冷鲜鸡冷藏保存过程中菌群结构变化分析
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摘要
为了探索冷鲜鸡冷藏保存过程中微生物菌群结构的变化,于华东地区一家禽定点屠宰场随机采集25只冷鲜鸡样品,置于4℃冷藏保存,在第0、1、3、5、7天,分别取5只冷鲜鸡,检测挥发性盐基氮、菌落总数、大肠埃希菌数量的变化,并进一步通过高通量测序技术分析菌群结构的变化。结果显示,随着冷藏时间的延长,挥发性盐基氮由5. 51 mg·(100 g)~(-1)增至29. 84 mg·(100 g)~(-1)(P <0. 05),菌落总数、大肠埃希菌总数分别由4. 46×10~6、1. 48×10~6CFU·g~(-1)增至1. 87×10~8、5. 18×10~6CFU·g~(-1)(P <0. 05)。高通量测序分析表明,在门水平上,变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)为优势菌门,其相对丰度在各个试验组均超过99%,变形菌门的相对丰度随着冷蔵天数的增加而增加,厚壁菌门的相对丰度随着冷藏天数的增加而减少。在属水平上,乳酸杆菌属(Lactobacillus)、不动杆菌属(Acinetobacter)、嗜冷杆菌属(Psychrobacter)、肠球菌属(Enterococcus)、假单胞菌属(Pseudomonas)、埃希氏菌属(Escherichia)为主要的优势菌属。随着冷藏天数的增加,在相对丰度大于0. 1%的属中,有9个属的相对丰度显著增加,包括肉食杆菌属(Carnobacterium)、乳球菌属(Lactococcus)、巨球菌属(Macrococcus)、假单胞菌属(Pseudomonas)、嗜冷杆菌属(Psychrobacter)、希瓦氏菌属(Shewanella)等典型的腐败菌; 10个菌属的相对丰度显著降低,包括拟杆菌属(Bacteroides)、布劳特氏菌属(Blautia)、肠球菌属(Enterococcus)、粪杆菌属(Faecalibacterium)、瘤胃球菌属(Ruminococcus)和SMB53等典型肉鸡肠道菌属。综上,冷鲜鸡肉的污染微生物起始阶段主要是肉鸡自身携带微生物,而后逐渐演替成腐败菌为优势菌群,导致鸡肉的腐败变质。
To explore the changes of microbial community structure in chilled chicken during cold storage,25 chilled chicken samples were obtained from poultry slaughterhouse in east China and stored in 4 ℃. Five chilled chickenswere used to measure the concentration of total volatile basic nitrogen (TVBN),total number of bacterial colonies and total E. coli counts on day 0,1,3,5,7,respectively. The bacterial community structures were analyzed by high-throughput sequencing. Results showed that the concentration of TVBN enhanced with the increasing cold storage days,from 5. 51 mg· (100 g)~(-1)to 29. 84 mg· (100 g)~(-1) (P < 0. 05). The total number of bacterial colonies were also increased,from 4. 46 × 10~6CFU·g~(-1)to 1. 87 × 10~8CFU·g~(-1) (P < 0. 05),while the total E. coli counts increased,from 1. 48 × 10~6CFU·g~(-1)g to 5. 18 × 10~6CFU·g~(-1) (P < 0. 05). High-throughput sequencing showed that Proteobacteria,Firmicutes,Actinobacteria and Bacteroidetes were the dominant phyla,the relative abundance of these four phyla were more than 99% in chilled chickens of different storage days. At genus level,Lactobacillus,Acinetobacter,Psychrobacter,Enterococcus,Pseudomonas,and Escherichia were dominant. The genera with relative abundance more than 0. 1% were selected for significance analysis among chilled chickens of different storage time.There were 9 genera of increased relative abundance,including Carnobacterium,Lactococcus,Macrococcus,Pseudomonas,Psychrobacter,and Shewanella,which were typical spoilage bacteria. Otherwise,10 genera were of reduced relative abundance,including Bacteroides,Blautia,Enterococcus,Faecalibacterium,Ruminococcus,and SMB53,which were typical intestinal bacteria in broilers. These findings suggested that the contaminant microorganisms in chilled chicken were from broilers in the startup phase,and then the spoilage bacteria became the dominant microorganisms,which could result in the spoilage of chicken meat.
