Screening and Identification of Lactic Acid Bacteria with High Yield of γ-Aminobutyric Acid in Traditionally Fermented Yak Yogurt in Western Sichuan Plateau
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摘要: 为筛选高产γ-氨基丁酸（γ-aminobutyric acid，GABA）的乳酸菌，以川西高原传统发酵牦牛乳中分离出的300株乳酸菌为研究对象，采用纸层析法和高效液相色谱法分别对其GABA生产能力进行定性和定量分析，对高产菌株进行体外耐受力实验和肠道黏附性实验，并确定优势菌株的种属。结果表明：300株待测菌株中，有24株具有生成GABA的能力，其中产量较高的5株菌分别为：菌株84（1.587 g/L）、菌株155（1.352 g/L）、菌株32（1.124 g/L）、菌株146（1.115 g/L）和菌株172（1.007 g/L）。菌株84在不同pH环境中处理3 h后有20%以上存活率，最高达到50.83%，为5株菌株中存活率最高。菌株32的存活率均能保持在10%~20%，在各pH环境中变化平稳，表明菌株84和菌株32具有良好的胃液耐受力，其余3株乳酸菌不耐酸性环境。在模拟肠液中反应4 h后，菌株84的乳酸菌存活率为37.57%，菌株32为12.8%，表明菌株84对人工肠液的耐受力稍高于菌株32。菌株32对胆盐的耐受力低于菌株84，而菌株84虽对NaCl有较好的耐受力，但较菌株32有一定差距，稳定性较差。在对肠道细胞的黏附实验中，菌株84的黏附能力高于菌株32。通过形态学特征、生理生化实验和16S rDNA基因鉴定，确定菌株84为发酵乳杆菌（Lactobacillus fermentum）。本研究为川西地区传统发酵牦牛酸乳中功能性益生菌研究提供了参考。Abstract: In order to screen out a lactic acid bacterium with high yield of γ-aminobutyric acid(GABA), 300 strains of lactic acid bacteria isolated from traditional fermented yak milk in the Western Sichuan Plateau were used as the research object. The qualitative and quantitative analysis of its GABA production capacity was carried out by paper chromatography and high performance liauid chromatogram, and then the high-yielding strains were subjected to in vitro tolerance experiments and intestinal adhesion experiments, and the species of dominant strains were determined. The results showed that among the 300 strains to be tested, 24 strains had the ability to produce GABA. The five strains with higher yields are: Strain 84(1.587 g/L), strain 155(1.352 g/L), strain 32(1.124 g/L), strain 146(1.115 g/L) and strain 172(1.007 g/L). Strain 84 had a survival rate of more than 20% after being treated for 3 hours in different pH environments, with a maximum of 50.83%, which was the highest survival rate among the 5 strains. The survival rate of strain 32 can be maintained at 10% to 20%, and it changes steadily in various pH environments. This indicates that strain 84 and strain 32 have good gastric juice tolerance, and the remaining three lactic acid bacteria were not tolerant to acidic environments. After reacting in simulated intestinal juice for 4 hours, the survival rate of lactic acid bacteria of strain 84 was 37.57%, and that of 32. The tolerance of strain 32 to bile salts was lower than that of strain 84. Although strain 84 had better tolerance to NaCl, it had a certain gap compared with strain 32 and it was less stable. In the adhesion experiment to intestinal cells, the adhesion ability of strain 84 was higher than that of strain 32. Through morphological characteristics, physiological and biochemical experiments and 16S rDNA gene identification, the strain 84 was determined to be Lactobacillus fermentum. This study provides a reference for the research on functional probiotics in traditional fermented yak yogurt in western Sichuan.
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Keywords:
- γ-aminobutyric acid /
- Yak yogurt /
- Lactobacillus fermentum /
- screening /
- identification
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[1] Zhang Heping,Jie Xu,Junguo Wang,et al. A survey on chemical and microbiological composition of kurut,naturally fermented yak milk from Qinghai in China[J]. Food Control,2008,19:578-586.
