CN117004503B - 一株唾液联合乳杆菌mb1及其在制备助睡眠和调理肠胃食品药品中的应用 - Google Patents
一株唾液联合乳杆菌mb1及其在制备助睡眠和调理肠胃食品药品中的应用 Download PDFInfo
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- CN117004503B CN117004503B CN202310234523.1A CN202310234523A CN117004503B CN 117004503 B CN117004503 B CN 117004503B CN 202310234523 A CN202310234523 A CN 202310234523A CN 117004503 B CN117004503 B CN 117004503B
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- salivarius
- saliva
- ligilactobacillus
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Abstract
本发明属于益生菌及其应用技术领域,具体涉及一株唾液联合乳杆菌MB1及其在制备助睡眠和调理肠胃食品药品中的应用。为进一步开发利用唾液联合乳杆菌的益生功能,本发明从一名健康成年人的粪便中分离得到一株唾液联合乳杆菌Ligilactobacillussalivarius MB1菌株,菌株MB1具有产蛋白酶、3‑羟基丁酸、γ‑氨基丁酸、透明质酸,以及抑制α‑葡萄糖苷酶活性和抑制乙酰胆碱酯酶活性等多种益生功效。因此,唾液联合乳杆菌MB1菌株具有可以促进对蛋白食物的消化吸收而改善蛋白过敏、抗炎和改善肠道菌群、抗抑郁和解酒、抗衰和抗皱、降糖、促进肠道吸收和蠕动等多种功能,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,具体涉及一株唾液联合乳杆菌MB1及其在制备助睡眠和调理肠胃食品药品中的应用。
背景技术
唾液联合乳杆菌(Ligilactobacillussalivarius),又名唾液乳杆菌,为革兰氏阳性杆菌,属于乳杆菌科、乳杆菌属,大小一般为0.6~0.9μm×1.5~5μm,两端圆,具有单个、双生和成长度不一的链,不运动,无鞭毛。唾液联合乳杆菌因首次在人体口腔唾液中被分离获得而得名,其对氧气无特殊要求,适宜生长的温度是37℃,几乎在人体所有的分泌物中都能被检测到,因此是人体内广泛存在的乳杆菌,也说明了唾液联合乳杆菌是存在于人体内的正常菌株。此外,唾液联合乳杆菌具有良好的耐酸和耐胆盐特性,能在人和动物的肠胃中生存。
唾液联合乳杆菌广泛地存在于人和动物的肠道中,并随粪便排出。作为人体正常菌群的一部分,唾液联合乳杆菌是肠道正常微生物体系的重要组成之一并终生伴随宿主,对于维持肠道微生态平衡有重要意义。作为肠道菌群中的益生菌,也是一种可食用型的乳酸菌,唾液联合乳杆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以及提高免疫力等。近些年来,唾液联合乳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,并且正在不断被用来制作成适用于人和动物的益生菌制剂。
唾液联合乳杆菌的不同菌株已被报道具有不同的益生功能:(1)抗菌作用:有研究表明,口服含有唾液联合乳杆菌WB21的片剂,能减少牙周炎病原菌的数量,调节口腔微生态平衡,并对牙周环境有一定的保护作用。(2)唾液联合乳杆菌具有一定的吸附作用,能够很好地在肠道表面定殖生长进而发挥免疫调节作用。有研究表明,唾液联合乳杆菌B1可以改善仔猪肠道微生物并增加肠道中免疫活性细胞的数量,有益于完善仔猪的免疫系统和激活局部免疫活性。(3)抗衰老作用:有研究表明,给线虫饲喂来自广西巴马百岁老人粪便中的唾液联合乳杆菌FDB89,可延长线虫的平均寿命,具有抗衰老的作用。(4)降胆固醇作用:随着人们生活水平的不断提高,摄入的营养也越加丰富,致使体内血清胆固醇的含量升高,从而引发高血压、冠心病、动脉硬化等一系列的疾病。有研究表明,唾液联合乳杆菌FBC05在体外对胆固醇具有降解作用。
唾液联合乳杆菌来源的多样性导致其具有基因的多样性和功能的多样性。然而,目前针对唾液联合乳杆菌的分离鉴定、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唾液联合乳杆菌的开发利用。因此,有必要根据唾液联合乳杆菌的不同来源使其更好地发挥作用,如根据菌株的功能或者益生代谢产物确定其功效,明确其应用前景。综上所述,益生性唾液联合乳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,本发明从一名健康成年人的粪便中分离得到一株唾液联合乳杆菌Ligilactobacillus salivarius MB1菌株,菌株MB1具有的多种益生功效,主要包括产蛋白酶、3-羟基丁酸、γ-氨基丁酸等,具有重要的潜在应用价值。
