CN114672434A - 一株嗜热链球菌gyx-8及其应用 - Google Patents
一株嗜热链球菌gyx-8及其应用 Download PDFInfo
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- streptococcus thermophilus
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- fermented milk
- helicobacter
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Abstract
本发明涉及微生物技术领域，本发明公开了一株嗜热链球菌GYX‑8及其应用，其调节肠道菌群，主要具备增加有益菌颤螺菌属（Oscillospira）和减少有害菌螺杆菌属（Helicobacter）；其调节短链脂肪酸的产生，主要有增加异丁酸、异戊酸和戊酸的产生，维持乙酸、丙酸和己酸的水平。嗜热链球菌CGMCC No.24357具备基本的益生特性，按照5‑8%菌种接种量用其制备的发酵乳具有良好的调节肠道菌群和调节短链脂肪酸产生的作用。具体地，接种按照5‑8%接种量嗜热链球菌CGMCC No.24357发酵乳具有调节肠道菌群、增加肠道中短链脂肪酸含量等功能。
Description
技术领域
本发明涉及一株嗜热链球菌GYX-8及其应用，属于微生物技术领域。
背景技术
大部分肠道菌群由严格的厌氧菌组成，它们的数量是兼性厌氧菌和需氧菌的两到三个数量级。尽管迄今为止已发现的细菌门有 50 多种，但人类肠道菌群的主要细菌门类是厚壁菌门（Firmicutes）和拟杆菌门（Bacteroidetes），疣状微生物菌（Verrucomicrobia）、放线菌（Actinobacteria）、变形杆菌（Proteobacteria）等。益生菌是活的微生物，当给予适量时，对宿主的健康有益，具有调节肠道菌群组成、促进正常肠道菌群的恢复的功能。在生物医学研究中，对益生菌种类的研究大多着眼于乳酸菌群。在肠道微生物群的研究中，乳酸菌被报道为乳酸菌群中最突出的益生菌。利用包括靶向、培养依赖方法和宏基因组测序在内的工具和技术，研究了益生菌种类在肠道菌群组成、多样性和功能方面的变化。然而，一些研究已经证明了益生菌种类与肠道菌群组成改变的关系，并且影响健康，研究表明，颤螺菌属（Oscillospira）作为有益菌，依赖于其他物种分泌的发酵产物，或者依赖于从宿主释放的糖粘蛋白，与体重指数 (BMI)呈负相关，与健康呈正相关；另外研究表明，基于颤螺菌属（Oscillospira）代谢物与炎症性疾病和BMI呈负相关，推测出颤螺菌属（Oscillospira）可能产生短链脂肪酸丁酸。但是并非所有益生菌都可以直接或间接增加颤螺菌的丰度，现有技术中有记载，长期补充鼠李糖乳杆菌降低了大鼠的体重，而它们的肠道颤螺旋体丰度显着下调。同样，丁酸梭菌能够产生丁酸，丁酸已被证明可以限制肝脏中的脂质沉积，恢复肠道屏障功能，并改善肝脏炎症，并具有益生菌潜力。丁酸梭菌缓解了结肠炎小鼠肠道炎症并伴随Oscillospira丰度降低。
而螺杆菌属（Helicobacter）是人类最常见的细菌感染之一，研究表明，受螺杆菌属（Helicobacter）的感染，通常是慢性的，如果不进行特殊治疗就不会痊愈。胃肠道黏膜可起到很好的保护、免受细菌感染，然而螺杆菌属（Helicobacter）高度适应这种生态位，具有一系列独特的特征，允许进入粘液、在粘液中定向附着于上皮细胞、逃避免疫反应，能够引起炎症；此外研究中发现，螺杆菌属（Helicobacter）与许多短链脂肪酸的产生呈现负相关。
