藏羊肉中优势腐败不动杆菌的分离鉴定及其生物膜形成特性
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摘要
本研究从冷鲜藏羊肉中共分离获得40株腐败菌,其中10株为不动杆菌,经结晶紫染色定量检测发现10株不动杆菌均具有中等或强粘附成膜能力。对1株成膜能力最强的不动杆菌A3的成膜特性进行深入分析,发现该菌在培养3 d时生物膜内菌数最高,随后培养5 d至7 d时菌数无显著(P>0. 05)变化。扫描电镜、激光共聚焦显微镜观察及胞外多糖质量浓度测定结果表明,该菌形成生物膜的过程中培养1 d到3 d细菌迅速繁殖,培养3 d到5 d,菌体聚集成团,并且此时不可溶性多糖的含量最高,培养7 d时胞外分泌物质减少,生物膜出现解离状态。因此,在藏羊肉加工过程中,针对此菌生物膜抑制的研究可着重关注控制胞外物质的分泌,并且在生物膜未成熟前实施消毒措施,有助于彻底杀灭生物膜内的腐败菌。
In this study,forty food spoilage strains were isolated and identified from the Tibetan mutton,and ten strains were Acinetobacter sp. Quantitative detection results by crystal violet staining showed that ten strains of Acinetobacter had neutral or strong adhesion ability. The biofilm formation characteristics of Acinetobacter A3 with the best biofilm forming ability were analyzed. It was found that the bacterial count in biofilm was the highest when biofilm was cultured for three days,and there was no significant change in the number of bacteria on the 5 th to 7 th day after culture. The results of scanning electron microscopy,confocal laser scanning microscopy and extracellular polysaccharide determination concentration showed that bacteria propagated rapidly from one to three days in the process of biofilm formation. When the biofilm was cultured for three to five days,the bacteria aggregated into groups,and the insoluble polysaccharide content was highest. The extracellular substances were decreased and the biofilm became dissociated when it was cultured for seven days. During the processing of Tibetan mutton,the secretion of extracellular substances should be paid more attention for biofilm inhibition research,and disinfection measures should be implemented before the biofilms matured in order to kill the spoilage bacteria.
In this study,forty food spoilage strains were isolated and identified from the Tibetan mutton,and ten strains were Acinetobacter sp. Quantitative detection results by crystal violet staining showed that ten strains of Acinetobacter had neutral or strong adhesion ability. The biofilm formation characteristics of Acinetobacter A3 with the best biofilm forming ability were analyzed. It was found that the bacterial count in biofilm was the highest when biofilm was cultured for three days,and there was no significant change in the number of bacteria on the 5 th to 7 th day after culture. The results of scanning electron microscopy,confocal laser scanning microscopy and extracellular polysaccharide determination concentration showed that bacteria propagated rapidly from one to three days in the process of biofilm formation. When the biofilm was cultured for three to five days,the bacteria aggregated into groups,and the insoluble polysaccharide content was highest. The extracellular substances were decreased and the biofilm became dissociated when it was cultured for seven days. During the processing of Tibetan mutton,the secretion of extracellular substances should be paid more attention for biofilm inhibition research,and disinfection measures should be implemented before the biofilms matured in order to kill the spoilage bacteria.
引文
[1]巴叶尔·阿不力克木,帕提姑·阿布都克热.肉孜阿吉巴什拜羊肉不同部位的品质特性分析[J].新疆农业科学,2012,49(9):1734-1741.
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[13]周文渊,张宏梅,姜燕,等.腐败牛奶中三种细菌生物被膜特性探究[J].现代食品科技,2014(1):87-90.
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[16] MITRAKUL K,SRISATJALUK R,SRISUKH V,et al. Citrushystrix(makrut oil)oral sprays inhibit Streptococcus mutans bio-film formation[J].Scienceasia,2016,42(1):12.
[17] LUO J,DONG B,WANG K,et al. Baicalin inhibits biofilm for-mation,attenuates the quorum sensing-controlled virulence and en-hances Pseudomonas aeruginosa clearance in a mouse peritonealimplant infection model[J]. PLoS ONE,2017,12(4):e0176883.
[18]杨子贤,王洪星,易小平.激光扫描共聚焦显微镜在生物科学研究中的应用[J].热带生物学报,2013,4(1):99-104.
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[21] KARUNAKARAN E,MUKHERJEE J,RAMALINGAM B,et al.‘Biofilmology’:a multidisciplinary review of the study of microbialbiofilms[J]. Applied Microbiology&Biotechnology,2011,90(6):1869-1881.
[22] GU H,FAN D,GAO J,et al. Effect of Zn Cl2on plaque growthand biofilm vitality[J]. Archives of Oral Biology,2012,57(4):369-375.
[23] SCHAUDINN C,CARR G,GORUR A,et al.Imaging of endodon-tic biofilms by combined microscopy(FISH/c LSM-SEM)[J].Journal of Microscopy,2009,235(2):124-127.
[24] REUVEN A,AVSHALOM T,MOSHE S,et al.Genetic adaptationof Streptococcus mutans during biofilm formation on different typesof surfaces[J]. BMC Microbiology,2010,10(1):51-61.
