CN117467586A - 一株具有延缓慢性肾病进程功效的鼠李糖乳酪杆菌 - Google Patents
一株具有延缓慢性肾病进程功效的鼠李糖乳酪杆菌 Download PDFInfo
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- CN117467586A CN117467586A CN202311827760.5A CN202311827760A CN117467586A CN 117467586 A CN117467586 A CN 117467586A CN 202311827760 A CN202311827760 A CN 202311827760A CN 117467586 A CN117467586 A CN 117467586A
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- lactobacillus rhamnosus
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- indole
- rhamnosus
- intestinal
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Abstract
本发明提供了一株具有延缓慢性肾病进程功效的鼠李糖乳酪杆菌，属于益生菌筛选与应用技术领域。所述鼠李糖乳酪杆菌的保藏号为CCTCC NO：M20221669，对肠道吲哚具有很强的降解效果，能在体外显著降解吲哚。该菌株对人工肠胃液具有较强的耐受性；对红霉素和氨苄西林等常见的抗生素敏感，不产生溶血素，不能够溶解血细胞，具有良好的生物安全性。
Description
技术领域
本发明属于益生菌筛选与应用技术领域，具体涉及一株具有延缓慢性肾病进程功效的鼠李糖乳酪杆菌。
背景技术
慢性肾衰CKD又称慢性肾功能不全、尿毒症，是各种原发性肾病或继发性于其他疾病引起的肾脏进行性损伤和肾功能的逐渐恶化。临床表现主要为尿毒素潴留、水电解质紊乱、肾性贫血和钙磷代谢紊乱等, 尿毒素能够加剧功能性肾损伤、肾纤维化及氧化应激等,导致慢性肾衰 CKD 病情的加速发展。到目前为止，慢性肾衰竭还缺乏有效的治疗手段，主要依靠血液透析及肾移植，但是血液透析需要患者终身透析, 费用高昂。并且尿毒素中如硫酸吲哚酚 (IS)、硫酸对甲酚 (PCS) 等蛋白结合型尿毒素, 极难经血液透析清除,而肾脏移植同样面临着肾源紧缺、费用更加高昂的困境。已有证据表明, 透析患者的体内 IS水平高达正常水平的 20 倍, 并且目前无经济有效的降低蛋白结合型尿毒素在体内蓄积的治疗方法。
在 CKD 患者体内, 血中硫酸吲哚酚IS水平与其肾功能呈负相关, 并且与主动脉硬化、第一次心力衰竭事件及心血管死亡有着直接的关系。近年来，消化道细菌治疗法成为了继消化道酶疗法的清除肠道毒素的小疗法。其治疗方法是使用自然菌种，如低毒土壤菌，体外诱导产生尿素酶，口服进入肠道，分解肠道尿毒素；或将高产酶基因导入另一种细菌中，构建工程菌，将该工程菌摄入肠道，降解尿毒素。但这类方法摄入的菌存在安全隐患，可能导致肠道菌群失调，并诱发感染。
