咸鳓鱼中产组胺菌的分离与鉴定
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摘要
针对细菌引起的腌制水产品中生物胺残留问题,胺以传统工艺腌制的咸鳓鱼为对象,利用组胺菌筛选培养基,从不同腌制时期的咸鳓鱼样品中对产组胺微生物进行了筛选,通过16S r DNA测序完成了分离菌株种属鉴定,采用PCR扩增检测了分离菌株组氨酸脱羧酶基因携带情况,并用高效液相色谱(high performance liquid chromatography,HPLC)检测了分离菌株发酵液中组胺的含量。结果表明,分离纯化后共获得32株疑似产生物胺细菌,分布于3个属8个种。葡萄球菌属细菌是优势分离菌株,共计29株,以腐生葡萄球菌数量最多,共16株;24株分离菌株携带组氨酸脱羧酶基因; 23株分离菌株发酵液中测出不同浓度的组胺含量,产组胺菌发酵液组胺平均含量达38. 10μg/m L,其中,1株腐生葡萄球菌菌株发酵液中组胺质量浓度高达100. 21μg/m L,是分离菌株中组胺积累能力最强的菌株。咸鳓鱼生产中产组胺菌的研究,对评估咸鳓鱼的安全性,科学指导其发酵菌株筛选具有理论指导意义。
Accumulation of biogenic amines in fermented seafood products caused by bacterial activity is a serious food safety problem. This study used selective plates to screen histamine-producing bacteria from traditional salted Chinese herring that were collected from different fermentation periods during its production. The isolates were identified by 16 S r DNA sequencing. Histidine decarboxylase genes were amplified using polymerase chain reaction(PCR),while the histamine content in the culture supernatant was detected using high performance liquid chromatography(HPLC). The results indicated that thirty-two presumptive histamine-producing isolates were obtained,which were classified into three genera and eight species. Staphylococcus was the predominant genus(twenty-nine strains),with S. saprophyticus was the dominant species(sixteen strains). Histidine decarboxylase genes were detected among twenty-four isolates,and different concentrations of histamine were detected in the cultures of twenty-three isolates.The average content of histamine produced was 38. 10 μg/m L. Among which,one S. saprophyticus strain produced the highest histamine of 100. 21 μg/m L. Studying histamine-producing bacteria in salted Chinese herring during its production has theoretical significances in evaluating the safety of salted Chinese herring as well as guiding the selection of fermentation strains scientifically.
Accumulation of biogenic amines in fermented seafood products caused by bacterial activity is a serious food safety problem. This study used selective plates to screen histamine-producing bacteria from traditional salted Chinese herring that were collected from different fermentation periods during its production. The isolates were identified by 16 S r DNA sequencing. Histidine decarboxylase genes were amplified using polymerase chain reaction(PCR),while the histamine content in the culture supernatant was detected using high performance liquid chromatography(HPLC). The results indicated that thirty-two presumptive histamine-producing isolates were obtained,which were classified into three genera and eight species. Staphylococcus was the predominant genus(twenty-nine strains),with S. saprophyticus was the dominant species(sixteen strains). Histidine decarboxylase genes were detected among twenty-four isolates,and different concentrations of histamine were detected in the cultures of twenty-three isolates.The average content of histamine produced was 38. 10 μg/m L. Among which,one S. saprophyticus strain produced the highest histamine of 100. 21 μg/m L. Studying histamine-producing bacteria in salted Chinese herring during its production has theoretical significances in evaluating the safety of salted Chinese herring as well as guiding the selection of fermentation strains scientifically.
引文
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[7] JEONG D W,LEE B,HER J Y,et al. Safety and technological characterization of coagulase-negative staphylococci isolates from traditional Korean fermented soybean foods for starter development[J]. International Journal of Food Microbiology,2016,236:9-16.
[8] KORAL S,TUFAN B,SCAVNICAR A,et al. Investigation of the contents of biogenic amines and some food safety parameters of various commercially salted fish products[J]. Food Control,2013,32(2):597-606.
[9] RABIE M,SIMON S L,SILIHA H,et al. Changes in free amino acids and biogenic amines of Egyptian salted-fermented fish(Feseekh)during ripening and storage[J]. Food Chemistry,2009,115(2):635-638.
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[11]郝淑贤,魏涯,周婉君,等.鲭鱼生物胺生成菌的分离与鉴定[J].食品科学,2015,36(7):97-100.
[12]袁开,吴佳佳,朱诚,等.养殖鱼塘底泥微生物抗生素耐药基因分布分析[J].环境科学学报,2017,37(10):3 649-3 655.
[13] JAW Y M,CHEN Y Y,LEE Y C,et al. Histamine content and isolation of histamine-forming bacteria in fish meal and fish soluble concentrate[J]. Fisheries Science,2012,78(1):155-162.
[14]杨健,吴祖芳,周秀锦,等.冷冻鲣鱼中产组胺菌的分离筛选及其生物学特性研究[J].中国食品学报,2012,12(8):25-31.
[15] ANGENLIKI S D,PANAGIOTA F,STEFAAN D S,et al. Effect of temperature and p H on the community dynamics of coagulase-negative staphylococci during spontaneous meat fermentation in a model system[J]. Food Microbiology,2018,76:180-188.
[16] JANSSENS M,MIJNSBRUGGE A V D,MAINAR M S,et al. The use of nucleosides and arginine as alternative energy sources by coagulase-negative staphylococci in view of meat fermentation[J]. Food Microbiology,2014,39(5):53-60.
