响应面法研究温度、盐度、pH及磷酸盐、硝氮、氨氮对副溶血弧菌生长的影响
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摘要
副溶血弧菌(Vibrio parahaemolyticus,VP)在海洋环境中普遍存在,是能够引起人类中毒的重要食源性病原菌之一,研究其生长受环境因子的影响规律对科研和社会生活都具有重要意义。本研究采用响应面法研究温度、盐度、pH、磷酸盐、硝氮和氨氮对VP的生长影响,以探讨、评估这6种理化因子的综合作用结果。首先用Design-Expert软件中的Box Behnken中心组合原则设计三因素三水平响应面实验,再进行响应面分析,以培养基光密度(OD_(600 nm))值为生长状态参数,获得该菌的最佳培养参数:温度为34.5℃,盐度为3%,酸碱度pH为8.0。在此基础上,考察NaNO_3、NH_4Cl和Na_3PO_4对VP生长的影响并建立模型。回归方程决定系数R~2=0.9217,校正系数R~2=0.8211,回归模型F=9.16, P=0.004,表明该模型具有较好的准确性。实验所得实测值与模型演算理论值拟合良好,皆表明磷酸盐、硝氮和氨氮对VP的生长都具有抑制作用。
Vibrio parahaemolyticus(VP), which is ubiquitous in the marine environment, is one of the key foodborne pathogens affecting humans. It is thus of great significance for both scientific research and public safety that we understand how and what environmental factors affect its growth. To determine the functional relationship between a combination of environmental factors and VP growth, in this study the effects of temperature, salinity, pH, phosphate, nitrate, and ammonium on the growth of this kind of bacteria were investigated using response surface methodology(RSM). First, a Box-Behnken design was used to optimize the three critical internal factors of temperature, salinity and pH for this species, using optical density(OD_(600 nm)) as a marker for the growth of VP. It was found that for these factors, the optimum parameters were 34.5℃, 3%, and 8.0, respectively. Based on these results, the combined effects of phosphate, nitrate, and ammonium on the growth of VP were then further studied with the same method to develop a response model. The coefficient of determination(R~2) of the model equation was 0.9217, while the correction coefficient(R~2) was 0.8211. The regression model had an F-value of 9.16, and was thus statistically significant(P=0.004). These results indicated that the model proposed herein had satisfactory accuracy. Results obtained from verified testing were also in good agreement with those obtained with the model. Clearly, phosphate, nitrate, and ammonium all exhibited negative effects on the growth of VP. These outcomes are beneficial to the culture of VP, and even to the establishment of early warning systems for marine environments and seafood safety.
Vibrio parahaemolyticus(VP), which is ubiquitous in the marine environment, is one of the key foodborne pathogens affecting humans. It is thus of great significance for both scientific research and public safety that we understand how and what environmental factors affect its growth. To determine the functional relationship between a combination of environmental factors and VP growth, in this study the effects of temperature, salinity, pH, phosphate, nitrate, and ammonium on the growth of this kind of bacteria were investigated using response surface methodology(RSM). First, a Box-Behnken design was used to optimize the three critical internal factors of temperature, salinity and pH for this species, using optical density(OD_(600 nm)) as a marker for the growth of VP. It was found that for these factors, the optimum parameters were 34.5℃, 3%, and 8.0, respectively. Based on these results, the combined effects of phosphate, nitrate, and ammonium on the growth of VP were then further studied with the same method to develop a response model. The coefficient of determination(R~2) of the model equation was 0.9217, while the correction coefficient(R~2) was 0.8211. The regression model had an F-value of 9.16, and was thus statistically significant(P=0.004). These results indicated that the model proposed herein had satisfactory accuracy. Results obtained from verified testing were also in good agreement with those obtained with the model. Clearly, phosphate, nitrate, and ammonium all exhibited negative effects on the growth of VP. These outcomes are beneficial to the culture of VP, and even to the establishment of early warning systems for marine environments and seafood safety.
