Microbes Quality Changes Of Quick-Frozen Dumpling And Establishment Of A Multiplex PCR For The Simultaneous Detection Of Important Pathogens

In this dissertation, the quick-frozen dumpling was used a materialsto elucidate the effect of different conditions on total bacteria colonies of quick-frozendumpling, the dominant spoilage organisms composition and its relationship betweenthe quality change of dumplings during storage, the effect of repeated freeze-thaw onthe the microbiological and physical quality of quick-frozen dumpling. Lastly, asimply multiplex PCR assays was established to sensitively and accurately detectSalmonella spp and Staphylococcus aureus, which were two important foodbornepathogens in frozen food. The details of this research were as follows:For frozen food, the objective level of microbiological pollution could bereflected actually only when samples was thawed out completely before inspection.Among the two plate count method including spread plate and6x6plate countmethods, the spread plate method was more suitable for quick-frozen food. The timeinterval between samples dilution and plate spread had better less than20min when alot of samples were detected.During the whole96d frozen storage, the total viable count of dumplingsincrease slightly, no apparent changes could be detected in pH value, althoughhardness, springiness, cohesiveness, chewiness all declined slightly.Combined withthe traditionally culture-based isolation and16S rDNA identification method,17strains of bacteria from quick-frozen dumpling were classified to belong to4generaand6kinds. These strains are Streptococcus equines, Streptococcus bovis,Streptococcus suis, Pseudomonas argentinensis, Enterobacter cloacae, Pantoeaagglomerans, respectively.Simulation temperature fluctuations in cold chain, the effect of repeatedfreeze-thaw on frozen dumpling quality were studied, the results showed thattemperature fluctuations could accelerate acid value, peroxide value to exceeds thenational standard; With the increase of repeated freeze-thaw, the dumpling meatstuffing acid value, peroxide value, thiobarbituric acid were gradually increase, totalbacteria count, coliform group count increases slightly, Total yeast and mold count first increased and then declined.After optimization of multiple polymerase chain reaction condition, the bestannealing temperature was60℃, the best primer concentration was200nM, the bestcycles was35times. Among3kinds of Genomic DNA extraction methods, the kitsmethod is more sensitive, accurate when multiple PCR detect a large number ofsamples. The sensitivity of boiled method was lower than kit method, however, it cansave time and cost when was used in multiple PCR detection. Before DNA extractionand multiple PCR detection, the bacteria should be enriched to make up for the lowsensitivity of the boiled method.When evalute the multiple PCR detection sensitivity combined with the DNAgenome kit based extraction method, as low as approximately31and26genomicDNA copies/reaction from Salmonella spp and Staphylococcus aureus could bedetected, respectively. When presently established multiple PCR was applied in thetwo food-borne pathogens detection in artificially contaminated frozen food,after only4h of culture period, as low to10CFU/10g of starting Salmonella sppand Staphylococcus aureus colonies in frozen dumpling could be detectedsimultaneously.