1株副干酪乳酸菌S-4对致病弧菌的抑制特性研究
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摘要
针对弧菌病害的防治,选取了一株副干酪乳酸菌(Lactobacillus paracasei) S-4对4种致病弧菌进行体外拮抗实验,并在此基础上比较了不同发酵时长及稀释倍数的上清液对弧菌的抑制作用,同时探究了抑菌物质特性及进行共培养拮抗实验.结果显示:副干酪乳酸菌S-4对4株致病弧菌均具有显著的抑制作用.副干酪乳酸菌S-4生长过程中,0～16 h的上清液无抑菌活性,20 h后开始表现出对4株弧菌的抑制作用; 36 h时的上清液对创伤弧菌、哈维氏弧菌、副溶血弧菌的抑制率大于95.42%,且该上清液3倍稀释后的抑菌活性无显著降低,为最佳收获时间;72 h时的上清液对溶藻弧菌的抑制率达到95.72%,6倍稀释后的抑菌活性无显著降低,为抑制溶藻弧菌的最佳收获时间.进一步探究菌株间的相互拮抗表明:副干酪乳酸菌S-4在共培养4～6 h就表现出明显的抑菌活性,比上清液提前12 h产生抑制效果,在培养初期就能有效抑制弧菌的生长.生化分析表明:抑菌物质对过氧化氢酶、蜗牛酶、胃蛋白酶不敏感,与多肽类物质无关,酸碱中和后抑菌活性完全消失,推测为有机酸.本研究表明:副干酪乳酸菌S-4对4株常见致病弧菌具有较强抑制作用,在水产养殖中具有潜在应用价值.
A strain of lactic acid bacteria S-4,identified as Lactobacillus paracasei S-4 according to the nucleotide sequence of its 16 S r DNA,was used to conduct experiments to observe its antagonistic effect on four strains of vibrio.This study compared the antibacterial effect of supernatant with different culture durations and dilution ratios on vibrio and explored the competition of L. paracasei S-4 in co-cultivation with vibrio in vitro. The results showed that the supernatant had no inhibitory activity at 16 h of fermentation but showed inhibitory activity to 4 strains of vibrio after 20 h culture. And 36 h supernatant was the best harvest time for inhibiting V. harveyi,V. vulnificus and V. parahaemolyticus,when the inhibition percentage reached more than 95.42% and there was no significant decrease of inhibitory effect after 3-fold dilution. But to V. alginolyticus,72 h supernatant was the best harvest time and showed a 95.72% inhibition ratio,and 6-fold dilution of it exhibited no significant difference. L. paracasei S-4 in co-cultivation with vibrio started to reduce the 4 strains of vibrio at 4-6 h,its inhibitory activity appearing 12 h ahead of supernatant,indicating that L. paracasei S-4 was able to inhibit the growth of vibrio at the beginning of culture. Biochemical assay showed that the inhibitory compounds were insensitive to catalase,pepsase and snailase,and ultrafiltration and cold ethanol precipitation could not eliminate their inhibitory activity. But the inhibitory activity disappeared completely when supernatant pH was adjusted to neutral,indicating that organic acid may play an important role in bacteriostatic activity. The results of this study showed that L. paracasei can strongly inhibit 4 strains of vibrio and has potential value of application in aquaculture.
