CN102753701A - 驴奶中分离的益生微生物 - Google Patents
驴奶中分离的益生微生物 Download PDFInfo
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- CN102753701A CN102753701A CN2010800199439A CN201080019943A CN102753701A CN 102753701 A CN102753701 A CN 102753701A CN 2010800199439 A CN2010800199439 A CN 2010800199439A CN 201080019943 A CN201080019943 A CN 201080019943A CN 102753701 A CN102753701 A CN 102753701A
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Abstract
本发明公开了分离自驴奶的乳杆菌属的益生乳细菌,及其在食品或饮料组合物如发酵奶制产品中的应用。
Description
技术领域
本发明涉及已知为益生菌的微生物领域及其在食品工业、人或动物食品中的使用。特别地,本发明涉及从生驴奶中分离的益生菌。
背景技术
益生微生物,或简称为益生菌,可定义为:当以适当的剂量给予时可能赋予健康或营养益处的微生物。它们属于所谓的功能食品的范畴。
已知益生菌在乳制品中,如DANONE出售的Actimel或Activia牌产品、Yakult Honsha出售的Yakult牌产品、或NESTLE出售的LC1牌产品。这些细菌属于乳酸细菌的不同属种,例如双歧杆菌属某些种(Bifidus spp.)、干酪乳杆菌(Lactobacillus casei)、鼠李糖乳杆菌(Lactobacillus rhamnosus)或约氏乳杆菌(Lactobacillus johnsonii)
尽管不知道益生菌作用的具体机制,但是广泛认为益生菌通过其能够对肠道菌群平衡具有影响而有益于健康或营养。
近年来,对驴奶的兴趣大大提高,主要是因为其组分,所以认为驴奶是婴幼儿营养奶粉、豆奶粉或其他配方的有效替换物。事实上,由于其多不饱和脂肪酸的组成、其钙/磷比率及其蛋白质含量,驴奶被认为非常接近于人奶。此外,驴奶富含溶菌酶,一种能够水解微生物细胞壁多糖的糖苷酶。多种科学证据证明了溶菌酶对营养和健康的重要性。高含量的乳糖对于钙的肠吸收具有积极效果,并负责口味。即使在生命的最初几个月后,驴奶也可增强骨质矿化并在那些严重Ig-E介导的牛奶蛋白过敏的儿童中构成重要的营养支持,因此在有效的免疫系统形成中发挥作用。在较大的年纪中,驴奶可发挥对抗不同心血管疾病和低血胆固醇饮食中的积极效果。
发明内容
本发明的目的是提供新颖的益生乳细菌。
为此目的,本发明的实施例提出益生乳细菌,尤其分离自驴奶并选自保藏在布达佩斯条约下的“德国微生物与细胞培养物保藏中心”(DSMZ)(德国布伦瑞克)中的下列编录号的菌株:在2008年12月8日的DSM 22098、DSM 22099、DSM 22100、DSM 22102、DSM22101,以及在2010年4月26日的DSM 23558、DSM 23559和DSM23560,可从所述菌株获得的突变菌株,以及属于植物乳杆菌群簇的菌株,其呈现出与选自保藏在DSM 22102、DSM 22101、DSM 23558、DSM23559、和DSM 23560下的菌株的菌株至少70%的DNA-DNA同源性和至少99.5%的16S RNA同一性。这种群簇定义了下文称为驴奶乳杆菌(Lactobacillus asini)的新种。
突变体可通过推断本发明菌株遗传物质改变或推断本发明菌株遗传物质与其他分子重组的基因工程技术获得。典型地,为了获得这种突变菌株,本领域技术人员可使用常用的诱变技术,如UV照射或暴露于化学诱变产品。
本发明的另一个实施例提出包括一种或众多上述的本发明益生乳细菌种类或菌株的组合物。所述的组合物可以是人或动物食用的食品组合物或饮料组合物。
本发明的进一步实施例提供制造食品组合物或饮料组合物的方法。所述的方法可至少包括下列步骤:
—用上述本发明的活的多种益生乳细菌接种食品,
—将接种后的食品放置在利于所述益生乳细菌代谢的条件下,
—将所述食品发酵直至得到至少[106]CFU/mL菌群的食品。
在另一个实施例中,制造食品组合物或饮料组合物的方法至少包含下列步骤:
—将上述益生乳细菌发酵直至达到至少[106]CFU/mL的菌群,
—例如,通过包裹的方式,保护所述益生乳细菌,
—将食品与所述被保护的益生乳细菌混合。
当达到发酵稳定期时终止发酵,优选在将所述的益生乳细菌放在利于其代谢的条件下24小时内终止。
本发明的另一个实施例提出益生乳细菌在制备用于治疗与病原微生物粘膜定植关联的疾病的组合物中的用途。粘膜包括但不局限于,肠粘膜、胃粘膜。例如,病原微生物可以是肠病原体。例如,根据本发明所述的乳杆菌菌株的抑制活性可对抗:肠病原体(例如肠炎沙门氏菌(Salmonella enteriditis)、霍乱弧菌(Vibrio cholereae)、大肠杆菌(Escherichia coli))。
