CN110452829B - 长双歧杆菌菌株及其应用 - Google Patents
长双歧杆菌菌株及其应用 Download PDFInfo
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- CN110452829B CN110452829B CN201910259092.8A CN201910259092A CN110452829B CN 110452829 B CN110452829 B CN 110452829B CN 201910259092 A CN201910259092 A CN 201910259092A CN 110452829 B CN110452829 B CN 110452829B
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- bifidobacterium longum
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Abstract
本发明涉及一株长双歧杆菌菌株LTBL16及其应用。该长双歧杆菌菌株的保藏编号是CCTCC M 2019028，包含SEQ ID NO：3和SEQ ID NO：4所示的特征序列，可以应用于发酵乳制品，或添加至食品、保健品、饲料、药品、食品补充剂中，用于调节人和动物的肠道菌群、提高机体免疫力，其优点是：对胃肠道环境适应能力突出，具有非常强的耐酸、耐胃蛋白酶、耐胆汁、耐胰蛋白酶和耐氧能力，具有非常高的益生潜质和应用价值。
Description
技术领域
本发明属于生物技术领域；具体涉及一株长双歧杆菌菌株及其应用。
背景技术
双歧杆菌是健康人体肠道内的优势菌群之一，被认为是益生菌。当足量摄入时，除提供 基本的营养成分外，还提供健康有益的活体生物。摄取双歧杆菌能够改善胃肠通过并可预防 或辅助治疗由缺乏微生物区系或有害微生物区系引起的疾病，例如胃肠道感染、便秘、过敏 性肠综合症、炎性肠疾病、克隆氏病和溃疡性结肠炎、食物过敏、抗生素诱发的腹泻、心脏 血管疾病、和某些癌症如结直肠癌，等等。
不同双歧杆菌菌株之间观察到了耐酸和胆盐的显著差异。指示存活能力是选择潜在的益 生菌菌株的重要判据。双歧杆菌在肠道内的数量随年龄变化而变化，从而影响着人体的生理 病理状况，一般随年龄增长逐渐减少，有文献报道在成人和婴儿中完成的体内研究指示一些 双歧杆菌菌株能够在通过胃肠道后依然存活。所以在百岁老人肠道中能够生存下来的双歧杆 菌尤为珍贵。广西巴马是世界公认的长寿之乡，分离该地区长寿老人肠道内的双歧杆菌，对 其特性进行研究，并根据其特性进行开发应用，或许能够对改善一般人群的健康状况提供新 的举措。
发明内容
本发明的第一个目的是提供一株益生效果更好的长双歧杆菌菌株。
本发明的第二个目的是提供上述长双歧杆菌菌株的应用。
该长双歧杆菌菌株的保藏编号是CCTCC NO：M 2019028，包含SEQ ID NO：3和SEQID NO：4所示的特征序列，可以活体形式存在，也可以非活体形式存在，优选以活体形式存在，进一步优选以冻干粉形式存在。
所述长双歧杆菌菌株LTBL16可以应用于发酵乳制品，或添加至食品、保健品、饲料、药 品、食品补充剂中，调节人和动物的肠道菌群、提高机体免疫力。
本发明长双歧杆菌菌株LTBL16的优点是：对胃肠道环境适应能力突出，具有非常强的 耐酸、耐胃蛋白酶、耐胆汁、耐胰蛋白酶和耐氧能力，具有非常高的益生潜质和应用价值。
附图说明
图1 LTBL16革兰氏染色的细胞形态图。
图2 LTBL16基因组DNA扩增产物琼脂糖凝胶电泳图。
图3 LTBL16全基因组图谱。
生物材料保藏信息
LTBL16，分类命名为长双歧杆菌LTBL16(Bifidobacterium longum LTBL16)，已于2019 年1月8日在位于中国.武汉.武汉大学的中国典型培养物保藏中心保藏，保藏编号为CCTCC NO：M 2019028。
具体实施方式