A strain of lactic acid bacteria S-4,identified as Lactobacillus paracasei S-4 according to the nucleotide sequence of its 16 S r DNA,was used to conduct experiments to observe its antagonistic effect on four strains of vibrio.This study compared the antibacterial effect of supernatant with different culture durations and dilution ratios on vibrio and explored the competition of L. paracasei S-4 in co-cultivation with vibrio in vitro. The results showed that the supernatant had no inhibitory activity at 16 h of fermentation but showed inhibitory activity to 4 strains of vibrio after 20 h culture. And 36 h supernatant was the best harvest time for inhibiting V. harveyi,V. vulnificus and V. parahaemolyticus,when the inhibition percentage reached more than 95.42% and there was no significant decrease of inhibitory effect after 3-fold dilution. But to V. alginolyticus,72 h supernatant was the best harvest time and showed a 95.72% inhibition ratio,and 6-fold dilution of it exhibited no significant difference. L. paracasei S-4 in co-cultivation with vibrio started to reduce the 4 strains of vibrio at 4-6 h,its inhibitory activity appearing 12 h ahead of supernatant,indicating that L. paracasei S-4 was able to inhibit the growth of vibrio at the beginning of culture. Biochemical assay showed that the inhibitory compounds were insensitive to catalase,pepsase and snailase,and ultrafiltration and cold ethanol precipitation could not eliminate their inhibitory activity. But the inhibitory activity disappeared completely when supernatant pH was adjusted to neutral,indicating that organic acid may play an important role in bacteriostatic activity. The results of this study showed that L. paracasei can strongly inhibit 4 strains of vibrio and has potential value of application in aquaculture.
引文
[1] CHANDRAKALA N,PRIYA S. Vibriosis in shrimp aquaculture a review[J]. International Journal of Scientific Research in Science,Engineering and Technology,2017,3:27-33.
[2]赵永泉．我国对虾病研究现状[J]．华南师范大学学报(自然科学版)，1997(3):103-113.ZHAO Y Q. Status of study on penaeid shrimp diseases in China[J]. Journal of South China Normal University(Natural Science Edition),1997(3):103-113.
[3]沈桂明，李晨，史成银，等．哈维氏弧菌(Vibrio harveyi)ML01株胞外产物及分泌性蛋白的分离与特性分析[J]．渔业科学进展，2017,38(4):25-33.SHEN G M,LI C,SHI C Y,et al. Isolation and characterization of the extracellular products(ECPs)and secretory proteins of the pathogenic Vibrio harveyi strain ML01[J]. Progress in Fishery Sciences,2017,38(4):25-33.
[4]夏青，王宝杰，刘梅，等．哈维氏弧菌浸浴后凡纳滨对虾肠道组织病理变化及转录水平的免疫应答[J]．水产学报，2015,39(10):1521-1529.XIA Q,WANG B J,LIU M,et al. Pathological changes and transcriptional response to immersion infection by Vibrio harveyi in shrimp(Litopenaeus vannamei)gut[J].Journal of Fisheries of China,2015,39(10):1521-1529.
[5] MARTINEZ J L. Environmental pollution by antibiotics and by antibiotic resistance determinants[J]. Environmental Pollution,2009,157(11):2893-2902.
[6] AJITHA S,SRIDHAR M,SRIDHAR N,et al. Probiotic effects of lactic acid bacteria against Vibrio alginolyticus in Penaeus(Fenneropenaeus)indicus(H. Milne Edwards)[J]. Asian Fisheries Society,2004,17:71-80.
[7] CHIU C H,GUU Y K,LIU C H,et al. Immune responses and gene expression in white shrimp,Litopenaeus vannamei,induced by Lactobacillus plantarum[J]. Fish&Shellfish Immunology,2007,23(2):364-377.
[8] VASEEHARAN B,RAMASAMY P. Control of pathogenic Vibrio spp. by Bacillus subtilis BT23,a possible probiotic treatment for black tiger shrimp Penaeus monodon[J]. Letters in Applied Microbiology,2003,36(2):83-87.
[9]叶海斌，樊英，严芳，等．益生菌制剂对虹鳟血清生化指标和免疫功能的影响[J]．华南师范大学学报(自然科学版)，2018,50(3):65-71.YE H B,FAN Y,YAN F,et al. Effects of dietary probiotics on serum biochemistry and immunity of rainbow trout,Oncorhynchus mykiss[J]. Journal of South China Normal University(Natural Science Edition),2018,50(3):65-71.
