樊丹,邓福容,李绍戊,卢彤岩,刘红柏,王荻

• 1:中国水产科学研究院黑龙江水产研究所
• 2:黑龙江省水生动物病害与免疫重点实验室
• 3:上海海洋大学水产科学国家级实验教学示范中心
• 4:上海海洋大学国家水生动物病原库

摘要(Abstract)：

为筛选对虹鳟(Oncorhynchus mykiss)有潜在益生效果的菌株,本研究从健康虹鳟幼鱼肠道中分离到1株潜在益生菌株RT-BS07,采用形态学观察和分子特征分析对该菌株进行了鉴定,并评价了该菌株的抑菌性、耐受性、黏附性和安全性。形态学观察显示,菌株RT-BS07为革兰氏阳性杆菌,有圆形或椭圆形芽孢。16S rRNA比对结果显示,该菌株与枯草芽孢杆菌(Bacillus subtilis)NCBI3610株的同源性为99.86%。结合16S rRNA基因序列分析和形态学观察结果,确定菌株RT-BS07为枯草芽孢杆菌。菌株RT-BS07具备产蛋白酶和纤维素酶能力,其酶活力分别为(0.039±0.002)和(0.393±0.002) U/mg;该菌株可有效抑制温和气单胞菌(Aeromonas sobria)、杀鲑气单胞菌(A.salmonicida)和鲁氏耶尔森氏菌(Yersinia ruckeri)等致病菌。菌株RT-BS07在pH为2～10 LB液体培养基中均可存活;在盐度为2%～8%条件下存活率为5.70%～93.28%,其存活率随盐度升高而降低。体外黏附试验结果表明,菌株RT-BS07可大量黏附在虹鳟前肠黏膜,不破坏肠道组织完整性,对左氟沙星、环丙沙星、氧氟沙星和链霉素等18种抗菌药物敏感。本研究分离筛选得到的枯草芽孢杆菌RT-BS07株具有产消化酶性能,其抗逆性能强,对虹鳟无毒害作用,可作为虹鳟健康养殖及益生菌制剂研发源益生菌候选株。

关键词(KeyWords)： 虹鳟;枯草芽孢杆菌;产酶性能;抑菌特性;黏附特性

Abstract:

Keywords:

基金项目(Foundation): 中央级公益性科研院所基本科研业务费专项(HSY202007M);; 现代农业产业技术体系项目(CARS-46);; 冷水性鱼类病害防控创新团队项目(2020TD43)资助~~