短链脂肪酸（SCFAs）是一种代谢产物，主要包括丁酸、丙酸和乙酸等，它们可以作为组蛋白去乙酰化酶抑制剂影响基因表达，参与肠道肿瘤的发生与发展。另外，在炎症疾病中，SCFAs 也可以影响白细胞的迁移和T 细胞的分化。以往的研究大多集中在益生菌及其代谢产物对肠道健康和肠道肿瘤的作用，但肠道菌群及其代谢产物可以全身性地影响免疫系统进而参与肠道外肿瘤的进程。据研究，通过益生菌的摄入可以调节肠道菌群的组成，可间接调节SCFAs产生。
因此，现有技术中，对于益生菌的研究仍存在很多未知。在未进行实验之前，研究人员并不清楚其是否对益生菌是否有直接或间接的作用，而对于开发出既能够增加有益菌颤螺菌并且还能减少有害菌螺杆菌属仍是一大挑战。
发明内容
发明目的：本发明所要解决的问题是提供一株嗜热链球菌（Streptococcus thermophilus）GYX-8。
本发明还要解决的技术问题是提供了含有所述嗜热链球菌（Streptococcus thermophilus）GYX-8的乳酸菌发酵乳。
本发明还要解决的技术问题是提供了嗜热链球菌Streptococcus thermophilus）GYX-8或乳酸菌发酵乳在调节肠道菌群和/或调节短链脂肪酸产生的应用。
技术方案：为了解决上述技术问题，本发明提供了一株嗜热链球菌（Streptococcus thermophilus）GYX-8，所述嗜热链球菌（Streptococcus thermophilus）GYX-8于2022年1月21日保藏于中国微生物菌株保藏管理委员会普通微生物中心，保藏编号为CGMCC No.24357，保藏日期为2022年1月21日。
本发明内容还包括一种乳酸菌发酵乳，所述乳酸菌发酵乳包含所述的嗜热链球菌（Streptococcus thermophilus）GYX-8。
其中，所述嗜热链球菌（Streptococcus thermophilus）GYX-8添加量为5-8%。
本发明内容还包括所述的嗜热链球菌（Streptococcus thermophilus）GYX-8或所述的发酵乳在调节肠道菌群和/或调节短链脂肪酸产生的应用。
优选地，上述发酵乳在门水平调节肠道菌群的应用，主要调节厚壁菌门、拟杆菌门
放线菌门和变形菌门，其中使用嗜热链球菌GYX-8发酵乳可以增加拟杆菌门的相对丰度。
优选地，上述发酵乳进一步在属水平上对肠道菌群变化进行分析，在属水平增加有益菌的应用，嗜热链球菌GYX-8可以增加颤螺菌属（Oscillospira）的相对丰度。
优选地，上述发酵乳进一步在属水平上对肠道菌群变化进行分析，在属水平增加有益菌的应用，嗜热链球菌GYX-8可以减少螺杆菌属（Helicobacter）、理研菌属（Rikenella）、萨特氏菌（Sutterella）和臭气杆菌（Odoribacter）的相对丰度，其中主要减少螺杆菌属（Helicobacter）的相对丰度。
优选地，所述调节菌群中的应用主要为增加颤螺菌（Helicobacter）和减少螺杆菌（Helicobacter）。
其中，所述调节短链脂肪酸产生的应用包括增加异丁酸、异戊酸和戊酸的产生，维持乙酸、丙酸和己酸的水平。
有益效果：与现有技术相比，本发明具备以下优点：本发明的嗜热链球菌GYX-8乳酸菌具有优良的益生特性，制备的发酵乳，具有调节肠道菌群和短链脂肪酸产生的作用。
本发明的发酵乳能够调节肠道菌群潜在的机制可能是：嗜热链球菌GYX-8乳酸菌制备的发酵乳通过调整肠道菌群，通过抑制肠道中革兰氏阴性菌的生长，改善肠屏障功能。在门水平上分析，厚壁菌门、拟杆菌门、变形菌门和放线菌门是盲肠微生物群落的优势菌门；进一步在属水平上对肠道菌群变化进行分析，对于有益菌的增加，部分菌株定植于人体肠道粘膜层并特异性降解粘蛋白，产生低聚糖和短链脂肪酸，在宿主的先天和获得性免疫系统中发挥重要作用，并具有抗炎作用，它主要可以有效增加粘液厚度和肠道屏障功能，再如，颤螺菌（Oscillospira）与许多慢性疾病相关，或能够产生丁酸盐等SCFAs的菌属，它已被定性为下一代益生菌候选物之一；对于有害菌减少，如，别样芽孢杆菌（Allobaculum）参与高脂饮食治疗小鼠肠道调节。