[10] SCHOLZ,DIAZ G G,RICQUE,et al. Enhancement of vibriosis resistance in juvenile Penaeus vannamei by supplementation of diets with different yeast products[J].Aquaculture,1999,176(3/4):271-283.
[11]相晨曦，王安利，廖绍安，等．不同破壁方法获取海洋毕赤酵母提取物及其应用[J]．华南师范大学学报(自然科学版)，2018,50(6):61-68.XIANG C X,WANG A L,LIAO S A,et al. Yeast extraction marine-derived Pichia guilliermondii with different cell wall disruption methods and its application[J]. Journal of South China Normal University(Natural Science Edition),2018,50(6):61-68.
[12] KHOUADJA S,HADDAJI N,HANCHI M,et al. Selection of lactic acid bacteria as candidate probiotics for Vibrio parahaemolyticus depuration in pacific oysters(Crassostrea gigas)[J]. Aquaculture Research,2016,48(4):1885-1894.
[13]柯杨勇，赖秋明，苏树叶，等．1株乳酸菌在凡纳滨对虾养殖中的应用效果[J]．热带生物学报，2015,6(1):18-24.HE Y Y,LAI Q M,SU S Y,et al. Application of lactic acid bacteria in shrimp farming[J]. Journal of Tropical Biology,2015,6(1):18-24.
[14] CHARERNJIRATRAGUL W,BHOOPONG P,KANTACHOTE D,et al. Inhibitory activity of lactic acid bacteria isolated from thai fermented food against pandemic strains of Vibrio parahaemolyticus[J]. Journal of Food Safety,2010,30(1):67-82.
[15] YANG Z Q,JIN C J,GAO L,et al. Alleviating effects of Lactobacillus strains on pathogenic Vibrio parahaemolyticusinduced intestinal fluid accumulation in the mouse model[J]. FEMS Microbiology Letters,2013,339(1):30-38.
[16]蔡秋杏，吴燕燕，李来好，等．来源于腌干鱼的乳酸菌中抗氧化酶及胞外多糖研究[J]．水产学报，2017,41(6):952-961.CAI Q X,WU Y Y,LI L H,et al. Study on antioxidant enzymes and exopolysaccharides of lactic acid bacteria separated from salt-dried fish products[J]. Journal of Fisheries of China,2017,41(6):952-961.
[17] WANG Y B. Effect of probiotics on growth performance and digestive enzyme activity of the shrimp Penaeus vannamei[J]. Aquaculture,2007,269(1):259-264.
[18]耿文超，关今韬，程申，等．副干酪乳杆菌的功能特性及其应用研究进展[J]．生物加工过程，2018,16(4):1-7.GENG W C,GUAN J T,CHENG S,et al. Progress in function characterization and application of Lactobacillus paracasei[J]. Chinese Journal of Bioprocess Engineering,2018,16(4):1-7.
[19] WANG Y,LU Z,WU H,et al. Study on the antibiotic activity of microcapsule curcumin against foodborne pathogens[J]. International Journal of Food Microbiology,2009,136(1):71-74.
[20]杨赟，杨柳，李晓静，等．蛇床子素通过促进胃癌细胞N87凋亡和细胞周期阻滞而抑制细胞增殖[J]．中国生物化学与分子生物学报，2019,35(1):74-80.YANG Y,YANG L,LI X J,et al. Osthole inhibits cell proliferation by promoting apoptosis and cell cycle arrest in gastric cancer cell line N87[J]. Chinese Journal of Biochemistry and Molecular Biology,2019,35(1):74-80.
[21] SEQUEIROS C,GARCS M E,VALLEJO M,et al. Potential aquaculture probiont Lactococcus lactis TW34 produces nisin Z and inhibits the fish pathogen Lactococcus garvieae[J]. Archives of Microbiology,2015,197(3):449-458.
