基于下一代测序技术分析巴氏杀菌乳中残留细菌在贮藏期间的动态变化
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摘要
采用高通量测序技术,分析巴氏杀菌乳在常见贮藏温度0、4、10℃条件下分别贮藏0、3、6、9、12、15 d内细菌16S rRNA的V3-V4区基因序列,进而比较不同贮藏条件下巴氏杀菌乳中微生物的群落组成及动态变化。结果共获得1 887个可操作分类单元,共检测到支原菌属(Mycoplasma)、草酸杆菌属(Oxalobacteraceae)、假单胞菌属(Pseudomonas)、不动细菌属(Actinetobacter)、链球菌(Streptococcus)等34类细菌菌属。多样性分析表明,不同贮藏温度条件下巴氏杀菌乳中微生物的组成及多样性在门、属水平上有明显差异。在0℃贮藏15 d内的巴氏杀菌乳微生物多样性保持最完整,并且0℃其营养品质也保持较好;而在4、10℃贮藏期间巴氏杀菌乳的微生物多样性及营养品质都有较大影响,菌群构成及优势菌群都发生了变化,主要菌群也随贮藏温度的升高变为假单胞菌属、气单胞菌属等。研究表明,在4℃贮藏3 d和9 d、10℃贮藏6 d作为巴氏杀菌乳品质腐败的关键时间点,都出现了之前未被检测出的类芽孢菌属(Paenibacillus)、沙雷氏菌属(新Serratia),初步推测此两种菌属是导致巴氏杀菌乳品质腐败的关键决定因素。
In this study, high-throughput sequencing technology was adopted to analyzed the V3-V4 region of the bacterial 16 S rRNA gene in pasteurized milk stored at different temperature(0, 4, and 10 ℃) for 0, 3, 6, 9, 12 or 15 d. Our aim was to reveal the dynamic changes in the microbial community composition. The results showed that a total of 1 887 operational taxonomic units(OTUs) were obtained and 34 genera of bacteria, including Mycoplasma, Oxalobacteraceae, Pseudomonas,Actinetobacter, and Streptococcus were detected. Bacterial diversity analysis showed that the composition and diversity of the bacterial community in pasteurized milk were significantly different at both the phylum and genus levels. The bacterial diversity in pasteurized milk stored at 0 ℃ for day was the most intact of all samples and its nutritional quality was also maintained well. Nonetheless, both the bacterial diversity and the nutritional quality were greatly affected by storage at 4 and 10 ℃. Under this circumstance, the flora composition and the dominant bacterial community both changed. The dominant bacteria became Pseudomonas and Aeromonas with the increase of storage temperature. Furthermore, this study found that days 3 and 9 of storage at 4 ℃ and day 6 at 10 ℃ were key points of time for pasteurized milk spoilage because Paenibacillus and Serratia were detected at this time point but not earlier. It is speculated that these two genera are the key determinants of pasteurization dairy product spoilage.
In this study, high-throughput sequencing technology was adopted to analyzed the V3-V4 region of the bacterial 16 S rRNA gene in pasteurized milk stored at different temperature(0, 4, and 10 ℃) for 0, 3, 6, 9, 12 or 15 d. Our aim was to reveal the dynamic changes in the microbial community composition. The results showed that a total of 1 887 operational taxonomic units(OTUs) were obtained and 34 genera of bacteria, including Mycoplasma, Oxalobacteraceae, Pseudomonas,Actinetobacter, and Streptococcus were detected. Bacterial diversity analysis showed that the composition and diversity of the bacterial community in pasteurized milk were significantly different at both the phylum and genus levels. The bacterial diversity in pasteurized milk stored at 0 ℃ for day was the most intact of all samples and its nutritional quality was also maintained well. Nonetheless, both the bacterial diversity and the nutritional quality were greatly affected by storage at 4 and 10 ℃. Under this circumstance, the flora composition and the dominant bacterial community both changed. The dominant bacteria became Pseudomonas and Aeromonas with the increase of storage temperature. Furthermore, this study found that days 3 and 9 of storage at 4 ℃ and day 6 at 10 ℃ were key points of time for pasteurized milk spoilage because Paenibacillus and Serratia were detected at this time point but not earlier. It is speculated that these two genera are the key determinants of pasteurization dairy product spoilage.
