CN117384788B - 一株唾液联合乳杆菌sm4及其在制备美白和降胆固醇食品药品中的应用 - Google Patents
一株唾液联合乳杆菌sm4及其在制备美白和降胆固醇食品药品中的应用 Download PDFInfo
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Abstract
本发明属于益生菌及其应用技术领域,具体涉及一株唾液联合乳杆菌SM4及其在制备美白和降胆固醇食品药品中的应用。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唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株,该菌株具有的多种益生功效,包括产生和分泌3‑羟基丁酸,产生和分泌γ‑氨基丁酸,产生和分泌透明质酸,产生和分泌谷胱甘肽,可清除DPPH自由基,可清除羟自由基,具有超氧化物歧化酶活性,可降解胆固醇。因此,该菌株具有抗氧化美白,延缓衰老,抗炎和抗过敏,抗氧化和抗炎,降胆固醇和改善心血管健康等多种功能,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SM4及其在制备美白和降胆固醇食品药品中的应用。
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检测到,因此是人体内广泛存在的乳杆菌,也说明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,并广泛地存在于人和动物的肠道中。
唾液联合乳杆菌是食品生产加工的菌种之一,广泛地存在于人和动物的肠道中,并随粪便排出。乳杆菌是人体正常菌群的一部分,其作为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具有重要意义。近年来,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近年研究的热点,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。
研究表明,不同的唾液联合乳杆菌菌株具有不同的益生功能,比如:(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。
由于唾液联合乳杆菌的来源具有多样性,导致其具有基因多样性和功能多样性。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,已发现具有益生功能的唾液联合乳杆菌还不是很多,且虽然目前针对唾液联合乳杆菌的功能及应用已经取得了一定的研究进展,但其潜在功效仍远未得到较完整的开发。因此,有必要挖掘更多具有益生功能的唾液联合乳杆菌,并根据唾液联合乳杆菌的不同来源进一步挖掘其功能,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,深入研究益生性唾液联合乳杆菌及其应用有很广阔的发展空间。
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唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株,该菌株具有产生和分泌3-羟基丁酸,产生和分泌γ-氨基丁酸,产生和分泌透明质酸,产生和分泌谷胱甘肽等多种功能,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唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株,所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4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唾液联合乳杆菌(Ligilactobacillussalivarius)SM4菌株在产谷胱甘肽中的应用。
经研究发现,益生菌唾液联合乳杆菌SM4菌株可产透明质酸(HA),提示唾液联合乳杆菌SM4菌株有望用于生产HA,并通过产HA的这一特性应用于抗氧化美白、延缓衰老、增强免疫力、抗肿瘤和抗过敏等领域。
本发明第三方面提供了第一方面所述的唾液联合乳杆菌(Ligilactobacillussalivarius)SM4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SM4菌株可产3-羟基丁酸(3-HB),提示唾液联合乳杆菌SM4菌株有望用于生产3-HB,并通过产3-HB的这一特性应用于抗骨质疏松,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)SM4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SM4菌株可产γ-氨基丁酸(GABA),提示唾液联合乳杆菌SM4菌株有望用于生产GABA,并通过产GABA的这一特性应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)SM4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SM4菌株可产透明质酸(HA),提示唾液联合乳杆菌SM4菌株有望用于生产HA,并通过产HA的这一特性应用于抗炎和抗血管生成作用,抗衰老、水润、抚平皱纹,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)SM4菌株在制备DPPH自由基清除剂中的应用。
