CN116496938B - 一株产透明质酸的嗜酸乳杆菌my2及其在制备抗衰老和美白食品药品中的应用 - Google Patents
一株产透明质酸的嗜酸乳杆菌my2及其在制备抗衰老和美白食品药品中的应用 Download PDFInfo
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Abstract
本发明属于益生菌及其应用技术领域，具体涉及一株产透明质酸的嗜酸乳杆菌MY2及其在制备抗衰老和美白食品药品中的应用。为进一步开发利用嗜酸乳杆菌的益生功能，本发明从一名健康成年人的粪便中分离出一株嗜酸乳杆菌(Lactobacillus acidophilus)MY2菌株，所述嗜酸乳杆菌MY2菌株可产生和分泌透明质酸(HA)，证明MY2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、抗皱、美白、促进伤口消炎及愈合等领域，比如，制备成抗衰老和美白食品药品，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，具体涉及一株产透明质酸的嗜酸乳杆菌MY2及其在制备抗衰老和美白食品药品中的应用。
背景技术
嗜酸乳杆菌(Lactobacillus acidophilus)，属于乳杆菌科乳杆菌属，菌体呈杆状，两端为圆形，大小为(0.6～0.9)μm×(1.5～6.0)μm，以单个、成双或短链排列，不运动，无鞭毛，无芽孢，革兰染色为阳性，主要存在于小肠中，释放乳酸、乙酸以及一些对有害菌起作用的抗菌素。该菌的最适生长温度为35～38℃，20℃以下不生长，耐热性差；最适pH为5.5～6.0，耐酸性强，能在其它乳酸菌不能生长的环境中生长繁殖；还能利用葡萄糖、果糖、乳糖、蔗糖进行同型发酵，发酵产生DL型乳酸。但该菌对蛋白质分解力弱。嗜酸乳杆菌是公认的安全性食品微生物，在营养、免疫等方面具有重要的益生功能，且已被证明是一种很有潜力的癌症预防剂。
嗜酸乳杆菌是乳酸菌家族中极受重视的益生菌之一，被视为第三代酸乳发酵剂菌种。嗜酸乳杆菌是人体肠道中的重要微生物，与人体健康息息相关，当其达到一定数量时，可以起到健康促进效果。此外，嗜酸乳杆菌还具有改善肠道健康、调节免疫、减轻肥胖、治疗肠道疾病等众多益生功能,在食品和药品中均有应用，市场需求量巨大。
嗜酸乳杆菌是一种可用于食品的乳杆菌，食用安全无毒。众所周知，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并终生伴随宿主，对于维持肠道微生态平衡有重要意义。而嗜酸乳杆菌也是一种常见的肠道益生菌，广泛地存在于人和动物的肠道并随粪便排出，并参与调节肠道健康。近些年来，嗜酸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。
目前，嗜酸乳杆菌的不同菌株已被报道具有不同的益生功能：(1)可以降低血液中的胆固醇含量；(2)增加宿主对铁的利用度；(3)减少氧化应激和炎症反应，减轻动脉粥样硬化病变；(4)增加宿主体内的乳杆菌和双歧杆菌的数量，增加乙酸、丁酸和丙酸的含量，改善肠道健康；(5)改善腹泻症状；(6)缓解肠易激综合征(IBS)症状；(7)抑制皮炎、湿疹等疾病；(8)抑制过敏反应；(9)改善糖尿病以及抗衰老；(10)降低病毒及细菌感染，抑制宿主炎症反应；(11)改善胃溃疡及胃肠道感染。
嗜酸乳杆菌的来源多样性导致其具有基因的多样性和功能的多样性。然而，目前针对嗜酸乳杆菌的分离鉴定、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嗜酸乳杆菌的开发利用。因此，有必要根据嗜酸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，益生性嗜酸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出一株嗜酸乳杆菌(Lactobacillus acidophilus)MY2菌株，所述嗜酸乳杆菌菌株MY2可产生和分泌透明质酸(HA)，可用于抗衰老、抗皱和美白等领域，具有巨大的潜在应用价值。
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嗜酸乳杆菌(Lactobacillus acidophilus)MY2菌株，所述嗜酸乳杆菌MY2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 M 20221498；所述嗜酸乳杆菌MY2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嗜酸乳杆菌(Lactobacillusacidophilus)MY2菌株在产透明质酸中的应用。
经研究发现，益生菌嗜酸乳杆菌菌株MY2菌株可产生透明质酸(HA)，提示嗜酸乳杆菌菌株MY2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，抗皱，美白，促进伤口消炎及愈合等领域。
本发明第三方面提供了一种制备透明质酸的方法，具体为：将第一方面所述的嗜酸乳杆菌(Lactobacillus acidophilus)MY2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透明质酸。
优选地，培养的条件为：37℃恒温厌氧。
优选地，接种前，先用MRS固体培养基将嗜酸乳杆菌(Lactobacillusacidophilus)MY2菌株培养至稳定期，接种时，再以1:30的稀释倍数扩培至MRS液体培养基。
本发明第四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嗜酸乳杆菌(Lactobacillus acidophilus)MY2菌株。
优选地，所述嗜酸乳杆菌(Lactobacillus acidophilus)MY2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：
本发明从一名健康成年人的粪便中分离出一株嗜酸乳杆菌(Lactobacillusacidophilus)MY2菌株，所述嗜酸乳杆菌MY2菌株可产生和分泌透明质酸(HA)，证明MY2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，抗皱，美白，促进伤口消炎及愈合等领域，比如，制备成抗衰老和美白食品药品，具有重要的应用价值和经济价值。
附图说明
图1为嗜酸乳杆菌MY2与其他同源性菌株的系统发育树；
