CN114958694B - 一种联产共轭亚油酸和γ-氨基丁酸的鼠李糖乳杆菌及应用 - Google Patents
一种联产共轭亚油酸和γ-氨基丁酸的鼠李糖乳杆菌及应用 Download PDFInfo
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Abstract
本发明公开一种联产共轭亚油酸和γ‑氨基丁酸的鼠李糖乳杆菌及其应用，该菌株于2022年5月13日保藏于中国典型培养物保藏中心，保藏编号为CCTCCM 2022615；本发明通过采用常压室温等离子体诱变技术，经过筛选得到能够高效联产共轭亚油酸和γ‑氨基丁酸的鼠李糖乳杆菌SG906，将此鼠李糖乳杆菌SG906接种至含有游离亚油酸和谷氨酸的培养基中发酵72h，发酵液中共轭亚油酸含量高达7.95g/L，γ‑氨基丁酸的含量高达178.32g/L，大大降低生产成本；本发明的鼠李糖乳杆菌SG906属于食品乳杆菌属，具有很好的安全性，可广泛应用于药物、保健品、饮料等领域，具有巨大的应用价值和市场开发潜力。
Description
技术领域
本发明具体涉及一种联产共轭亚油酸苯和γ-氨基丁酸的鼠李糖乳杆菌及应用，属于微生物技术领域。
背景技术
随着经济发展和人民生活水平的提高，肥胖引发的高血脂、Ⅱ型糖尿病、高血压和动脉粥样硬化、甚至恶性肿瘤等一系列疾病，对人体健康危害极大。因此，低价安全高效的营养保健用品是当前的研究热点。
共轭亚油酸(Conjugated linoleic acid，简称CLA)是含有共轭双键的十八碳二烯酸的多种位置异构体和几何异构体的总称，CLA的双键在碳链上主要有四种位置的排列方式：8,10-、9,11-、10,12-和11,13-，且由于共轭双键两端的碳原子都具有顺式(cis)和反式(trans)两种几何构型，即每种位置异构又具有cis-cis、cis-trans、trans-cis、trans-trans四种几何异构体，所以CLA异构体的种类众多，其中，c9,t11-CLA和t10,c12-CLA是含量最多且已经被证实具有生理活性的两种主要异构体。大量研究表明，它具有多种营养和保健功能，如抗癌、抗糖尿病、抗动脉粥样硬化、降低体脂含量、胰岛素抵抗、调节机体免疫等多种营养和保健功能。
天然的CLA主要存在于牛、羊等反刍动物的肉和乳制品中，含量极低，目前工业上普遍采用化学法合成CLA，但是存有试剂残留，成本高等问题。利用微生物合成CLA不需要高温高压，分离纯化步骤相对简单。已发现一些瘤胃菌、丙酸菌和乳酸菌等能合成CLA，但是瘤胃菌、痤疮丙酸杆菌、溶纤维丁酸弧菌、埃氏巨球形菌等细菌没有列入食用菌名录，不能直接食用。用食品安全级乳酸菌生产CLA可以直接应用于食品，因此，应用乳酸菌生产CLA将有广阔的应用前景，但是目前已报道的一些益生菌，如罗伊氏乳酸杆菌、乳酸乳球菌和短双歧杆菌生产CLA的效率很低。虽然CLA早在2009年就被中国卫计委批准列入新食品名录。但是，化学合成或生物合成的CLA价格昂贵，限制其在常规食品添加剂上的规模化应用。
γ-氨基丁酸(γ-amino butyric acid，简称GABA)，是一种天然的功能性非蛋白质氨基酸，具有治疗糖尿病、降血压、防止肥胖、降血氨、改善睡眠、活化肝功能，改善更年期综合症等重要生理功能，在功能食品中的应用已成为研究热点，广泛应用于食品、药品和化妆品中。日本厚生省、欧洲食品***(EFSA)和美国食品药品管理局(FDA)承认乳酸菌发酵生产的GABA为天然食品添加剂，我国***2009年批准此类GABA为新资源食品。目前，合成GABA的方法分有化学法和生物法；化学法存在吡咯烷酮残留、副反应多等问题，不能应用于食品领域；生物法包括植物富集法和微生物法，发芽的糙米和豆类均含有大量的γ-氨基丁酸，但是提取成本高无法大规模生产。微生物由于生长周期短，繁殖速度快，近些年广泛被应用于生产GABA，已发现一些大肠杆菌、短乳杆菌、副断乳杆菌、酵母、霉菌等能合成GABA，但是由于发酵液成分复杂，提取高品质GABA成本高，价格昂贵，不利于工业化生产。
