代谢物以及二者组合对变形链球菌MTCC 1943 生物膜的生长/pH下降和水不溶性葡聚糖的形成的作用。
最佳实施方案的详细描述
在最佳实施方案中，本发明涉及用于从益生菌菌株凝结芽孢杆菌 SBC37-01(保藏于微生物菌种保藏与基因库，菌株号MTCC 5856)中产生部分纯化的细胞外代谢物制剂的纯化方法，所述菌株表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌NBRC3887和凝结芽孢杆菌 ATCC 7050具有99％遗传同源性，所述纯化方法包括以下步骤：
1.将表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌 NBRC 3887和凝结芽孢杆菌ATCC 7050具有99％遗传同源性的凝结芽孢杆菌MTCC 5856的培养物接种到1.0升的葡萄糖酵母提取物醋酸盐肉汤培养基(HiMedia,Mumbai India)或含0.5％tween80的MRS肉汤培养基或玉米浆干粉培养基中；
2.在37℃，120rpm下在步骤1的接种过的培养基中进行发酵24-48h；
3.在4000-7000rpm下离心步骤2的发酵肉汤；
4.在50℃下通过利用旋转蒸发步骤3的离心的上清液10倍。
5.向步骤4的10倍浓缩的100ml的上清液滴加150ml冰冷的丙酮，然后混合；
6.将步骤5的混合物在0℃下孵育30分钟，然后在7000-8000rpm下离心；
7.弃去步骤6中获得的沉淀物并收集60％丙酮饱和的上清液(～200ml)。
8.通过旋转蒸发浓缩步骤7中的丙酮饱和的上清液至50ml。
9.利用4N HCl调节pH至5.0，过滤(0.22micron；Millex,Millipore,India) 并在进一步使用之前贮存在-20℃下。
10.冷冻干燥/喷雾干燥/托盘干燥步骤8的上清液。
在另一最优选的实施方案中，本发明涉及微生物控制的方法，所述方法包括将来自益生菌菌株凝结芽孢杆菌SBC37-01(保藏于微生物菌种保藏与基因库，菌株号MTCC5856)的有效浓度的部分纯化的细胞外代谢物制剂与靶微生物细胞相接触，所述益生菌菌株凝结芽孢杆菌SBC37-01表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌ATCC 7050具有99％遗传同源性。在更具体的实施方案中，微生物细胞可以是选自以下的一种：铜绿假单胞菌，大肠杆菌，金黄色葡萄球菌，表皮葡萄球菌，变形链球菌，痤疮丙酸杆菌，蜡状芽孢杆菌和阿博尼沙门氏菌。
在又一个另外的最优选的实施方案中，本发明涉及微生物控制的方法，所述方法包括将微生物细胞与有效浓度的基本上由来自益生菌菌株凝结芽孢杆菌SBC37-01(保藏于微生物菌种保藏与基因库，菌株号MTCC 5856) 中的部分纯化的细胞外代谢物组成的制剂和协同防腐剂共混物相接触，所述益生菌菌株凝结芽孢杆菌SBC37-01表现出与已知细菌菌株凝结芽孢杆菌 ATCC 31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌ATCC 7050具有99％遗传同源性，所述协同防腐剂共混物包括约61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。在更具体的实施方案中，微生物细胞可以是选自以下的一种：铜绿假单胞菌，大肠杆菌，金黄色葡萄球菌，表皮葡萄球菌，变形链球菌，痤疮丙酸杆菌，蜡状芽孢杆菌和阿博尼沙门氏菌。
在又一个另外的最优选的实施方案中，本发明还涉及抑制微生物生物膜形成的方法，所述方法包括将产生物膜微生物细胞和单独的基本上由来自益生菌菌株凝结芽孢杆菌SBC37-01(保藏于微生物菌种保藏与基因库，菌株号MTCC 5856)中的部分纯化的细胞外代谢物组成的制剂相接触，或与同协同防腐剂共混物组合的所述制剂相接触，所述益生菌菌株凝结芽孢杆菌 SBC37-01表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌ATCC 7050具有99％遗传同源性，所述协同防腐剂共混物包括约61％w/w的百里酚，约38％的甘油一月桂酸酯和获自木兰的超临界流浸膏的约1％w/w的木兰醇。
本文示出了以下的实施例以阐释本发明的示例性实施方案
实施例1
微生物和培养条件
