冷藏鲣鱼优势腐败产胺菌分离鉴定及其致腐产胺能力分析

刘洋帆; 王迪; 李春生; 相欢; 陈胜军; 杨贤庆; 李来好; 邓建朝

doi:10.13386/j.issn1002-0306.2022040070

冷藏鲣鱼优势腐败产胺菌分离鉴定及其致腐产胺能力分析

doi: 10.13386/j.issn1002-0306.2022040070

刘洋帆^{1, 2,},
王迪^{1, 3},
李春生^{1, 3},
相欢^{1, 3},
陈胜军^{1, 3},
杨贤庆^{1, 3},
李来好^{1, 3},
邓建朝^{1, 3, ,}

1.
中国水产科学研究院南海水产研究所，农业农村部水产品加工重点实验室，国家水产品加工技术研发中心，广东广州 510300
2.
上海海洋大学食品学院，上海 201306
3.
大连工业大学海洋食品精深加工关键技术省部共建协同创新中心，辽宁大连 116034

基金项目: 广东省重点领域研发计划（2021B0202060001, 2019B020225001）；财政部和农业农村部财政专项（NFZX2021）；国家现代农业产业技术体系（CARS－47）；广东省现代农业产业技术体系创新团队建设专项（2022KJ151）；中国水产科学研究院基本科研业务费资助（2020TD73）。

详细信息

作者简介:
刘洋帆（1998−），女，硕士研究生，研究方向：水产品加工与质量安全，E-mail：18742026042@163.com

通讯作者:
邓建朝（1977−），男，博士，副研究员，研究方向：水产品加工与质量安全，E-mail：djc9801@foxmail.com

中图分类号: TS201.3
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出版历程
- 收稿日期: 2022-04-08
- 网络出版日期: 2022-10-21
- 刊出日期: 2022-11-23

Isolation and Identification of Dominant Spoilage Biogenic Amine Producing Bacteria from Refrigerated Skipjack Tuna and Analysis of Their Production Ability

LIU Yangfan^{1, 2
,},
WANG Di^{1, 3},
LI Chunsheng^{1, 3},
XIANG Huan^{1, 3},
CHEN Shengjun^{1, 3},
YANG Xianqing^{1, 3},
LI Laihao^{1, 3},
DENG Jianchao^{1, 3
, ,}

1.
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, National Research and Development Center for Aquatic Product Processing, Guangzhou 510300, China
2.
College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
3.
Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Dalian 116034, China

