鲫肠道乳酸菌的分离及益生特性
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摘要
为获得鱼源益生乳酸菌,本研究从健康鲫肠道内分离鉴定得到38株乳酸菌,并选择其中8株乳酸菌进行体外益生特性分析。结果显示,8株乳酸菌均能在p H=4.5和10%鲫胆汁环境中存活,对嗜水气单胞菌、豚鼠气单胞菌、无乳链球菌和副溶血弧菌均有较强的抑菌能力,但菌体表面疏水性和自凝集能力具有菌株特异性。选择5株疏水性较高(63%~89%)、自凝集能力强(37%~45%)的乳酸乳球菌进行体外黏附能力测定,结果显示,黏附能力在菌株间具有差异性(4.5%~7.9%),但均能显著降低嗜水气单胞菌NJ-35的体外黏附率,黏附抑制率达30%~35%;最后对筛选出的3株高黏附力的乳酸乳球菌进行安全性评价,发现3株菌对鲫均无致病力。鱼源乳酸菌的筛选,为鲫养殖生产中益生菌的实际应用提供了理论依据。
As an important freshwater aquaculture species in China, Carassius auratus culture is currently suffering from serious losses due to infectious diseases. The use of antibiotics in preventing and controlling the bacterial diseases has caused the evolution of resistant strains of aquatic pathogens. This is a serious constraint on aquaculture development. Therefore, some alternative measures need to be developed to improve the quality and sustainability of aquaculture production. Probiotics can be a substitute for antibiotics. Lactobacillus sp. from the host or the culture environment have shown to have ability to better adapt to the culture environment and colonize in the host gut, which help them exhibit their probiotic effects better. This study intends to screen probiotic candidates from C. auratus according to their in vitro probiotic properties, which include acid and bile tolerance ability, antimicrobial activity, cell surface hydrophobicity, aggregation ability, in vitro adhesive ability and pathogenicity in crucian carp. Based on biochemical tests and 16 S r RNA gene sequence analysis, thirty-eight strains of Lactobacillus sp. were isolated from the gut of C. auratus, which belonged to Lactococcus, Leuconostos, Carnobacterium and Enterococcus faecium, respectively, and eight of them were selected to evaluate the in vitro probiotic properties. The results showed that all eight strains of Lactobacillus sp. bacteria could tolerate acid(p H 4.6) and bile(10% bile of C. auratus), and exhibit good antibacterial activities against four pathogens including Aeromonas hydrophila, A. caviae, Streptococcus agalactiae and Vibrio parahaemolyticus. Five of eight strains showed high cell surface properties, i.e. hydrophobicity(63%-89%) and autoaggregation(37%-45%), and their adhesive rates to C.auratus intestinal mucus were ranged from 4.5% to 7.9%. These five isolates differed in coaggregation ability, but exhibited good adhesion inhibition rates for the binding of A. hydrophila to C. auratus intestinal mucus, with the inhibition rates of 30% to 35%. Three L. lactis isolates 16-7, 16-19 and16-24 with a higher adhesive ability showed no pathogenicity when administered by intraperitoneal injection to C. auratus. Based on our data, the three L. lactis isolates could be considered as potential probiotic candidates for C. auratus farming. Further in vivo evaluation needs to be performed in the future study.
As an important freshwater aquaculture species in China, Carassius auratus culture is currently suffering from serious losses due to infectious diseases. The use of antibiotics in preventing and controlling the bacterial diseases has caused the evolution of resistant strains of aquatic pathogens. This is a serious constraint on aquaculture development. Therefore, some alternative measures need to be developed to improve the quality and sustainability of aquaculture production. Probiotics can be a substitute for antibiotics. Lactobacillus sp. from the host or the culture environment have shown to have ability to better adapt to the culture environment and colonize in the host gut, which help them exhibit their probiotic effects better. This study intends to screen probiotic candidates from C. auratus according to their in vitro probiotic properties, which include acid and bile tolerance ability, antimicrobial activity, cell surface hydrophobicity, aggregation ability, in vitro adhesive ability and pathogenicity in crucian carp. Based on biochemical tests and 16 S r RNA gene sequence analysis, thirty-eight strains of Lactobacillus sp. were isolated from the gut of C. auratus, which belonged to Lactococcus, Leuconostos, Carnobacterium and Enterococcus faecium, respectively, and eight of them were selected to evaluate the in vitro probiotic properties. The results showed that all eight strains of Lactobacillus sp. bacteria could tolerate acid(p H 4.6) and bile(10% bile of C. auratus), and exhibit good antibacterial activities against four pathogens including Aeromonas hydrophila, A. caviae, Streptococcus agalactiae and Vibrio parahaemolyticus. Five of eight strains showed high cell surface properties, i.e. hydrophobicity(63%-89%) and autoaggregation(37%-45%), and their adhesive rates to C.auratus intestinal mucus were ranged from 4.5% to 7.9%. These five isolates differed in coaggregation ability, but exhibited good adhesion inhibition rates for the binding of A. hydrophila to C. auratus intestinal mucus, with the inhibition rates of 30% to 35%. Three L. lactis isolates 16-7, 16-19 and16-24 with a higher adhesive ability showed no pathogenicity when administered by intraperitoneal injection to C. auratus. Based on our data, the three L. lactis isolates could be considered as potential probiotic candidates for C. auratus farming. Further in vivo evaluation needs to be performed in the future study.
