CN114891706B - 高耐酸醋杆菌及其应用 - Google Patents

高耐酸醋杆菌及其应用 Download PDF

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CN114891706B
CN114891706B CN202210816813.2A CN202210816813A CN114891706B CN 114891706 B CN114891706 B CN 114891706B CN 202210816813 A CN202210816813 A CN 202210816813A CN 114891706 B CN114891706 B CN 114891706B
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柏映国
黄火清
罗会颖
姚筱暄
唐洪亮
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Beijing Yixing Biotechnology Co ltd
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Abstract

本发明涉及微生物领域，具体涉及高耐酸醋杆菌及其应用。与野生型醋杆菌WCM23相比，本发明的突变醋杆菌菌株有较高的耐酸能力和更高的产酸能力；更高的乙醇脱氢酶和乙醛脱氢酶活。

Description

高耐酸醋杆菌及其应用
技术领域
本发明涉及微生物领域，具体涉及高耐酸醋杆菌及其应用。
背景技术
醋杆菌（Acetobacter sp.）广泛分布于自然界的含糖或酸性物质中，是快速适应不同环境的典型生物。醋杆菌能够氧化乙醇或糖类并生成乙酸或葡萄糖酸等有机酸，是一类重要的工业微生物，主要用于食醋与果醋饮料的生产。醋杆菌能在有氧条件下，通过与膜结合的乙醇脱氢酶、乙醛脱氢酶以及辅酶吡咯喹啉醌（PQQ）的复合体作用，把酵母菌在无氧条件下生成的乙醇氧化为乙酸，乙酸具有独特的风味，这也是我国传统酿醋工艺路线的生化原理。醋杆菌除生成醋酸以外，还能生成羟基酸，如羟基乙酸、草酸、酒石酸、己二酸、琥珀酸、甘露糖酸和葡萄糖酸等，这些酸也是形成醋的复合主要香味物质。虽然，与其他多数细菌相比较，醋杆菌具有较强的耐酸性，以菌体细胞的膜蛋白为载体，通过消耗能量将乙酸转运出细胞，维持了胞内的低乙酸水平，但是在食醋发酵过程中，由于持续产酸，pH值快速降低，最终影响了其生长和代谢。
由于醋杆菌菌体胞内本身含有丰富的乙醇脱氢酶、乙醛脱氢酶以及辅酶吡咯喹啉醌酶系，这些酶的共同作用可以在体外将乙醇快速氧化为乙酸，因此可以通过诱导培养醋杆菌发酵技术和细胞破碎技术制备乙醇脱氢酶、乙醛脱氢酶以及辅酶吡咯喹啉醌酶复合物。这种复合物在很多领域具有重要应用价值，比如，可以和其他中药组分复配，提升其解酒醒酒能力。人体饮酒时摄入的酒精约90%在肝脏中进行代谢。当酒精代谢速度小于摄入速度时，酒精就会在体内短时间大量蓄积，使肝脏谷胱甘肽巯基转移酶，谷胱甘肽过氧化物酶等含量显著降低，进而对肝脏产生损伤。而且过量的酒精会扰乱人体脂质代谢。
由于现有醋杆菌胞内含有乙醇脱氢酶、乙醛脱氢酶以及辅酶吡咯喹啉醌复合物浓度仍然较低，因此筛选高耐酸高含量脱氢酶系的醋杆菌仍具有巨大的应用价值。
发明内容
本发明的目的是提供一种高耐酸醋杆菌。
本发明的再一目是提供上述高耐酸醋杆菌的应用。
根据本发明的高耐酸醋杆菌（Acetobacter sp.）CM-067，其保藏编号为CGMCCNo.24906。
本发明提供了上述高耐酸醋杆菌的以下应用：
发酵制备乙醇脱氢酶或乙醛脱氢酶；
制备食醋；或
制备饮料。
本发明经突变获得醋杆菌（Acetobacter sp.）CM-067，与野生型醋杆菌WCM23相比，醋杆菌（Acetobacter sp.）CM-067在发酵培养基中有较高的耐酸能力和更高的产酸能力；醋杆菌（Acetobacter sp.）CM-067具有更高的乙醇脱氢酶和乙醛脱氢酶活力。
附图说明
图1显示诱变菌株067号透明圈；
图2显示野生型菌和本申请的诱变菌株的乙醇降解率。
醋杆菌CM-067，分类命名：醋杆菌Acetobacter sp.，保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC)，保藏地址为北京市朝阳区北辰西路1号院3号，邮政编码100101，保藏日期为 2022年05月18日，保藏号为CGMCC No.24906。