经研究发现,益生菌唾液联合乳杆菌SM4菌株可清除DPPH自由基,提示唾液联合乳杆菌SM4菌株有望用于制备DPPH自由基清除剂,并通过清除DPPH自由基发挥抗氧化的能力,进而避免DPPH自由基给机体带来的危害。
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)SM4菌株在制备羟自由基清除剂中的应用。
经研究发现,益生菌唾液联合乳杆菌SM4菌株可清除羟自由基(·OH),提示唾液联合乳杆菌SM4菌株有望用于制备·OH自由基清除剂,并通过清除·OH自由基发挥抗氧化的能力,进而避免·OH自由基给机体带来的危害。
本发明第八方面提供了第一方面所述的唾液联合乳杆菌(Ligilactobacillussalivarius)SM4菌株在制备超氧化物歧化酶活性增强剂中的应用。
经研究发现,益生菌唾液联合乳杆菌SM4菌株具有超氧化物歧化酶(SOD)活性,提示唾液联合乳杆菌SM4菌株有望用于制备SOD活性增强剂,并通过SOD活性应用于抗氧化、抗炎、增强免疫力、抗衰老、降血脂、增强肝功能、改善视力、降血糖等领域。
本发明第九方面提供了第一方面所述的唾液联合乳杆菌(Ligilactobacillussalivarius)SM4菌株在制备降解或降低胆固醇的制剂中的应用。
经研究发现,益生菌唾液联合乳杆菌SM4菌株可降解胆固醇,提示唾液联合乳杆菌SM4菌株有望用于制备降解或降低胆固醇的制剂,并通过降解胆固醇的功能,从而避免胆固醇过量给机体带来的危害。
本发明第十方面提供了一种制备谷胱甘肽的方法,具体为:将第一方面所述的唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株接种于MRS液体培养基中,培养至稳定期后收取菌悬液,最后对菌悬液进行分离即得谷胱甘肽。
优选地,培养的条件为:37℃恒温厌氧。
优选地,先用MRS液体培养基将唾液联合乳杆菌(Ligilactobacillussalivarius)SM4菌株培养至稳定期,再以1:20-1000的稀释倍数扩培至新的MRS液体培养基中。
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唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株,菌株SM4具有的多种益生功效,包括产生和分泌3-羟基丁酸,产生和分泌γ-氨基丁酸,产生和分泌透明质酸,产生和分泌谷胱甘肽,可清除DPPH自由基,可清除羟自由基,具有超氧化物歧化酶活性,可降解胆固醇。因此,唾液联合乳杆菌SM4菌株具有抗炎和改善肠道菌群;抗抑郁、解酒和助睡眠;抗衰和抗皱;抗氧化美白、延缓衰老、抗炎和抗过敏;抗氧化和抗炎;抗衰老;增强免疫力和护肝;降胆固醇和改善心血管健康等多种功能。可见,本发明新分离得到的唾液联合乳杆菌SM4菌株具有多种益生功效,可用于美白和降胆固醇等领域,比如,制作成美白和降胆固醇药品,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SM4菌株的系统发育树(菌株来源于NCBI的Genome数据库,其中Ligilactobacillus salivarius MB1为发明人之前申报的另一个菌株,保藏号为CCTCC NO:M20221502,申请号为202310234523.1);
图2为唾液联合乳杆菌SM4菌株可以产生和分泌3-羟基丁酸;
图3为唾液联合乳杆菌SM4菌株可以产生和分泌γ-氨基丁酸;
图4为唾液联合乳杆菌SM4菌株可以产生和分泌透明质酸;
图5为唾液联合乳杆菌SM4菌株可以产生和分泌GSH;
图6为唾液联合乳杆菌SM4菌株可以清除DPPH自由基;
图7为唾液联合乳杆菌SM4菌株可以清除羟自由基;
图8为唾液联合乳杆菌SM4菌株的发酵液具有SOD活性;
图9为胆固醇的标准曲线;
图10为唾液联合乳杆菌SM4菌株可降胆固醇。