引文
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[19]Cebeci A,Gürakan C.Properties of potential probiotic Lactobacillus plantarum strains[J].Food Microbiology,2003,20(5):511-518.
[20]Kumar R V J,Seo B J,Mun M R,et al.Putative probiotic Lactobacillus spp.from porcine gastrointestinal tract inhibit transmissible gastroenteritis coronavirus and enteric bacterial pathogens[J].Tropical Animal Health and Production,2010,42(8):1855-1860.
[21]Sahoo T K,Jena P K,Nagar N,et al.In vitro evaluation of probiotic properties of lactic acid bacteria from the gut of Labeo rohita and Catla catla[J].Probiotics and Antimicrobial Proteins,2015,7(2):126-136.
[22]Grze?kowiak?,Collado M C,Salminen S.Evaluation of aggregation abilities between commensal fish bacteria and pathogens[J].Aquaculture,2012,356-357:412-414.
[23]Collado M C,Meriluoto J,Salminen S.Measurement of aggregation properties between probiotics and pathogens:in vitro evaluation of different methods[J].Journal of Microbiological Methods,2007,71(1):71-74.
[24]Naito Y,Tohda H,Okuda K,et al.Adherence and hydrophobicity of invasive and noninvasive strains of Porphyromonas gingivalis[J].Molecular Oral Microbiology,1993,8(4):195-202.
[25]Pérez P F,Minnaard Y,Disalvo E A,et al.Surface properties of bifidobacterial strains of human origin[J].Applied and Environmental Microbiology,1998,64(1):21-26.
[26]Wadstroum T,Andersson K,Sydow M,et al.Surface properties of lactobacilli isolated from the small intestine of pigs[J].Journal of Applied Microbiology,1987,62(6):513-520.
[27]靳彩娟.高粘附性乳酸菌的筛选、鉴定及其表面疏水特性研究[D].扬州:扬州大学,2013.Jin C J.Screening,identification and surface hydrophobic properties of high adhesive lactic acid bacteria[D].Yangzhou:Yangzhou University,2013(in Chinese).
[28]Chabrillón M,Rico R M,Balebona M C,et al.Adhesion to sole,Solea senegalensis Kaup,mucus of microorganisms isolated from farmed fish,and their interaction with Photobacterium damselae subsp.piscicida[J].Journal of Fish Diseases,2005,28(4):229-237.
[29]Gueimonde M,Jalonen L,He F,et al.Adhesion and competitive inhibition and displacement of human enteropathogens by selected lactobacilli[J].Food Research International,2006,39(4):467-471.
[2]Balcázar J L,De Blas I,Ruiz-Zarzuela I,et al.The role of probiotics in aquaculture[J].Veterinary Microbiology,2006,114(3-4):173-186.
[3]Lee H I,Kim M H,Kim K Y,et al.Screening and selection of stress resistant Lactobacillus spp.isolated from the marine oyster(Crassostrea gigas)[J].Anaerobe,2010,16(5):522-526.
[4]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.
[5]Ring?E,Bendiksen H R,Wesmajervi M S,et al.Lactic acid bacteria associated with the digestive tract of Atlantic salmon(Salmo salar L.)[J].Journal of Applied Microbiology,2000,89(2):317-322.
[6]Mathur S,Singh R.Antibiotic resistance in food lactic acid bacteria-a review[J].International Journal of Food Microbiology,2005,105(3):281-295.
[7]Mu?oz-Atienza E,Araújo C,Magadán S,et al.In vitro and in vivo evaluation of lactic acid bacteria of aquatic origin as probiotics for turbot(Scophthalmus maximus L.)farming[J].Fish&Shellfish Immunology,2014,41(2):570-580.
[8]TouréR,Kheadr E,Lacroix C,et al.Production of antibacterial substances by bifidobacterial isolates from infant stool active against Listeria monocytogenes[J].Journal of Applied Microbiology,2003,95(5):1058-1069.
[9]Won K M,Park S I.Pathogenicity of Vibrio harveyi to cultured marine fishes in Korea[J].Aquaculture,2008,285(1-4):8-13.
