CN110106119B - 一株分离自母乳的鼠李糖乳杆菌m9及其应用 - Google Patents
一株分离自母乳的鼠李糖乳杆菌m9及其应用 Download PDFInfo
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- CN110106119B CN110106119B CN201910453691.3A CN201910453691A CN110106119B CN 110106119 B CN110106119 B CN 110106119B CN 201910453691 A CN201910453691 A CN 201910453691A CN 110106119 B CN110106119 B CN 110106119B
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Abstract
本申请提供一株分离自母乳的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9),其微生物保藏编号为CGMCC No.16002,所述鼠李糖乳杆菌M9具有优异的胃肠液耐受性和良好的胆盐耐受性,具备作为益生菌的基本条件;并且具有增强免疫抵抗作用、提高肠道菌群多样性、增加肠道菌群稳定性的益生特点;所述鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)可以作为益生菌添加到各类普通食品、保健品等中。
Description
技术领域
本申请涉及鼠李糖乳杆菌(Lactobacillus rhamnosus),特别涉及一株分离自母乳的鼠李糖乳杆菌M9及其应用。
背景技术
健康人的胃肠道内寄居着种类繁多的微生物,这些微生物称为肠道菌群。肠道菌群按一定的比例组合,各菌间互相制约,互相依存,在质和量上形成一种生态平衡,一旦机体内外环境发生变化,特别是长期应用广谱抗生素,敏感肠菌被抑制,未被抑制的细菌而乘机繁殖,从而引起菌群失调,其正常生理组合被破坏,而产生病理性组合、引起临床症状就称为肠道菌群失调症。
肠道菌群失调症以严重腹泻或慢性腹泻为主要临床表现,有些旅游者可能因气候和环境的改变而发生肠道菌丛失调,俗称水土不服。近年来,由于冰箱的普及使用,有的家庭储存大量的肉食品及疏菜,过久的储存使食物变质,食用后引起肠道菌群失调,造成呕吐、腹泻,有的出现精神不集中甚至精神恍惚。
肠道菌群的平衡一旦被破坏,则很难重新恢复原有的平衡状态,导致肠道菌群失调难以调整和治愈。
发明内容
本申请的目的之一在于提供一株从中国内蒙古、新疆、湖北、北京等地区100份健康妇女母乳样中分离得到的540株乳酸菌和双歧杆菌中筛选出的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9),其微生物保藏编号为CGMCC No.16002,分类命名为:鼠李糖乳杆菌;保藏单位为中国微生物菌种保藏管理委员会普通微生物中心;保藏时间为2018年6月22日;保藏地址:中国北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所。
在本申请中,采用PacBio SMRT RSII第三代测序平台对Lactobacillusrhamnosus M9全基因组进行测定,其基因组长度3002913bp,GC含量46.77%(其中,GC含量是指在DNA4种碱基中,鸟嘌呤和胞嘧啶所占的比率),染色体基因组含有2965个编码基因,76个RNA基因。
在本申请中,所述鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)可以根据包括以下方法分离得到:将采集到的100份健康妇女的母乳样品进行倍比稀释,得到10-1~10-2稀释梯度,吸取各梯度母乳样品200μL均匀涂布于MRS固体培养基平皿内,37℃厌氧培养48-72h。挑取形态、大小、颜色不同的单克隆接种于液体培养基中,于37℃恒温培养箱培养24h。待菌株生长良好后,进行革兰氏染色、镜检。保存分离株,并提取菌株基因组DNA用于后续测定分析。
