基于Illumina MiSeq技术比较二种多脂鱼在腌干过程中的菌相变化
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摘要
通过比较研究多脂红肉鱼(蓝圆鲹)和白肉鱼(带鱼)腌干加工中菌相的变化规律,以探讨加工过程对菌相的影响并寻找具有抗氧化作用的优势菌。在腌干加工过程中采用Illumina平台的MiSeq技术比较分析了两种多脂鱼的菌相变化情况。结果显示,两种鱼的菌相主要分布在拟杆菌门、变形菌门;在科水平上,初始原料的蓝圆鲹和带鱼分别含7个和15个科的细菌,带鱼包括了蓝圆鲹的所有菌群,肠杆菌科作为共同的优势菌,在蓝圆鲹和带鱼中分别占47%和26%。从腌制开始,两种鱼的菌群数都大量减少,弧菌和芽孢杆菌科作为共同优势菌,前者平均占蓝圆鲹和带鱼的40.3%和42.2%,后者则平均占16.7%和13.3%。原料中,蓝圆鲹和带鱼都包含了肠杆菌科、假单胞菌、弧菌科和希瓦氏菌科这4种腐败菌,加工阶段,两种鱼的优势腐败菌都为弧菌科。乳酸菌包括链球菌科和乳杆菌科,仅出现在带鱼中。研究表明,在腌干加工中,带鱼的细菌减少程度大于蓝圆鲹,总体上均呈现下降趋势,两种鱼含共同的菌群和优势菌,却表现出明显的差异。腌干后两种鱼的腐败菌大大减少,说明腌干加工有利于降低鱼类腐败的可能性。可选择带鱼作乳酸菌的分离以进行后续的抗氧化研究。
By comparing the changes of the microflora during salted-dried processing of the red-fleshed fish(Decapterus maruadsi) and the white-fleshed fish(Trichiurus lepturus), the dominant bacterium with antioxidant activity was also studied. In this paper, MiSeq sequencing technology was utilized in two kinds of fish in different processing phases. The results showed that the microflora of the two fishes was mainly distributed in Bacteroidetes and Proteobacteria. At the family level of the initial material, there were 7 and 15 colonies in D. maruadsi and T.lepturus, respectively. All the species in round scad were totally in T. lepturus and Enterbacteriaceae was as common dominant bacteria, accounted for 47% and 26% in D. maruadsi and T. lepturus respectively. From the start of salting, the number of bacteria in both fishes was greatly reduced. Vibrionaceae and Bacillaceae were the common dominant bacteria, the former accounted for an average of 40.3% and 42.2% of D. maruadsi and T.lepturus, and the latter accounted for an average of 16.7% and 13.3%. Among the raw materials, four spoilage bacteria of Enterobacteriaceae, Pseudomonas, Vibrionaceae, and Shewanellaceae were showed in both fishes. The dominant spoilage bacteria in both fishes during salt-dried processing was Vibrionaceae. Lactic acid bacteria(LAB), including Streptococcaceae and Lactobacillaceae, only appeared in T. lepturus. Therefore, during salt-dried processing, the degree of reduction of bacteria in hairtail was greater than that of round scad, and they all showed a decrease. The two fishes contained common bacteria and dominant bacteria, but showed significant differences.The spoilage bacteria of both fishes have been greatly reduced, suggesting that salt-dried processing is beneficial to reducing the possibility of fish corruption. T. lepturus could be selected for LAB isolation for subsequent antioxidant studies.
By comparing the changes of the microflora during salted-dried processing of the red-fleshed fish(Decapterus maruadsi) and the white-fleshed fish(Trichiurus lepturus), the dominant bacterium with antioxidant activity was also studied. In this paper, MiSeq sequencing technology was utilized in two kinds of fish in different processing phases. The results showed that the microflora of the two fishes was mainly distributed in Bacteroidetes and Proteobacteria. At the family level of the initial material, there were 7 and 15 colonies in D. maruadsi and T.lepturus, respectively. All the species in round scad were totally in T. lepturus and Enterbacteriaceae was as common dominant bacteria, accounted for 47% and 26% in D. maruadsi and T. lepturus respectively. From the start of salting, the number of bacteria in both fishes was greatly reduced. Vibrionaceae and Bacillaceae were the common dominant bacteria, the former accounted for an average of 40.3% and 42.2% of D. maruadsi and T.lepturus, and the latter accounted for an average of 16.7% and 13.3%. Among the raw materials, four spoilage bacteria of Enterobacteriaceae, Pseudomonas, Vibrionaceae, and Shewanellaceae were showed in both fishes. The dominant spoilage bacteria in both fishes during salt-dried processing was Vibrionaceae. Lactic acid bacteria(LAB), including Streptococcaceae and Lactobacillaceae, only appeared in T. lepturus. Therefore, during salt-dried processing, the degree of reduction of bacteria in hairtail was greater than that of round scad, and they all showed a decrease. The two fishes contained common bacteria and dominant bacteria, but showed significant differences.The spoilage bacteria of both fishes have been greatly reduced, suggesting that salt-dried processing is beneficial to reducing the possibility of fish corruption. T. lepturus could be selected for LAB isolation for subsequent antioxidant studies.