用于本研究的细菌菌株包括变形链球菌MTCC 1943，金黄色葡萄球菌 ATCC29213，表皮葡萄球菌ATCC 14990，大肠杆菌ATCC 25922，铜绿假单胞杆菌ATCC 9027，阿博尼沙门氏菌NCIM 2257和蜡样芽孢杆菌ATCC 14579。参照菌株购自ATCC(American TypeCulture Collection,Manassas,VA, USA)，MTCC(IMTECH,Chandigarh,India)和NCIM(National Collection of Industrial Microorganisms,Pune,India)。变形链球菌和痤疮丙酸杆菌分别培养于脑-心灌注琼脂(BHI；Difco Laboratories,Detroit,MI,USA)和增强梭菌琼脂 (RCA；HiMedia,Mumbai,India)。金黄色葡萄球菌、表皮葡萄球菌、大肠杆菌、铜绿假单胞杆菌、阿博尼沙门氏菌和蜡样芽孢杆菌培养于37℃下的胰酶解酪蛋白大豆琼脂(Difco Laboratories)。变形链球菌和痤疮丙酸杆菌在37℃下在厌氧室(Coy LaboratoryProducts rnc,Michigan)中厌氧(80％N2，10％H2和 10％CO2)孵育达48小时。用于本发明的凝结芽孢杆菌SBC37-01表征并保藏于微生物菌种保藏与基因库(Changdigarh,India)，并且菌株被分配菌株号凝结芽孢杆菌MTCC 5856。
技术-用于协同研究的棋盘方法
这是获得体外抗菌组合的最常用的方法。术语“棋盘”指所检验的两种药物的多个稀释形成的图案(试管或微量滴定板孔的图案)(Eliopoulos GM, Moellering RC:Antimicrobial combinations.In Antibiotics in laboratory medicine.Edited byLorian V.Baltimore,The Williams&Wilkins Co； 1991:432-492)。在本研究中，棋盘由列组成，其中每个试管(或孔)包含相同量的来自益生菌菌株凝结芽孢杆菌MTCC 5856的部分纯化的细胞外代谢物制剂，其沿X-轴(行)稀释，其中每个管(或孔)包含在Y-轴上稀释的相同的量的防腐剂共混物(图2)。结果，棋盘的每个正方形(其代表一个试管、孔或培养板)包含来自益生菌菌株凝结芽孢杆菌MTCC 5856的部分纯化的细胞外代谢物制剂和防腐剂共混物的独特组合。本研究的防腐剂共混物的浓度范围为2000μg/ml–31.25μg/ml，且在某些情况下更低，而且在从 8％(v/v)至0.015％(v/v)的范围中检验了来自益生菌菌株凝结芽孢杆菌 MTCC 5856的部分纯化的细胞外代谢物制剂。该棋盘技术可利用液体或半固体(琼脂)培养基来进行。对于变形链球菌和痤疮丙酸杆菌分别使用了 BHI肉汤和RC肉汤，并在厌氧室内在37℃下厌氧孵育(80％N2，10％H2和10％CO2)培养板48小时。Mueller hinton肉汤(Difco)用于金黄色葡萄球菌、表皮葡萄球菌、大肠杆菌、铜绿假单胞菌、阿博尼沙门氏菌和蜡样芽孢杆菌，并将培养板在37℃下孵育18小时。
杀伤动力学
针对铜绿假单胞菌、大肠杆菌、金黄色葡萄球菌、表皮葡萄球菌、变形链球菌、痤疮丙酸杆菌、蜡样芽孢杆菌和阿博尼沙门氏菌进行了防腐剂共混物和从凝结芽孢杆菌MTCC5856中部分纯化的细胞外代谢物制剂的时间-杀伤研究，并如先前所描述的(EliopoulusGM,Moellering RCJ.Antimicrobial combinations.In:Lorian V,ed.Antibiotics inLaboratory Medicine,4th edn. Baltimore:Williams&Willdns,1996；330-6.)利用时间-杀伤曲线方法进行了评估。使用对数期(1×106cfu/ml)的细菌悬浮液作为接种体。如棋盘测定中所确定的，检验了不同浓度下单独的和与防腐剂共混物组合的从凝结芽孢杆菌MTCC5856中部分纯化的细胞外代谢物制剂。通过连续稀释方法在各自培养基上以三个重复确定了以cfu/ml为单位的细胞群，并在各自生长条件下进行孵育。在37℃下孵育的0小时(未处理对照)和24小时取得了活菌计数和吸光度(OD610)。将活菌计数表示为Log10cfu/ml，吸光度表示为610nm 下的OD值。
生物膜易感性测定
通过微稀释法(Wei等人.Journal of Antimicrob.Chemother.2006.57: 1100-1109)检查了单独的和与防腐剂共混物组合的从凝结芽孢杆菌MTCC 5856中部分纯化的细胞外代谢物制剂对于变形链球菌、大肠杆菌、金黄色葡萄球菌、表皮葡萄球菌和铜绿假单胞菌的生物膜抑制作用。该方法与用于浮游生物细胞的棋盘法类似。对于变形链球菌生物膜测定，使用了添加了2％蔗糖的BHI肉汤。在其他生物体的情况下，在研究中使用了添加2％葡萄糖的TSB。从过夜生长培养物中制备的细菌悬浮液，并将悬浮液的浑浊度调节至610nm下的光密度(A610)为0.7(1×109CFU/ml)。本研究中防腐剂共混物的浓度范围为2000μg/ml至31.25μg/ml和在某些情况下更低的浓度，而从凝结芽孢杆菌MTCC 5856中部分纯化的细胞外代谢物制剂在8％(v/v)至 0.015％(v/v)的范围被检验。