副溶血性弧菌拮抗菌的筛选鉴定及抑菌物质特性研究
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摘要
为获取既可抑制食源性致病菌又可抑制水产致病菌的益生菌株,以人畜共患致病菌副溶血性弧菌(Vibrio parahaemolyticus ATCC17802)为指示菌,采用点种法初筛以及打孔扩散法复筛,得到1株来源于上海东海海水的H19菌株,其抑菌物抑菌圈直径达(17.16±0.28)mm。通过形态观察及16SrDNA序列分析可知,菌株H19为枯草芽孢杆菌(Bacillus subtilis H19,GenBank登录号:MG383451)。将菌株H19进行抑菌谱试验,探究菌株H19抑菌物的理化性质,并且抑菌物通过硫酸铵沉淀及不同孔径透析袋截留粗提,粗提物经蛋白酶处理及Tricine-SDS-PAGE蛋白质凝胶电泳可知,该抑菌物具有广谱抑菌活性,既可抑制革兰氏阳性菌又可抑制大部分革兰氏阴性菌,且具有较好的热、pH以及紫外线稳定性,该抑菌物可能为2种新型抗菌肽,分子量分别在6.5~9.5kDa以及27.0~35.0kDa。由此可见,枯草芽孢杆菌H19所产抗菌肽具有防治食源性疾病以及水产疾病的应用潜力。
In order to screen a probiotic strain against foodborne pathogens and aquatic pathogens,the main zoonotic pathogenic bacteria of mariculture Vibrio parahaemolyticus ATCC17802 was used as indicator bacterium.The target probiotics was screened using the spot-on-lawn method and rescreened by the agar diffusion method,and the diameter of inhibition zone of antibacterial substances reached(17.16±0.28)mm.Through the combination of morphology and 16S rDNA sequence analysis,strain H19 was identified as Bacillus subtilis(GenBank accession number: MG383451). Antimicrobial spectrum of B.subtilis H19 was tested,meanwhile the physical and chemical properties of antimicrobial substances produced by H19 was studied.The supernatant of the H19 fermentation broth was subjected to ammonium sulfate precipitation and dialysis against several different pore-size membranes,and two novel kinds of antibacterial peptides with molecular weight of 6.5~9.5 kDa and 27.0~35.0 kDa respectively were obtained based on Tricine-SDS-PAGE protein gel electrophoresis and protease treatment.It had broad-spectrum antimicrobial activity,which could inhibit both Gram-positive bacteria and most of Gram-negative bacteria,and showed good heat,pH and UV stability.Consequently,the antibacterial peptide produced by B.subtilis H19 had the potential to antagonize the pathogenic bacteria for the prevention and control of foodborne and aquatic diseases.
In order to screen a probiotic strain against foodborne pathogens and aquatic pathogens,the main zoonotic pathogenic bacteria of mariculture Vibrio parahaemolyticus ATCC17802 was used as indicator bacterium.The target probiotics was screened using the spot-on-lawn method and rescreened by the agar diffusion method,and the diameter of inhibition zone of antibacterial substances reached(17.16±0.28)mm.Through the combination of morphology and 16S rDNA sequence analysis,strain H19 was identified as Bacillus subtilis(GenBank accession number: MG383451). Antimicrobial spectrum of B.subtilis H19 was tested,meanwhile the physical and chemical properties of antimicrobial substances produced by H19 was studied.The supernatant of the H19 fermentation broth was subjected to ammonium sulfate precipitation and dialysis against several different pore-size membranes,and two novel kinds of antibacterial peptides with molecular weight of 6.5~9.5 kDa and 27.0~35.0 kDa respectively were obtained based on Tricine-SDS-PAGE protein gel electrophoresis and protease treatment.It had broad-spectrum antimicrobial activity,which could inhibit both Gram-positive bacteria and most of Gram-negative bacteria,and showed good heat,pH and UV stability.Consequently,the antibacterial peptide produced by B.subtilis H19 had the potential to antagonize the pathogenic bacteria for the prevention and control of foodborne and aquatic diseases.
引文
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[13]张孟,王艳婷,汪立平,等.海洋抑菌放线菌Y15的筛选、鉴定及其代谢产物理化性质[J].微生物学通报,2017,44(3):513-524.
[14]郑苗,邓泽元,任志青,等.东方伊萨酵母高密度培养的研究[J].中国食品学报,2016(4):96-103.
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[20]陈夕军,李娟,孙启利,等.水稻内生枯草芽孢杆菌G87抗菌蛋白的分离纯化及理化特性[J].微生物学报,2010,50(10):1 353-1 357.