[25] BRIDIER A,MEYLHEUC T,BRIANDET R. Realistic represen-tation of Bacillus subtilis biofilms architecture using combined mi-croscopy(CLSM,ESEM and FESEM)[J]. Micron,2013,48(3):65-69.
[26] HATHROUBI S,HANCOCK M A,BOSSJ T,et al. Surfacepolysaccharide mutants reveal that absence of O antigen reducesbiofilm formation of Actinobacillus pleuropneumoniae[J]. Infection&Immunity,2015,84(1):127-137.
[27]戚韩英,汪文斌,郑昱,等.生物膜形成机理及影响因素探究[J].微生物学通报,2013,40(4):677-685.
[28]刘红艳,韦曦,凌均棨.饥饿状态粪肠球菌生物膜胞外多糖的合成能力[J].中华口腔医学研究杂志,2012,6(5):16-20.
[29] COMTE S,GUIBAUD G,BAUDU M. Relations between extrac-tion protocols for activated sludge extracellular polymeric sub-stances(EPS)and EPS complexation properties:Part I. Compari-son of the efficiency of eight EPS extraction methods[J].Enzyme&Microbial Technology,2006,38(2):237-245.
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[31]李京宝,韩峰,于文功.细菌生物膜研究技术[J].微生物学报,2007,47(3):558-561.
[32] EIGHMY T T,MARATEA D,BISHOP P L. Electron microscopicexamination of wastewater biofilm formation and structural compo-nents[J].Appl Environ Microbiol,1983,45(6):1921-1931.
[33] FRLUND B,PALMGREN R,KEIDING K,et al. Extraction ofextracellular polymers from activated sludge using a cation ex-change resin[J]. Water Research,1996,30(8):1749-1758.
[34] WINGENDER J,STRATHMANN M,RODE A,et al. Isolationand biochemical characterization of extracellular polymeric sub-stances from Pseudomonas aeruginosa[J].Methods in Enzymology,2001,336:302-314.
[35] MA LM,CONOVER M,LU H,et al. Assembly and developmentof the Pseudomonas aeruginosa biofilm matrix[J]. PLoS Patho-gens,2009,5(3):e1000354.
[36] WATNICK P I,KOLTER R. Steps in the development of a Vibriocholerae EI Tor biofilm[J]. Molecular Microbiology,1999,34(3):586-595.
[37] DANESE PN,PRATT LA,KOLTER R,et al. Exopolysaccharideproduction is required for development of Escherichia coli K-12biofilm architecture[J]. Journal of Bacteriology,2000,182(12):3593-3596.
[38] SUTHERLAND IW. The biofilm matrix-an immobilized but dynam-ic microbial environment[J].Trends in Microbiology,2001,9(5):222-227.
[2] BAS P,MORAND-FEHR P. Effect of fatty acid composition oflamb fat deposits[J].Meat Science,2000,64:61-79.
[3]余群力,韩玲,杨勤,等.甘加藏羊肉营养成分及风味物质的测定与评价[J].营养学报,2009,31(6):609-671.
[4] ROWE A,MACEDO F A F,VISENTAINER J V,et al. Musclecomposition and fatty acid profile in lambs fattened in dry lot orpasture[J].Meat Science,1999,51:283-288.
[5]闫忠心,靳义超.基于氨基酸和脂肪酸的藏羊肉质量评价[J].食品工业科技,2016,37(3):351-353,363.
[6] DRENKARD E. Antimicrobial resistance of Pseudomonas aerugi-nosa biofilms[J]. Microbes and Infection,2003,5(13):1213-1219.
[7] O'TOOLE G,KAPLAN H B,KOLTER R. Biofilm formation asmicrobial development.[J]. Annual Review of Microbiology,2000,54(1):49-79.
[8] BRIDIER A,SANCHEZ-VIZUETE P,GUILBAUD M,et al. Bio-film-associated persistence of food-borne pathogens[J]. Food Mi-crobiology,2015,45:167-178.
[9]张秋勤.生鲜鸡肉中腐败菌群体感应信号分子研究[D].南京:南京农业大学,2014.
[10] JUL M,LAHELLEC C. Evaluation of the hygienic problems relat-ed to the chilling of poultry carcasses[J]. Information on Agricul-ture,1976,22:1-121.
[11]刘芳,都立辉,沙莎,等.利用16S rRNA基因同源性分析鉴定两株明串珠菌[J].中国微生态学杂志,2006,18(6):427-429.
[12]崔海月,刘芳,王道营,等.传统肉制品中一株植物乳杆菌的分离筛选及鉴定[J].江苏农业科学,2012,40(11):272-273.
[13]周文渊,张宏梅,姜燕,等.腐败牛奶中三种细菌生物被膜特性探究[J].现代食品科技,2014(1):87-90.
[14] LIU F,DU L,ZHAO T,et al. Effects of phenyllactic acid as sani-tizing agent for inactivation of listeria monocytogenes,biofilms[J].Food Control,2017,78:72-78.
