CN114634885B - 一株高产γ-氨基丁酸的植物乳杆菌及其应用 - Google Patents
一株高产γ-氨基丁酸的植物乳杆菌及其应用 Download PDFInfo
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Abstract
本发明涉及一株高产γ‑氨基丁酸的植物乳杆菌，命名为植物乳杆菌(Lactobacillusplantarum)HG‑06，于2022年01月21日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCCNO：M2022109。本发明得到的植物乳杆菌γ‑氨基丁酸产量更高，5L发酵罐转化实验的最终产量为204.37g/L，底物摩尔转化率为97.22％，比出发菌株产量提高了56.83％，为产业化的提高提供了坚实的理论基础和数据参考。
Description
技术领域
本发明涉及微生物技术领域，尤其涉及一株高产γ-氨基丁酸的植物乳杆菌及其应用。
背景技术
γ-氨基丁酸(GABA)作为一种天然的非蛋白质氨基酸，广泛分布于动植物中，其具有多种生理功能，主要有调节血压与心率、改善肝脏、肾脏功能、抗衰老、调节激素分泌等，因此，它既可以作为一种药效良好的药物，又可以被视为具有保健功能的新型功能因子，广泛应用于食品和医疗卫生行业。
GABA的制备方法主要有三种，分别是化学合成法、植物富集法和微生物发酵法。化学合成法成本高，副反应多，环境污染严重；植物富集法产量太低，不适合工业化生产；而微生物发酵法成本低，安全性高，环保，具有很好的开发前景，因此成为GABA生产的首选方法。在早期的研究中，微生物发酵法生产GABA以大肠杆菌为生产菌，然而若要进行食品开发，使用大肠杆菌无疑存在安全性方面的种种问题。而乳酸菌作为一种食品安全级(GRAS)微生物，在食品工业中应用历史悠久，被公认为最具代表性的有益菌。利用乳酸菌属的植物乳杆菌生产GABA，具有专一性高、设备简单、环保、成本低等优点，适用于大规模产业化生产，所以引起的关注众多。
迄今为止，人们已从鲜奶、泡菜、酸菜、清酒、植物叶片及各种传统的发酵乳制品和肉制品中分离得到多种具有GABA合成能力的乳酸菌。专利CN202111126098.1公开了一株产γ-氨基丁酸的植物乳杆菌LHP710，该菌从野生蓝莓果渣发酵物中分离纯化得到，以L-谷氨酸为底物催化制备γ-氨基丁酸的产量可达44.3g/1L生物催化反应体系；专利CN201610968493.7公开了一株产γ-氨基丁酸的植物乳杆菌SG5，发酵48hGABA的含量可达59μg/mL；专利CN201310045139.3公开了一株高产γ-氨基丁酸的植物乳杆菌LW106，经发酵培养可以获得γ-氨基丁酸含量为20-25g/L的发酵液。然而整体来看乳酸菌产GABA的能力还不足以达到食品工业生产的目标，因此，对于适用于食品工业的高产GABA乳酸菌的选育工作显得尤为重要。
发明内容
为解决上述技术问题，本发明克服了目前选育的植物乳杆菌产γ-氨基丁酸能力不强等问题，以一株具有生产GABA能力的植物乳杆菌为出发菌株，利用ARTP诱变系统进行诱变，筛选得到一株GABA转化率高的突变株，为GABA的工业化生产提供了潜在优良菌种。
本发明的第一个目的是提供一株高产γ-氨基丁酸的植物乳杆菌，命名为植物乳杆菌(Lactobacillusplantarum)HG-06，于2022年01月21日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCCNO：M2022109。
本发明的第二个目的是提供上述植物乳杆菌在催化L-谷氨酸或L-谷氨酸钠生产γ-氨基丁酸中的应用。
进一步地，上述应用包括将植物乳杆菌接种至种子培养基中进行培养获得种子液，再将种子液转接至发酵培养基中获得大量含有谷氨酸脱羧酶的植物乳杆菌菌体，收集菌体悬浮在缓冲液中投加底物L-谷氨酸或L-谷氨酸钠生产γ-氨基丁酸的步骤。
进一步地，种子培养基为MRS培养基，配方为蛋白胨8-10g/L、牛肉浸粉4-5g/L、酵母浸粉4-5g/L、葡萄糖15-20g/L、磷酸氢二钾1.8-2.2g/L、柠檬酸三铵1.8-2.2g/L、醋酸钠4.8-5.2g/L、硫酸镁0.15-0.2g/L、硫酸锰0.03-0.06g/L、吐温80 0.5-1.0mL/L、pH6.0-6.5。