作者(Author): 樊丹,邓福容,李绍戊,卢彤岩,刘红柏,王荻

参考文献(References)：

• [1]刘帅，卢彤岩，李绍戊．冷水性鱼类疫苗研究进展[J]．中国预防兽医学报，2017,39(1):74-79.Liu S,Lu T Y,Li S W.Vaccine research progress of cold water fish[J].Chinese Journal of Preventive Veterinary Medicine,2017,39(1):74-79．
• [2]Liu X,Steele J C,Meng X Z.Usage,residue,and human health risk of antibiotics in Chinese aquaculture:A review[J].Environmental Pollution,2017,223:161-169．
• [3]闫波，刘宁．乳酸菌及其在食品工业中的应用与展望[J]．食品研究与开发，2004(4):22-25.Yan B,Liu N.Lactobacillus and its application and prospect in food industry[J].Food Research and Development,2004(4):22-25．
• [4]González-Félix M L,Gatlin D M,Urquidez-Bejarano P,et al.Effects of commercial dietary prebiotic and probiotic supplements on growth,innate immune responses,and intestinal microbiota and histology of totoaba macdonaldi[J].Aquaculture,2018,491:239-251．
• [5]Nawaz A,Bakhsh Javaid A,Irshad S,et al.The Functionality of prebiotics as immunostimulant:Evidences from trials on terrestrial and aquatic animals[J].Fish and Shellfish Immunology,2018,7(6):272-278．
• [6]Kechagia M,Basoulis D,Konstantopoulou S,et al.Health benefits of probiotics[J].Nutrition,2013,2(1):13-19．
• [7]Balcázar J L,Blas I D,Ruiz-Zarzuela I,et al.The role of probiotics in aquaculture[J].Veterinary Microbiology,2006,114(3):173-186．
• [8]Kiadaliri M,Firouzbakhsh F,Deldar H.Effects of feeding with red algae(Laurencia caspica)hydroalcoholic extract on antioxidant defense,immune responses,and immune gene expression of kidney in rainbow trout(Oncorhynchus mykiss)infected with Aeromonas hydrophila[J].Aquaculture,2020,529(73):53-61．
• [9]魏媛，龚国利．抗菌肽与革兰氏阳性菌的互作及其机制[J]．动物营养学报，2017,29(8):2636-2642.Wei Y,Gong G L.Intereaction and its mechanism between peptides and gram-positive bacteria[J].Chinese Journal of Animal Nutrition,2017,29(8):2636-2642．
• [10]Ray A K,Ghosh K,Ringo E,et al.Enzyme-producing bacteria Isolated from fish gut:A review[J].Aquaculture Nutrition,2012,18(5):465-492．
• [11]Kornbluth A,Sachar D B.Ulcerative colitis practice guidelines in adults:American college of gastroenterology,practice parameters committee[J].Am Gastroenterol,2010,105(3):501-524．
• [12]Merrifield D L,Dimttrogou A,Bradley G,et al.Probiotic applications for rainbow trout(Oncorhynchus mykiss Walbaum)I.effects on growth performance,feed utilization,intestinal microbiota and related health criteria[J].Aquaculture Nutrition,2010,16(5):504-510．
• [13]Sharifuzzaman S M,Austin B.Influence of probiotic feeding duration on disease resistance and immune parameters in rainbow trout[J].Fish and Shellfish Immunology,2009,27(3):440-445．
• [14]Chen Y,Hua X,Ren X,et al.Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6and VP2induces protection against infectious pancreatic necrosis in rainbow trout(Oncorhynchus mykiss)[J].Fish and Shellfish Immunology,2020,100:18-26．
• [15]Huyben D,Sun L,Moccia R,et al.Dietary live yeast and increased water temperature influence the gut microbiota of rainbow trout[J].Journal of Applied Microbiology,2018,124(6):1377-1392．
• [16]Vazirzadeh A,Roosta H,Masoumi H,et al.Long-term effects of three probiotics,singular or combined,on serum innate immune parameters and expressions of cytokine genes in rainbow trout during grow-out[J].Fish and Shellfish Immunology,2020(98):748-757．
• [17]Merrifield D L,Dimitroglou A,Foey A,et al.The current status and future focus of probiotic and prebiotic applications for salmonids[J].Aquaculture,2010,302(1-2):1-18．
• [18]Mohammadian T,Nasirpour M,Tabandeh M R,et al.Administrations of autochthonous probiotics altered juvenile rainbow trout Oncorhynchus mykiss health status,growth performance and resistance to Lactococcus garvieae,an experimental onfection[J].Fish and Shellfish Immunology,2019(86):269-279．
• [19]Reyes Cerpa S,Vallejos Vidal E,Gonzalez Bown M J,et al.Effect of yeast(Xanthophyllomyces dendrorhous)and plant(Saint John′s wort,lemon balm,and rosemary)extract based functional diets on antioxidant and immune status of Atlantic salmon(Salmo salar)subjected to crowding stress[J].Fish and Shellfish Immunology,2018(74):250-259．
• [20]Safari R,Adel M,Lazado C C,et al.Host-derived probiotics Enterococcus casseliflavus improves resistance against streptococcus iniae infection in rainbow trout(Oncorhynchus mykiss)via immunomodulation[J].Fish and Shellfish Immunology,2016(52):198-205．
