CN117946938A - 一株植物乳植杆菌sm3及其在制备降血糖和助睡眠食品药品中的应用 - Google Patents
一株植物乳植杆菌sm3及其在制备降血糖和助睡眠食品药品中的应用 Download PDFInfo
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Abstract
本发明属于益生菌及其应用技术领域,具体涉及一株植物乳植杆菌SM3及其在制备降血糖和助睡眠食品药品中的应用。为了挖掘更多具有益生功能的植物乳植杆菌,本发明从广东地区一名健康成年人的粪便样品中分离纯化得到一株植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株,该菌株可产生和分泌γ‑氨基丁酸,可抑制α‑葡萄糖苷酶活性,具有优越的蛋白酶活性,可抑制乙酰胆碱酯酶活性,可产生和分泌谷胱甘肽,可抑制肾素活性,具有可以抗抑郁、解酒和助睡眠;降糖;促进对蛋白食物的消化吸收,改善蛋白过敏;促进肠道吸收和蠕动等多种功能,在降血糖和助睡眠等领域具有重要的应用价值和经济价值。
Description
技术领域
本发明属于益生菌及其应用技术领域,具体涉及一株植物乳植杆菌SM3及其在制备降血糖和助睡眠食品药品中的应用。
背景技术
植物乳植杆菌(Lactiplantibacillus plantarum),又名植物乳杆菌,属于乳杆菌科乳杆菌属,革兰氏阳性菌,厌氧或兼性厌氧,最适培养pH为6.5,菌体呈直或弯的杆状,单个、有时成对或呈链状。植物乳杆菌是人体胃肠道中的益生菌,可以产生有机酸、细菌素、过氧化氢、双乙酞等多种天然抑菌物质,具有维持肠道菌群平衡,降低胆固醇水平,提高机体免疫力,促进营养物质吸收等多种功能。
植物乳杆菌是一种可食用的乳杆菌,与人类的生活密切相关,常见于乳制品、肉类及许多蔬菜的发酵制品中,其能通过胃部定植于肠道发挥有益作用,对肠道微生物有重要影响,无论是在食品发酵,还是工业乳酸发酵以及医疗保健等领域,都有着广泛的应用。众所周知,乳杆菌是人体正常菌群的一部分,作为肠道正常微生物体系的重要组成并终生伴随宿主,对于维持肠道微生态平衡有重要意义。而植物乳植杆菌作为可食用型的乳酸菌,也是一种重要的肠道益生菌,可以起到帮助肠道蠕动的作用,并且还可以增强人体的消化能力,并提高免疫力等。近些年来,植物乳植杆菌作为一种具有极大潜力的益生性乳杆菌已成为研究的热点,并且正在不断被制作成适用于人和动物的益生菌制剂。
研究发现,植物乳植杆菌的不同菌株具有不同的益生功能,比如:(1)抗病原细菌:有研究发现,植物乳植杆菌对沙门氏菌具有强烈的抑制作用,且对幽门螺杆菌也有抑制作用。(2)调节免疫:有研究表明,植物乳植杆菌HK-LP对小鼠的IL-12具有很强诱导作用,饲喂HK-LP可抑制免疫球蛋白的产生,抵抗小鼠因自然饮食而引起的食物过敏,还可以抑制肿瘤的增长以及肿瘤细胞在小鼠体内的移植。(3)排镉作用:有研究表明,植物乳植杆菌CCFM8610具有耐酸性,在体外对镉离子有良好的耐受功能和吸附能力,能降低镉暴露小鼠肝脏和肾脏中的镉含量,并具有促进镉暴露小鼠通过粪便排镉的作用。(4)抑制大肠埃希氏菌:有研究表明,植物乳植杆菌N13具有耐酸性、耐胆盐的能力,对肠上皮细胞株HT-29细胞具有很高的黏附性,可以抑制肠侵袭性大肠埃希氏菌的生长,并防止器对HT-29发生侵袭。(5)具有蛋白水解活性:有研究表明,植物乳植杆菌CW006具有蛋白水解活性,并能将原料乳发酵生成血管紧张素转换酶抑制肽,进而达到抗高血压的作用。
