CN116555115B - 一种植物乳杆菌nxu0011及其用途 - Google Patents
一种植物乳杆菌nxu0011及其用途 Download PDFInfo
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Abstract
本发明属于微生物技术领域，具体涉及一种植物乳杆菌NXU0011及其用途。该植物乳杆菌NXU0011保藏于中国普通微生物菌种保藏管理中心，保藏编号为CGMCCNO：26970。该菌对大肠杆菌以及金黄色葡萄球菌具有良好的抑制效果，植物乳杆菌NXU0011还具有良好的抗氧化性能，并可用于制备降血糖药物；同时植物乳杆菌NXU0011在唾液、胃液、肠液中的存活率较高，具有成为良好的益生效应调节剂的潜力。
Description
技术领域
本发明属于微生物技术领域，具体涉及一种植物乳杆菌NXU0011及其用途。
背景技术
益生菌是通过定殖在人体内，改变宿主某一部位菌群组成的一类对宿主有益的活性微生物。通过调节宿主黏膜与***免疫功能或通过调节肠道内菌群平衡，促进营养吸收保持肠道健康的作用，从而产生有利于健康作用的单微生物或组成明确的混合微生物。人体、动物体内有益的细菌或真菌主要有酵母菌、益生芽孢菌、丁酸梭菌、乳杆菌、双歧杆菌、放线菌等。目前研究发现酵母菌具有调节肠道平衡、促进饲料转化以及提高机体免疫功能等益生特性；酵母菌有酿酒酵母属、德尔布有孢圆酵母属、假丝酵母属、威克汉姆酵母属、毕赤酵母属、布拉氏酵母属、白球拟酵母属、薛瓦酵母属、深红酵母属、粟酒裂殖酵母属、鲍氏酵母属等，酵母菌可以作为食品发酵的益生菌使用，也可以药用，或者添加到饲料中用以促进动物的生长发育、缩短饲养期、增加肉量和蛋量、改良肉质和提高瘦肉率、改善皮毛的光泽度、增强幼禽畜的抗病能力等；益生芽孢杆菌是一类对机体具有益生特性的、好氧或兼性厌氧、产芽孢的杆状细菌，为革兰氏染色阳性菌，芽孢杆菌在工农业及医药生产中均有广泛应用；丁酸梭菌，是从健康人和动物肠道中分离出的一种厌氧的革兰氏阳性芽孢杆菌，研究发现丁酸梭菌具有维持肠道菌群平衡、增强机体免疫功能、生成营养物质、防治肠炎等良好的益生特性；双歧杆菌是人体内存在的一种生理性细菌，对人体健康具有生物屏障、营养作用、抗肿瘤作用、免疫增强作用、改善胃肠道功能、抗衰老等多种重要的生理功能；乳酸菌指发酵糖类主要产物为乳酸的一类无芽孢、革兰氏染色阳性细菌的总称，乳酸菌是最常见的益生菌，已经被广泛应用并认为对人和动物是安全的，乳酸菌能促进动物生长，调节胃肠道正常菌群、维持微生态平衡，从而改善胃肠道功能、提高食物消化率和生物效价、降低血清胆固醇、控制内毒素、抑制肠道内腐败菌生长、提高机体免疫力等。
糖尿病是一种以高血糖为特征的代谢性疾病。高血糖则是由于胰岛素分泌缺陷或其生物作用受损，或两者兼有引起。长期存在的高血糖，导致各种组织，特别是眼、肾、心脏、血管、神经的慢性损害、功能障碍。乳酸菌由于其益生作用和安全性，被广泛应用于发酵食品，潜在的防治糖尿病功能也已得到广泛认可，已被作为一种安全有效降血糖的生物效应调节剂。现有发现的具有降血糖功能的乳酸菌不胜枚举，其筛选来源广泛涉及各地传统乳酸菌发酵制品。但多数乳酸菌对于DPPH的清除率为82.92％左右；或在人工肠液中存活率不高，对模拟人工胃肠液中耐受效果不够理想。因此，研究效果更佳的乳杆菌是十分必要的。
发明内容
鉴于以上技术问题，本发明提供一种植物乳杆菌NXU0011，其对DPPH自由基清除率高达95.17％，模拟唾液、胃液、肠液存活率分别为119.81％、39.27％、122.52％。
本发明第一方面，提供一种植物乳杆菌NXU0011，其保藏编号为CGMCC NO：26970。
本发明第二方面，提供一种所述植物乳杆菌NXU0011的发酵方法，包括以下步骤：
将所述植物乳杆菌NXU0011接种于MRS液体培养基中，在37±5℃下培养24～48h，得到植物乳杆菌NXU0011发酵液。
