高通量测序结合传统方法分析4℃下鲜切菠菜的菌群变化
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摘要
采用Illumina Miseq~(TM)高通量测序技术结合传统检测方法,对4℃下贮藏的鲜切菠菜在贮藏过程中存在的腐败菌进行研究。结果表明:在科的分类标准下,在贮存初期,假单胞菌科和肠杆菌科为优势菌科,丰度分别占比48. 99%、48. 62%,从菌群变化的角度来看,随着贮藏时间的推移,假单胞菌科的丰度从初始丰度的48. 99%到贮藏末期的70. 27%;而肠杆菌科则从初始丰度的48. 62%不断递减至21. 75%。此时,假单胞菌科为贮藏末期的优势菌科;在属的分类标准下,假单胞菌属、泛菌属为贮存初期的优势菌属,丰度分别占比47. 84%、30. 7%,欧文氏菌属、布丘氏菌为次优势菌,丰度为8. 09%和4. 64%;贮存中期,假单胞菌属为优势菌属,其丰度上升至58. 25%,而泛菌属丰度下降至13. 94%,欧文氏菌属丰度基本保持不变达8. 69%、布丘氏菌属丰度小幅上升至9. 73%;在贮存末期,假单胞菌属占绝对优势,丰度占比68. 97%,而泛菌属丰度仅占5. 4%,欧文氏菌属占比10. 7%,布丘氏菌属占比4%,假单胞菌属成为优势腐败菌属。高通量检测技术和传统检测方法相比,在鉴定样品DNA组成成分时可精确定量,提高了精度,节约了时间。
In order to analyze the types and quantities of spoilage bacteria in fresh-cut spinach stored at 4 ℃,high-throughput sequencing and traditional methods( physiological,biochemical,and morphological characterization)were used. The results showed that at early stage of storage,Pseudomonadaceae( 48. 99% abundance) and Enterobacteriaceae( 48. 62% abundance) were dominant species. Moreover,the abundance of Pseudomonadaceae rose to70. 27% at the end of storage while the abundance of Enterobacteriaceae decreased to 21. 75%,therefore,Pseudomonadaceae was dominant at the end of storage. Furthermore,Pseudomonas and Pantoea were dominant bacteria during early storage period,as their abundances reached 47. 84% and 30. 7%,respectively. Erwinia and Buttiauxella were the second dominant bacteria with abundances of 8. 09% and 4. 64%,respectively. At middle stage of storage,the abundances of Pseudomonas,Pantoea,Erwinia,and Buttiauxella reached 58. 25%,13. 94%,8. 69%,and9. 73%,respectively. At the end of storage,Pseudomonas was dominant and accounted for 68. 97%,while Pantoea,Erwinia,and Buttiauxella only accounted for 5. 4%,10. 7%,and 4%,respectively. Therefore,Pseudomonas was the main dominant spoilage bacteria. Additionally,compared with traditional methods,high-throughput detection technology can accurately quantify and identify DNA components,and also improve precisions and save time.
In order to analyze the types and quantities of spoilage bacteria in fresh-cut spinach stored at 4 ℃,high-throughput sequencing and traditional methods( physiological,biochemical,and morphological characterization)were used. The results showed that at early stage of storage,Pseudomonadaceae( 48. 99% abundance) and Enterobacteriaceae( 48. 62% abundance) were dominant species. Moreover,the abundance of Pseudomonadaceae rose to70. 27% at the end of storage while the abundance of Enterobacteriaceae decreased to 21. 75%,therefore,Pseudomonadaceae was dominant at the end of storage. Furthermore,Pseudomonas and Pantoea were dominant bacteria during early storage period,as their abundances reached 47. 84% and 30. 7%,respectively. Erwinia and Buttiauxella were the second dominant bacteria with abundances of 8. 09% and 4. 64%,respectively. At middle stage of storage,the abundances of Pseudomonas,Pantoea,Erwinia,and Buttiauxella reached 58. 25%,13. 94%,8. 69%,and9. 73%,respectively. At the end of storage,Pseudomonas was dominant and accounted for 68. 97%,while Pantoea,Erwinia,and Buttiauxella only accounted for 5. 4%,10. 7%,and 4%,respectively. Therefore,Pseudomonas was the main dominant spoilage bacteria. Additionally,compared with traditional methods,high-throughput detection technology can accurately quantify and identify DNA components,and also improve precisions and save time.