摘要

摘要: 为探究鲣鱼中优势腐败产胺菌的种类，从冷藏的鲣鱼中筛选出3种优势腐败产胺菌，通过16S rDNA分子鉴定技术对菌株进行鉴定为荧光假单胞菌（Pseudomonas fluorescens）、弗氏柠檬酸杆菌（Citrobacter freundii）和嗜水气单胞菌（Aeromonas hydrophila），将鉴定出的优势腐败产胺菌接种至无菌鱼肉中4 ℃条件下贮藏，通过测定菌落总数和挥发性盐基氮（TVB-N）变化，以腐败代谢产物产量因子（Y_TVB-N/CFU）分析3种优势腐败产胺菌对鲣鱼的致腐能力，并通过样品中生物胺的含量比较3种优势腐败产胺菌的产胺能力。结果表明：在贮藏8 d后接种荧光假单胞菌、弗氏柠檬酸杆菌和嗜水气单胞菌组的菌落总数分别达到8.36、8.27和8.13 lg CFU/g，TVB-N值分别为28.21、30.30和31.29 mg/100 g，3种优势腐败产胺菌对鲣鱼的致腐能力大小为嗜水气单胞菌>弗氏柠檬酸杆菌>荧光假单胞菌。接种荧光假单胞菌组、弗式柠檬酸杆菌组和嗜水气单胞菌组的组胺含量分别为196.23、83.43和261.22 mg/kg，3组样品中总生物胺含量为嗜水气单胞菌>荧光假单胞菌>弗氏柠檬酸杆菌。综合比较得出，嗜水气单胞菌对4 ℃冷藏鲣鱼致腐产胺能力最强，本研究增加对冷藏鲣鱼中优势腐败产胺菌种类的了解并提供了部分理论基础。
- 鲣鱼 /
- 优势腐败菌 /
- 产胺菌 /
- 致腐能力 /
- 产胺能力
Abstract: In order to explore the types of dominant spoilage biogenic amine-producing bacteria in skipjack tuna, three dominant bacteria were screened from refrigerated skipjack tuna. The strains were identified by 16S rDNA molecular identification technology, which were Pseudomonas fluorescens, Citrobacter freundii and Aeromonas hydrophila. The dominant spoilage biogenic amine-producing bacteria were inoculated onto sterile fish and stored at 4 ℃. By measuring the total number of colonies and the value of volatile base nitrogen (TVB-N), we used the yield factor (Y_TVB-N/CFU) of spoilage metabolites to analyze the spoilage ability of the three dominant spoilage biogenic amine-producing bacteria. The production ability of bacteria was compared by detecting the content of biogenic amines in the samples. The results showed that the total number of colonies in the groups inoculated with P. fluorescens, C. freundii and A. hydrophila were 8.36, 8.27 and 8.13 lg CFU/g at the end of storage, respectively. The TVB-N values reached 28.21, 30.30 and 31.29 mg/100 g, respectively. The spoilage ability of the three dominant spoilage amine-producing bacteria were A. hydrophila>C. freundii>P. fluorescence. On the 8th day of storage, the histamine contents of the P. fluorescens, C. freundii and A. hydrophila groups reached 196.23, 83.43 and 261.22 mg/kg, respectively. The total biogenic amine contents of the three groups of samples at the end of storage were A. hydrophila>P. fluorescens>C. freundii. The combined comparison concluded that A. hydrophila had the highest spoilage biogenic amine-producing ability in skipjack tuna. This study increased the knowledge and provided a partial theoretical basis for the dominant spoilage biogenic amine producing species in refrigerated skipjack tuna.
- skipjack tuna /
- dominant spoilage bacteria /
- biogenic amine-producing bacteria /
- spoilage ability /
- biogenic amine-producing ability

HTML全文

图 1 8种菌株的16S rDNA电泳图谱

Figure 1. 16S rDNA electrophoresis map of 8 strains

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图 2 3种菌株16S rDNA序列的系统发育树

Figure 2. Phylogenetic tree of 16S rDNA sequences from 3 strains

注：A为T4的系统发育树；B为T6的系统发育树；C为T7的系统发育树。

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图 3 鲣鱼接种3种优势腐败产胺菌的菌落总数变化

Figure 3. Changes in colony counts of 3 dominant spoilage biogenic amine-producing bacteria inoculated to skipjack tuna

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图 4 鲣鱼接种3种优势腐败产胺菌的TVB-N值变化

Figure 4. Changes in TVB-N values of 3 dominant spoilage biogenic amine-producing bacteria inoculated to skipjack tuna

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图 5 贮藏第8天4组样品的总生物胺含量

Figure 5. Total biogenic amine content of the four groups of samples on the 8th day storage

注：不同小写字母表示组别之间存在显著性差异（P<0.05）。

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表 1 鲣鱼接种3种优势腐败产胺菌致腐因子的比较

Table 1. Comparison of yield factors in skipjack tuna inoculated with 3 dominant spoilage biogenic amine-producing bacteria

菌名	菌落总数（CFU/g）		腐败代谢产物含量（mg N/100 g）		腐败代谢产物产量因子（mg/CFU）
菌名	N₀	N_i	（TVB-N）₀	（TVB-N）_i	Y_TVB-N/CFU
荧光假单胞菌	5.57×10⁵	2.60×10⁸	6.71	28.21	8.39×10⁻⁸
弗氏柠檬酸杆菌	3.87×10⁵	2.00×10⁸	6.22	30.30	1.20×10⁻⁷
嗜水气单胞菌	5.63×10⁵	1.7×10⁸	6.67	31.29	1.48×10⁻⁷