为实现上述目的,本发明是通过以下技术方案来实现的:
本发明第一方面提供了一株唾液联合乳杆菌(Ligilactobacillussalivarius)MB1菌株,所述唾液联合乳杆菌MB1菌株于2022年09月27日保藏于中国典型培养物保藏中心,保藏号为:CCTCC NO:M 20221502;所述唾液联合乳杆菌MB1菌株的16S rDNA全序列如SEQ ID No:1所示。
本发明第二方面提供了第一方面所述的唾液联合乳杆菌(Ligilactobacillussalivarius)MB1菌株在产蛋白酶中的应用。
经研究发现,益生菌唾液联合乳杆菌MB1菌株可产蛋白酶,提示唾液联合乳杆菌MB1菌株有望用于生产蛋白酶,并通过产蛋白酶的这一特性用于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,抗过敏等领域。
本发明第三方面提供了第一方面所述的唾液联合乳杆菌(Ligilactobacillussalivarius)MB1菌株在产3-羟基丁酸中的应用。
经研究发现,益生菌唾液联合乳杆菌MB1菌株可产3-羟基丁酸(3-HB),提示唾液联合乳杆菌MB1菌株有望用于生产3-HB,并通过产3-HB的这一特性用于为身体各种活动提供能量,抗骨质疏松,预防及治疗慢性综合征,改善脑部认知功能,改善脂代谢等领域。
本发明第四方面提供了第一方面所述的唾液联合乳杆菌(Ligilactobacillussalivarius)MB1菌株在产γ-氨基丁酸中的应用。
经研究发现,益生菌唾液联合乳杆菌MB1菌株可产γ-氨基丁酸(GABA),提示唾液联合乳杆菌MB1菌株有望用于生产GABA,并通过产GABA的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、增强记忆能力,提高脑活力等领域。
本发明第五方面提供了第一方面所述的唾液联合乳杆菌(Ligilactobacillussalivarius)MB1菌株在产透明质酸中的应用。
经研究发现,益生菌唾液联合乳杆菌MB1菌株可产透明质酸(HA),提示唾液联合乳杆菌MB1菌株有望用于生产HA,并通过产HA的这一特性用于抗炎,抗血管生成,抗衰老,促进伤口消炎及愈合等领域。
本发明第六方面提供了第一方面所述的唾液联合乳杆菌(Ligilactobacillussalivarius)MB1菌株在制备α-葡萄糖苷酶抑制剂中的应用。
经研究发现,益生菌唾液联合乳杆菌MB1菌株可有效抑制α-葡萄糖苷酶活性,而a-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,提示唾液联合乳杆菌MB1菌株有望用于降血糖和抑制肥胖等领域。
本发明第七方面提供了第一方面所述的唾液联合乳杆菌(Ligilactobacillussalivarius)MB1菌株在制备乙酰胆碱酯酶抑制剂中的应用。
经研究发现,益生菌唾液联合乳杆菌MB1菌株可有效抑制乙酰胆碱酯酶活性,提示唾液联合乳杆菌MB1菌株有望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,促进胃肠道吸收和蠕动等领域。
本发明第八方面提供了一种用于具有益生功能的菌剂,所述菌剂包括第一方面所述的唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株。
优选地,所述唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株为经发酵后的菌体混合物。发酵所用的培养基为MRS培养基。
优选地,在医药应用领域,所述菌剂还包括药学上可接受的载体和/或赋形剂。
更优选地,上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。上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注射剂、片剂、颗粒剂、胶囊剂、滴丸剂、缓释剂、口服液制剂。
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:
本发明从一名健康成年人的粪便中分离得到一株唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株,菌株MB1具有的多种益生功效,包括具有优越的蛋白酶活性,产生和分泌3-羟基丁酸,产生和分泌γ-氨基丁酸,产生和分泌透明质酸,抑制α-葡萄糖苷酶活性,抑制乙酰胆碱酯酶活性。因此,唾液联合乳杆菌MB1菌株具有可以促进对蛋白食物的消化吸收,改善蛋白过敏;抗炎和改善肠道菌群;抗抑郁和解酒;抗衰和抗皱;降糖;促进肠道吸收和蠕动等多种功能。可见,本发明新分离得到的唾液联合乳杆菌菌株MB1菌株具有多种益生功效,可用于助消化、调理肠胃和美白抗皱等领域,比如,制作成助睡眠和调理肠胃食品药品,具有重要的应用价值和经济价值。
附图说明
图1为唾液联合乳杆菌MB1与其他同源乳酸菌的系统发育树;