引文
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[14]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.
[15]储建军,傅玲琳,王翀,等.缢蛏冰温保活过程中菌相变化及优势腐败菌生长动力学建模[J].食品科学,2017,38(1):110-115.
[16]李晓然,李洁,刘晓峰,等.利用高通量测序分析云南豆豉中细菌群落多样性[J].食品与生物技术学报,2014,33(2):137-141.
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[18]关统伟,向慧平,王鹏昊,等.基于高通量测序的郫县豆瓣不同发酵期细菌群落结构及其动态演替[J].食品科学,2018,39(4):106-111.
[19]余丹,毛娉,宋颀,等.基于高通量测序的传统甜面酱自然发酵过程中的微生物群落结构及其动态演替[J].微生物学通报,2018,45(5):1 061-1 072.
[20] BOLIN H R,STAFFORD A E,KING A D,et al. Factors affecting the storage stability of shredded lettuce[J].Journal of Food Science,2010,42(5):1 319-1 321.
[21]杨秋月.芽苗菜优势腐败菌生长规律及控制技术研究[D].天津:天津科技大学,2013.
[22]张一敏,朱立贤,张万刚,等.生鲜牛肉中的腐败微生物概述[J].食品科学,2018,39(13):289-296..
[23] WELLS J M,LIAO C H. The Diversity of the Pectolytic Fluorescent Pseudomonads[M]. Plant Pathogenic Bacteria. Springer Netherlands,1987:273-276.
[24] PINTOA L,IPPOLITOB A,BARUZZIA F. Control of spoiler Pseudomonas spp. on fresh cut vegetables by neutral electrolyzed water[J]. Food Microbiology,2015,50:102-108.
[25] LIAO C H. Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida[J]. The Journal of Bacteriology,1991,173(14):4 386-4 393.
[26] ZUCKER M,HANKIN L,SANDS D. Factors governing pectate lyase synthesis in soft rot and non-soft rot bacteria[J]. Physiological Plant Pathology,1972,2(1):59-67.
[27] LOCASCIO S J,BARTZ J A,WEINGARTNER D P.Calcium and potassium fertilization of potatoes grown in north Florida. 1. Effects on potato yield and tissue caconcentration and K-concentrations[J]. American Potato Journal,1992,69(2):95-104.
[28] BARRAS F,HARRIS S. Extracellular enzymes and their role in Erwinia virulence[J]. Methods in Microbiology,1998,27:157-168.
[2] GUTIERREZ J,BOURKE P,LONCHAMP J,et al. Impact of plant essential oils on microbiological,organoleptic and quality markers of minimally processed vegetables[J].Innovative Food Science&Emerging Technologies,2009,10(2):195-202.
[3] POIMENIDOU S V,BIKOULI V C,GARDELI C,et al.Effect of single or combined chemical and natural antimicrobial interventions on Escherichia coli O157∶H7,total microbiota and color of packaged spinach and lettuce[J].International Journal of Food Microbiology, 2016, 220(2):6-18.
[4]徐晓霞,陈安均,桑伟娜,等.不同温度贮藏鲜切生菜腐败细菌的分离及鉴定[J].食品与发酵工业,2016,42(1):53-58.
[5] UFUK M,KASl M REZZAN. Yellowing of fresh-cut spinach(Spinacia oleracea,L.)leaves delayed by UV-B applications[J]. Information Processing in Agriculture,2017,4(3):214-219.
[6]宋晓雪,胡文忠,毕阳,等.鲜切果蔬中致腐微生物污染及其非热杀菌的研究进展[J].食品工业科技,2014,35(10):351-354.
