|
|
|
| Microbiological Safety Evaluation of Edible Bucket Worms |
| CAI Yang-Lian, QIU Fan, HUANG Kuan-Guan, ZHANG Xin-Qi, WU Hong, HUANG Jun-Hao* |
| College of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, China |
|
|
|
|
Abstract Bucket worms are larva of longhorn beetles Apriona germari and A. swainsoni living in the trunk of Caesalpinia decapetala (Leguminosae). These insects are considered of high nutritional and medicinal value, and therefore widely consumed. The purpose of this study was to characterize the microflora of fresh edible bucket worms by the methods of high-throughput sequencing and traditional culture, and further evaluate the food safety based on relative abundance of Enterobacteriaceae, Saccharomycete, molds, and lactic acid bacteria (Lactobacillales) according to GB4789.1-2016 and ISO standards, and culture-dependent method was used to analyze thermoduric bacteria. The results showed that there were no significant differences between the microbial community compositions of 2 bucket worm species. The bacterial community of A. germari was mainly composed of Proteobacteria, Oxyphotobacteria and Bacteroides, while A. swainsoni was mainly composed of Proteobacteria, Firmicutes and Actinobacteria. Ascomycota and Basidiomycota were dominant fungal phyla in 2 bucket worm species, in which Ascomycetes were the absolutely dominant group. The analysis of safety-related indicators showed that relative abundance of Enterobacteriaceae in A. germari and A. swainsoni accounted for 60.28% and 39.82%, while Lactobacillus accounted for 1.24% and 3.65% respectively in bacteria; the molds accounted for 40.26% and 6.74% in fungal, while the yeast accounted for 59.74% and 93.26% respectively. Among yeast, 21 genera were found common in bucket worms, which were considered to be toxic or harmful to human beings (Homo sapien). In addition, 4 genera of potentially poisonous thermoduric bacteria including Staphylococcus, Streptomyces, Acinetobacter, and Pseudomonas were identified in 2 insect species, the colony number was significantly reduced after treatment at 50 ℃, 60 ℃ and 70 ℃, and almost inactivated completely at 80 ℃. Overall, the results of this study indicated that there were a variety of potential spoilage bacteria and food pathogens in the bucket worms, and the food safety risk could be eliminated or minimized through processing steps such as a shock heat treatment. The microbial safety evaluation based on high-throughput sequencing in present study could provide reference for hygiene criteria of edible insects.
|
|
Received: 21 January 2020
|
|
|
|
Corresponding Authors:
* , huangjh@zafu.edu.cn
|
|
|
|
[1] 董寅初. 1997. 低温肉制品是我国肉制品发展的总趋势[J]. 肉类研究, 11(1): 3-5.
(Dong Y C.1997. Low temperature meat products is the general trend of the development of meat products in China[J]. Meat Research, 11(1): 3-5.)
[2] 高冠群. 2018. 克里角梢小蠹发生规律与寄主选择机制的研究[D]. 博士学位论文, 西北农林科技大学, 导师: 陈辉, pp. 33-34.
(Gao G Q.2018. Occurrence and host selection mechanism of Trypophloeus klimeschi eggers[D]. Thesis for Ph.D., Northwest A&F University, Supervisor: Chen H, pp. 33-34.)
[3] 谷立慧, 钟青萍, 方祥, 等. 2016. “活的非可培养态”食源致病细菌的研究进展[J]. 食品与发酵工业, 42(2): 270-274.
(Gu L H, Zhong Q P, Fang X, et al.2016. Research progress on the viable but non-culturable state of food-borne pathogenic bacteria[J]. Food and Fermentation Industries, 42(2): 270-274.)
[4] 国家卫生和计划生育委员会, 国家食品药品监督管理总局. 2016. 《GB4789.1-2016食品安全国家标准食品微生物学检验总则》[M]. 北京, 中国标准出版社, 1-5.
(National Health and Family Planning Commission, State Food and Drug Administration. 2016. GB4789.1-2016 National Food Safety Standard-Food Microbiology Test-General Principles[M]. Beijing: China Standard Publishing House, 1-5.)