其中MRS培养基的组成是:大豆蛋白胨10g、牛肉膏5g、酵母粉4g、葡萄糖20g、Tween80 1ml、磷酸二氢钠2g、无水乙酸钠5g、柠檬酸三胺2g、硫酸锰0.02g、硫酸镁0.1g,蒸馏水1L,调节pH至6.2左右,琼脂15g,121℃、15min灭菌。
本申请对所述鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)分子生物学鉴定方法包括:
将冷冻保存的供试菌株接种于TPY增菌液体培养基中,于30℃恒温培养24h,经TPY传代培养2~3代后,取2mL对数生长末期的菌体培养物置于无菌EP管内离心,经8000×g离心3min(4℃)后收集菌体,弃上清,采用乳酸菌专用CTAB冻融法提取菌株的基因组DNA。
其中,TPY增菌培养液的组成是:乳糖10g、牛肉膏5g、酵母粉5g、酪蛋白胨10g、大豆蛋白胨5g、磷酸氢二钾2.5g、磷酸二氢钾2.5g、硫酸镁0.1g、吐温80 0.25g、L-半胱氨酸盐酸盐0.5g、15g琼脂、蒸馏水1L,121℃、15min灭菌。
采用PacBio SMRT RSII第三代测序平台对Lactobacillus rhamnosus M9全基因组进行测定,其基因组长度3,002,913bp,GC含量46.77%,染色体基因组含有2965个编码基因,76个RNA基因。
本发明提供的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)具有如下形态学特征:菌体在显微镜(5000倍电镜)下呈杆状,细胞成单、成对或成链状,如图1所示,不具鞭毛或纤毛,不会移动。
本发明的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)在MRS培养基上形成的明显菌落且较大,直径在0.5-1mm之间,边缘整齐,呈奶油白色的不透明菌落,如图2所示。
本申请的另一目的是提供所述鼠李糖乳杆菌M9在制备用作肠道菌群调节剂中的用途。本申请所分离的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)的发酵代谢产物具有调节肠道菌群,从而缓解腹泻的作用。
进一步地,所述鼠李糖乳杆菌M9还可以用作发酵乳制品以及发酵谷物制品的发酵剂。具体地,所述鼠李糖乳杆菌M9可以作为乳制品发酵剂,例如可以作为酸奶、饮用型酸奶、发酵乳饮料或者发酵豆乳的发酵剂。所述鼠李糖乳杆菌M9可以作为发酵谷物制品的发酵剂,例如,可以作为发酵燕麦、糙米或者薏米的发酵剂。
世界卫生组织定义的益生菌是指“摄入一定数量,能够对宿主健康起有益作用的活的微生物制剂”。
本申请提供的鼠李糖乳杆菌M9能够对胃肠道消化液和胆盐有良好的耐受性,因此,本申请提供的鼠李糖乳杆菌M9属于益生菌。
本申请还提供一种益生菌菌剂,所述益生菌菌剂由所述鼠李糖乳杆菌M9制备。
本申请还提供一种复合益生菌,所述复合益生菌包括前述鼠李糖乳杆菌M9,其中,所述鼠李糖乳杆菌M9可以为主要成分也可以为辅助成分。
在本申请中,所述复合益生菌可以为粉剂、片剂、溶液剂或者乳剂等所有医学可接受剂型。
在本申请中,所述复合益生菌可以用作调节肠道菌群平衡的调节剂。所述复合益生菌用作调节肠道菌群平衡的调节剂时,可以单独使用,也可以与其它调节肠道菌群的物质配合使用。
可选地,在所述复合益生菌中,还可以包括所述鼠李糖乳杆菌M9的代谢产物。
本申请还提供一种鼠李糖乳杆菌菌剂,该菌剂由所述鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)发酵而得,具体包括鼠李糖乳杆菌M9(Lactobacillusrhamnosus M9)及其发酵代谢产物。该菌剂也可以用作缓解腹泻的药物。
进一步地,本申请还提供一种包括所述鼠李糖乳杆菌菌剂的复合菌剂,在该复合菌剂中,所述鼠李糖乳杆菌菌剂可以为主要成分,也可以为辅助成分。该复合菌剂也可以用作缓解腹泻的药物。
