CN101541947A - 分离自母乳的具有益生菌活性和抑制体重增加活性的乳酸菌 - Google Patents
分离自母乳的具有益生菌活性和抑制体重增加活性的乳酸菌 Download PDFInfo
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- CN101541947A CN101541947A CNA2007800291132A CN200780029113A CN101541947A CN 101541947 A CN101541947 A CN 101541947A CN A2007800291132 A CNA2007800291132 A CN A2007800291132A CN 200780029113 A CN200780029113 A CN 200780029113A CN 101541947 A CN101541947 A CN 101541947A
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- acid bacteria
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Abstract
本发明涉及一种分离自人母乳的乳酸菌,更准确地说,涉及一种分离自韩国人母乳的加氏乳杆菌BNR17菌株,所述菌株具有优良的益生菌活性,例如耐酸性、胆汁酸抗性和抗菌活性,并且还具有抑制体重增加的作用。此外,本发明的加氏乳杆菌BNR17除了通过将包含在摄入食品中的单糖合成不可消化的多糖并将合成的多糖释放出体外的抑制体重增加的作用外,还具有优良的耐酸性、胆汁酸抗性、肠吸收活性和对致病微生物具有的抗菌活性。因此,由于这些有益作用,本发明的菌株不但可以有效用于生产发酵乳、其它的发酵食品产品和动物饲料,而且可以有效用于生产抑制体重增加的活细胞产品和食品添加剂。
Description
技术领域
本发明涉及一种益生乳酸菌,更准确地说,涉及一种新的属于乳杆菌属的乳酸菌,所述乳酸菌分离自人母乳,并具有优良的益生菌活性,例如耐酸性、胆汁酸抗性和抗菌活性以及抑制体重增加的活性。
背景技术
乳酸菌与人类共有悠久的历史,是对人类健康非常有益的微生物,因此需求量不断提高。根据乳酸菌研究的最新进展,其应用已经从普通的食品扩大到保健食品和药品。乳酸菌例如有链球菌属、小球菌属、明串珠菌属、乳杆菌属、芽孢乳杆菌属和双歧杆菌属微生物。
乳酸菌存在于动物肠内,在其中分解宿主动物摄入的营养和纤维素,然后将其作为能源来制备乳酸和抗菌素,以抑制致病菌在肠内的生长,保持肠内健康。还将乳酸菌用于刺激动物生长、改善饲料利用、增强对疾病的抵抗力、抑制致病菌生长、降低死亡率、抑制有毒物质产生以及产生各种维生素。
然而,为了在肠内有效,从体外引入的乳酸菌必须安全到达肠内并附着于粘膜上来发挥功能。为此,乳酸菌必须能直接粘着于肠粘膜上,当其口服时必须较少被胃酸破坏,必须对胆汁酸具有强的抗性,并且必须对病原体具有强的抗菌活性。
当将乳酸菌用于对人类健康的食品或药品时,认为其分离自人体以得到更好的效果。尤其是已经确认分离自母乳的乳酸菌更有效并且更安全。然而,人体来源的乳酸菌大部分分离自成人粪便或母乳哺育的婴儿的粪便。分离自母乳的乳酸菌大部分是罗伊氏乳杆菌(Lactobacillusreuteri)以及其它几乎未曾报道过的乳酸菌。
同时,肥胖病是慢性病,其原因尚未完全揭示,但认为其发展归因于几种不同因素的共同作用。肥胖病可能导致高血压、糖尿病、心血管疾病、胆结石(galstone)、骨关节炎、睡眠呼吸暂停综合症、呼吸紊乱、前列腺癌、乳癌、结肠癌等。用于预防和治疗肥胖病的惯用方法主要有饮食-锻炼疗法、外科手术、药物疗法等。饮食-锻炼疗法鼓励摄取低热值、低脂肪的食品和耗氧的体育锻炼。该方法要求耐性,因为其需要重复并坚持地进行,这就是该方法似乎对于公众无效的原因。外科手术通过手术除去体脂肪。该方法具有在短时间内获得理想结果的优点,但同时具有承受痛苦的外科手术、对其效果的持续有质疑以及费用昂贵的缺点。药物疗法需要用心观察,因为其带来很多副作用。
最近,已经积极进行了由乳酸菌产生多糖的研究。已知乳酸菌产生胞外多糖的机制很复杂。根据乳酸菌的种类,多糖的产率和结构存在巨大差异。已经报道了由乳酸菌产生的多糖具有抗癌活性和免疫增强活性(Kitazawa,H.Int.J.Food Microbiol.,1998,40,169-175;Hosono,A.Biosci.Biotechnol.Biochem.,1997,61,312-316,Chabot,S.Lait.2001,81,683-697)。因为乳酸菌本身列为GRAS(公认是安全的,GenerallyRecognized As Safe),预计由乳酸菌产生的多糖是非常安全的。
发明内容
本发明涉及一种分离自人母乳的乳酸菌,本发明的一个目的是提供具有对酸、pH和胆汁酸具有强的抗性,以及对肠具有强附着的乳酸菌,使得由消化酶分解的低分子量碳水化合物转化为高分子量的多糖,并排泄多糖而不是使其被身体吸收。
为了实现上述目的,本发明提供了一种加氏乳杆菌(Lactobacillusgasseri)BNR17菌株,该乳酸菌分离自人母乳。
本发明的乳酸菌菌株具有以下特征:
①形态学-在乳酸菌MRS琼脂平板培养基上,于37℃培养24小时之后的形态学:
i.菌落的形状、大小和颜色:圆形、0.5mm×2mm,乳白色,光滑表面。
ii.革兰氏染色:阳性。
iii.类型:杆式(杆菌)。
iv.孢子形成:无。
v.迁移性:无。
②生理学性质
i.生长温度:25-45℃
ii.生长pH:pH 4.0-10.0
iii.最适生长温度:37-40℃
iv.最适生长pH:pH 6.0-8.0
③氧气的影响:兼性厌氧。
④糖类的可利用性:
甘油-、核糖-、阿东糖醇-、半乳糖+、D-葡萄糖+、D-果糖+、D-甘露糖+、甘露醇-、山梨醇-、N-乙酰葡糖苷+、七叶苷+、水杨苷+、纤维二糖+、麦芽糖+、乳糖+、蜜二糖-、蔗糖+、海藻糖+、菊粉-、松三糖-、棉子糖-、淀粉-、β-龙胆二糖-、D-松二糖+、D-塔格糖+
⑤耐酸性:在pH 2.0下生存。
⑥胆汁酸抗性:在0.3%的胆汁酸中生存。
⑦附着于肠:附着于人肠上皮细胞Caco-2细胞。
⑧抗生素抗性:对庆大霉素、卡那霉素、链霉素、杆菌肽、新霉素、萘啶酮酸、环丙沙星、多粘菌素B和甲氧苄啶有抗性。对红霉素、青霉素、四环素、氨苄青霉素、氯霉素、万古霉素和头孢西丁、利福平敏感。
⑨对于致病菌的抗菌活性:对于大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、蜡状芽孢杆菌、单核细胞增生李斯特菌和奇异变形杆菌具有抗菌活性。
⑩抗菌肽的存在:由PCR检测到对应于细菌素的加氏菌素T(gassericin T)(乳酸菌的抗菌肽成分之一)的基因。