[5] 侯梦婷, 胡家香, 刘爱军. 2019. 世界昆虫食品产业发展现状及问题研究[J]. 世界农业, (04): 13-19.
(Hou M T, Hu J X, Liu A J. 2019. Research on development status and problems of world insect food industry[J]. World Agriculture, (04): 13-19.)
[6] 李克柱. 2005.天然食品防腐剂乳酸链球菌素(Nisin)在低温肉类食品中的应用[J]. 肉类研究, (9): 31-34.
(Li K Z. 2005. Application of natural food preservative nisin in low temperature meat food[J]. Meat Research, (9): 31-34.)
[7] 李晓丹, 屈建航, 张璐洁, 等. 2017. 环境微生物可培养性影响因素及培养方法研究进展[J]. 生命科学研究, 21(2): 154-158.
(Li X D, Qu J H, Zhang L J, et al.2017. Progresses on the influence factors of culturability and cultivation strategies of environmental microorganisms[J]. Life Science Research, 21(2): 154-158.)
[8] 李仲兴, 张新华, 王永祥. 2005. 乳球菌及其感染的研究进展[J]. 国外医学(临床生物化学与检验学分册), 26(12): 928-931.
(Li Z X, Zhang X H, Wang Y X.2005. Research progress of Lactococcus and its infection[J]. Foreign Medical Sciences (Clinical Biochemistry & Laboratory Medicine), 26(12): 928-931.)
[9] 刘松. 2017. 竹虫(Omphisa fuscidentalis)肠道微生物多样性及纤维素酶学特性研究[D]. 硕士学位论文, 中国农业科学院, 导师: 胡国全, pp. 12-13.
(Liu S.2017. Study on the microbial diversity and enzymatic activity of Omphisa fuscidentalis Hampson[D]. Thesis for M.D., Chinese Academy of Agricultural Sciences Dissertation, Supervisor: Hu G Q, pp. 12-13.)
[10] 刘小改, 杨亚军, 廖秋菊, 等. 2016. 稻纵卷叶螟肠道细菌群落结构与多样性分析[J]. 昆虫学报, 59(9): 965-976.
(Liu X G, Yang Y J, Liao Q J, et al.2016. Analysis of the bacterial community structure and diversity in the intestine of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)[J]. Acta Entomologica Sinicam, 59(9): 965-976.)
[11] 刘泽玉, 李龙, 武国华. 2019. 几种常见食用昆虫营养功能概况[J]. 中国蚕业, 40(3): 48-51.
(Liu Z Y, Li l, Wu G H.2019. Nutritional function of several common edible insects[J]. China Sericulture, 40(3): 48-51.)
[12] 史黎央. 2018. 桑天牛和锈色粒肩天牛人工饲养技术的研究[D]. 硕士学位论文, 浙江农林大学, 导师: 徐华潮, p. 10.(Shi L Y. 2018. Research on artificial breeding technology of Apriona germari and A. swainsoni[D]. Thesis for M.D., Zhejiang A&F University, Supervisor: Xu H C, p.10.)
[13] 孙新新. 2017. 黄翅大白蚁肠道微生物多样性及几丁质降解微生物的分离与鉴定[D]. 硕士学位论文, 山东大学, 导师: 申玉龙, pp. 23-24.
(Sun X X.2017. Studies on the gut microbial diversity and isolation, identification of chitin-degrading bacteria from the hindgut of Macrotermes barneri[D]. Thesis for M.D., Shandong University, Supervisor: Shen Y L, pp. 23-24.)
[14] 王天召, 王正亮, 朱杭锋, 等. 2019. 基于高通量测序的褐飞虱肠道微生物多样性分析[J]. 昆虫学报, 62(3): 323-333.
(Wang T Z, Wang Z L, Zhu H F, et al.2019. Analysis of the gut microbial diversity of the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) by high throughput sequencing[J]. Acta Entomologica Sinica, 62(3): 323-333.)
[15] 王震杰. 2014. 扶桑绵粉蚧共生微生物的分子鉴定及分析[D]. 硕士学位论文, 浙江师范大学, 导师: 阮永明, pp. 18-19.