进一步地，发酵培养基的配方为蛋白胨15-20g/L、酵母膏6-8g/L、葡萄糖20-30g/L、丁二酸钠5-8g/L、L-谷氨酸钠3-6g/L、硫酸镁0.15-0.2g/L、硫酸锰0.03-0.06g/L、pH6.0-6.5。
进一步地，转化体系中，缓冲液为含有1-10‰曲拉通X-100的pH4.5-5.0的醋酸/醋酸钠缓冲液或含有1-10‰曲拉通X-100的pH4.5-5.0的柠檬酸磷酸盐缓冲液。
进一步地，底物投加方式为分批补料，使L-谷氨酸或L-谷氨酸钠的浓度保持在80-100g/L。
进一步地，转化温度为30-40℃，优选为37℃。
进一步地，转化时间为12-24h。
进一步地，上述植物乳杆菌的接种量为1-20％。
本发明的第三个目的是提供一种微生物菌剂，该微生物菌剂含有上述植物乳杆菌。
进一步地，微生物菌剂为固态菌剂或液态菌剂。
进一步地，上述微生物菌剂具有改善睡眠的功能。
借由上述方案，本发明至少具有以下优点：
本发明所筛选到的植物乳杆菌，γ-氨基丁酸产量比目前报道过的植物乳杆菌有明显提高，并且具有稳定的产量，在5L罐转化实验的最终产量为204.37g/L，底物摩尔转化率为97.22％，比出发菌株产量提高了56.83％。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
生物材料保藏
植物乳杆菌(Lactobacillusplantarum)HG-06，所述植物乳杆菌(Lactobacillusplantarum)HG-06已于2022年01月21日保藏于中国典型培养物保藏中心，保藏编号为CCTCCNO：M2022109，保藏地址为中国武汉武汉大学。
附图说明
为了使本发明的内容更容易被清楚的理解，下面根据本发明的具体实施例并结合附图，对本发明作进一步详细的说明。
图1为致死率随诱变时间的变化数值关系；
图2为突变菌株植物乳杆菌HG-06的生长曲线；
图3为5L发酵罐分批补料产量图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
下述实施例中涉及的培养基如下：
MRS培养基：蛋白胨10g/L、牛肉浸粉5g/L、酵母浸粉4g/L、葡萄糖20g/L、磷酸氢二钾2g/L、柠檬酸三铵2g/L、醋酸钠5g/L、硫酸镁0.2g/L、硫酸锰0.05g/L、吐温80 1mL/L、pH6.2。
发酵培养基：蛋白胨15g/L、酵母膏6g/L、葡萄糖25g/L、丁二酸钠5g/L、L-谷氨酸钠3g/L、硫酸镁0.2g/L、硫酸锰0.05g/L、pH6.2。
筛选培养基：MRS固体培养基中添加溴甲酚绿指示剂。
实施例1常压室温等离子体诱变仪对植物乳杆菌进行诱变
利用常压室温等离子体诱变仪对植物乳杆菌进行诱变，包括以下步骤：
(1)从平板挑取一环活化后的菌种接于装有40mLMRS液体培养基的250mL三角瓶中，37℃静置培养8～10h，使菌体处于对数生长期。取1mL对数生长期菌液测其OD600nm，根据OD600nm及菌落数的大体关系估算菌体浓度，取1mL菌液12000r/min离心2min，收集菌体并用无菌生理盐水洗涤2～3次，将其稀释至106CFU/mL的菌悬液。
(2)吸取10μL菌液均匀涂在灭菌冷却后的载片上，以He为工作气体，射频功率为100W，照射距离2mm，气体流速10L/min，照射距离2mm，气体流速10L/min，样品量10uL，通入气体为氦气，诱变时间分别为0s、10s、30s、50s、70s、90s、110s。
(3)将诱变过的载片置于装有1mL无菌生理盐水的1mL离心管中，用漩涡振荡仪进行振荡洗脱，制成菌悬液，稀释到1×10-4梯度后，取100uL均匀涂到平板上，37℃培养1d，制作致死率曲线，确定致死率随诱变时间的变化数值关系。如图1所示，处理时间在70s以上时，致死率达到了90％以上，由现代育种理论可知，菌体致死率在90％～95％时正突变率最高，因此选择70s为最佳诱变时间，致死率为93.80％。
实施例2诱变后高产GABA的突变菌株的筛选