本发明的另一个实施例提出益生乳细菌用于使发酵食品防护食品病原体的用途,所述食品病原体如:李斯特菌(Lysteria)或沙门氏菌(Salmonella)、弯曲菌(Campylobacter)或梭菌(Clostridium),通过抑制所述食品病原体滋生的进行。
本发明的另一个实施例提出如上定义的本发明益生乳细菌用于在发酵期间利用所述细菌生产丁酸的用途。
本发明的另一个实施例提供生产丁酸的方法,包括下列步骤:在适当的条件下发酵本发明的益生乳细菌和收集上述细菌的发酵过程中产生的丁酸。
本领域技术人员通过阅读此处提供的公开内容与附图,本发明的这些方面以及其他方面、特征和优点将变的明显。详细的描述,同时指出本发明的优选实施例,将仅以举例说明的方式给出。
附图简述
图1显示驴奶中本发明的克隆和参比菌株在24h温育过程中pH的演变。
图2显示从生驴奶中分离的8个克隆的DNA指纹图谱。
图3a至3e分别显示DNA指纹分析后克隆37、38、41、43和48的电泳图谱。
图4显示使用dUTP地高辛作为探针标记的植物乳杆菌DNA,克隆37、38和48之间DNA-DNA杂交的结果。
图5显示克隆37、38和48之间DNA-DNA杂交的结果是在植物乳杆菌(L.plantarum)、类植物乳杆菌(L.paraplantarum)、戊糖乳杆菌(L.pentosus)菌株类型DNA内与克隆37中标记的DNA探针的DNA-DNA杂交。
详细说明
在过去的几十年里,益生菌在人类健康中的作用通过引起其市场需求的显著增长,以及其更大规模的产品生产和消费,在科学水平和工业水平都取得了紧密关联。在生存力方面,益生菌菌株具有克服胃酸的极低pH和胆汁盐的清洗效果并以存活生理状态到达作用位点:肠上皮的能力,这是极为重要的。益生细菌能够在结肠定植,以积极影响病原细菌的感染效果,从而刺激免疫系统并减少不利的代谢物浓度。此外,一些菌株,包括本发明的细菌,能够一定程度地减少胆固醇和甘油三酯的血浆浓度。
在通过肠胃道的过程中,要求益生菌培养物耐受胃部胃蛋白酶和低pH的存在、十二指肠中富含蛋白酶的环境、和胆汁盐的抗微生物活性。尽管胃部的pH在摄取食物后可升高至6.0或更高,但是其主要范围是从2.5至3.5。通过胃部以后,小肠是肠胃道中的第二个主要障碍。尽管小肠的pH(即,7.0至8.5)更有利于细菌生存,但是胆汁盐的存在可具有不利效果。传统地,益生菌候选物在消化道运输中的生存能力使用常规镀饰技术进行评估,该技术在模拟消化道液中进行温育的过程中,提供活的并且可再生的细胞数目的信息,例如,如Charteris等《Development of an in vitro methodology to determine the transittolerance of potentially probiotic Lactobacillus and Bifidobacteriumspecies in the upper gastrointestinal tract》,J.Appl.Microbiol 84,759-768(1998)中描述的。
因而,多种基于培养的方法可用于表征益生微生物,如:胃液抗性、胆汁盐抗性、胆汁盐水解能力、疏水性评价、抗微生物活性、抗生素敏感性。这些测试方法在下列实例部分充分描述。
在本发明的实施例中,益生细菌符合至少一项下列标准:
—至少75%CFU/mL在此处描述的胃液抗性评价后得以保留,
—至少75%CFU/mL在此处描述的胃液抗性和胆汁盐抗性评价后得以保留,
—在此处描述的疏水性测试中,具有0.5M硫酸铵或更少,优选0.1M硫酸铵或更少的显微镜玻片上可见微生物聚集,
—对其他乳杆菌某些种的抗微生物活性低,即,用晕圈法(halo test)测量,小于0.5cm的晕圈,优选如此处所述的小于0.2cm,
—对绿脓杆菌(Pseudomonas aeruginosa)、蜡状杆菌(Bacilluscereus)、或大肠杆菌(Escherichia coli)的抑制活性,即,用晕圈法测量,即,大于0.5cm的晕圈,优选如此处所述的大于0.8cm。
克隆37、38、48、43、41、32、34和57代表本发明的特定实施例。它们已于2008年12月8日(克隆37、38、48、43、41)和2010年4月26日(克隆32、34、57)保藏在符合布达佩斯条约的“德国微生物与细胞培养物保藏中心”(DSMZ)中,编录号为:
克隆37 DSM 22098
克隆38 DSM 22099
克隆48 DSM 22100
克隆43 DSM 22102
克隆41 DSM 22101
克隆32 DSM 23558
克隆34 DSM 23559
克隆57 DSM 23560。
克隆37、38和48属于植物乳杆菌驴奶亚种(Lactobacillus plantarumasini),并且克隆43、32、34、57和41属于驴奶乳杆菌种。更具体地,克隆32属于称为驴奶乳杆菌丁酸菌(Lactobacillus asini ssp.butyricus)的亚种、克隆34属于称为驴奶乳杆菌乳酸菌(Lactobacillus asini ssp.lactis)的亚种、以及克隆57属于称为驴奶乳杆菌尾状菌(Lactobacillusasini ssp.caudatus)的亚种。
所述的益生细菌可作为鲜培养物或干燥食品补充物提供,如膳食补充物。就后者而言,生物质可以是冷冻干燥或喷雾干燥的,以提供高质量的培养物粉末,包括例如至少108CFU/g,优选超过109CFU/g。