益生菌是一种对宿主有益的活性微生物，可以定植于人体肠道内，改善肠道菌群平衡，控制肠道感染。激活肠淋巴细胞能启动全身免疫系统提高免疫力，还有降低患者血压、血脂等作用。近年来, 有大量研究证实, 肠道微生态失调与CKD 发病机制及病情进展密切相关。CKD 患者肠道内约190多种细菌丰度发生改变, 这一现象已在CKD模型动物上得到验证。经由肠道进入体内的尿毒素完全依赖于肾脏清除, 随着CKD患者肾功能的丧失,尿毒素不断积累, 会扰乱肠道菌群、破坏肠道屏障的完整性使屏障通透性增加, 从而导致肠道细菌进入血液, 肠道内有毒物质吸收增加, 引发全身性炎症及多种并发症, 进而导致患者死亡。因此, 靶向肠道细菌、减少尿毒素蓄积这一新的CKD治疗靶点应被广泛重视。之前的研究中Suzuki等从慢性肾衰患者的粪便中分离到一株产脲酶的双歧杆菌，CHOW等发现德式乳杆菌在高浓度尿素培养基中多次传代后，表现出对尿素的降解能力。研究发现，口服嗜酸乳杆菌可以降低透析病人尿毒症毒素水平。在哺乳动物体内，尿毒素 IS始于食物蛋白质的降解, 肠道微生物将色氨酸代谢转化为 IS前体吲哚, 吲哚在肝脏进一步合成为IS, 最终通过肾脏排出体外。因此, 靶向肠道细菌中的代谢通路, 抑制肠道细菌中吲哚的生成, 降低体内尿毒素硫酸吲哚酚的水平, 减轻吲哚及硫酸吲哚酚对机体的损伤, 对于慢性肾衰竭患者的治疗具有重大意义。
发明内容
本发明的目的是提供一株新的鼠李糖乳酪杆菌（Lacticaseibacillusrhamnosus）及其应用，所提供的鼠李糖乳酪杆菌是从自然发酵的泡菜发酵液中分离得到的，对肠道中色氨酸代谢产物吲哚具有很强的降解能力，而吲哚是蛋白结合型尿毒素硫酸吲哚酚的前体物质，吲哚含量的降低会直接减少患者体内的硫酸吲哚酚水平，从而减轻肾衰患者症状，可应用于肾衰患者的辅助治疗，减轻痛苦，延长生命。
本发明所提供的鼠李糖乳酪杆菌为（Lacticaseibacillus rhamnosus）VHProbiM12株，已于2022年10月26日保藏在位于中国武汉武汉大学的中国典型培养物保藏中心，其保藏号为CCTCC NO：M20221669。
本发明所提供的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ），其Riboprinter 指纹图谱如图2所示；其RAPD指纹图谱如图3所示，rep-PCR指纹图谱如图4所示，MALDI-TOF-MS蛋白质指纹图谱如图5所示。
本发明还提供所述的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）株的一种用途，是在制备用于清除尿毒素、缓解慢性肾病的制品中的应用。
本发明还提供所述的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）株的另一种用途，是在制备具有降低胆固醇功能的制品中的应用。
本发明再一个方面还提供所述的鼠李糖乳酪杆菌（Lacticaseibacillusrhamnosus ）株的另一种用途，是在制备抗氧化制品中的应用。
本发明还提供了一种用于降解肠道吲哚的制品，包含鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）株的活菌和/或其发酵产物。
所述的制品，为药品。
本发明提供的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）对肠道吲哚具有很强的降解效果，能在体外显著降解吲哚。该菌株对人工肠胃液具有较强的耐受性；对红霉素和氨苄西林等常见的抗生素敏感，不产生溶血素，不能够溶解血细胞，具有良好的生物安全性。
本发明所筛选的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）株含胆盐水解酶，具有较强的抗氧化能力和降解胆固醇能力，DPPH清除率达到41.76%，胆固醇降解率达到19.58%。该菌株具有较好的表面疏水性，为37.23%。且可以有效降解吲哚，48h降解率为52.38%±1.49%。
本发明所筛选的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）可用于制备清除尿毒素，缓解慢性肾病的药品，应用前景广阔。