本发明具体实施方式中的试验结果以平均值±标准差(Means±SD)表示，或平行试验三 次测值，取平均值，n.d表示未检测到，“+”表示检测结果为阳性，“-”表示检测结果为阴性。 统计分析使用单因子变异数分析【One-way analysis of variance(One-wayANOVA)】，并以 Tukey多重比较检验(Tukey′s Multiple Comparison Test)比较组间差异，当组间差异达到统 计学意义，以上标a表示p＜0.05、ab表示p＜0.01。
MTPY固态培养基、TPY固体培养基、TPY液态培养基为本领域常用培养基。NCBI表示美国国家生物技术信息中心。BLAST(Basic Local Alignment Search Tool)生物信息分析软 件名称。DNA测序单位是生工生物工程(上海)股份有限公司。
本发明菌种来源：本发明粪便样品来源于中国广西省巴马县16名年龄在100岁～128岁 之间的健康百岁以上老人，其中男性5名，女性11名，采样前2周之内，没有服用胃酸抑制 剂、泻药、反腹泻药、抗生素、益生菌、益生元、合生元等影响实验结论的食品或药品。
实施例1菌种筛选与初步鉴定
1.菌种筛选与鉴定
解冻的粪便样品用无菌磷酸盐缓冲溶液(PBS)混匀，涂布于MTPY固态培养基，37℃厌氧培养，挑取表面光滑、边缘完整、凸圆、闪亮、柔软且呈蓝色菌落画线转接至TPY固体 培养基，将镜检呈棍棒状或匙状、过氧化氢酶试验呈阴性且需氧条件下不能在TPY固体培养基上生长的革兰氏阳性菌暂定为疑似双歧杆菌(图1)。将挑取的疑似双歧杆菌在TPY固体培养基上进一步画线厌氧培养，重复多次进行纯化，直至菌落形态一致且显微镜检菌体形态 一致。挑取单个菌落接种至TPY液体培养基，厌氧培养，菌液保存，编号为LTBL16。
LTBL16的过氧化氢酶实验、明胶液化实验、硝酸盐还原试验、石蕊牛奶实验、淀粉实 验、吲哚试验、葡萄糖产酸产气实验等生理生化特性试验结果见表1，糖发酵试验结果见表 2。表1说明LTBL16能够产酸但不能产气，能分泌胞外淀粉酶、还原硝酸盐。表2表明LTBL16能够利用葡萄糖、麦芽糖、果糖、***糖、葡萄糖酸盐、蔗糖、半乳糖和乳糖， 不能够利用甘露醇、木糖、山梨醇、鼠李糖、纤维二糖、七叶灵、海藻糖。根据《伯杰士细 菌学手册》及《乳酸细菌分类鉴定及实验方法》，可初步认为菌株LTBL16为双歧杆菌(Bifidobacteriumlongum)。
表1 LTBL16生理生化特性
表2 LTBL16糖发酵试验结果
2. 16S rDNA序列测定
提取LTBL16基因组DNA，将LTBL16基因组DNA作为模板进行聚合酶链反应(Polymerase Chain Reaction，PCR)扩增。扩增反应的正向引物是5’-GCAAGGCTAAAACTCAAAGA-3’，反向引物是5’-CGGGTGCTGCCCACTTTCAT-3’。扩增反应完毕后，扩增 产物经琼脂糖凝胶电泳，电泳结果见图2。图2中约500bp处有明亮条带，表明PCR扩增成 功。LTBL16的16S rDNA序列如SEQ ID NO：1所示。将测得的LTBL16 16S rDNA序列在 NCBI上使用BLAST进行比对，再采用MEGA6软件以邻位连接法显示双歧杆菌属与相关种 的16S rRNA序列***发育树，进行1000次相似度重复计算，结果显示LTBL16的16S rDNA序列与长双歧杆菌KCTC3128序列完全一致，相似比例达100％，与双岐杆菌短亚种 DSM 20213同源比例达96％。综合菌株LTBL16的形态特征、生理生化实验、糖发酵实验， 可以确认LTBL16为长双歧杆菌(Bifidobacteriumlongum subsp.longum)。
实施例2 LTBL16全基因组序列测定
1.基本特征
提取菌株LTBL16基因组DNA，进行全基因组序列测定。结果显示LTBL16有1条染色体，不含质粒。染色体基因组为环状，鸟嘌呤(G)和胸嘧啶(C)之和平均含量为 60.26％，长度为2,430,682bp，含2,071个独立编码区(CDSs)，平均长度为1013bp，编码 区总长度占全基因组的比例为86.33％。
2.基因功能
在所有的2,071个独立编码区中，有1,605个基因能够归类至直系同源群(Clusterof Orthologous Group，COG)功能分类数据库中，其中参与复制、重组和修复的基因有88个， 参与氨基酸转运和代谢的基因有177个，参与能量代谢的基因有46个，参与无机盐转运和代 谢的基因有69个，有68个基因功能未知，被定义为假定蛋白，有466个编码基因与COG数 据库中的基因无任何匹配。