引文
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[33]CHEN T R,WEI Q K,CHEN Y J.Pseudomonas spp.and Hafnia alvei growth in UHT milk at cold storage[J].Food Control,2011,22(5):697-701.
[34]CHEN L,DANIEL R M,COOLBEAR T.Detection and impact of protease and lipase activities in milk and milk powders[J].International Dairy Journal,2003,13(4):255-275.DOI:10.1016/S0958-6946(02)00171-1.
[2]王加启.建议我国实施优质乳工程[J].中国畜牧兽医,2013,40(增刊1):1-8.
[3]顾佳升,张书义,韩荣伟.巴氏杀菌工艺是引领我国奶业出困境的核心技术[J].中国奶牛,2016(8):60-65.DOI:10.19305/j.cnki.11-3009/s.2016.08.016.
[4]谢爱英,党亚丽,李芳芳,等.不同巴氏杀菌条件对酸奶品质的影响[J].食品科学,2011,32(9):14-17.
[5]张竟丰,王丽,陈洵,等.乳及乳制品中常见“活的非可培养态”食源致病菌研究进展[J].食品科学,2019,40(3):300-306.DOI:10.7506/spkx1002-6630-20171022-233.
[6]ROSSVOLL E,RONNING H T,GRANUM P E,et al.Toxin production and growth of pathogens subjected to temperature fluctuations simulating consumer handling of cold cuts[J].International Journal of Food Microbiology,2014,185(18):82-92.DOI:10.1016/j.ijfoodmicro.2014.05.020.
[7]SCHMIDT V S J,KAUFMANN V,KULOZIK U,et al.Microbial biodiversity,quality and shelf life of microfiltered and pasteurized extended shelf life(ESL)milk from Germany,Austria and Switzerland[J].International Journal of Food Microbiology,2012,154(1/2):1-9.DOI:10.1016/j.ijfoodmicro.2011.12.002.
[8]CHRISTIANSEN P,NIELSEN E W,VOGENSEN F K,et al.Heat resistance of Lactobacillus paracasei isolated from semi-hard cheese made of pasteurized milk[J].International Dairy Journal,2006,16(10):1196-1204.
[9]夏红英.酶在乳制品加工中的应用[J].食品科学,1991,12(12):29-31.
[10]OUWEHAND A C,SALVADORI B B,FONDEN R,et al.Health effects of probiotics and culture-containing dairy products in humans[J].Bulletin-International Dairy Federation,2003.
[11]SOGIN M L,MORRISON H G,HUBER J A,et al.Microbial diversity in the deep sea and the underexplored rare biosphere[J].International Journal of Food Microbiology,2006,103(32):12115-12120.DOI:10.1073/pnas.0605127103.
[12]ROESCH L F,FULTHORPE R R,RIVA A,et al.Pyrosequencing enumerates and contrasts soil microbial diversity[J].ISME Journal,2007,1(4):283-290.DOI:10.1038/ismej.2007.53.
[13]ANDERSSON A F,LINDBERG M,JAKOBSSON H,et al.Comparative analysis of human gut microbiota by barcoded pyrosequencing[J].PLoS ONE,2008,3(7):2836.DOI:10.1371/journal.pone.0002836.
[14]CLAESSON M J,O’SULLIVAN O,WANG Q,et al.Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine[J].PLoS ONE,2009,4(8):e6669.DOI:10.1371/journal.pone.0006669.
[15]QUIGLEY L,MCCARTHY R,O’SULLIVAN O,et al.The microbial content of raw and pasteurized cow milk as determined by molecular approaches[J].Journal of Dairy Science,2013,96:4928-4937.DOI:10.3168/jds.2013-6688.
[16]张哲,刘小鸣,陈卫.内蒙古传统发酵驼乳中乳酸菌和酵母菌的分离鉴定及其生物多样性分析[J].中国食品学报,2018,18(7):230-238.DOI:10.16429/j.1009-7848.2018.07.028.