[22]曹琛，黄金勇，李举鹏，等．TCBS培养基在虾病防治监测中的应用[J]．水产科学，2003,22(6):46-47.CAO S,HUANG J Y,LI J P,et al. The use of TCBS medium in prevention and supervision of shrimp diseases[J]. Fisheries Science,2003,22(6):46-47.
[23]贾芳芳，孟祥晨．乳酸菌中共培养诱导细菌素产生机制的研究进展[J]．食品安全质量检测学报，2017(4):222-228.JIA F F,MENG X C. Research progress of co-culture-inducible bacteriocin production in lactic acid bacteria[J].Journal of Food Safety and Quality,2017(4):222-228.
[24] ELMAHDI S,DASILVA L V,PARVEEN S. Antibioticresistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries:a review[J]. Food Microbiology,2016,57:128-134.
[25]周海平，李卓佳，杨莺莺，等．乳酸杆菌LH对几种水产养殖病原弧菌的抑制作用[J]．应用海洋学学报，2006,25(3):388-395.ZHOU H P,LI Z J,YANG Y Y,et al. Antagonistic action of Lactobacillaceae on pathogen Vibrio spp[J]. Journal of Applied of Oceanography,2006,25(3):388-395.
[26]王伟，丛丽娜，黄君，等．海参肠道中乳酸菌的分离鉴定及活性物质研究[J]．工业微生物，2018,48(4):24-29.WANG W,CONG L N,HUANG J,et al. Isolation and identification of lactic acid bacteria in sea cucumber and analysis of active substances[J]. Industrial Microbiology,2018,48(4):24-29.
[27] KONGNUM K,HONGPATTARAKERE T. Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp(Litopenaeus vannamei)challenged with Vibrio harveyi[J]. Fish&Shellfish Immunology,2012,32(1):170-177.
[28] SHA Y,WANG B,LIU M,et al. Interaction between Lactobacillus pentosus HC-2 and Vibrio parahaemolyticus E1in Litopenaeus vannamei in vivo and in vitro[J]. Aquaculture,2016,465:117-123.
[29] OGAWA M,SHIMIZU K,NOMOTO K,et al. Inhibition of in vitro growth of Shiga toxin-producing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid[J]. International Journal of Food Microbiology,2001,68(1/2):135-140.
[30] MESSAOUDI S,MANAI M,KERGOURLAY G,et al. Lactobacillus salivarius:bacteriocin and probiotic activity[J].Food Microbiology,2013,36(2):296-304.
[31] ARAUZ L J D,JOZALA A F,MAZZOLA P G,et al. Nisin biotechnological production and application:a review[J].Trends in Food Science&Technology,2009,20(3/4):146-154.
[2]赵永泉．我国对虾病研究现状[J]．华南师范大学学报(自然科学版)，1997(3):103-113.ZHAO Y Q. Status of study on penaeid shrimp diseases in China[J]. Journal of South China Normal University(Natural Science Edition),1997(3):103-113.
[3]沈桂明，李晨，史成银，等．哈维氏弧菌(Vibrio harveyi)ML01株胞外产物及分泌性蛋白的分离与特性分析[J]．渔业科学进展，2017,38(4):25-33.SHEN G M,LI C,SHI C Y,et al. Isolation and characterization of the extracellular products(ECPs)and secretory proteins of the pathogenic Vibrio harveyi strain ML01[J]. Progress in Fishery Sciences,2017,38(4):25-33.
[4]夏青，王宝杰，刘梅，等．哈维氏弧菌浸浴后凡纳滨对虾肠道组织病理变化及转录水平的免疫应答[J]．水产学报，2015,39(10):1521-1529.XIA Q,WANG B J,LIU M,et al. Pathological changes and transcriptional response to immersion infection by Vibrio harveyi in shrimp(Litopenaeus vannamei)gut[J].Journal of Fisheries of China,2015,39(10):1521-1529.
[5] MARTINEZ J L. Environmental pollution by antibiotics and by antibiotic resistance determinants[J]. Environmental Pollution,2009,157(11):2893-2902.
