副溶血性弧菌是一种常见的食源性病原菌和水产致病菌。该研究通过琼脂扩散法从芦潮港海泥中筛选对副溶血性弧菌具有较强抑制作用的菌株,对其进行形态观察和16S rDNA分子鉴定,并对其所产抑菌物质进行抑菌谱和理化特性研究。结果表明,筛选出的枯草芽胞杆菌H5对副溶血性弧菌ATCC 17802具有强烈的抑制作用,抑菌圈直径达(32.05±0.10)mm,其产生的抑菌物质具有广谱抑菌活性,对革兰氏阳性菌的抑制作用大于革兰氏阴性菌。通过理化性质研究发现,H5所产抑菌物质在121 ℃处理30 min和较宽的pH值范围内(2~12)可保持良好的稳定性,并具有良好的紫外线稳定性和有机溶剂稳定性,且Mn2+和Zn2+可提高其抑菌活性而Ca2+和Mg2+可抑制其活性。H5所产抑菌物质经过蛋白酶处理后活性下降,证明其是一种抗菌肽。综上所述,枯草芽胞杆菌H5所产抗菌肽不仅具有良好的稳定性,而且抑菌谱广、抑菌活性强,在食品工业和医药行业具有广阔的开发和应用前景。
Vibrio parahaemolyticus is a common food-borne pathogen and aquatic pathogen. In this study, an isolate H5 with strong inhibitory effect on V.parahaemolyticus was screened and systemically identified as Bacillus subtilis.B.subtilis H5 had strong antimicrobial activity against V.parahaemolyticus ATCC17802 with the diameter of inhibition zone of (32.05±0.10) mm.Moreover, its antibacterial substance exhibited a broad-spectrum antimicrobial activity and its inhibitory effect on Gram-positive bacteria was stronger than that of Gram-negative bacteria.The antibacterial substance produced by B.subtilis H5 could maintain good activity at of 121 ℃ for 30 min and a wide pH range from 2 to 12.In addition, the antimicrobial substance had good stability of temperature, pH, UV and organic solvent.Mn2+ and Zn2+ could improve the antibacterial activity while Ca2+ and Mg2+ inhibit it.Besides, the antibacterial substance was sensitive to proteases, which indicated that it was a kind of antimicrobial peptide.In conclusion, the antimicrobial peptide produced by B.subtilis H5 is stable, with broad-spectrum antimicrobial activity and strong inhibition effect, which has a broad application prospect in food industry and pharmaceutical industry.
[1] 刘雪飞. 副溶血性弧菌拮抗菌的分离及其抑菌机理研究[D].沈阳:沈阳农业大学,2016.
LIU X F.Isolation and antibacterial mechanism of antagonistic bacteria against Vibrio parahaemolyticus[D].Shenyang:Shenyang Agricultural University,2016.
[2] MOK J S,RYU A,KWON J Y,et al.Distribution of Vibrio species isolated from bivalves and bivalve culture environments along the Gyeongnam coast in Korea:Virulence and antimicrobial resistance of Vibrio parahaemolyticus isolates[J].Food Control,2019,106:106697.
[3] BIERBAUM G,SAHL H G.Lantibiotics:Mode of action,biosynthesis and bioengineering[J].Current Pharmaceutical Biotechnology,2009,10(1):2-18.
[4] ALEXANDRE Y,LE BLAY G,BOISRAMÉ-GASTRIN S,et al.Probiotics:A new way to fight bacterial pulmonary infections?[J].Mèdecine et Maladies Infectieuses,2014,44(1):9-17.
[5] 孙力军, 王雅玲,陈宏.芽孢杆菌抗菌肽应用于海产品食源性病原菌控制研究展望[J].食品与发酵工业,2010,36(10):127-130.
SUN L J,WANG Y L,CHEN H.A prospect of research on Bacillus antimicrobial peptide application in the seafood safety control[J].Food and Fermentation Industries,2010,36(10):127-130.
[6] PU Y H,SUN L J,WANG Y L,et al.Modeling inhibitory activity of a novel antimicrobial peptide AMPNT-6 from Bacillus subtilis against Vibrio parahaemolyticus in shrimp under various environmental conditions[J].Food Control,2013,33(1):249-253.
[7] CHENG A C,LIN H L,SHIU Y L,et al.Isolation and characterization of antimicrobial peptides derived from Bacillus subtilis E20-fermented soybean meal and its use for preventing Vibrio infection in shrimp aquaculture[J].Fish and Shellfish Immunology,2017,67:270-279.
[8] LANDY M,ROSENMAN S B,WARREN G H.An antibiotic from Bacillus subtilis active against pathogenic fungi[J].Journal of Bacteriology,1947,54(1):24.
[9] 郝彦利, 汪立平,黄宇良,等.副溶血性弧菌拮抗菌的筛选鉴定及抑菌物质特性研究[J].食品与机械,2018,34(6):39-45;191.