由于植物乳植杆菌具有来源多样性,导致其具有基因多样性和功能多样性,表现为不同的植物乳植杆菌菌株具有不同的益生功能。目前,虽然有少部分研究开始关注植物乳植杆菌的开发利用,但针对植物乳植杆菌的分离鉴定、益生特性和代谢机制的研究依然较少,这在一定程度上也影响了对植物乳植杆菌的开发利用。因此,有必要根据植物乳植杆菌的不同来源深入开发其益生功能,明确其应用前景,进一步丰富益生性植物乳植杆菌的数量,使其在人类健康方面发挥更大的作用。综上所述,益生性植物乳植杆菌的研究及应用具有较为广阔的发展空间。
发明内容
为了克服上述现有技术的不足,本发明从中国广东地区一名健康成年人的粪便样品中分离纯化得到一株植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株,该菌株可产生和分泌γ-氨基丁酸,可抑制α-葡萄糖苷酶活性,具有优越的蛋白酶活性,可抑制乙酰胆碱酯酶活性,可产生和分泌谷胱甘肽,可抑制肾素活性,在降血糖和助睡眠等领域具有重要的潜在应用价值。
为了实现上述目的,本发明所采用的技术方案是:
本发明第一方面提供了一株植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株,所述副干酪乳酪杆菌SM3菌株于2023年7月3日保藏于中国典型培养物保藏中心,保藏号为:CCTCC NO:M 20231156;所述副干酪乳酪杆菌SM3菌株的16S rDNA全序列如SEQID No:1所示。
本发明第二方面提供了第一方面所述的植物乳植杆菌(Lactiplantibacillusplantarum)SM3菌株在制备肾素抑制剂中的应用。
经研究发现,益生菌植物乳植杆菌SM3菌株可以有效的抑制肾素活性,提示植物乳植杆菌SM3菌株可作为肾素抑制剂用于抑制肾素活性,进而预防和治疗高血压以及其他心血管疾病与肾病。
本发明第三方面提供了第一方面所述的植物乳植杆菌(Lactiplantibacillusplantarum)SM3菌株在产γ-氨基丁酸中的应用。
经研究发现,益生菌植物乳植杆菌SM3菌株可产γ-氨基丁酸(GABA),提示植物乳植杆菌SM3菌株可用于生产GABA,并通过产GABA这一特性用于改善机体睡眠质量、抗抑郁、抗焦虑、降血压、改善脂质代谢等领域。
本发明第四方面提供了第一方面1所述的植物乳植杆菌(Lactiplantibacillusplantarum)SM3菌株在制备α-葡萄糖苷酶抑制剂中的应用。
经研究发现,益生菌植物乳植杆菌SM3菌株可有效抑制α-葡萄糖苷酶活性,而α-葡萄糖苷酶与2型糖尿病有关,抑制α-葡萄糖苷酶是控制餐后高血糖的方法之一,提示植物乳植杆菌SM3菌株有望用于降血糖和抑制肥胖等领域。
本发明第五方面提供了第一方面所述的植物乳植杆菌(Lactiplantibacillusplantarum)SM3菌株在产蛋白酶中的应用。
优选地,所述蛋白酶包括(但不限于)降解牛奶蛋白的蛋白酶。
经研究发现,益生菌植物乳植杆菌SM3菌株可产蛋白酶,提示植物乳植杆菌SM3菌株有望用于生产蛋白酶,并通过产蛋白酶的这一特性应用于促进人体对食物中蛋白质的消化吸收,抗过敏,帮助动物消化吸收营养等领域。
本发明第六方面提供了第一方面所述的植物乳植杆菌(Lactiplantibacillusplantarum)SM3菌株在制备乙酰胆碱酯酶抑制剂中的应用。
经研究发现,益生菌植物乳植杆菌SM3菌株可有效抑制乙酰胆碱酯酶活性,提示植物乳植杆菌SM3菌株可通过抑制乙酰胆碱酯酶活性用于增强认知能力和记忆力,扩张血管,兴奋骨骼肌和平滑肌等领域。
本发明第七方面提供了第一方面所述的植物乳植杆菌(Lactiplantibacillusplantarum)SM3菌株在产还原型谷胱甘肽中的应用。
经研究发现,益生菌植物乳植杆菌SM3菌株可产还原型谷胱甘肽(GSH),提示植物乳植杆菌SM3菌株可用于生产GSH,并通过产GSH这一特性用于抗氧化美白、延缓衰老、增强免疫力、抗肿瘤和抗过敏等领域。
