In this study, plated streaks were used to separate and purify cultures to obtain epiphytic bacteria, genomic DNA was extracted, 16S rDNA was amplified by PCR, the gene was sequenced, phylogenetic tree was constructed for cluster analysis, and morphological observation was performed to determine the species of strains. The epiphyte bacteria with antibacterial activity were screened. After fermenting the selected target strains, crude extracts were obtained by solvent extraction, and the fermentation products were analyzed by electrospray ionization mass spectrometry (ESI-MS) to determine their structures. The results showed that nine strains of epiphyte were isolated and identified from the Xiahei grapes. Among them, BDG-5 and BDD-2 had significant antibacterial activities. They were Pseudomonas paralactis and Bacillus velezensis, respectively. ESI-MS showed that P.paralactis fermentation products contained cyclolipopeptides surfactin and iturin C. B.velezensis produced lipopeptides surfactin, iturin A, and bacillinomycin D. These cyclolipopeptides were the main substances possessed antibacterial activity.
FENG Yuyan
,
LIU Xiaofan
,
FAN Qing
,
CHEN Kaiye
,
HU Yimin
,
XIN Zhihong
. Screening,identification,and structure characterization of grape epiphytic bacteria[J]. Food and Fermentation Industries, 2019
, 45(5)
: 31
-37
.
DOI: 10.13995/j.cnki.11-1802/ts.017826
[1] 刘万振,武婷婷,生吉萍,等.有机和常规红提葡萄附生菌的比较分析[J].食品科学,2011,32(3):126-129.
[2] ANDREWS J H,HARRIS R F.The ecology and biogeography of microorganisms on plant surfaces[J].Annual Review of Phytopathology,2000,38(1):145-180.
[3] 刘丽,杨瑞娟,王桥美,等.云南大叶种古茶树表皮附生菌群的研究[J].农村实用技术,2017(2):30-33.
[4] 张克坤,程传云,徐卫东,等.夏黑葡萄在中国的发展历程与展望[J].中外葡萄与葡萄酒,2017(6):57-59+65.
[5] 杨雨蒙,徐敬国,胡伊旻,等.夏黑葡萄附生酵母菌分离及多相分类鉴定[J].食品工业科技,2017,38(22):100-104;113.
[6] 张俊杰,郭晨,尚益民,等.市售夏黑无核葡萄果表微生物多样性研究[J].轻工学报,2017,32(6):20-26.
[7] 王傲,杨柯,吕曼,等.基于NRPS基因筛选鉴定产生环脂肽葡萄附生细菌的研究[J].食品工业科技,2017,38(12):164-170.
[8] 李代婧,殷跃,刘梦婕,等.基于NRPS基因筛选与鉴定海芦笋内生细菌[J].食品与发酵工业,2017,43(9):58-63.
[9] 东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:318-419.
[10] 布坎南R E,吉本斯N E,著,中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组,译.伯杰鉴定手册(第八版)[M].北京:科学出版社,1995:591-611.
[11] 美国Omega公司.D3350-01 E.Z.N.A细菌DNA微量提取试剂盒说明书[EB/OL].https://wenku.baidu.com/view/3c2ed972b7360b4c2f3f644d.html.
[12] 谭才邓,朱美娟,杜淑霞,等.抑菌试验中抑菌圈法的比较研究[J].食品工业科技,2016,37(11):122-125.
[13] WEISBURG W G,BARNS S M,PELLETIER D A,et al.16S ribosomal DNA amplification for phylogenetic study[J].Journal of Bacteriology,1991,173(2):697-703.
[14] 刘丽霞.Surfactin合成酶相关基因研究[D].南京:南京农业大学,2012.
[15] 李慧.解淀粉芽孢杆菌及其生物合成环脂肽抗生素条件研究[D].南京:南京大学,2012.
[16] LEE M H,LEE J,NAM Y D,et al.Characterization of antimicrobial lipopeptides produced by Bacillus sp.LM7 isolated from chungkookjang,a Korean traditional fermented soybean food[J].International Journal of Food Microbiology,2016(2 221):12-18.
[17] 金疏桐,王祖华,徐启燃,等.内生贝莱斯芽胞杆菌生物拮抗活性的研究[J].中国微生态学杂志,2017,29(4):385-389.
[18] 宗英,赵月菊,刘阳,等.一株贝莱斯芽孢杆菌抑制禾谷镰刀菌的研究[J].核农学报,2018,32(2): 310-317.
[19] LECLRE V,BCHET Max, ADAMS A,de,la,le.Mycosubtilin overproduction by Bacillus subtilis BBG100 enchances the Organism’s antagonistic and biocontrol activites[J].Applied and Environmental Microbiology,2005,71(8):4 577-4 584.
[20] KRACHT M,ROKOS H,OZEL M,et al.Antiviral and hemolytic activities of surfactin insoforms and their methyl ester derivatives[J].The Journal of Antibiotics,1999,52(7):613-619.
[21] 刘京兰.解淀粉芽抱杆菌CC09产Iturin A的发酵条件及Iturin A的提取工艺研究[D]南京:南京大学,2014.
[22] 杨柯,王傲,吕曼,等.基于NRPS功能基因筛选鉴定盐生海芦笋内生细菌的研究[J].食品工业科技,2017,38(1):175-181.
[23] ONGENA M,JACQUES P.Bacillus lipopeptides:versatile weapons for plant disease biocontrol[J].Trends in Microbiology,2008,16(3):115-125.
[24] NEUBECK M,HUPTAS C,GLCK C,von,der,am.Pseudomonas lactis sp. nov. and Pseudomonas paralactis sp.nov.,isolated from bovine raw milk[J].Int J Syst Evol Microbiol,2017(67):1 656-1 664.
[25] 鲍瑞峰,秦丹.果蔬采后病害生物防治的研究进展[J].保鲜与加工,2009,9(3):1-5.