附图说明
图1为M12菌株在含吲哚的培养基中的生长曲线图；
图2为M12菌株Riboprinter 指纹图谱；
图3为M12菌株的RAPD指纹图谱；
图4为M12菌株的rep-PCR指纹图谱；
图5为M12菌株的MALDI-TOF-MS蛋白质指纹图谱；
图6为M12菌株的胆盐酶活性图。
具体实施方式
本发明提供的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）符合法规要求，可以作为一种药品原料来源使用，长期服用不会有副作用及过量的风险。经多相分类学鉴定，鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）为一株新发现的菌株。本发明提供的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ） 能有效降解肠道中的吲哚，进而达到降低尿毒素硫酸吲哚酚含量、缓解肾病的功效，单独使用该菌株且无需与益生元和/或其它益生菌复配即可降解肠道吲哚，具有重要的应用价值。
本发明所述筛选方法并不局限于实施例所述，已知的能够达到筛选目的的方法均可以，实施例的筛选说明只是对本发明的说明，并不是对本发明保护范围的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
下面结合具体实施例对本发明做详细的描述。
实施例1 菌株的筛选
1、乳酸菌的初步分离纯化
配制MRS (Man Rogosa Sharpe) 肉汤：纯水1L，蛋白胨10g，牛肉浸取物10g，酵母提取物5.0g, 乙酸钠5g，葡萄糖5g，磷酸二氢钾2g，吐温80 1.0mL，柠檬酸二胺2.0g, 碳酸钙20g，七水硫酸镁0.58g，七水硫酸锰0. 25g，调pH 6.2-6.5。
配置MRS琼脂培养基：1LMRS肉汤添加15g琼脂。
取1g新鲜泡菜发酵液，用无菌生理盐水稀释后放入无菌样品袋，用匀浆机拍打混匀，取100μl混合液梯度稀释，涂布于MRS琼脂培养基后于37℃厌氧培养48h，待平板长出单菌落镜检。根据镜检结果，申请人共筛选出14株（类）杆菌，并反复纯化，确定得到的是纯菌株，分别命名为M01、M02、……、M13、M14。挑取单菌落在液体MRS培养基中扩大培养，并进一步进行涂布分离，进行3-5次纯化、分离，最后得到不同菌落形态的纯平板；挑取纯平板的单菌落于MRS液体培养基中，37℃恒温培养过夜，得分离菌悬液，得到初筛菌悬液。
2、体外可降解吲哚的菌株筛选
2.1 准备工作
配制吲哚溶液：用70%乙醇配制30mg/ml的吲哚母液，0.22um滤膜除菌备用。使用时用菌株培养基稀释至所需浓度。
菌株准备：乳酸菌划线活化。取冷冻保藏的乳酸菌甘油管，无菌操作，划线至MRS平板，37℃培养48~72h。
乳酸菌菌体溶液准备：待平板长出单菌落后，无菌下挑至MRS肉汤，37℃静置培养24h。按1%接种量接种至新鲜MRS肉汤，每株菌约6ml。
2.2 菌株对不同浓度吲哚的耐受性试验。
将吲哚用菌株培养基稀释为0、50、100、150、200、300、400、600ug/ml，分别按1%接种量接种M12，用酶标仪监测M12的生长曲线。
结果显示，培养基中含有低浓度吲哚时菌株活性不受影响，甚至有稍微促进菌株生长的现象，当培养基中吲哚含量接近300ug/ml时，菌株活性受到抑制，同时根据资料显示人肠道或粪便吲哚0.25-2.6mM，约为45-468ug/ml，因此，300ug/ml可以作为验证菌株吲哚降解能力的吲哚添加浓度。
2.3 菌株代谢吲哚能力检测：