(Wang Z J.2014. Molecule identification and analyse of the symbiont microorganism in mealybug Phenacoccus solenopsis Tinsley[D]. Thesis for M.D., Zhejiang Normal University, Supervisor: Luan Y M, pp. 18-19.)
[16] 相辉, 黄勇平. 2008. 肠道微生物与昆虫的共生关系[J]. 昆虫知识, 45(5): 687-693.
(Xiang H, Huang Y P.2008. Symbiosis between gut microbiota and insects[J]. Chinese Bulletin of Entomology, 45(5): 687-693.)
[17] 相辉, 李木旺, 赵勇, 等. 2007. 家蚕幼虫中肠细菌群落多样性的PCR-DGGE和16S rDNA文库序列分析[J]. 昆虫学报, 50(3): 222-233.
(Xang H, Li M W, Zhao Y, et al.2007. Bacterial community in midguts of the silkworm larvae estimated by PCR DGGE and 16S rDNA gene library analysis[J]. Acta Entomologica Sinicam, 50(3): 222-233.)
[18] 杨紫薇, 刘庆华, 熊海容, 等. 2019. 昆虫蛋白的开发应用现状与展望[J]. 现代农业科技, (12): 206-208.
(Yang Z W, Liu Q H, Xiong H R, et al. 2019. Status and prospects of development and application of insect proteins[J]. Modern Agricultural Science and Technology, (12): 206-208.)
[19] 张号杰, 郭爱红, 秦江帆, 等. 2015. 昆虫蛋白的营养价值及其在动物生产中的应用[J]. 中国饲料, (3): 28-30.
(Zhang H J, Guo A H, Qin J F, et al. 2015. Nutritional values of insect protein and its application in animal production [J]. China Feed, (3): 28-30.)
[20] 张佳兰, 王靖, 任广旭, 等. 2019. 昆虫蛋白质的功能制备方法及相关食品的开发现状[J]. 农产品加工, (8): 75-77.
(Zhang J L, Wang J, Ren G X, et al. 2019. Function of insect protein preparation method and development status of related foods[J]. Farm Products Processing, (8): 75-77.)
[21] 张凯华, 刘双全, 任林, 等. 2012. 鲍曼不动杆菌感染的临床调查与耐药情况分析.当代医学, (33): 26-28.
(Zhang K H, Liu S Q, Ren L, et al. 2012. Investigation and drug resistance of Acinetobacter baumannii[J]. Contemporary Medicine, (33): 26-28.)
[22] 张帅帅, 南小宁, 王云果, 等. 2017. 基于PCR-DGGE技术的3种植食性叶蜂幼虫肠道细菌群落结构分析[J]. 西北林学院学报, 32(5): 154-160.
(Zhang S S, Nan X N, Wang Y G, et al.2017.Gut bacteria flora from three kinds of herbivorous sawfly larvae based on PCR-DGGE technology[J]. Journal of Northwest Forestry University, 32(5): 154-160.)
[23] 郑思敏, 黄先智, 沈以红. 2019. 可食性昆虫的食用安全性和营养评价及开发利用研究进展[J]. 蚕业科学, 45(02): 293-299.
(Zheng S M, Huang X Z, Shen Y H.2019. A review on food safety, nutrition evaluation and utilization of edible insects[J]. Science of Sericulture, 45(02): 293-299.)
[24] 周佳. 2015. 水-沉积物界面细菌可培养性研究[D]. 硕士学位论文, 河南工业大学, 导师: 屈建航, pp. 5-6.
(Zhou J.2015. Study on the bacterial culturability of water-sediment interface[D]. Thesis for M.D., Henan University of Technology, Supervisor: Qu J H, pp. 5-6.)
[25] Ahasan M S, Waltzek T B, Huerlimann R, et al.2018. Comparative analysis of gut bacterial communities of green turtles (Chelonia mydas) prehospitalization and post-rehabilitation by high-throughput sequencing of bacterial 16S rRNA gene[J]. Microbiological Research, 207: 91-99.
[26] Anderson M J.2001. A new method for non-parametric multivariate analysis of variance[J]. Austral Ecology, 26(1): 32-46.
[27] Banjo A D, Lawal O A, Adeyemi A I.2006. The microbial fauna associated with the larvae of Oryctes monocerus[J]. Journal of Applied Sciences Research, 2(11): 837-843.