[17] SEITTER M,NERZ C,ROSENSTEIN R,et al. DNA microarray based detection of genes involved in safety and technologically relevant properties of food associated coagulase-negative staphylococci[J]. International Journal of Food Microbiology,2011,145(2-3):449-458.
[18] FUKAMI K,SATOMI M,FUNATSU Y,et al. Characterization and distribution of Staphylococcus sp. implicated for improvement of fish sauce odor[J]. Fisheries Science,2004,70(5):916-923.
[19] FENG Yarong. Significance of biogenic amines to food safety and human health[J]. Meat Research,2005,29(7):675-690.
[20]王颖,邱璠,韩北忠,等.食品中的生物胺及其检测方法[J].中国酿造,2011,30(10):1-5.
[21] COTON E,COTON M. Evidence of horizontal transfer as origin of strain to strain variation of the tyramine production trait in Lactobacillus brevis[J]. Food Microbiology,2009,26(1):52-57.
[2] MARUI'CN,VIDAˇCEK S,JANˇCI T,et al. Determination of volatile compounds and quality parameters of traditional Istrian dry-cured ham[J]. Meat Science,2014,96(4):1 409.
[3]欧昌荣,管娟,汤海青,等.鲭鱼中组胺降解菌的筛选鉴定和发酵条件初探[J].中国食品学报,2014,14(8):158-164.
[4] CZAJKOWSKA M A,LESZCAYNSKA J. Risk assessment related to biogenic amines occurrence in ready-to-eat baby foods[J]. Food&Chemical Toxicology,2017,105:82-92.
[5] HSIUHUA H,TINCHEN C,LIN H C,et al. Histamine content and histamine-forming bacteria in dried milkfish(Chanos chanos)products[J]. Food Chemistry,2009,114(3):933-938.
[6] TAKHASHI H,KIMURA B,YOSHIKAWA M,et al. Cloning and sequencing of the histidine decarboxylase genes of gram-negative,histamine-producing bacteria and their application in detection and identification of these organisms in fish[J]. Applied&Environmental Microbiology,2003,69(5):2 568-2 579.
[7] JEONG D W,LEE B,HER J Y,et al. Safety and technological characterization of coagulase-negative staphylococci isolates from traditional Korean fermented soybean foods for starter development[J]. International Journal of Food Microbiology,2016,236:9-16.
[8] KORAL S,TUFAN B,SCAVNICAR A,et al. Investigation of the contents of biogenic amines and some food safety parameters of various commercially salted fish products[J]. Food Control,2013,32(2):597-606.
[9] RABIE M,SIMON S L,SILIHA H,et al. Changes in free amino acids and biogenic amines of Egyptian salted-fermented fish(Feseekh)during ripening and storage[J]. Food Chemistry,2009,115(2):635-638.
[10] ZHANG He,LI Yan,XU Kunhua,et al. Microbiological changes and biodiversity of cultivable indigenous bacteria in sanbao larger yellow croaker(Pseudosciaena crocea),a Chinese salted and fermented seafood[J]. Journal of Food Science,2015,80(4):M776-M781.
[11]郝淑贤,魏涯,周婉君,等.鲭鱼生物胺生成菌的分离与鉴定[J].食品科学,2015,36(7):97-100.
[12]袁开,吴佳佳,朱诚,等.养殖鱼塘底泥微生物抗生素耐药基因分布分析[J].环境科学学报,2017,37(10):3 649-3 655.
[13] JAW Y M,CHEN Y Y,LEE Y C,et al. Histamine content and isolation of histamine-forming bacteria in fish meal and fish soluble concentrate[J]. Fisheries Science,2012,78(1):155-162.
[14]杨健,吴祖芳,周秀锦,等.冷冻鲣鱼中产组胺菌的分离筛选及其生物学特性研究[J].中国食品学报,2012,12(8):25-31.
[15] ANGENLIKI S D,PANAGIOTA F,STEFAAN D S,et al. Effect of temperature and p H on the community dynamics of coagulase-negative staphylococci during spontaneous meat fermentation in a model system[J]. Food Microbiology,2018,76:180-188.
[16] JANSSENS M,MIJNSBRUGGE A V D,MAINAR M S,et al. The use of nucleosides and arginine as alternative energy sources by coagulase-negative staphylococci in view of meat fermentation[J]. Food Microbiology,2014,39(5):53-60.
[17] SEITTER M,NERZ C,ROSENSTEIN R,et al. DNA microarray based detection of genes involved in safety and technologically relevant properties of food associated coagulase-negative staphylococci[J]. International Journal of Food Microbiology,2011,145(2-3):449-458.
[18] FUKAMI K,SATOMI M,FUNATSU Y,et al. Characterization and distribution of Staphylococcus sp. implicated for improvement of fish sauce odor[J]. Fisheries Science,2004,70(5):916-923.
[19] FENG Yarong. Significance of biogenic amines to food safety and human health[J]. Meat Research,2005,29(7):675-690.
[20]王颖,邱璠,韩北忠,等.食品中的生物胺及其检测方法[J].中国酿造,2011,30(10):1-5.
[21] COTON E,COTON M. Evidence of horizontal transfer as origin of strain to strain variation of the tyramine production trait in Lactobacillus brevis[J]. Food Microbiology,2009,26(1):52-57.