引文
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Beuchat LR.Interacting effects of pH,temperature,and salt concentration on growth and survival of Vibrio parahaemolyticus.Applied Microbiology,1973,25(5):844
Bjk D,Jacobs JM,Davis MF,et al.Environmental determinants of Vibrio parahaemolyticus in the Chesapeake Bay.Applied&Environmental Microbiology,2017,83(21):e01147-17
Blackwell KD,Oliver JD.The ecology of Vibrio vulnificus,Vibrio cholerae,and Vibrio parahaemolyticus in North Carolina estuaries.Journal of Microbiology,2008,46(2):146-153
Bonninjusserand M,Copin S,Le CB,et al.Vibrio species involved in seafood-borne outbreaks(Vibrio cholerae,V.parahaemolyticus and V.vulnificus):Review of microbiological versus recent molecular detection methods in seafood products.Critical Reviews in Food Science and Nutrition,2018,in press,DOI:10.1080/10408398.2017.1384715
Chen XT,Huang LP,Sun M,et al.Optimization of conversion process ofα-cyclodextrin with enzyme using response surface methodology.Progress in Fishery Sciences,2017,38(5):143-151[陈晓彤,黄立萍,孙谧,等.响应面分析法优化α-环糊精的酶转化工艺.渔业科学进展,2017,38(5):143-151]
Ching B,Chew SF,Wong WP,et al.Environmental ammonia exposure induces oxidative stress in gills and brain of Boleophthalmus boddarti(mudskipper).Aquatic Toxicology,2009,95(3):203-212
Criminger JD,Hazen TH,Sobecky PA,et al.Nitrogen fixation by Vibrio parahaemolyticus and its implications for a new ecological niche.Applied&Environmental Microbiology,2007,73(18):5959-5961
Ham H,Orth K.The role of typeⅢsecretion System 2 in Vibrio parahaemolyticus pathogenicity.Journal of Microbiology,2012,50(5):719
Hermenegildo C,Monfort P,Felipo V.Activation of N-methyl-D-aspartate receptors in rat brain in vivo following acute ammonia intoxication:Characterization by in vivo brain microdialysis.Hepatology,2000,31(3):709
Han JR,Zhang FH.Optimization of submerged fermentation medium for agaricus bisporus by rseponsesurface methodology.Journal of ShanxiUniversity(Natural Science Edition),2017,40(3):602-608[韩建荣,翟飞红.响应面法优化双孢蘑菇液态发酵培养基.山西大学学报(自然科学版),2017,40(3):602-608]
Huang MS,Yang QQ,Zhang Y et al.Rapid detection method of Vibrio parahaemolyticus in seawater based on visual LAMP.Chinese Fishery Quality and Standards,2017,7(6):58-65[黄梦诗,杨倩倩,张艳,等。海水中副溶血弧菌的可视化LAMP快速检测方法.中国渔业质量与标准,2017,7(6):58-65]
Jiang B,Wang CG,Du X,et al.Optimization of fermentation medium for keratinase production from Bacillus sp.CJPE209using reponse surfsce methodology.China Brewing,2017,36(5):76-80[蒋彪,王常高,杜馨,等.响应面法优化芽孢杆菌CJPE209产角蛋白酶发酵培养基的研究.中国酿造,2017,36(5):76-80]
Johnson CN.Influence of environmental factors on Vibrio spp.in coastal ecosystems.Microbiology Spectrum,2015,3(3):2008-2014
Johnson CN,Bowers JC,Griffitt KJ,et al.Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the coastal and estuarine waters of louisiana,Maryland,Mississippi,and Washington(United States).Applied&Environmental Microbiology,2012,78(20):7249-7257
Johnson CN,Flowers AR,Iii NFN,et al.Relationships between environmental factors and pathogenic Vibrios in the Northern Gulf of Mexico.Applied&Environmental Microbiology,2010,76(21):7076-7084
Kaper J,Lockman H,Colwell RR,et al.Ecology,serology,and enterotoxin production of Vibrio cholerae in Chesapeake Bay.Applied&Environmental Microbiology,1979,37(1):91
Karami HR,Keyhani M,Mowla D.Experimental analysis of drag reduction in the pipelines with response surface methodology.Journal of Petroleum Science&Engineering,2016,138(2):104-112
Kumar M,Jain AK,Ghosh M,et al.Statistical optimization of physical parameters for enhanced bacteriocin production by L.casei.Biotechnology&Bioprocess Engineering,2012,17(3):606-616
Liu N,Zou D,Dong D,et al.Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp.and Vibrio parahaemolyticus.Scientific Reports,2017,7:45601
Liu DW,Ning XB,Zhang JL,Optimization of growth condition of Vibrio parahaemolyticus via response surface methodology.Microbiology China,2008,35(2):306-310[刘代新,宁喜斌,张继伦.响应面分析法优化副溶血性弧菌生长条件.微生物学通报,2008,35(2):306-310]