本申请人发现,本申请提供的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9),具有优异的胃肠液耐受性和良好的胆盐耐受性,具备作为益生菌的基本条件;并且具有增强免疫抵抗作用、提高肠道菌群多样性、增加肠道菌群稳定性的益生特点;所述鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)可以作为益生菌添加到各类普通食品、保健品等中。
附图说明
图1示出鼠李糖乳杆菌M9的电镜下形态图;
图2示出鼠李糖乳杆菌M9的菌落形态图;
图3示出鼠李糖乳杆菌M9在模拟胃肠液中存活率变化图;
图4示出鼠李糖乳杆菌M9发酵稳定性结果。
具体实施方式
下面通过对本发明进行详细说明,本发明的特点和优点将随着这些说明而变得更为清楚、明确。
以下详述本发明。
实施例
实施例1菌株益生特性
耐酸、耐胆盐和粘附性是用来筛选益生菌的首要标准,因为益生菌被摄入人体后,在肠道内存活能力高低直接关系到益生菌能否发挥健康的促进作用,因此益生菌耐酸、耐胆盐和粘附性特性是益生菌作为功能性食补因子的重要指标。
(1)菌种供试菌液的制备
将连续传代培养的培养周期末期的菌体离心(2500×g、7min)收集,用PBS(pH=7.2,0.8%NaCl、0.02%KH2PO4、0.115%Na2HPO4,W/V,PBS下同)洗涤2次后,菌体悬浮于10mLPBS中,然后用灭菌PBS梯度稀释至合适倍数后,采用MRS琼脂培养基平板倾注法计菌落总数,同时测定相应稀释度菌悬液在600nm处OD值。
依据上述各稀释度OD值与所对应的近似菌落数(cfu/ml)的线性关系,即可通过调整各菌体在600nm处OD值,使供试菌液菌体最终浓度为1~2×109cfu/ml。
(2)人工胃液和肠液的制备
模拟胃肠液的制备方法:将PBS灭菌后,用1mol/L HCL调节pH值至2.5,加入3.0mg/ml胃蛋白酶,用0.22μm微孔滤膜过滤除菌,制成模拟人工胃液;将PBS灭菌后,用0.1mol/LNaOH调节pH值至8.0,加入0.1%胰蛋白酶和1.8%牛胆盐,用0.22μm微孔滤膜过滤除菌,制成人工模拟肠液。
(3)供试菌株对人工胃液和肠液耐受能力测定
(3.1)胃液耐受性:取1.0ml各供试菌液加入到9.0ml(2)中制得的人工胃液中,37℃消化3h,同时分别于0h、3h取样平板倾注法计数测定活菌数,每个样品做3个平行,将分离纯化得到的菌株活化培养两代,离心洗菌两次,收集菌体。
(3.2)肠液耐受性:取1.0ml已经胃液消化3h的人工含菌胃液加入到9.0ml(2)中制得的人工肠液中,继续于37℃水浴培养,分别于进入肠液的4h和8h,即,与胃液消化的3h累加,如图4所示,所述菌液分别于7h和11h再次采样,用MRS琼脂培养基倾注法计数测定活菌数,每个样品做3个平行。
菌株存活率用菌株存活率可以使用以下公式I进行计算:
其中,
N1表示菌株处理后活菌数;
N0表示菌株初始活菌数。
试验结果如图3所示,由图3可知:所述鼠李糖乳杆菌M9(Lactobacillusrhamnosus M9)在pH=2.5的人工胃液中消化3小时的存活率83.72%,之后继续在pH=8.0的人工消化液中消化11小时,其存活率高达78.33%。
由此可知,所述鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)具有良好的胃肠道消化液耐受性,能够以活的状态进入人体肠道,发挥健康功效,所述特性是作为益生菌的基础。
本申请人认为:本发明提供的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)能够抵御消化道环境的不利影响并在肠道内很好的黏附和定植,其活菌体具有增强免疫抵抗作用、提高肠道菌群多样性、增加肠道菌群稳定性的效果,用于人以及动物时,能够密集、停留在肠内的消化管道壁上,从而起到使有害菌不能够停留的作用,并且产生乳酸来降低肠内的pH值,抑制有害细菌的繁殖。并且,所述鼠李糖乳杆菌M9(Lactobacillus rhamnosusM9)活菌菌体能够生成细菌素和过氧化物,从而能够抑制病原菌的繁殖,对负责吸收营养成分的肠绒毛的活动起到帮助作用。