含1%菌株的新鲜培养基添加吲哚使其终浓度为300ug/ml，添加吲哚0h、24h、48h分别取1.5ml培养基至新的EP管，10000g/min,离心3min，收上清，使用吲哚检测试剂盒检测上清中的吲哚含量。操作方法及结果判定具体参见Indole Assay Kit说明书。
吲哚降解率计算：吲哚降解率=（初浓度-终浓度）/初浓度*100%。
结果显示，本发明分离得到的14株潜在乳酸菌中 24h的吲哚的降解率超过30%的有6株，其中M12的24h吲哚的降解率为33.67%±3.19%；48h吲哚的降解率超过50%的有3株，其中M12的48h吲哚的降解率为52.38%±1.49%，是所有菌株中降解率最高的。
M12菌株在不同浓度吲哚中的生长曲线如图1所示。
实施例2 菌株M12鉴定
1、菌落形态鉴定
将M12菌株接种于MRS琼脂培养基上，37℃厌氧培养24h后，可见M12菌落直径为0.5-1mm，圆形，边缘整齐，不透明，正面乳白色，中间凸起，表面光滑，表面明亮，显微镜下菌体呈长杆状，长短不均一，成单或成对、成链排列。
2、生理生化特性鉴定
接种液的准备如下：在无菌条件下，取适量新鲜M12菌液，5000rpm/min离心5 min，用PBS缓冲液洗2次，再用同体积PBS缓冲液重菌体后稀释50倍，作为接种液。
2.1、盐度耐受性试验
在无菌条件下，向96孔板中分别加入190μL盐浓度为1%、2%、3%、4% 、5%、6%、7%、8%的BSM液体培养基，每个盐浓度做3个平行，然后再加入10μL接种液，不接菌的孔作为对照。每孔加入50μL高压灭菌过的石蜡油以防止培养过程中水分蒸发。置于37℃恒温培养，观察培养基是否变浑浊。结果显示M12菌株最大耐受盐浓度≧8%。
2.3、碳源代谢试验
利用API 50CHL试剂条对M12菌株进行碳源代谢实验，实验方法及结果判读具体参见API 50CHL试剂盒说明书。M12菌株鉴定结果为：%ID=81.3且T值=0.44，API结果为鼠李糖乳酪杆菌，结果见表1。
表1：M12的API 50CHL检测结果表
| 底物 | 结果 | 底物 | 结果 | 底物 | 结果 |
| 阴性对照 | - | 肌醇 | - | D-松三糖 | + |
| 丙三醇 | - | 甘露醇 | + | D-棉子糖 | - |
| 赤藻糖醇 | - | 山梨醇 | + | 淀粉 | - |
| D-阿拉伯糖 | - | 甲基-α-D-吡喃甘露糖苷 | - | 糖原 | - |
| L-阿拉伯糖 | - | 甲基-α-D-吡喃葡萄糖苷 | - | 木糖醇 | - |
| D-核糖 | + | N-乙酰葡萄糖胺 | + | D-龙胆二糖 | + |
| D-木糖 | - | 苦杏仁甙 | + | D-土伦糖 | - |
| L-木糖 | - | 熊果苷 | + | D-来苏糖 | - |
| D-核糖醇 | - | 七叶灵柠檬酸铁 | + | D-塔格糖 | + |
| 甲基-β-D吡喃木糖苷 | - | 水杨苷 | + | D-岩藻糖 | - |
| D-半乳糖 | + | D-纤维二糖 | + | L-岩藻糖 | - |
| D-葡萄糖 | + | D-麦芽糖 | - | D-阿拉伯醇 | - |
| D-果糖 | + | D-乳糖 | + | L-阿拉伯醇 | - |
| D-甘露糖 | + | D密二糖 | - | 葡萄糖酸钾 | + |
| L-山梨糖 | - | D-蔗糖 | - | 2-酮基葡萄糖酸钾 | - |
| L-鼠李糖 | + | D-海藻糖 | + | 5-酮基葡萄糖酸钾 | - |
| 卫矛醇 | + | 菊粉 | - | / | / |
2.4、葡萄糖产酸产气试验
本实施例中所用的培养基配方如下：
蛋白胨0.5g；酵母提取物0.3g；吐温80 0.1mL；盐溶液A 0.5ml；盐溶液B 0.5ml；乙酸钠 0.5g；葡萄糖2.5g；2%溴甲酚绿（w/v）0.05mL；蒸馏水100ml；pH6.8～7.0。