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)唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株,分离自中国广东广州地区一名健康成年人(BMI=18.8)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: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),微量还原型谷胱甘肽(GSH)测定试剂盒(南京建成,Cat:A006-2-1),DPPH自由基清除能力试剂盒(南京建成,Cat:A153-1-1),羟自由基(·OH)测定试剂盒(南京建成,Cat:A018-1-1),超氧化物歧化酶(SOD)测定试剂盒(南京建成,Cat:A001-3)。
(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MRSMRS液体培养基。
实施例1唾液联合乳杆菌(Ligilactobacillus salivarius SM4)菌株的分离鉴定
唾液联合乳杆菌Ligilactobacillus salivarius SM4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,然后观察菌落的生长状况:生长形态、是否有菌丝、颜色是否均匀一致,菌落个体表面及边缘。最终初步分离得到一株纯化菌株,菌株编号为SM4,培养24小时后观察到菌株的菌落表面光滑湿润,呈乳白色,边缘整齐,隆起不透明的菌落。
接着,通过16S rDNA通用引物(27F:AGAGTTTGATCCTGGCTCAG,1492R:TACGGCTACCTTGTTACGACTT)对分离得到的SM4菌株进行分子鉴定后,由北京百迈客生物科技有限公司对SM4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,结果表明,SM4菌株与已知的唾液联合乳杆菌(Ligilactobacillus salivarius)16S rDNA序列的同源性>99%,并与同源菌株进行进化分析(图1),确认菌株SM4是同种不同株的唾液联合乳杆菌。
最后,对菌株SM4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 20231168;保藏单位地址:中国.武汉.武汉大学;分类命名:Ligilactobacillussalivarius。
唾液联合乳杆菌是一种益生菌菌株,其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分离得到的一株新的唾液联合乳杆菌SM4可以作为益生菌使用,并可能具有新的功效和功能。
Ligilactobacillussalivarius SM416S rDNA sequence(1434bp,SEQ ID No:1):
ATACTGCAGTCGACGAACTTTCTTACACCGAATGCTTGCATTCANCGTAAGAAGTTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTAAAAGAAGGGGATAACACTTGGAAANAGGTGCTAATACCGTATATCTCTAAGGATCGCATGATCCTTAGATGAAAGATGGTTCTGCTATCGCTTTTAGATGGACCCGCGGCGTATTAACTAGTTGGTGGGGTAACGGCCTACCAAGGTGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACACGAGTGAGAGTAACTGTTCATTCGATGACGGTATCTAACCAGCAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAACGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGTAGTGCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGTTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGATTAGGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAAGTTGGGCACTCTGGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAGACCGCGAGGTTTAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCGCAAGGAGCCAGCCGTCTA。
实施例2唾液联合乳杆菌(Ligilactobacillus salivarius SM4)菌株的功能及其应用
(1)唾液联合乳杆菌SM4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SM4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SM4的上清液中3-HB的浓度为0.49μg/mL,说明唾液联合乳杆菌SM4可以在稳定期产生并分泌3-羟基丁酸(图2)。
