Carbon Utilization And Growth Kinetics Of Shewanella Putrefaciens Isolated From Fish

Shewanella putrefaciens is a common spoilage bacteria in aquatic products,even in the ice temperature it can also translate oxidation of trimethylamine to trimethylamine,dimethylamine,formaldehyde and H2 S,which can change the texture and sensory properties of aquatic products.Studies have shown it has a strong ability of spoilage,and it can adhere to the surface of the food and forms biofilm,increases pathogenic potential,and so on.However,the mechanism of spoilage is still not very clear,and aquatic product shelf life prediction model development will be the focus of future research areas.This paper sets out the metabolic potential and activity of SSOs and the estimation of the growth of spoilage in different environmental conditions,providing us with a tool for rapid assessment of fish freshness/spoilage status and shelf life.In this paper,the effects of environmental factors on its growth by establishing the growth model and the effect of environmental factors on the carbon source metabolism are studied.The main contents of this paper include the following three aspects:(1)The Shewanella separated from corruption large yellow croaker was used as the research object.S.putrefaciens' growth data under different pH,NaCl and temperature was measured using viable count and optical density methods,and Gompertz model was built.The effect of environmental factors on growth kinetic parameters was compared and analyzed.The results showed that at pH6.0 and NaCl3.5%,S.putrefaciens couldn't grow during 3-8?.The differences of maximum specific growth rate(?max)and generation time were not significant(p>0.05),but the differences of lag phase(?)and most probable density(MPD)were significant(p<0.05).Environmental factors had significant effect on the growth kinetic parameters.The higher temperature or lower NaCl concentration resulted from shorter generation time.During 3-25 ?,the higher temperature resulted from larger ?max.The ?max increased with the speed of 0.014±0.003h-1/?.When temperature was higher,? would be shorter.The ? decreased at a speed of 1.5h/? using viable count method,while during 8-15?,it decreased at a speed of approximate 2.5h/? by optical density method.The ?max was influenced significantly by NaCl and pH,while lag time showed no significant changes by viable count method.Using optical density method,the changes of ?max were effected no significant by NaCl and pH,while lag times' were apparent.Thus,two counting methods could both judge the Shewanella growth/non-growth state,and using opticaldensity methods,the error of estimates of ? and MPD increased with more harsh environments.(2)In order to study the effect of different temperature,pH,NaCl and ?-polylysine on growth of S.putrefaciens from fish,the turbidity method was used to obtain the data under different experimental conditions after the best inoculation size was determined.Then the modified Gompertz equation was fitted to get kinetic parameters,and the influence of the environment on the kinetic parameters was analyzed.At last,the square root equation was fitted and the influence of temperature on kinetic parameters was analyzed.The results showed that:(1)the best inoculation size was106-107 CFU/mL,and the OD value with inoculation sizes in the range of106-109CFU/mL showed a good linear relationship.the maximum specific growth rate(?max)was not affected by inoculation size,but the lag time was opposite.At5? and pH ? 5,S.putrefaciens did not grow,at 10-30? and pH 4.0 S.putrefaciens did not grow;at 5? and Aw ? 0.951 S.putrefaciens did not grow,at 15-30? and Aw?0.927,S.putrefaciens did not grow;at 5-15? and NaCl concentration ? 5.5%,S.putrefaciens did not grow,and at 20-30 ?,NaCl concentration ?8.5% S.putrefaciens did not grow;at 5? ?-polylysine concentration?0.8% or at 15-30? ?-polylysine concentration ?0.4%,S.putrefaciens did not grow.Besides ?-polylysine concentration ?0.05%,the inhibitory effect is not obvious.The temperature had a great effect on the growth parameters of S.putrefaciens under different pH,Aw,NaCl and ?-polylysine conditions: 5-15?,Aw/ NaCl with ?max and ?-polylysine with ? showed a good linear relationship.Aw/ NaCl with ? and ?-polylysine with ?max showed a good binomial relationship.In condition of pH,?max changes were affected by temperature,but in condition of Aw,NaCl and ?-polylysine,?max changes were not affected by temperature;In condition of pH,Aw and NaCl,? changes were affected by temperature,but in condition of?-polylysine,? changes were not affected by temperature.(3)In order to investigate carbon source utilization of S.putrefaciens under different temperatures,growth data of S.putrefaciens at 5,15,25 and 33? were obtained by porous plate technique.Then growth kinetics parameters(?max and ?)were obtained by fitting the modified Gompertz equation.Combining growth kinetics parameters,average well color development(AWCD)and utilization area,carbon source utilization of S.putrefaciens was analyzed.The results showed that at 25 ? S.putrefaciens had the highest carbon utilization and activity,15? followed,33? lowest.The utilization proportion of carbohydrates and carboxylic acids were 30% ± 0.014 and 29% ± 0.027 at5-33?,respectively.Temperature had a great influence on the utilization of different types of carbon source of Shewanella.At the same temperature,the ? of every kind of carbon source didn't change obvious,but the ?max changed obviously.In carbohydrates,at 5 ? dextrin and D-maltose could be better utilizated,at 15 ? dextrin and?-D-glucose could be better utilizated,at 25? D-Arabinol,stachyose could be better utilizated,at 33? no one could be better utilizated.In carboxylic acid,at 5?,L-malic acid and L-lactic acid could be better utilizated,at 15? propionic acid and acetic acid could be better utilizated,at 25? propionic acid,acetoacetic acid and D-malic acidcould be better utilizated,at 33? only carboxylic acid could be better utilizated.In amino acids,at 5 ? only L-serine could be better utilizated,at 15 ? L-serine and amino-acetyl-L-proline could be better utilizated,at 25 ? aminoacyl-L-proline,LSerine,L-aspartic acid and L-glutamic acid could be better utilizated,and at 33 ?L-aspartic acid could be better utilizated.In At 5? gelatin and inosine could be better utilizated,at 15? gelatin and methyl pyruvate could be better utilizated,at 25?Tween 40 and inositol could be better utilizated,at 33? only L-histamine and bromine-succinic acid could be better utilizated.