CN112300955A - 一种γ-胺基丁酸高产乳酸菌株及应用 - Google Patents
一种γ-胺基丁酸高产乳酸菌株及应用 Download PDFInfo
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Abstract
一株γ‑胺基丁酸高产乳酸菌株（Lactobacillus sp.）CPC202，经鉴定为短乳杆菌（Lactobacillus brevis），保藏号为CCTCC NO:M2019232。该菌株接种至添加有5‑9%的麸胺酸（glutamate）的MRS液体培养基中发酵培养后，能获取每公升含量达20公克（含）以上的γ‑胺基丁酸（γ‑aminobutyric acid,GABA），因而能以单一乳酸菌提高γ‑胺基丁酸的产量。
Description
技术领域
本发明有关于一种乳酸菌株，尤其涉及一株产出高产量γ-胺基丁酸能力的乳酸菌（Lactobacillus sp.）CPC202，γ-胺基丁酸的产生含量达29.8g/L。
背景技术
据中国台湾近几年民众营养健康状况变迁调查显示，18岁以上民众 高血压盛行率为24.1％。高血压是心脏病、中风、肾脏病、及眼疾等疾病 的重大危险因子，世界高血压联盟估计全球约有18％的人口(约9百40 万人)死亡的主要原因与高血压有关。高血压通常没有症状，许多人虽然 知道自己有高血压，却未采取任何控制血压的措施，任由其造成人体器官 的伤害。
此外，现代人生活压力大，直接影响睡眠，2013年中国台湾睡眠医学学会显示中国台湾失眠症盛行率为19.3%，2017年的调查更是指出“高年长者”及“女性”有较多的睡眠问题。长期的睡眠不足会导致焦虑的发生，如果长期暴露在失眠与焦虑下，久而久之就会发展为忧郁症，由数据看来使用抗忧虑药物的人数逐年增加，中国台湾忧郁症盛行率约7.3%（约150万人），但这仅仅是症状严重而求助医生的部分，大部分皆有轻度症状，只是不愿就医，所以以保健食品来舒缓失眠焦虑及忧虑的潜在市场非常的大，忧郁症已被联合国世界卫生组织视为新世纪三大疾病之一。
γ-胺基丁酸（γ-aminobutyric acid, GABA）是一种由四个碳所组成非蛋白质的水溶性天然胺基酸，分布非常广泛，在动植物中均有存在，以游离态存于原核与真核细胞的游离胺基酸群中。在动物体内，γ-胺基丁酸不会穿透血脑屏障，仅存在于神经组织中，它是由大脑组织自行合成的，其中脑组织中的含量为0.1～0.6 mg/g组织。γ-胺基丁酸具有抑制多巴胺即交感神经系统神经传导物质的作用，可抑制神经的兴奋性，降低癫痫的发生、以及高血压与焦虑的生成等，对心血管功能及舒压、镇定功效扮演重要角色。而γ-胺基丁酸的生成，则是由克氏循环（TCA cycle）中的麸胺酸（glutamate）被L-麸氨酸脱羧酶（Glutamate decarboxylase, GAD）与维生素B6所催化，通过一个叫γ-胺基丁酸的代谢分流途径（GABA shunt）而产生。γ-胺基丁酸是一种新型功能性原料，广泛应用于保健食品、医药化工等领域中。
γ-胺基丁酸可经由化学合成、自天然物中萃取、或经由微生物代谢产生。其中化学合成法的缺点是成本较高，得率较低，合成方法条件剧烈，且有化学物质残留，即使得到纯品也不属于天然产物，并且在生产过程中使用危险溶剂，甚至是有毒溶剂，因此该法所制得的γ-胺基丁酸主要应用于化工与医药领域，不能用于食品，也不能作为一种天然的食品添加剂。自天然物中提取γ-胺基丁酸的方法费时费工，产率不高。而微生物发酵是目前在产业界生产γ-胺基丁酸广为使用的方法，其产品具有成本低及含量高，可安全用于食品等优点。
就微生物发酵而言，目前已有多项相关专利及文献提出，例如：中国台湾专利公告号TW I460272 B揭示了“发酵虫草属真菌米基而制备γ-胺基丁酸的方法及其应用”，此案是以复合菌株，依一定程序及菌种比例生产γ-胺基丁酸，另中国专利公告号CN 105482981A揭示了“一种降血压保健醋的生产方法”，此案亦是一种结合两种乳杆菌以制造γ-胺基丁酸的制备方法；中国专利公告号CN 103013879 B揭示了“一株具有高产γ-胺基丁酸能力的菌株”，此案是以植物乳杆菌作为生产γ-胺基丁酸菌种；以及Jang EK等人在2015年于Pubmed期刊提出文献（Jang EK et al., "γ-aminobutyric acid(GABA) production andangiotensin-1 converting enzyme (ACE) inhibitory activity of fermentedsoybean containing Sea Tangle by the co-culture of Lactobacillus brevis withAspergillus oryzae", Journal of Microbiology and Biotechnology, 2015, 25:1315-20.），其是以米曲菌结合短乳杆菌共同发酵产出含有1.9g/kg的γ-胺基丁酸。
鉴于上述分析结果，因此，一般无法符合使用者于实际使用时直接提供以单一短乳杆菌高产γ-胺基丁酸能力的菌株。
发明内容
本发明的主要目的在于，克服已知技术所遭遇的上述问题，并提供一种高产γ-胺基丁酸的乳酸菌株，该乳酸菌株能用于发酵高产γ-胺基丁酸；并能用于制备具改善血压、舒眠、镇定解压功效的保健食品、营养补充品或医药组成物。