多糖的产生:本发明的乳酸菌在补充有2%葡萄糖的MRS培养基上培养24小时后产生大约520mg/L的多糖。多糖主要由葡萄糖、甘露糖、半乳糖、岩藻糖、阿拉伯糖和D-葡糖胺组成。由本发明的乳酸菌所产生的多糖没有被消化酶例如α-淀粉酶和胰酶分解。
韩国人与西方人具有不同的饮食习惯。因此,很清楚且自然的是,分离自韩国人的乳酸菌最适合于韩国人。分离自韩国人母乳的乳酸菌适合益生乳酸菌所需要的每个基本条件,使得对韩国人具有最佳的健康增强作用。本发明者将具有上述特征的这种乳酸菌命名为“加氏乳杆菌BNR17”,并于2006年1月23日保藏在韩国生物技术和生物科学研究所(KRIBB)的韩国典型菌种保藏中心(KCTC)(保藏编号:KCTC10902BP)。
本发明还提供了一种包含有效剂量的乳酸菌的组合物。本发明的组合物可以按照食品、食品添加剂、动物饲料和动物饲料添加剂的形式提供。
由本发明提供的新的乳酸菌,加氏乳杆菌BNR17(保藏编号:KCTC10902BP)具有优良的耐酸性、胆汁酸抗性和抗菌活性,使其成为作为生产各种发酵乳产品及其它发酵食品的种子的优良候选者。本文所述的发酵乳产品可列举酸乳酪、可尔必思(calpis)、干酪和黄油,而本文所述的其它发酵食品可列举豆腐、黄豆酱、清国酱(Chungkukjang)、果冻和韩国泡菜(Kimchi),然而并不总限于此。用本发明的乳酸菌代替菌株,仅通过常规方法就可以容易地生产发酵乳产品和发酵食品。
根据本发明的优选实施方式,与给予PBS(磷酸盐缓冲盐水)的对照组的大鼠相比,在给予加氏乳杆菌BNR17的实验组大鼠中,观察到大约7.7%抑制体重增加的作用(参见表6)。除抑制体重增加的作用外,实验组的饮食功效与对照组相比也显著降低。检测了实验组和对照组的粪便中包含的多糖。结果是实验组的粪便中包含的多糖含量高于对照组(参见图6)。这些结果表明加氏乳杆菌BNR17的不可消化多糖的生产能力在体重调节中起到一定作用。同时,在摄取加氏乳杆菌BNR17的实验组大鼠中,未发现明显的副作用,而每个器官的重量都没有明显不同于对照组(参见表7和表8)。没有观察到作为人摄取微生物时主要关心的问题之一的微生物转换,表明本发明的乳酸菌对于人摄取来说非常安全(参见图7)。
可以按照仅包含加氏乳杆菌BNR17或含有任何可接受的载体的组合物的可食用形式来生产本发明的乳酸菌食品产品。可以将本发明的乳酸菌加入到不包含任何益生菌的食品中或已经包含几种益生菌的食品中。可与本发明的乳酸菌共用来生产乳酸菌食品的微生物必须适合于人或动物摄入,并具有益生菌活性,例如抑制致病菌或改善哺乳动物肠内的微生物平衡,但并不总限于此。益生菌微生物可列举酵母,例如酵母菌属、假丝酵母属、毕赤氏酵母属(Pichia)和球拟酵母属(Torulopsis);真菌,例如曲霉菌属、根霉菌属、毛霉菌属和青霉菌属;以及细菌,所述细菌属于乳杆菌属、双歧杆菌属、明串珠菌属、乳球菌属、芽孢杆菌属、链球菌属、丙酸杆菌属、肠球菌属和小球菌属。优选地,所述益生菌微生物可选自酿酒酵母(Saccharomyces cerevisiae)、凝结芽孢杆菌(Bacillus coagulans)、地衣芽孢杆菌、枯草杆菌、两歧双歧杆菌(Bifidobacterium bifidum)、婴儿双歧杆菌(Bifidobacterium infantis)、长双歧杆菌(Bifidobacterium longum)、屎肠球菌(Enterococcus faecium)、粪肠球菌(Enterococcus faecalis)、嗜酸乳杆菌(Lactobacillus acidophilus)、消化乳杆菌(Lactobacillus alimentarius)、干酪乳杆菌(Lactobacillus casei)、弯曲乳杆菌(Lactobacillus curvatus)、德氏乳杆菌(Lactobacillus delbruckii)、约氏乳杆菌(Lactobacillus johnsonii)、香肠乳杆菌(Lactobacillusfarciminus)、加氏乳杆菌(Lactobacillus gasseri)、瑞士乳杆菌(Lactobacillushelveticus)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、罗伊氏乳杆菌(Lactobacillus reuteri)、清酒乳杆菌(Lactobacillus sakei)、乳酸乳球菌(Lactococcus lactis)和乳酸片球菌(Pediococcus acidilactici)所组成的组。更优选将具有优良益生菌活性和免疫增强活性以及抗癌活性的益生菌微生物混合物加入到本发明的乳酸菌食品中以获得更强的效果。适用于本发明的乳酸菌食品的载体例如有稀释剂、高纤维添加剂、胶囊用材料和脂类,其为本领域技术人员所熟知。可将本发明的乳酸菌加氏乳杆菌BNR17制成胶囊剂、培养悬浮液或干粉。
另外,可以将包含本发明乳酸菌的组合物制为动物饲料或动物饲料添加剂。
本发明的动物饲料添加剂可制成干燥形式或液体形式,除了加氏乳杆菌BNR17之外,可以包含其它非致病微生物。可添加的微生物可以选自产生蛋白酶、脂肪酶和糖类转换酶的枯草杆菌、具有有机分解活性并在厌氧条件下保持生理活性的乳杆菌菌株、有助于提高家畜的乳产量和体重并增加饲料可消化性的丝状真菌例如米曲霉(Aspergillus oryzae)(Slyter,L.L.J.Animal Sci.1976,43,910-926)以及酵母例如酿酒酵母(Johnson,D.E等人,J.Anim.Sci.,1983,56,735-739;Williams,P.E.V.等人,1990,211)所组成的组。
除了加氏乳杆菌BNR17之外,本发明的动物饲料添加剂还可以包括一种或多种酶制剂。可添加的酶制剂可以是干燥形式或液体形式,其选自脂肪酶、将肌醇六磷酸分解为磷酸盐和磷酸肌醇酯的肌醇六磷酸酶、水解包含在淀粉和糖原内的α-1,4-糖苷键的淀粉酶、水解有机磷酸酯的磷酸酶、分解纤维素的羧甲基纤维素酶、分解木糖的木糖酶、将麦芽糖水解为两个葡萄糖的麦芽糖酶以及将蔗糖水解为葡萄糖-果糖的转化酶所组成的组。
将本发明的乳酸菌作为添加剂加入到动物饲料时,适宜的饲料原料选自农作物、大豆蛋白、花生、豌豆、制糖甜菜、纸浆、农作物副产品、动物肠粉和鱼粉所组成的组。此时,可将这些原料原样使用或在加工后使用。为了加工动物饲料,例如将用于饲料的原料通过压力压缩来排出,但不总限于此。就使用蛋白作为原料而言,优选为挤出。特别地,挤出使蛋白通过热处理变性,使抗酶因子受到破坏。更具体地说,就使用大豆蛋白而言,挤出改善了蛋白的可消化性,钝化了抗营养因子例如作为蛋白酶抑制剂之一的胰蛋白酶抑制剂,增加了由蛋白酶引起的消化性,使得蛋白的营养价值提高。
本发明进一步提供了用于预防和治疗肥胖病的药物组合物,其包含有效剂量的加氏乳杆菌BNR17(保藏编号:KCTC 10902BP)。