乳酸菌作为一种食品安全级(GRAS)的微生物具有各种保健功效，常作为益生菌添加于食品中以增进人体健康，其中鼠李糖乳杆菌是人体肠道菌群之一，乳杆菌属鼠李糖乳杆菌(Lactobacillus rhamnosus)，是人体正常菌群之一，肠道黏着率高，定植能力强，并具有高效降胆固醇，促进细胞***，可起到调节肠道菌群、预防和治疗腹泻、排除毒素、预防龋齿、提高机体免疫力及抗癌等重要的生理保健功能，也是功能研究较多的乳杆菌，广泛应用于酸奶、奶酪和固体饮料等食品的生产中。
现有技术中，一般的功能食品都是通过外添加的方式使其富含CLA或者GABA，而天然或微生物合成的高纯CLA和GABA价格较贵，限制了CLA和GABA的大规模应用。大量研究表明乳酸菌具有合成CLA或者GABA的能力，现有技术中尝试将产CLA或GABA的菌种应用到食品中，但是大部分学者筛选到的产CLA或GABA的菌株有的产量过低，有的只能生产CLA或GABA，有的菌株非安全菌株，不允许在食品中应用或者在食品培养配方中不能生长。
发明内容
为了解决上述技术问题，本发明提供一种联产共轭亚油酸和γ-氨基丁酸的鼠李糖乳杆菌及其应用，本发明的鼠李糖乳杆菌在高产CLA的同时GABA的产量也能达到较高的水平，有效促进功能活性成分多优并举，达到功能多效叠加的目的，在食品、保健品和药品领域具有重要的应用价值。
本发明的目的一在于一种联产共轭亚油酸和γ-氨基丁酸的鼠李糖乳杆菌，所述发酵联产共轭亚油酸和γ-氨基丁酸的鼠李糖乳杆菌SG906于2022年5月13日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC M 2022615，保藏地址为中国武汉，武汉大学，邮编：430072。
本发明提供的发酵联产共轭亚油酸和γ-氨基丁酸的鼠李糖乳杆菌的形态学及生理化学特征如下：
菌落颜色：乳白色
需氧方式：兼性厌氧
适宜生长温度：35℃-37℃
适宜生长pH：5-6
菌落形态：杆状
革兰氏染色：阳性。
本发明目的二在于提供一种鼠李糖乳杆菌在发酵联产共轭亚油酸和γ-氨基丁酸中的应用。
本发明的目的三在于提供一种联产共轭亚油酸和γ-氨基丁酸的方法，采用本发明提供的鼠李糖乳杆菌SG906依次经过活化培养、种子培养、发酵罐补料反馈培养高效联产共轭亚油酸和γ-氨基丁酸。
进一步的，所述发酵罐内液体培养基pH为6.0～6.5，包括如下按质量百分比计的组份：葡萄糖0.5％，牛大骨蛋白胨2％，酵母粉2％，七水硫酸镁0.03％，一水硫酸锰0.01％，吐温-80 0.2％，谷氨酸5％，亚油酸0.2％，其余为水。
进一步的，所述发酵罐培养周期划分为两个阶段：
(1)在发酵培养起始阶段，初始葡萄糖的浓度控制在30g/L，通过流加70％的葡萄糖控制发酵体系中的残糖(以葡萄糖计)为0.5-1.0g/L；
(2)在发酵罐内OD600达到25后，通过流加亚油酸和谷氨酸控制发酵体系中的亚油酸浓度保持在0.01-0.1g/L和谷氨酸的浓度保持在1-5g/L；
其中，所述发酵罐培养全过程控制溶解氧在20-45％。
本发明目的四在于提供一种鼠李糖乳杆菌在复合功能发酵茶饮料中的应用。
相较于现有技术，本发明的有益效果在于：
1、本发明采用常温常压等离子体诱变技术，首次发现能够高效联产共轭亚油酸和γ-氨基丁酸的鼠李糖乳杆菌SG906，该菌株经过分批补料发酵，发酵液中共轭亚油酸的浓度最高可达7.95g/L，以亚油酸为底物转化率达到80.72％，发酵液中γ-氨基丁酸的浓度最高可达178.30g/L，转化率达98.56％，证实了本发明提供的鼠李糖乳杆菌SG906极具工业化运用价值。