[17]WATANABE K,FUJIMOTO J,SASAMOTO M,et al.Diversity of lactic acid bacteria and yeasts in Airag and Tarag,traditional fermented milk products of Mongolia[J].World Journal of Microbiology&Biotechnology,2008,24(8):1313-1325.DOI:10.1007/s11274-007-9604-3.
[18]PORCELLATO D,ASPHOLM M,SKEIE S B,et al.Microbial diversity of consumption milk during processing and storage[J].International Journal of Food Microbiology,2018,266:21-30.DOI:10.1016/j.ijfoodmicro.2017.11.004.
[19]NIEMINEN T T,KOSKINEN K,LAINE P,et al.Comparison of microbial communities in marinated and unmarinated broiler meat by metagenomics[J].International Journal of Food Microbiology,2012,157(2):142-149.DOI:10.1016/j.ijfoodmicro.2012.04.016.
[20]张和平,郑艺.高通量测序技术在乳制品研究中的应用[J].中国食品学报,2015,15(3):1-7.DOI:10.16429/j.1009-7848.2015.03.001.
[21]孙雪姣.不同贮藏温度对巴氏杀菌乳品质和微生物的影响[D].沈阳:沈阳农业大学,2018.
[22]姚新奎,车驰.巴氏杀菌乳营养价值及发展前景[J].新疆畜牧业,2010(6):10-13.DOI:10.3969/j.issn.1003-4889.2010.06.005.
[23]JAY J M,LOESSNER M J,GOLDEN D A.Modern food microbiology[M].US:Springer,2015.
[24]QUIGLEY L,O’SULLIVAN O,STANTON C.The complex microbiota of raw milk[M].FEMS Microbiology Reviews,2013,37:664-698.DOI:10.1111/1574-6976.12030.
[25]ELWELL M W,BARBANO D M.Use of microfiltration to improve fluid milk quality[J].Journal of Dairy Science,2006,89:20-30.DOI:10.3168/jds.S0022-0302(06)72361-X.
[26]陈庆华,王欣.冷藏温度及时间对巴氏杀菌乳品质的影响研究[J].食品科技,2009,34(1):84-87.DOI:10.13684/j.cnki.spkj.2009.01.039.
[27]BOOR K J.Fluid dairy product quality and safety:looking to the future[J].Journal of Dairy Science,2001,84(1):1-11.
[28]DOUGHARI H J,NDAKIDEMI P A,HUMAN I S,et al,The ecology,biology and pathogenesis of Acinetobacter spp.:an overview[J].Microbes Environments,2011,26(2):101-112.DOI:10.1264/jsme2.ME10179.
[29]ENEROTH A,SVENSSON B,MOLIN G,et al.Contamination of pasteurized milk by Bacillus cereus in the filling machine[J].Journal of Dairy Research,2001,68(2):189-196.DOI:10.1017/S002202990100485X.
[30]NOVAK J S,CALL J,TOMASULA P,et al.An assessment of pasteurization treatment of water,media,and milk with respect to Bacillus spores[J].Journal of Food Protection,2005,68(4):751-757.DOI:10.4315/0362-028X-68.4.751.
[31]BUEHNER K P,ANAND S,DJIRA G D,et al.Prevalence of thermoduric bacteria and spores on 10 Midwest dairy farms[J].Journal of Dairy Science,2014,97(11):6777-6784.DOI:10.3168/jds.2014-97-12-8009.
[32]DOYLE C J,GLEESON D,O’TOOLE P W,et al.Impacts of seasonal housing and teat preparation on raw milk microbiota:a high-throughput sequencing study[J].Applied and Environmental Microbiology,2017,83(2).DOI:10.1128/AEM.02694-16.
[33]CHEN T R,WEI Q K,CHEN Y J.Pseudomonas spp.and Hafnia alvei growth in UHT milk at cold storage[J].Food Control,2011,22(5):697-701.
[34]CHEN L,DANIEL R M,COOLBEAR T.Detection and impact of protease and lipase activities in milk and milk powders[J].International Dairy Journal,2003,13(4):255-275.DOI:10.1016/S0958-6946(02)00171-1.