CN113046276B - 一种母乳源鼠李糖乳杆菌及其应用 - Google Patents
一种母乳源鼠李糖乳杆菌及其应用 Download PDFInfo
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- CN113046276B CN113046276B CN202110486399.9A CN202110486399A CN113046276B CN 113046276 B CN113046276 B CN 113046276B CN 202110486399 A CN202110486399 A CN 202110486399A CN 113046276 B CN113046276 B CN 113046276B
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Abstract
本发明涉及微生物技术领域，公开了一种母乳源鼠李糖乳杆菌及其应用。本发明所述鼠李糖乳杆菌保藏编号为CGMCC NO.21815。本发明提供的鼠李糖乳杆菌AUH2101来源于中国健康母乳，不仅能产生较高浓度的乳酸和过氧化氢两种抑菌物质，对大多数抗生素敏感，还具有胃肠道条件耐受能力和良好的重金属镉耐受性及去除能力，进而使其具有良好的抑菌潜力、较低的潜在风险和良好的排镉功能，有望被开发并应用于食品行业。
Description
技术领域
本发明涉及微生物技术领域，具体的说是涉及一种母乳源鼠李糖乳杆菌及其应用。
背景技术
母乳被认为是婴儿成长的最佳营养来源，对婴儿的成长发育起着至关重要的作用。大量研究证实母乳中含有多种微生物，其中不乏乳酸菌、双歧杆菌等益生菌，目前已成为益生菌的一个重要来源。目前母乳来源的益生菌相对较少，有发酵乳杆菌CECT5716、唾液乳杆菌CECT5713等，这些菌株均表现出一定的益生功能，有必要不断从母乳中发掘益生菌。
镉是一种对人体健康极其有害的重金属，广泛并稳定地存在于环境中，为已知的最易在体内积累的毒物。然而目前对于镉中毒引起的疾病的治疗药物都存在一定的毒副作用。因此，具有镉去除能力的益生菌的使用是一种更为安全有效的选择。已有报道的具有镉中毒治疗效果的菌株均具有较好的体外镉耐受性及去除能力。因此，通过体外试验筛选具有镉去除能力的益生菌具有重要意义。
发明内容
鉴于此，本发明的目的在于提供一种母乳源鼠李糖乳杆菌，使其不仅对镉有较强的吸收能力和耐受能力，同时还具备较佳的胃肠道耐受力；
本发明的另外一个目的在于提供上述鼠李糖乳杆菌在制备食品中的应用。
为了实现上述目的，本发明提供如下技术方案：
本发明从中国健康母乳中分离获得一株细菌菌株，命名为AUH2101，其具有良好的胃肠道条件耐受性，对阿奇霉素、青霉素、罗红霉素、四环素在内的多种抗生素敏感，对重金属镉离子有良好的耐受性和吸附能力；
利用细菌基因组DNA提取试剂盒(购买自天根生化科技(北京)有限公司，型号为DP302)提取该菌株基因组DNA，将提取的菌株基因组DNA作为16S rDNA-PCR的模板进行扩增，将该菌株的16S rDNA基因序列(SEQ ID NO.1所示)与GenBank数据库中的基因序列进行同源性比较，并从分子生物学水平上确定该菌的种属；结果显示，本发明的鼠李糖乳杆菌AUH2101的16S rRNA与目前已知的其它所有鼠李糖乳杆菌的相应序列均不完全相同。其中，有些鼠李糖乳杆菌与本发明的鼠李糖乳杆菌AUH2101同源性可达99.52％，进而可以说明本发明的鼠李糖乳杆菌AUH2101是一株以往未被分离得到的新鼠李糖乳杆菌。本发明提供的鼠李糖乳杆菌(Lactobacillus rhamnosus)AUH2101已于2021年2月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.21815，保藏地址为中国北京市朝阳区北辰西路1号院3号。
本发明提供的鼠李糖乳杆菌AUH2101，不仅能产生较高浓度的乳酸和过氧化氢两种抑菌物质，对大多数抗生素敏感，还具有胃肠道条件耐受能力和良好的重金属镉耐受性及去除能力，进而使其具有良好的抑菌潜力、较低的潜在风险和良好的排镉功能。基于此，本发明提出了保藏编号为CGMCC NO.21815的鼠李糖乳杆菌在制备食品或药品中的应用。