本发明第三方面，提供一种根据上述方法发酵得到的发酵液。
本发明第四方面，提供一种所述植物乳杆菌NXU0011或所述发酵液在制备降血糖产品中的用途。
优选地，所述植物乳杆菌NXU0011或所述发酵液用于制备α-淀粉酶抑制剂；
所述植物乳杆菌NXU0011或所述发酵液用于制备α-葡萄糖苷酶抑制剂。
本发明第五方面，提供一种所述植物乳杆菌NXU0011或所述发酵液在制备抗氧化产品中的用途。
本发明第六方面，提供一种所述植物乳杆菌NXU0011或所述发酵液在制备抑菌产品中的用途。
优选地，所述抑菌是抑制大肠杆菌、金黄色葡萄球菌中的一种或两种。
对比现有技术，本发明的有益效果为：
本发明提供一株植物乳杆菌菌株NXU0011，植物乳杆菌NXU0011对大肠杆菌以及金黄色葡萄球菌具有良好抑菌性能，其抑菌直径分别达16.83mm和16.28mm。其DPPH自由基清除率高达95.17％，具有良好的抗氧化性能。模拟唾液、胃液、肠液存活率分别为119.81％、39.27％、122.52％。α-淀粉酶抑制率为85.35％，α-葡萄糖苷酶抑制率为51.74％，整体上对于降血糖仍具有较明显效果。
生物材料保藏信息：
植物乳杆菌菌株NXU0011，分类命名为植物乳杆菌，拉丁名：Lactobacillusplantarum。该菌株NXU0011于2023年3月31日保藏在中国普通微生物菌种保藏管理中心，保藏编号为CGMCC NO：26970，保藏地址为北京市朝阳区北辰西路1号院3号。
附图说明
图1是植物乳杆菌NXU0011在MRS培养基上菌落形态；
图2是植物乳杆菌NXU0011在光学显微镜下菌体形态；
图3是植物乳杆菌NXU0011在NCBI同源性比对结果；
图4是植物乳杆菌NXU0011的生长曲线；
图5是植物乳杆菌NXU0011对大肠杆菌K12的抑菌效果；图中不同编号表示三组平行试验；
图6是植物乳杆菌NXU0011对金黄色葡萄球菌ATCC6538的抑菌效果，图中1、2、3表示三组平行试验。
具体实施方式
本发明结合实施例和相应附图做进一步阐释说明，以下实施例仅用于说明目的，不用于限制本发明范围。
实施例1
植物乳杆菌NXU0011分离与筛选
1、样品稀释
取25g宁夏银川市贺兰县塞尚金河乳业周边土壤样本于225mL无菌水中，均质操作，得到10-1的土壤菌悬液。吸取1mL该土壤菌悬液于9mL无菌水中进行充分混匀得到10-2梯度，以此类推，得到其余10-3、10-4、10-5、10-6、10-7梯度稀释液备用。
2、浇注培养
取10-5、10-6、10-7梯度土壤菌悬液各1mL，分别注入各平板，每个梯度做三组平行。于高压蒸汽灭菌的MRS培养基中添加质量分数5％碳酸钙作为指示剂，加热融化，冷却至45℃左右，每个平板浇注约20mL培养基。操作完毕后，将平板倒置于37℃恒温培养箱中培养48h。
3、菌落观察
选取菌落分布较为均匀的平板进行观察。选择在培养基表面或者内部生长情况良好，有透明圈，菌落边缘整齐，白色有光泽，并且易挑起不粘连培养基的菌落。
4、划线分离
接种环挑取菌株，在平板上划线分离操作。操作完毕后，将平板倒置于37℃恒温培养箱中培养48h。
5、液体培养
挑取菌落于MRS液体培养基中富集培养。37℃恒温培养48h，得到富集菌悬液。
6、光学显微镜镜检
接种环挑取一环液体培养的富集菌悬液于载玻片制片，革兰氏染色后，100倍油镜观察菌体形态。
菌株NXU0011的最适生长温度为35～37℃，最适pH为6.5左右。其菌落生物特征为：菌落呈圆形，光滑湿润且凸起，边缘整齐，呈乳白色，如图1。厌氧或兼性厌氧，革兰氏制片染色后观察为革兰氏阳性菌，无芽孢鞭毛，且菌体形态均为直的短杆菌，两端钝圆，单个、一对、或短链状，如图2。
实施例2
植物乳杆菌NXU0011鉴定