[10]Santos Y,Bandin I,Nieto T P,et al.Comparison of the cell surface hydrophobicity of bacterial fish pathogens by different procedures[M]//Perkins F O,Cheng T C.Pathology in Marine Science.Amsterdam:Elsevier,1990:101-115.
[11]Del Re B,Sgorbati B,Miglioli M,et al.Adhesion,autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum[J].Letters in Applied Microbiology,2000,31(6):438-442.
[12]Irianto A,Austin B.Use of probiotics to control furunculosis in rainbow trout,Oncorhynchus mykiss(Walbaum)[J].Journal of Fish Diseases,2002,25(6):333-342.
[13]Ghosh S,Sinha A,Sahu C.Isolation of putative probionts from the intestines of Indian major carps[J].The Israeli Journal of Aquaculture-Bamidgeh,2007,59(3):127-132.
[14]房海,陈翠珍,张晓君.牙鲆格氏乳球菌感染症及其病原[J].中国水产科学,2006,13(3):403-409.Fang H,Chen C Z,Zhang X J.Lactococcus garvieae as a pathogen in flounder(Paralichthys olivaceus Temminck et Schlegel)[J].Journal of Fishery Sciences of China,2006,13(3):403-409(in Chinese).
[15]Ugajin M.Studies on Streptococcus sp.as a causative agent of an epizootic among the cultured ayu(Plecoglossus altivelis)in Tochigi Prefecture,Japan,1980[J].Fish Pathology,1981,16(3):119-127.
[16]Leisner J J,Laursen B G,Prévost H,et al.Carnobacterium:positive and negative effects in the environment and in foods[J].FEMS Microbiology Reviews,2007,31(5):592-613.
[17]曾东,王益平,倪学勤,等.鲤益生菌筛选及部分菌株对鲤前肠黏液的体外黏附作用[J].中国水产科学,2009,16(3):427-433.Zeng D,Wang Y P,Ni X Q,et al.Screening of carp probiotic bacteria and adhesion to carp foregut mucus in vitro[J].Journal of Fishery Sciences of China,2009,16(3):427-433(in Chinese).
[18]Balcázar J L,Vendrell D,de Blas I,et al.Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish[J].Aquaculture,2008,278(1-4):188-191.
[19]Cebeci A,Gürakan C.Properties of potential probiotic Lactobacillus plantarum strains[J].Food Microbiology,2003,20(5):511-518.
[20]Kumar R V J,Seo B J,Mun M R,et al.Putative probiotic Lactobacillus spp.from porcine gastrointestinal tract inhibit transmissible gastroenteritis coronavirus and enteric bacterial pathogens[J].Tropical Animal Health and Production,2010,42(8):1855-1860.
[21]Sahoo T K,Jena P K,Nagar N,et al.In vitro evaluation of probiotic properties of lactic acid bacteria from the gut of Labeo rohita and Catla catla[J].Probiotics and Antimicrobial Proteins,2015,7(2):126-136.
[22]Grze?kowiak?,Collado M C,Salminen S.Evaluation of aggregation abilities between commensal fish bacteria and pathogens[J].Aquaculture,2012,356-357:412-414.
[23]Collado M C,Meriluoto J,Salminen S.Measurement of aggregation properties between probiotics and pathogens:in vitro evaluation of different methods[J].Journal of Microbiological Methods,2007,71(1):71-74.
[24]Naito Y,Tohda H,Okuda K,et al.Adherence and hydrophobicity of invasive and noninvasive strains of Porphyromonas gingivalis[J].Molecular Oral Microbiology,1993,8(4):195-202.
[25]Pérez P F,Minnaard Y,Disalvo E A,et al.Surface properties of bifidobacterial strains of human origin[J].Applied and Environmental Microbiology,1998,64(1):21-26.
[26]Wadstroum T,Andersson K,Sydow M,et al.Surface properties of lactobacilli isolated from the small intestine of pigs[J].Journal of Applied Microbiology,1987,62(6):513-520.
[27]靳彩娟.高粘附性乳酸菌的筛选、鉴定及其表面疏水特性研究[D].扬州:扬州大学,2013.Jin C J.Screening,identification and surface hydrophobic properties of high adhesive lactic acid bacteria[D].Yangzhou:Yangzhou University,2013(in Chinese).
[28]Chabrillón M,Rico R M,Balebona M C,et al.Adhesion to sole,Solea senegalensis Kaup,mucus of microorganisms isolated from farmed fish,and their interaction with Photobacterium damselae subsp.piscicida[J].Journal of Fish Diseases,2005,28(4):229-237.
[29]Gueimonde M,Jalonen L,He F,et al.Adhesion and competitive inhibition and displacement of human enteropathogens by selected lactobacilli[J].Food Research International,2006,39(4):467-471.