根据本发明,益生细菌可用于制备食品或饮料,以供人或动物消费。食品和饮料包括发酵的食品,如鲜乳制品、发酵奶、酸奶。奶通常指牛奶。在本发明的语境中,尽管其他奶也可用于制备乳制品,包含马奶、山羊奶、骆驼奶、绵羊奶,但是优选驴奶。
制备这种发酵乳制食品的方法是本领域普通技术人员力所能及的。优选地,以存活形式提供或保持所述益生细菌,直到使用。可使用鲜奶或奶粉并用水重新调和。可向奶中加入各种成分,以改进发酵或向消费者提供额外的健康益处(如添加维生素或矿物质)。
发酵后,奶可转化为白干酪(cottage-cheese)或夸克蛋糕(quark),例如通过向发酵的奶中添加凝乳酶(rennet)。本领域技术人员熟知这些过程。
在另一个实施例中,益生细菌作为成分用于食品或饮料组合物中,而不对所述组合物进行发酵。换句话说,所述的益生细菌是膳食或营养补充物。在这种情形中,益生细菌优选以活菌形式提供。
根据本发明所述的益生乳细菌也可用于保护食品,如发酵食品,通过抑制上述食品病原体的滋生而抵御食品病原体,如:李斯特菌或沙门氏菌、弯曲菌或梭菌。在上述情形中,食品或饮料中混合了包括如此处所述的期望益生细菌的组合物。混合后的产品独立于包括期望益生细菌的组合物而发酵。无论如何,发酵持续时间优选不超过约24小时。通常,益生菌株在上述时间内达到发酵的稳定期。
在本发明的实施例中,发酵在达到发酵稳定期时停止,优选在将接种后的食品放在有利于所述益生乳细菌代谢的条件下24小时内。
本发明也涉及益生乳细菌在制备用于治疗与病原微生物粘膜定植关联的疾病的组合物中的用途。粘膜包含但不局限于,肠粘膜和胃粘膜。例如,原微生物可以是肠病原体。例如,根据本发明所述的乳杆菌菌株的抑制活性可对抗:肠病原体(例如沙门氏菌、霍乱弧菌、大肠杆菌)。这与益生细菌附着上皮细胞,如肠道细胞的能力相关联,如目前已知的,能力可在一定程度上将此类病原细菌逐出肠道。相应地,这可能与益生细菌的疏水性有关。
本发明也涉及本发明的益生乳细菌用于在发酵期间利用所述细菌生产丁酸的用途,例如发酵已接种有这种细菌的食品,然后由用户摄取,菌落在结肠内的内部发酵或在适当条件中合适培养基的发酵,允许收集所述细菌发酵过程中生产的丁酸。优选的细菌是DSM 23558、DSM 22098、DSM 22100、和DSM 22099。
具体实施方式
基于培养的方法(益生菌测试、胆汁盐水解能力、疏水性评价、抗微生物活性、抗生素敏感性、有机酸的生产、温育过程中pH的演变)
用无菌生理液(NaCl 0.85%)连续稀释从有机饲养获得的生驴奶,并接种在特用于乳杆菌属分离的MRS(Man,de Rogosa and Sharpe)琼脂平板上。在不同温度下将平板在厌氧环境(Anaerogen,Oxoid)中温育48h。从最高稀释度的MRS琼脂平板中任意选择乳酸细菌分离物(约150个)。分离物在MRS液体培养基中次培养,并划线至MRS琼脂上。
益生性测试
1)胃液抗性
生长后,将每个菌落离心,并在无菌生理液中洗涤,并用相同的溶液重新悬浮至原始体积。胃液抗性的评价根据De Giulio等《Use ofalginate and cryo-protective sugars to improve the viability of lactic acidbacteria after freezing and freeze-drying》World Journal of Microbiology &Biotechnology 2005年21期,739-746页中的描述进行评估,但作一些修改。等分样品在模拟胃液(胃蛋白酶3g/L,pH 2.5)中温育,在温育的不同时间取出,直至180分钟,并进行铺平板。使用并接种未处理的细胞作为阴性对照。对胃液的抗性通过菌落形成单位(CFU)/mL进行评价并与阴性对照比较。
2)对胃液和胆汁盐的抗性
对胃液具有的抗性的菌落在由含有0.3%胆汁盐的MRS组成的模拟胆汁液中温育,最多3h,然后接种到MRS琼脂平板上,根据De Giulio等(2005)所述。未处理的菌落用作阴性对照。对胆汁盐的抗性通过菌落形成单位(CFU)/mL进行评价并与阴性对照比较。
结果
在从生驴奶中分离的150个细菌菌落中,只有8个鉴定为属于乳杆菌属(暂时命名为克隆32、克隆34、克隆37、克隆38、克隆41、克隆43、克隆48、和克隆57),展示出对胃液和胆汁盐的抗性,并显示形成约75%的CFU/mL,对比于未处理的对照乳杆菌的100%。其最佳生长温度约为30至31℃。
150个菌落中只出现8个展示了对模拟胃液和对胆汁盐存在的良好抗性。然后研究这些菌落的下列活性:疏水性测试、筛选具有胆汁盐水解活性的培养物、抗微生物活性和抗生素敏感性。
3)疏水性测试
菌落潜在地附着到肠上皮细胞的能力通过使用Strus等《The invitro activity of vaginal Lactobacillus with probiotic properties againstCandida》Infectious Diseases in obstetrics and Gynaecology,2005年13卷第2期69-75页,以及Ljungh等《High surface hydrophobicity ofautoaggregating Staphylococcus aureus strains isolated from humaninfections studied with the salt aggregation test》Infection and immunity,1985年47期522-526页中的间接疏水性方法进行评价。菌落在MRS液体培养基中生长24h。微生物悬浮液与等体积硫酸铵混合,硫酸铵预先以不同摩尔浓度制备(范围从20mM至4M)。能够引起显微镜载玻片上可见微生物聚集的硫酸铵最小浓度与盐聚集测试成反比。等渗溶液用作对照。