将配制好的培养基分装至含有倒置小试管的大试管中，3mL/管，121℃，高压灭菌15min。
盐溶液 A 成分：KH2PO4 10g、K2HPO4 1.0g，溶于蒸馏水，定容至100mL。
盐溶液 B 成分：MgSO4·7H2O 11.5g、MnSO4·2H2O 2.4g、FeSO4·7H2O 0.68g，溶于蒸馏水，定容至100mL。
在无菌条件下，将接种液按10%的接种量接种培养基，不接菌的培养基作为对照，然后用2mL无菌液体石蜡封住顶部，置于37℃培养24h，观察培养基颜色有无变化。
结果显示：37℃培养24h后，培养基由绿色变为黄色，小倒管内无气体，说明M12菌株发酵葡萄糖产酸，不产气。
3、分子生物学鉴定
3.1 16s rDNA基因序列分析
1、基因组DNA提取
参照天根细菌基因组DNA提取试剂盒（目录号：DP302）操作。
2、16s rDNA 基因扩增
引物序列：
27F：AGAGTTTGATCCTGGCTCA（SEQ ID NO:2）；
1492R：GGTTACCTTGTTACGACTT（SEQ ID NO:3）。
通过测序获得M12菌株的16s rDNA序列（SEQ ID NO:1），并将该序列在NCBI 数据库中进行比对，初步确定M12菌株为鼠李糖乳酪杆菌。
3.2 Riboprinter指纹图谱
用一根取菌棒从琼脂培养基平板上沾取已纯化好的单菌落，将其放入有缓冲液的样品管中，用手持搅拌器搅拌使其在缓冲液中悬浮，然后将样品架放入加热器中灭活后放入Riboprinter系统中，样品经过DNA制备、转膜、成像检测及数据处理后，得到细菌鉴定结果。鉴定结果显示，M12菌株为鼠李糖乳酪杆菌，其Riboprinter指纹图谱结果见图2。
3.3 RAPD和rep-PCR指纹图谱鉴定
1、RAPD指纹图谱鉴定
引物序列： GAGGGTGGCGGTTCT（SEQ ID NO:4）。
表2：RAPD 反应体系表
| 反应成分 | 体积 |
| TaqDNA 聚合酶(5U/μL) | 0.2 μl |
| 10×Buffer（含Mg2+） | 2 μl |
| 引物（10 uM） | 1 μl |
| dNTPs（2.5 mM） | 0.8 μl |
| DNA模板 | 2 μl |
| 无菌双蒸水 | 14 μl |
| 总体积 | 20 μl |
制备1.5%的琼脂糖凝胶板，DL2000DNA Marker 作为结果对照，稳压100V电80min，最后利用凝胶成像系统检测电泳图。M12菌株的RAPD指纹图谱如图3所示。
2、rep-PCR指纹图谱
引物序列：CTACGGCAAGGCGACGCTGACG（SEQ ID NO:5）。
rep-PCR的反应体系如表3所示。
表3：rep-PCR 的反应体系表
| 反应成分 | 体积 |
| r TaqDNA聚合酶 | 0.2 μl |
| 10×Ex Taq DNA Buffer（含Mg2+） | 2 μl |
| 引物（10 uM） | 1 μl |
| dNTPs（2.5 mM） | 2 μl |
| DNA模板 | 2 μl |
| 无菌双蒸水 | 12.8 μl |
DL2000 DNA Marker 作为结果对照。电压100 V，电泳时间80min 检测扩增结果。M12菌株的rep-PCR 指纹图谱如图4所示。
3.4 MALDI-TOF-MS检测菌株核糖体蛋白表达