图2为唾液联合乳杆菌MB1对牛奶平板的降解实验(左边,空白对照组;右边,实验组);
图3为唾液联合乳杆菌MB1可以产生和分泌3-羟基丁酸;
图4为唾液联合乳杆菌MB1可以产生和分泌γ-氨基丁酸;
图5为唾液联合乳杆菌MB1可以产生和分泌透明质酸;
图6为唾液联合乳杆菌MB1发酵液可分泌物质显著抑制α-葡萄糖苷酶活性;
图7为唾液联合乳杆菌MB1发酵液可分泌物质显著抑制乙酰胆碱酯酶活性。
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:唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株,由中山大学生命科学学院肠道微生物组研究润泽实验室分离自中国广东一名健康的成年人的粪便中,保存于甘油管内-80℃低温冷冻保藏。一般情况下,将其接种于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以获得菌落,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以获得发酵液。
(2)试剂盒:3-羟基丁酸(3-HB)检测试剂盒(Cloud-Clone Corp.,Cat:CEB022Ge),γ-氨基丁酸(GABA)检测试剂盒(Cloud-Clone Corp.,Cat:CEA900Ge),透明质酸(又名玻尿酸,HA)检测试剂盒(Cloud-Clone Corp.,Cat:CEA182Ge),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)。
(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唾液联合乳杆菌(Ligilactobacillus salivarius)MB1的分离鉴定
唾液联合乳杆菌Ligilactobacillus salivarius MB1从中国广东省广州市海珠区一名健康成年人(男性,34岁,BMI=24.4)的粪便中分离得到,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,彻底研磨成匀浆,然后用移液器吸取适量研磨液,涂布于MRS平板上,室温培养3天。用记号笔将分离实验平板中待划线纯化的菌落编号,并相应地在平板上标明菌株编号。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MRS平板上。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MB1,培养24小时后观察到菌株的落菌落较小,无色,表面光滑,不透明,凸起,边缘整齐。
接着,通过16S rDNA通用引物(27F:AGAGTTTGATCCTGGCTCAG,1492R:TACGGCTACCTTGTTACGACTT)进行分子鉴定后,由北京百迈客生物科技有限公司对分离得到的L.salivarius MB1菌株进行全基因组测序。将得到的序列16S rDNA sequence(SEQ IDNo:1)在NCBI的Genome数据库进行BLAST比对。结果表明,菌株MB1与已知的唾液联合乳杆菌16S rDNA序列的同源性>99%,并与同源菌株进行进化树分析(图1),确认菌株MB1是同种不同株的唾液联合乳杆菌。
最后,对菌株MB1进行保藏,保藏信息如下:保藏时间:2022年09月27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 20221502;保藏单位地址:中国.武汉.武汉大学;分类命名:Ligilactobacillus salivarius。
唾液联合乳杆菌是国家批准可用于食品的益生菌菌株,具有广泛的益生功效,如抗炎、抗衰老和降胆固醇等,但不同来源的菌株其功效不同,说明本发明从人体粪便中分离得到的新的唾液联合乳杆菌MB1可以作为益生菌使用,并可能具有新的功效和功能。
L.salivarius 16S rDNA sequence(1436bp,SEQ ID No:1):
ATACTGCAGTCGACGAAACTTTCTTACACCGAATGCTTGCATTCACCGTAAGAAGTTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTAAAAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATATCTCTAAGGATCGCATGATCCTTAGATGAAAGATGGTTCTGCTATCGCTTTTAGATGGACCCGCGGCGTATTAACTAGTTGGTGGGGTAACGGCCTACCAAGGTGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACACGAGTGAGAGTAACTGTTCATTCGATGACGGTATCTAACCAGCAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAACGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGTAGTGCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGTTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGATTAGGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAAGTTGGGCACTCTGGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAGACCGCGAGGTTTAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCGCAAGGAGCCAGCCGTCTAA。
实施例2唾液联合乳杆菌(Ligilactobacillus salivarius)MB1的功能及其应用
(1)唾液联合乳杆菌MB1菌株可产降解牛奶蛋白的蛋白酶
唾液联合乳杆菌MB1分泌蛋白酶水解蛋白能力的鉴定和测量是根据琼脂孔扩散试验进行的,使用的培养基为脱脂牛奶平板培养基(MP平板)。试验时,往实验组滴加3uL浓度为10Abs的唾液联合乳杆菌MB1菌液,对照组滴加3uL空白MRS培养基。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,MB1可以显著的降解蛋白,并形成明显的降解圈(图2),说明唾液联合乳杆菌MB1可产蛋白酶。
可见,唾液联合乳杆菌MB1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领域的生产;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。
(2)唾液联合乳杆菌MB1菌株可产生和分泌3-羟基丁酸(3-HB)
将用MRS液体培养基培养至稳定期的唾液联合乳杆菌MB1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,通过3-HB特定ELISA试剂盒(CEB022Ge)测定发酵液上清的3-HB浓度。结果显示,与空白培养基MRS中不存在3-HB相比,菌株MB1的上清液中3-HB的浓度为1.658μg/mL,说明唾液联合乳杆菌MB1可以在稳定期产生并分泌3-羟基丁酸(图3)。
3-HB可以为身体各种活动提供能量,是潜在的能量/功能型食物,已被添加到运动员饮料中,因此益生菌唾液联合乳杆菌MB1可用作为能量型食品的添加剂。同时,鉴于3-HB可以有效的抗骨质疏松,预防及治疗慢性综合征(高血压,酒精性脂肪肝,肠炎,肠癌等),改善脑部认知功能(提高学习记忆的能力,保护神经胶质细胞,改善阿尔兹海默症等),改善脂代谢。因此,益生菌唾液联合乳杆菌MB1菌株可通过产生3-羟基丁酸的功效发挥以上多个用途。
此外,3-羟基丁酸是机体天然产生的一种内源性小分子物质,对维持结肠直肠组织完整性具有重要作用,并且具有维持肠道健康、防止结肠疾病和消炎产能的作用。3-HB可以促进肠道有益菌增殖,缓解多发性硬化的症状,在调节菌群改善健康方面具有巨大的潜力。因此,益生菌唾液联合乳杆菌MB1菌株也有助于改善肠道菌群,缓解肠道炎症。
(3)唾液联合乳杆菌MB1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唾液联合乳杆菌MB1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,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MB1的发酵上清液中GABA的浓度显著性增加,可积累量为90.81pg/mL,说明唾液联合乳杆菌MB1可以在稳定期产生并分泌γ-氨基丁酸(图4)。
γ-氨基丁酸是一种重要的中枢神经系统抑制性神经递质,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,益生菌唾液联合乳杆菌MB1菌株可通过产生γ-氨基丁酸的功效发挥以上多个用途。
(4)唾液联合乳杆菌MB1菌株可产生和分泌透明质酸(HA)
将用MRS液体培养基培养至稳定期的唾液联合乳杆菌MB1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,通过透明质酸(又名玻尿酸,HA)特定ELISA试剂盒(CEA182Ge)测定发酵液上清的HA浓度。结果显示,与空白培养基MRS的中低浓度HA相比,菌株MB1的发酵上清液中HA的浓度显著性增加,可积累量为156.8ng/mL,说明唾液联合乳杆菌MB1可以在稳定期产生并分泌透明质酸(图5)。