结果
—0.1M硫酸铵的可见聚集:克隆32、37、43和57
—0.5M硫酸铵的可见聚集:克隆34、38、41和48。
从这些数据,可假设克隆32、37、43和57对于肠上皮细胞具有更强的体外附着能力。
4)筛选具有胆汁盐水解活性的培养物
胆汁盐水解是哺乳动物胆汁盐代谢中重要的代谢反应。近年来,使用胆汁盐水解以影响人和动物的胆固醇代谢的兴趣升高。胆汁盐的水解和胆固醇向细胞膜的掺入具有降低人血清胆固醇浓度的潜力。经由小肠中结合胆汁盐水解的游离胆汁盐的释放导致粪便中更多胆汁盐的排泄。胆固醇从人体移除的主要方式是以水解胆汁盐的形式排泄。大部分结合胆汁盐经由进入肝循环而再循环。排泄了的胆汁盐必须由新的胆酸代替,新的胆酸从人体中的胆固醇形成。因此,排泄的胆汁盐越多,人体库中使用的胆固醇越多。此外,游离胆汁盐不支持从小肠吸收胆固醇和其他脂质,但是结合胆汁盐支持。
菌落的胆汁盐水解活性遵照Minelli等《Assessment of novelprobiotic Lactobacillus casei strains for the production of functional dairyfoods》Int.Dairy J.,2004年14期723-726页中描述的方法定性评价。简言之,菌株的过夜液体培养物(5μL)在含有0.5%结合胆汁盐混合物和0.37g/L CaCl2的MRS琼脂上形成斑点,并如上所述进行温育。斑点周围析出胆酸(不透明晕圈)的出现被认为是阳性结果,显示菌落水解胆汁盐的能力。
结果
定性上评价了水解胆汁提取物的能力。所有菌株均能水解胆汁提取物。
5)抗微生物活性
使用琼脂平板分析上的抑制晕圈测试以研究微生物菌株的抗微生物活性。测试样本对抗下列细菌:
—非病原株:嗜酸乳杆菌(Lactobacillus acidophilus)DSM 20079、干酪乳杆菌(Lactobacillus casei)ATCC 9595、保加利亚乳杆菌(Lactobacillus bulgaricus)DSM 20081、沙克乳杆菌(Lactobacillus sakei)DSM 20494、鼠李糖乳杆菌(Lactobacillus rhamnosus)DSM 20711;
—革兰氏阳性病原株:蜡状杆菌GN 101、DSM 4313和DSM4384,以及粪肠球菌(Enterococcus faecalis)ATCC 29212,
—革兰氏阴性病原株:大肠杆菌(DSM 8579)和绿脓杆菌(DSM50071)。
所有菌株均购自德国微生物与细胞培养物保藏中心(德国DSMZ)。每种菌株在其特定生长培养基中37℃温育18h:乳酸细菌在Man deRogosa Sharpe(MRS)液体培养基(Oxoid)中生长,大肠杆菌、粪肠球菌、绿脓杆菌和蜡状杆菌在营养液体培养基(Oxoid)中生长。微生物悬浮液(1×108CFU/mL)均匀涂布在特定固体培养基平板上。50μL的每种培养物单个地放在接种的平板上。室温下无菌环境中静置30min后,取决于菌株,在37℃平板温育24至48h。准确测量平板上显示的清澈区直径,其为以cm表示的抗微生物活性。使用无菌去离子水作为阴性对照;标准抗微生物试剂氯霉素用作阳性对照,如Dall′Agnol等《Antimicrobial activity of some Hypericum species》Phytomedicine 10:511-516.(2003)中的描述。
结果
a)对其他乳杆菌属某些种的抗微生物活性
克隆不展示对沙克乳杆菌20494的抗微生物活性(除了克隆57以非常低的程度外)。
—克隆37、38、和48展示对干酪乳杆菌ATCC 9595、保加利亚乳杆菌ATCC 11842、发酵乳杆菌DSM 20052、鼠李糖乳杆菌DSM20711的低抑制活性;
—克隆41展示对发酵乳杆菌DSM 20052、鼠李糖乳杆菌DSM20711、嗜酸乳杆菌DSM 20079的低抑制活性;
—克隆43展示对发酵乳杆菌DSM 20052和嗜酸乳杆菌DSM20079的低抑制活性。
这种结果被认为是正常的,这是因为通过所有乳酸细菌生产的抗微生物物质,如细菌素,其不仅能够对致病细菌进行作用,同时作为“领土防御机制”,也对其他乳杆菌进行作用。
b)对致病细菌的抗微生物活性
下表显示结果:
说明:
ND 不可检测
OX 低抑制活性(晕圈:<5mm)
× 中等程度抑制活性(晕圈:6-8mm)
×× 不连续的抑制活性(晕圈从8至10mm)
××× 强抑制活性(晕圈>10mm)
观察到克隆37展示的对绿脓杆菌的强抗微生物活性是令人感兴趣的。克隆37、38和48对产生毒素的菌株——大肠杆菌DSM 8579展示了一定的抗微生物活性。
6)抗生素敏感性
菌落对不同抗生素的敏感性通过使用30mg至40mg的下列抗生素进行评价:
—作为细胞壁合成抑制剂的氨苄青霉素和羟基氨苄青霉素;
