两株茶叶籽水浆发酵杆菌的分离鉴定及其数量动态分析
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摘要
【目的】鉴定茶叶籽水浆发酵分层过程中后期的微生物种类,并研究其数量动态,为优化茶叶籽油生物发酵生产工艺及揭示茶叶籽水浆发酵分层机理提供参考依据。【方法】利用添加放线菌酮的MRS固体培养基对发酵5 h的茶叶籽水浆发酵液划线培养,并进行微生物分离与提纯;通过形态学观察、酶学反应、碳源反应、16S rDNA及pheS基因测序分析、系统发育树分析,对分离的微生物进行鉴定;再利用涂布培养法观察菌体的数量动态变化情况。【结果】从茶叶籽水浆发酵液中分离纯化获得两株菌株,分别编号为JJZ12和JJZ21。两株菌株的菌落均呈圆形,白色,凸起,表面光滑湿润,边缘整齐。其中,JJZ12菌株的菌体呈杆状,大小0.6~1.2μm×1.0~3.0μm,成对或短链排列,革兰氏阳性;JJZ21菌株的菌体大小为0.5~0.7μm×0.8~1.7μm,单个或成对排列,革兰氏阳性。经鉴定,JJZ12和JJZ21分别为Gluconacetobacter liquefaciens(产液葡糖酸醋杆菌)和Lactobacillus plantarum subsp. plantarum(植物乳杆菌)的一个菌株;结合本研究编号,两种杆菌分别命名为G. liquefaciens JJZ12和L. plantarum subsp. plantarum JJZ21。在茶叶籽水浆发酵过程中,发酵液中JJZ12和JJZ21菌体的总数量在发酵5 h后逐渐快速增加,至15 h时达最大值,之后又逐渐回落进入稳定状态。【结论】JJZ12和JJZ21两株杆菌是茶叶籽水浆发酵中后期起主要作用的发酵微生物,该结论为解释茶叶籽水浆发酵分层现象的发生机理提供了理论依据。
【Objective】The species and count dynamic of microorganism in fermentation layering of Camellia sinensis water milk were clarified to provide reference for optimization of fermentation technique of C. sinensis and revealing the fermentation layering mechanism of C. sinensis water milk.【Method】C. sinensis water milk that fermented for 5 h was cultured on MRS solid culture medium with cycloheximide. The microorganism was isolated and purified. Through morphological observation,enzymatic reaction,carbon source reaction gene sequencing and comparison of 16 S rDNA and pheS,establishment and analysis of phylogenetic tree,the isolated microorganism were identified. Spread plate method was used to observe the dynamic amount of bacteria.【Result】Two strains were isolated and purified from fermented C. sinensis water milk,their serial numbers were JJZ12 and JJZ21 respectively. The colony of JJZ12 was round,white and convex,with smooth and moist surface and neat edges;and its thallus were rod-shaped,with a size of 0.6-1.2 μm×1.0-3.0 μm,and in pairs or short chains,gram-positive. The morphology of JJZ21 colony was the same as JJZ12. However,its size of thallus was 0.5-0.7 μm×0.8-1.7 μm,and single or paired,gram positive. JJZ12 and JJZ21 were authenticated as a strain of Gluconacetobacter liquefaciens and Lactobacillus plantarum subsp. plantarum respectively. Combined with theresearch number,the full name of these two zymobacter strains were G. liquefaciens JJZ12 and L. plantarum subsp. plantarum JJZ21. In the fermentation process of C. sinensis water milk,the total number of JJZ12 strains and JJZ21 strains in fermented milk increased rapidly after 5 h,and peaked at 15 h,and after that gradually reduced to a stable status.【Conclusion】JJZ12 and JJZ21 are two strains that play a major role in the middle and later periods of C. sinensis water milk fermentation as the fermentation microorganism. The conclusion afford preliminary evidences for explaining occurrence mechanism of fermentation layering of C. sinensis water milk.