引文
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[16]蔡秋杏,吴燕燕,李来好,等.厦门白姑鱼腌制加工过程中的脂肪酸变化分析[J].食品科学,2015,36(12):76-81.Cai Q X,Wu Y Y,Li L H,et al.Changes in fatty acids during salting and drying of white croaker(Argyrosomus amoyensis)[J].Food Science,2015,36(12):76-81(in Chinese).
[17]陈胜军,杨贤庆,樊丽琴,等.蓝圆鲹在不同腌制条件下三甲胺和二甲胺含量变化规律[J].食品科学,2012,33(13):58-61.Chen S J,Yang X Q,Fan L Q,et al.Change regularity of trimethylamine and dimethylamine contents in Decapterus maruadsi during salting under varying conditions[J].Food Science,2012,33(13):58-61(in Chinese).
[18]陈泽斌,李冰,王定康,等.Illumina MiSeq高通量测序分析核桃内生细菌多样性[J].江苏农业学报,2015,31(5):1129-1133.Chen Z B,Li B,Wang D K,et al.Diversity of endophytic bacteria in walnut analyzed by Illumina MiSeq high-throughput sequencing technology[J].Jiangsu Journal of Agricultural Sciences,2015,31(5):1129-1133(in Chinese).
[19]陈泽斌,李冰,王定康,等.应用Illumina MiSeq高通量测序技术分析玉米内生细菌多样性[J].现代食品科技,2016,32(2):113-120.Chen Z B,Li B,Wang D K,et al.Study on the diversity of endophytic bacteria in maize using Illumina MiSeq high-throughput sequencing system[J].Modern Food Science and Technology,2016,32(2):113-120(in Chinese).
[20]Mago?T,Salzberg S L.FLASH:fast length adjustment of short reads to improve genome assemblies[J].Bioinformatics,2011,27(21):2957-2963.
[21]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.
[22]Bokulich N A,Subramanian S,Faith J J,et al.Qualityfiltering vastly improves diversity estimates from Illumina amplicon sequencing[J].Nature Methods,2013,10(1):57-59.
[23]DeSantis T Z,Hugenholtz P,Larsen N,et al.Greengenes,a chimera-checked 16S rRNA gene database and workbench compatible with ARB[J].Applied and Environmental Microbiology,2006,72(7):5069-5072.
[24]曾燕,简平,倪学勤,等.Illumina MiSeq测序平台测定蒙古羊瘤胃液相和固相菌群多样性[J].动物营养学报,2015,27(10):3256-3262.Zeng Y,Jian P,Ni X Q,et al.Bacteria community diversity of liquid and solid phases of ruminal contents of Mongolian sheep analyzed by Illumina MiSeq platform[J].Chinese Journal of Animal Nutrition,2015,27(10):3256-3262(in Chinese).
[25]Wang Q,Garrity G M,Tiedje J M,et al.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J].Applied and Environmental Microbiology,2007,73(16):5261-5267.
[26]Sogin M L,Morrison H G,Huber J A,et al.Microbial diversity in the deep sea and the underexplored"rare biosphere"[J].Proceedings of the National Academy of Science of the United States of America,2006,103(32):12115-12120.
[27]Hayat K,Zhang X M,Farooq U,et al.Effect of microwave treatment on phenolic content and antioxidant activity of citrus mandarin pomace[J].Food Chemistry,2010,123(2):423-429.
[28]杨培周,钱静,姜绍通,等.臭鳜鱼的质构特性、特征气味及发酵微生物的分离鉴定[J].现代食品科技,2014,30(4):55-62.Yang P Z,Qian J,Jiang S T,et al.Texture properties,characteristic smell,microbial isolation and identification of smelly mandarin fish[J].Modern Food Science and Technology,2014,30(4):55-62(in Chinese).
[29]刘书臣,廖明涛,赵巧灵,等.不同贮藏温度下大目金枪鱼鲜度及组胺变化[J].食品与发酵工业,2013,39(5):213-218.Liu S C,Liao M T,Zhao Q L,et al.Study on the freshness and histamine variation of big eye tuna during storage temperatures[J].Food and Fermentation Industries,2013,39(5):213-218(in Chinese).