—作为蛋白质合成抑制剂的硫酸庆大霉素、林可霉素、硫酸链霉素、四环素、氯霉素和螺旋霉素;
—作为核酸合成抑制剂的利福霉素。
每种抗生素粉末都小心地称重、溶解、用合适的稀释剂稀释并在添加到MRS培养基前过滤灭菌。平板接种LAB菌株,并如上述般温育。通过抑制晕圈测试评价其对抗生素的敏感性或抗性。
结果
克隆对测试中使用的抗生素展示变化程度的敏感性。如下表所示,克隆32对所有的抗生素都敏感;另一方面,其他克隆或多或少对链霉素和林可霉素具有抗性。
7)生产有机酸
发酵后有机酸的生产由过滤后上清液的高效液相色谱来测定(Vulevic,Rastall & Gibson,2004;Nazzaro et al.2009),使用装备紫外线检测器的Gold System设备(Beckman,CA,USA)。样品装载在预包装Aminex HPX-87柱(3007.8mm,Bio-Rad,Hercules,CA,USA)上,并用0.005mM硫酸洗脱。总运行时间为40min;流速为0.6ml/min;注射体积为20μl;并且检测波长为210nm。
结果
在下表中报告了在驴奶中生长的本发明菌株和一些参比菌株在发酵过程中生产的丁酸和柠檬酸的量。数据以绝对面积(mm2)表示。
菌株 | 丁酸的量 |
嗜酸乳杆菌(对比) | 26.21 |
植物乳杆菌(对比) | 21.5 |
发酵乳杆菌(对比) | 33.56 |
克隆32(本发明) | 60.3 |
克隆37(本发明) | 65.41 |
克隆38(本发明) | 42.91 |
克隆48(本发明) | 60.37 |
菌株 | 柠檬酸的量 |
嗜酸性乳杆菌(对比) | 51.37 |
植物乳杆菌(对比) | 2.95 |
发酵乳杆菌(对比) | 57.97 |
克隆32(本发明) | 65.81 |
克隆37(本发明) | 82.1 |
克隆38(本发明) | 83.11 |
克隆41(本发明) | 76.2 |
克隆43(本发明) | 79.98 |
克隆48(本发明) | 85.71 |
丁酸的生产可通过发酵已接种本发明细菌的食品在外部进行,然后这种食品可被用户摄取以增加消化系统中的丁酸。
丁酸是短链脂肪酸,也可用结肠中的细菌从发酵中生产,并符合结肠上皮细胞能量需要的60%-70%。而且,丁酸刺激结肠、小肠中段、回肠上皮细胞的再生,并抑制结肠中微生物的生长。上皮细胞不佳的丁酸代谢可引起结肠溃疡性炎症。
上表显示本发明的菌株能够生产比参比菌株更高量的丁酸。因而,本发明的菌株允许刺激肠屏障并增强免疫系统。
柠檬酸在食品和饮料中用作天然防腐剂。上表显示本发明的菌株能够生产比参比菌株更高量的柠檬酸。因而,其允许改进包括本发明菌株的组合物的储存特性。
8)温育中pH的演变
测试了驴奶中微生物的生长。驴奶从Eurolactis农场以喷雾干燥的形式获得。奶重新悬浮在无菌去离子水中(比率为100gr+600ml水)。42ml的这种悬浮物接种0.840ml分离的微生物培养物(2%接种物,在λ600nm处的初始吸收度=1)。样品在30℃温育。
驴奶在相同的条件中接种植物乳杆菌、类植物乳杆菌(Lactobacillusparaplantarum)和戊糖乳杆菌类型的菌株,同时37℃的温度用于保加利亚乳杆菌和鼠李糖乳杆菌。
菌株生长进驴奶的能力和发酵能力分别通过菌落形成单位(CFU)/ml计数微生物细胞(通过3、6和24小时的温育后在MRS琼脂上厌氧培养测定)和测量结果pH来评价。
结果
初始pH值为7.14。
图1显示驴奶中本发明克隆和参比菌株在24小时的温育过程中pH降低。参比A指明本发明不同菌株的组。参比B至F分别对应保加利亚乳杆菌、鼠李糖乳杆菌、类植物乳杆菌、戊糖乳杆菌、和植物乳杆菌。
图1显示参比菌株与本发明克隆相比,展示更高的酸性发酵能力。产品的口味受低pH负影响。因而,本发明的克隆允许保留包括这些克隆的组合物的口味。
表型和基因型方法(糖代谢、DNA指纹、DNA-DNA杂交、16 S RNA序列比对)
9)糖代谢
分离物使用碳水化合物发酵API 50CHL(BioMérieux)鉴别系统鉴别至物种水平。用于鉴别乳杆菌属和相关生物体的API 50CHL培养基,是现成可用的培养基,使得待研究的49种碳水化合物在API 50CH条上的发酵。在具有待测微生物的培养基中制备悬浮液,并且每管条带都被接种。在温育的过程中,碳水化合物发酵成酸,使pH降低,通过指示物的颜色改变来检测。其结果构成了菌株的生化曲线,并在其鉴别或分类中使用。假定的益生菌菌株接种在API 50CHL条带中并在24和48h的37℃温育后进行颜色改变的评价。
每个条带都由下表所示组成。其结果通过使用API BioMérieux软件分析。
发酵测试的结果(API)
克隆32:不够辨别
克隆34:不够辨别
克隆37:优良鉴别植物乳杆菌99.5%
克隆38:疑惑鉴别
克隆41:初步疑惑鉴别
克隆43:初步疑惑鉴别
克隆48:优良鉴别植物乳杆菌99.5%
克隆57:不够辨别
基因型方法
下列方法学应用于分子鉴别:DNA指纹、DNA-DNA杂交和16 SRNA测序。
10)DNA指纹