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表 2 鲣鱼接种3种优势腐败产胺菌的生物胺含量

Table 2. Biogenic amines content of 3 dominant spoilage biogenic amine-producing bacteria inoculated to skipjack tuna

菌株	时间（d）	生物胺含量（mg/kg）
菌株	时间（d）	色胺	酪胺	苯乙胺	组胺	尸胺	腐胺
空白对照	0	ND	ND	0.28±0.02^d	0.67±0.01^a	1.95±0.15^a	ND
	1	ND	ND	0.23±0.00^c	0.88±0.00^a	2.44±0.16^b	ND
	2	ND	ND	0.24±0.00^c	0.86±0.00^a	3.16±0.17^c	ND
	3	ND	ND	0.19±0.00^c	1.57±0.01^b	5.11±0.27^f	ND
	4	ND	ND	0.17±0.01^a	1.79±0.06^bc	5.49±0.04^f	ND
	5	ND	ND	0.18±0.02^ab	1.99±0.23^c	4.65±0.02^e	ND
	6	ND	ND	0.17±0.00^a	2.13±0.26^d	4.43±0.29^e	ND
	7	ND	0.43±0.34^a	0.23±0.00^c	3.35±0.10^e	3.52±0.41^cd	ND
	8	ND	0.74±0.02^b	0.49±0.01^e	5.40±0.13^f	3.74±0.20^d	ND
荧光假单胞菌	0	ND	ND	0.28±0.04^c	7.53±0.42^a	2.65±0.27^a	ND
	1	ND	ND	0.25±0.03^c	9.31±1.16^a	4.46±0.40^a	0.21±0.09^a
	2	ND	ND	0.18±0.05^ab	5.68±1.06^a	4.77±0.65^a	5.73±0.95^b
	3	ND	ND	0.23±0.03^bc	17.95±0.96^ab	5.27±0.24^a	10.29±1.12^c
	4	ND	ND	0.16±0.02^a	27.50±1.59^b	23.50±2.42^b	16.95±1.38^d
	5	ND	ND	0.16±0.04^a	45.58±3.82^c	33.72±2.60^c	26.46±3.28^e
	6	ND	0.48±0.18^a	0.17±0.01^a	76.99±0.57^d	45.43±4.36^d	33.10±0.32^f
	7	ND	0.75±0.06^c	0.26±0.02^c	138.83±4.88^e	54.34±0.59^e	45.89±1.36^g
	8	ND	0.69±0.16^b	0.27±0.02^c	196.23±7.21^f	65.35±1.21^f	57.22±1.71^h
弗氏柠檬酸杆菌	0	ND	ND	0.24±0.01^bcd	8.26±0.76^a	3.11±0.22^a	ND
	1	ND	ND	0.24±0.03^bcd	9.22±0.59^a	5.60±0.33^a	0.30±0.22^a
	2	ND	ND	0.15±0.02^ab	5.94±0.70^a	11.02±0.84^b	3.34±0.99^ab
	3	ND	ND	0.27±0.12^d	14.08±0.62^a	15.74±3.12^c	8.67±0.86^b
	4	ND	ND	0.16±0.02^abc	18.27±4.86^ab	20.60±2.68^d	18.45±1.27^c
	5	ND	ND	0.14±0.02^a	28.71±1.63^bc	28.69±3.41^e	26.26±6.18^d
	6	ND	0.49±0.03^b	0.16±0.02^abc	38.10±6.54^c	39.71±1.53^f	38.33±3.94^e
	7	ND	0.28±0.47^a	0.24±0.01^bcd	50.67±2.66^d	43.93±3.52^g	45.45±0.93^f
	8	ND	0.61±0.28^c	0.25±0.03^cd	83.43±7.92^e	55.49±4.24^h	55.39±2.65^g
嗜水气单胞菌	0	ND	ND	0.24±0.03^ab	8.98±0.58^a	3.80±0.37^a	ND
	1	ND	ND	0.22±0.02^ab	14.50±1.62^a	5.60±0.22^a	0.30±0.09^a
	2	ND	ND	0.14±0.04^a	36.74±3.59^b	13.50±2.68^b	4.67±0.33^b
	3	ND	ND	0.20±0.01^ab	53.12±1.67^c	19.58±3.09^c	8.34±1.07^c
	4	ND	ND	0.26±0.05^bc	71.69±6.00^d	28.58±0.37^d	13.40±2.81^d
	5	ND	ND	0.34±0.04^c	107.84±2.96^e	35.21±2.22^e	22.63±2.63^e
	6	ND	ND	0.63±0.12^d	149.78±4.97^f	45.48±1.96^f	32.13±1.08^f
	7	ND	ND	0.78±0.07^e	204.29±7.48^g	58.76±2.00^g	45.84±3.69^g
	8	ND	0.25±0.04	0.86±0.04^e	261.22±11.42^h	69.99±2.83^h	54.81±3.01^h
注：ND表示未检出，同列不同小写字母表示同一组别不同贮藏时间之间存在显著性差异（P<0.05）。