本发明的加氏乳杆菌BNR17通常以片剂或胶囊剂的形式来给药,所述片剂或胶囊剂通过将乳酸菌与药学上可接受的载体、赋形剂或另一有效的辅助组分混合来制备。
用于本发明药物组合物的可接受的载体、赋形剂或稀释剂可列举乳糖、右旋糖、蔗糖、山梨醇、甘露醇、淀粉、阿拉伯树胶、磷酸钙、藻酸盐、黄蓍胶、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水、糖浆、甲基纤维素、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁或矿物油。本发明的微生物组合物还可包括润滑剂、润湿剂、乳化剂、悬浮剂、防腐剂、甜味剂或调味剂。本发明的组合物可根据本领域技术人员众所周知的常规方法以肠溶包衣制剂的形式制备,以使该组合物经过胃安全地到达小肠,并快速而容易地在其中释放作为活性成分的微生物。
本发明的微生物组合物还可根据通用的胶囊生产方法制成胶囊形式。例如,将标准载体用于制备包含本发明冷冻干燥微生物的小球,所述小球填充软胶囊剂。另一个例子是将本发明的微生物与药学上可接受的载体例如可溶性胶、纤维素、硅酸盐或油混合来制备悬浮液或分散溶液,所述悬浮液或分散溶液填充软胶囊剂。
本发明的药物组合物可以按照用于口服的单位药物形式作为肠溶包衣制剂提供。本文的“肠溶包衣”表示药物不被胃酸分解而保持包衣状态,但在小肠中分解以在其中释放活性成分,其包括药学上可接受的各种包衣。本发明的“肠溶包衣”在模拟胃液例如HCl溶液(pH 1)于36-38℃维持至少两个小时,但优选在模拟肠液例如KH2PO4缓冲液(pH6.8)中30分钟内将包衣分解。
进行本发明的肠溶包衣,其中每个片芯上包覆16-30mg、优选16-20mg或少于25mg的组合物。包衣的优选厚度为5-100μm,为了获得最佳结果,更优选为20-80μm。肠溶包衣的原料可以选自众所周知的高分子物质。那些高分子物质记载于大量的参考文献中(L.Lachman等人,The Theory and Practice of Industrial Pharmacy,第三版,1986,第365-373页;H.Sucker等人,Pharmazeutische Technologie,Thieme,1991,第355-359页;Hagers Handbuch der pharmazeutischen Praxis,第四版,第7卷,第739-742页和第766-778页(SpringerVerlag,1971);以及Remington′sPharmaceutical Sciences,第13版,第1689-1691页(Mack Publ.,Co.,1970)),其可列举出纤维素酯衍生物、纤维素醚、丙烯酸树脂的丙烯酸甲酯共聚物以及马来酸和苯二甲酸衍生物的共聚物。
本发明的肠溶包衣可由常规方法进行,所述常规方法是将包衣溶液喷雾到片芯上。肠溶包衣可接受的溶剂选自酒精例如乙醇、酮例如丙酮、卤化烃例如CH2Cl2及其混合物所组成的组。可将软化剂例如邻苯二甲酸二丁酯或三醋酸甘油酯以1∶0.05-0.3(包衣材料∶软化剂)的比例加入到包衣溶液中。优选连续喷雾,并且喷雾量视包衣状况而定。可以调节喷雾压力,并且通常认为1-1.5bar获得最佳效果。
本发明的“药学上的有效剂量”指能降低低糖碳水化合物吸收到哺乳动物肠中的本发明微生物的最低量。可以根据给药途径和受试者来调节由本发明的组合物输送给人体的微生物的剂量。
可以将本发明的组合物每天至少一天一次给予受试者。单位剂量指物理上独立的、适于向受试者(人或其它哺乳动物)单位给药的单位,并且每个单位包含需要量的可接受载体和需要量的用于治疗作用的本发明的微生物。用于口服给予的本发明组合物的单位剂量优选为0.1-10g,更优选为0.5-5g。本发明微生物药学上有效的剂量是0.1-10g/天。然而,剂量可根据患者的体重、肥胖病的严重程度以及有效的辅助成分和微生物而变化。可将一天的剂量分成几个亚单位,使得必要时可以连续给药。因此,本发明组合物的剂量不以任何方式限制本发明的精神和范围。
本发明组合物的定期给予通过释放微生物来竞争和形成阻断吸收的微生物群落,从而导致糖类在人体内吸收的阻断,并进一步参与单糖例如碳水化合物转换为多糖以抑制其吸收。另外,由微生物产生的膳食纤维提供了适于有益肠道菌生长并刺激肠运动的较好条件。所以,本发明的组合物可有效用于肥胖病的预防和治疗。
为了使药物组合物的体重减轻作用或肥胖病预防效果最大化,可将本领域技术人员已知的任何体重减轻剂以适当的量包含于该组合物中。可在多次试验之后由本领域技术人员确定该量。作为添加剂有效成分的体重减轻剂优选选自共轭亚油酸、聚右旋糖、菊粉、瓜耳胶、阿拉伯树胶、左旋肉碱、葡萄籽提取物、低聚果糖、低聚木糖、棉子糖、葡糖酸、食用伞菌、聚花青素、乳果糖、乳糖醇、低聚乳果糖、朝鲜当归(Angelicagigas)提取物、枳棋子(Hovenia dulcis)提取物和陈皮提取物组成的组,但并不总限于此。
本发明还提供了通过培养加氏乳杆菌BNR17(保藏编号:KCTC10902BP)而制备的培养液。对用于制备培养液的培养基并没有限制,可使用包含用于微生物培养的培养基的任何培养基。如果需要,本发明的培养液还可以包含用于特定用途的任何添加剂。例如为了使体重减轻作用最大化,可将本领域技术人员众所周知的任何体重减轻剂加入培养液中,此时可通过重复试验测定有效剂量范围后,由本领域技术人员确定该体重减轻剂的含量。
本发明还提供了由本发明的乳酸菌产生的细菌素肽(bacteriocinpeptide)以及编码该肽的基因。本申请的发明者将该细菌素肽命名为“加氏菌素BNR17(gassericin BNR17)”,证实其具有SEQ.ID.NO:5所示的核苷酸序列。将加氏菌素BNR17的核苷酸序列与通用的抗微生物肽加氏菌素T的核苷酸序列(NCBI Blast检索号AB029612,SEQ.ID.NO:6)相比较,结果加氏菌素BNR17与加氏菌素T具有大约98%的同源性。
本发明进一步提供了包含加氏菌素BNR17基因的重组载体。
可通过将具有SEQ.ID.NO:5所示的核苷酸序列的基因插入大肠杆菌的通用表达载体中来制备本发明的重组载体。用于构建重组载体的母载体不局限于具体的一种,几乎可以使用任何微生物表达载体,但优选大肠杆菌表达载体。
本发明还提供了用重组载体转化的转化体。
可以通过将上述重组载体导入任意的宿主细胞容易地制备本发明的转化体。本文的宿主细胞可以选自真核细胞或原核细胞以及多细胞动物来源的细胞系组成的组,但并不总限于此。
附图说明
参考附图可最好地理解本发明优选实施方式的应用,其中:
图1是显示本发明的加氏乳杆菌BNR17和加氏乳杆菌KC26(NCBIGENBANK登录号:AF243156)16S rRNA的序列比较的图。
图2是显示本发明的加氏乳杆菌BNR17的肠附着的显微照片。
图3是显示本发明的加氏乳杆菌BNR17对各种致病菌具有抗菌活性的一组照片。