[7]雷振河.采用高通量测序技术分析清香型白酒酿造微生物[J].食品与发酵工业,2015,41(9):164-167.
[8] TASSIOS P T,MORAN-GILAD J. Bacterial next generation sequencing(NGS)made easy[J]. Clinical Microbiology and Infection,2018,24(4):332-334.
[9]陈泽斌,李冰,徐胜光,等.高通量测序分析食用玫瑰花瓣内生细菌多样性[J].中国食品学报,2017,17(9):227-235.
[10] PAILLARTM J M,et al. Bacterial population dynamics and sensorial quality loss in modified atmosphere packed fresh-cut iceberg lettuce[J]. Postharvest Biology and Technology,2017,124:91-99.
[11]崔慧玲,陈安均,罗婵,等.鲜切生菜贮藏过程中优势腐败细菌的多样性分析[J].食品与发酵工业,2014,40(7):223-229.
[12]中华人民共和国卫生部,中国国家标准化管理委员会. GB 4789. 2—2016,食品微生物学检验,菌落总数测定[S].北京:中国标准出版社,2016.
[13] 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.
[14]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.
[15]储建军,傅玲琳,王翀,等.缢蛏冰温保活过程中菌相变化及优势腐败菌生长动力学建模[J].食品科学,2017,38(1):110-115.
[16]李晓然,李洁,刘晓峰,等.利用高通量测序分析云南豆豉中细菌群落多样性[J].食品与生物技术学报,2014,33(2):137-141.
[17]雷昊,谢晶,乔永祥,等.臭氧水清洗结合气调包装对鲜切杭白菜保鲜效果的研究[J].食品与机械,2017,33(6):110-113.
[18]关统伟,向慧平,王鹏昊,等.基于高通量测序的郫县豆瓣不同发酵期细菌群落结构及其动态演替[J].食品科学,2018,39(4):106-111.
[19]余丹,毛娉,宋颀,等.基于高通量测序的传统甜面酱自然发酵过程中的微生物群落结构及其动态演替[J].微生物学通报,2018,45(5):1 061-1 072.
[20] BOLIN H R,STAFFORD A E,KING A D,et al. Factors affecting the storage stability of shredded lettuce[J].Journal of Food Science,2010,42(5):1 319-1 321.
[21]杨秋月.芽苗菜优势腐败菌生长规律及控制技术研究[D].天津:天津科技大学,2013.
[22]张一敏,朱立贤,张万刚,等.生鲜牛肉中的腐败微生物概述[J].食品科学,2018,39(13):289-296..
[23] WELLS J M,LIAO C H. The Diversity of the Pectolytic Fluorescent Pseudomonads[M]. Plant Pathogenic Bacteria. Springer Netherlands,1987:273-276.
[24] PINTOA L,IPPOLITOB A,BARUZZIA F. Control of spoiler Pseudomonas spp. on fresh cut vegetables by neutral electrolyzed water[J]. Food Microbiology,2015,50:102-108.
[25] LIAO C H. Cloning of pectate lyase gene pel from Pseudomonas fluorescens and detection of sequences homologous to pel in Pseudomonas viridiflava and Pseudomonas putida[J]. The Journal of Bacteriology,1991,173(14):4 386-4 393.
[26] ZUCKER M,HANKIN L,SANDS D. Factors governing pectate lyase synthesis in soft rot and non-soft rot bacteria[J]. Physiological Plant Pathology,1972,2(1):59-67.
[27] LOCASCIO S J,BARTZ J A,WEINGARTNER D P.Calcium and potassium fertilization of potatoes grown in north Florida. 1. Effects on potato yield and tissue caconcentration and K-concentrations[J]. American Potato Journal,1992,69(2):95-104.
[28] BARRAS F,HARRIS S. Extracellular enzymes and their role in Erwinia virulence[J]. Methods in Microbiology,1998,27:157-168.