To explore the changes of microbial community structure in chilled chicken during cold storage,25 chilled chicken samples were obtained from poultry slaughterhouse in east China and stored in 4 ℃. Five chilled chickenswere used to measure the concentration of total volatile basic nitrogen (TVBN),total number of bacterial colonies and total E. coli counts on day 0,1,3,5,7,respectively. The bacterial community structures were analyzed by high-throughput sequencing. Results showed that the concentration of TVBN enhanced with the increasing cold storage days,from 5. 51 mg· (100 g)~(-1)to 29. 84 mg· (100 g)~(-1) (P < 0. 05). The total number of bacterial colonies were also increased,from 4. 46 × 10~6CFU·g~(-1)to 1. 87 × 10~8CFU·g~(-1) (P < 0. 05),while the total E. coli counts increased,from 1. 48 × 10~6CFU·g~(-1)g to 5. 18 × 10~6CFU·g~(-1) (P < 0. 05). High-throughput sequencing showed that Proteobacteria,Firmicutes,Actinobacteria and Bacteroidetes were the dominant phyla,the relative abundance of these four phyla were more than 99% in chilled chickens of different storage days. At genus level,Lactobacillus,Acinetobacter,Psychrobacter,Enterococcus,Pseudomonas,and Escherichia were dominant. The genera with relative abundance more than 0. 1% were selected for significance analysis among chilled chickens of different storage time.There were 9 genera of increased relative abundance,including Carnobacterium,Lactococcus,Macrococcus,Pseudomonas,Psychrobacter,and Shewanella,which were typical spoilage bacteria. Otherwise,10 genera were of reduced relative abundance,including Bacteroides,Blautia,Enterococcus,Faecalibacterium,Ruminococcus,and SMB53,which were typical intestinal bacteria in broilers. These findings suggested that the contaminant microorganisms in chilled chicken were from broilers in the startup phase,and then the spoilage bacteria became the dominant microorganisms,which could result in the spoilage of chicken meat.
引文
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[19]向文良,罗海,梁华忠,等.基于16S-23S r RNA ITS AFLP对米酒发酵过程中原核微生物的演替分析[J].酿酒科技,2010,188(2):43-46.XIANG W L,LUO H,LIANG H Z,et al.Prokaryotic microbial community succession analysis of rice wine during its fermentation by use of 16S-23S r RNA ITS AFLP[J].LiquorMaking Science&Technology,2010,188(2):43-46.(in Chinese with English abstract)
[20]诸永志,姚丽娅,王道营,等.冷却羊肉微生物菌群分析与鉴定[J].江西农业学报,2009,21(3):124-126.ZHU Y Z,YAO L Y,WANG D Y,et al.Isolation and identification of spoilage bacteria in chilled mutton[J].Acta Agriculturae Jiangxi,2009,21(3):124-126.(in Chinese with English abstract)
[21]刘莹莹,罗瑞明,卢君逸,等.冷却羊肉贮藏中品质变化及假单胞菌生长预测模型的建立[J].肉类研究,2013,27(5):5-9.LIU Y Y,LUO R M,LU J Y,et al.Predictive modelling of quality changes and Pseudomonas growth during chilled storage of mutton[J].Meat Research,2013,27(5):5-9.(in Chinese with English abstract)
[22]江芸,高峰,徐幸莲,等.冷却猪肉不同前处理对细菌DNA提取及PCR-DGGE的影响[J].食品科学,2010,31(6):258-262.JIANG Y,GAO F,XU X L,et al.Dilution-PCR and PCR-DGGE for evolution of effect of pre-treatment methods on extraction of bacterial DNA from chilled pork[J].Food Science,2010,31(6):258-262.(in Chinese with English abstract)
[23]梁荣蓉.生鲜鸡肉调理制品菌群结构分析和产品货架期预测模型的研究[D].泰安:山东农业大学,2010.LIANG R R.Bacterial community and shelf-life predictive model of freshly prepared chicken products[D].Tai'an:Shandong Agricultural University,2010.(in Chinese with English abstract)
[24]蓝蔚青,谢晶,周会,等.不同时期鲳鱼冷藏期间优势腐败菌的多样性变化[J].食品科学,2015,26(2):226-231.LAN W Q,XIE J,ZHOU H,et al.Diversity of dominant spoilage bacteria in pomfret(Pampus argenteus)from different growing seasons during chilled storage[J].Food Science,2015,26(2):226-231.(in Chinese with English abstract)
[25]江芸.托盘和真空包装冷却猪肉冷藏过程中菌相变化规律研究[D].南京:南京农业大学,2010.JIANG Y.Changes in the bacterial community on tray-and vacuum-packaged pork during chilled storage[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese with English abstract)
[26]倪萍,纪文营,黄洁洁,等.冷却猪肉中热死环丝菌的分离、鉴定及致腐能力[J].食品科学,2013,34(21):140-144.NI P,JI W Y,HUANG J J,et al.Isolation,identification and spoilage capability of Brochothrix thermosphacta isolated from chilled pork[J].Food Science,2013,34(21):140-144.(in Chinese with English abstract)
[27]PENNACCHIA C,ERCIKUBU D,VILLANI F.Development of a real-time PCR assay for the specific detection of Brochothrix thermosphacta in fresh and spoiled raw meat[J].International Journal Food Microbiology,2009,134(3):230-236.