[28] Chen B, Du K, Sun C, et al.2018. Gut bacterial and fungal communities of the domesticated silkworm (Bombyx mori) and wild mulberry-feeding relatives[J]. The ISME Journal, 12(9): 2252-2262.
[29] Clarke K R.1993. Non-parametric multivariate analyses of changes in community structure[J]. Australian Journal of Ecology, 18(1): 117-143.
[30] Dasen G H.1998. Molecular identification and applied genetics of Propionibacteria[D]. Thesis for Ph.D., ETH Zurich, Supervisor: Michael Teuber, pp.1-7.
[31] Dijk R, van den Berg D, Beumer R R, et al.2007. Microbiologie van voedingsmiddelen. Methoden, principes en criteria[M]. Noordervliet, Houten, pp. 1-685.
[32] Dillon R, Charnley K.2002. Mutualism between the desert locust Schistocerca gregaria and its gut microbiota[J]. Research in Microbiology, 153(8): 503-509.
[33] Edgar R C, Haas B J, Clemente J C, et al.2011. UCHIME improves sensitivity and speed of chimera detection[J]. Bioinformatics, 27(16): 2194-2200.
[34] Edgar R C.2013. UPARSE: Highly accurate OTU sequences from microbial amplicon reads[J]. Nature Methods, 10(10): 996-998.
[35] Engel P, Martinson V G, Moran N A.2012. Functional diversity within the simple gut microbiota of the honey bee[J]. Proceedings of the National Academy of Sciences of the USA, 109(27): 11002-11007.
[36] Engel P, Moran N A.2013. The gut microbiota of insects-diversity in structure and function[J]. FEMS Microbiology Reviews, 37(5): 699-735.
[37] Finley S J, Benbow M E, Javan G T.2015. Potential applications of soil microbial ecology and next-generation sequencing in criminal investigations[J]. Applied Soil Ecology, 88(): 69-78.
[38] Garofalo C, Osimani A, Milanović V, et al.2017. The microbiota of marketed processed edible insects as revealed by high-throughput sequencing[J]. Food Microbiology, 62(): 15-22.
[39] Garofalo C, milanović V, Cardinali F, et al.2019. Current knowledge on the microbiota of edible insects intended for human consumption: A state-of-the-art review[J]. Food Research International, 108527. DOI: 10.1016/j.foodres.2019.108527
[40] Grabowski N T, Jansen W, Klein G.2014. Microbiological status of edible insects sold as pet feed in Germany[J]. Insects to Feed the World, 14-17. DOI: 10.2376/0003-925X-67-4
[41] Grabowski N T, Klein G.2017. Microbiology of processed edible insect products-Results of a preliminary survey[J]. International Journal of Food Microbiology, 243: 103-107.
[42] Haynes S, Darby A C, Daniell T J, et al.2003. Diversity of bacteria associated with natural aphid populations[J]. Applied and Environmental Microbiology, 69(12): 7216-7223.
[43] Holzapfel W H.1992. Culture media for non-sporulating Gram-positive food spoilage bacteria[J]. International Journal of Food Microbiology, 17(2): 113-133.
[44] Jander G, Rahme L G, Ausubel F M.2000. Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects[J]. Journal of Bacteriology, 182(13): 3843-3845.
[45] Kembel S, Cowan P, Helmus MR, et al.2010. Picante: R tools for integrating phylogenies and ecology[J]. Bioinformatics, 26: 1463-1464.
[46] Liu W T, Marsh T L, Cheng H, et al.1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA[J]. Applied and Environmental Microbiology, 63(11): 4516-4522.
[47] Masson Y, Ainsworth P, Fuller D, et al.2002. Growth of Pseudomonas fluorescens and Candida sake in homogenized mushrooms under modified atmosphere[J]. Journal of Food Engineering, 54(2): 125-131.
[48] McCune B, Grace J B, Urban D L.2002. Analysis of ecological communities (vol. 28)[M]. MjM software design, Gleneden Beach, pp. 1-304.