(4)胆盐耐受能力
在菌株传代过程中,每培养200代测定菌株的胆盐耐受能力。
试验方法:将处于培养周期末期的试验菌株按1%接种量接入含牛胆盐MRS培养基(0.3%Oxgall+0.05%半胱氨酸+0.2%巯基乙酸钠)中,以不加牛胆盐的MRS为对照。将试验菌株置于37℃水浴培养,每小时取样于620nm测定其吸光值,至吸光值增加0.3个单位为止。
试验菌株耐受胆盐的能力以延滞期的长短为评价标准,其中试验组与空白组菌株吸光值增加0.3个单位所需时间的差值即为延迟时间(LT,lag time),每个菌株均做三个平行试验。
分离纯化得到的菌株活化培养三代后,以1%的接种量分别接种到含0.3%牛胆盐(培养基中添加0.2g/100mL巯基乙酸钠,0.3g/100mL牛胆盐)和不含牛胆盐的MRS培养基中,后者作为空白对照,置于37℃厌氧培养。培养期间,每隔一小时取样于620nm测定OD值,每组测定三个平行,直到OD值增加0.3个单位及以上时停止培养。并计算菌株在含有胆盐和不含胆盐的MRS培养基中生长,OD值增加0.3个单位所需的时间,二者之差被称为延迟时间(LT)。
本发明的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)在不含胆盐的培养基中培养,OD620nm值增加0.3个单位所需的时间为2.51h,在含有胆盐的培养基中所需的时间为2.17h,延迟时间为0.34h。
在本领域中,一般认为,抗性菌株(延迟时间≤0.25h),耐受性菌株(0.25h<延迟时间≤0.67h),弱耐受性菌株(0.67h<延迟时间<1.00h),敏感菌株(延迟菌株≥1.00h)。因此,鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)的延迟时间为0.34h,为耐受性菌株。
对于活细胞来说,胆盐能破坏活细胞的细胞膜,并且,在小肠中对益生菌存活性影响最大的因素就是胆盐,所以,对胆盐的耐受性是评价益生菌的重要指标之一。由本实施例可知,分离自母乳的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)具有良好的益生特性。
(6)菌株糖发酵实验
糖发酵试验是常用的鉴别微生物的生化反应,在肠道细菌的鉴定上尤为重要。绝大多数细菌都能利用糖类作为碳源和能源,但是它们在分解糖类物质的能力上有很大的差异。根据API 50CH标准化鉴定系统的规范流程进行操作。
本申请提供的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)可利用乳糖及发酵多种单糖,如,葡萄糖、阿拉伯糖以及麦芽糖等。
具体实验结果如表1所示:
表1 Lactobacillus rhamnosus M9糖发酵实验结果
其中,“+”表示能够被利用的底物,“-”表示不能被利用的底物。由表2可知,鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)可利用D/L-阿拉伯糖、D-葡萄糖、D-半乳糖、木糖醇等,不可利用甘露醇、L-木糖、淀粉和糖原等等。
综上所述,本发明的鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)可以很好的利用乳糖并具有良好的胃肠液和胆盐耐受性,能够以活的状态进入人体肠道,发挥健康功效,是一株可以广泛应用于普通食品和保健品等的益生菌。
实施例2 Lactobacillus rhamnosus M9在发酵豆乳中的应用
豆乳中富含蛋白质、膳食纤维、矿物质、维生素,以及大量人体必需的亚油酸和亚麻酸。发酵豆乳是豆制品的重要组成部分,是在豆浆基础上接种乳酸菌发酵而成的植物性发酵食品。经乳酸菌发酵作用,发酵豆乳可提高脂质代谢,不会引起肥胖;发酵后的大豆蛋白质更容易消化吸收,且能提高机体免疫力并有效降低血清中总胆固醇含量;被分解的大豆低聚糖能促进益生菌增殖,保持肠道健康。
