Study On Preservation Effect Of Compound Green Food Preservatives On Carassius Auratus

Carassius auratus is a traditional freshwater fish in China,the output of which is at the forefront among all the freshwater fishes.Carassius auratusis are riching in calcium,iron,vitamins,riboflavin and many other nutrients.However,a spoilage problem raises for carassius auratus due to the high contents of water,proteins,unsaturated fatty acids and endogenous enzymes.The spoilage of aquatic products can be attributed to the microorganisms,enzymes and fat oxidation.Among them,microbial contamination is the main contributor.Therefore,it is important to study the evolutions of spoilage microorganisms in carassius auratus and develop novel technologies to inhibit the microbial growth.The results can provide guidance for aquatic products fishing,processing,storage,transportation and marketing.In this thesis,the dominant spoilage bacteria were selected through analyzing thr microbial community in spoiled carassius auratus.Meanwhile,a green antimicrobial peptide with a wide antibacterial spectrum was developed according to the previous studies in our laboratory.To study the effect of the aforementioned peptide on the preservation of Carassius auratus,single factor experiment and orthogonal experiments were performed.The optimal addition of antimicrobial peptide was obtained consequently.These results are helpful to apply antimicrobial peptide for the preservation of Carassius auratus in practical application.1.Analysis of microflora changes in spoiled Carassius auratus.Three methods,including traditional physiological and biochemical analysis combined with 16SrDNA molecular biology method,polymerase chain reaction-amplification and denaturing gradient gel electrophors(PCR-DGGE),and high-throughput sequencing were used to analyze the evolution of bacteria and identify dominant spoilage bacteria in Carassius auratus during storage.The results showed that Pseudomonas,Acinetobacter,Flavobacterium and Aeromonas were the dominant spoilage bacteria in spoiled Carassius auratus according to traditional physiological and biochemical analysis.Next,the PCR-DGGE result revealed that Pseudomonas,Brochothrix thermosphacta,Acinetobacter and Aeromonas were the dominant spoilage bacteria.Furthermore,through the high-throughput sequencing,365568 sequences were obtained from 12 different samples,which indicating that Brochothrix thermosphacta,Pseudomonas and Aeromonas were the dominant spoilage bacteria in Carassius auratus.Comparing the aforementioned three analyzing methods,it was found that the results obtained by high throughput sequencing technology were more objective,comprehensive and accurate than that of other methods.2.Screening of Green Preservatives and Preparation of mixed Preservative.The inhibitory effect of 12 preservatives(sodium erythorbate,citric acid,EDTA-2Na,potassium sorbate,sodium dehydroacetate,sodium acetate,sodium propionate,sodium lactate,sodium benzoate,Nisin,kojic acid and thymol)on dominant spoilage bacteria(Pseudomonas fluorescens,Aeromonas hydrophila and Brochothrix thermosphacta)in Carassius auratus were assessed.The preservatives with high inhibitory effects were then selected.Next,the selected preservatives were mixed with two antimicrobial peptides.One was the crude extract from Bacillus amyloliquefaciens broth(ALE),while the other one was the crude extract from Lactobacillus plantarum 163 broth(ALP).Carassius auratus was soaked the prepared solutions containing preservatives.The preservation effect was evaluated through the determination of total number of colonies,total volatile basic nitrogen(TVB-N),thiobarbituric acid reactive substance(TBA),pH value and sensory evaluation indexes.Single factor experiments and orthogonal experiments were carried out to optimize the combination of different preservatives.The results showed that thymol had the highest antibacterial capacity,followed by EDTA-2Na,kojic acid,citric acid and sodium dehydroacetate.Meanwhile,all the three types of preservatives could decrease the the total number of colonies,TVB-N and TBA values,as well as increase the pH value of Carassius auratus.The shelf-lifes of Carassius auratus were extended by 2,6 and 4 days after the treatments of thymol(2.5%),ALE(67608 IU/mL)and ALP(35418 IU/mL),respectively.According to the orthogonal experiment results,the optimal combination of thymol,ALE and ALP was 2%thymol,32359 IU/mL antibacterial lipopeptide crude extract ALE+35418 IU/mL bacteriocin crude extract ALP,which was named as Preservative A.The ANOVA analysis exhibited that thymol,ALE and ALP all could affect the total colony number in Carassius auratus during storage significantly,demonstrating that all of them can delay the spoilage and deterioration of Carassius auratus.3.Application of Preservative A in preservation of Carassius auratus.Carassius auratus were soaked in the solution containing Preservative A at the aforementioned optimal concentration.Meanwhile,Carassius auratus treated by sterile water and 2%Nisin were regarded as control samples.The total number of colonies,the main dominant spoilage bacteria colony number,TVB-N,TBA,pH value and sensory evaluation indexes were measured to evaluate the preservative effect of Preservative A on Carassius auratus during storage.The results showed that Preservative A effectively inhibited the growth of bacteria including dominant spoilage bacteria(Brochothrix thermosphacta,Aeromonas and Pseudomonas),was well as decreased the TVB-N,pH and TBA values.The shelf-life of Carassius auratus was extended by 9 d,which was 8 d longer than that of Carassius auratus treated by 2%Nisin.This result showed that Preservative A was a better preservative for the preservation of crucian carp than nisin within the selected experimental range.