Microbial analysis of Litopenaeus vannamei during partial freezing storage by Illumina high throughput sequencing
-
摘要: 文章以微冻贮藏的凡纳滨对虾 (Litopenaeus vannamei) 整虾、去头尾虾和虾仁的表面微生物作为研究对象，采用高通量测序技术鉴定不同贮藏时期样品菌群的组成及变化情况。结果显示：1) 各组样品的操作分类单元 (Operational taxonomic unit, OTU) 随贮藏时间的推移呈下降趋势，贮藏4周后虾仁的OTU数量最少，整虾与去头尾虾的OTU数量相对较多；2) 各组样品的赵氏指数 (Chao)、艾斯指数 (ACE) 和香农指数 (Shannon) 逐渐减小，辛普森指数 (Simpson) 增加，即贮藏过程中样品菌群丰度和多样性逐渐降低；3) 在属水平上，弧菌属 (Vibrio) 是各组样品的最初优势菌，弓形菌属 (Arcobacter) 在去头尾虾和整虾中存在较多。微冻4周后，假单胞菌属 (Pseudomonas)、希瓦氏菌属 (Shewanella) 和嗜冷杆菌属 (Psychrobacter)成为优势腐败菌；4) 属水平上的热图和关键物种分析显示，3种形式的对虾菌种组成和比例有所差异。综上，凡纳滨对虾贮藏时间和形态不同，其表面微生物种类、丰度等差异显著，在冷链流通过程中需要针对性地抑制微生物优势以保障产品品质。Abstract: Taking Litopenaeus vannamei of different forms (whole shrimp, decapitated shrimp, shrimp meat) as research objects, we applied Illumina high throughput sequencing technology to identify the surface microbial composition and changes of samples during partial freezing storage. The results show that: 1) The operational taxonomic units (OTUs) number of all samples decreased with the extension of storage time. After four weeks of storage, the whole shrimp and the decapitated shrimp had more OTUs, and the OTU number in shrimp meat was the least. 2) Chao, ACE and Shannon indices of all samples decreased but Simpson index increased, which indicates that the abundance and diversity of samples reduced during the storage. 3) At genus level, Vibrio was the main microorganism of all samples at the beginning, and Arcobacter existed more in the decapitated shrimp and the whole shrimp. After four weeks of storage, Pseudomonas, Shewanella and Psychrobacter became the dominant spoilage bacteria. 4) The heatmap and key species analysis at genus level show that the composition and proportion of three forms of shrimp samples were different. Therefore, different forms of L. vannamei had different microbial compositions and abundance in various storage periods. It is necessary to inhibit the advantages of microorganisms specifically to ensure the quality of the products during the cold chain process.
-
图 1 不同形式凡纳滨对虾的韦恩图
R. 虾仁样品；Q. 去头尾虾样品；Z. 整虾样品；后图同此
Figure 1. Venn diagram of L. vannamei with different forms
R. Shrimp samples; Q. Decapitated shrimp samples; Z. Whole shrimp samples; the same below.
图 2 不同形式凡纳滨对虾多样性指数的稀释曲线
Figure 2. Rarefaction curves of Alpha diversity indices of L. vannamei with different forms
图 3 不同形式对虾样品微生物物种组成 (目水平)
数字0、2、4分别表示贮藏0周、2周和4周；后图同此
Figure 3. Bacterial community composition at order level of L. vannamei with different forms
The number 0, 2 and 4 indicate the storage time of 0 week, 2 weeks and 4 weeks. The same below.
图 4 不同形式对虾样品微生物物种组成 (属水平)
Figure 4. Bacterial community composition at genus level of L. vannamei with different forms
图 5 不同形式对虾样品微生物丰度聚类热图 (属水平)
同一行中颜色深浅表示该微生物在不同样品中的丰度差异；图上方进化树表明样本间的相似程度；图左方进化树表明微生物间的相似程度
Figure 5. Microbial community heatmap analysis at genus level of L. vannamei with different forms
The shade of color in the same row indicates the difference in the abundance of the microorganism in different samples; the evolutionary tree on the top of the figure indicates the degree of similarity between samples; the evolutionary tree on the left of the figure indicates the degree of similarity between microorganisms.