使用细菌基因组DNA提取试剂盒对不同乳酸菌进行基因提取，采用乳酸菌的16SrDNA的通用引物进行PCR扩增。PCR扩增引物序列为FA-27F:5’GCAG AGTTCTCGGAGTCACGAAGAGTTTGATCCTGGCTCAG-3’(如SEQ ID NO：1所示)、RA-1495R:5’AGCGGATCACTTCACACAGGACTACGGGTACCTTGTT ACGA-3’(如SEQ ID NO：2所示)，以提取菌株的基因组DNA作为扩增模板，其PCR扩增体系为10×PCR buffer 5μL、dNTP 4μL、引物(F+R)3μL、Ta q DNA酶0.5μL、基因组DNA模板1.5μL，双蒸水6μL。扩增设定条件为：94℃预变性5min，94℃变性1min、58℃退火1min、72℃延伸2min、经过30个循环后，72℃末端延伸10min，4℃保温。PCR产物点样于1％的琼脂糖凝胶中跑胶。取2μL PCR产物与6×Loading Buffer混合后加入点样孔，电泳跑胶，于凝胶成像仪进行观察，在1500bp处观察到一条清晰的条带，并未见明显的非特异性扩增。
通过16S rDNA的扩增产物测序，如SEQ ID NO：3所示，具体如下：
GGGTGTGACTATAGATGCAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGCAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGAGCAGA
登录NCBI网站，BLAST序列比对结果显示，菌株与植物乳杆菌的16S rDNA同源性达到99.72％，可鉴定该菌为植物乳杆菌，命名为植物乳杆菌NXU0011，如图3所示。
实施例3
植物乳杆菌NXU0011的生长曲线测定
将植物乳杆菌NXU0011菌株以2.0％的接种量接种于MRS液体培养基中，置于37℃恒温培养箱，培养24h。每隔2h取一次发酵菌液，以无菌的MRS的液体培养基为对照组，使用紫外分光光度计测定发酵液中菌株生长量。OD600nm处，平行测定3次，记录菌的吸光值，得到生长曲线如图4所示。
生长曲线图显示，植物乳杆菌NXU0011在0～8h为迟滞期，OD值变化幅度不大；8～12h为对数生长期，OD值呈指数增长；12h～24h进入稳定期，OD值增长缓慢，后呈一定稳定趋势。
实施例4
植物乳杆菌NXU0011的益生功能试验
1、消化应激能力测定：
将菌种于8000×g离心10min，无菌PBS(pH 7.2)洗涤后将菌体沉淀悬浮于模拟唾液中，37℃培养5min，8000×g离心10min后将菌体沉淀重悬于模拟胃液中，37℃培养3h，8000×g离心10min后再将菌体沉淀重悬于模拟肠液中，37℃培养2h；每次应激后均采用平板菌落计数法测定乳杆菌的活菌数，每组设置三个平行，37℃培48h，按公式计算存活率：
结果显示，植物乳杆菌NXU0011在模拟唾液中37℃培养5min后，存活率达119.81％；在模拟胃液中37℃培养3h后，存活率为39.27％；在模拟肠液中37℃培养2h后，存活率达122.52％。
2、DPPH自由基清除能力测定
分别取2mL DPPH无水乙醇溶液、0.2mL菌液及1.8mL无水乙醇溶液混合在一起，混匀后黑暗中避光反应30min，8000r/min离心10min后取上清，测定其在波长517nm处的吸光度值记为A1。以无水乙醇代替所测样品溶液作为对照组，并将吸光度值记为A0。以无水乙醇取代DPPH无水乙醇溶液作为空白对照组，并将吸光度值记录为A2。每组样品平行测定3次取平均值。测定DPPH自由基清除率能力的计算公式：
结果测得DPPH自由基清除率为95.17％。
3、抑菌能力测定
选用常见的2种病原菌:大肠杆菌和金黄色葡萄球菌作为指示菌，将大肠杆菌菌悬液及金黄色葡萄球菌的菌悬液浓度均调整为108～109CFU/mL，采用牛津杯法测定菌株发酵液对大肠杆菌和金黄色葡萄球菌的体外抑菌效果。