图4是显示根据本发明的加氏乳杆菌BNR17的生长,葡萄糖消耗量和多糖产量的图。■:细胞生长,◆:葡萄糖浓度,▲:EPS(多糖)浓度
图5是显示摄取本发明的加氏乳杆菌BNR17的大鼠粪便的量的图。
图6是显示摄取本发明的加氏乳杆菌BNR17的大鼠粪便的EPS(多糖)浓度的图。
图7是显示分离自摄取本发明的加氏乳杆菌BNR17的大鼠的、除小肠外的其它器官的菌落的RAPD-PCR图谱的一组电泳照片,其中使用SEQ.ID.NO:7(A)、NO:8(B)和NO:9(C)所示的引物。泳道1:加氏乳杆菌BNR17,泳道2-5:分离自摄取加氏乳杆菌BNR17的大鼠的、除小肠外的其它器官的菌落,M:DNA长度标记物。
实施例
如以下实施例所示来说明本发明的应用和目前优选的实施方式。
然而应当理解,本领域技术人员通过考虑该公开内容可以在本发明的精神和范围之内做出修改和改进。
实施例1:分离自人母乳的乳酸菌
从分娩婴儿不超过两周的妇女获取人母乳。然后,用PBS稀释母乳,并将未稀释的乳汁和稀释的乳汁分别分配到乳酸菌选择培养基中。在37℃下将培养基培养2-3天,然后通过形态和颜色将其中产生的菌落拣选。将分离的菌落进行革兰氏染色,然后在显微镜下观察来选择格兰氏阳性并具有杆状结构的那些菌落。37℃下在MRS液体培养基(pH 6.8)中将选择的菌落培养24小时。选择pH低于4.5的培养液中的菌落。在MRS培养基(pH 2.0)中将菌落培养2小时,随后在补充有0.3%牛胆汁的MRS培养基中进一步培养9小时。分离显示耐酸性和胆汁酸抗性的存活的乳酸菌菌株,并通过16S rRNA测序进行鉴定。结果证实该菌株属于加氏乳杆菌种(SEQ.ID.NO:1,图1),并命名为“加氏乳杆菌BNR17”。
实施例2:分离的乳酸菌的糖类利用
通过应用API50CHL试剂盒(Biomerieux,法国)与其它标准菌株相比较来研究以上分离的本发明加氏乳杆菌BNR17的糖类利用,结果如表1所示。在表1中,5314表示加氏乳杆菌CECT5714;5315表示加氏乳杆菌CECT5715;11413表示加氏乳杆菌LMG11413;18194表示加氏乳杆菌LMG18194;4479表示加氏乳杆菌CECT4479;18176表示加氏乳杆菌LMG18176;而13047表示加氏乳杆菌LMG13047。
表1
BNR17 | 5714 | 5715 | 11413 | 18194 | 4479 | 18176 | 13047 | |
甘油 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
赤藓醇 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
D-阿拉伯糖 | 0 | 0 | 4 | 0 | 0 | 0 | 0 | 0 |
L-阿拉伯糖 | 0 | 0 | 4 | 0 | 0 | 0 | 0 | 0 |
核糖 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
D-木糖 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
L-木糖 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
阿东糖醇 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
β-甲基木糖苷 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
半乳糖 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
D-葡萄糖 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
D-果糖 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
D-甘露糖 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
L-山梨糖 | 0 | 0 | 0 | 5 | 0 | 0 | 0 | 0 |
鼠李糖 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
半乳糖醇 | 0 | 0 | 0 | 4 | 0 | 0 | 0 | 0 |
肌醇 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
甘露醇 | 0 | 0 | 3 | 5 | 0 | 0 | 0 | 0 |
山梨醇 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
α-甲基-D-甘露糖苷 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
α-甲基-D-葡糖苷 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
N-乙酰葡糖胺 | 5 | 4 | 5 | 5 | 5 | 5 | 5 | 5 |
扁桃苷 | 5 | 5 | 5 | 0 | 0 | 5 | 5 | 0 |
熊果苷 | 5 | 5 | 5 | 1 | 1 | 5 | 5 | 4 |
七叶苷 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
水杨苷 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 3 |
纤维二糖 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
麦芽糖 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
乳糖 | 5 | 0 | 4 | 5 | 5 | 5 | 5 | 0 |
蜜二糖 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 0 |
蔗糖 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
海藻糖 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
菊粉 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
松三糖 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
D-棉子糖 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 0 |