[49] Mlcek J, Rop O, Borkovcova M, et al.2014. A comprehensive look at the possibilities of edible insects as food in Europe-a review[J]. Polish Journal of Food and Nutrition Sciences, 64(3): 147-157.
[50] Oksanen J, Blanchet F G, Kindt R, et al.2011. Vegan: Community Ecology Package. http://CRAN.R-project.org/package=vegan.
[51] Orsi L, Voege L L, Stranieri S.2019. Eating edible insects as sustainable food? Exploring the determinants of consumer acceptance in Germany[J]. Food Research International, 125: 108573.
[52] Osimani A, Milanović V, Garofalo C, et al.2018. Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR[J]. International Journal of Food Microbiology, 276(): 54-62.
[53] Rumpold B A, Fröhling A, Reineke K, et al.2014. Comparison of volumetric and surface decontamination techniques for innovative processing of mealworm larvae (Tenebrio molitor)[J]. Innovative Food Science & Emerging Technologies, 26(): 232-241.
[54] Sant'anna M R V, Darby A C, Brazil R P, et al.2012. Investigation of the bacterial communities associated with females of Lutzomyia sand fly species from South America[J]. PLOS ONE, 7(8): e42531.
[55] Schloss P D, Delalibera Jr I, Handelsman J O, et al.2006. Bacteria associated with the guts of two wood-boring beetles: Anoplophora glabripennis and Saperda vestita (Cerambycidae)[J]. Environmental Entomology, 35(3): 625-629.
[56] Song Z Q, Wang F P, Zhi X Y, et al.2013. Bacterial and archaeal diversities in Yunnan and Tibetan hot springs, China[J]. Environmental Microbiology, 15(4): 1160-1175.
[57] Stoops J, Maes P, Claes J, et al.2012. Growth of Pseudomonas fluorescens in modified atmosphere packaged tofu[J]. Letters in Applied Microbiology, 54(3): 195-202.
[58] Stoops J, Crauwels S, Waud M, et al.2016. Microbial community assessment of mealworm larvae (Tenebrio molitor) and grasshoppers (Locusta migratoria migratorioides) sold for human consumption. Food microbiology, 53: 122-127.
[59] Stull V J, Finer E, Bergmans R S, et al.2018. Impact of edible cricket consumption on gut microbiota in healthy adults, a double-blind, randomized crossover trial[J]. Scientific Reports, 8(1): 10762.
[60] Suenami S, Nobu M K, Miyazaki R.2019. Community analysis of gut microbiota in hornets, the largest eusocial wasps, Vespa mandarinia and V. simillima[J]. Scientific Reports, 9(1): 9830.
[61] van Huis A.2013. Potential of insects as food and feed in assuring food security[J]. Annual Review of Entomology, 58(1): 563-583.
[62] Wang A, Yao Z, Zheng W, et al.2014. Bacterial communities in the gut and reproductive organs of Bactrocera minax (Diptera: Tephritidae) based on 454 pyrosequencing[J]. PLOS ONE, 9(9): e106988.
[63] Yu V L.1979. Serratia marcescens: Historical perspective and clinical review[J]. New England Journal of Medicine, 300(16): 887-893.
[64] Yun J H, Roh S W, Whon T W, et al.2014. Insect gut bacterial diversity determined by environmental habitat, diet, developmental stage, and phylogeny of host[J]. Applied and Environmental Microbiology, 80(17): 5254-5264.
[65] Zhang B, Zhang J, Liu Y, et al.2018. Co-occurrence patterns of soybean rhizosphere microbiome at a continental scale[J]. Soil Biology and Biochemistry, 118: 178-186.
[66] Zhang Q, Xiao X, Li M, et al.2017. Vildagliptin increases butyrate-producing bacteria in the gut of diabetic rats[J]. PLOS ONE, 12(10): e0184735.
[67] Zhang Z, Jiao S, Li X, et al.2018. Bacterial and fungal gut communities of Agrilus mali at different developmental stages and fed different diets[J]. Scientific Reports, 8(1): 15634.
[68] Zimmerman N B, Vitousek P M.2012. Fungal endophyte communities reflect environmental structuring across a Hawaiian landscape[J]. Proceedings of the National Academy of Sciences of the USA, 109(32): 13022-13027. |
|
|
|