• [21]秦春秀，李琪敏，周婷婷，等．枯草芽孢杆菌Hab-8菌株分离筛选鉴定及抑菌机理初步研究[J]．黑龙江农业科学，2019(2):28-36.Qin C X,Li Q M,Zhou T,et al.Isolation,screening,identification and antimicrobial mechanism of Bacillus amyloliquefaciens Strain HAB-8[J].Heilongjiang Agricultural Sciences,2019(2):28-36．
• [22]肖立，金晶晶，顾晏，等．肾活检免疫组织化学法与免疫荧光直接法染色结果比较[J]．诊断学理论与实践，2017,16(1):79-83.Xiao L,Jin J,Gu Y,et al.Comparison of immunohistochemistry and immunofluorescence in renal core biopsy specimen[J].Journal of Diagnostics Concepts and Practice,2017,16(1):79-83．
• [23]Williams M L,Lawrence M L.Identification and characterization of a two-component hemolysin from Edwardsiella ictaluri[J].Veterinary Microbiology,2005,108(3-4):281-289．
• [24]谭瑶，赵清，舒为群，等．K-B纸片扩散法药敏试验[J]．检验医学与临床，2010,7(20):2290-2291.Tang Y,Zhao Q,Shu W Q,et al.Drug sensitivity test of K-Bpaper diffusion method[J].Laboratory Medicine and Clinic,2010,7(20):2290-2291．
• [25]National Committee for Clinical Laboratory Standards.Analysis and Presentation of Cumulative Antimicrobial Susceptibility Test data;Proposed Guideline,M39-P[M].Wayne:National Committee for Clinical Laboratory Standards,2002．
• [26]Huyben D,Nyman A,Vidakovi′c A,et al.Effects of dietary inclusion of the yeasts Saccharomyces cerevisiae and Wickerhamomyces anomalus on gut microbiota of rainbow trout[J].Aquaculture,2017,473:528-537．
• [27]Hemarajata P,Versalovic J.Effects of probiotics on gut microbiota:Mechanisms of intestinal immunomodulation and neuromodulation[J].Therapeutic Advances in Gastroenterology,2013,6(1):39-51．
• [28]窦春萌，左志晗，刘逸尘，等．凡纳滨对虾肠道内产消化酶益生菌的分离与筛选[J]．水产学报，2016,40(4):537-546.Dou C M,Zuo Z H,Liu Y C,et al.Isolation and screeing of digestive enzyme producing probiotics from intestine of Litopenaeus vannamei[J].Journal of Fisheries of China,2016,40(4):537-546．
• [29]韩旭凯．一株饲用枯草芽孢杆菌的体外研究[D]．上海：华东理工大学，2020.Han X K.In Vitro Study on a Strain of Bacillus subtilis for Feeding[D].Shanghai:East China University of Science and Technology,2020．
• [30]巴翠玉，张林波，张培军，等．2株枯草芽孢杆菌的分离鉴定及特性研究[J]．华南农业大学学报，2017,38(3):46-51.Ba C Y,Zhang L B,Zhang P J,et al.Isolation,identification and characterization of two strains of Bacillus subtilis[J].Journal of South China Agricultural University,2017,38(3):46-51．
• [31]王成强，王际英，黄炳山，等．珍珠龙胆石斑鱼肠道枯草芽孢杆菌的分离鉴定及产酶能力分析[J]．渔业研究，2019,41(5):366-373.Wang C Q,Wang J Y,Huang B S,et al.Study on Bacillus subtilis from hybrid grouper(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus♂）intestine and its zymogenicities[J].Journal of Fisheries Research,2019,41(5):366-373．
• [32]任雨薇．枯草芽孢杆菌对嗜水气单胞菌毒力的影响以及群体感应基因luxS的表达[D]．武汉：华中农业大学，2017.Ren Y W.The Influences of Bacillus subtilis on the Virulence of Aeromonas hydrophila and the Expression of Quorum Sensing luxS gene[D].Wuhan:Huazhong Agricultural University,2017．
• [33]刘晓燕，王玲玲，栾会妮，等．一株枯草芽孢杆菌的分离鉴定、生物学特性及其对水质净化的作用[J]．微生物学通报，2021(1):1-15.Liu X Y,Wang L,luan H L,et al.Isolation,identification,and biological characteristics of Bacillus subtilis and its effect on water purification[J].Microbiology China,2021(1):1-15．
• [34]秦瑶，王苇，郭秉娇，等．2株枯草芽孢杆菌对大肠杆菌和沙门氏菌的体外抑菌试验研究[J]．中国畜牧兽医，2014,41(1):207-210.Qin Y,Wang W,Guo B J,et al.Study on Bacillus subtilis strains Q4and Q10inhibiting the growth of Escherichia coli and Salmonellain vitro[J].China Animal Husbandry and Veterinary Medicine,2014,41(1):207-210．
• [35]Markowiak P,Slizewska K.The role of probiotics,prebiotics and synbiotics in animal nutrition[J].Gut Pathog,2018,10:21-26．
• [36]Chaucheyras Durand F,Durand H.Probiotics in animal nutrition and health[J].Beneficial Microbes,2010,1(1):3-9．
• [37]Giarma E,Amanetidou E,Toufexi A,et al.Defense systems in developing Artemia franciscana nauplii and their modulation by probiotic bacteria offer protection against a Vibrio anguillarum challenge[J].Fish and Shellfish Immunol,2017,66:163-172．
• [38]Verschuere L,Rombaut G,Sorgeloos P,et al.Probiotic bacteria as biological control agents in aquaculture[J].Microbiology and Molecular Biology Reviews,2000,64(4):13-18．
• [39]李旭，李雪，胡秀彩，等．鲫鱼肠道中枯草芽孢杆菌的分离鉴定及药敏试验[J]．江苏农业科学，2014,42(7):244-245.Li X,Li X,Hu X C,et al.Isolation,identification and drug sensitivity test of a strain Bacillus subtilis from intestine of Carassius auratus[J].Jiangsu Agricultural Sciences,2014,42(7):244-245．
• [40]贺刚，何力，谢从新，等．草鱼肠道枯草芽孢杆菌的耐药性分析[J]．现代农业科技，2008(22):219-222.He G,He L,Xie C X,et al.Drug resistance analysis of a strain Bacillus subtilis from intestine of Ctenopharyngodon idella[J].Modern Agricultural Science and Technology,2008(22):219-222.

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