将40μl的新鲜肉汤培养基添加至每个孔，然后添加60μl的上述悬浮液至培养板的每个孔。这产生了每个孔的6x 107CFU/ml的最终接种体；在37℃下孵育48小时之后，从每个孔倾去培养培养上清液，并通过利用磷酸盐缓冲盐水(PBS；pH 7.2)洗涤孔去除浮游生物细胞。利用甲醇固定生物膜15分钟，然后在室温下风干。利用0.1％(w/v) 结晶紫(Sigma Chemical Co.,St Louis,MO)将干燥的培养板的孔染色10分钟并用水彻底洗涤，直至阴性对照孔表现出无色。通过向结晶紫染色的孔添加200μl的95％的乙醇计量生物膜形成，并记录595nm(A595)处的吸光度。利用算式(用检验剂处理的生物膜的A595/未处理的对照的A595)×100计算生物膜抑制的百分比。使用无检验剂的培养作为未处理的对照。
水不溶性葡聚糖中针对粘附的变形链球菌的抗微生物活性
通过先前所描述的方法(Katsura et al.,Antimicrob.Agents Chemother.2001.45:3009-3013)进行了由变形链球菌MTCC 1943进行的水不溶性葡聚糖的形成。简言之，将100μl的变形链球菌MTCC 1943(～1×108细胞/ml) 的培养物的等分试样接种到试管中的10ml的含2％蔗糖(w/v)的新鲜BHI 肉汤中并在30°倾斜下在37℃下孵育24小时。轻轻去除含浮游生物细胞的流体。用10ml的无菌水轻轻洗涤含水不溶性葡聚糖的变形链球菌MTCC 1943细胞，并重悬于10ml的磷酸盐缓冲液(10mM，pH 5.0)中，所述磷酸盐缓冲液含有单独的和与防腐剂共混物组合的从凝结芽孢杆菌MTCC 5856菌株中部分纯化的细胞外代谢物制剂，然后在37℃下孵育5分钟。将氯己定0.12％(v/v)(Sigma Chemical Co.,St Louis,MO)用作本研究的内参。用无菌的盐溶液(0.89％NaCl,w/v)轻轻洗涤混合物，随后在含2％蔗糖(w/v) 的10ml的BHI肉汤中重悬处理过的细胞。在37℃下孵育细胞6小时、12 小时、18小时和24小时后，利用pH计测量培养物产生的酸。轻轻去除含变形链球菌MTCC的游离细胞的流体。将水不溶性葡聚糖重悬于10ml的1 N NaOH溶液中并使其均匀；测量610nm处的浑浊度。
结果
表1显示了当与仅含部分纯化的细胞外代谢物(PPEM)或天然防腐剂共混物(NPB)的A制剂，和混合细胞外代谢物(PPEM)或天然防腐剂共混物(NPB)的B制剂接触时人类微生物病原体的倍数降低。可注意到混合细胞外代谢物(PPEM)或天然防腐剂共混物(NPB)的制剂对于病原体铜绿假单胞菌引起了显著的八倍降低，和对于病原体大肠杆菌引起了显著的四倍降低。
表1天然防腐剂共混物和益生菌菌株凝结芽孢杆菌MTCC 5856的部分纯化的细胞外代谢物针对人类病原体的棋盘检验的结果
虽然已参照优选的实施方案描述了本发明，本领域技术人员应清楚本发明并不局限于此。而且，本发明的范围仅结合所附权利要求来理解。
Claims (8)
1.用于从益生菌菌株凝结芽孢杆菌(Bacillus coagulans)MTCC 5856中产生部分纯化的细胞外代谢物制剂的纯化方法，所述菌株表现出与已知细菌菌株凝结芽孢杆菌ATCC31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌ATCC 7050具有99％遗传同源性，所述纯化方法包括以下步骤：
a.将表现出与已知细菌菌株凝结芽孢杆菌ATCC 31284、凝结芽孢杆菌NBRC 3887和凝结芽孢杆菌ATCC 7050具有99％遗传同源性的凝结芽孢杆菌MTCC 5856接种到1.0升的葡萄糖酵母提取物醋酸盐肉汤培养基或含0.5％tween 80的MRS肉汤培养基或玉米浆干粉培养基中；
b.在37℃，120rpm下在步骤a的接种过的培养基中进行发酵24-48h；
c.在4000-7000rpm下离心步骤b的发酵肉汤；
d.在50℃下通过利用旋转蒸发器浓缩步骤c的上清液10倍；
e.向步骤d的100ml的10倍浓缩上清液中逐滴加入150ml冰冷的丙酮，然后混合；
f.将步骤e的混合物在0℃孵育30分钟，然后在7000-8000rpm下离心；
g.弃去步骤f中获得的沉淀物并收集60％丙酮饱和的上清液200ml；
h.通过旋转蒸发器浓缩步骤g中的丙酮饱和的上清液至50ml；
i.利用4N HCl调节pH至5.0，过滤并在进一步使用之前贮存在-20℃，其中所述过滤步骤使用0.22微米的由印度生产的Millex Millipore；
j.冷冻干燥/喷雾干燥/托盘干燥步骤h的上清液；