[30]蒋倩倩,戴志远,赵巧灵,等.冰藏鲐鱼中组胺的产生及腐败微生物的变化[J].中国食品学报,2013,13(8):219-224.Jiang Q Q,Dai Z Y,Zhao Q L,et al.Histamine production and spoilage microorganisms variation of the ice-stored mackerel[J].Journal of Chinese Institute of Food Science and Technology,2013,13(8):219-224(in Chinese).
[2]Sampaio G R,Saldanha T,Soares R A M,et al.Effect of natural antioxidant combinations on lipid oxidation in cooked chicken meat during refrigerated storage[J].Food Chemistry,2012,135(3):1383-1390.
[3]Sanches-silva A,Costa D,Albuquerque T G,et al.Trends in the use of natural antioxidants in active food packaging:a review[J].Food Additives&ContaminantsPart A:Chemistry Analysis Control Exposure&Risk Assessment,2014,31(3):374-395.
[4]曾雪峰.淡水鱼发酵对酸鱼品质影响的研究[D].无锡:江南大学,2013:13-21.Zeng X F.Study on the effect of Suan Yu property of fermented freshwater fish[D].Wuxi:Jiangnan University,2013:13-21(in Chinese).
[5]Matamoros S,Pilet M F,Gigout F,et al.Selection and evaluation of seafood-borne psychrotrophic lactic acid bacteria as inhibitors of pathogenic and spoilage bacteria[J].Food Microbiology,2009,26(6):638-644.
[6]Kuda T,Kawahara M,Nemoto M,et al.In vitro antioxidant and anti-inflammation properties of lactic acid bacteria isolated from fish intestines and fermented fish from the Sanriku Satoumi region in Japan[J].Food Research International,2014,64:248-255.
[7]王悦齐,李来好,蔡秋杏,等.分离自腌干鱼的抗氧化发酵菌株的筛选及鉴定[J].南方水产科学,2016,12(3):74-83.Wang Y Q,Li L H,Cai Q X,et al.Screening and identification of antioxidant starter culture strains from salted dried fish[J].South China Fisheries Science,2016,12(3):74-83(in Chinese).
[8]游刚,吴燕燕,李来好,等.添加复合乳酸菌再发酵对腌干鱼肉微生物、亚硝酸盐和亚硝胺的影响[J].南方水产科学,2015,11(4):109-115.You G,Wu Y Y,Li L H,et al.Effect of inoculating compound lactic acid bacteria on microbial,nitrites and nitrosamines of salted fish[J].South China Fisheries Science,2015,11(4):109-115(in Chinese).
[9]农业农村部渔业渔政管理局.2018中国渔业统计年鉴[M].北京:中国农业出版社,2018.Ministry of Agriculture and Rural Affairs of the People’Republic of China.2018 China fishery statistical yearbook[M].Beijing:China agriculture press,2018(in Chinese).
[10]李燕,吴佳佳,张井,等.PCR-DGGE技术分析传统臭鳜鱼发酵过程中细菌群落结构[J].食品科学,2017,38(18):29-34.Li Y,Wu J J,Zhang J,et al.PCR-DGGE analysis of bacterial community structure in stinky mandarin fish(Siniperca chuatsi)[J].Food Science,2017,38(18):29-34(in Chinese).
[11]Kouakou A C,N'Guessan K F,Durand N,et al.Molecular bacterial characterization and free amino acids composition in Ivorian traditional fermented fish produced by two methods[J].Fisheries Science,2012,78(5):1125-1136.
[12]Marui J,Boulom S,Panthavee W,et al.Cultureindependent analysis of the bacterial community during fermentation of pa-som,a traditional fermented fish product in Laos[J].Fisheries Science,2014,80(5):1109-1115.
[13]张家松,段亚飞,张真真,等.对虾肠道微生物菌群的研究进展[J].南方水产科学,2015,11(6):114-119.Zhang J S,Duan Y F,Zhang Z Z,et al.Research progress of intestinal microbial flora in shrimp[J].South China Fisheries Science,2015,11(6):114-119(in Chinese).
[14]Handelsman J,Rondon M R,Brady S F,et al.Molecular biological access to the chemistry of unknown soil microbes:a new frontier for natural products[J].Chemistry&Biology,1998,5(10):R245-R249.
[15]谢萍,徐明生,尹忠平,等.MiSeq测序研究散装酱卤鸭肉贮藏期间微生物群落多样性[J].现代食品科技,2015,31(11):120-126.Xie P,Xu M S,Yun Z P,et al.MiSeq sequencing to study the diversity of Microflora in Bulk-marinated duck meat during storage[J].Modern Food Science and Technology,2015,31(11):120-126(in Chinese).