作为优选，所述食品或药品为具有本发明记载的AUH2101任意功效或组合功效的食品或药品；其中，食品优选为益生菌食品，包括但不限于乳制品、豆制品、果蔬制品或活菌制剂。
本发明还提供了一种益生菌食品，其在食品原料中添加有保藏编号为CGMCCNO.21815的鼠李糖乳杆菌。作为优选，所述食品原料包括乳粉、配方奶粉、液态乳、米粉、果蔬粉和果蔬浆。此外，可以根据需要对本发明鼠李糖乳杆菌进行扩大化培养再添加，同时也可以根据需要在添加完本发明鼠李糖乳杆菌后进行发酵，进一步制备成发酵产品。
在本发明具体实施方式中，本发明提供了一种发酵乳，其利用液态乳品添加AUH2101进行发酵制备获得；本发明还提供了一种果蔬发酵饮料，其利用打浆后的果蔬添加AUH2101进行发酵制备获得；另外，本发明提供的鼠李糖乳杆菌AUH2101还可以用于制备配方奶粉、米粉等食品中。例如，将鼠李糖乳杆菌AUH2101进行大规模培养，将培养物进行冷冻干燥，按照活菌数>106CFU/g添加至配方奶粉、米粉等食品中。活菌数在此仅是举例说明，可根据实际需求调整，食品原料也并不局限于上述提及的几种食品原料。
此外，本发明还提供了一种活菌制剂，其为保藏编号为CGMCC NO.21815的鼠李糖乳杆菌经冻干或扩繁后冻干的活菌制剂。
由以上技术方案可知，本发明提供的鼠李糖乳杆菌AUH2101来源于中国健康母乳，不仅能产生较高浓度的乳酸和过氧化氢两种抑菌物质，对大多数抗生素敏感，还具有胃肠道条件耐受能力和良好的重金属镉耐受性及去除能力，进而使其具有良好的抑菌潜力、较低的潜在风险和良好的排镉功能，有望被开发并应用于食品行业。
生物保藏材料信息说明
AUH2101，分类命名：鼠李糖乳杆菌Lactobacillus rhamnosus；已于2021年2月5日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.21815，保藏地址为中国北京市朝阳区北辰西路1号院3号。
附图说明
图1所示为本发明鼠李糖乳杆菌AUH2101的菌落形态图；
图2所示为本发明鼠李糖乳杆菌AUH2101的***进化树图。
具体实施方式
本发明实施例公开了一种母乳源鼠李糖乳杆菌及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明内。本发明所述鼠李糖乳杆菌及其应用已通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述鼠李糖乳杆菌及其应用进行改动或适当变更与组合，来实现和应用本发明技术。
公开于本发明背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
以下就本发明所提供的一种母乳源鼠李糖乳杆菌及其应用做进一步说明。
实施例1：菌株的筛选
1、样品采集及菌株分离
采集样品前，对健康志愿者的***进行严格的卫生处理，然后收集10mL左右母乳置于无菌离心管中。取2mL母乳样品倾注于提前准备好的MRS琼脂培养基上，晃动使之平铺于培养基表面，用封口膜将培养皿封口，于37℃培养24～48h至长出单个菌落，挑选具有乳酸菌典型形态如白色或乳白色的特征单菌落。
2、菌株筛选
将分离得到的菌种划线接种于含溴甲酚紫的MRS琼脂培养基中，37℃培养48h。长出单菌落后，选择使培养基变黄的单菌落，于显微镜下观察菌体形态，将选取的具有典型乳酸菌形态的菌株进行后续实验。图1为本实施例培养所得的菌株的菌落形态图。
从来源于母乳的样品中共分离出52个菌株，经纯化后在MRS琼脂培养基菌落形态呈白色且表面光滑；经革兰氏染色后，筛选后得到16株革兰氏阳性菌，其编号和名称如表1所示。
在上述菌株初筛和复筛过程中所使用的培养基组成如下：
MRS琼脂培养基的组成如下：
蛋白胨10.0g/L，牛肉浸粉8.0g/L，酵母浸粉4.0g/L，葡萄糖20.0g/L，磷酸氢二钾2.0g/L，柠檬酸氢二铵2.0g/L，乙酸钠5.0g/L，硫酸镁0.2g/L，硫酸锰0.04g/L，琼脂14.0g/L，吐温80 1.0g/L，水。
3、菌株的鉴定