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SM4可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),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),改善脂代谢。因此,益生菌唾液联合乳杆菌SM4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。通过3-HB处理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SM4菌株也有助于改善肠道菌群,缓解肠道炎症。
(2)唾液联合乳杆菌SM4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SM4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MRS对照相比,菌株SM4的发酵上清液中GABA的浓度显著性增加,可积累量为540.27pg/mL,说明唾液联合乳杆菌SM4可以在稳定期产生并分泌γ-氨基丁酸(图3)。
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具备改善机体睡眠质量、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SM4菌株可通过产生γ-氨基丁酸的功效发挥以上多个用途。
(3)唾液联合乳杆菌SM4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SM4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MRS对照相比,菌株SM4的发酵上清液中HA的浓度显著性增加,可积累量为60.15ng/mL,说明唾液联合乳杆菌SM4可以在稳定期产生并分泌透明质酸(图4)。
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SM4菌株可通过产生透明质酸的功效发挥以上多个用途。
(4)唾液联合乳杆菌SM4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唾液联合乳杆菌SM4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MRS对照相比,菌株SM4的发酵上清液中GSH的浓度为261.9μmol/L,经SM4发酵后GSH的浓度显著增加(***P<0.001),说明唾液联合乳杆菌SM4可以在稳定期产生并分泌谷胱甘肽(GSH)(图5)。
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系统功能,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,谷胱甘肽可以用于药物,在抗氧化美白、延缓衰老、增强免疫力、抗肿瘤和抗过敏等领域具有广泛的应用价值。
由此,益生菌唾液联合乳杆菌SM4菌株可以通过产生的GSH的功能,从而发挥以上多个功效。
(5)唾液联合乳杆菌SM4菌株可清除DPPH自由基
将用MRS液体培养基培养至稳定期的唾液联合乳杆菌SM4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DPPH自由基清除能力试剂盒(南京建成,Cat:A153-1-1)测定发酵液上清对DPPH的清除能力。结果显示,与空白对照MRS相比,菌株SM4的发酵上清液对DPPH自由基清除率为21.5%(***P<0.001),说明唾液联合乳杆菌SM4具有较好的清除DPPH自由基的能力,表现出良好的抗氧化能力(图6)。
自由基,化学上也称为“游离基”,是指化合物的分子在光热等外界条件下,共价键发生均裂而形成的具有不成对电子的原子或基团。由于自由基含未配对的电子,所以极不稳定(特别是羟自由基),因此会从邻近的分子(包括脂肪、蛋白质和DNA)上夺取电子,让自己处于稳定的状态。这样一来,邻近的分子又变成一个新的自由基,然后再去夺取电子。如此连锁反应会让细胞的结构受到破坏,造成细胞功能丧失、基因突变,甚至死亡。
自由基有诸多坏处。诸如:(1)削弱细胞的抵抗力,使身体易受细菌和病菌感染;(2)产生破坏细胞的化学物质,形成致癌物质;(3)阻碍细胞的正常发展,干扰其复原功能,使细胞更新率低于枯萎率;(4)破坏体内的遗传基因(DNA)组织,扰乱细胞的运作及再生功能,造成基因突变,演变成癌症;(5)破坏细胞内的线粒体(能量储存体),造成氧化性疲劳;(6)破坏细胞膜,干扰细胞的新陈代谢,使细胞膜丧失保护细胞的功能;(7)侵袭细胞组织及荷尔蒙所必须的氨基酸,干扰体内系统的运作,导致恶性循环,以致产生更多自由基,其连锁反应会导致自由基危害遍及全身;(8)破坏蛋白质,破坏体内的酶,导致炎症和衰老;(9)破坏脂肪,使脂质过氧化,导致动脉粥样硬化,发生心脑血管疾病;(10)破坏碳水化合物,使透明质酸降解,导致关节炎等。