[6] AJITHA S,SRIDHAR M,SRIDHAR N,et al. Probiotic effects of lactic acid bacteria against Vibrio alginolyticus in Penaeus(Fenneropenaeus)indicus(H. Milne Edwards)[J]. Asian Fisheries Society,2004,17:71-80.
[7] CHIU C H,GUU Y K,LIU C H,et al. Immune responses and gene expression in white shrimp,Litopenaeus vannamei,induced by Lactobacillus plantarum[J]. Fish&Shellfish Immunology,2007,23(2):364-377.
[8] VASEEHARAN B,RAMASAMY P. Control of pathogenic Vibrio spp. by Bacillus subtilis BT23,a possible probiotic treatment for black tiger shrimp Penaeus monodon[J]. Letters in Applied Microbiology,2003,36(2):83-87.
[9]叶海斌，樊英，严芳，等．益生菌制剂对虹鳟血清生化指标和免疫功能的影响[J]．华南师范大学学报(自然科学版)，2018,50(3):65-71.YE H B,FAN Y,YAN F,et al. Effects of dietary probiotics on serum biochemistry and immunity of rainbow trout,Oncorhynchus mykiss[J]. Journal of South China Normal University(Natural Science Edition),2018,50(3):65-71.
[10] SCHOLZ,DIAZ G G,RICQUE,et al. Enhancement of vibriosis resistance in juvenile Penaeus vannamei by supplementation of diets with different yeast products[J].Aquaculture,1999,176(3/4):271-283.
[11]相晨曦，王安利，廖绍安，等．不同破壁方法获取海洋毕赤酵母提取物及其应用[J]．华南师范大学学报(自然科学版)，2018,50(6):61-68.XIANG C X,WANG A L,LIAO S A,et al. Yeast extraction marine-derived Pichia guilliermondii with different cell wall disruption methods and its application[J]. Journal of South China Normal University(Natural Science Edition),2018,50(6):61-68.
[12] KHOUADJA S,HADDAJI N,HANCHI M,et al. Selection of lactic acid bacteria as candidate probiotics for Vibrio parahaemolyticus depuration in pacific oysters(Crassostrea gigas)[J]. Aquaculture Research,2016,48(4):1885-1894.
[13]柯杨勇，赖秋明，苏树叶，等．1株乳酸菌在凡纳滨对虾养殖中的应用效果[J]．热带生物学报，2015,6(1):18-24.HE Y Y,LAI Q M,SU S Y,et al. Application of lactic acid bacteria in shrimp farming[J]. Journal of Tropical Biology,2015,6(1):18-24.
[14] CHARERNJIRATRAGUL W,BHOOPONG P,KANTACHOTE D,et al. Inhibitory activity of lactic acid bacteria isolated from thai fermented food against pandemic strains of Vibrio parahaemolyticus[J]. Journal of Food Safety,2010,30(1):67-82.
[15] YANG Z Q,JIN C J,GAO L,et al. Alleviating effects of Lactobacillus strains on pathogenic Vibrio parahaemolyticusinduced intestinal fluid accumulation in the mouse model[J]. FEMS Microbiology Letters,2013,339(1):30-38.
[16]蔡秋杏，吴燕燕，李来好，等．来源于腌干鱼的乳酸菌中抗氧化酶及胞外多糖研究[J]．水产学报，2017,41(6):952-961.CAI Q X,WU Y Y,LI L H,et al. Study on antioxidant enzymes and exopolysaccharides of lactic acid bacteria separated from salt-dried fish products[J]. Journal of Fisheries of China,2017,41(6):952-961.
[17] WANG Y B. Effect of probiotics on growth performance and digestive enzyme activity of the shrimp Penaeus vannamei[J]. Aquaculture,2007,269(1):259-264.