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参考文献(30)

[1]	童铃, 金毅, 徐坤华, 等. 3种鲣鱼背部肌肉的营养成分分析及评价[J]. 南方水产科学,2014,10(5):51−59. [TONG L, JIN Y, XU K H, et al. Analysis of nutritional components in back muscle of skipjacks[J]. South China Fisheries Science,2014,10(5):51−59. doi: 10.3969/j.issn.2095-0780.2014.05.008
[2]	李海波, 杨雪, 白冬, 等. 鲣鱼 (Katsuwonus pelamis) 肌肉蛋白在热处理过程中的营养变化及功能性评价[J]. 海洋与湖沼,2017,48(1):155−160. [LI H B, YANG X, BAI D, et al. The nutrinent content and evaluation of skipjack's back muscle before and after poached[J]. Oceanologia Et Limnologia Sinica,2017,48(1):155−160.
[3]	刘爱芳, 谢晶, 钱韻芳. 冷藏金枪鱼优势腐败菌致腐败能力[J]. 食品科学,2018,39(3):7−14. [LIU A F, XIE J, QIAN Y F. Spoilage potential of dominant spoilage bacteria from chilled tuna (Thunnus obesus)[J]. Food Science,2018,39(3):7−14. doi: 10.7506/spkx1002-6630-201803002
[4]	励建荣, 杨兵, 李婷婷. 水产品优势腐败菌及其群体感应系统研究进展[J]. 食品科学,2015,36(19):5. [LI J R, YANG B, LI T T. Advances in quorum sensing of dominant spoilage bacteria from aquatic products[J]. Food Science,2015,36(19):5.
[5]	冯豪杰, 蓝蔚青, 臧一宇, 等. 优势腐败菌对暗纹东方鲀冷藏期间品质变化影响及致腐能力分析[J]. 食品科学,2022,43(1):1191−1197. [FENG H J, LAN W Q, ZANG Y Y, et al. Effects of dominant spoilage bacteria on quality change in obscure pufferfish (Takifugu obscurus) during cold storage and analysis of their spoilage ability[J]. Food Science,2022,43(1):1191−1197. doi: 10.7506/spkx1002-6630-20210131-363
[6]	刘光明, 梁一巍, 李传勇, 等. 海洋中上层鱼类产品中生物胺的调查与控制[J]. 中国食品学报,2019,19(8):1−12. [LIU G M, LIANG Y W, LI C Y, et al. Investigation and control of biogenic amines in marine pelagic fishes products[J]. Journal of Chinese Institute of Food Science and Technology,2019,19(8):1−12.
[7]	HOUICHER A, BENSID A, REGENSTEIN J M, et al. Control of biogenic amine production and bacterial growth in fish and seafood products using phytochemicals as biopreservatives: A review[J]. Food Bioscience,2020,39(2):100807.
[8]	ZOGUL F, HAMED I. The importance of lactic acid bacteria for the prevention of bacterial growth and their biogenic amines formation: A review[J]. Critical Reviews in Food Science & Nutrition,2017:1.
[9]	国家卫生和计划生育委员会. GB 2733-2015 食品安全国家标准鲜、冻动物性水产品[S]. 北京:中国标准出版社, 2016. National Health and Family Planning Commission. GB 2733-2015 National food safety standard fresh and frozen aquatic products of animal origin[S]. Beijing: China Standards Press, 2016.
[10]	JSKELINEN E, JAKOBSEN L, HULTMAN J, et al. Metabolomics and bacterial diversity of packaged yellowfin tuna (Thunnus albacares) and salmon (Salmo salar) show fish species-specific spoilage development during chilled storage[J]. International Journal of Food Microbiology,2018:293.