Liang Y,Lu GH,Qi PD,et al.Influence of nutrients on absorption and ecotoxicological effects of contaminants in aquatic ecosystem:A review of recent researches.Journal of Environment and Health,2011,28(2):181-184[梁艳,陆光华,齐鹏德,等.水体中营养盐对污染物生物吸收及生物毒性的影响.环境与健康杂志,2011,28(2):181-184]
Mccarter LL,Silverman M.Phosphate regulation of gene expression in Vibrio parahaemolyticus.Journal of Bacteriology,1987,169(8):34-41
Oliver JD,Warner RA,Cleland DR.Distribution of Vibrio vulnificus and other lactose-fermenting vibrios in the marine environment.Applied&Environmental Microbiology,1983,45(3):985-998
Randa MA,Polz MF,Lim E.Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR.Applied&Environmental Microbiology,2004,70(9):5469-5476
Rehnstam-Holm AS,Atnur V,Godhe A.Defining the niche of Vibrio parahaemolyticus during pre-and post-monsoon seasons in the Coastal Arabian Sea.Microbial Ecology,2014,67(1):57-65
Sun BY,Yang DD,Chen LM.A multivariate reciprocity analysis of Vibrio parahaemolyticus in the inshore waters along China East Coast.Journal of Microbiology,2015(2):109-112[孙博怿,杨丹丹,陈兰明.我国东部海域近岸水体中副溶血性弧菌的多元相关性分析.微生物学杂志,2015(2):109-112]
Young I,Gropp K,Fazil A,et al.Knowledge synthesis to support risk assessment of climate change impacts on food and water safety:A case study of the effects of water temperature and salinity on Vibrio parahaemolyticus in raw oysters and harvest waters.Food Research International,2015,68:86-93
Yin SL,Zhang YX,Zhang Q,et al.The Effect of inorganic phosphate on the biosynthesis of secondary metabolites in streptomyces.China Biotechnology,2015,35(9):105-113[尹守亮,张玉秀,张琪,等.无机磷酸盐对链霉菌合成次级代谢产物的影响.中国生物工程杂志,2015,35(9):105-113]
Zhang C,Yuan Q,Lu Y.Inhibitory effects of ammonia on methanogen mcrA transcripts in anaerobic digester sludge.FEMS Microbiology Ecology,2014,87(2):368-377
Beuchat LR.Interacting effects of pH,temperature,and salt concentration on growth and survival of Vibrio parahaemolyticus.Applied Microbiology,1973,25(5):844
Bjk D,Jacobs JM,Davis MF,et al.Environmental determinants of Vibrio parahaemolyticus in the Chesapeake Bay.Applied&Environmental Microbiology,2017,83(21):e01147-17
Blackwell KD,Oliver JD.The ecology of Vibrio vulnificus,Vibrio cholerae,and Vibrio parahaemolyticus in North Carolina estuaries.Journal of Microbiology,2008,46(2):146-153
Bonninjusserand M,Copin S,Le CB,et al.Vibrio species involved in seafood-borne outbreaks(Vibrio cholerae,V.parahaemolyticus and V.vulnificus):Review of microbiological versus recent molecular detection methods in seafood products.Critical Reviews in Food Science and Nutrition,2018,in press,DOI:10.1080/10408398.2017.1384715
Chen XT,Huang LP,Sun M,et al.Optimization of conversion process ofα-cyclodextrin with enzyme using response surface methodology.Progress in Fishery Sciences,2017,38(5):143-151[陈晓彤,黄立萍,孙谧,等.响应面分析法优化α-环糊精的酶转化工艺.渔业科学进展,2017,38(5):143-151]
Ching B,Chew SF,Wong WP,et al.Environmental ammonia exposure induces oxidative stress in gills and brain of Boleophthalmus boddarti(mudskipper).Aquatic Toxicology,2009,95(3):203-212
Criminger JD,Hazen TH,Sobecky PA,et al.Nitrogen fixation by Vibrio parahaemolyticus and its implications for a new ecological niche.Applied&Environmental Microbiology,2007,73(18):5959-5961
Ham H,Orth K.The role of typeⅢsecretion System 2 in Vibrio parahaemolyticus pathogenicity.Journal of Microbiology,2012,50(5):719
Hermenegildo C,Monfort P,Felipo V.Activation of N-methyl-D-aspartate receptors in rat brain in vivo following acute ammonia intoxication:Characterization by in vivo brain microdialysis.Hepatology,2000,31(3):709
Han JR,Zhang FH.Optimization of submerged fermentation medium for agaricus bisporus by rseponsesurface methodology.Journal of ShanxiUniversity(Natural Science Edition),2017,40(3):602-608[韩建荣,翟飞红.响应面法优化双孢蘑菇液态发酵培养基.山西大学学报(自然科学版),2017,40(3):602-608]