但是,发酵豆乳目前存在菌种适应性不强、组织状态差、发酵风味不佳以及具有豆腥味等问题。将Lactobacillus rhamnosus M9添加到发酵豆乳中可以很好的改善豆乳发酵状态和风味,有效去除豆腥味。
实验方法:水预热至65℃后加入大豆粉,混料均匀后55℃水合30min,加入8%的白砂糖和稳定剂,均质后杀菌(90℃10min),冷却41~43℃接菌发酵,其中,接菌所用菌种为嗜热链球菌ST-6(5×106CFU/g)和益生菌Lactobacillus rhamnosus M9(1×104CFU/g),发酵终点至pH=4.50及以下,约发酵6小时,后熟后,10℃留样每间隔7天测定货架期内酸度和益生菌活菌数变化,结果如表2所示:
表2发酵豆乳在10℃贮藏期间酸度和M9活菌数的变化
由表2的测定结果可知,发酵豆乳中添加益生菌Lactobacillus rhamnosus M9没有加重豆乳的后酸化现象,几乎没有影响;发酵结束后M9的活菌数达到109CFU/g以上且在贮藏期间均保持较高的活菌数,证明分离自母乳的Lactobacillus rhamnosus M9具有良好的益生特性。
实施例3 Lactobacillus rhamnosus M9在发酵薏米饮料中的应用
目前,用于谷物饮料发酵的益生菌以保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌和双歧乳杆菌等乳酸菌为主。
本实施例用鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)与嗜热链球菌S10组合物发酵薏米来制备薏米饮料,并以单嗜热链球菌S10发酵薏米制备薏米饮料作为对照组。
具体实验方法:将薏米清洗并浸泡过夜后按1:10的比例加水,90℃熬制2小时,并加入耐高温淀粉酶进行水解,水解后待冷却至65℃,加入6wt%的白砂糖,混合均匀后在30Mpa条件下均质,再在95℃条件下杀菌10分钟,冷却至37℃时接菌发酵。
实验组接入鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)(1×106CFU/g)和嗜热链球菌S10(2×106CFU/g)进行发酵;对照组接入嗜热链球菌S10(2×106CFU/g),发酵终点至pH=4.0,发酵约20~24小时,发酵结束后进行感官评定和稳定性比较,结果如表3和图4所示:
表3发酵薏米饮料感官评鉴结果
由表3和图4结果表明,添加鼠李糖乳杆菌M9(Lactobacillus rhamnosus M9)的实验组能够改善发酵薏米饮料的口感,酸中带甜,滋气味丰富柔和,而且在一定程度上增强发酵薏米的稳定性。
本申请人发现,谷物饮料最大的问题是稳定性,有研究表明,益生菌发酵可降解支链淀粉提高淀粉保水性,形成强凝胶特性;另外,微生物对糖的消耗,多种分泌酶系对蛋白质持水性和乳化性的提高、代谢物对黏度的增加,均能促进谷物饮料的稳定性。薏米是我国传统的药食兼用谷物资源,含多种必需氨基酸及矿物质,我国薏米产量较大,把薏米加工成方便食用易吸收的谷物饮料,既符合现代饮料潮流,又有利于开发薏米深加工食品。
以上结合具体实施方式和范例性实例对本申请进行了详细说明,不过这些说明并不能理解为对本申请的限制。本领域技术人员理解,在不偏离本申请精神和范围的情况下,可以对本申请技术方案及其实施方式进行多种等价替换、修饰或改进,这些均落入本申请的范围内。本申请的保护范围以所附权利要求为准。
Claims (1)
1.一株鼠李糖乳杆菌M9在制备用作肠道菌群调节剂中的用途,其中,所述鼠李糖乳杆菌M9(Lactobacillus rhamnosus)M9分离自母乳,其微生物保藏编号为CGMCC No.16002,所述鼠李糖乳杆菌M9的基因组长度3002913bp,GC含量46.77%,染色体基因组含有2965个编码基因,76个RNA基因。
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