冷吃兔特定腐败菌的分离与鉴定
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摘要
对冷吃兔的腐败细菌进行分离、生理生化鉴定及分子生物学鉴定。通过稀释平板划线法分离腐败微生物,获得纯菌落。将分离的纯菌株接种到无菌冷吃兔中富集培养,测定菌落总数和挥发性盐基氮含量,确定特定腐败菌;通过生理生化实验结果,以《伯杰氏细菌鉴定手册》和《常见细菌系统鉴定手册》为参考进行菌种初步鉴定;通过DNA提取、聚合酶链式反应扩增和凝胶电泳将菌株准确鉴定到种。结果表明:引起冷吃兔腐败变质的特定腐败菌为沃氏葡萄球菌和枯草芽孢杆菌。
Bacteria from spoiled cold spicy rabbit meat were isolated and identified by physiological, biochemical and molecular biological methods. The pure strains, obtained by the streak plate method, were re-inoculated onto sterile spicy rabbit meat and enriched. The aerobic bacterial count and total volatile basic nitrogen(TVB-N) content were measured to identify the specific spoilage organisms(SSO). These strains were tentatively identified according to the Bergey's Manual of Determinative Bacteriology and the Manual for Systematic Identification of Common Bacteria and further identified to the species level by sequential DNA extraction, polymerase chain reaction amplification and gel electrophoresis. The SSO in cold spicy rabbit meat were Staphylococcus warneri and Bacillus subtilis.
Bacteria from spoiled cold spicy rabbit meat were isolated and identified by physiological, biochemical and molecular biological methods. The pure strains, obtained by the streak plate method, were re-inoculated onto sterile spicy rabbit meat and enriched. The aerobic bacterial count and total volatile basic nitrogen(TVB-N) content were measured to identify the specific spoilage organisms(SSO). These strains were tentatively identified according to the Bergey's Manual of Determinative Bacteriology and the Manual for Systematic Identification of Common Bacteria and further identified to the species level by sequential DNA extraction, polymerase chain reaction amplification and gel electrophoresis. The SSO in cold spicy rabbit meat were Staphylococcus warneri and Bacillus subtilis.
引文
[1]陈静霞,余静.兔肉的营养价值及其加工业现状[J].肉类研究,2008,22(12):74-76.
[2]杨绍斌,王国泽,曾浪,等.四川省兔肉产业发展现状浅谈[J].肉类工业,2017(6):42-45.DOI:10.3969/j.issn.1008-5467.2017.06.010.
[3]谷子林,陈宝江,黄玉亭,等.我国兔肉消费市场瓶颈分析及对策[J].中国养兔,2014(4):27-30;10.DOI:10.3969/j.issn.1005-6327.2014.04.007.
[4]武拉平.“十三五”以来中国兔产业发展报告(2016-2018年)[C]//2018第8届兔业发展大会论文集.北京:中国畜牧业协会,2018:17.
[5]胡伟峰.川南·自贡冷吃兔&火边子牛肉:不得不吃的盐都美食[J].四川党的建设(城市版),2013(3):65-66.
[6]史磊.中国城市居民兔肉及兔产品消费需求:基于2017年消费者调研数据[C]//2018第8届兔业发展大会论文集.北京:中国畜牧业协会,2018:5.
[7]李光辉,钟世荣,冯治平.冷吃兔复合调味料的研究[J].江苏调味副食品,2009,26(2):9-11.DOI:10.3969/j.issn.1006-8481.2009.02.003.
[8]刘学铭,方少钦,唐道帮,等.我国熟肉制品微生物安全现状与控制技术[J].现代食品科技,2012(1):99-103.DOI:10.3969/j.issn.1673-9078.2012.01.024.
[9]石振兴,朱仁俊.控制肉制品微生物污染方法的研究进展[J].肉类研究,2009,23(7):30-32.
[10]GRAM L,DALGAARD P.Fish spoilage bacteria:problems and solutions[J].Current Opinion in Biotechnology,2002,13(3):262-266.DOI:10.1016/S0958-1669(02)00309-9.
[11]HUIS IN’T VELD J H.Microbial and biochemical spoilage of foods:an overview[J].International Journal of Food Microbiology,1996,33(1):1-18.DOI:10.1016/0168-1605(96)01139-7.
[12]SAMELIS J,GEORGIADOU K G.The microbial association of Greek taverna sausage stored at 4 and 10℃in air,vacuum or 100%carbon dioxide,and its spoilage potential[J].Journal of Applied Microbiology,2000,88(1):58-68.DOI:10.1046/j.1365-2672.2000.00936.x.
[13]CHENOLL E,MACIáN M C,ELIZAQUíVEL P,et al.Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage:population analysis by rDNA-based methods[J].Journal of Applied Microbiology,2007,102(2):498-508.DOI:10.1111/j.1365-2672.2006.03081.x.