根据Ronimus等人(1997)所述使用随机扩增多态性DNA-PCR(RAPD-PCR)分析产生指纹模式。DNA扩增在50μL PCR反应混合物中执行,该反应混合物包括:50-200ng基因组DNA、1倍PCR缓冲液(作为DNA聚合酶试剂盒的成分供应)、3mM MgCl2、250μMdNTPs、0.5μM OPR-2引物(5′-CACAGCTGCC-3′,序列SEQ ID NO:1)或OPR-13引物(5′-GGACGACAAG-3′,序列SEQ IDNO:2)和2.5单位PlatinumTaq DNA聚合酶(INVITROGEN)。上述混合物在iCycler热循环仪(BIO RAD)中进行扩增。扩增过程由下列组成:92℃初始变性2min和35次如下循环:94℃下15sec,36℃下15sec退火(通过温度梯度扩增预先优化)以及72℃下2min延伸。在72℃下进行7min的最终延伸。10-20μL PCR产物在使用2100型生物分析仪的DNA 7500微晶片(Agilent)上进行电泳,该生物分析仪装备2100EXPERT软件(Agilent)。
结果
【0096】API测试后进行基因型研究。DNA指纹分析(图2)显示克隆37、38和48似乎属于同一种。在图3a至3e,提供了关于克隆37、38、41、43和48的电泳图谱。
11)16s RNA序列比对
总RNA按照制造商的说明通过RiboPure-Bacteria试剂盒(Ambion)进行抽提。RNA溶解在RNA储存液(Ambion)中,通过Bio-Photometer(Eppendorf)紫外定量,并储存在-80℃。RNA等分试样(6μg)在20μL终体积反应混合物中用无RNA酶的DNA酶I(Ambion无DNATM试剂盒)消化,以去除污染性基因组DNA。DNA酶消化后,RNA样品的浓度和纯度通过RNA-6000-Nano微晶片分析进行评价,使用装备2100-Expert-Software(Agilent)的2100Bioanalyzer按照制造商的说明进行操作。对于所有测试样品,RNA完整性数(R.I.N)大于7(相当于0-10级)的样品在20μL反应混合物中被逆转录。含有3μg总RNA的混合物,如2100 Bioanalyzer所评价的,2皮摩尔的1517R正向引物(见前文)、10μL总体积中的2mM dNTPs在70℃下温育2min,并迅速冷却至40℃。向混合物中加入10μL含有2倍的合适缓冲液(Invitrogen)、20mM二硫苏糖醇、20单位的RNA酶抑制剂(Invitrogen)、和200单位的MoMuLV SuperscriptIII逆转录酶(Invitrogen)的混合物。反应混合物(终体积20μL)在55℃下温育60min,并在70℃下持续15min以便终止反应和进行RNA降解。16S cDNA的扩增在50μL含有下列物质的反应混合物中通过iCycler-iQ5执行:1倍的合适缓冲液(Invitrogen)、200皮摩尔dNTPS、300nM在大肠杆菌16S RNA序列编录(8-正向和1517-反向)上设计的的细菌16S通用引物。扩增过程由下列程序组成:94℃下3min的初始变性、25次下列循环:94℃下30sec、57℃下30sec退火和72℃下1min的延伸,以及72℃下7min的最终延伸。5μL的PCR产物在1%琼脂糖凝胶上(Agarose-1000,Invitrogen)在1倍TAE缓冲液中以5V/cm电泳4h。凝胶和电泳缓冲液中都包含溴化乙锭(0.1μg/mL),并且PCR产物由紫外可视化检测并记录在Polapan 55膜上(Polaroid)。100-150μL的每种扩增混合物经空气干燥并送至MWG(Ebersberg-Germany)进行测序。使用基于第一次测序数据设计的引物8F、1517R和内部引物368F进行测序。序列重叠(sequence-conting)用DNA-Baservers.2.0软件利用“.scf”序列文件执行。生成的“.fasta”序列(约1400个碱基长度)在NBCI-BLAST和“Ribosome Data Base Project”-RDP数据库中进行比对。
结果
核糖体RNA经抽提和反转录。获得的cDNA如上所述进行扩增和测序。获得了1400个碱基的序列重叠群并在数据库中比较。下列为在Ribosomal Database Project(RDP)中序列比较的合成。出现这样的图片,将我们的克隆包括在植物乳杆菌、类植物乳杆菌和戊糖乳杆菌种的非常紧密的群簇中,这些种呈现大于99.9%的16 S RNA同一性。下列给出的结果是本发明的克隆与这些种比较的16 S RNA同一性百分数。
克隆41
细菌域(domain bacteria)(20)(匹配序列)
厚壁菌门(20)“杆菌”纲(20)“乳杆菌”目(20)乳杆菌科(20)乳杆菌属(20)
0.994 1400类植物乳杆菌(T);DSM 10667T;A5306297
0.979 1405植物乳杆菌(T);JCM 1149;D79210
0.984 1410戊糖乳杆菌(T);JCM 1558;D79211
克隆43
细菌域(20)(匹配序列)
壁菌门(20)“杆菌”纲(20)“乳杆菌”目(20)乳杆菌科(20)乳杆菌属(20)
1400类植物乳杆菌(T);DSM 10667T;AJ306297
0.985 1405植物乳杆菌(T);JCM 1149;D79210
0.990 1410戊糖乳杆菌(T);JCM 1558;D79211
克隆48
细菌域(20)(匹配序列)