本发明第八方面提供了一种具有益生功能的菌剂,所述菌剂包括第一方面所述的植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株。
优选地,所述菌剂为植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株经发酵后的产物。
优选地,所述菌剂还包括辅料。
更优选地,所述辅料包括载体和赋形剂。所述赋形剂是指可用于药学领域的稀释剂、黏合剂、润滑剂、崩解剂、助溶剂、稳定剂等以及一些药用基质。所述载体是药物领域中可得到的功能性药用辅料,包括表面活性剂、助悬剂、乳化剂以及一些新型药用高分子材料,如环糊精、壳聚糖、聚乳酸(PLA)、聚乙醇酸聚乳酸共聚物(PLGA)、透明质酸等。
优选地,在医药应用领域,所述菌剂的剂型包括片剂、颗粒剂、胶囊剂、滴丸剂、缓释剂、口服液制剂、注射剂。
更优选地,上述剂型是指临床上常用的剂型。药物制剂可以经口服或胃肠外方式(例如静脉、皮下、腹膜内或局部)给药,如果某些药物在胃部条件下是不稳定的,可将其制备成肠衣片剂。
与现有技术相比,本发明的有益效果是:
本发明从中国广东地区一名健康成年人的粪便样品中分离纯化得到一株植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株,该菌株具有多种益生功效,包括可产生和分泌γ-氨基丁酸,可抑制α-葡萄糖苷酶活性,具有优越的蛋白酶活性,可抑制乙酰胆碱酯酶活性,可产生和分泌谷胱甘肽,可抑制肾素活性。因此,植物乳植杆菌SM3菌株具有可以抗抑郁、解酒和助睡眠;降糖;促进对蛋白食物的消化吸收,改善蛋白过敏;促进肠道吸收和蠕动;可抗氧化美白、延缓衰老、抗炎和抗过敏;降血压和保护肾脏等多种功能。可见,本发明新分离得到的植物乳植杆菌SM3菌株具有多种益生功效,可用于降血糖和助睡眠等领域,比如,制作成降血糖和助睡眠的药品,具有重要的应用价值和经济价值。
附图说明
图1为植物乳植杆菌SM3菌株的***发育树(植物乳植杆菌JF1来源于中国发明专利“CN116555075B”,植物乳植杆菌JF4来源于中国发明专利“CN116656526B”,其余建树菌株均来源于NCBI的Genome数据库);
图2为植物乳植杆菌SM3菌株可以产生和分泌γ-氨基丁酸;
图3为植物乳植杆菌SM3发酵液可分泌物质显著抑制α-葡萄糖苷酶活性;
图4为植物乳植杆菌SM3菌株对牛奶平板的降解实验(左边,空白对照组;右边,实验组);
图5为植物乳植杆菌SM3发酵液可分泌物质显著抑制乙酰胆碱酯酶活性;
图6为植物乳植杆菌SM3菌株可以产生和分泌GSH;
图7为植物乳植杆菌SM3发酵液可分泌物质显著抑制肾素活性。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例中的实验方法,如无特殊说明,均为常规方法,下述实施例中所用的试验材料,如无特殊说明,均为可通过常规的商业途径购买得到。
下列实施例涉及的实验材料如下:
(1)菌株:植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株,委托润泽实验室分离自中国广东广州地区一名健康成年人(BMI=19.5)的肠道粪便样品中,于甘油管内置于-80℃低温冷冻保藏。一般情况下,将该菌株接种于MRS固体培养基平板表面并在37℃恒温厌氧培养箱中倒置培养24h可获得菌落,或在37℃恒温厌氧培养箱内于MRS液体培养基中振荡培养24-48h可获得发酵液。
(2)试剂盒:γ-氨基丁酸(GABA)检测试剂盒(Cloud-Clone Corp.,Cat:CEA900Ge),α-葡萄糖苷酶抑制剂筛选试剂盒(abcam,Cat:ab284520),乙酰胆碱酯酶抑制剂筛选试剂盒(abnova,Cat:KA6219),微量还原型谷胱甘肽(GSH)测定试剂盒(南京建成,Cat:A006-2-1),肾素(Renin)抑制剂筛选试剂盒(abnova,Cat:KA1361)。
