发酵肠中一株细菌的16S rDNA鉴定及抗菌肽Surfactin对其芽孢灭活的效果
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摘要
经过杀菌的发酵肠,仍有部分会变质,为探究其原因并进行控制而进行了以下试验。从商业发酵肠中分离获得1株耐热芽孢菌株B1,为构建控制技术,提高发酵肠安全水平,对该芽孢菌进行鉴定,并研究抗菌肽surfactin对其芽孢生长曲线的影响及盐类对surfactin灭活芽孢效果的影响。采用16S rDNA序列分析对该菌进行鉴定,并研究了不同浓度的Na Cl和Na_2HPO_4·12H_2O对surfactin灭活芽孢效果的影响。结果表明,该菌属蜡样芽孢杆菌属,同源性相似度达99%,仅有surfactin对芽孢作用时,不同浓度(20、25、30、35μg/mL)的surfactin对其芽孢没有显著的杀灭作用,与Na Cl和Na_2HPO_4·12H_2O协同作用时,3%、5%、9%的Na Cl可配合50μg/mL和100μg/mL的surfactin可有效杀灭芽孢,Na_2HPO_4·12H_2O无此效果。该菌属于蜡样芽孢杆菌属,Na Cl与surfactin共同作用可有效杀灭蜡样芽孢杆菌的芽孢,Na_2HPO_4·12H_2O无显著作用。
To control the spoilage of fermented sausages after sterilization and assure safety of fermented sausage were, the microbes in spoiled sausages were detected and a microorganism strain B1 was isolated from the commercial fermented sausage. 16 S r DNA sequence analysis showed that the B1 strain belongs to Bacillus cereus. The effect of antibacterial peptide surfactin on the spore growth curve and the effects of two salts NaCl and Na_2 HPO_4·12 H_2 O on surfactin inactivation of spore were studied. No significant inactivating effect of surfactin with different concentrations(20～35 μg/mL) on the spores was found. However, the combined use of surfactin(50 and 100 μg/mL) and NaCl(3%,5% and 9%) showed inactivation effects. The combined use of surfactin with Na_2 HPO_4·12 H_2 O had no such effect.
To control the spoilage of fermented sausages after sterilization and assure safety of fermented sausage were, the microbes in spoiled sausages were detected and a microorganism strain B1 was isolated from the commercial fermented sausage. 16 S r DNA sequence analysis showed that the B1 strain belongs to Bacillus cereus. The effect of antibacterial peptide surfactin on the spore growth curve and the effects of two salts NaCl and Na_2 HPO_4·12 H_2 O on surfactin inactivation of spore were studied. No significant inactivating effect of surfactin with different concentrations(20～35 μg/mL) on the spores was found. However, the combined use of surfactin(50 and 100 μg/mL) and NaCl(3%,5% and 9%) showed inactivation effects. The combined use of surfactin with Na_2 HPO_4·12 H_2 O had no such effect.
引文
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[16]黄现青,别小妹,陆兆新,等.抗微生物脂肽体外抗PRV和PPV的研究[J].中国病毒学,2006,5:426-429HUANG Xian-qing,BIE Xiao-mei,LU Zhao-xin,et al.In vitro assays of an antimicrobial lipopeptide produced by Bacillus subtilis fmb J against pseudorabies virus and porcine parvovirus[J].Virologica Sinica,2006,5:426-429
[17]Huang X,Lu Z,Bie X,et al.Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method[J].Applied Microbiology and Biotechnology,2007,74(2):454-461
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[21]范秀容,沈萍.微生物学实验方法[M]北京:高等教育出版社,1980,58-76FAN Xiu-rong,SHEN Ping.Microbiology Experimental Method[M].Beijing:Higher Education Press,1980,58-76
[22]徐叔云,卞如濂,陈修.药理实验方法学[M]北京:人民卫生出版社,1982,1063-1114XU Shu-yun,BIAN Ru-lian,Chen Xiu.Experimental Methodology of Pharmacology[M].Beijing:People's Medical Publishing House(PMPH),1982,1063-1114
[23]马绪荣,苏德模.药品微生物学检验手册[M]北京:科学出版社,2001,210-215MA Xu-rong,SU De-mo.Drug Microbiology Testing Manual[M].Beijing:Science Press,2001,210-215