厚壁菌门(20)“杆菌”纲(20)“乳杆菌”目(20)乳杆菌科(20)乳杆菌属(20)
1.000植物乳杆菌(T);JCM 1149;D79210
克隆57
细菌域(0/20/5164)(选择/匹配/总RDP序列)
厚壁菌门(0/20/1178)“杆菌”纲(0/20/697)“乳杆菌”目(0/20/289)乳杆菌科(0/20/114)乳杆菌属(0/20/104)
0.987 1400类植物乳杆菌(T);DSM 10667T;A5306297
0.990 1405植物乳杆菌(T);JCM 1149;D79210
0.994 1410戊糖乳杆菌(T);JCM 1558;D79211
12)DNA-DNA杂交
为了进行DNA-DNA杂交和同源百分数计算,DNA使用Genomic-DNA-Buffer Set和Genomic-tip-100/G柱(QIAGEN SPA,MilanoItaly),按照制造商的说明但做微小修改,从细菌细胞培养物(每菌株约250mg干粒)中抽提并纯化。DNA溶解在TE缓冲液(10mM pH为8的Tris,1mM EDTA)中,并连续稀释至50μg/mL的终浓度(WS),使用生物分光光度计(Eppendorf,Germany)通过紫外吸收进行评价。WS DNA浓度使用Quant-iT DNA分析试剂盒(INVTTROGEN,MilanoItaly)通过荧光测量结果进行确认;DNA大小使用DNA作为分子重量标记物(DNA大小约32kD)通过0.8%DNA分级琼脂糖(BIO-RAD)电泳估计。WS溶液稀释至0.1×SSC中2ng/mL的终浓度,0.1×SSC含有5ng/mL鲱鱼精子DNA。DNA在100℃下变性10min,并迅速浸入冰水浴中。将每种菌株50-100ng的DNA在尼龙膜上点四次,所述尼龙膜通过使用连接至软真空的Dot-Blot设备(Bio-Rad,Ca USA)充正电(Roche,Germany)。点中包含标准曲线20至200ng DNA/斑点的同源DNA(与探针连接的DNA)。DNA经过3min的UV暴露和120℃下1h的真空下烘烤,与尼龙交联。膜在-20℃下冷冻,直到分析。
按照制造商的说明,使用随机引发的DNA标记试剂盒(Roche,Germany),在20μL反应混合物中将1μg与探针连接的DNA用地高辛-dUTP进行过夜标记。膜在40℃下在DIG Easy-Hyb溶液(Roche,Germany)中预杂交3h,并在40℃下在DIG Easy-Hyb溶液中过夜杂交,DIG Easy-Hyb溶液含有20pg/mL地高辛标记的探针(如上述热变形的),使用试管旋转式杂交温育箱(GFL)。严格洗涤为:室温下在含有0.1倍SDS的2倍SSC溶液中冲洗两次,每次5min;68℃下在含有0.1倍SDS的0.1倍SSC溶液中冲洗两次,每次15min。免疫检测通过使用抗地高辛AP抗体(抗地高辛FAB片段与碱性磷酸酶结合)、CDP-Star化学发光底物和地高辛洗液(DIG Wash)及阻断缓冲液套装试剂盒进行,所有试剂盒及说明均来自Roche。化学发光通过使用装备Quantity-one软件的VersaDOC 4000设备(BIO-RAD)在时间暴露线性的条件中进行定量。DNA-DNA同源百分数根据Jahnke(1994)所述,通过将从同源DNA斑点获得的化学发光值(调整柱强度×mm2)的中值假定为100%,并考虑同源DNA标准曲线的线性反应来计算。重复样品的中间标准偏差不超过中值的5%。
结果
利用dUTP-地高辛作为探针标记的植物乳杆菌DNA,通过DNA-DNA杂交确认代谢数据。如图4所示,克隆37、38、48分别显示与植物乳杆菌(图4上的L.P1)86%、99%、99%的DNA-DNA同源性,已知不同种的分类学DNA-DNA限制低于DNA-DNA同源性的70%。
为了从三种可能性中区别开并确认先前的发现,克隆37、38和48在植物乳杆菌(图5上的PL)、类植物乳杆菌(图5上的PPL)和戊糖乳杆菌(图5上的PE)菌种类型的DNA内进行与克隆37中获得的标记DNA探针的DNA-DNA杂交(图5)。N-Ctr为阴性对照。
前述的本发明优选实施例不是穷尽性的或将本发明限制于公开的实施例。对于本领域技术人员来说,在本发明范围内的多种修改是显而易见的,并可从本发明的实践中获得。
基于DNA-DNA杂交和16S RNA分析的实验结果,菌株37、38和48属于单一群簇,该群簇也存在植物乳杆菌、戊糖乳杆菌和类植物乳杆菌,但与它们不同。认为菌株37、38和48代表植物乳杆菌的亚种,将被命名为驴奶植物乳杆菌。同样的理由,菌株41和43明显属于新种——驴奶乳杆菌。事实上,菌株41和43表现出与其他乳杆菌40-50%的相似性,主要是与戊糖乳杆菌、植物乳杆菌和类植物乳杆菌。
菌株37、38和48,及菌株41和43,是属于乳杆菌属的微生物,其与植物乳杆菌、类植物乳杆菌和戊糖乳杆菌紧密群簇的16S RNA的同源性高于99.2%,并且具有与这种同源性成比例的相对指纹和蛋白质特性。
序列列表
SEQ IDNO:1
长度:10个碱基
类型:核酸
链型:单链
拓扑学:线性
分子类型:DNA
序列:
CACAGCTGCC
SEQ IDNO:2
长度:10个碱基
类型:核酸
链型:单链
拓扑学:线性
分子类型:DNA
序列:
GGACGACAAG
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Claims (12)