实施例3 LTBL16的特征序列
LTBL16全基因序列在NCBI上使用BLAST比对，发现2段序列体现了LTBL16基因组的专有特征。因此，将这段2段序列作为长双歧杆菌LTBL16的标记基因，序列分别为SEQ IDNO：4和SEQ ID NO：5。
实施例4菌株LTBL16的体外益生能力
益生菌在肠道内定植，首先要能抵御低pH值的胃酸环境，所以耐酸及耐胃蛋白酶性能 评价是益生菌筛选的重要指标。测定这些指标，可以判断所筛选菌株通过胃后的存活能力。
1.LTBL16耐酸及耐胃蛋白酶性能评价
LTBL16以1％的接种量接种于含1200U/mL胃蛋白酶、pH 3.0的TPY液态培养基，置于厌氧条件下培养2h后稀释、涂布，进行活菌计数，计算存活率。
N0：0小时的活菌数；N：耐酸和胃蛋白酶实验2小时的活菌数。
LTBL16的耐酸及耐胃蛋白酶性能测试结果以及和参考菌株——长双歧杆菌CICC6188菌 株(购自中国工业微生物菌种保藏中心)的对比结果见表3。
表3 LTBL16在酸和胃蛋白酶中的活菌数和存活率
通常人进食后，胃液pH为3左右，胃蛋白酶含量约为1200U/mL，食物在胃中停留时间 与摄入的食物组成有关，一般流体在胃中停留时间大概为1～2小时。由表3可知，LTBL16在耐酸和胃蛋白酶实验后，存活率比参考菌株CICC6188高，且存在显著差异(p＜0.01)，证明LTBL16通过人体胃液作用后仍然保持较高的存活率，具有作为益生菌的良好潜质。
2.LTBL16耐胆汁及耐胰蛋白酶性能评价
LTBL16以1％接种量接种于含0.3％胆盐、75U/mL胰蛋白酶、pH 8.0的TPY液态培养基，置于厌氧条件下培养24h后稀释涂布进行活菌计数，计算存活率，结果见表4。
N0：0小时活菌数；N：耐胆汁和胰蛋白酶24小时后活菌数。
表4 LTBL16在胆汁和胰蛋白酶中的活菌数和存活率
LTBL16与参考菌株CICC6188对胆汁和胰蛋白酶都有一定的耐受性。在胆盐含量为0.3％、胰蛋白酶含量75U/mL的条件下培养24小时，LTBL16能保持106cfu/mL以上的活菌数，存活率在79％以上，显著高于参考菌株CICC6188。通常人体小肠内的胆盐含量为 0.03～0.3％，胰蛋白酶含量约为14～72U/mL，而食物通过小肠的时间很短，因此以上测试的 结果具有良好的代表性，可以判定LTBL16具有较强的耐胆汁和胰蛋白酶能力，能顺利通过 小肠。
3.LTBL16耐氧性评价
双歧杆菌大多数来源于人或动物胃肠道，一直在厌氧条件下生长和繁殖，分离后暴露在 氧气中会导致其死亡，这是双歧杆菌等厌氧性益生菌活力丧失的主要原因之一，限制了它的 商业化的广泛应用。将LTBL16和参考菌株CICC6188同时置于转速为200rpm的摇床中分别 处理12h和24h，测它们的存活率，评价耐氧性能。实验结果见表5。
表5 LTBL16耐氧性测定
LTBL16经耐氧处理12h后的存活率显著高于参考菌株CICC6188；耐氧处理24h后，存 活率仍然相当高，而参考菌株CICC618却没有检测到存活率，说明菌株LTBL16具有超乎寻 常的耐氧能力。
实施例5 LTBL16在食品领域的应用
LTBL16可以直接用于发酵乳制品，也可以冻干粉形式添加至食品、饲料、药品中，调节 人和动物的肠道菌群、提高机体免疫力。LTBL16也可以以非活体形式食用。
冻干粉加工方法如下：
取LTBL16发酵菌液于4℃离心，去上清，获得菌泥，用原发酵液1/10体积的生理盐水重 悬，将灭菌保护剂(脱脂乳12％，海藻糖7.0％，L-谷氨酸钠1.0％，吐温801.0％)与菌泥按 照10∶1的比例混合，置-80℃冰箱中预冻6h后，取出冷冻干燥，即得LTBL16冻干菌粉。
以上所述仅是本发明的优选实施方式，本技术领域普通技术人员在不脱离本发明原理的 前提下，还可以做出若干改进，这些改进也应视为本发明的保护范围。
Claims (1)
1.一株长双歧杆菌(Bifidobacterium Longum)菌株，其特征是：其保藏编号是CCTCCNO：M 2019028。
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