(3)MRS平板:牛肉膏10g,蛋白胨10g,酵母膏5g,柠檬酸三铵2g,乙酸钠5g,葡萄糖20g,磷酸氢二钾2g,吐温80 1mL,硫酸镁0.58g,硫酸锰0.25g,琼脂15g,ddH2O补齐至1L,调pH至6.2-6.6,121℃高压灭菌20min,配置成MRS平板。
(4)MRS液体培养基:牛肉膏10g,蛋白胨10g,酵母膏5g,柠檬酸三铵2g,乙酸钠5g,葡萄糖20g,磷酸氢二钾2g,吐温80 1mL,硫酸镁0.58g,硫酸锰0.25g,ddH2O补齐至1L,调pH至6.2-6.6,121℃高压灭菌20min,配置成MRS液体培养基。
(5)MP平板:脱脂奶粉10g,氯化钠1g,牛肉膏10g,蛋白胨10g,酵母膏5g,葡萄糖20g,柠檬酸三铵2g,乙酸钠5g,磷酸氢二钾2g,吐温80 0.5mL,硫酸镁0.58g,硫酸锰0.25g,琼脂15g,ddH2O补齐至1L,调pH至6.2-6.6,121℃高压灭菌20min,配置成MP平板。
实施例1植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株的分离鉴定
植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株从中国广东地区一名健康成年人的粪便样品中分离得到,具体如下:
用无菌水将粪便样品反复清洗3次,置于研钵中,每100mg粪便加入500uL无菌水,彻底研磨匀浆,用移液器吸取适量研磨液,涂布于MRS平板上,室温培养3天。然后,用记号笔对分离实验平板中待划线纯化的菌落进行编号,并相应地在平板上标明菌株编号。标记后,挑取菌落接种到MRS平板上,用平板划线法纯化菌株。若此法不能将菌株分离则需要从富集平板上挑取菌落,经MRS液体培养基梯度稀释后,涂布于培养基上。最后,参考《伯杰细菌鉴定手册》(第八版)和《真菌分类鉴定手册》,首先分辨属于细菌的菌株。初步分离得到一株纯化菌株,菌株编号为SM3,培养24小时后观察到其菌落呈白色圆形、中央凸起,菌落表面细密而光滑。
接着,通过16S rDNA通用引物(27F:AGAGTTTGATCCTGGCTCAG,1492R:TACGGCTACCTTGTTACGACTT)分离得到的SM3菌株进行分子鉴定后,由北京百迈客生物科技有限公司对SM3菌株进行全基因组测序。将得到的16S rDNA sequence(SEQ ID No:1)在NCBI的Genome数据库进行BLAST比对。结果显示,SM3菌株与已知的植物乳植杆菌(Lactiplantibacillus plantarum)16S rDNA序列的同源性>99%,并与同源菌株进行进化分析(图1),确认SM3是同种不同株的植物乳植杆菌。
最后,对菌株SM3进行保藏,保藏信息如下:保藏时间:2023年7月3日;保藏单位名称:中国典型培养物保藏中心(CCTCC);保藏号:CCTCC NO:M 20231156;保藏单位地址:中国.武汉.武汉大学;分类命名:Lactiplantibacillus plantarum。
植物乳植杆菌是可用于食品的益生菌菌株,具有广泛的益生功效,如抗炎、抗高血压和抗病原细菌等,但不同来源的菌株其功效不同,说明本发明从广东地区一名健康成年人的粪便样品中分离得到的新的植物乳植杆菌SM3菌株可以作为益生菌使用,并可能具有新的功效和功能。
Lactiplantibacillus plantarum SM3 16S rDNA sequence(1434bp,SEQ ID No:1):
GTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAA。
实施例1植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株的功能及其应用
(1)植物乳植杆菌SM3菌株可产生和分泌γ-氨基丁酸(GABA)
将用MRS液体培养基培养至稳定期的植物乳植杆菌SM3以1:30的稀释倍数扩培至新的MRS液体培养基中,在培养至稳定期24h时收取菌悬液,10,000×g、4℃离心10min后收取发酵液上清,然后通过GABA特定ELISA试剂盒(CEA900Ge)测定发酵液上清的GABA浓度。结果显示,与空白培养基MRS中低浓度GABA相比,菌株SM3的发酵上清液中GABA的浓度显著性增加,可积累量为546.0pg/mL,说明植物乳植杆菌SM3可以在稳定期产生并分泌γ-氨基丁酸(图2)。
γ-氨基丁酸是一种重要的中枢神经***抑制性神经递质,广泛存在于动植物、微生物体内。现已证实,作为小分子量非蛋白质氨基酸的GABA具备食用安全性,可作为食品添加剂。研究表明,摄入一定量的GABA具有改善机体睡眠质量、抗抑郁、抗焦虑、降血压、改善脂质代谢、增强记忆能力并提高脑活力、加快大脑新陈代谢、健肝利肾、促进乙醇代谢(解酒)、改善更年期综合症等生理功效。
因此,益生菌植物乳植杆菌SM3菌株可通过产生γ-氨基丁酸的功效发挥以上多个用途。
(2)植物乳植杆菌SM3菌株发酵液可有效抑制α-葡萄糖苷酶活性
将用MRS液体培养基培养至稳定期的植物乳植杆菌SM3以1:30的稀释倍数扩培至新的MRS液体培养基中,在培养至稳定期24h时收取菌悬液,10,000×g、4℃离心10min后收取发酵液上清,然后通过α-葡萄糖苷酶抑制剂筛选试剂盒(ab284520)测定发酵液上清对α-葡萄糖苷酶水解葡萄糖的酶活性能力的影响。结果显示,与空白培养基MRS的无抑制作用相比,菌株SM3的发酵上清液显著抑制了α-葡萄糖苷酶水解葡萄糖的能力,其抑制率约为18.48%,说明植物乳植杆菌SM3的发酵液可以有效的抑制α-葡萄糖苷酶的活性(图3)。
a-葡萄糖苷酶是一种在碳水化合物分解中起作用的酶,与2型糖尿病有关,抑制α-葡萄糖苷酶是控制餐后高血糖的方法之一,从而有助于糖尿病的治疗。因此,α-葡萄糖苷酶抑制剂有助于维持血糖平,能够改善糖尿病并发症。此外,抑制α-葡萄糖苷酶的活性可以控制肥胖。
因此,益生菌植物乳植杆菌SM3菌株具有抑制a-葡萄糖苷酶活性的能力,这使它成为潜在的降血糖和抑制肥胖的益生菌。
(3)植物乳植杆菌SM3菌株可产蛋白酶
根据琼脂孔扩散试验使用脱脂牛奶平板培养基(MP平板)鉴定和测量植物乳植杆菌SM3分泌蛋白酶水解蛋白的能力。试验时,往实验组的MP平板中滴加3uL浓度为10Abs的植物乳植杆菌SM3菌液,对照组则滴加3uL空白MRS培养基,并在37℃恒温厌氧培养箱中倒置培养3天。结果表明,与滴加空白培养基的对照相比,菌株SM3可以显著的降解蛋白,形成明显的降解圈(图4),说明植物乳植杆菌SM3菌株可产降解牛奶蛋白的蛋白酶。
植物乳植杆菌SM3可产蛋白酶,其作为益生菌菌株使用可以促进人体对食物中蛋白质的消化吸收,提高对小肽和氨基酸的吸收。同时,也可用于抗过敏(改善蛋白消化不良或不吸收导致的食物过敏)。此外,也可以用于蛋白酶的提取,应用于食品工业或洗涤工业等蛋白酶的生产中;也可用于微生物饲料中以帮助动物消化吸收营养,提高对饲料的利用率。
(4)植物乳植杆菌SM3菌株发酵液可有效抑制乙酰胆碱酯酶活性