1.益生乳细菌,所述益生乳细菌特别地从驴奶中分离,并选自以编录号DSM 22098、DSM 22099、DSM 22100、DSM 22102、DSM 22101、DSM23558、DSM 23559、和DSM 23560保藏在DSMZ中的菌株,可从所述菌株获得的突变菌株,并且所述菌株属于植物乳杆菌(Lactobacillus Plantarum)群簇,表现出与选自以编录号DSM 22102、DSM 22101、DSM 23558、DSM23559、和DSM 23560保藏的菌株的菌株至少70%的DNA-DNA同源性和至少99.5%的16S RNA同一性。
2.组合物,包含根据权利要求1所述的一种或众多益生乳细菌种类或菌株。
3.根据权利要求2所述的组合物,其中所述的组合物为食品组合物或饮料组合物。
4.制造食品组合物或饮料组合物的方法,至少包含下列步骤:
—用根据权利要求1所述的活益生乳细菌接种食品,
—将接种后的食品放置在有利于所述益生乳细菌代谢的环境中,
—发酵所述食品直至获得至少[106]CFU/mL菌群的食品。
5.制造食品组合物或饮料组合物的方法,至少包含下列步骤:
—发酵根据权利要求1所述的益生乳细菌直至获得至少[106]CFU/mL的菌群,
—保护所述的益生乳细菌,
—将食品与所述被保护的益生乳细菌混合。
6.根据权利要求4或5所述的方法,其中所述的发酵在其到达发酵稳定期时终止,优选在将所述益生乳细菌放置在利于其代谢的条件下约24小时内。
7.根据权利要求1所述的益生乳细菌在制备用于治疗与病原微生物粘膜定植关联的疾病的组合物中的用途。
8.根据权利要求7所述的用途,其中所述的粘膜选自肠粘膜和胃粘膜。
9.根据权利要求7或8所述的用途,其中所述的病原微生物是肠病原体,如:肠炎沙门氏菌(Salmonella enteriditis)、霍乱弧菌(Vibrio cholereae)、大肠杆菌(Escherichia coli)。
10.根据权利要求1所述的益生乳细菌用于使发酵食品防护食品病原体的用途,所述食品病原体如:李斯特菌(Lysteria)或沙门氏菌(Salmonella)、弯曲菌(Campylobacter)或梭菌(Clostridium),通过抑制所述食品病原体滋生的进行。
11.根据权利要求1所述的益生乳细菌用于在发酵期间利用所述细菌生产丁酸的用途。
12.用于生产丁酸的方法,包括下列步骤:在适当的条件下发酵根据权利要求1所述的益生乳细菌和收集所述细菌在发酵期间产生的所述丁酸。
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- 2010-05-05 ES ES10718580.3T patent/ES2562702T3/es active Active
- 2010-05-05 US US13/319,124 patent/US20120128821A1/en not_active Abandoned
- 2010-05-05 CA CA2761141A patent/CA2761141C/en active Active
- 2010-05-05 EP EP10718580.3A patent/EP2427566B1/en active Active
- 2010-05-05 CN CN201080019943.9A patent/CN102753701B/zh not_active Expired - Fee Related
- 2010-05-05 JP JP2012509030A patent/JP5875975B2/ja active Active
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CN105764352A (zh) * | 2013-10-14 | 2016-07-13 | 国家研究会议 | 食品组合物 |
CN107151638A (zh) * | 2017-05-25 | 2017-09-12 | 中驭(北京)生物工程有限公司 | 一株改善肝功能的植物乳杆菌zy001及其在发酵乳中的应用 |
Also Published As
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CA2761141C (en) | 2020-08-04 |
CA2761141A1 (en) | 2010-11-11 |
WO2010128084A1 (en) | 2010-11-11 |
EP2427566A1 (en) | 2012-03-14 |
EP2248908A1 (en) | 2010-11-10 |
EP2427566B1 (en) | 2015-10-28 |
US20120128821A1 (en) | 2012-05-24 |
CN102753701B (zh) | 2015-01-21 |
ES2562702T3 (es) | 2016-03-07 |
JP2012525828A (ja) | 2012-10-25 |
JP5875975B2 (ja) | 2016-03-02 |
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