利用细菌基因组DNA提取试剂盒(购买自天根生化科技(北京)有限公司，型号为DP302)提取上述16种菌株基因组DNA，将提取的菌株基因组DNA作为16S rDNA-PCR的模板进行扩增。其中，引物为通用引物27F(5′-AGAGTTTGATCCTGGCTCAG-3′)和1492R(5′-TACGGCTACCTTGTTACGACTT-3′)，扩增的产物进行电泳(120V，30min)以获得纯化的菌株基因片段。将上述纯化的菌株基因片段进行测序，将菌株的16S rDNA基因序列与GenBank数据库中的基因序列进行同源性比较，并从分子生物学水平上确定该菌的种属，具体测定结果如表1所示。
结果发现，本发明的鼠李糖乳杆菌AUH2101的16S rDNA(SEQ ID NO.1所示)与目前已知的其它所有鼠李糖乳杆菌的相应序列均不完全相同。其中，有些鼠李糖乳杆菌与本发明的鼠李糖乳杆菌AUH2101同源性可达99.52％，进而可以说明本发明的鼠李糖乳杆菌AUH2101是一株以往未被分离得到的新鼠李糖乳杆菌。
通过NCBI Blast比对，并结合菌株镜检形态和来源，初步确定16株乳杆菌，具体请参见表1。其中，16株菌株包括13株鼠李糖乳杆菌(Lactobacillus rhamnosus)，3株副干酪乳杆菌(Lactobacillus paracasei)。
根据上述比对结果，下载多株鼠李糖乳杆菌的序列文件，借助Mega 5软件进行***进化树构建，如图2所示。
表1
实施例2：鼠李糖乳杆菌AUH2101的模拟胃肠道条件耐受能力
1)模拟胃肠道条件耐受力的测试
菌株发酵酸奶的配制：按14.7％(wt/v)的比例冲泡奶粉，充分溶解后110℃灭菌12min，冷却至室温(约20℃)，将灭菌牛奶分装为40mL每管。将筛选得到16种菌株分别进行厌氧培养活化后，按3％的接种量接种到灭菌牛奶中，37℃发酵约4h，之后4℃过夜，乳酸菌含量为109cfu/ml。
模拟唾液的配制：6.2g/L NaCl,2.2g/L KCl,0.22g/L CaCl2,1.2g/L NaHCO3；
模拟胃液的配制：0.21g NaCl，80.2mg胃蛋白酶，用浓盐酸调节pH＝5.0，用双蒸水定容至100ml，胃蛋白酶的活力为370U/ml；
模拟十二指肠液的配制：5g/L NaCl，0.6g/L KCl，0.25g/L CaCl2，0.3％猪胆汁，7％胰酶，混匀，胰蛋白酶的活力为10010U/mg Pro；
试验处理方法：准备含有109cfu/mL乳酸菌的发酵牛奶(30mL)，用5mL模拟唾液混匀，然后加入5mL模拟胃液，37℃震荡培养。该环节pH需经历一个不断降低的过程，通过添加HCl溶液来调节。人胃模拟区的pH曲线控制为与人食用酸奶后的变化曲线一致：起始pH5.0，40min时pH 3.0，60min时pH 2.1，80-180min期间维持pH 1.8。过程中，每次pH降低前分别取一份样品，备用检测。此后，用1M NaHCO3将pH调整为6.5±0.2时，加入10mL模拟十二指肠液并混匀，120min后，取样用于检测。所有采集的样品，进行10倍梯度系列稀释，分别取1mL倾注于MRS平板表面，水平晃动培养皿使样品均匀分布，并于37℃需氧培养48h，利用平板计数法测定活菌数，以计算存活率(％)，以发酵乳杆菌CECT5716株(CECT5716)为对照，结果如表2所示。
表2
由表2可知，副干酪乳杆菌LHL1、鼠李糖乳杆菌LHL2、鼠李糖乳杆菌LHL3、鼠李糖乳杆菌LHL6、鼠李糖乳杆菌LHL7和鼠李糖乳杆菌AUH2101经模拟胃肠道条件处理后活菌数大于1×106CFU/mL，其中菌株LHL6存活率最高，达到了7.13％；其余菌株存活率均小于1％，进而能够表明来源母乳的乳杆菌菌株的耐酸耐胆盐能力较弱。鼠李糖乳杆菌LHL2、鼠李糖乳杆菌LHL7、鼠李糖乳杆菌AUH2101与发酵乳杆菌CECT5716存活率相当。
实施例3：菌株对抗生素耐药性的测试
参照EFSA微生物抗药物敏感试验执行标准，本实施例对筛选到的6株菌株和CECT5716(发酵乳杆菌CECT5716株)进行耐药性测试，结果如表3所示。
表3
注：S表示敏感；I表示中介；R表示耐药。
由表3可知，各菌株在耐药性方面存在差异。其中菌株LHL1对青霉素G和罗红霉素敏感，菌株LHL7对青霉素G和罗红霉素敏感，菌株LHL2、LHL3、LHL6和AUH2101同时对青霉素G、四环素和罗红霉素敏感，具有良好的安全性。此外，菌株AUH2101还对阿奇霉素敏感。与婴幼儿准用菌株CECT5716相比，本发明提供的鼠李糖乳杆菌AUH2101对抗生素敏感的种类更多。