Armidon | 5 | 3 | 5 | 1 | 1 | 5 | 3 | 3 |
糖原 | 0 | 0 | 0 | 4 | 4 | 0 | 0 | 0 |
木糖醇 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
β-龙胆二糖 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
D-松二糖 | 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
D-来苏糖(Lycose) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
D-塔格糖 | 5 | 5 | 5 | 3 | 3 | 5 | 5 | 5 |
D-岩藻糖 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
L-岩藻糖 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
D-阿糖醇(Arabinol) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
如表1所示,与其它乳酸菌菌株相比,本发明的加氏乳杆菌BNR17在糖类利用上可与其它相区别(如粗斜体字所示)。
实施例3:分离的乳酸菌的酶活性
应用APIZYM试剂盒(Biomerieux,法国)将实施例1中分离的加氏乳杆菌BNR17的酶活性与其它标准菌株的酶活性相比较,结果如表2所示。在表2中,13134表示加氏乳杆菌LMG13134。
表2
BNR17 | 11413 | 13047 | 13134 | 18176 | 18194 | 4479 | 5714 | 5715 | |
对照 | - | - | - | - | - | - | - | - | - |
碱性磷酸盐 | - | - | - | - | - | - | - | - | - |
酯酶(C4) | 1 | 2 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
酯酶脂肪酶(C8) | - | - | - | 1 | - | - | - | - | - |
脂肪酶(C4) | - | - | - | - | - | - | - | - | - |
亮氨酸芳香基酰胺酶 | + | + | + | + | + | + | + | + | + |
缬氨酸芳香基酰胺酶 | 1 | 1 | 1 | 3 | 2 | - | - | - | 1 |
胱氨酸芳香基酰胺酶 | 1 | 1 | 1 | 4 | 2 | 3 | 4 | 1 | 1 |
胰蛋白酶 | - | - | - | - | - | - | - | - | - |
α-胰凝乳蛋白酶 | - | - | 1 | - | - | - | - | - | - |
酸性磷酸酶 | + | 1 | 2 | + | - | 1 | 2 | 1 | 1 |
萘酚-As-二磷酸水解酶 | + | 1 | + | + | + | - | + | + | + |
α-半乳糖苷酶 | + | + | - | - | + | - | + | + | + |
β-半乳糖苷酶 | + | 4 | 1 | 3 | - | - | - | - | - |
β-葡糖醛酸酶 | - | + | + | - | - | - | - | - | - |
α-葡糖苷酶 | 1 | 1 | + | - | 1 | 2 | 1 | - | 1 |
β-葡糖苷酶 | 3 | + | - | 3 | + | - | 3 | 4 | + |
N-乙酰-β-氨基葡糖苷酶 | 1 | + | 1 | - | + | - | - | - | 1 |
α-甘露糖苷酶 | - | - | - | - | - | - | - | - | - |
α-岩藻糖苷酶 | - | - | - | - | - | - | - | - | - |
如表2所示,本发明的加氏乳杆菌BNR17在酶活性上可与其它菌株相区别(如粗斜体字所示)。
实施例4:耐酸性和胆汁酸抗性
为了研究本发明菌株的耐酸性和胆汁酸抗性,将加氏乳杆菌BNR17接种到4ml的MRS液体培养基中,并在37℃下培养18-20小时。将一些培养液再接种到具有调节的pH 2.0、107CFU/ml浓度的另一MRS培养基中,并在37℃下培养2小时。应用MRS琼脂平板对活细胞进行计数。再次使用测定耐酸性的培养液来离心。回收细胞,并接种到补充有0.3%牛胆汁的MRS液体培养基(pH 6.8)中,随后在37℃下培养9小时。也应用MRS琼脂平板对活细胞进行计数。结果如表3所示。
表3
处理之前 | 在pH 2.0下处理 | 0.3%牛胆汁处理 | |
BNR17 | 3.1×107 | 2.1×107 | 1.5×107 |
结果是即使用强酸(pH 2.0)处理之后,加氏乳杆菌BNR17仍然显示高的存活率,在补充有0.3%牛胆汁的培养基中也是如此。
实施例5:肠附着
将本发明的菌株接种到平板上,在该平板上,人肠上皮细胞系CaCo-2以107CFU/ml的浓度培养于PRMI1640(Gibco)中。在37℃下培养菌株一小时,随后用PBS冲洗三次来除去未粘着的细胞。将样品用甲醇固定,然后用结晶紫染色,随后在显微镜下观察。结果证实本发明的加氏乳杆菌BNR17非常好地粘着于CaCo-2细胞上(图2)。
实施例6:对致病菌的抗菌活性
在37℃下,在BHI液体培养基(Difco)中培养大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、蜡状芽孢杆菌、单核细胞增生李斯特菌和奇异变形杆菌18小时,然后以105CFU/ml的浓度分别接种到6份5ml BHI琼脂培养基(琼脂含量:0.7%)中。这些培养基叠置于其上固定有BHI琼脂培养基(琼脂含量:1.5%)的6块平板上。在将这6块平板培养基固化后,在每个培养基中形成直径4mm的孔,在其中加入40μl在37℃下培养24小时的乳酸菌培养液的上清液(2X),随后在37℃下培养5小时。
结果是在孔周围观察到清楚的生长抑制环,表明本发明的菌株对各种致病菌具有抗菌活性(表4和图3)。
表4
致病菌 | 生长抑制环的直径(mm) |
大肠杆菌KCTC1039 | 16 |
蜡状芽孢杆菌KCTC1526 | 16 |
单核细胞增生李斯特菌KCTC3710 | 12 |
奇异变形杆菌KCTC2510 | 14 |
金黄色葡萄球菌KCTC1928 | 6 |
鼠伤寒沙门氏菌KCTC2421 | 18 |
实施例7:抗生素抗性