[24]郑乾魏.发酵肠优质乳酸菌的分离,筛选与鉴定[D].河北农业大学,2014ZHEN Qian-wei.Selecting isolation and identification of the lactic acid bacteria from the natural fermented sausages.[D].Agricultural University of Hebei,2014
[25]陈忠杰,李存法,王璐.引起罐藏食品腐败变质的微生物探讨[J].郑州牧业工程高等专科学校学报,2012,32(1):19-21CHEN Zhong-jie,LI Cun-fa,WANG Lu.Discussion on microorganisms causing spoilage and deterioration of canned food[J].Modern Animal Husbandry,2012,32(1):19-21
[26]黄现青,王玉芬,赵改名,等.一株玉米肠致腐菌的16S r DNA鉴定及其防腐剂复配优化[J].中国农业科学,2010,43(24):5112-5120HUANG Xian-qing,WANG Yu-fen,ZHAO Gai-ming,et al.Identification of A Microorganism Strain Isolated from the Corn Sausage by 16S r DNA Sequence Analysis and Optimization of Preservative Mixture.[J].Scientia Agricultura Sinica,2010,43(24):5112-5120
[2]帅瑾.传统自然发酵四川香肠中乳酸菌的分离,鉴定及其应用[D].四川农业大学,2013SHUAI Jin.Isolation,Identification and application of lactic acid bacteria from traditional natural fermentation sausage in Sichuan[D].Sichuan Agricultural University,2013
[3]Claus D,Berkley RCW.Genus Bacillus Cohn 1872,174AL[C]//Sneath P H A,Mair N S,Sharpe M E,Holt JG(ed).Bergey’s mannual of systermatics bacteriology.Baltimore:Williams&Wilkins,1986,2:1105-1139
[4]王阶平,刘波,刘国红,等.芽胞杆菌系统分类研究最新进展[J].福建农业学报,2017,32(7):784-800WANG Jie-ping,LIU Bo,LIU Guo-hong,et al.Recent advancements in taxonomy of bacillus-like bacteria[J].Fujian Journal of Agricultural Sciences,2017,32(7):784-800
[5]Ahn J,Balasubramaniam V M,Yousef A E.Inactivation kinetics of selected aerobic and anaerobic bacterial spores by pressure-assisted thermal processing[J].International Journal of Food Microbiology,2007,113(3):321-329
[6]Cho M,Gandhi V,Hwang T M,et al.Investigating synergism during sequential inactivation of MS-2 phage and Bacillus subtilis spores with UV/H2O2 followed by free chlorine[J].Water Research,2011,45(3):1063-1070
[7]De Lamo-Castellvi S,Ratphitagsanti W,Balasubramaniam VM,et al.Inactivation of Bacillus amyloliquefaciens spores by a combination of sucrose laurate and pressure-assisted thermal processing[J].Journal of Food Protection,2010,73(11):2043-2052
[8]Handler F A,Edmonds J M.Quantitative analysis of effects of UV exposure and spore cluster size on deposition and inhalation hazards of Bacillus spores[J].Aerosol Science and Technology,2015,49(11):1121-1130
[9]Margosch D,Ehrmann M A,Buckow R,et al.High-pressure-mediated survival of Clostridium botulinum and Bacillus amyloliquefaciens endospores at high temperature[J].Applied and Environmental Microbiology,2006,72(5):3476-3481
[10]刘洁,林影,吴晓英,等.高静压对枯草杆菌芽孢灭活作用的研究[J].工业微生物,2008,4:35-39LIU Jie,LIN Ying,WU Xiao-ying,et al.Study on the inactivation of Bacillus subtilis spores by high static pressure[J].Industrial Microbiology,2008,4:35-39
[11]张永吉,刘文君,张琳.氯对紫外线灭活枯草芽孢杆菌的协同作用[J].环境科学,2006,2:329-332ZHANG Yong-ji,LIU Wen-jun,ZHANG Lin.Synergistic disinfection of Bacillus subtilis spores by UV irradiation and chlorine[J].Environmental Science,2006,2:329-332
[12]许能锋,林立旺,徐文体,等.邻苯二甲醛杀灭枯草杆菌黑色变种芽孢效果及其影响因素的研究[J].中国消毒学杂志,2005,2:125-127,124XU Neng-feng,LIN Li-wang,XU Wen-ti,et al.Study on efficacy of ortho-phthalaldehyde in killing spores of Bacillus subtilis var.niger and its influencing factors[J].Chinese Journal of Disinfection,2005,2:125-127,124