本发明的发酵乳能够调节短链脂肪酸产生潜在的机制可能是：短链脂肪酸是碳原子数小于6的羧酸，在宿主的健康和疾病中起着至关重要的作用，是结肠和回肠细胞的关键能量来源，通过调控相关基因表达影响肠道上皮屏障和防御功能，并且是肠道微生物的重要代谢产物，所以会随肠道菌群的变化而变化。分析肠道微生物和短链脂肪酸的相关性，如Helicobacter属与乙酸和丙酸水平呈负相关。
附图说明
图1为本发明中嗜热链球菌GYX-8乳酸菌制备的发酵乳对小鼠肠道多样性的影响图。
图2为本发明中嗜热链球菌GYX-8乳酸菌制备的发酵乳在门水平对小鼠肠道菌群的影响图。
图3为本发明中嗜热链球菌GYX-8乳酸菌制备的发酵乳在属水平上分析，部分有益菌的增加，主要为颤螺菌（Oscillospira）的增加，对小鼠肠道菌群的影响图。
图4为本发明中嗜热链球菌GYX-8乳酸菌制备的发酵乳在属水平上分析，部分有害菌的减少，主要为理研菌属（Rikenella）、萨特氏菌（Sutterella）和臭气杆菌（Odoribacter）对小鼠肠道菌群的影响图。
图5为本发明中嗜热链球菌GYX-8乳酸菌制备的发酵乳在属水平上分析，部分有害菌的减少，主要为螺杆菌属（Helicobacter）的减少，对小鼠肠道菌群的影响图。
图6为本发明中嗜热链球菌GYX-8乳酸菌制备的发酵乳在调节短链脂肪酸产生的应用，主要有增加异丁酸、异戊酸和戊酸的产生，维持乙酸、丙酸和己酸的水平影响图。
具体实施方式
以下结合附图做进一步的说明。
1、材料与方法
1.1实验材料
1.1.1菌株：嗜热链球菌GYX-8，筛选至新疆农家自制奶疙瘩。
1.1.2脱脂乳（澳大利亚德运）：2%能量、6%蛋白质、2%碳水化合物、3%钠、15%钙。
1.1.3实验动物：下述实施例中涉及SPF级8周龄雄性C57BL/6小鼠购自江苏省艾菱菲实验动物有限公司。
1.1.4培养基
1.1.4.1嗜热链球菌GYX-8的培养基
MRS固体培养基(g/L)：蛋白胨10g/L、牛肉浸粉8g/L、酵母浸粉4g/L、葡萄糖20g/L、磷酸氢二钾2g/L、柠檬酸氢二铵2g/L、乙酸钠5g/L、硫酸镁0.2g/L、硫酸锰0.04g/L、吐温801g/L、琼脂20g/L，pH为5.7±0.2。
MRS液体培养基(g/L)：蛋白胨10g/L、牛肉浸粉8g/L、酵母浸粉4g/L、葡萄糖20g/L、磷酸氢二钾2g/L、柠檬酸氢二铵2g/L、乙酸钠5g/L、硫酸镁0.2g/L、硫酸锰0.04g/L、吐温801g/L，pH为5.7±0.2。
1.2实验方法
1.2.1菌株培养
活菌数的检测方法：采用国标《GB 4789.35-2016食品安全国家标准食品微生物学检测乳酸菌检测》。
1.2.2乳杆菌菌悬液的制备
将乳杆菌嗜热链球菌GYX-8（-80℃保藏的甘油管）划线于MRS固体培养基上，37℃条件下培养48h，得到单菌落；挑取单菌落接种于MRS液体培养基中，37℃条件下培养18h进行活化，连续活化两代，得到活化液；将活化液按2％（v/v）的接种量接种于MRS液体培养基中，37℃条件下培养18h，得到菌液。
1.2.3发酵乳的制备
以5%接种量将上述菌液接种于20mL澳大利亚德运脱脂乳，放置42℃培养，直至凝乳。
1.2.4实验小鼠分组及实验设计