【Objective】The species and count dynamic of microorganism in fermentation layering of Camellia sinensis water milk were clarified to provide reference for optimization of fermentation technique of C. sinensis and revealing the fermentation layering mechanism of C. sinensis water milk.【Method】C. sinensis water milk that fermented for 5 h was cultured on MRS solid culture medium with cycloheximide. The microorganism was isolated and purified. Through morphological observation,enzymatic reaction,carbon source reaction gene sequencing and comparison of 16 S rDNA and pheS,establishment and analysis of phylogenetic tree,the isolated microorganism were identified. Spread plate method was used to observe the dynamic amount of bacteria.【Result】Two strains were isolated and purified from fermented C. sinensis water milk,their serial numbers were JJZ12 and JJZ21 respectively. The colony of JJZ12 was round,white and convex,with smooth and moist surface and neat edges;and its thallus were rod-shaped,with a size of 0.6-1.2 μm×1.0-3.0 μm,and in pairs or short chains,gram-positive. The morphology of JJZ21 colony was the same as JJZ12. However,its size of thallus was 0.5-0.7 μm×0.8-1.7 μm,and single or paired,gram positive. JJZ12 and JJZ21 were authenticated as a strain of Gluconacetobacter liquefaciens and Lactobacillus plantarum subsp. plantarum respectively. Combined with theresearch number,the full name of these two zymobacter strains were G. liquefaciens JJZ12 and L. plantarum subsp. plantarum JJZ21. In the fermentation process of C. sinensis water milk,the total number of JJZ12 strains and JJZ21 strains in fermented milk increased rapidly after 5 h,and peaked at 15 h,and after that gradually reduced to a stable status.【Conclusion】JJZ12 and JJZ21 are two strains that play a major role in the middle and later periods of C. sinensis water milk fermentation as the fermentation microorganism. The conclusion afford preliminary evidences for explaining occurrence mechanism of fermentation layering of C. sinensis water milk.
引文
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刘英男,王光强,夏永军,张汇,熊智强,艾连中.2007.一株具有良好粘附性的植物乳杆菌的生物学特性及应用[J].工业微生物,47(2):43-47.[Liu Y N,Wang G Q,Xia YJ,Zhang H,Xiong Z Q,Ai L Z.2007.Biological characteristics and application of a Lactobacillus plantarum with good adhesion[J].Industrial Microbiology,47(2):43-47.]
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马国善.2018.食品微生物检验菌落总数测定方法的效果观察[J].中国医药指南,16(20):47.[Ma G S.2018.Observation of effectiveness of the method for determining total number of bacterial colonies in food microbiological testing[J].Guide of China Medicine,16(20):47.]
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王颜颜,夏茂宁,欧维正,黄劲,明春艳,刘涛华,吕倩,周兵,康颖倩.2017.16S rRNA和secA1基因构建临床诺卡菌的系统发育树比较[J].贵州医科大学学报,42(4):409-415.[Wang Y Y,Xia M N,Ou W Z,Huang J,Ming CY,Liu T H,LüQ,Zhou B,Kang Y Q.2017.Comparison of phylogeny of clinical Nocardia species based on 16SrRNA and secA1 gene sequences[J].Journal of Guizhou Medical University,42(4):409-415.]
辛若竹,丁梅,孟宪志,孙健.2008.活性酸乳中每种益生菌总数的同时计数法[J].食品科学,29(4):244-248.[Xin RZ,Ding M,Meng X Z,Sun J.2008.Simultaneous counting method for total probiotics species in active yoghurt[J].Food Science,29(4):244-248.]
张峥,张戈,杨蓉,龙宣杞.2017.培养基不同营养水平对一株植物乳杆菌发酵生长及保存的影响[J].新疆农业科学,54(6):1108-1113.[Zhang Z,Zhang G,Yang R,Long XQ.2017.Effects of different nutritional levels on the growth and preservation of a strain medium:Lactobacillus plantarum[J].Xinjiang Agricultural Sciences,54(6):1108-1113.]
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Amr A S,Jabay O A.2004.Effect of salt iodization on the quality of pickled vegetables[J].Journal of Food,Agriculture&Environment,41(2):402-408.
Brown R M,Willison J H,Richardson C L.1976.Cellulose biosynthesis in Acetobacter xylinum:Visualization of the site of synthesis and direct measurement of the in vivo process[J].Proceedings of the National Academy of Sciences of the United States of America,73(12):4565-4569.
Erkkil?S,Pet?j?E,Eerola S,Lilleberg L,Mattila-Sandholm T,Suihko M-L.2001.Flavor profiles of dry sausages fermented by selected novel meat starter cultures[J].Meat Science,58(2):111-116.
Fotana J D,Franco V C,Souza S J,Lyra I N,de Souza A M.1991.Nature of plant stimulators in the production of Acetobacter xylinum(tea fungus)biofilm used in skin therapy[J].Applied Biochemistry&Biotechnology,28-29(1):341-351.
Gobbetti M,Corsetti A,Rossi J.1994.The sourdough microflora.Interactions between lactic acid bacteria and yeasts:Metabolism of amino acids[J].World Journal of microbiology and biotechnology,41(4):456-460.