将加氏乳杆菌BNR17培养液使用药签涂在MRS琼脂平板上,其上放置圆片,所述圆片包含红霉素、青霉素、庆大霉素、卡那霉素、链霉素、杆菌肽、氯霉素、万古霉素、四环素、氨苄青霉素、头孢西丁、利福平、新霉素、萘啶酮酸、环丙沙星、多粘菌素B或甲氧苄啶,随后在37℃下培养24小时。
结果证实本发明的加氏乳杆菌BNR17对庆大霉素、链霉素和甲氧苄啶具有抗性。
实施例8:抗菌肽基因的检测
通过PCR研究细菌素基因,所述PCR通过使用加氏乳杆菌BNR17基因组DNA作为模板,并使用SEQ.ID.NO:3和NO:4所示的引物来进行。所述的SEQ.ID.NO:3和NO:4特异于细菌素基因的核苷酸序列,已知所述细菌素由加氏乳杆菌种产生的抗菌肽。
结果证实了对应于加氏菌素的PCR产物,并如SEQ.ID.NO:5所示。将该核苷酸序列与SEQ.ID.NO:6所示的加氏菌素T(NCBI Blast检索号:AB029612)相比较,证实具有大约98%的同源性。
实施例9:β-葡糖醛酸酶活性
已知肠道菌产生的β-葡糖醛酸酶是一种致癌酶,因此认为具有该酶活性的菌株是有害菌株。为了研究本发明的加氏乳杆菌BNR17是否具有β-葡糖醛酸酶活性,使用API ZYM试剂盒(Biomerieux,法国)来测定加氏乳杆菌BNR17的酶活性。
结果证实本发明的菌株不具有β-葡糖醛酸酶活性,表明该菌株是安全的(表5)。
表5
酶 | 活性 |
α-半乳糖苷酶 | 阳性 |
β-半乳糖苷酶 | 阳性 |
β-葡糖醛酸酶 | 阴性 |
α-葡糖苷酶 | 阴性 |
β-葡糖苷酶 | 阳性 |
实施例10:葡萄糖消耗量和多糖产量
将加氏乳杆菌BNR17以浓度106CFU/ml接种到通过加入2%葡萄糖(w/v)而制备的MRS培养基(Difco)中,培养96小时,其间逐步测定细胞数目,同时还测定葡萄糖消耗量和胞外多糖(EPS)产量。
结果是在第12小时的培养基上观察到最高水平的,并且从那时起,该水平降低。在培养7小时之后,葡萄糖浓度迅速减低,在36小时之后,没有检测到葡萄糖浓度的变化,表明自培养开始起36小时之内,大部分葡萄糖耗尽。在第24小时EPS产量达到最大(最高水平:520mg/l),并在36小时稍微减低,但此后再次提高。推测将其归因于引起细胞中各种多糖释放到培养液中的细胞自溶作用(图4)。
实施例11:由加氏乳杆菌BNR17生产的多糖的通过消化酶的分解
将100mg的α-淀粉酶(Sigma)和胰酶(Sigma)各自溶于0.05M磷酸盐缓冲液(pH 7.0)。将50μl的上述酶溶液和150μl的0.05M磷酸盐缓冲液(pH 7.0)加入200μl的多糖(EPS)溶液中,所述的多糖(EPS)溶液从加氏乳杆菌BNR17培养液的上清液中提取,随后在37℃下反应一小时。反应混合物在100℃下加热15分钟以钝化其中的酶,随后在室温下冷却。用葡萄糖试剂盒(Sigma)测定葡萄糖浓度。
结果是在处理每种消化酶之前,在多糖溶液中检测不到葡萄糖,而在处理胰酶和α-淀粉酶之后,分别检测到3.70mg/l和19.1mg/l的葡萄糖。这些结果表明由加氏乳杆菌BNR17生产的多糖几乎不被消化酶分解。
实施例12:加氏乳杆菌BNR17抑制体重增加的作用
将8周龄的雄性SD大鼠分为两组。一组8周每天仅仅口服给予PBS(pH 7.4),而另一组8周每天口服给予悬浮有109CFU/ml的加氏乳杆菌BNR17的PBS。每周测定体重、摄入食物和血液化学值例如胆固醇水平的变化。还测定粪便量和粪便中的EPS量来研究加氏乳杆菌BNR17抑制体重增加的作用与其多糖生产能力的关系。8周以后,处死全部试验动物,并解剖摘取肝、肾、脾、MLN(肠系膜淋巴结),测定其重量。将每个摘取的器官的少许匀浆,涂在作为乳酸菌选择培养基的LBS琼脂上,然后培养,并且考察产生菌落的RAPD(随机扩增多态性DNA)-PCR图谱。将结果与加氏乳杆菌BNR17的RAPD-PCR图谱相比较来研究该菌株是否转入其它的器官中。
结果8周后在仅口服给予PBS的对照组中观察到大约179.1%的体重增加,而在口服给予加氏乳杆菌BNR17的实验组中观察到大约171.6%的体重增加(表6)。与对照组相比,实验组还显示出较低的每日体重增加率和食物功效比。
表6
在表6中,食物功效比(FER)表示体重增加(g/天)/食物摄入(g/天)。*P<0.05,**P<0.05。
对照组和实验组的粪便量和粪便中的EPS量的测定结果如图5和图6所示。在对照组和实验组之间,粪便量没有很大不同,但在给予加氏乳杆菌BNR17的实验组中ESP的量显著增加。该结果表明加氏乳杆菌BNR17将摄入体内的糖类组分转化为不可消化的多糖,从而将多糖释放出体外,导致体内吸收率降低和抑制体重增加的作用。
为了检测用于人体给药的菌株的安全性,测定血液化学值和器官重量。在对照组和实验组中每个水平和值都相似,表明该菌株不引起副作用(表7和表8)。
表7
组 | 胆固醇(mg/dL) | 葡萄糖(mg/dL) | HDL(mg/dL) | LDL(mg/dL) | 总蛋白(g/dL) | 甘油三酯(mg/dL) |
对照 | 99.7±11.7 | 88.9±12.1 | 38.5±3.5 | 21.5±1.6 | 8.6±0.3 | 115.9±11.0 |
BNR17 | 96.3±6.3 | 79.2±4.1 | 40.8±1.8 | 20.8±3.1 | 9.0±0.3 | 132.6±5.5 |
表8
组 | 肝 | 肾 | 脾 |
对照 | 0.029±0.001 | 0.007±0.000 | 0.002±0.000 |
BNR17 | 0.027±0.003 | 0.007±0.001 | 0.002±0.000 |
在表8中,每个数字表示器官重量(g)/大鼠体重(g)。
为了研究该菌株是否转入到其它器官中,通过使用分别由SEQ.ID.NO:7-NO:9所示的引物p1、p2和OPL5来研究在LBS琼脂平板上培养的每个器官组织的菌落的RAPD-PCR图谱。如下进行使用引物p1和p2的PCR:94℃(2分钟)、36℃(5分钟)、72℃(5分钟)-4个循环/94℃(1分钟)、36℃(1分钟)、72℃(2分钟)-36个循环。如下进行使用引物OPL5的PCR:94℃(2分钟)-1个循环/94℃(40秒)、45℃(1分钟)、72℃(1分钟)-2个循环/94℃(40秒)、52℃(1分钟)、72℃(3分钟)-30个循环/70℃(5分钟)-1个循环。
结果没有菌落显示出与BNR17类似的图谱(图7)。因此,证实当摄取时,BNR17是安全的菌株,其不会转入除小肠之外的其它器官。
制备实施例1:发酵乳的制备
使用脱脂奶粉将原料奶将非脂乳固体含量调节为8-20%,并在72-75℃下灭菌15秒。将灭菌的原料奶冷却到适当温度,将本发明的加氏乳杆菌BNR17以106CFU/ml的浓度接种,随后培养直到pH达到4-5为止。当培养完成时,冷却培养液。同时,将0.1-50重量%的浓缩果汁、0.1-20重量%的膳食纤维、0.5-30重量%的葡萄糖、0.1-15重量%的低聚糖、0.01-10重量%的钙和0.001-5重量%的维生素全部溶解来制备糖浆。将糖浆灭菌,冷却,并与上述培养液混合,随后搅拌均匀。将生成的混合物包装,结果制备得到发酵乳。测定发酵乳产品的风味、物理性质和味道,结果令人满意。