[14]BJ?RKROTH K J,KORKEALA H.Lactobacillus fructivorans spoilage of tomato ketchup[J].Journal of Food Protection,1997,60(5):505-509.DOI:10.1002/(SICI)1097-4660(199705)69:1<130::AID-JCTB686>3.0.CO;2-X.
[15]SAMELIS J,KAKOURI A,GEORGIADOU K G,et al.Evaluation of the extent and type of bacterial contamination at different stages of processing of cooked ham[J].Journal of Applied Microbiology,1998,84(4):649-660.DOI:10.1046/j.1365-2672.1998.00392.x.
[16]PAPADOPOULOU O S,PANAGOU E Z,MOHAREB F R,et al.Sensory and microbiological quality assessment of beef fillets using a portable electronic nose in tandem with support vector machine analysis[J].Food Research International,2013,50(1):241-249.DOI:10.1016/j.foodres.2012.10.020.
[17]中华人民共和国国家卫生和计划生育委员会,国家食品药品监督管理总局.食品安全国家标准食品微生物学检验菌落总数测定:GB 4789.2-2016[S].北京:中国标准出版社,2016.
[18]中华人民共和国国家卫生和计划生育委员会.食品安全国家标准食品中挥发性盐基氮的测定:GB 5009.228-2016[S].北京:中国标准出版社,2016.
[19]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.
[20]周贤轩,杨波,陈新华.几种分子生物学方法在菌种鉴定中的应用[J].生物技术,2004,14(6):35-38.DOI:10.3969/j.issn.1004-311X.2004.06.018.
[21]中华人民共和国国家卫生和计划生育委员会,国家食品药品监督管理总局.食品安全国家标准熟肉制品:GB 2726-2016[S].北京:中国标准出版社,2016.
[22]上海市食品卫生监督检验所.肉与肉制品卫生标准的分析方法:GB 5009.44-2003[S].北京:中国标准出版社,2003.
[23]马跃龙,俞淑,薛峰,等.调理肉制品挥发性盐基氮的研究[J].农产品加工(学刊),2012(5):112-113.DOI:10.3969/j.issn.1671-9646(X).2012.05.035.
[24]WIEGEL J,QUANDT L.Determination of the gram type using the reaction between polymyxin B and lipopolysaccharides of the outer cell wall of whole bacteria[J].Microbiology,1982,128(10):2261-2270.DOI:10.1099/00221287-128-10-2261.
[25]布坎南R E,吉本斯N E.伯杰氏细菌鉴定手册[M].8版.北京:科学出版社,1984.
[26]鞠慧萍,宋白薇.沃氏葡萄球菌VITEK鉴定分析[J].畜牧兽医科技信息,2010(9):44-47.DOI:10.3969/J.ISSN.1671-6027.2010.09.030.
[27]杨雪莲,张明苏,苏晓琳,等.隐形眼镜所携带细菌种类及对人眼的危害[J].世界最新医学信息文摘,2018,18(89):48;50.
[28]LISTGARTEN M A.Microbiological composition and pattern of antibiotic resistance in subgingival microbial samples from patients with refractory periodontitis[J].Journal of Periodontology,64(3):155-161.DOI:10.1902/jop.1993.64.3.155.
[29]刘志恒.现代微生物学[J].北京:科学出版社,2008:92-99.
[30]杨彪,周刚,王倩倩,等.枯草芽孢杆菌在家禽生产中的应用[J].饲料博览,2014(4):31-34.DOI:10.3969/j.issn.1001-0084.2014.04.008.
[31]朱玥妍,刘姣,杜春梅.芽孢杆菌生物防治植物病害研究进展[J].安徽农业科学,2012,40(34):16635-16638.DOI:10.13989/j.cnki.0517-6611.2012.34.112.
[2]杨绍斌,王国泽,曾浪,等.四川省兔肉产业发展现状浅谈[J].肉类工业,2017(6):42-45.DOI:10.3969/j.issn.1008-5467.2017.06.010.
[3]谷子林,陈宝江,黄玉亭,等.我国兔肉消费市场瓶颈分析及对策[J].中国养兔,2014(4):27-30;10.DOI:10.3969/j.issn.1005-6327.2014.04.007.
[4]武拉平.“十三五”以来中国兔产业发展报告(2016-2018年)[C]//2018第8届兔业发展大会论文集.北京:中国畜牧业协会,2018:17.
[5]胡伟峰.川南·自贡冷吃兔&火边子牛肉:不得不吃的盐都美食[J].四川党的建设(城市版),2013(3):65-66.
[6]史磊.中国城市居民兔肉及兔产品消费需求:基于2017年消费者调研数据[C]//2018第8届兔业发展大会论文集.北京:中国畜牧业协会,2018:5.
[7]李光辉,钟世荣,冯治平.冷吃兔复合调味料的研究[J].江苏调味副食品,2009,26(2):9-11.DOI:10.3969/j.issn.1006-8481.2009.02.003.
[8]刘学铭,方少钦,唐道帮,等.我国熟肉制品微生物安全现状与控制技术[J].现代食品科技,2012(1):99-103.DOI:10.3969/j.issn.1673-9078.2012.01.024.