[2] 廉桂芳,谢婕,赵欣,等. 传统发酵牦牛酸乳的品质形成规律[J]. 食品与发酵工业,2017,43(5):56-63. [3] 杨洁,薛建岗,陈永福,等. 西藏地区两种传统发酵乳的氨基酸含量分析[J]. 中国奶牛,2013,8:50-52. [4] 张和平,孟和毕力格,土俊国,等.分离白内蒙古传统发酵酸马奶中L.casei Zhang潜在益生特性的研究[[J]. 中国乳品工业,2006,34(4):4-10. [5] 张和平,张七斤,任贵强,等.乳酸杆菌对攻毒小鼠的保护作用和对肠道菌群的影响[J]. 微生物学通报,2007,34(3):447-450. [6] 赵欣,蓦宇. 耗牛酸乳分离发酵乳杆菌发酵豆浆的胃溃疡预防效果研究[J].食品科学,2014,35(17):236-240. [7] 杨天予,刘一倩,马挺军. 富含γ-氨基丁酸藜麦发酵饮料工艺优化[J]. 食品工业科技,2019,40(16):169-175 ,180.
[8] Kook M C,Cho S C,Kang,J Y,et al. Effect of gamma-aminobutyric acid produced by Lactobacillus sakei B2-16 on diet and exercise in high fat diet-induced Obese rats[J]. Food Sci Biotechnol,2014,23(6):1965.
[9] Bhandage A K,Hellgren C,Jin Z,et al. Expression of GABA receptors subunits in peripheral blood mononuclear cells is gender dependent,altered in pregnancy and modified by mental health[J]. Acta Physiol,2015,213(3):575.
[10] 梁恒宇,邛立康,林海龙,等. 新资源食品氨基丁酸(GABA)的研究进展[J]. 食品研究与开发,2013,15:119-123. [11] 王建峰,任举. γ-氨基丁酸的生理作用与制备方法综述[J]. 山东化工,2013,42(2):251-252. [12] Benarroch E E. Pedunculopontine nucleus functional organizationand clinical implication[J]. Neurology,2013,80(12):1148-1155.
[13] Park K B,Oh S H. Production of yogurt with enhanced levels ofγ-aminobutyric acid and valuable nutrients using lactic acid bacteriaand germinated soybean extract[J]. Bioresource Technology,2007,9(8):1675-1679.
[14] 刘佳荣,梁金钟. 黄浆水中高产γ-氨基丁酸乳酸菌的筛选及鉴定[J]. 食品科学,2014,35(23):221-225. [15] 贤乾隆. 产γ-氨基丁酸乳酸菌的筛选及其功能性发酵酸奶的研制[D].柳州:广西科技大学,2013. [16] 熊京京,任佳秀,周姝含,等. 淡豆豉炮制过程中产γ-氨基丁酸微生物的筛选和鉴定[J]. 中国中药杂志,2019,44(11):2266-2273. [17] 赵宏飞,宋伟,裴家伟,等. 食品中三种γ-氨基丁酸检测方法比较[J]. 中国乳品工业,2008,36(11):52;54. [18] 中华人民共和国卫生部. 中华人民共和国药典[M]. 广州:广东科技出版社,1995:附录64. [19] 辛羚,郭本恒,吴正钧. 3株乳杆菌在模拟消化环境中存活性能的研究[J]. 中国乳品工业,2005,33(5):15-17. [20] 王翔宇,王琳琳,陈炼红. 传统发酵牦牛乳中2株高产胞外多糖乳酸菌在模拟消化道中耐受力的研究[J]. 食品科技,2019,44(4):12-17. [21] Bernet M F,Brassart D,Neeser J R,et al. Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cellninteractions[J]. Appl Environ Microbiol,1993,59:4121-4128.