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[22]王艳婷,汪立平,唐潮,等.蒙古干酪中产细菌素戊糖片球菌的筛选及其细菌素理化特性研究[J].食品与发酵工业,2015,41(10):14-19.
[23]孔维嘉,王洋,尚楠,等.扩展青霉拮抗菌的筛选鉴定及抗菌物质分析[J].食品科学,2011,32(15):153-157.
[24]XIE Jian-hua,ZHANG Ri-jun,SHANG Chang-jiang,et al.Isolation and characterization of a bacteriocin produced by an isolated Bacillus subtilis LFB112that exhibits antimicrobial activity against domestic animal pathogens[J].African Journal of Biotechnology,2009,8(20):283-288.
[25]李云,杨胜远,林晓东,等.产细菌素苏云金芽孢杆菌的鉴定及其所产抗菌物质性质[J].食品科学,2010,31(11):147-152.
[26]孙苗苗,赵昆,武志芳,等.枯草芽孢杆菌LL18-4产类细菌素的分离纯化及特性研究[J].四川大学学报:自然科学版,2009,46(6):1 862-1 866.
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[30]STEIN T,BORCHERT S,CONRAD B,et al.Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3[J].Journal of Bacteriology,2002,184(6):1 703-1 711.
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[34]赵朋超,王建华,权春善,等.枯草芽孢杆菌抗菌肽生物合成的研究进展[J].中国生物工程杂志,2010,30(10):108-113.
[35]IL P,RYU J,KIM Y H,et al.Production of Biosurfactant Lipopeptides Iturin A,Fengycin,and SurfactinA from Bacillus subtilis CMB32for Control of Colletotrichum gloeosporioides[J].Journal of Microbiology&Biotechnology,2010,20(1):138-145.
[36]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(11):110-112.
[2]储卫华,朱卫,康春涛.海洋蛭弧菌的分离鉴定及其对副溶血弧菌的作用[J].微生物学通报,2009,36(1):20-24.
[3]ALEXANDRE Y,LE B G,BOISRAME-GASTRIN S,et al.Probiotics:a new way to fight bacterial pulmonary infections?[J].Medecine Et Maladies Infectieuses,2014,44(1):9-17.
[4]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(10):127-130.
[5]KOZASA M.Toyocerin(Bacillus toyoi)as growth promotor for animal feeding[J].Microbiologie Aliments Nutrition,1986,4(2):121-135.
[6]XU De-feng,WANG Ya-ling,SUN Li-jun,et al.Inhibitory activity of a novel antibacterial peptide AMPNT-6from Bacillus subtilis,against Vibrio parahaemolyticus,in shrimp[J].Food Control,2013,30(1):58-61.
[7]石广举,孙力军,王雅玲,等.NT-6抗菌脂肽对凡纳滨对虾生长性能及养殖源头弧菌数的影响[J].广东农业科学,2014,41(12):119-122.
[8]王娇,曲朋,孙飞,等.弧菌颉颃菌的筛选及其在对虾养殖中的初步应用[J].中国畜牧兽医,2014,41(6):203-206.
[9]NATH S,CHOWDHURY S,SARKAR S,et al.Detection and characterisation of antimicrobial peptide produced by Bacillus subtilis FPTB23isolated from curd[J].Asian Journal of Animal Science,2015,10(2):115-123.
[10]阮乾坤.产海洋生物活性物质的微生物筛选及鉴定[D].大连:大连工业大学,2012:11-22.
[11]中国微生物菌种保藏委员会.中国菌种目录[M].北京:轻工业出版社,1983:404-422.
[12]赵延存,李鹏霞,黄开红,等.适于解淀粉芽孢杆菌BGP20菌体生长的培养基响应面优化[J].食品科学,2014,35(3):157-162.
[13]张孟,王艳婷,汪立平,等.海洋抑菌放线菌Y15的筛选、鉴定及其代谢产物理化性质[J].微生物学通报,2017,44(3):513-524.
[14]郑苗,邓泽元,任志青,等.东方伊萨酵母高密度培养的研究[J].中国食品学报,2016(4):96-103.
[15]汪攀,易敢峰,孙自博,等.一株凝结芽孢杆菌的分离鉴定及益生性研究[J].中国畜牧兽医,2017,44(4):1 195-1 202.
[16]CHAHAD O B,BOUR M E,CALO-MATA P,et al.Discovery of novel biopreservation agents with inhibitory effects on growth of food-borne pathogens and their application to seafood products[J].Research in Microbiology,2012,163(1):44-54.