[13]曾庆梅,潘见,谢慧明,等.超高压灭活枯草芽孢杆菌(AS1.140)的参数优化[J].农业工程学报,2005,4:158-162ZENG Qing-mei,PAN Jian,XIE Hui-ming,et al.Parameter optimization of inactivated Bacillus subtilis(AS1.140)by ultra-high pressure[J].Transactions of the Chinese Society of Agricultural Engineering,2005,4:158-162
[14]高晓平,胡惠,黄现青.Surfactin抑制乳中大肠杆菌O157活性研究[J].食品科学,2009,30(11):91-94GAO Xiao-ping,HU Hui,HUANG Xian-qing.Sterilization effect of Surfactin on escherichia coli O157 in fresh milk[J].Food Science,2009,30(11):91-94
[15]黄现青,别小妹,吕凤霞,等.枯草芽孢杆菌fmb J产脂肽抑制点青霉效果及其桃防腐试验[J].农业工程学报,2008,1:263-267HUANG Xian-qing,BIE Xiao-mei,LYU Feng-xia,et al.Inhibitory effect of lipopeptides from Bacillus subtilis fmb Jon Penicillium notatum and its antisepsis to peach[J].Transactions of the Chinese Society of Agricultural Engineering,2008,1:263-267
[16]黄现青,别小妹,陆兆新,等.抗微生物脂肽体外抗PRV和PPV的研究[J].中国病毒学,2006,5:426-429HUANG Xian-qing,BIE Xiao-mei,LU Zhao-xin,et al.In vitro assays of an antimicrobial lipopeptide produced by Bacillus subtilis fmb J against pseudorabies virus and porcine parvovirus[J].Virologica Sinica,2006,5:426-429
[17]Huang X,Lu Z,Bie X,et al.Optimization of inactivation of endospores of Bacillus cereus by antimicrobial lipopeptides from Bacillus subtilis fmbj strains using a response surface method[J].Applied Microbiology and Biotechnology,2007,74(2):454-461
[18]东秀珠,蔡妙英.常见细菌系统鉴定手册.[M]北京:科学出版社,2001:256-323DONG Xiu-zhu,CAI Miao-ying.Common Bacterial System Identification Manual[M].Beijing:Science Press,2001:256-323
[19]Macrae A.The use of 16S r DNA methods in soil microbial ecology[J].Brazilian Journal of Microbiology,2000,31(2):77-82
[20]黄现青.Bacillus subtilis fmb J产生的脂肽抗微生物效果及安全性评价[D].南京农业大学,2006HUANG Xian-qing.Antimicrobial effect and safety of lipopeptide produced by Bacillus subtilis fmbj[D].Nanjing Agricultural University,2006
[21]范秀容,沈萍.微生物学实验方法[M]北京:高等教育出版社,1980,58-76FAN Xiu-rong,SHEN Ping.Microbiology Experimental Method[M].Beijing:Higher Education Press,1980,58-76
[22]徐叔云,卞如濂,陈修.药理实验方法学[M]北京:人民卫生出版社,1982,1063-1114XU Shu-yun,BIAN Ru-lian,Chen Xiu.Experimental Methodology of Pharmacology[M].Beijing:People's Medical Publishing House(PMPH),1982,1063-1114
[23]马绪荣,苏德模.药品微生物学检验手册[M]北京:科学出版社,2001,210-215MA Xu-rong,SU De-mo.Drug Microbiology Testing Manual[M].Beijing:Science Press,2001,210-215
[24]郑乾魏.发酵肠优质乳酸菌的分离,筛选与鉴定[D].河北农业大学,2014ZHEN Qian-wei.Selecting isolation and identification of the lactic acid bacteria from the natural fermented sausages.[D].Agricultural University of Hebei,2014
[25]陈忠杰,李存法,王璐.引起罐藏食品腐败变质的微生物探讨[J].郑州牧业工程高等专科学校学报,2012,32(1):19-21CHEN Zhong-jie,LI Cun-fa,WANG Lu.Discussion on microorganisms causing spoilage and deterioration of canned food[J].Modern Animal Husbandry,2012,32(1):19-21
[26]黄现青,王玉芬,赵改名,等.一株玉米肠致腐菌的16S r DNA鉴定及其防腐剂复配优化[J].中国农业科学,2010,43(24):5112-5120HUANG Xian-qing,WANG Yu-fen,ZHAO Gai-ming,et al.Identification of A Microorganism Strain Isolated from the Corn Sausage by 16S r DNA Sequence Analysis and Optimization of Preservative Mixture.[J].Scientia Agricultura Sinica,2010,43(24):5112-5120