由此,益生菌唾液联合乳杆菌SM4菌株可以通过清除DPPH自由基的功能,从而避免该自由基给机体带来的危害。
(6)唾液联合乳杆菌SM4菌株可清除羟自由基(·OH)
将用MRS液体培养基培养至稳定期的唾液联合乳杆菌SM4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,然后通过羟自由基(·OH)测定试剂盒(南京建成,Cat:A018-1-1)测定发酵液上清对羟自由基的清除能力。结果显示,与空白对照MRS相比,菌株SM4的发酵上清液对羟自由基清除率为24.37%(**P<0.01),说明唾液联合乳杆菌SM4具有较好的清除羟自由基的能力,表现出良好的抗氧化能力(图7)。
羟基自由基(·OH)是一种重要的活性氧,从分子式上看是由氢氧根(OHˉ)失去一个电子形成的。羟基自由基具有极强的得电子能力,也就是氧化能力,氧化电位为2.8V,是自然界中仅次于氟的氧化剂。
羟基自由基几乎可以破坏所有类型的大分子,包括碳水化合物、核酸(突变)、脂质(脂质过氧化)和氨基酸(例如苯丙氨酸转化为间酪氨酸和邻酪氨酸)等。羟自由基对人体的攻击首先是从细胞膜开始的,导致细胞膜失去弹性和功能,引发心血系统疾病。大量资料证明,炎症、肿瘤、衰老、血液病,以及心、肝、脾、肺和皮肤等各方面疾病的发生机理均与体内自由基产生过多或者清除自由基能力下降有着密切的关系。
由此,益生菌唾液联合乳杆菌SM4菌株可以通过清除羟自由基的功能,从而避免该自由基给机体带来的危害。
(7)唾液联合乳杆菌SM4菌株具有超氧化物歧化酶(SOD)活性
将用MRS液体培养基培养至稳定期的唾液联合乳杆菌SM4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,然后通过超氧化物歧化酶(SOD)测定试剂盒(南京建成,Cat:A001-3)测定发酵液上清的SOD活性。其中,每毫升反应液中SOD抑制率达50%时所对应的SOD量为一个SOD活力单位(U)。结果显示,与空白对照MRS相比,菌株SM4的发酵上清液中SOD的活性为32.23U/mg,说明唾液联合乳杆菌SM4发酵液具有较好的SOD活性,表现出良好的抗氧化能力(图8)。
超氧化物歧化酶(SOD)是一种抗氧化酶,具有抗氧化、抗炎、增强免疫力等功效与作用。主要表现在:(1)抗氧化:SOD能够清除细胞内的自由基,减轻氧化应激对细胞的损伤,保护细胞免受自由基的攻击。(2)抗炎:SOD能够抑制炎症反应,减少炎症细胞的释放,降低炎症的程度和持续时间。(3)增强免疫力:SOD能够促进免疫细胞的产生,提高身体的免疫力,预防疾病的发生和恢复身体的健康。(4)抗衰老:可防止脂质过氧化,有效清除自由基,使细胞保持活性,防止机体过快衰老。(5)降血脂:能清除血管内阻力,软化血管,降低血液粘稠度,使血脂水平逐渐恢复正常。(6)增强肝功能:可以减少血液中的毒素,减轻氧自由基对肝脏造成的损伤。(7)改善视力:有助于清除眼内过多的自由基,从而提高视力,预防白内障。(8)降血糖:可使胰岛细胞恢复活力,加快分裂速度,进而达到降血糖的目的。
由此,益生菌唾液联合乳杆菌SM4菌株可以通过具有超氧化物歧化酶(SOD)活性的功能,从而发挥以上多个功效。
(8)唾液联合乳杆菌SM4菌株可降解胆固醇
制备邻苯二甲醛溶液:准确称量0.05g邻苯二甲醛试剂,用冰乙酸溶解并定容至100mL,备用。同时制备胆固醇标准储备液(0.1mg/mL),4℃冷藏备用。按2倍梯度稀释胆固醇储备液并加入冰乙酸至2mL,然后加入邻苯二甲醛溶液4mL,室温放置10min,再于冷水浴中缓慢加入18.4M浓硫酸4mL,涡旋混匀后,自然冷却,静置10min后,在20min内测定550nm处的吸光度值,构建胆固醇的标准曲线(图9)。
将用MRS液体培养基培养至稳定期的唾液联合乳杆菌SM4菌株以1:30的稀释倍数扩培至新的MRS-胆固醇培养基中,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,然后测定上清液在550nm处的吸光度值,并根据胆固醇的标准曲线,计算上清液中的胆固醇含量。结果显示,与空白对照MRS相比,菌株SM4的发酵上清液对胆固醇的清除率为12.70%(***P<0.001),说明唾液联合乳杆菌SM4菌株具有降胆固醇的能力(图10)。
胆固醇是身体组织的重要组成部分,过高的胆固醇摄入已经成为诱发冠心病、动脉粥样硬化、脑中风等心脑血管疾病的重要因素。一项大规模的研究结果显示,控制胆固醇含量的升高不仅能够减少患心脏病的风险,而且还有助于避免患肾衰竭之类的肾脏疾病。