按照0.1%的接种量在MRS液体培养基中接种M12新鲜菌液，37℃，150rpm培养48h后，收集菌体，无菌水洗涤4次，晾干表面水分。然后取少量新鲜菌体以薄膜的形式均匀涂布于靶板上，加1μL裂解液覆盖样品，晾干后，再加1μL基质溶液覆盖样品，晾干后，将样品靶放入质谱仪进行鉴定。用激光照射样品与基质形成的共结晶薄膜，使样品中蛋白质电离，离子在10～20KV电场作用下加速飞过飞行管道，根据到达检测器的飞行时间不同检测蛋白质的分子量。利用Autofms 1000 分析软件Autof Analyzer v1.0获取蛋白指纹图谱，M12菌株主要核糖体蛋白的离子峰为：m/z3477.337、4693.108、5892.517、9383.546。鉴定结果如图5所示。
3.5全基因组测序
按照 0.1%的接种量在MRS液体培养基中接种新鲜菌液，37℃培养20 h，8000rpm离心10 min，收集菌体。菌体送到测序中心，得到该菌的全基因组序列，基因组序列已上传至NCBI基因数据库，GenBank登录号为CP134216。
结合M12菌株的菌落形态、生理生化特性以及分子生物学的鉴定结果，确定该菌株为一株新的鼠李糖乳酪杆菌，将其命名为鼠李糖乳酪杆菌（Lacticaseibacillusrhamnosus）。
实施例3 鼠李糖乳酪杆菌对人工胃液和人工肠液的耐受性试验
1、人工胃液的配制
分别称取蛋白胨5g、酵母提取物2.5g、葡萄糖1g和NaCl 2g，加入1000mL蒸馏水，用稀盐酸调pH3.0，然后115℃灭菌20min。然后使用前加入3.2g猪粘膜胃蛋白酶，摇匀溶解，置37℃水浴摇床中温水浴1h，以模拟人体温度。
2、人工肠液的配制
分别称取蛋白胨5g、 酵母提取物2.5g、葡萄糖1g、KH2PO4 6.8g和牛胆盐 3.0g，加入77mL 的0.2mol/L的NaOH溶液，定容至1000mL，用稀盐酸或者氢氧化钠溶液调pH6.8±0.1，115℃灭菌20min。然后使用前加入1g胰酶，摇匀溶解，置37℃水浴摇床中温水浴1h，以模拟人体温度。
3、实验方法
取2 mL新鲜菌液，5000rpm/min 离心5min 收集菌体，菌体用生理盐水洗涤3次，再用2mL 生理盐水重悬，作为接种液。取1mL接种液，加入到24 mL人工胃液或肠液中，置于37℃水浴摇床（200rpm/min）3h，取样1mL，检测活菌量。
活菌计数方法按照国标《GB4789.35-2016-食品微生物检验乳酸菌检验》测定菌量，该菌株经过人工胃液或肠液消化后的活菌量（Log CFU/mL）见表4。
表4：人工胃肠液消化后的活菌量表
| 消化前 | 人工胃液消化后 | 人工肠液消化后 |
| 8.12±0.06 | 8.02±0.01 | 7.65±0.03 |
从表4可知，本发明筛选到的鼠李糖乳酪杆菌经人工胃液消化后，活菌量基本没有变化；经人工肠液消化3h后，只下降了0.47个Log值。从而说明该菌株对人工胃肠液具有很强的耐受性。
实施例4 鼠李糖乳酪杆菌的抗生素耐受性试验
微量肉汤稀释法测定抗生素对鼠李糖乳酪杆菌的最小抑菌浓度MIC值具体结果见表5。
表5：鼠李糖乳酪杆菌的抗生素MIC值表
MIC单位μg/mL。
从表5的结果可以看出，本发明提供的鼠李糖乳酪杆菌对红霉素、氨苄西林等常见抗生素敏感，生物安全性良好。
实施例5 鼠李糖乳酪杆菌的抗氧化功能测定
1、菌株清除DPPH（1，1-二苯基-2-三硝基苯肼）能力测定
将生长状态优良的鼠李糖乳酪杆菌单菌落接种于3mL的MRS液体培养基中，37℃条件下培养18-20h，以此培养液为接种液，按照2%的接种量接种于50mL的MRS液体培养基中，静置培养18h，获得菌株的培养液。吸取1mL菌液收集菌体后，用1mLPBS缓冲液洗涤菌体2遍后，再加入2mL PBS溶液重悬菌体，得菌悬液。
取1mL鼠李糖乳酪杆菌的菌悬液，加入1mL 0.4 mM的现配的DPPH自由基溶液，混合均匀后然后置于室温温度下遮光反应30 min，然后测定样品在波长 517nm处的吸光度A样本，测3次平行。对照组样品以等体积PBS溶液和DPPH·乙醇混合液，并以等体积鼠李糖乳酪杆菌的菌悬液和乙醇混合液空白调零。