Iurkevich D I,Kutyshenko V P.2002.Medusomyces(tea fungus):Scientific history,composition,physiology,and metabolism[J].Biofizika,47(6):16-29.
Jiang J Z,Ren C J,Wang Z B.2013.New process for extracting oil and starch from tea seeds[J].Journal of Food Science and Engineering,3(12):699-703.
Lee S G,Lee Y J,Kim M K,Ham J.2010.A study on the yogurt manufacture suitability and antimicrobial activity of Lactobacillus plantarum LHB55 isolated from Kimchi[J].Journal of Animal Science&Technology,52(2):141-148.
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东秀珠,蔡妙英.2001.常见细菌系统鉴定手册[M].北京:科学出版社:373-380.[Dong X Z,Cai M Y.2001.Common Bacterial System Identification Manual[M].Beijing:Science Press:373-380.]
冯静,施庆珊,欧阳友生,陈仪本.2009.葡糖醋杆菌的研究进展[J].化学与生物工程,26(5):10-13.[Feng J,Shi Q S,Ouyang Y S,Chen Y B.2009.Research progress on Gluconacetobacter[J].Chemistry&Bioengineering,26(5):10-13.]
冯静,施庆珊,欧阳友生,陈仪本.2010.葡糖杆菌属分类及其主要应用的研究进展[J].微生物学杂志,30(2):86-90.[Feng J,Shi Q S,Ouyang Y S,Chen Y B.2010.Advance in classification and main application of Gluconobacter[J].Journal of Microbiology,30(2):86-90.]
郭翔,艾连中,吴艳,张灏,陈卫,郭本恒.2009.植物乳杆菌Lactobacillus plantarum GUO安全性研究[J].食品科学,30(23):436-438.[Guo X,Ai L Z,Wu Y,Zhang H,Chen W,Guo B H.2009.Mutagenicity and virulence evaluation of Lactobacillus plantarum GUO[J].Food Science,30(23):436-438.]
江和源,张建勇,高晴晴.2007.茶皂素的性质、制备与应用[J].中国茶叶,29(3):14-15.[Jiang H Y,Zhang J Y,Gao Q Q.2007.Character,preparation and utilization of tea saponion[J].China Tea,29(3):14-15.]
姜金仲,韩宝银,王自布.2013.一种茶叶籽油生物发酵法生产工艺(ZL2013102932241):中国,CN103351940A[P].2013-10-16.[Jiang J Z,Han B Y,Wang Z B.2013.A process technique by biological fermentation for tea seed oil(ZL2013102932241):China,CN103351940A[P].2013-10-16.]
姜金仲,王超英,韩晗,刘娟,吴杰.2015.茶叶籽仁水浆静置发酵分层生产茶叶籽油及淀粉[J].中国油脂,40(3):74-78.[Jiang J Z,Wang C Y,Han H,Liu J,Wu J.2015.Productions of tea seed oil and starch by static fermentation of tea seed milk[J].China Oils and Fats,40(3):74-78.]
姜金仲,杨鹏鸣,王超英,韩晗,刘建芳.2017a.茶叶籽水浆发酵分层过程及其间总质量动态研究[J].中国粮油学报,32(2):104-108.[Jiang J Z,Yang P M,Wang C Y,Han H,Liu J F.2017a.Layering process and total weight dynamic of tea seed water milk in fermentation[J].Journal of the Chinese Cereals and Oils Association,32(2):104-108.]
姜金仲,杨鹏鸣,王兴春,穆安德,刘寨生.2017b.茶叶籽酵母的分离鉴定及其发酵特性研究[J].食品工业科技,38(21):105-108.[Jiang J Z,Yang P M,Wang X C,Mu AD,Liu Z S.2017b.Studies on identification and fermentation characteristics of tea seed yeast[J].Science and Technology of Food Industry,38(21):105-108.]
金雷,陈瑜,严忠雍,郭远明,祝银,龙举,颜蓓乔.2014.邻苯二甲酸二丁酯高效降解菌H-2的分离鉴定及其降解特性研究[J].食品科学,35(15):202-206.[Jin L,Chen Y,Yan Z Y,Guo Y M,Zhu Y,Long J,Yan B Q.2014.Isolation and identification of a di-n-butyl phthalate(DBP)-degrading strain H-2 and its degradation characteristics[J].Food Science,35(15):202-206.]