制备实施例2:乳酸菌粉末的制备
将本发明的加氏乳杆菌BNR17以106CFU/ml的浓度接种到MRS液体培养基中,随后在37℃下控制pH发酵18-24小时。通过使用30体积%的NaOH作为中和剂使pH达到5.7±0.2来控制pH。当培养完成时,在4℃下以10,000×g进行离心来回收细胞。制备补充有5重量%的脱脂奶、2.5重量%的乳清和5重量%的蔗糖(相对于组合物的总重而言)的保护剂。将等量的回收细胞和保护剂混合,随后通过使用冻干机来研成粉末。产生的加氏乳杆菌BNR17干燥粉末包含超过1×1011CFU/g活细胞。所述保护剂还可以包括10重量%的海藻糖、10重量%的麦芽糊精和7.5重量%的乳糖。
制备实施例3:乳酸菌产品的制备
由制备实施例2生产的乳酸菌粉末制备乳酸菌产品例如乳酸菌食品和消化药。将10重量%的低聚糖、20重量%的无水葡萄糖、5重量%的结晶果糖、2重量%的维生素C、5重量%的果粉香精、5重量%的芦荟、15重量%的膳食纤维和18重量%的车前籽壳(Psyllium Husk)加入到20重量%的加氏乳杆菌BNR17的干粉中,并将混合物包装在棒状物或瓶子中。由此制备的乳酸菌产物中,活细胞超过5×108CFU/g。
制备实施例4:用于饲料添加剂的组合物的制备
由表9所示的下列成分制备用于饲料添加剂的包含加氏乳杆菌BNR17的组合物。
表9用于饲料添加剂的组合物的构成比率(重量%)
加氏乳杆菌BNR17 | 酶制剂 | 非致病微生物 | 氨基酸 | 其它 | |
制备实施例<4-1> | 100 | - | - | - | - |
制备实施例<4-2> | 90 | 10 | - | - | - |
制备实施例<4-3> | 80 | 10 | 10 | - | - |
制备实施例<4-4> | 70 | 10 | 10 | 10 | - |
制备实施例<4-5> | 60 | 15 | 15 | 8 | 2 |
制备实施例<4-6> | 50 | 20 | 15 | 8 | 2 |
本文所用的酶制剂是肌醇六磷酸酶、纤维素酶、木糖酶、麦芽糖酶和转化酶的混合物,并且所述非致病微生物是米曲霉。
工业实用性
如上文所说明的,本发明的加氏乳杆菌BNR17具有较宽的最适生长温度和pH范围。而且,本发明的菌株除了具有抑制体重增加的作用外,不仅具有优良的耐酸性、胆汁酸抗性和肠附着能力,而且对致病微生物具有强烈的抗菌活性。所以,本发明的菌株可以有效用于生产发酵乳及其它发酵产品,而且作为动物饲料添加剂也非常有用。
序列表
随同附上序列表。
序列表
<110>Bioneer Corporation
株式会社 百奥尼
<120>Lactic Acid Bacteria Isolated from Mother′s Milk with Probiotic
Activity and Inhibitory Activity against Body Weight Augmentation
分离自母乳的具有益生菌活性和抑制体重增加活性的乳酸菌
<130>2007-opa-3710
<160>9
<170>KopatentIn 1.71
<210>1
<211>840
<212>DNA
<213>Lactobacillus gasseri BNR17
加氏乳杆菌BNR17
<400>1
gctgactcct ataaaggtta tcccaccggc tttgggtgtt acagactctc atggtgtgac 60
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc gcgattacta 120
gcgattccag cttcgtgtag gcgagttgca gcctacagtc cgaactgaga acggctttca 180
nagatccgct tgccttcgca ggttcgcttc tcgttgtacc gtccattgta gcacgtgtgt 240
agcccaggtc ataaggggca tgatgacttg acgtcatccc caccttcctc cggtttgtca 300
ccggcagtct cattagagtg cccaacttaa tgatggcaac taatgacaag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacagccatg caccacctgt 420
ctcagcgtcc ccgaagggaa ctcctaatct cttaggtttg cactggatgt caagacctgg 480
taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggagtgc ttaatgcgtt 600
agctgcagca ctgagaggcg gaaacctccc aacacttagc actcatcgtt tacggcatgg 660
actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttgca 720
gaccagagag ccgccttcgc cactggtgtt cttccatata tctacgcatt ccaccgctac 780
acatggagtt ccactctcct cttctgcact caagttcaac agtttctgat gcaattctcc 840
840
<210>2
<211>840
<212>DNA
<213>Lactobacillus gasseri KC26
加氏乳杆菌KC26
<400>2
gctgactcct ataaaggtta tcccaccggc tttgggtgtt acagactctc atggtgtgac 60
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc gcgattacta 120
gcgattccag cttcgtgtag gcgagttgca gcctacagtc cgaactgaga acggctttca 180
gagatccgct tgccttcgca ggttcgcttc tcgttgtacc gtccattgta gcacgtgtgt 240
agcccaggtc ataaggggca tgatgacttg acgtcatccc caccttcctc cggtttgtca 300
ccggcagtct cattagagtg cccaacttaa tgatggcaac taatgacaag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacagccatg caccacctgt 420