[16]蔡秋杏,吴燕燕,李来好,等.厦门白姑鱼腌制加工过程中的脂肪酸变化分析[J].食品科学,2015,36(12):76-81.Cai Q X,Wu Y Y,Li L H,et al.Changes in fatty acids during salting and drying of white croaker(Argyrosomus amoyensis)[J].Food Science,2015,36(12):76-81(in Chinese).
[17]陈胜军,杨贤庆,樊丽琴,等.蓝圆鲹在不同腌制条件下三甲胺和二甲胺含量变化规律[J].食品科学,2012,33(13):58-61.Chen S J,Yang X Q,Fan L Q,et al.Change regularity of trimethylamine and dimethylamine contents in Decapterus maruadsi during salting under varying conditions[J].Food Science,2012,33(13):58-61(in Chinese).
[18]陈泽斌,李冰,王定康,等.Illumina MiSeq高通量测序分析核桃内生细菌多样性[J].江苏农业学报,2015,31(5):1129-1133.Chen Z B,Li B,Wang D K,et al.Diversity of endophytic bacteria in walnut analyzed by Illumina MiSeq high-throughput sequencing technology[J].Jiangsu Journal of Agricultural Sciences,2015,31(5):1129-1133(in Chinese).
[19]陈泽斌,李冰,王定康,等.应用Illumina MiSeq高通量测序技术分析玉米内生细菌多样性[J].现代食品科技,2016,32(2):113-120.Chen Z B,Li B,Wang D K,et al.Study on the diversity of endophytic bacteria in maize using Illumina MiSeq high-throughput sequencing system[J].Modern Food Science and Technology,2016,32(2):113-120(in Chinese).
[20]Mago?T,Salzberg S L.FLASH:fast length adjustment of short reads to improve genome assemblies[J].Bioinformatics,2011,27(21):2957-2963.
[21]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.
[22]Bokulich N A,Subramanian S,Faith J J,et al.Qualityfiltering vastly improves diversity estimates from Illumina amplicon sequencing[J].Nature Methods,2013,10(1):57-59.
[23]DeSantis T Z,Hugenholtz P,Larsen N,et al.Greengenes,a chimera-checked 16S rRNA gene database and workbench compatible with ARB[J].Applied and Environmental Microbiology,2006,72(7):5069-5072.
[24]曾燕,简平,倪学勤,等.Illumina MiSeq测序平台测定蒙古羊瘤胃液相和固相菌群多样性[J].动物营养学报,2015,27(10):3256-3262.Zeng Y,Jian P,Ni X Q,et al.Bacteria community diversity of liquid and solid phases of ruminal contents of Mongolian sheep analyzed by Illumina MiSeq platform[J].Chinese Journal of Animal Nutrition,2015,27(10):3256-3262(in Chinese).
[25]Wang Q,Garrity G M,Tiedje J M,et al.Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J].Applied and Environmental Microbiology,2007,73(16):5261-5267.
[26]Sogin M L,Morrison H G,Huber J A,et al.Microbial diversity in the deep sea and the underexplored"rare biosphere"[J].Proceedings of the National Academy of Science of the United States of America,2006,103(32):12115-12120.
[27]Hayat K,Zhang X M,Farooq U,et al.Effect of microwave treatment on phenolic content and antioxidant activity of citrus mandarin pomace[J].Food Chemistry,2010,123(2):423-429.
[28]杨培周,钱静,姜绍通,等.臭鳜鱼的质构特性、特征气味及发酵微生物的分离鉴定[J].现代食品科技,2014,30(4):55-62.Yang P Z,Qian J,Jiang S T,et al.Texture properties,characteristic smell,microbial isolation and identification of smelly mandarin fish[J].Modern Food Science and Technology,2014,30(4):55-62(in Chinese).
[29]刘书臣,廖明涛,赵巧灵,等.不同贮藏温度下大目金枪鱼鲜度及组胺变化[J].食品与发酵工业,2013,39(5):213-218.Liu S C,Liao M T,Zhao Q L,et al.Study on the freshness and histamine variation of big eye tuna during storage temperatures[J].Food and Fermentation Industries,2013,39(5):213-218(in Chinese).
[30]蒋倩倩,戴志远,赵巧灵,等.冰藏鲐鱼中组胺的产生及腐败微生物的变化[J].中国食品学报,2013,13(8):219-224.Jiang Q Q,Dai Z Y,Zhao Q L,et al.Histamine production and spoilage microorganisms variation of the ice-stored mackerel[J].Journal of Chinese Institute of Food Science and Technology,2013,13(8):219-224(in Chinese).