清除率按下列公式计算：清除率%=[1-（A样品-A空白）/A对照]×100%。
结果见表 6。
表6： DPPH自由基清除率表
| 菌株 | 清除率 | 标准差 |
| 鼠李糖乳酪杆菌 | 41.76% | 2.55% |
2、菌株抗脂质过氧化实验鉴定
1）菌株培养及发酵上清液、菌体、胞内提取物的制备：
鼠李糖乳酪杆菌在MRS液体培养基中37℃培养24h，传3代后，6000 r/min，4℃离心10min，收集上清液即为发酵上清液。收集的菌体用PBS缓冲液（pH 7.4）于6000 r/min 离心10min，洗涤3次。将菌体重悬于PBS缓冲液，调整菌体浓度为1.0×109cells/mL，获得菌悬液。菌悬液用超声破碎仪超声破碎20分钟，得到胞内提取物。
（2）亚油酸乳化液的制备：
0.1mL亚油酸，0.2mL Tween 20，19.7mL去离子水。
（2）实验过程
0.5 mL的PBS溶液（pH 7.4）中加入1 mL亚油酸的乳化液，1 mLFeSO4（1%），再加入0.5 mL样品，37℃水浴1.5 h，混合液加入0.2 mL TCA（4%），2 mL TBA（0.8%），100 ℃水浴30min，迅速冷却，4000 rpm/min离心15 min，收集上清液在532 nm下测吸光度即为A；对照组以0.5 mL蒸馏水代替样品即为A0。
抑制率/% ＝(A0－A)/A0×100%
注：A为样品组吸光度；A0为对照组吸光度。
结果见表7。
表7：鼠李糖乳酪杆菌抗脂质过氧化抑制率表
| 鼠李糖乳酪杆菌 | 抑制率 | 标准差 |
| 发酵上清液 | 5.93% | 1.15% |
| 胞内提取物 | 9.68% | 3.28% |
实施例5 鼠李糖乳酪杆菌的表面疏水性测试
将鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）在液体MRS培养基中进行活化；吸取1 ml菌液6000 rmp离心2 min，收集菌体后用PBS溶液冲洗2次。用灭菌的0.1MKNO3 1ml溶液重悬菌体。将菌悬液稀释三倍后记录OD600为A0。加入菌悬液三分之一体积的二甲苯，混匀后在室温下静置10min（此时形成两相体系）。将两相体系涡旋振荡2min后再静置20min, 重新形成水相和有机相。小心吸取水相，在OD600nm测量吸光度A1。细胞疏水性按下列公式计算，测三次实验取平均值。
疏水性（%）=(A0-A1)/A1×100%。
结果显示，鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）菌株的表现疏水性为37.23%±0.25%。
实施例6 鼠李糖乳酪杆菌体外胆固醇降解试验
1、胆盐酶活性定性试验
在新鲜配制的MRS液体培养基中添加0.2%TCA、0.2%的巯基乙酸钠、0.37 g/LCaCl2和1.5%琼脂。121℃灭菌15min，倒入平板中，直至平板中MRS凝固倒置放入厌氧罐中备用。把无菌滤纸片均匀的放入制作好的平板中，用移液枪在滤纸片上滴加10 μL 新鲜培养的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）菌液，平板再次正放入厌氧罐中，37℃培养72 h后观察结果。
结果如图6所示，滤纸片周围出现钙圈，说明鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）的胆盐酶活性为阳性。
2、胆固醇降解试验
胆固醇胶束溶液的配制：准确称取1g 胆固醇，溶于无水乙醇中，并定容至100 mL，在无菌条件下用 0.22 µm 微孔滤膜过滤除菌。