取健康的C57BL/6小鼠24只（雄性，7-8周龄，体重20-23 g）将小鼠圈养22±2℃的受控环境中，湿度为40-60%，光/暗周期为12小时。小鼠适应性喂养期间自由饮水和标准饲料，并对其体重和总体健康状况进行密切监测。经过1周的适应期后，随机分为2组，每组6只，2组分别为：正常组（PF）、嗜热链球菌GYX-8发酵乳组（ST）。
实验共24天，第一周为小鼠适应期，适应期小鼠圈养在22±2℃的受控环境中，湿度为40-60%，光/暗周期为12小时。小鼠适应性喂养期间自由饮水和标准饲料，并对其体重和总体健康状况进行密切监测。经过1周的适应期后开始实验，正常喂饲料，发酵乳组灌胃发酵乳（1.5 mL/100g，108 CFU/mL），记录灌胃时间，连续喂养14天。
1.2.5样品收集
灌胃结束后，收集小鼠新鲜盲肠内容物样品。对灌胃结束的小鼠禁食12小时后，麻醉小鼠，辅以颈椎脱臼法处死。于无菌条件下解剖小鼠，在冰上将小鼠的盲肠内容物收集入无菌的冻存管中，置于-80℃冰箱中，用于检测肠道菌群组成和短链脂肪酸。
1.2.9肠道菌群分析
微生物组总DNA提取：小鼠盲肠内容物中的总微生物按照DNA提取试剂盒（OmegaBio-tek, Norcross, GA, USA）说明书进行提取，同时采用Nanodrop对DNA进行定量，并通过1.2%琼脂糖凝胶电泳检测DNA提取质量。细菌16SrRNA基因高变区 V3-V4 被扩增用于高通量扩增子测序。PCR产物用Qiagen Gel Extraction Kit (Germantown, MD, USA)纯化，并通过Quant-iTPicoGreen dsDNA Assay Kit 和酶标仪 (BioTek, FLx800) 进行定量。使用Illumina（美国加利福尼亚州圣地亚哥）TruSeq Nano DNA LT Library Prep Kit制备测序文库，并在Qubit荧光计和Agilent Bioanalyzer 2100系统上评估文库质量。
首先对高通量测序的原始下机数据根据序列质量进行初步筛查；通过质量初筛的原始序列按照index和Barcode信息，进行文库和样本划分，并去除barcode序列；按照QIIME2 dada2分析流程或Vsearch软件的分析流程进行序列去噪或OTU聚类；对各样本在不同物种分类学水平的具体组成进行展示；根据ASV/OTU在不同样本中的分布，评估每个样本的Alpha多样性水平，并通过稀疏曲线反映测序深度是否合适；通过聚类，结合相应统计学检验方法，衡量不同样本（组）间的物种丰度组成差异。
1.2.10短链脂肪酸的测定
取适量样本，加50μL15%磷酸，加入100mg玻璃珠，再加125 μg/mL的内标（异己酸）溶液100 μL和乙醚400 μL；放入高通量组织研磨仪中，55Hz研磨60s，重复2次；于4℃12000rpm离心10 min，取上清上机测试。
GC-MS检测：色谱条件：色谱柱 Agilent HP-INNOWAX毛细管柱（30 m*0.25 mm ID*0.25 μm）；分流进样，进样量1 μL，分流比10:1。进样口温度250℃；离子源温度230℃；传输线温度250℃，四极杆温度150℃。程序升温起始温度90℃；然后以10℃/min升温至120℃；再以5℃/min升温至150℃；最后以25℃/min升温至250℃维持2 min。载气为氦气，载气流速1.0 mL/min。MS条件：电子轰击电离（EI）源，SIM扫描方式，电子能量70 eV。
2、实验结果与分析
2.1 肠道菌群分析