以处理时间为70s利用ARTP进行诱变，将诱变过的载片置于装有1mL无菌生理盐水的1mL离心管中，用漩涡振荡仪进行振荡洗脱，制成菌悬液，稀释到1×10-4梯度后，取100uL均匀涂到含有溴甲酚绿指示剂的筛选培养基表面，37℃培养1d，挑选菌落直径大并且颜色更偏向蓝绿色的菌株，转接到液体种子培养基中，37℃静置培养12h进行活化，于20％(V/V)甘油管中-20℃保存。
对从筛选平板上得到的菌株进行活化，将活化后的菌液以5％(V/V)的接种量接种到装有5mLMRS培养基的试管中，37℃静置培养24h后，利用HPLC对发酵液中的GABA含量进行检测，以具有较高产GABA能力的菌株再次进行ARTP诱变，最终得到一株高产量的植物乳杆菌HG-06突变株。
实施例3诱变菌株摇瓶转化能力测试
对产量最高的HG-06菌株进行10次传代，以检测突变株的遗传稳定性，并且在每次传代后，对突变菌进行活化。将活化后的菌液以5％的接种量接种到装有200mL培养基的500mL三角瓶中，37℃静置培养24h后，收集菌体，重悬于pH4.8的醋酸/醋酸钠缓冲液中，添加100g/LL-谷氨酸钠以及1‰的曲拉通X-100，在37℃条件下转化12h，通过HPLC检测转化液中的GABA含量，发现突变菌株连续传代10代以后产量均比较稳定，产量平均为66.27g/L，底物摩尔转化率为94.57％。
对突变菌株进行生长曲线测定(结果见图2)发现该突变菌株的对数生长期很短，猜想可能与其高产GABA有关。
实施例45L罐转化能力测试
将传代稳定的植物乳杆菌HG-06菌株进行活化后，并以15％接种量接种至装有3L发酵培养基的5L罐中，于0.2vvm，37℃静置培养30-36h，收集湿菌体，悬浮在终体积为1L、pH4.8的醋酸/醋酸钠缓冲溶液中，菌量OD600nm控制为20，添加300g/LL-谷氨酸以及1‰的曲拉通X-100，在37℃下转化24h，从0h起，在6h、14h、24h对GABA的浓度进行测定，结果如图3。最终产量为204.37g/L，底物摩尔转化率为97.22％，比出发菌株产量提高了56.83％，说明诱变菌株具有较强的GABA转化生产能力。
显然，上述实施例仅仅是为清楚地说明所作的举例，并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一株高产γ-氨基丁酸的植物乳杆菌（Lactobacillus plantarum）HG-06，其特征在于：于2022年01月21日保藏于中国典型培养物保藏中心，保藏地址为中国武汉大学，保藏编号为CCTCC NO：M 2022109。
2.权利要求1所述的植物乳杆菌（Lactobacillus plantarum）HG-06在催化L-谷氨酸或L-谷氨酸钠生产γ-氨基丁酸中的应用。
3.根据权利要求2所述的应用，其特征在于：所述的应用包括将植物乳杆菌（Lactobacillus plantarum）HG-06接种至种子培养基中进行培养获得种子液，再将种子液转接至发酵培养基中获得菌体，收集菌体悬浮在缓冲液中，投加L-谷氨酸或L-谷氨酸钠转化为γ-氨基丁酸的步骤。
4.根据权利要求3所述的应用，其特征在于：所述缓冲液为含有1-10‰曲拉通X-100的pH 4.5-5.0的醋酸/醋酸钠缓冲液或含有1-10‰曲拉通X-100的pH 4.5-5.0的柠檬酸磷酸盐缓冲液。
5.根据权利要求3所述的应用，其特征在于：投加L-谷氨酸或L-谷氨酸钠的方式为分批补料。
6.根据权利要求3所述的应用，其特征在于：转化温度为30-40℃。
7.根据权利要求3所述的应用，其特征在于：转化时间为12-24h。
8.根据权利要求3所述的应用，其特征在于：所述植物乳杆菌（Lactobacillusplantarum）HG-06的接种量为1-20%。
9.一种微生物菌剂，其特征在于：所述微生物菌剂含有权利要求1所述的植物乳杆菌（Lactobacillus plantarum）HG-06。
10.权利要求1所述的植物乳杆菌（Lactobacillus plantarum）HG-06或权利要求9所述的微生物菌剂在制备具有改善睡眠功能的药物组合物中的应用，其特征在于：所述药物组合物中含有植物乳杆菌生产的γ-氨基丁酸。
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