称取蛋白胨10.0g、牛肉膏10.0g、酵母膏5.0g、柠檬酸氢二铵2.0g、葡萄糖20.0g、吐温80 1.0 Ml、乙酸钠5.0g、硫酸镁0.1g、硫酸锰 0.05g、磷酸氢二钾2.0g、胆盐1g，蒸馏水1000Ml，调节pH值7.3，115℃灭菌30min，然后加入胆固醇溶液使胆固醇终浓度为0.1%。
按照0.1%的接种量接种新鲜菌液，37℃静止培养48h，然后取0.2mL菌液，加入1.8mL无水乙醇，混匀，静止10分钟，3000转离心5分钟，取上清液用于测定胆固醇含量。胆固醇测定方法按照GB/T 5009.128-2003 <食品中胆固醇的测定>。
结果显示：本发明提供的鼠李糖乳酪杆菌对胆固醇的降解率达到19.58 %（此为不含胆盐的数据）。
实施例7 鼠李糖乳酪杆菌的肠道细胞粘附性测试
Caco-2细胞以2×106cells/孔的接种量接种于六孔板，二氧化碳培养箱培养24h，用于细胞粘附实验；将稳定期的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）菌株用MRS培养基重悬至5×108CFU/mL；取1mL鼠李糖乳酪杆菌（Lacticaseibacillusrhamnosus ）菌悬液加入已有细胞贴壁的六孔板，二氧化碳培养箱培养2h；PBS反复洗涤3次，去除未粘附的细菌；加入500µl胰酶消化3分钟，再加入1.5mL细胞培养液终止消化，反复吹打，并将所得溶液收集至无菌EP管中，并将收集的溶液进行10倍、100倍、1000倍、10000倍梯度稀释，涂板计数。同时对空白组的细胞进行计数。根据以下公式计算供试菌株粘附能力：
粘附能力（CFU/cells）=每个培养孔内粘附的细菌总数/每个培养孔的总细胞数。
结果显示，鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）黏附能力为3.1，标准差为1.41%。
综上所述，本发明提供的鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）对常见抗生素敏感，不产溶血素，生物安全性良好。经体外实验验证了鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）对吲哚具有较强的降解能力，能够用于清除尿毒素，缓解慢性肾病。所述鼠李糖乳酪杆菌（Lacticaseibacillus rhamnosus ）可广泛用于制备具有降解肠道吲哚、缓解慢性肾病功能的药品。
Claims (8)
1.一种鼠李糖乳酪杆菌，其特征在于，所述的鼠李糖乳酪杆菌（Lacticaseibacillusrhamnosus）的保藏编号为CCTCC NO：M20221669。
2.如权利要求1所述的鼠李糖乳酪杆菌，其特征在于，所述的鼠李糖乳酪杆菌的Riboprinter 指纹图谱如图2所示，RAPD指纹图谱如图3所示。
3.如权利要求1所述的鼠李糖乳酪杆菌，其特征在于，所述鼠李糖乳酪杆菌的rep-PCR指纹图谱如图4所示，MALDI-TOF-MS蛋白质指纹图谱如图5所示。
4.权利要求1所述的鼠李糖乳酪杆菌在制备具有清除尿毒素、缓解慢性肾病功效的药品中的应用。
5.权利要求1所述的鼠李糖乳酪杆菌在制备具有降低胆固醇功效的药品中的应用。
6.权利要求1所述的鼠李糖乳酪杆菌在制备抗氧化的药品中的应用。
7.一种用于降解肠道吲哚、缓解慢性肾病的药品，其特征在于，所述的药品中包含权利要求1所述的鼠李糖乳酪杆菌的活菌。
8.如权利要求7所述的药品，其特征在于，所述的药品中包含权利要求1所述的鼠李糖乳酪杆菌的发酵产物。
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