其中所述凝结芽孢杆菌(Bacillus coagulans)MTCC 5856保藏于微生物菌种保藏与基因库(Microbial Type Culture Collection and Gene Bank)，并分配菌株号MTCC 5856。
2.一种微生物控制的方法，所述方法包括步骤：将有效浓度的根据权利要求1所述方法制备的部分纯化的细胞外代谢物制剂与靶微生物细胞相接触。
3.权利要求2所述的方法，其中所述微生物细胞是选自包含以下的组中之一：铜绿假单胞菌，大肠杆菌，金黄色葡萄球菌，表皮葡萄球菌，变形链球菌，痤疮丙酸杆菌，蜡状芽孢杆菌和阿博尼沙门氏菌。
4.一种微生物控制的方法，所述方法包括步骤：将微生物细胞与有效浓度的基本上由权利要求1所述方法制备的部分纯化的细胞外代谢物组成的制剂和防腐剂共混物相接触，所述防腐剂共混物包括61％w/w的百里酚，38％w/w的甘油一月桂酸酯和获自木兰(Magnolia officinalis)的超临界流浸膏的1％w/w的木兰醇。
5.权利要求4所述的方法，其中所述微生物细胞是选自包含以下的组中之一：铜绿假单胞菌，大肠杆菌，金黄色葡萄球菌，表皮葡萄球菌，变形链球菌，痤疮丙酸杆菌，蜡状芽孢杆菌和阿博尼沙门氏菌。
6.一种抑制微生物生物膜形成的方法，所述方法包括步骤：将产生物膜的微生物细胞与基本上由权利要求1所述方法所制备的的部分纯化的细胞外代谢物组成的制剂单独地相接触，或与防腐剂共混物组合的所述制剂相接触，所述防腐剂共混物包括61％w/w的百里酚，38％w/w的甘油一月桂酸酯和获自木兰的超临界流浸膏的1％w/w的木兰醇。
7.权利要求6所述的方法，其中所述产生物膜的微生物细胞是选自包含以下的组中之一：铜绿假单胞菌，大肠杆菌，金黄色葡萄球菌，表皮葡萄球菌，变形链球菌。
8.一种在蔗糖存在下防止pH下降并进一步防止变形链球菌的生物膜聚集的方法，所述方法包括步骤：将产生物膜/斑变形链球菌与基本上由权利要求1所述方法所制备的部分纯化的细胞外代谢物组成的制剂单独相接触，或与防腐剂共混物组合的所述制剂相接触，所述防腐剂共混物包括61％w/w的百里酚，38％w/w的甘油一月桂酸酯和获自木兰的超临界流浸膏的1％w/w的木兰醇。
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| RU2617965C2 (ru) | 2017-04-28 |
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| EP2989197A4 (en) | 2016-04-06 |
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| AU2014369819B2 (en) | 2017-09-28 |
| KR101770902B1 (ko) | 2017-08-24 |
| US20160324161A1 (en) | 2016-11-10 |
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| JP6126307B2 (ja) | 2017-05-10 |
| CA2899511C (en) | 2018-11-27 |
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| WO2015100405A1 (en) | 2015-07-02 |
| US9681665B2 (en) | 2017-06-20 |
| AU2014369819A1 (en) | 2015-09-10 |
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| PH12015502025A1 (en) | 2016-01-18 |
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| EP2989197B1 (en) | 2018-08-29 |
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| AU2017228561B2 (en) | 2018-12-13 |
| US9635862B2 (en) | 2017-05-02 |
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| US20160324162A1 (en) | 2016-11-10 |
| US9596861B2 (en) | 2017-03-21 |
| PH12015502025B1 (en) | 2016-01-18 |
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