HAO Y L,WANG L P,HUANG Y L,et al.Isolation and identification of antagonistic bacteria aganist Vibrio parahaemolyticus and its characteristics of antibacterial substances[J].Food & Machinery,2018,34(6):39-45;191.
[10] HUANG T P,ZHANG X J,PAN J R,et al.Corrigendum:Purification and characterization of a novel cold shock protein-like bacteriocin synthesized by Bacillus thuringiensis[J].Scientific Reports,2017,7:40975.
[11] WAN H F,LIU T,SU C W,et al.Evaluation of bacterial and fungal communities during the fermentation of Baixi sufu,a traditional spicy fermented bean curd[J].Journal of the Science of Food and Agriculture,2020,100(4):1 448-1 457.
[12] JAMALUDDIN N,ARIFF A B,WONG F W F.Purification of a bacteriocin-like inhibitory substance derived from Pediococcus acidilactici Kp10 by an aqueous micellar two-phase system[J].Biotechnology Progress,2019,35(1):e2719.
[13] ZHU X,ZHAO Y Z,SUN Y L,et al.Purification and characterisation of plantaricin ZJ008,a novel bacteriocin against Staphylococcus spp. from Lactobacillus plantarum ZJ008[J].Food Chemistry,2014,165:216-223.
[14] WANG Z X,WANG Y P,ZHENG L,et al.Isolation and characterization of an antifungal protein from Bacillus licheniformis HS10[J].Biochemical and Biophysical Research Communications,2014,454(1):48-52.
[15] 赵述淼. 猪源益生芽孢杆菌的分离筛选与应用研究[D].武汉:华中农业大学,2009.
ZHAO S M.Screening and application of a beneficial Bacillus from pigs[D].Wuhan:Huazhong Agricultural University,2009.
[16] GE J P,SUN Y Y,XIN X,et al.Purification and partial characterization of a novel bacteriocin synthesized by Lactobacillus paracasei HD1-7 isolated from Chinese sauerkraut juice[J].Scientific Reports,2016,6:19366.
[17] 朱晶婷. 副溶血性弧菌拮抗菌的筛选及其拮抗效果研究[D].舟山:浙江海洋大学,2019.
ZHU J T.Screening and antagonism characteristic of antagonistic bacteria against Vibrio parahaemolyticus[D].Zhoushan:Zhejiang Ocean University,2019.
[18] 孙珊, 郝林,王倩.枯草芽孢杆菌W321产抑菌物质的理化性质及抑菌谱研究[J].食品工业,2012,33(11):110-112.
SUN S,HAO L,WANG Q.Physical and chemical properties and antimicrobial spectrum of antibacterial substances from Bacillus subtilis W321[J].The Food Industry,2012,33(11):110-112.
[19] SHARMA N,KAPOOR R,GAUTAM N,et al.Purification and characterization of bacteriocin produced by Bacillus subtilis R75 isolated from fermented chunks of mung bean(Phaseolus radiatus)[J].Food Technology and Biotechnology,2011,49(2):169-176.
[20] 白杰, 贠建民,祝发明,等.枯草芽孢杆菌菌株B3抗菌肽的分离纯化与鉴定[J].食品与发酵工业,2018,44(8):78-85.
BAI J,YUN J M,ZHU F M,et al.Isolation,purification and identification of antibacterial peptide from Bacillus subtilis strain B3[J].Food and Fermentation Industries,2018,44(8):78-85.
[21] KARAGIOTA A,TSITSOPOULOU H,TASAKIS R N,et al.Characterization and quantitative determination of a diverse group of Bacillus subtilis subsp.subtilis NCIB 3610 antibacterial peptides[J].Probiotics and Antimicrobial Proteins,2021,13(2):555-570.
[22] WU Y B,ZHOU L B,LU F X,et al.Discovery of a novel antimicrobial lipopeptide,brevibacillin V,from Brevibacillus laterosporus fmb70 and its application on the preservation of skim milk[J].Journal of Agricultural and Food Chemistry,2019,67(45):12 452-12 460.
[23] BURIANEK L L,YOUSEF A E.Solvent extraction of bacteriocins from liquid cultures[J].Letters in Applied Microbiology,2000,31(3):193-197.
[24] 尚伟. 抗兔腹泻芽孢杆菌Tu-569菌株的筛选、发酵及应用[D].保定:河北农业大学,2012.
SHANG W.Screening,fermentation and application of Bacillus velezensis strain Tu-569 against diarrhea of rabbits[D].Baoding:Hebei Agricultural University,2012.
[25] 刘静, 王军,姚建铭,等.枯草芽孢杆菌JA抗菌物特性的研究及抗菌肽的分离纯化[J].微生物学报,2004,44(4):511-514.
LIU J,WANG J,YAO J M,et al.Properties of the crude extract of Bacillus subtilis and purification of antimicrobial peptides[J].Acta Microbiologica Sinica,2004,44(4):511-514.
[26] STEIN T.Bacillus subtilis antibiotics:Structures,syntheses and specific functions[J].Molecular Microbiology,2005,56(4):845-857.