表 1 不同形式凡纳滨对虾的测序覆盖率和操作分类单元数量
Table 1. Coverage and OTU number of L. vannamei with different forms
时间
Time虾仁
Shrimp meat去头尾虾
Decapitated shrimp整虾
Whole shrimp覆盖率
Coverage操作分类单元数量
OTU number覆盖率
Coverage操作分类单元数量
OTU number覆盖率
Coverage操作分类单元数量
OTU number0周 0 week 0.998 7 281 0.998 4 281 0.998 8 316 1周 1 week 0.998 5 189 0.999 0 401 0.999 1 322 2周 2 weeks 0.999 2 113 0.998 8 297 0.998 8 333 3周 3 weeks 0.999 9 36 0.999 8 50 0.999 8 50 4周 4 weeks 0.999 9 31 0.999 8 54 0.999 7 59 
下载: 导出CSV
表 2 不同形式凡纳滨对虾的Alpha丰富度指数表
Table 2. Alpha abundance indices of L. vannamei with different forms
时间
Time虾仁
Shrimp meat去头尾虾
Decapitated shrimp整虾
Whole shrimp赵氏指数 Chao 艾斯指数 ACE 赵氏指数 Chao 艾斯指数 ACE 赵氏指数 Chao 艾斯指数 ACE 0周 0 week 341 338 342 336 375 378 1周 1 week 231 236 429 437 363 365 2周 2 weeks 138 143 382 377 397 401 3周 3 weeks 45 50 80 69 80 120 4周 4 weeks 42 48 61 70 96 112
下载: 导出CSV
表 3 不同形式凡纳滨对虾的Alpha多样性指数表
Table 3. Alpha diversity indices of L. vannamei with different forms
时间
Time虾仁
Shrimp meat去头尾虾
Decapitated shrimp整虾
Whole shrimp香农指数
Shannon辛普森指数
Simpson香农指数
Shannon辛普森指数
Simpson香农指数
Shannon辛普森指数
Simpson0周 0 week 2.61 0.152 1 2.96 0.099 7 3.42 0.061 2 1周 1 week 1.91 0.278 3 3.43 0.086 9 2.49 0.213 1 2周 2 weeks 1.50 0.357 5 2.84 0.104 3 3.08 0.094 4 3周 3 weeks 1.08 0.436 6 1.43 0.345 3 1.59 0.358 4 4周 4 weeks 1.06 0.446 2 1.23 0.480 9 1.41 0.361 5
下载: 导出CSV
表 4 不同形式对虾样品关键物种差异 (属水平)
Table 4. Key species differences at genus level of L. vannamei with different forms
% 前10个物种
Top 10 species虾仁
Shrimp meat去头尾虾
Decapitated shrimp整虾
Whole shrimp假单胞菌属 Pseudomonas 36.730 4 35.589 3 15.183 3 希瓦氏菌属 Shewanella 33.741 4 27.325 8 11.265 6 嗜冷杆菌属 Psychrobacter 0.339 6 8.251 5 31.040 7 弧菌属 Vibrio 13.798 0 4.175 6 2.838 0 不动杆菌属 Acinetobacter 0.745 0 2.723 2 4.240 6 黄杆菌属 Flavobacterium 0.363 5 1.416 2 5.580 7 假交替单胞菌属 Pseudoalteromonas 0.210 7 0.391 1 6.549 9 弓形菌属 Arcobacter 0.567 3 1.984 1 4.513 1 假红杆菌属 Pseudorhodobacter 0.013 7 2.440 6 1.598 1 肉杆菌属 Carnobacterium 0.671 0 0.158 8 1.567 2
下载: 导出CSV
-
[1] ZENG D G, CHEN X L, XIE D X, et al. Analysis on differential gene expression in shrimp Litopenaeus vannamei induced by IHHNV infection using high-throughput sequencing[J]. J South Agr, 2014, 45(11): 1899-1903. [2] 农业农村部渔业渔政管理局. 2020中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2020: 1-182. [3] NIRMAL N P, BENJAKUL S. Effect of ferulic acid on inhibition of polyphenoloxidase and quality changes of Pacific white shrimp (Litopenaeus vannamei) during iced storage[J]. Food Chem, 2009, 116(1): 323-331. doi: 10.1016/j.foodchem.2009.02.054 [4] 刘金昉, 刘红英, 齐凤生, 等. 复合生物保鲜剂结合冰温贮藏对南美白对虾的保鲜效果[J]. 食品科学, 2014, 35(20): 286-290. doi: 10.7506/spkx1002-6630-201420056 [5] ZHANG B, MA L, DENG S, et al. Shelf-life of Pacific white shrimp (Litopenaeus vannamei) as affected by weakly acidic electrolyzed water ice-glazing and modified atmosphere packaging[J]. Food Control, 2015, 51: 114-121. [6] 沈辉, 万夕和, 何培民, 等. 脊尾白虾肠道微生物菌群结构[J]. 微生物学通报, 2015, 42(10): 1922-1928. [7] DI BELLA J M, BAO Y, GLOOR G B, et al. High throughput sequencing methods and analysis for microbiome research[J]. J Microbiol Methods, 2013, 95(3): 401-414. doi: 10.1016/j.mimet.2013.08.011 [8] 曹荣, 刘淇, 赵玲, 等. 