[28]XIAO Y P,XIANG Y,ZHOU W D,et al.Microbial community mapping in intestinal tract of broiler chicken[J].Poultry Science,2017,96(5):1387-1393.
[2]黄强力,闵成军.冷鲜肉的推广势在必行[J].肉类工业,2012(3):51-53.HUANG Q L,MIN C J.It is necessary to extend chilled meat[J].Meat Industry,2012(3):51-53.(in Chinese with English abstract)
[3]叶藻,谢晶.冷鲜鸡保鲜剂的研究进展[J].食品与机械,2015,31(2):248-251.YE Z,XIE J.Research progress on cold fresh chicken preservation[J].Food&Machinery,2015,31(2):248-251.(in Chinese with English abstract)
[4]肖英平,何祥祥,戴宝玲,等.采样方法对冷鲜鸡表面细菌DNA提取及高通量测序结果的影响[J].食品科学,2017,38(24):260-264.XIAO Y P,HE X X,DAI B L,et al.Effects of different sampling methods on microbial DNA extraction from chilled chicken and the high-throughput sequencing of amplification products[J].Food Science,2017,38(24):260-264.(in Chinese with English abstract)
[5]CAPORASO J G,KUCZYNSKI J,STOMBAUGH J,et al.QIIME allows analysis of high-throughput community sequencing data[J].Nature Methods,2010,7(5):335-336.
[6]BOKULICH N A,SUBRAMANIAN S,FAITH J J,et al.Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing[J].Nature Methods,2013,10(1):57-59.
[7]EDGAR R C,HAAS B J,CLEMENTE J C,et al.UCHIMEimproves sensitivity and speed of chimera detection[J].Bioinformatics,2011,27(16):2194-2200.
[8]HAAS B J,GEVERS D,EARL A M,et al.Chimeric 16Sr RNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons[J].Genome Research,2011,21(3):494-504.
[9]EDGAR R C.UPARSE:highly accurate OTU sequences from microbial amplicon reads[J].Nature Methods,2013,10(10):996-998.
[10]NYCHAS G J,SKANDAMIS P N,TASSOU C C,et al.Meat spoilage during distribution[J].Meat Science,2008,78(1/2):77-89.
[11]王虎虎,徐幸莲.冰鲜鸡肉中致病菌三重PCR检测方法的建立[J].中国农业科学,2010,43(17):3608-3615.WANG H H,XU X L.Detection of pathogenic microorganism in fresh chicken meat by multiplex PCR[J].Scientia Agricultura Sinica,2010,43(17):3608-3615.(in Chinese with English abstract)
[12]HINTON J R,CASON J A,INGRAM K D.Tracking spoilage baeteria in commereial poultry processing and refrigerated storage of poultry carcasses[J].Food Microbiology,2004,91(2):155-165.
[13]高磊,谢晶,叶藻,等.冷鲜鸡腿肉中优势腐败菌的分离鉴定及腐败能力研究[J].食品与发酵工业,2015,41(8):48-53.GAO L,XIE J,YE Z,et al.Identification of dominant spoilage bacteria isolated from leg of chilled chicken and study on its spoilage capability[J].Food and Fermentation Industries,2015,41(8):48-53.(in Chinese with English abstract)
[14]GEERAERTS W,POTHAKOS V,DE VUYST L,et al.Variability within the dominant microbiota of sliced cooked poultry products at expiration date in the Belgian retail[J].Food Microbiology,2018,73:209-215.