图2为嗜酸乳杆菌MY2可以产生和分泌透明质酸。
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：嗜酸乳杆菌(Lactobacillus acidophilus)MY2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：透明质酸(又名玻尿酸，HA)检测试剂盒(Cloud-Clone Corp.,Cat:CEA182Ge)。
(3)MRS平板的制备：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。
(4)MRS液体培养基的制备：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。
实施例1嗜酸乳杆菌(Lactobacillus acidophilus)MY2的分离鉴定
嗜酸乳杆菌Lactobacillus acidophilus MY2菌株从从中国广东省广州市海珠区一名健康成年人(男性，32岁，BMI＝22.5)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MY2，培养24小时后观察该菌的菌落为乳白色、圆形凸起、边缘光滑整齐。
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.acidophilus MY2菌株进行全基因组测序。将得到的序列16S rDNA sequence(SEQIDNo：1)在NCBI的Genome数据库进行BLAST比对，结果表明，MY2菌株与已知的嗜酸乳杆菌16S rDNA序列的同源性>99％(图1)；并与同源菌株进行进化分析，确认MY2菌株是同种不同株的嗜酸乳杆菌。
最后，对菌株MY2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 M 20221498；保藏单位地址：中国.武汉.武汉大学；分类命名：Lactobacillus acidophilus。
嗜酸乳杆菌是一种可用于食品的益生菌菌株，具有广泛的益生功效，如可降低胆固醇、改善肠道健康、抑制过敏等，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新嗜酸乳杆菌MY2可以作为益生菌使用，并可能具有新的功效和功能。
L.acidophilus MY2的16S rDNA sequence(1436bp，SEQ ID No：1)：
TACTGCAGTCGAGCGAGCTGAACCaACAGATTCACTTCGGTGATGACGTTGGGAACGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTATAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAGGGTAACGGCCTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCCGAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGCAATCCGTAGAGATACGGAGTTCCCTTCGGGGACACTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGTACAACGAGGAGCAAGCCTGCGAAGGCAAGCGAATCTCTTAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTCTGCAATGCCCAAAGCCGGGGCCTAACTTCGGGAAGGAGCCGTCTAA。
实施例2嗜酸乳杆菌(Lactobacillus acidophilus)MY2的功能及其应用
将用MRS液体培养基培养至稳定期的嗜酸乳杆菌MY2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，MY2的发酵上清液中HA的浓度显著性增加，可积累量为7.78ng/mL，说明嗜酸乳杆菌MY2可以在稳定期产生并分泌透明质酸(图2)。
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。
综上可见，本发明新分离的嗜酸乳杆菌菌株MY2可产生和分泌透明质酸(HA)，并可通过产生透明质酸的功效发挥以上多个用途，比如，抗衰老、抗皱和美白等，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 (6)
1.一株嗜酸乳杆菌(Lactobacillus acidophilus)MY2菌株在产透明质酸中的应用，其特征在于，所述嗜酸乳杆菌MY2菌株于2022年10月4日保藏于中国典型培养物保藏中心，保藏号为：CCTCC M 20221498；所述嗜酸乳杆菌MY2菌株的16S rDNA全序列如SEQ ID No：1所示。
2.一种制备透明质酸的方法，其特征在于，将嗜酸乳杆菌(Lactobacillusacidophilus)MY2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透明质酸；所述嗜酸乳杆菌MY2菌株于2022年10月4日保藏于中国典型培养物保藏中心，保藏号为：CCTCC M 20221498；所述嗜酸乳杆菌MY2菌株的16S rDNA全序列如SEQ ID No：1所示。
3.根据权利要求2所述的一种制备透明质酸的方法，其特征在于，培养的条件为：37℃恒温厌氧。
4.根据权利要求2所述的一种制备透明质酸的方法，其特征在于，接种前，先用MRS固体培养基将嗜酸乳杆菌(Lactobacillus acidophilus)MY2菌株培养至稳定期，接种时，再以1:30的稀释倍数扩培至MRS液体培养基。
5.一种用于具有益生功能的菌剂，其特征在于，所述菌剂包括嗜酸乳杆菌(Lactobacillus acidophilus)MY2菌株，所述嗜酸乳杆菌MY2菌株于2022年10月4日保藏于中国典型培养物保藏中心，保藏号为：CCTCC M 20221498；所述嗜酸乳杆菌MY2菌株的16SrDNA全序列如SEQ ID No：1所示。
6.根据权利要求5所述的一种用于具有益生功能的菌剂，其特征在于，所述菌剂为嗜酸乳杆菌(Lactobacillus acidophilus)MY2菌株发酵后的菌体混合物。
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