取大肠杆菌菌悬液lmL、金黄色葡萄球菌悬液lmL，与经融化后保持在45℃左右的MRS培养基15mL迅速混合均匀倒入平板，待其充分冷却凝固后，放入已灭菌的牛津杯数个，并一定次序排列整齐。取发酵液180μL加入牛津杯中，37℃温箱培养18h后用游标卡尺测量抑菌圈直径，每株益生菌设3个重复，取其抑菌圈直径的平均值作为最终抑菌圈直径，根据美国临床实验室标准化委员会药敏试验判断标准，将抑菌圈直径＞15mm认定为具有较强抑菌能力，可用于后续菌株的筛选。
测量得到植物乳杆菌NXU0011对大肠杆菌抑菌圈直径为16.83mm，对金黄色葡萄球菌的抑菌圈直径为16.28mm，如图5(图中显示三组平行实验)、图6(图中显示三组平行实验)。
4、α-淀粉酶抑制率测定
用0.25mL植物乳杆菌NXU0011菌液与1mg/mL的α-淀粉酶溶液等体积混合，于37℃孵育10min，然后将反应液加入至37℃的0.5mL质量分数1.5％的可溶性淀粉溶液中，于37℃反应5min，再加入1mL DNS溶液，在沸水浴中反应5min后迅速冷却至室温，稀释10倍后静置30min，并于540nm处测定吸光值。用PBS溶液(0.1mol/L，pH＝6.8)作为空白对照，每组设定3个平行。
式中：
A为样品组，含有样品溶液和α-淀粉酶溶液；
B为样品空白组，含有样品溶液不含α-淀粉酶溶液；
C为对照组，不含样品溶液含α-淀粉酶溶液；
D为空白对照组，不含样品溶液不含α-淀粉酶溶液。
植物乳杆菌NXU0011与α-淀粉酶作用后，其催化效率大大下降，抑制率达85.35％。
5、α-葡萄糖苷酶抑制率测定
在96孔板210μL的反应体系中，加入30μL的菌液、50μLPBS溶液(0.1mol/L，pH＝6.8)和50μL浓度为2.5mmol/L的PNPG溶液，37℃孵育10min。然后加入30μL浓度为0.4U/mL的α-葡萄糖苷酶溶液37℃继续反应30min，最后加入50μL的浓度为1mol/L的Na2CO3溶液终止反应。反应完成后，于酶标仪405nm处测定吸光值。用PBS溶液(0.1mol/L，pH＝6.8)作为空白对照，每组设定3个平行。
式中：
A为样品组，含有样品溶液和α-葡萄糖苷酶溶液；
B为样品空白组，含有样品溶液不含α-葡萄糖苷酶溶液；
C为对照组，不含样品溶液含α-葡萄糖苷酶溶液；
D为空白对照组，不含样品溶液不含α-葡萄糖苷酶溶液。
试验以卡拉波糖的抑制作为阳性对照试验，其抑制率为62.34％，植物乳杆菌NXU0011的抑制率为51.74％。
实施例中所使用的实验方法如无特殊说明，均为常规方法。实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
应当理解的是，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (2)
1.一种植物乳杆菌NXU0011或其发酵液在制备降血糖产品中的用途，其特征在于，所述植物乳杆菌NXU0011的保藏编号为CGMCC NO：26970；
所述发酵液的发酵方法包括以下步骤：将所述植物乳杆菌NXU0011接种于MRS液体培养基中，在37±5℃下培养24～48h，得到植物乳杆菌NXU0011发酵液；
所述植物乳杆菌NXU0011或其发酵液用于制备α-淀粉酶抑制剂或α-葡萄糖苷酶抑制剂，所述植物乳杆菌NXU0011在肠液中的存活率为122.52％，所述植物乳杆菌NXU0011的发酵液对α-淀粉酶的抑制率为85.35％、对α-葡萄糖苷酶的抑制率为51.74％。
2.一种权利要求1所述的植物乳杆菌NXU0011或其发酵液在制备抗氧化产品中的用途，所述抗氧化包括用于清除自由基DPPH，所述植物乳杆菌NXU0011对自由基DPPH的清除率为95.17％。
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