Huang MS,Yang QQ,Zhang Y et al.Rapid detection method of Vibrio parahaemolyticus in seawater based on visual LAMP.Chinese Fishery Quality and Standards,2017,7(6):58-65[黄梦诗,杨倩倩,张艳,等。海水中副溶血弧菌的可视化LAMP快速检测方法.中国渔业质量与标准,2017,7(6):58-65]
Jiang B,Wang CG,Du X,et al.Optimization of fermentation medium for keratinase production from Bacillus sp.CJPE209using reponse surfsce methodology.China Brewing,2017,36(5):76-80[蒋彪,王常高,杜馨,等.响应面法优化芽孢杆菌CJPE209产角蛋白酶发酵培养基的研究.中国酿造,2017,36(5):76-80]
Johnson CN.Influence of environmental factors on Vibrio spp.in coastal ecosystems.Microbiology Spectrum,2015,3(3):2008-2014
Johnson CN,Bowers JC,Griffitt KJ,et al.Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the coastal and estuarine waters of louisiana,Maryland,Mississippi,and Washington(United States).Applied&Environmental Microbiology,2012,78(20):7249-7257
Johnson CN,Flowers AR,Iii NFN,et al.Relationships between environmental factors and pathogenic Vibrios in the Northern Gulf of Mexico.Applied&Environmental Microbiology,2010,76(21):7076-7084
Kaper J,Lockman H,Colwell RR,et al.Ecology,serology,and enterotoxin production of Vibrio cholerae in Chesapeake Bay.Applied&Environmental Microbiology,1979,37(1):91
Karami HR,Keyhani M,Mowla D.Experimental analysis of drag reduction in the pipelines with response surface methodology.Journal of Petroleum Science&Engineering,2016,138(2):104-112
Kumar M,Jain AK,Ghosh M,et al.Statistical optimization of physical parameters for enhanced bacteriocin production by L.casei.Biotechnology&Bioprocess Engineering,2012,17(3):606-616
Liu N,Zou D,Dong D,et al.Development of a multiplex loop-mediated isothermal amplification method for the simultaneous detection of Salmonella spp.and Vibrio parahaemolyticus.Scientific Reports,2017,7:45601
Liu DW,Ning XB,Zhang JL,Optimization of growth condition of Vibrio parahaemolyticus via response surface methodology.Microbiology China,2008,35(2):306-310[刘代新,宁喜斌,张继伦.响应面分析法优化副溶血性弧菌生长条件.微生物学通报,2008,35(2):306-310]
Liang Y,Lu GH,Qi PD,et al.Influence of nutrients on absorption and ecotoxicological effects of contaminants in aquatic ecosystem:A review of recent researches.Journal of Environment and Health,2011,28(2):181-184[梁艳,陆光华,齐鹏德,等.水体中营养盐对污染物生物吸收及生物毒性的影响.环境与健康杂志,2011,28(2):181-184]
Mccarter LL,Silverman M.Phosphate regulation of gene expression in Vibrio parahaemolyticus.Journal of Bacteriology,1987,169(8):34-41
Oliver JD,Warner RA,Cleland DR.Distribution of Vibrio vulnificus and other lactose-fermenting vibrios in the marine environment.Applied&Environmental Microbiology,1983,45(3):985-998
Randa MA,Polz MF,Lim E.Effects of temperature and salinity on Vibrio vulnificus population dynamics as assessed by quantitative PCR.Applied&Environmental Microbiology,2004,70(9):5469-5476
Rehnstam-Holm AS,Atnur V,Godhe A.Defining the niche of Vibrio parahaemolyticus during pre-and post-monsoon seasons in the Coastal Arabian Sea.Microbial Ecology,2014,67(1):57-65
Sun BY,Yang DD,Chen LM.A multivariate reciprocity analysis of Vibrio parahaemolyticus in the inshore waters along China East Coast.Journal of Microbiology,2015(2):109-112[孙博怿,杨丹丹,陈兰明.我国东部海域近岸水体中副溶血性弧菌的多元相关性分析.微生物学杂志,2015(2):109-112]
Young I,Gropp K,Fazil A,et al.Knowledge synthesis to support risk assessment of climate change impacts on food and water safety:A case study of the effects of water temperature and salinity on Vibrio parahaemolyticus in raw oysters and harvest waters.Food Research International,2015,68:86-93
Yin SL,Zhang YX,Zhang Q,et al.The Effect of inorganic phosphate on the biosynthesis of secondary metabolites in streptomyces.China Biotechnology,2015,35(9):105-113[尹守亮,张玉秀,张琪,等.无机磷酸盐对链霉菌合成次级代谢产物的影响.中国生物工程杂志,2015,35(9):105-113]
Zhang C,Yuan Q,Lu Y.Inhibitory effects of ammonia on methanogen mcrA transcripts in anaerobic digester sludge.FEMS Microbiology Ecology,2014,87(2):368-377