[17]LIU Xue-fei,LI Ya,LI Jian-rong,et al.Isolation and characterisation of Bacillus,spp.antagonistic to Vibrio parahaemolyticus,for use as probiotics in aquaculture[J].World Journal of Microbiology&Biotechnology,2015,31(5):795-803.
[18]张梅梅,索化夷,赵欣,等.传统发酵牦牛酸乳中细菌素产生菌的筛选与鉴定[J].食品与机械,2017,33(3):21-26.
[19]SABATE D C,AUDISIO M C.Inhibitory activity of surfactin,produced by different Bacillus subtilis subsp.subtilis strains,against Listeria monocytogenes sensitive and bacteriocin-resistant strains[J].Microbiological Research,2013,168(3):125-129.
[20]陈夕军,李娟,孙启利,等.水稻内生枯草芽孢杆菌G87抗菌蛋白的分离纯化及理化特性[J].微生物学报,2010,50(10):1 353-1 357.
[21]贾洁.枯草芽孢杆菌R21-4抗菌蛋白的分离提取与应用[D].北京:中国农业大学,2005:20-24.
[22]王艳婷,汪立平,唐潮,等.蒙古干酪中产细菌素戊糖片球菌的筛选及其细菌素理化特性研究[J].食品与发酵工业,2015,41(10):14-19.
[23]孔维嘉,王洋,尚楠,等.扩展青霉拮抗菌的筛选鉴定及抗菌物质分析[J].食品科学,2011,32(15):153-157.
[24]XIE Jian-hua,ZHANG Ri-jun,SHANG Chang-jiang,et al.Isolation and characterization of a bacteriocin produced by an isolated Bacillus subtilis LFB112that exhibits antimicrobial activity against domestic animal pathogens[J].African Journal of Biotechnology,2009,8(20):283-288.
[25]李云,杨胜远,林晓东,等.产细菌素苏云金芽孢杆菌的鉴定及其所产抗菌物质性质[J].食品科学,2010,31(11):147-152.
[26]孙苗苗,赵昆,武志芳,等.枯草芽孢杆菌LL18-4产类细菌素的分离纯化及特性研究[J].四川大学学报:自然科学版,2009,46(6):1 862-1 866.
[27]SUMI C D,YANG B W,YEO I C,et al.Antimicrobial peptides of the genus Bacillus:a new era for antibiotics[J].Revue Canadienne De Microbiologie,2015,61(2):93-103.
[28]CHATTERJEE S,CHATTERJEE S,LAD S J,et al.Mersacidin,a new antibiotic from Bacillus.Fermentation,isolation,purification and chemical characterization[J].Journal of Antibiotics,1992,45(6):832-838.
[29]KIM S Y,LEE N K,HAN E J,et al.Characterization of subtilin KU43Produced by Bacillus subtilis,KU43isolated from traditional Korean fermented foods[J].Food Science&Biotechnology,2012,21(5):1 433-1 438.
[30]STEIN T,BORCHERT S,CONRAD B,et al.Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3[J].Journal of Bacteriology,2002,184(6):1 703-1 711.
[31]PAIK S H,CHAKICHERLA A,HANSEN J N.Identification and characterization of the structural and transporter genes for,and the chemical and biological properties of,sublancin 168,a novel lantibiotic produced by Bacillus subtilis 168[J].Journal of Biological Chemistry,1998,273(36):23-34.
[32]ZHENG Guo-lu,SLAVIK M F.Isolation,partial purification and characterization of a bacteriocin produced by a newly isolated Bacillus subtilis strain[J].Letters in Applied Microbiology,1999,28(5):363-367.
[33]MARX R,STEIN T,ENTIAN K D,et al.Structure of the Bacillus subtilis peptide antibiotic subtilosin A determined by1H-NMR and matrix assisted laser desorption/ionization timeof-flight mass spectrometry[J].Journal of Protein Chemistry,2001,20(6):501-506.
[34]赵朋超,王建华,权春善,等.枯草芽孢杆菌抗菌肽生物合成的研究进展[J].中国生物工程杂志,2010,30(10):108-113.
[35]IL P,RYU J,KIM Y H,et al.Production of Biosurfactant Lipopeptides Iturin A,Fengycin,and SurfactinA from Bacillus subtilis CMB32for Control of Colletotrichum gloeosporioides[J].Journal of Microbiology&Biotechnology,2010,20(1):138-145.
[36]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(11):110-112.