[9]石振兴,朱仁俊.控制肉制品微生物污染方法的研究进展[J].肉类研究,2009,23(7):30-32.
[10]GRAM L,DALGAARD P.Fish spoilage bacteria:problems and solutions[J].Current Opinion in Biotechnology,2002,13(3):262-266.DOI:10.1016/S0958-1669(02)00309-9.
[11]HUIS IN’T VELD J H.Microbial and biochemical spoilage of foods:an overview[J].International Journal of Food Microbiology,1996,33(1):1-18.DOI:10.1016/0168-1605(96)01139-7.
[12]SAMELIS J,GEORGIADOU K G.The microbial association of Greek taverna sausage stored at 4 and 10℃in air,vacuum or 100%carbon dioxide,and its spoilage potential[J].Journal of Applied Microbiology,2000,88(1):58-68.DOI:10.1046/j.1365-2672.2000.00936.x.
[13]CHENOLL E,MACIáN M C,ELIZAQUíVEL P,et al.Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage:population analysis by rDNA-based methods[J].Journal of Applied Microbiology,2007,102(2):498-508.DOI:10.1111/j.1365-2672.2006.03081.x.
[14]BJ?RKROTH K J,KORKEALA H.Lactobacillus fructivorans spoilage of tomato ketchup[J].Journal of Food Protection,1997,60(5):505-509.DOI:10.1002/(SICI)1097-4660(199705)69:1<130::AID-JCTB686>3.0.CO;2-X.
[15]SAMELIS J,KAKOURI A,GEORGIADOU K G,et al.Evaluation of the extent and type of bacterial contamination at different stages of processing of cooked ham[J].Journal of Applied Microbiology,1998,84(4):649-660.DOI:10.1046/j.1365-2672.1998.00392.x.
[16]PAPADOPOULOU O S,PANAGOU E Z,MOHAREB F R,et al.Sensory and microbiological quality assessment of beef fillets using a portable electronic nose in tandem with support vector machine analysis[J].Food Research International,2013,50(1):241-249.DOI:10.1016/j.foodres.2012.10.020.
[17]中华人民共和国国家卫生和计划生育委员会,国家食品药品监督管理总局.食品安全国家标准食品微生物学检验菌落总数测定:GB 4789.2-2016[S].北京:中国标准出版社,2016.
[18]中华人民共和国国家卫生和计划生育委员会.食品安全国家标准食品中挥发性盐基氮的测定:GB 5009.228-2016[S].北京:中国标准出版社,2016.
[19]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.
[20]周贤轩,杨波,陈新华.几种分子生物学方法在菌种鉴定中的应用[J].生物技术,2004,14(6):35-38.DOI:10.3969/j.issn.1004-311X.2004.06.018.
[21]中华人民共和国国家卫生和计划生育委员会,国家食品药品监督管理总局.食品安全国家标准熟肉制品:GB 2726-2016[S].北京:中国标准出版社,2016.
[22]上海市食品卫生监督检验所.肉与肉制品卫生标准的分析方法:GB 5009.44-2003[S].北京:中国标准出版社,2003.
[23]马跃龙,俞淑,薛峰,等.调理肉制品挥发性盐基氮的研究[J].农产品加工(学刊),2012(5):112-113.DOI:10.3969/j.issn.1671-9646(X).2012.05.035.
[24]WIEGEL J,QUANDT L.Determination of the gram type using the reaction between polymyxin B and lipopolysaccharides of the outer cell wall of whole bacteria[J].Microbiology,1982,128(10):2261-2270.DOI:10.1099/00221287-128-10-2261.
[25]布坎南R E,吉本斯N E.伯杰氏细菌鉴定手册[M].8版.北京:科学出版社,1984.
[26]鞠慧萍,宋白薇.沃氏葡萄球菌VITEK鉴定分析[J].畜牧兽医科技信息,2010(9):44-47.DOI:10.3969/J.ISSN.1671-6027.2010.09.030.
[27]杨雪莲,张明苏,苏晓琳,等.隐形眼镜所携带细菌种类及对人眼的危害[J].世界最新医学信息文摘,2018,18(89):48;50.
[28]LISTGARTEN M A.Microbiological composition and pattern of antibiotic resistance in subgingival microbial samples from patients with refractory periodontitis[J].Journal of Periodontology,64(3):155-161.DOI:10.1902/jop.1993.64.3.155.
[29]刘志恒.现代微生物学[J].北京:科学出版社,2008:92-99.
[30]杨彪,周刚,王倩倩,等.枯草芽孢杆菌在家禽生产中的应用[J].饲料博览,2014(4):31-34.DOI:10.3969/j.issn.1001-0084.2014.04.008.
[31]朱玥妍,刘姣,杜春梅.芽孢杆菌生物防治植物病害研究进展[J].安徽农业科学,2012,40(34):16635-16638.DOI:10.13989/j.cnki.0517-6611.2012.34.112.