实施例4：菌株对镉的耐受能力实验
称取27.44g Cd(NO3)2·4H2O溶于100mL无菌蒸馏水中，配制成镉浓度为100g/L的镉水溶液母液，经0.45μm滤膜过滤后，储存于4℃冰箱中备用。配制不同镉浓度的MRS固体培养基，使镉离子浓度依次为5、10、50、100、200、500和1000mg/L，将各培养基平分为4块区域，每个区域接种一个菌株，留空一块为对照，于37℃需氧培养48～72h观察并记录各菌种生长情况。同时准备含镉浓度分别为5、10、50、100、200、500、1000和2000mg/L的MRS肉汤培养基，按1％的接种量接种过夜培养菌液，接种后37℃培养20h，测定各培养液在600nm处的吸光值(OD600nm)。获得各菌株在含镉MRS固体培养基和含镉MRS肉汤培养基中的最小抑菌浓度(MIC，minimum inhibition concentration)，评价镉耐受能力，结果如表4所示。
表4
由表4可以看出，其中菌株LHL6、LHL7、AUH2101和对照菌株CECT5716在MRS固体培养基上可耐受1000mg/L镉浓度，在MRS肉汤培养基中可耐受500mg/L镉浓度，具有良好的镉耐受能力，其他菌株镉耐受能力相对较弱。
实施例5：菌株的镉吸附能力
将表4中4株对镉具有高耐受性(MIC≥500mg/L)的实验菌株和1株在镉耐受性实验中MIC最低(10mg/L)的乳酸菌LHL1接种于MRS培养基上，置于37℃培养48h，挑取单菌落接种于MRS肉汤中，37℃培养20h后，取10mL菌液12000g离心20min收集菌体，用灭菌超纯水洗涤2次，称取菌体重量。取100g/L的镉溶液母液用无菌水配制成浓度为50mg/L的Cd2+水溶液，加入离心得到的菌体中，使菌体终浓度为1g/L，置于37℃摇床培养60min。于8000g离心20min使吸附后的菌体沉淀，收集上清液，采用电感耦合等离子光谱发生仪检测乳酸菌吸附后溶液中残余的Cd2+浓度，计算镉去除率，以表征菌株的镉吸附能力，结果见表5。
表5
由表5可以看出，菌株AUH2101的镉去除率最高，可达7.81％，说明该菌株具有较好的镉吸附能力。
综合以上各实施例结果，本发明提供的鼠李糖乳杆菌，不仅对大多数抗生素敏感，具有较好的耐胃酸和耐胆盐能力，还具有良好的镉耐受能力和吸附能力，具有较为广泛的应用前景。
实施例6：发酵乳制品
将14.7g脱脂乳粉溶于水中，充分溶解后115℃灭菌15min，待其冷却至60～70℃加入，接种鼠李糖乳杆菌AUH2101菌粉，在37℃培养至凝乳，转移至4℃进行后熟，得到发酵乳。
实施例7：活菌制剂
将鼠李糖乳杆菌AUH2101进行大规模培养，将培养物进行冷冻干燥。
实施例8：果蔬发酵饮料
将果蔬与水1:1～5混合，打浆，95～105℃灭菌10min，冷却至32～37℃，接种2％的鼠李糖乳杆菌AUH2101，发酵12～48h；离心，得发酵果蔬汁，添加稳定剂，均质，得到发酵果蔬饮料。
以上所述只是用于理解本发明的方法及其核心思想，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利的保护范围。
序列表
<110> 澳优乳业（中国）有限公司
中南大学
<120> 一种母乳源鼠李糖乳杆菌及其应用
<130> MP21006172
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1467
<212> DNA
<213> 人工序列(Artificial Sequence)
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tgggggataa catttggaaa cagatgctaa taccgcataa atccaagaac cgcatggttc 180
ttggctgaaa gatggcgtaa gctatcgctt ttggatggac ccgcggcgta ttagctagtt 240
ggtgaggtaa cggctcacca aggcaatgat acgtagccga actgagaggt tgatcggcca 300
cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga atcttccaca 360
atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag aaggctttcg ggtcgtaaaa 420
ctctgttgtt ggagaagaat ggtcggcaga gtaactggtg tcggcgtgac ggtatccaac 480
cagaaagcca cggctaacta cgtgccagca gccgcggaat acgtaggtgg caagcgttat 540
ccggatttat tgggcgaaag cgagcgcagg cggtttttta agtctgatgg ggaagccctc 600
ggcttaaccg aggaagtgca tcggaaactg ggaaacttga gtgcagaaga ggacagtgga 660
actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg cgaaggcggc 720
tgtctggtct gtaactgacg ctgaggctcg aaagcatggg tagcgaacag gattagatac 780
cctggtagtc catgccgtaa acgatgaatg ctaggtgttg gagggtttcc gcccttcagt 840
gccgcagcta acgcattaag cattccgcct ggggagtacg accgcaaggt tgaaactcaa 900
aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga 960
agaaccttac caggtcttga catcttttga tcacctgaga gatcaggttt ccccttcggg 1020
ggcaaaatga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag 1080
tcccgcaacg agcgcaaccc ttatgactag ttgccagcat ttagttgggc actctagtaa 1140
gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg 1200
acctgggcta cacacgtgct acaatggatg gtacaacgag ttgcgagacc gcgaggtcaa 1260
gctaatctct taaagccatt ctcagttcgg actgtaggct gcaactcgcc tacacgaagt 1320
cggaatcgct agtaatcgcg gatcagcacg ccgcggtgaa tacgttcccg ggccttgtac 1380
acaccgcccg tcacaccatg agagtttgta acacccgaag ccggtggcgt aaccctttta 1440
gggagcgagc cgtctaaggg acaaagt 1467
Claims (9)
1.一种母乳源鼠李糖乳杆菌(Lactobacillus rhamnosus)，其特征在于，保藏编号为CGMCCNO.21815。
2.根据权利要求1所述鼠李糖乳杆菌，其特征在于，其16S rDNA序列如SEQ ID NO.1所示。
3.保藏编号为CGMCC NO.21815的鼠李糖乳杆菌在制备食品中的应用。
4.根据权利要求3所述应用，其特征在于，所述食品为益生菌食品。
5.根据权利要求4所述应用，其特征在于，所述益生菌制品为乳制品、豆制品、果蔬制品或活菌制剂。
6.一种益生菌食品，其特征在于，食品原料中添加有保藏编号为CGMCC NO.21815的鼠李糖乳杆菌。
7.根据权利要求6所述益生菌食品，其特征在于，所述食品原料包括乳粉、配方奶粉、液态乳、米粉、果蔬粉和果蔬浆。
8.一种发酵制品，其特征在于，使用保藏编号为CGMCC NO.21815作为发酵菌。
9.一种活菌制剂，其特征在于，其为保藏编号为CGMCC NO.21815的鼠李糖乳杆菌经冻干或扩繁后冻干的活菌制剂。
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