基于高通量测序的牡蛎冷藏过程中微生物群落分析[J]. 农业工程学报, 2016, 32(20): 275-280. doi: 10.11975/j.issn.1002-6819.2016.20.036 [9] 周涛, 吴晓营, 罗海波, 等. 贮藏温度对即食小龙虾品质及微生物菌群多样性的影响[J]. 食品与机械, 2019, 35(9): 141-146. [10] 邓晓影, 张宾, 汤贺, 等. 基于高通量测序的南美白对虾中微生物群落分析[J]. 食品科学, 2018, 39(24): 149-155. doi: 10.7506/spkx1002-6630-201824023 [11] 翁丽华, 江芸, 徐幸莲, 等. PCR-DGGE研究热鲜肉贮藏过程中的菌相变化[J]. 食品科学, 2012, 33(23): 199-203. [12] 王建辉, 杨晶, 刘永乐, 等. 基于 PCR-DGGE技术对冷藏过程中草鱼肌肉的细菌群落结构分析[J]. 中国食品学报, 2014, 14(10): 203-209. [13] RONG C, LING Z, HUI S, et al. Characterization of microbial community in high-pressure treated oysters by high-throughput sequencing technology[J]. Innov Food Sci Emerg Technol, 2018, 45: 241-248. doi: 10.1016/j.ifset.2017.11.001 [14] 江杨阳, 杨水兵, 余海霞, 等. 基于培养基法和高通量测序法分析冷藏小龙虾优势腐败菌[J]. 食品科学, 2019, 40(16): 130-136. doi: 10.7506/spkx1002-6630-20180718-229 [15] 赵海鹏, 谢晶, 严文蓉. 南美白对虾冷藏过程中的细菌分离初步鉴定及菌相分析[J]. 江苏农业学报, 2011, 27(1): 164-168. doi: 10.3969/j.issn.1000-4440.2011.01.030 [16] 郭红, 董士远, 刘尊英, 等. 南美白对虾冰温下菌相变化[J]. 中国海洋大学学报, 2010, 40(6): 77-80, 166. [17] 凌萍华, 谢晶. 冰温技术结合保鲜剂对南美白对虾品质的影响[J]. 食品科学, 2010, 31(14): 280-284. [18] PREIDIS G A, AJAMI N J, WONG M C, et al. Composition and function of the undernourished neonatal mouse intestinal microbiome[J]. J Nutr Biochem, 2015, 26(10): 1050-1057. doi: 10.1016/j.jnutbio.2015.04.010 [19] 曹荣, 张井, 孟辉辉, 等. 高通量测序与传统纯培养方法在牡蛎微生物群落分析中的应用对比[J]. 食品科学, 2016, 37(24): 137-141. doi: 10.7506/spkx1002-6630-201624021 [20] ZHENG R, XU X, XING J, et al. Quality evaluation and characterization of specific spoilage organisms of Spanish mackerel by high-throughput sequencing during 0 ℃ cold chain logistics[J]. Foods, 2020, 9(3): 312. doi: 10.3390/foods9030312 [21] RAHIMI E, SHAKERIAN A, RAISSY M. Prevalence of Listeria species in fresh and frozen fish and shrimp in Iran[J]. Ann Microbiol, 2012, 62(1): 37-40. [22] 刘柳, 孔保华. 温度及气调包装对冷却猪肉中单核细胞增生性李斯特菌生长的影响[J]. 食品科学, 2008, 29(1): 334-337. doi: 10.3321/j.issn:1002-6630.2008.01.075 [23] LI K, ZHUO C, TENG C, et al. Effects of Ganoderma lucidum polysaccharides on chronic pancreatitis and intestinal microbiota in mice[J]. Int J Biol Macromol, 2016, 93: 904-912. [24] 江艳华, 姚琳, 李风铃, 等. 基于高通量测序的冷冻南极磷虾中细菌菌群结构分析[J]. 食品安全质量检测学报, 2016, 7(7): 2840-2845. [25] CAPORASO J G, LAUBER C L, WALTERS W A, et al. Ultra-highthroughput microbial community analysis on the Illumina HiSeq and MiSeq platforms[J]. ISME J, 2012, 6(8): 1621-1624. doi: 10.1038/ismej.2012.8 [26] 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. doi: 10.7506/spkx1002-6630-201724042 [27] 崔宏博, 薛勇, 宿玮, 等. 南美白对虾即食虾仁加工工艺和贮藏研究[J]. 食品科学, 2012, 33(4): 257-261. [28] 田凤, 王玲. 湛江地区养殖南美白对虾冷藏期间的菌相分析及优势腐败菌的初步鉴定[J]. 食品与发酵工业, 2013, 39(2): 24-28. [29] 吴海虹, 孙芝兰, 张新笑, 等. 不同包装方式下冷鲜青虾的菌群多样性分析[J]. 食品科学, 2019, 40(3): 251-258. doi: 10.7506/spkx1002-6630-20171027-318 [30] 黄佳奇. 小黄鱼优势腐败菌的分离鉴定及其与品质的相关性研究[D]. 杭州: 浙江大学, 2018: 12-29. [31] 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]. Appl Environ Microbiol, 2007, 73(16): 5261-5267. -
点击查看大图
计量
- 文章访问数: 81
- HTML全文浏览量: 29
- PDF下载量: 20
- 被引次数: 0
粤公网安备 44010502001741号