将用MRS液体培养基培养至稳定期的植物乳植杆菌SM3以1:30的稀释倍数扩培至新的MRS液体培养基中,在培养至稳定期24h时收取菌悬液,10,000×g、4℃离心10min后收取发酵液上清,然后通过乙酰胆碱酯酶抑制剂筛选试剂盒(KA6219)测定发酵液上清对乙酰胆碱酯酶(AchE)活性的抑制能力。结果显示,与空白培养基MRS的无抑制作用相比,菌株SM3的发酵上清液显著抑制了乙酰胆碱酯酶的活性,其抑制率约为37.02%,说明植物乳植杆菌MY3的发酵液可以有效的抑制乙酰胆碱酯酶的活性(图5)。
乙酰胆碱酯酶抑制剂通过对乙酰胆碱酯酶的可逆性抑制,让胆碱能够在神经纤维末梢释放的乙酰胆碱的作用下不断堆积于突触处,兴奋胆碱受体,延长并且增加乙酰胆碱的作用。乙酰胆碱酯酶抑制剂可增强认知能力和记忆力(改善阿尔茨海默症、改善认知障碍),扩张血管(改善心率过速),兴奋骨骼肌和平滑肌(改善便秘、术后腹胀、术后尿潴留)。
因此,益生菌植物乳植杆菌MY3菌株可抑制乙酰胆碱酯酶活性,可以兴奋骨骼肌和肠道的平滑肌,这使它成为潜在的促进胃肠道吸收和蠕动的益生菌。
(5)植物乳植杆菌SM3菌株可产生和分泌还原型谷胱甘肽(GSH)
将用MRS液体培养基培养至稳定期的植物乳植杆菌SM3菌株以1:30的稀释倍数扩培至新的MRS液体培养基中,在培养至稳定期24h时收取菌悬液,10,000×g、4℃离心10min后收取发酵液上清,然后通过还原型谷胱甘肽(GSH)测定试剂盒(A006-2-1)测定发酵液上清GSH的浓度。结果显示,与空白培养基MRS中低浓度的GSH相比,菌株SM3的发酵上清液中GSH的浓度为284.04μmol/L,提示经SM3发酵后GSH的浓度显著增加(****P<0.0001),说明植物乳植杆菌SM3可以在稳定期产生并分泌谷胱甘肽(GSH)(图6)。
谷胱甘肽(glutathione,GSH)是由谷氨酸、半胱氨酸和甘氨酸组成,且含有γ-酰胺键和巯基的三肽,具有抗氧化作用和整合解毒作用。半胱氨酸上的巯基为谷胱甘肽活性基团(故谷胱甘肽常简写为GSH)。谷胱甘肽有助于维持正常的免疫***功能,并具有抗氧化作用和整合解毒作用,在多种细胞生化过程中发挥重要作用,如自由基中和、解毒、半胱氨酸的运输和储存、细胞氧化还原作用的维持、抗坏血酸和维生素E的再生等。主要包括以下几个方面:
①解毒作用:与毒物或药物结合,消除其毒性作用;
②参与氧化还原反应:作为重要的还原剂,参与体内多种氧化还原反应;
③保护巯基酶的活性:使巯基酶的活性基团(-SH)维持还原状态;
④维持红细胞膜结构的稳定:消除氧化剂对红细胞膜结构的破坏作用。
由此,GSH的多种生物学功能赋予了其多种功效和用途,主要表现在:
1)抗氧化剂:清除人体内的自由基,保护许多蛋白质和酶等分子中的巯基不被有害物质氧化,从而保证蛋白质和酶等分子生理功能的正常发挥;人体红细胞中谷胱甘肽的含量很多,这对于保护红细胞膜上蛋白质的巯基处于还原状态,防止溶血具有重要意义;还可防止皮肤老化及色素沉着,减少黑色素的形成,改善皮肤抗氧化能力,并使皮肤产生光泽。
2)临床药物:利用其巯基螯合重金属、氟化物、芥子气等毒素而防止中毒;还可以在肝炎、溶血性疾病以及角膜炎、白内障和视网膜疾病等方面作为治疗或辅助治疗的药物;还可以纠正乙酰胆碱、胆碱酯酶的不平衡,起到抗过敏作用。
3)食品添加剂:强化食品营养、稳定维生素C、强化风味。
综上所述,谷胱甘肽不仅可以用于药物,而且可以作为功能性食品的基料,在抗氧化美白、延缓衰老、增强免疫力、抗肿瘤和抗过敏等功能性食品领域具有广泛的应用价值。
由此,益生菌植物乳植杆菌SM3菌株可以通过产生的GSH的功能,从而发挥以上多个功效。
(6)植物乳植杆菌SM3菌株发酵液可有效抑制肾素活性
将用MRS液体培养基培养至稳定期的植物乳植杆菌SM3以1:30的稀释倍数扩培至新的MRS液体培养基中,在培养至稳定期24h时收取菌悬液,10,000×g、4℃离心10min后收取发酵液上清,然后通过肾素抑制剂筛选试剂盒(KA1361)测定发酵液上清对肾素的抑制能力。结果显示,与空白培养基MRS的无抑制作用相比,菌株SM3的发酵上清液具有抑制肾素的能力,其抑制率约为21.14%(*P<0.05),说明植物乳植杆菌SM3的发酵液可以有效的抑制肾素(图7)。