具体实施方式
实施例1 野生型醋杆菌WCM23诱变筛选获得高耐酸醋杆菌CM-067
从甘油管取出野生型菌种WCM23，接种于种子培养基：1%葡萄糖，2%的酵母粉，35℃培养16小时，离心收集菌体，然后用生理盐水重选清洗3次后，用生理盐水稀释成OD600=2.0，经过ARTP等离子诱变仪器，通过调整参数，使其致死率在99.0%-99.9%，然后稀释涂板筛选。筛选培养基为：0.5%葡萄糖，2%的酵母粉，碳酸钙1%，乙醇5%，2%琼脂，35℃培养48小时后，根据透明圈大小进行筛选。经过4轮筛选，初步获得43株透明圈较大的突变体。
将这43株透明圈较大的突变体接种在复筛培养基，0.5%葡萄糖，2%的酵母粉，乙醇5%，35度，250rpm，装液量15%，培养24小时，测定培养结束pH值和发酵液中乙酸浓度，其中野生型菌种WCM23的发酵终了pH值为2.97，乙酸浓度为0.92%，编号为CM-067的诱变株pH最低为2.16，乙酸浓度为1.24%，比野生型菌株提高了29%，诱变菌株067号透明圈如图1所示。由于复筛培养基中没有碳酸钙作为缓冲，微生物在底物足量情况下，其生长代谢过程持续产酸，直至最后抑制自己生长。因此发酵终了pH值低表明其生长过程耐受酸的能力更强。
实施例2 细胞的破碎
从甘油管取出野生型菌种WCM23和诱变株CM-067，分别接种于种子培养基：1%葡萄糖，2%的酵母粉，35度培养16小时，然后以2%的接种量接种至发酵培养基为：0.5%葡萄糖，2%的酵母粉，碳酸钙1%， 200rpm，35度培养24小时后，添加无水乙醇至终浓度5%（V/V），继续培养24小时后，离心，取沉淀重悬于生理盐水中，然后缓慢滴加乙酸，调pH至2.0，目的是溶解菌泥当中的碳酸钙，离心获得纯菌体，然后用生理盐水清洗3次后，重悬于生理盐水中，并通过生理盐水精确调整使菌液OD600=5.0。样品置冰浴中，功率为300W，超声20秒间歇10秒，循环 40次的方式破碎菌体。离心取上清液进行乙醇降解能力测定。
实施例3细胞破碎液降解乙醇测定
首先用 0.1 mol/L 甘氨酸/HCL（pH4.5）缓冲液配制0.5%（V/V）的底物，然后取900ul上述配好的含有0.5%乙醇的底物溶液，加入稀释10倍的细胞破碎上清液，于 37℃反应 60 min，然后置冰水浴迅速降温，然后用截留分子量3k的超滤膜，4摄氏度下，12000rpm，离心10分钟，取滤过液稀释上气相质谱联用测定。
乙醇含量测定采用气相质谱联用（GC-MS）方法：
SPME条件（50/30 μm DVB/CAR/PDMS）：
孵化时间:3 min
萃取时间:15 min
萃取温度:60℃
解吸附时间:3 min
GC条件（VF-WAX ms 60 m×di 0.25 mm×0.25 μm）：
进样口温度:250℃
载气: He
载气流速:1 mL/min
进样方式：分流
分流比: 10:1
升温程序：初始温度60℃，保持1 min；以5℃/min，升温至100℃，保持0 min；以20℃/min，升温至230℃，保持3 min。
MS条件：
全扫描模式
MS传输线温度:250℃
离子源温度:280℃
扫描范围:30-400 m/z
结果表明（图2）：野生型菌种WCM23的乙醇降解率为36.4%，而CM-067的乙醇降解率为45.6%，比野生型提高25.3%。这也表明诱变使乙醇（乙醛）脱氢酶的活力提升幅度更大。
以上实施例仅用于解释本申请的技术方案，不限定本申请的保护范围。

Claims (3)

1. 野生型醋杆菌WCM23的 高耐酸醋杆菌（Acetobacter sp.）突变菌株，其特征在于，所述突变菌株的保藏编号为CGMCC No.24906。
2.权利要求1所述野生型醋杆菌WCM23的 高耐酸醋杆菌（Acetobacter sp.）突变菌株的以下应用：
发酵制备乙醇脱氢酶或乙醛脱氢酶；
制备食醋；或
制备饮料。
3.根据权利要求2所述的应用，其特征在于，所述野生型醋杆菌WCM23的 高耐酸醋杆菌（Acetobacter sp.）突变菌株在34-36℃温度下发酵。
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