2、本发明提供的鼠李糖乳杆菌SG906是食品安全乳酸菌，在商业上广泛应用的一种益生菌种，同时兼具联产共轭亚油酸和γ-氨基丁酸的能力，可广泛地应用于制备富含CLA和GABA的复合功能保健品、食品、食品添加剂或饮料，大幅降低制备成本，具有广阔的应用前景。
附图说明
图1为本发明实施例1中鼠李糖乳杆菌CICC 22152诱变致死率曲线；
图2为本发明实施例1中鼠李糖乳杆菌CICC 22152和优良突变株96孔板初筛发酵结果示意图；
图3为本发明实施例2中鼠李糖乳杆菌CICC 22152和优良突变株摇瓶复筛发酵结果示意图；
图4为本发明实施例2中鼠李糖乳杆菌SG906 20L发酵罐发酵曲线图。
具体实施方式
下面结合附图和较佳实施例对本发明做进一步的说明，在本发明中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值；对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开；
以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到；
以下实施例中的定量试验，均设置三次重复实验，结果取平均值；
下述实施例中共轭亚油酸的检测：照中华人民共和国轻工行业标准QB/T5403-2019共轭亚油酸检测方法进行检测；
γ-氨基丁酸的检测：参照中华人民共和国轻工行业标准QB/T4587-2013γ-氨基丁酸检测方法进行检测。
实施例1鼠李糖乳杆菌及其诱变筛选
一种鼠李糖乳杆菌，经由源于中国工业微生物菌种保藏管理中心的出发菌鼠李糖乳杆菌CICC 22152经常压室温等离子体诱变筛选，进而得到所述鼠李糖乳杆菌SG906；
其中，鼠李糖乳杆菌SG906的诱变筛选方法，包括如下步骤：
S1、诱变预处理：将出发菌鼠李糖乳杆菌CICC 22152的甘油菌100μL接种至5mLMRS液体培养基培养，在37℃下静置培养至菌液OD600值为1.0，在8000rpm的转速下离心处理10min，弃上清液收集菌体，用含5％(v/v)甘油的生理盐水洗涤菌体两遍，并重悬制成分散均匀的菌悬液，调整细菌的终浓度为100～110CFU·mL-1；
S2、常压室温等离子体诱变：取20μL经过步骤S1制得的菌悬液均匀涂于无菌载片上表面，干燥后用镊子将载片转移至载物台，采用高纯氦气作为等离子体的工作气体，设置电源功率50W，照射距离4mm，等离子体的温度为26℃，气流量为10L/min，处理菌物载片，进行常压室温等离子体诱变处理，照射时间分别取0(对照)、5、10、15、20、25、30、35、40、45、50、55s；处理后将载片转移到1.5mL的EP管中，在振荡器上再生培养60min，再用含5％(v/v)甘油的生理盐水稀释10倍，形成新的菌悬液；
其中，将菌悬液涂布于MRS固体培养基平板后置于37℃培养箱内培养，培养48h，观察菌株生长情况，统计菌落数，绘制致死率曲线(如图1所示)，选择致死率为75％的处理时间；
S3、诱变后培养：样品诱变处理完毕后，取经过步骤S2获得的新的菌悬液200μL涂布于MRS固体培养基，37℃静置培养48h分离单克隆；
S4、初筛：采用96孔板对诱变分离的突变株进行初筛，挑取单克隆到含1mL初筛液体培养基的96孔板培养，培养条件37℃，100rpm，培养72h，离心收集上清液，检测上清液中共轭亚油酸和γ-氨基丁酸含量，筛选共轭亚油酸和γ-氨基丁酸含量高的菌种，共筛选的到三株高产突变株作为初筛菌株，分别命名SG253、SG906、SG1158，并进行甘油保藏，三株菌株的共轭亚油酸和γ-氨基丁酸产量如图2所示；其中，初筛液体培养基包含如下质量百分数成分：葡萄糖0.3％，牛大骨蛋白胨1％，酵母粉1％，七水硫酸镁0.02％，一水硫酸锰0.005％，吐温-80 0.1％，谷氨酸6％，亚油酸0.02％，其余为水，pH调至6.0；