透明质酸,又名玻尿酸,是一种可生物降解、生物相容性、无毒、无致敏性的聚合物,具有多种生物学功能。既具有抗炎和抗血管生成作用,又具有较强的抗衰老、水润、抚平皱纹的能力。由于其有助于皮肤抗皱,促进伤口消炎及愈合,可用于抗皱剂,具有开发皮肤化妆品的潜力。此外,由于HA具有很高的润滑、吸水和保水能力,可以影响多种细胞功能,如迁移、粘附和增殖,使得HA也被广泛应用于眼科手术,关节炎治疗,伤口愈合支架,组织工程,以及植入材料等生物医学领域。
因此,益生菌唾液联合乳杆菌MB1菌株可通过产生透明质酸的功效发挥以上多个用途。
(5)唾液联合乳杆菌MB1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唾液联合乳杆菌MB1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,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,MB1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64.33%,说明唾液联合乳杆菌MB1的发酵液可以有效的抑制α-葡萄糖苷酶的活性(图6)。
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,益生菌唾液联合乳杆菌MB1菌株具有抑制a-葡萄糖苷酶活性,这使它成为潜在的降血糖和抑制肥胖的益生菌。
(6)唾液联合乳杆菌MB1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唾液联合乳杆菌MB1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,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,MB1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10.25%,说明唾液联合乳杆菌MB1的发酵液可以有效的抑制乙酰胆碱酯酶的活性(图7)。
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,延长并且增加了乙酰胆碱的作用。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,益生菌唾液联合乳杆菌MB1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。
综上可知,本发明新分离的唾液联合乳杆菌菌株MB1具有的多种益生功效:(1)具有优越的蛋白酶活性;(2)可产生和分泌3-羟基丁酸;(3)可产生和分泌γ-氨基丁酸;(4)可产生和分泌透明质酸;(5)可抑制α-葡萄糖苷酶活性;(6)可抑制乙酰胆碱酯酶活性。可见,本发明新分离得到的唾液联合乳杆菌菌株MB1菌株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)
1.一株唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株,其特征在于,所述唾液联合乳杆菌MB1菌株于2022年09月27日保藏于中国典型培养物保藏中心,保藏号为:CCTCC NO:M 20221502;所述唾液联合乳杆菌MB1菌株的16S rDNA全序列如SEQ ID No:1所示。
2.权利要求1所述的唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株在产降解牛奶蛋白的蛋白酶中的应用。
3.权利要求1所述的唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株在产3-羟基丁酸中的应用。
4.权利要求1所述的唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株在产γ-氨基丁酸中的应用。
5.权利要求1所述的唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株在产透明质酸中的应用。
6.权利要求1所述的唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株在制备α-葡萄糖苷酶抑制剂中的应用。
7.权利要求1所述的唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株在制备乙酰胆碱酯酶抑制剂中的应用。
8.一种用于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所述的唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株。
9.根据权利要求8所述的一种用于具有益生功能的菌剂,其特征在于,所述唾液联合乳杆菌(Ligilactobacillus salivarius)MB1菌株为经发酵后的菌体混合物。
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