总胆固醇过高会导致各种疾病。主要表现在:(1)胆固醇结石:肝胆固醇的高量分泌会导致胆固醇过饱和,胆固醇的结晶与晶体生长过快,进而破坏胆汁酸肝肠循环系统的平衡,引起机体胆固醇结石的形成。(2)肺癌:高胆固醇饮食可能与肺癌的发生风险增加相关,但饮食中胆固醇摄入量受个人体质、膳食组成或其他高脂饮食的影响,并不能准确反应体内胆固醇的真实水平,而肺内胆固醇水平与肺癌发病风险呈更为密切的正相关。(3)冠状动脉硬化:血清LDL和三酰甘油水平增高是动脉粥样硬化病变形成的重要原因。降低LDL胆固醇水平已成为减少动脉粥样硬化病变以及由此引发的心脑血管疾病等并发症的重要目标。动脉粥样硬化病变始于LDL的局部累积,LDL通过黏附于富含蛋白多糖的细胞外基质蛋白上而被隔离在内皮下,并通过内皮通透性的改变、细胞间转运以及活性受体介导的胞移等机制发生累积作用。(4)高胆固醇血症:膳食胆固醇摄入过量(≥300mg/d)的男女性人群发生高胆固醇血症的危险性明显增高。(5)冠心病和缺血性脑卒:胆固醇或低密度脂蛋白胆固醇升高是冠心病和缺血性脑卒中的独立危险因素。血浆高密度脂蛋白胆固醇可能与炎症、血栓形成等疾病相关。
由此,益生菌唾液联合乳杆菌SM4菌株可以通过降解胆固醇的功能,从而避免胆固醇过量给机体带来的危害。
综上可知,本发明新分离的唾液联合乳杆菌菌株SM4菌株具有的多种益生功效:(1)可产生和分泌3-羟基丁酸;(2)可产生和分泌γ-氨基丁酸;(3)可产生和分泌透明质酸;(4)可产生和分泌谷胱甘肽;(5)可清除DPPH自由基;(6)可清除羟自由基;(7)具有超氧化物歧化酶活性;(8)可降解胆固醇。可见,本发明新分离得到的唾液联合乳杆菌菌株SM4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 (10)
1.唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株在产谷胱甘肽中的应用,其特征在于,所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4菌株的16S rDNA全序列如SEQ ID No:1所示。
2.唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株在产3-羟基丁酸中的应用,其特征在于,所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4菌株的16S rDNA全序列如SEQ ID No:1所示。
3.唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株在产γ-氨基丁酸中的应用,其特征在于,所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4菌株的16S rDNA全序列如SEQ ID No:1所示。
4.权利要求1所述的唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株在产透明质酸中的应用,所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4菌株的16S rDNA全序列如SEQ ID No:1所示。
5.唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株在制备DPPH自由基清除剂中的应用,其特征在于,所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4菌株的16SrDNA全序列如SEQ ID No:1所示。
6.唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株在制备羟自由基清除剂中的应用,其特征在于,所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4菌株的16S rDNA全序列如SEQ ID No:1所示。
7.唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株在产超氧化物歧化酶中的应用,其特征在于,所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4菌株的16S rDNA全序列如SEQ ID No:1所示。
8.唾液联合乳杆菌(Ligilactobacillus salivarius)SM4菌株在制备降解或降低胆固醇的制剂中的应用,其特征在于,所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4菌株的16S rDNA全序列如SEQ ID No:1所示。
9.一种制备谷胱甘肽的方法,其特征在于,将唾液联合乳杆菌(Ligilactobacillussalivarius)SM4菌株接种于MRS液体培养基中,培养至稳定期后收取菌悬液,最后对菌悬液进行分离即得谷胱甘肽;所述唾液联合乳杆菌SM4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 20231168;所述唾液联合乳杆菌SM4菌株的16S rDNA全序列如SEQ ID No:1所示。
10.根据权利要求9所述的一种制备谷胱甘肽的方法,其特征在于,培养的条件为:37℃恒温厌氧。
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