S5、复筛：将经过步骤S4初筛获取的优良突变株和对照菌的甘油菌分别按5％接种量，接种至MRS固体培养基上培养，再取1mL甘油菌液接种至25mL复筛种子培养基培养，摇管装液量50％(v/v)，培养温度为37℃，静置培养时间48h；其中，复筛种子培养基包含如下质量百分数成分：葡萄糖0.2％，牛大骨蛋白胨0.5％，酵母粉0.5％，七水硫酸镁0.01％，一水硫酸锰0.002％，吐温-800.1％，其余为水，pH调至6.0；复筛种子培养基中的种液按15％接种量取18.75mL种子培养液接种至125mL复筛发酵培养基培养，250mL摇瓶装液量125mL，发酵温度37℃，静置发酵72h，然后离心收集上清液；其中，复筛发酵培养基包含如下质量百分数成分：葡萄糖0.3％，牛大骨蛋白胨2％，酵母粉1.5％，七水硫酸镁0.03％，一水硫酸锰0.01％，吐温-80 0.2％，谷氨酸6％，亚油酸0.1％，其余为水，pH调至6.0；参见图3，检测上清液共轭亚油酸和γ-氨基丁酸的含量，筛选出高产菌株鼠李糖乳杆菌SG906，该菌经72h摇瓶发酵，发酵液中共轭亚油酸达到0.76g/L，与对照菌相比提高7.6倍，γ-氨基丁酸浓度达到15.2g/L，与对照菌相比提高25.3倍；
上述诱变筛选方法中，MRS液体培养基包含如下质量百分数成分：酪蛋白胨1％，牛肉膏1％，酵母粉0.5％，葡萄糖0.5％，乙酸钠0.5％，柠檬酸二铵0.2％，吐温-80 0.1％，磷酸氢二钾0.2％，七水硫酸镁0.02％，一水硫酸锰0.005％，其余为水，pH调至6.0；
MRS固体培养基含如下质量百分数成分：酪蛋白胨1％，牛肉膏1％，酵母粉0.5％，葡萄糖0.5％，乙酸钠0.5％，柠檬酸二铵0.2％，吐温-80 0.1％，磷酸氢二钾0.2％，七水硫酸镁0.02％，一水硫酸锰0.005％，琼脂2％，其余为水，pH调至6.0；
最后，将筛选得到的优质鼠李糖乳杆菌突变株SG906进行传代培养以考察其遗传稳定性，每2天传代一次，传代10代，每隔一代进行摇瓶发酵测定菌株生物量、共轭亚油酸和γ-氨基丁酸的含量，结果表明鼠李糖乳杆菌突变株SG906传代过程中菌株生物量、共轭亚油酸和γ-氨基丁酸均无明显变化，具有良好的遗传稳定性；将获得遗传稳定的、能够同时高效积累共轭亚油酸和γ-氨基丁酸的鼠李糖乳杆菌SG906于2022年5月15日保藏于中国典型培养物保藏中心(简称CCTCC；地址：中国武汉，武汉大学；邮编：430072)，保藏编号为CCTCC NO：M 2022615。
实施例2鼠李糖乳杆菌在发酵联产共轭亚油酸和γ-氨基丁酸中的应用
发酵联产共轭亚油酸和γ-氨基丁酸的方法，采用本发明提供的鼠李糖乳杆菌SG906，包括如下步骤：
A1、菌种活化：取50μL鼠李糖乳杆菌SG906甘油菌，接种至含MRS固体培养基的试管斜面，培养箱中于37℃培养48h进行菌种活化，再用5mL无菌生理盐水洗下菌体细胞，制备菌株细胞悬液，为活化后的菌株悬液；
A2、种子培养：取75mL经过步骤A1制得的活化后的菌株悬液按5％的接种量转接于含1.5L种子培养基的5L摇瓶，于37℃进行静置培养48h，制备种子液，进一步提高菌株的数量，强化菌株活力；
A3、鼠李糖乳杆菌SG906补料反馈发酵：采用20L全自动发酵罐(装液量为10L)进行鼠李糖乳杆菌SG906补料反馈发酵；取经过步骤A2培养好的1.5L种子培养液按15％的接种量无菌接种于10L发酵罐内液体培养基中，在发酵培养起始阶段，通气比为0.1(V/V·m)，罐压为0.01Mpa，搅拌控制为100rpm，温度控制在37℃，初始葡萄糖的浓度控制在30g/L，通过流加70％的葡萄糖控制发酵体系中的残糖(以葡萄糖计)为0.5-1.0g/L；在发酵罐内OD600达到25后，通过流加亚油酸和谷氨酸控制发酵体系中的亚油酸浓度保持在0.01-0.1g/L和谷氨酸的浓度保持在1-5g/L；发酵罐培养全过程控制溶解氧在20-45％，pH为5.0-5.5发酵周期控制在72h，8000rpm离心10min，收集发酵上清液；