[11]	LAKSHMANAN R, SHAKILA R J, JEYASEKARAN G. Survival of amine-forming bacteria during the ice storage of fish and shrimp[J]. Food Microbiology,2002,19(6):617−625. doi: 10.1006/fmic.2002.0481
[12]	KULEY E, DURMUS M, BALIKCI E, et al. Fish spoilage bacterial growth and their biogenic amine accumulation: Inhibitory effects of olive by-products[J]. International Journal of Food Properties,2016,20(5-8):1029−1043.
[13]	WANG H, LUO Y, HUANG H, et al. Microbial succession of grass carp (Ctenopharyngodon idellus) filets during storage at 4 ℃ and its contribution to biogenic amines' formation[J]. International Journal of Food Microbiology,2014,190:66−71. doi: 10.1016/j.ijfoodmicro.2014.08.021
[14]	中华人民共和国国家卫生和计划生育委员会. GB 5009.228-2016 食品安全国家标准食品中挥发性盐基氮的测定[S]. 北京:中国标准出版社, 2017. National Health and Family Planning Commission of the People's Republic of China. GB 5009.228-2016 National food safety standards Determination of volatile basic nitrogen in food[S]. Beijing: China Standards Press, 2017.
[15]	许振伟, 李学英, 杨宪时, 等. 海水鱼优势腐败菌腐败能力分析[J]. 食品与机械,2011,27(4):71−74. [XU Z W, LI X Y, YANG X S, et al. Analysis on spoilage ability of dominant spoilage bacteria from marine fish[J]. Food & Machinery,2011,27(4):71−74. doi: 10.3969/j.issn.1003-5788.2011.04.019
[16]	刘洋帆, 李绪鹏, 冯阳, 等. 超高效液相色谱-串联质谱法测定鲣鱼中的生物胺[J]. 食品与发酵工业: 1−9 [2022-09-17]. DOI: 10.13995/j.cnki.11-1802/ts.029408. LIU Y F, LI X P, FENG Y, et al. UPLC-MS/MS method for detection of biogenic amines in skipjack tuna[J]. Food and Fermentation Industries: 1−9 [2022-09-17]. DOI: 10.13995/j.cnki.11-1802/ts.029408.
[17]	XIE J, ZHANG Z, YANG S P. et al. Study on the spoilage potential of Pseudomonas fluorescens on salmon stored at different temperatures[J]. Journal of Food Science and Technology,2018,55(1):217−225. doi: 10.1007/s13197-017-2916-x
[18]	SETTANNI L, MICELIA, FRANCESCA N, et al. Microbiological investigation of Raphanus sativus L. grown hydroponically in nutrient solutions contaminated with spoilage and pathogenic bacteria[J]. International Journal of Food Microbiology,2013,160(3):344−352. doi: 10.1016/j.ijfoodmicro.2012.11.011
[19]	聂芳红, 谢日东, 雷晓凌, 等. 湛江水产品中大肠菌群的分离鉴定与分类研究[J]. 广东农业科学,2014,41(6):166−170. [NIE F H, XIE R D, LEI X L, et al. Isolation, identification and classification of E. coliforms from aquatic products in Zhanjiang[J]. Guangdong Agricultural Sciences,2014,41(6):166−170. doi: 10.3969/j.issn.1004-874X.2014.06.043
[20]	ERDEM B, KARIPTAS E, KAYA T, et al. Factors influencing antibacterial activity of chitosan against Aeromonas hydrophila and Staphylococcus aureus[J]. International Current Pharmaceutical Journal,2016,5(5):45−48. doi: 10.3329/icpj.v5i5.27316