刘英男,王光强,夏永军,张汇,熊智强,艾连中.2007.一株具有良好粘附性的植物乳杆菌的生物学特性及应用[J].工业微生物,47(2):43-47.[Liu Y N,Wang G Q,Xia YJ,Zhang H,Xiong Z Q,Ai L Z.2007.Biological characteristics and application of a Lactobacillus plantarum with good adhesion[J].Industrial Microbiology,47(2):43-47.]
鲁战会,李里特,李再贵,辰巳英三.2002.自然发酵对淀粉凝胶的改性机理及发酵菌株的筛选[J].食品与发酵工业,28(4):1-6.[Lu Z H,Li L T,Li Z G,Eizo T.2002.Study on the modification mechanism of natural fermented starch gel and the isolation of microorganism[J].Food and Fermentation Industries,28(4):1-6.]
马国善.2018.食品微生物检验菌落总数测定方法的效果观察[J].中国医药指南,16(20):47.[Ma G S.2018.Observation of effectiveness of the method for determining total number of bacterial colonies in food microbiological testing[J].Guide of China Medicine,16(20):47.]
梅乐和,姚善泾,林东强.2000.生化生产工艺学[M].北京:科学出版社.[Mei L H,Yao S J,Lin D Q.2000.Biochemical Production Technology[M].Beijing:Science Press.]
王颜颜,夏茂宁,欧维正,黄劲,明春艳,刘涛华,吕倩,周兵,康颖倩.2017.16S rRNA和secA1基因构建临床诺卡菌的系统发育树比较[J].贵州医科大学学报,42(4):409-415.[Wang Y Y,Xia M N,Ou W Z,Huang J,Ming CY,Liu T H,LüQ,Zhou B,Kang Y Q.2017.Comparison of phylogeny of clinical Nocardia species based on 16SrRNA and secA1 gene sequences[J].Journal of Guizhou Medical University,42(4):409-415.]
辛若竹,丁梅,孟宪志,孙健.2008.活性酸乳中每种益生菌总数的同时计数法[J].食品科学,29(4):244-248.[Xin RZ,Ding M,Meng X Z,Sun J.2008.Simultaneous counting method for total probiotics species in active yoghurt[J].Food Science,29(4):244-248.]
张峥,张戈,杨蓉,龙宣杞.2017.培养基不同营养水平对一株植物乳杆菌发酵生长及保存的影响[J].新疆农业科学,54(6):1108-1113.[Zhang Z,Zhang G,Yang R,Long XQ.2017.Effects of different nutritional levels on the growth and preservation of a strain medium:Lactobacillus plantarum[J].Xinjiang Agricultural Sciences,54(6):1108-1113.]
周光燕,张小平,钟凯,孙桂丹,Kristina Lindstrom.2006.乳酸菌对泡菜发酵过程中亚硝酸盐含量变化及泡菜品质的影响研究[J].西南农业学报,19(2):290-293.[Zhou G Y,Zhang X P,Zhong K,Sun G D,Lindstrom K.2006.Effect of different lactic acid bacteria inoculation on the nitrite concentration and the quality of pickle[J].Southwest China Journal of Agricultural Sciences,19(2):290-293.]
Amr A S,Jabay O A.2004.Effect of salt iodization on the quality of pickled vegetables[J].Journal of Food,Agriculture&Environment,41(2):402-408.
Brown R M,Willison J H,Richardson C L.1976.Cellulose biosynthesis in Acetobacter xylinum:Visualization of the site of synthesis and direct measurement of the in vivo process[J].Proceedings of the National Academy of Sciences of the United States of America,73(12):4565-4569.
Erkkil?S,Pet?j?E,Eerola S,Lilleberg L,Mattila-Sandholm T,Suihko M-L.2001.Flavor profiles of dry sausages fermented by selected novel meat starter cultures[J].Meat Science,58(2):111-116.
Fotana J D,Franco V C,Souza S J,Lyra I N,de Souza A M.1991.Nature of plant stimulators in the production of Acetobacter xylinum(tea fungus)biofilm used in skin therapy[J].Applied Biochemistry&Biotechnology,28-29(1):341-351.
Gobbetti M,Corsetti A,Rossi J.1994.The sourdough microflora.Interactions between lactic acid bacteria and yeasts:Metabolism of amino acids[J].World Journal of microbiology and biotechnology,41(4):456-460.
Iurkevich D I,Kutyshenko V P.2002.Medusomyces(tea fungus):Scientific history,composition,physiology,and metabolism[J].Biofizika,47(6):16-29.
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