上述所提的乳酸菌株为Lactobacillus sp.CPC202，经鉴定为短乳杆菌（Lactobacillus brevis），于2019年4月3日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2019232，保藏地址为中国武汉武汉大学。同时，本菌株CPC202保藏于财团法人食品工业发展研究所，编号为2017ID048。
本菌株CPC202是从中国台湾的发酵酸菜中分离筛选得到。
本菌株CPC202于37℃在MRS培养基上培养；初步试验显示：本菌株CPC202为革兰氏阳性杆菌，不具触酶、氧化酶及运动性，于好氧环境及厌氧环境下生长，不产生内生孢子，结合参见图1。
根据16S rDNA序列进行比对分析，本菌株CPC202最接近Lactobacillus brevis，序列相似性高达100%， 结合菌株形态、生理生化特性分析结果，确认本菌株CPC202为短乳杆菌（Lactobacillus brevis）的一新种。
将本菌株CPC202接种至添加有5～9%（w/v）麸胺酸（glutamate，即谷氨酸）的MRS液体培养基中进行发酵培养，所产出的γ-胺基丁酸（γ-aminobutyric acid, GABA）每公升含量达29.82公克。较佳的，麸胺酸的添加量为7%（w/v）。
附图说明
图1是本发明菌株CPC202的显微照片图。
图2是本发明的GAD基因表现PCR产物电泳图。
图3是本发明菌株的GABA产生量示意图。
图2和图3中14065为BCRC编号，代表嗜酸乳杆菌；202为本发明菌株CPC202。
具体实施方式
以下实施例仅举例以供了解本发明的细节与内涵，但不用于限制本发明的申请专利范围。
实施例1：本菌株CPC202的制备
将取自中国台湾的发酵酸菜置于PBS溶液中，再将少量液体接种至MRS液体培养基中，在37℃静置20小时后，在MRS固态培养基上四曲划线纯化分离出单一菌株。
实施例2：γ-胺基丁酸生成调控基因GAD表现测试
本实施例以聚合酶链锁反应（polymerase chain reaction, PCR）侦测各菌株中GAD基因的表现情形，以初步筛选具有分泌GABA潜力之乳酸菌株，结果如图2所示。图中14065为BCRC编号，代表嗜酸乳杆菌（Lactobacillus acidophilus）标准菌株，用作阳性对照，M为DNA分子大小标准。
实施例3：γ-胺基丁酸产生方式
将本发明菌株CPC202以乳酸杆菌MRS培养基活化培养，在生产γ-胺基丁酸时，添加7%（w/v）麸胺酸到MRS液体培养基，置于37℃培养培养2天后，将发酵液在4℃条件下，以13000rpm/min离心15分钟，取上清液检测其γ-胺基丁酸生成量。
实施例4：γ-胺基丁酸产生量检测
本测试以液相层析串联质谱仪（LC/MC/MC）分析检测菌株γ-胺基丁酸的产生量（如图3所示），本菌株CPC202经发酵培养，所产出的γ-胺基丁酸产量为29.820 g/L（发酵液），曾有文献指出高血压患者每天18 mg γ-胺基丁酸，即有良好调节血压功效；故本方法所得产出量约为文献建议高血压患者每天使用量的1656倍。
藉此，本发明系经过筛选并以实验检测所得乳酸菌株其具备的特征以及可达成的性能，并发现获得的本菌株CPC202能产生高产量γ-胺基丁酸，γ-胺基丁酸具有抑制多巴胺即交感神经系统神经传导物质的作用，可抑制神经的兴奋性，降低癫痫的发生、高血压与焦虑的生成等，对心血管功能及舒压、镇定功效扮演重要角色。此菌株能应用于制备具改善血压、舒眠、镇定解压功效的保健食品、营养补充品或医药组成物，具有经济价值。
综上所述，本发明的一种γ-胺基丁酸高产乳酸菌株及其应用，可有效改善现有技术的种种缺点，直接提供以单一短乳杆菌（Lactobacillus brevis）产出高产量γ-胺基丁酸（GABA）能力的菌株，γ-胺基丁酸具有重要生理功能，是作为抗忧郁、抗焦虑或血压调节的医药及保健重要原料；本发明筛得具有产生高产量γ-胺基丁酸乳酸菌株CPC202，具有制备成改善血压、舒眠、镇定解压功效的保健食品、营养补充品或医药组成物的潜力，进而使本发明能产生更进步、更实用、更符合使用者所须，确已符合发明专利申请的要件，依法提出专利申请。
但以上所述，仅为本发明的较佳实施例而已，当不能以此限定本发明实施的范围。故，凡依本发明申请专利范围及发明说明书内容所作的简单的等效变化与修饰，皆应仍属本发明专利涵盖的范围内。
Claims (7)
1.一株γ-胺基丁酸高产乳酸菌株Lactobacillus sp. CPC202，保藏号为CCTCC NO:M2019232。
2.权利要求1所述的乳酸菌株CPC202在发酵生产γ-胺基丁酸中的应用。
3.权利要求1所述的乳酸菌株CPC202在制备改善血压、舒眠、镇定解压的保健食品中的应用。
4.权利要求1所述的乳酸菌株CPC202在制备改善血压、舒眠、镇定解压的营养补充品中的应用。
5.权利要求1所述的乳酸菌株CPC202在制备改善血压、舒眠、镇定解压的医药组成物中的应用。
6.一种利用乳酸菌株发酵生产γ-胺基丁酸的方法，其中，该乳酸菌株为权利要求1所述的乳酸菌株CPC202，所用培养基为添加有5-9%麸胺酸的MRS液体培养基。
7.如权利要求6所述的方法，其中，所述麸胺酸的添加量为7%。
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