[21]	BO H K, FERNA N N I C, BARROS V Z J, et al. Species differentiation of seafood spoilage and pathogenic gram-negative bacteria by MALDI-TOF mass fingerprinting[J]. Journal of Proteome Research,2010,9(6):3169−3183. doi: 10.1021/pr100047q
[22]	励建荣, 李婷婷, 李学鹏. 水产品鲜度品质评价方法研究进展[J]. 北京工商大学学报(自然科学版),2010,28(6):1−8. [LI J R, LI T T, LI X P. Advances in methods for evaluating freshness of aquatic products[J]. Journal of Beijing Technology and Business University: Natural Science Edition,2010,28(6):1−8.
[23]	国家市场监督管理总局, 中国国家标准化管理委员会. GB/T 18108-2019 食品安全国家标准鲜海水鱼通则[S]. 北京:中国标准出版社, 2019. State Administration for Market Regulation, Standardization Administration of the People's Republic of China. GB/T 18108-2019 National food safety standard General rules of fresh marine fish[S]. Beijing: China Standards Press, 2019.
[24]	WANG H, LIU X C, et al. Spoilage potential of three different bacteria isolated from spoiled grass carp (Ctenopharyngodon idellus) fillets during storage at 4 ℃[J]. LWT-Food Science & Technology,2017:10−17.
[25]	KAUFMANN M, KLINGER C. [Methods in Molecular Bio-logy] Functional genomics volume 815 \| \| multiple-gene silencing using antisense RNAs in Escherichia coli[J]. Journal of Marine Science and Engineering, 2022, 10.1007/978-1-61779-424-7(Chapter 23): 307−319.
[26]	钱韻芳, 杨胜平, 谢晶, 等. 气调包装凡纳滨对虾特定腐败菌致腐败能力研究[J]. 中国食品学报,2015,15(1):85−91. [QIAN Y F, YANG S P, XIE J, et al. Studies on the putrefaction potential of the specific spoilage organisms from modified atmosphere packaged Litopenaeus vannamei[J]. Journal of Chinese Institute of Food Science and Technology,2015,15(1):85−91.
[27]	于淑池, 杨毅, 冯紫蓝, 等. 冷藏卵形鲳鲹优势腐败菌的分离鉴定及致腐能力分析[J]. 食品工业科技, 2021, 42(1): 101-109. YU S C, YANG Y, FENG Z L, et al. Isolation and identification of dominant spoilage bacteria in Trachinotus ovatus during chilled storage and their spoilage capability[J]. Science and Technology of Food Industry, 2021, 42(1): 101-109.
[28]	王光强, 俞剑燊, 胡健, 等. 食品中生物胺的研究进展[J]. 食品科学,2016,37(1):269−278. [WANG G Q, YU J S, HU J, et al. Progress in research on biogenic amines in foods[J]. Food Science,2016,37(1):269−278. doi: 10.7506/spkx1002-6630-201601046
[29]	LIERKA P. Biogenic amines in fish, fish products and shellfish: A review[J]. Food Additives & Contaminants,2011,28(11):1547−1560.
[30]	KIM S H, FIELD K G, CHANG D S. Identification of bacteria crucial to histamine accumulation in pacific mackerel during storage[J]. Journal of Food Protection,2001,64(10):1556−1564. doi: 10.4315/0362-028X-64.10.1556