其中，发酵罐内液体培养基包括如下按质量百分比计的组份：葡萄糖0.5％，牛大骨蛋白胨2％，酵母粉2％，七水硫酸镁0.03％，一水硫酸锰0.01％，吐温-800.2％，谷氨酸5％，亚油酸0.2％，其余为水；
最终，测定发酵上清液中共轭亚油酸和γ-氨基丁酸含量，将经过步骤A3获得的发酵液上清进行共轭亚油酸和γ-氨基丁酸检测，参见图4，检测结果如下：鼠李糖乳杆菌SG906经72h补料反馈发酵，发酵液中共轭亚油酸的浓度最高可达7.95g/L，以亚油酸为底物转化率达到80.72％；发酵液中γ-氨基丁酸的浓度最高可达178.32g/L，转化率达98.56％，极具工业化运用价值。
实施例3鼠李糖乳杆菌SG906在制备复合功能发酵茶饮料中的应用
(1)制备茶叶浸提液：取1kg茶叶加入50kg蒸馏水，于100℃煮沸浸提15min，5000rpm离心20min取得茶叶浸提液液；
(2)鼠李糖乳杆菌SG906发酵茉莉花茶
取2.25L培养好的鼠李糖乳杆菌SG906种子液按5％的接种量接种至茉莉花茶浸提液发酵体系中，初始的鼠李糖乳杆菌SG906的菌落数控制在150CFU·mL-1，于37℃，100rpm进行发酵96h，得到复含共轭亚油酸和γ-氨基丁酸的益生菌发酵茶饮料，其CLA和GABA的含量分别为0.63g/L和2.14g/L。
茶叶浸提液发酵体系组成：葡萄糖5％，酵母提取物0.3％，大豆蛋白胨0.3％，奶粉1％，柠檬酸钠0.2％，谷氨酸5％，亚油酸0.08％，其余为茶叶浸提液，磷酸调节pH至5.0。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
Claims (6)
1.一种联产共轭亚油酸和γ-氨基丁酸的鼠李糖乳杆菌(Lactobacillus
rhamnosus)SG906，其特征在于：保藏编号为CCTCC M 2022615的鼠李糖乳杆菌SG906。
2.如权利要求1所述的鼠李糖乳杆菌(Lactobacillus rhamnosus)SG906在发酵联产共轭亚油酸和γ-氨基丁酸中的应用。
3.一种联产共轭亚油酸和γ-氨基丁酸的方法，其特征在于：采用如权利要求1所述的鼠李糖乳杆菌(Lactobacillus rhamnosus)SG906依次经过活化培养、种子培养、发酵罐补料反馈培养高效联产共轭亚油酸和γ-氨基丁酸。
4.如权利要求3所述的一种联产共轭亚油酸和γ-氨基丁酸的方法，其特征在于：所述发酵罐内液体培养基pH为6.0～6.5，包括如下按质量百分比计的组份：葡萄糖0.5％，牛骨蛋白胨2％，酵母粉2％，七水硫酸镁0.03％，一水硫酸锰0.01％，吐温-80 0.2％，谷氨酸5％，亚油酸0.2％，其余为水。
5.如权利要求4所述的一种联产共轭亚油酸和γ-氨基丁酸的方法，其特征在于：所述发酵罐补料反馈培养周期划分为两个阶段：
(1)在发酵培养起始阶段，初始葡萄糖的浓度控制在30g/L，通过流加70％的葡萄糖控制发酵体系中的残糖为0.5-1.0g/L；
(2)在发酵罐内OD600达到25后，通过流加亚油酸和谷氨酸控制发酵体系中的亚油酸浓度保持在0.01-0.1g/L和谷氨酸的浓度保持在1-5g/L；
其中，所述发酵周期为72h，发酵罐补料反馈培养全过程控制溶解氧在20-45％。
6.如权利要求1所述的鼠李糖乳杆菌(Lactobacillus rhamnosus)SG906在制备复合功能发酵茶饮料中的应用。
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