ctcagcgtcc ccgaagggaa ctcctaatct cttaggtttg cactggatgt caagacctgg 480
taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggagtgc ttaatgcgtt 600
agctgcagca ctgagaggcg gaaacctccc aacacttagc actcatcgtt tacggcatgg 660
actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttgca 720
gaccagagag ccgccttcgc cactggtgtt cttccatata tctacgcatt ccaccgctac 780
acatggagtt ccactctcct cttctgcact caagttcaac agtttctgat gcaattctcc 840
840
<210>3
<211>20
<212>DNA
<213>Artificial Sequence人工序列
<220>
<223>gaT-950 forward primer
gaT-950正向引物
<400>3
ggagtaggtg gagcgacagt 20
<210>4
<211>20
<212>DNA
<213>Artificial Sequence人工序列
<220>
<223>gaT-1075 reverse primer
gaT-1075反向引物
<400>4
tccaccagta gctgccgtta 20
<210>5
<211>110
<212>DNA
<213>Artificial Sequence人工序列
<220>
<223>gassericin BNR17 gene originated from Lactobacillus gasseri BNR17
来自加氏乳杆菌BNR17的加氏菌素BNR17基因
<400>5
tgccgttacg ccagcccatg ctattggaac atagtgtgct ccaacagagc cacaagcagg 60
accgcaaact gcatttccaa gagcccgtcc agcgactgtc gctccaccta 110
<210>6
<211>110
<212>DNA
<213>Artificial Sequence人工序列
<220>
<223>gassericin T gene disclosed in NCBI Blast Search No.AB029612
NCBI Blast检索号AB029612公开的加氏菌素T基因
<400>6
tgccgttacg ccagcccatg ctattggaac atagtgtgct ccaacaaagc cacaagcagg 60
accgcaaact gcatttccaa gagcccatcc agcgactgtc gctccaccta 110
<210>7
<211>10
<212>DNA
<213>Artificial Sequence人工序列
<220>
<223>primer p1
引物p1
<400>7
agcagcgtgg 10
<210>8
<211>10
<212>DNA
<213>Artificial Sequence人工序列
<220>
<223>primer p2
引物p2
<400>8
ggcatgacct 10
<210>9
<211>10
<212>DNA
<213>Artificial Sequence人工序列
<220>
<223>primer OPL5
引物OPL5
<400>9
acgcaggcac 10
Claims (13)
1.一种加氏乳杆菌BNR17菌株(保藏编号:KCTC 10902BP)。
2.根据权利要求1所述的加氏乳杆菌BNR17菌株(保藏编号:KCTC10902BP),其中所述菌株包含由SEQ.ID.NO:1所示的16S rRNA序列。
3.一种组合物,所述组合物包含有效剂量的权利要求1所述的加氏乳杆菌BNR17(保藏编号:KCTC 10902BP)。
4.根据权利要求3所述的组合物,其中所述组合物选自食品、食品添加剂、动物饲料和动物饲料添加剂所组成的组。
5.根据权利要求4所述的组合物,其中所述动物饲料添加剂包含选自其它非致病微生物、酶及其混合物所组成的组的一种或多种。
6.一种用于预防或治疗肥胖病的药物组合物,所述组合物包含有效剂量的权利要求1所述的加氏乳杆菌BNR17(保藏编号:KCTC10902BP)。
7.一种由权利要求1所述的菌株产生的细菌素肽。
8.一种编码权利要求7所述的细菌素肽的基因。
9.根据权利要求8所述的基因,其中所述基因如SEQ.ID.NO:5所示。
10.根据权利要求7所述的细菌素肽,其中所述细菌素肽由SEQ.ID.NO:5所示的序列编码。
11.一种包含权利要求8或9所述基因的重组载体。
12.一种由权利要求11所述的重组载体转化的转化体。
13.一种权利要求1所述的加氏乳杆菌BNR17(保藏编号:KCTC10902BP)的培养液。
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CN103260632A (zh) * | 2011-01-25 | 2013-08-21 | 株式会社明治 | 胃泌素生成抑制剂及含有该胃泌素生成抑制剂的食品组合物 |
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KR20080110447A (ko) | 2008-12-18 |
US8309076B2 (en) | 2012-11-13 |
CN101541947B (zh) | 2013-03-13 |
EP2046943A1 (en) | 2009-04-15 |
EP2046943A4 (en) | 2010-09-22 |
KR101108428B1 (ko) | 2012-01-31 |
WO2008016214A1 (en) | 2008-02-07 |
JP2009545311A (ja) | 2009-12-24 |
USRE46912E1 (en) | 2018-06-26 |
US20100203025A1 (en) | 2010-08-12 |
USRE48652E1 (en) | 2021-07-20 |
EP2046943B1 (en) | 2013-06-19 |
DK2046943T3 (da) | 2013-09-23 |
JP5081242B2 (ja) | 2012-11-28 |
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