[15]商宝娣,杨星,李正友,等.希瓦氏菌的研究进展[J].福建农业,2015(7):152-154.SHANG B D,YANG X,LI Z Y,et al.Research on Shewanella[J].Fujian Agriculture,2015(7):152-154.(in Chinese)
[16]OLOFSSON T C,AHRNE S,MOLIN G.Composition of the bacterial population of refrigerated beef,identified with direct16S r RNA gene analysis and pure culture technique[J].International Journal of Food Microbiology,2007,118(3):233-240.
[17]曾晓房,林惠珍,邝智祥,等.冰鲜肉中腐败菌的研究现状[J].安徽农业科学,2010,38(34):19550-19552.ZENG X F,LIN H Z,KUANG Z X,et al.Research progress of spoilage bacteria in chilled meat[J].Journal of Anhui Agricultral Sciences,2010,38(34):19550-19552.(in Chinese with English abstract)
[18]SALAZAR J K,WANG Y,YU S,et al.Polymerase chain reaction-based serotyping of pathogenic bacteria in food[J].Journal of Microbiological Methods,2015,110:18-26.
[19]向文良,罗海,梁华忠,等.基于16S-23S r RNA ITS AFLP对米酒发酵过程中原核微生物的演替分析[J].酿酒科技,2010,188(2):43-46.XIANG W L,LUO H,LIANG H Z,et al.Prokaryotic microbial community succession analysis of rice wine during its fermentation by use of 16S-23S r RNA ITS AFLP[J].LiquorMaking Science&Technology,2010,188(2):43-46.(in Chinese with English abstract)
[20]诸永志,姚丽娅,王道营,等.冷却羊肉微生物菌群分析与鉴定[J].江西农业学报,2009,21(3):124-126.ZHU Y Z,YAO L Y,WANG D Y,et al.Isolation and identification of spoilage bacteria in chilled mutton[J].Acta Agriculturae Jiangxi,2009,21(3):124-126.(in Chinese with English abstract)
[21]刘莹莹,罗瑞明,卢君逸,等.冷却羊肉贮藏中品质变化及假单胞菌生长预测模型的建立[J].肉类研究,2013,27(5):5-9.LIU Y Y,LUO R M,LU J Y,et al.Predictive modelling of quality changes and Pseudomonas growth during chilled storage of mutton[J].Meat Research,2013,27(5):5-9.(in Chinese with English abstract)
[22]江芸,高峰,徐幸莲,等.冷却猪肉不同前处理对细菌DNA提取及PCR-DGGE的影响[J].食品科学,2010,31(6):258-262.JIANG Y,GAO F,XU X L,et al.Dilution-PCR and PCR-DGGE for evolution of effect of pre-treatment methods on extraction of bacterial DNA from chilled pork[J].Food Science,2010,31(6):258-262.(in Chinese with English abstract)
[23]梁荣蓉.生鲜鸡肉调理制品菌群结构分析和产品货架期预测模型的研究[D].泰安:山东农业大学,2010.LIANG R R.Bacterial community and shelf-life predictive model of freshly prepared chicken products[D].Tai'an:Shandong Agricultural University,2010.(in Chinese with English abstract)
[24]蓝蔚青,谢晶,周会,等.不同时期鲳鱼冷藏期间优势腐败菌的多样性变化[J].食品科学,2015,26(2):226-231.LAN W Q,XIE J,ZHOU H,et al.Diversity of dominant spoilage bacteria in pomfret(Pampus argenteus)from different growing seasons during chilled storage[J].Food Science,2015,26(2):226-231.(in Chinese with English abstract)
[25]江芸.托盘和真空包装冷却猪肉冷藏过程中菌相变化规律研究[D].南京:南京农业大学,2010.JIANG Y.Changes in the bacterial community on tray-and vacuum-packaged pork during chilled storage[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese with English abstract)
[26]倪萍,纪文营,黄洁洁,等.冷却猪肉中热死环丝菌的分离、鉴定及致腐能力[J].食品科学,2013,34(21):140-144.NI P,JI W Y,HUANG J J,et al.Isolation,identification and spoilage capability of Brochothrix thermosphacta isolated from chilled pork[J].Food Science,2013,34(21):140-144.(in Chinese with English abstract)
[27]PENNACCHIA C,ERCIKUBU D,VILLANI F.Development of a real-time PCR assay for the specific detection of Brochothrix thermosphacta in fresh and spoiled raw meat[J].International Journal Food Microbiology,2009,134(3):230-236.
[28]XIAO Y P,XIANG Y,ZHOU W D,et al.Microbial community mapping in intestinal tract of broiler chicken[J].Poultry Science,2017,96(5):1387-1393.