[18]耿文超，关今韬，程申，等．副干酪乳杆菌的功能特性及其应用研究进展[J]．生物加工过程，2018,16(4):1-7.GENG W C,GUAN J T,CHENG S,et al. Progress in function characterization and application of Lactobacillus paracasei[J]. Chinese Journal of Bioprocess Engineering,2018,16(4):1-7.
[19] WANG Y,LU Z,WU H,et al. Study on the antibiotic activity of microcapsule curcumin against foodborne pathogens[J]. International Journal of Food Microbiology,2009,136(1):71-74.
[20]杨赟，杨柳，李晓静，等．蛇床子素通过促进胃癌细胞N87凋亡和细胞周期阻滞而抑制细胞增殖[J]．中国生物化学与分子生物学报，2019,35(1):74-80.YANG Y,YANG L,LI X J,et al. Osthole inhibits cell proliferation by promoting apoptosis and cell cycle arrest in gastric cancer cell line N87[J]. Chinese Journal of Biochemistry and Molecular Biology,2019,35(1):74-80.
[21] SEQUEIROS C,GARCS M E,VALLEJO M,et al. Potential aquaculture probiont Lactococcus lactis TW34 produces nisin Z and inhibits the fish pathogen Lactococcus garvieae[J]. Archives of Microbiology,2015,197(3):449-458.
[22]曹琛，黄金勇，李举鹏，等．TCBS培养基在虾病防治监测中的应用[J]．水产科学，2003,22(6):46-47.CAO S,HUANG J Y,LI J P,et al. The use of TCBS medium in prevention and supervision of shrimp diseases[J]. Fisheries Science,2003,22(6):46-47.
[23]贾芳芳，孟祥晨．乳酸菌中共培养诱导细菌素产生机制的研究进展[J]．食品安全质量检测学报，2017(4):222-228.JIA F F,MENG X C. Research progress of co-culture-inducible bacteriocin production in lactic acid bacteria[J].Journal of Food Safety and Quality,2017(4):222-228.
[24] ELMAHDI S,DASILVA L V,PARVEEN S. Antibioticresistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries:a review[J]. Food Microbiology,2016,57:128-134.
[25]周海平，李卓佳，杨莺莺，等．乳酸杆菌LH对几种水产养殖病原弧菌的抑制作用[J]．应用海洋学学报，2006,25(3):388-395.ZHOU H P,LI Z J,YANG Y Y,et al. Antagonistic action of Lactobacillaceae on pathogen Vibrio spp[J]. Journal of Applied of Oceanography,2006,25(3):388-395.
[26]王伟，丛丽娜，黄君，等．海参肠道中乳酸菌的分离鉴定及活性物质研究[J]．工业微生物，2018,48(4):24-29.WANG W,CONG L N,HUANG J,et al. Isolation and identification of lactic acid bacteria in sea cucumber and analysis of active substances[J]. Industrial Microbiology,2018,48(4):24-29.
[27] KONGNUM K,HONGPATTARAKERE T. Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp(Litopenaeus vannamei)challenged with Vibrio harveyi[J]. Fish&Shellfish Immunology,2012,32(1):170-177.
[28] SHA Y,WANG B,LIU M,et al. Interaction between Lactobacillus pentosus HC-2 and Vibrio parahaemolyticus E1in Litopenaeus vannamei in vivo and in vitro[J]. Aquaculture,2016,465:117-123.
[29] OGAWA M,SHIMIZU K,NOMOTO K,et al. Inhibition of in vitro growth of Shiga toxin-producing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid[J]. International Journal of Food Microbiology,2001,68(1/2):135-140.
[30] MESSAOUDI S,MANAI M,KERGOURLAY G,et al. Lactobacillus salivarius:bacteriocin and probiotic activity[J].Food Microbiology,2013,36(2):296-304.
[31] ARAUZ L J D,JOZALA A F,MAZZOLA P G,et al. Nisin biotechnological production and application:a review[J].Trends in Food Science&Technology,2009,20(3/4):146-154.