Study On Fatty Acids Profile Of Vibrio Parahaemolyticus By Gas Chromatography

Vibrio parahaemolyticus is the primary reason for bacterial food poisioning incoastal areas. It is found globally in marine products such as fish, shrimp, shelfish andso on. Pathogenic strains cause a great damage to human health and property. Rapidand accurate identification of V. parahaemolyticus in food is not only the concernedproblem, but also the important assignment to guarantee food safety. Fatty acid, oneof a microbial marker, has widely applied in bacterial identification and ecologicalanalysis. The cellular fatty acid in V. parahaemolyticus is comprehensively studied bygas chromatography, which is for the porpurse of detecting V. parahaemolyticus andbuilting a fingerprint. The results were as follows:(1) Nine strains of V. parahaemolyticus were isolated from Zhanqiao Bridge andthe Beaches through plate separation, PCR and gene sequencing. Among these strains,there were four strains of V. parahaemolyticus and three strains of Vibrio genus, whilethe other two strains were not been confirmed and discussed.(2) In the optimization on the conditions of fatty acid derivatization, a greatinfluence of different saponification and methyl esterification conditions on theanalysis results was discorvered. Therefore, appropriate derivatization conditions arevery necessary for fatty acid analysis. With the standard of the peak quantities, thepeak height and the percentage of fatty acids, it was found that saponification in lowtemperature was beneficial to the quantity and height of chromatogram; Methylesterification in high temperature easily damaged the structure of long-chain andunsaturated fatty acids. Moreover, the longer the time was, the worse the effect ofmethyl esterification was. Under the experimental conditions, the optimumderivatization conditions were as follows: saponification,2mL2mol/LNaOH-methanol, water bath at70℃for10min; methyl esterification,2mL10%H2SO4-methanol, water bath at70℃for15min.(3) The impact of culture temperature, medium pH, culture time and low temperature in4℃on fatty acid patterns of one strains of V. parahaemolyticus wasdiscussed. The result indicated that the percentage of saturated fatty acids increasedgradually concomitant with a decreased production of unsaturated fatty acids withrising temperature. In low pH environment, short-chain fatty acids had a greatadvantage over long-chain fatty acids, which was reverse in high pH temperature.Most unsaturated fatty acids, except C17:1, tended to decrease with prolonged culturetime, while the content of saturated fatty acids was gradually increasing.4℃is an adverse temperature for most bacteria to grow. From6d to24d, thecolony counts of V. parahaemolyticus were reduced, finally to zero. The alterations infatty acids composition were observered during the period of time. As a result of atemperature downshift to4℃,the cyclic change of C12:0, C13:0, C14:0, C14:1andC15:0was divided into three stages, taking on the trend of droping first, then risingand droing finally; whereas C18:1(9c) and C18:1(11c) appeared the tendency of risinginitially, then droping and rising ultimately; the percent of C17:0and C17:1wasdeclining.(4) Fatty acids of fifteen strains of V. parahaemolyticus were detected by gaschromatography. Eight strains of V. parahaemolyticus were similar in fatty acidscomposition. They were same in fatty acid types, including saturated fatty acids fromC12to C18, monounsaturated fatty acids from C14to C18and some branched chainfatty acids, whose percentage were28.23%～35.35%,57.48%～64.70%and1.19%～5.27%, respectively. C16:0, C16:1(9c) and C18:1(11c), with the percentage rangefrom77.74%to83.20%, were dominant fatty acids in V. parahaemolyticus.However, V. parahaemolyticus and other seven strains of marine bacteria differedin fatty acids profile. Relatively speaking, the diversity in cellular fatty acids patternwere not markedly in strains belong to same genus. Moreover, fatty acids profile wasmuch more different between Pseudomonas aeruginosa (P.a), Aeromonas hydrophila(A.h), Shewanella putrefaciens (YY) and V. parahaemolyticus. For example, thepreponderant fatty acids were C16:1(9c), C13:0and12methyl14:0in YY, which wasdifferent from V. parahaemolyticus. Additionally, their percentage was65.34%.(5) With the basis of fatty acids composition, the fingerprint of V. parahaemolyticus was built via similarity analysis and principal component analysis.The similarity analysis of each sample was processed compared with the average ofeight strains of V. parahaemolyticus and the conclusions were as follows: the strainswere considered as V. parahaemolyticus when the matching values of correlationcoefficient and vector cosine were higher than0.980; while bacteria, with scoresranging from0.900to0.980, respectively, were regarded as Vibrio; samples with lowcorrelation coefficient (<0.850) and vector cosine (<0.850) were categorized underother genera. In summary, analysis on FA profile is valuable for the physiologicalstudy of V. parahaemolyticus and its rapid identification.