[15]王虎虎.肉源沙门氏菌生物菌膜的形成及转移规律研究[D].南京:南京农业大学,2014.
[16] MITRAKUL K,SRISATJALUK R,SRISUKH V,et al. Citrushystrix(makrut oil)oral sprays inhibit Streptococcus mutans bio-film formation[J].Scienceasia,2016,42(1):12.
[17] LUO J,DONG B,WANG K,et al. Baicalin inhibits biofilm for-mation,attenuates the quorum sensing-controlled virulence and en-hances Pseudomonas aeruginosa clearance in a mouse peritonealimplant infection model[J]. PLoS ONE,2017,12(4):e0176883.
[18]杨子贤,王洪星,易小平.激光扫描共聚焦显微镜在生物科学研究中的应用[J].热带生物学报,2013,4(1):99-104.
[19] HARIMAWAN A,TINGY P. Investigation of extracellular poly-meric substances(EPS)properties of P. aeruginosa,and B. subti-lis,and their role in bacterial adhesion[J]. Colloids&SurfacesBiointerfaces,2016,146:459-467.
[20] HENRICI A T. Studies of freshwater bacteria[J].Journal of Bacte-riology,1933,25(3):277-287.
[21] KARUNAKARAN E,MUKHERJEE J,RAMALINGAM B,et al.‘Biofilmology’:a multidisciplinary review of the study of microbialbiofilms[J]. Applied Microbiology&Biotechnology,2011,90(6):1869-1881.
[22] GU H,FAN D,GAO J,et al. Effect of Zn Cl2on plaque growthand biofilm vitality[J]. Archives of Oral Biology,2012,57(4):369-375.
[23] SCHAUDINN C,CARR G,GORUR A,et al.Imaging of endodon-tic biofilms by combined microscopy(FISH/c LSM-SEM)[J].Journal of Microscopy,2009,235(2):124-127.
[24] REUVEN A,AVSHALOM T,MOSHE S,et al.Genetic adaptationof Streptococcus mutans during biofilm formation on different typesof surfaces[J]. BMC Microbiology,2010,10(1):51-61.
[25] BRIDIER A,MEYLHEUC T,BRIANDET R. Realistic represen-tation of Bacillus subtilis biofilms architecture using combined mi-croscopy(CLSM,ESEM and FESEM)[J]. Micron,2013,48(3):65-69.
[26] HATHROUBI S,HANCOCK M A,BOSSJ T,et al. Surfacepolysaccharide mutants reveal that absence of O antigen reducesbiofilm formation of Actinobacillus pleuropneumoniae[J]. Infection&Immunity,2015,84(1):127-137.
[27]戚韩英,汪文斌,郑昱,等.生物膜形成机理及影响因素探究[J].微生物学通报,2013,40(4):677-685.
[28]刘红艳,韦曦,凌均棨.饥饿状态粪肠球菌生物膜胞外多糖的合成能力[J].中华口腔医学研究杂志,2012,6(5):16-20.
[29] COMTE S,GUIBAUD G,BAUDU M. Relations between extrac-tion protocols for activated sludge extracellular polymeric sub-stances(EPS)and EPS complexation properties:Part I. Compari-son of the efficiency of eight EPS extraction methods[J].Enzyme&Microbial Technology,2006,38(2):237-245.
[30] WAGNER M,IVLEVA N P,HAISCH C,et al. Combined use ofconfocal laser scanning microscopy(CLSM)and Raman microsco-py(RM):investigations on EPS-Matrix[J]. Water Research,2009,43(1):63-76.
[31]李京宝,韩峰,于文功.细菌生物膜研究技术[J].微生物学报,2007,47(3):558-561.
[32] EIGHMY T T,MARATEA D,BISHOP P L. Electron microscopicexamination of wastewater biofilm formation and structural compo-nents[J].Appl Environ Microbiol,1983,45(6):1921-1931.
[33] FRLUND B,PALMGREN R,KEIDING K,et al. Extraction ofextracellular polymers from activated sludge using a cation ex-change resin[J]. Water Research,1996,30(8):1749-1758.
[34] WINGENDER J,STRATHMANN M,RODE A,et al. Isolationand biochemical characterization of extracellular polymeric sub-stances from Pseudomonas aeruginosa[J].Methods in Enzymology,2001,336:302-314.
[35] MA LM,CONOVER M,LU H,et al. Assembly and developmentof the Pseudomonas aeruginosa biofilm matrix[J]. PLoS Patho-gens,2009,5(3):e1000354.
[36] WATNICK P I,KOLTER R. Steps in the development of a Vibriocholerae EI Tor biofilm[J]. Molecular Microbiology,1999,34(3):586-595.
[37] DANESE PN,PRATT LA,KOLTER R,et al. Exopolysaccharideproduction is required for development of Escherichia coli K-12biofilm architecture[J]. Journal of Bacteriology,2000,182(12):3593-3596.
[38] SUTHERLAND IW. The biofilm matrix-an immobilized but dynam-ic microbial environment[J].Trends in Microbiology,2001,9(5):222-227.