[22] 缪存影. 高产γ-氨基丁酸乳酸菌的筛选鉴定、条件优化及谷氨酸脱羧酶基因的克隆[D]. 金华:浙江师范大学,2010. [23] 布坎南R E,吉布斯N E. 伯杰氏细菌鉴定手册[M]. 中国科学院微生物研究所《伯杰氏细菌鉴定手册》翻译组,译. 8版. 北京:科学出版社,1984:809-821. [24] 崔琴,贠建民,张丽,等. 甘南州牧区优良乳酸菌耐受特性的研究[J]. 食品工业科技,2010,31(3):216-219. [25] 杨桂梅,张永红,苏娜,等. 2株猪源益生性肠球菌对酸和胆盐及热的耐受性研究[J]. 北京农学院学报,2008,23(4):29-32. [26] 赵光侠. 胃液细菌培养在重症SIRS患儿的临床意义[J].小儿急救医学,2005,12(3):184-187. [27] 孟丽,唐善虎,杨蓉生,等. 干酪乳杆菌在模拟人体消化道环境及高盐环境中生长活力的研究[J]. 食品研究与开发,2009,30(4):46-49. [28] 王记成,郭壮,闫丽雅,等. 益生菌Lactobacillus casei Zhang与商业益生菌对胃肠转运耐受性及发酵特性的比较[J]. 中国食品学报,2009,9(5):14-23. [29] 陆艳琦,孙志红. 漫谈渗透现象和渗透压[J]. 郑州铁路职业技术学院学报,2006,18(1):65-67. [30] 李妍,张兰威. 几株乳酸菌益生潜力及降胆固醇的研究[J]. 微生物学通报,2007,34(6):1146-1149. [31] 谭霄,孙擎,曾林,等. 1株产γ-氨基丁酸植物乳杆菌谷氨酸脱羧酶基因的克隆与表达[J]. 食品科学,2018,39(18):159-165. [32] Luka M,Robert G,Kim T H,et al. Modulation of inhibitory plasticity in basal ganglia output nuclei of patients with Parkinson's disease[J]. Neurobiology of Disease,2019,124:46-56.
[33] Shabel S J,Proulx C D,Piriz J,et al. Mood regulation.GABA/glutamate co-release controls habenula output and is modified by antidepressant treatment.[J]. Science,2014,345(6203):1494-1498.
[34] 李理. 植物乳杆菌γ-氨基丁酸产量影响因素及代谢途径的研究[D]. 哈尔滨:东北农业大学,2015. [35] Toshiyuki H,Fatima R,Tomohide H,et al. GABA,γ-aminobutyric acid,protects against severe liver injury[J]. Journal of Surgical Research,2019,236:172-183.
[36] Hwang S,Ham S,Lee S E,et al. Hypoxia regulates the level of glutamic acid decarboxylase enzymes and interrupts inhibitory synapse stability in primary cultured neurons[J]. Neurotoxicology,2018,65:221-230.
[37] 徐林敏. 巨大芽孢杆菌X-1谷氨酸脱羧酶及γ-氨基丁酸转化条件的研究[D]. 南京:南京农业大学,2015. [38] 黄俊. 利用短乳杆菌制备γ-氨基丁酸相关过程研究田[D]. 杭州:浙江大学,2006. [39] Nomura M,Kimoto H,Someya Y,et al. Production of γ-aminobutyric acid by cheese starters during cheese ripening[J]. Journal of Dairy Science,1998,81(6):1486.
[40] Okada T,Sugishita T,Murakami T,et al. Effect of the defatted rice germ enriched with GABA for sleeplessness,depression,autonomic disorder by oral administration[J]. Nippon Shokuhin Kagaku Kogaku Kaishi,2000,47(8):596-603.
[41] 许建军,江波,许时婴. 生物合成γ-氨基丁酸的乳酸菌的筛选[J]. 食品科技,2002,10:7-10. [42] 傅元欣. 乳酸菌Lactococcus lactis SYFS 1.009生物法制备γ-氨基丁酸的研究[D]. 无锡:江南大学,2008. -
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