肾素是一种约40kDa的天冬氨酸蛋白酶,能够将血管紧张素原转化为血管紧张素I。血管紧张素转换酶(ACE)则是血管内皮中发现的一种单体锌金属酶,可以将血管紧张素Ⅰ转换为血管紧张素Ⅱ,而血管紧张素Ⅱ是肾素-血管紧张素***(RAS)通路的最终活性信使。血管紧张素II可以通过直接作用于肾小球旁细胞来抑制肾素的分泌。血管紧张素II有许多生理作用,其中最重要的是其可以作为一种强大的血管收缩剂,通过改变周围血管的阻力来增加血压。由于血管紧张素原是唯一已知的肾素底物,通过肾素切割血管紧张素原是影响RAS通路的最终活性的关键所在,故抑制肾素将是控制高血压的一个有吸引力的策略。此外,肾素抑制剂可以阻止血管紧张素I和血管素的形成。因此,可以利用肾素抑制剂抑制肾素活性的作用来预防和治疗高血压以及其他心血管疾病与肾病。
可见,益生菌植物乳植杆菌SM3菌株具有抑制肾素的能力,这使它成为潜在的降血压和保护肾脏的益生菌。
综上可知,本发明新分离的植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株具有多种益生功效:(1)可产生和分泌γ-氨基丁酸;(2)可抑制α-葡萄糖苷酶活性;(3)具有优越的蛋白酶活性;(4)可抑制乙酰胆碱酯酶活性;(5)可产生和分泌谷胱甘肽;(6)可抑制肾素活性。可见,本发明新分离得到的植物乳植杆菌SM3菌株在降血糖和助睡眠等领域具有重要的应用价值和经济价值。
以上对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (9)
1.一株植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株,其特征在于,所述副干酪乳酪杆菌SM3菌株于2023年7月3日保藏于中国典型培养物保藏中心,保藏号为:CCTCC NO:M 20231156;所述副干酪乳酪杆菌SM3菌株的16S rDNA全序列如SEQ ID No:1所示。
2.权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株在制备肾素抑制剂中的应用。
3.权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株在产γ-氨基丁酸中的应用。
4.权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株在制备α-葡萄糖苷酶抑制剂中的应用。
5.权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株在产蛋白酶中的应用。
6.权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株在制备乙酰胆碱酯酶抑制剂中的应用。
7.权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株在产还原型谷胱甘肽中的应用。
8.一种具有益生功能的菌剂,其特征在于,所述菌剂包括权利要求1所述的植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株。
9.根据权利要求8所述的一种具有益生功能的菌剂,其特征在于,所述菌剂为植物乳植杆菌(Lactiplantibacillus plantarum)SM3菌株经发酵后的产物。
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