肠道菌群被认为是身体的另一个器官，与宿主的新陈代谢和健康密切相关。随着肠道菌群研究的不断深入，肠道菌群在健康水平中起着至关重要的作用。因此，探究了嗜热链球菌GYX-8乳酸菌制备的发酵乳对肠道菌群的调节，利用16S rDNA测序联合PCR技术分析了小鼠肠道中微生物的组成。以Chao1指数来评估各组小鼠肠道中的微生物总数。香农（Shannon）指数来评估各组物种的均一性。Goods coverage代表测序深度。结果如图1所示，发酵乳组小鼠肠道中Chao1指数相对于正常组显著升高(P<0.05)，具有较高的物种丰富度。同时，所有样品的Goods coverage值均高于0.994，表明本次测序对样品的覆盖率很高。
为了进一步研究肠道菌群组成的差异，将对正常组和嗜热链球菌GYX-8发酵乳组的小鼠肠道菌群在不同的分类水平上进行了比较。在门水平上，厚壁菌门、拟杆菌门、放线菌门和变形菌门为主要的菌群，与正常组相比，嗜热链球菌GYX-8发酵乳组拟杆菌门水平增加，厚壁菌门、变形菌门和放线菌门基本维持不变（如图2）。进一步，检测了小鼠肠道菌群在属水平的变化。与正常组相比，部分有益菌的增加，增加颤螺菌属（Oscillospira）相对丰度，其与许多慢性疾病相关，或能够产生SCFAs的菌属，它已被定性为下一代益生菌候选物之一，ST组颤螺菌属（Oscillospira）增加的相对丰度是PF组的2倍以上（如图3）；与正常组相比，部分有害菌的减少，可以减少理研菌属（Rikenella）、萨特氏菌（Sutterella）和臭气杆菌（Odoribacter）相对丰度（图4），其中主要减少螺杆菌属（Helicobacter），螺杆菌属（Helicobacter）与许多短链脂肪酸的产生呈现负相关，ST组大大减少了螺杆菌属（Helicobacter），其相对丰度仅仅只有PF组的十分之一（如图5）。
2.11 肠道中短链脂肪酸含量分析
短链脂肪酸（Short-chain fatty acids, SCFAs）是碳原子数小于6的羧酸，SCFAs在宿主的健康和疾病中起着至关重要的作用。肠道菌群的改变会导致其代谢产物的变化，接下来利用GC-MS测定了肠道微生物代谢产物中的短链脂肪酸。如图6所示，相比于正常组，显著增加异丁酸、异戊酸和戊酸的产生（P＜0.01），并基本维持乙酸、丙酸和己酸的水平，说明肠道菌群的改变会导致短链脂肪酸的变化。
以上所述仅是本发明的优选实施方式，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，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.一株嗜热链球菌（Streptococcus thermophilus）GYX-8，其特征在于，所述嗜热链球菌（Streptococcus thermophilus）GYX-8于2022年1月21日保藏于中国微生物菌株保藏管理委员会普通微生物中心，保藏编号为CGMCC No.24357，保藏日期为2022年1月21日。
2.一种乳酸菌发酵乳，其特征在于，所述乳酸菌发酵乳包含权利要求1所述的嗜热链球菌（Streptococcus thermophilus）GYX-8。
3.根据权利要求2所述的乳酸菌发酵乳，其特征在于，所述嗜热链球菌（Streptococcus thermophilus）GYX-8添加量为5-8%。
4.权利要求1所述的嗜热链球菌（Streptococcus thermophilus）GYX-8或权利要求2或3所述的发酵乳在调节肠道菌群和/或调节短链脂肪酸产生的应用。
5.根据权利要求4所述的应用，其特征在于，所述调节菌群中的应用为增加颤螺菌和减少螺杆菌属。
6.权利要求1所述的4所述的应用，其特征在于，所述调节短链脂肪酸产生的应用包括增加异丁酸、异戊酸和戊酸的产生，维持乙酸、丙酸和己酸的水平。
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