Growth Kinetics Of Dominant Spoilage Bacteria And Mechanism Of Cold Tolerance In Sinonovacula Constricta During Controlled Freezing-point Storage

This study explored the effect of controlled freezing-point storage on the quality of Sinonovacula constricta.The structure and dynamics of the microbial community in Sinonovacula constricta during controlled freezing-point storage was investigated by Illumina high-throughput sequencing technology,and the growth kinetic models for dominant spoilage bacteria(Pseudomonas spp.)were also studied at different temperatures.To better understand the molecular mechanisms of cold tolerance of Sinonovacula constricta,two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS),which was coupled with isobaric tags for relative and absolute quantification(iTRAQ)labeling,was employed to detect the changes of proteome in Sinonovacula constricta.The main results obtained in this study were as follows:1.The ecological ice temperature of Sinonovacula constricta that found in this study was-1.9?1.5 °C,and the controlled freezing-point storage at-1.5 °C can prolong the survival time of Sinonovacula constricta up to 11 days.In this work,the freshness and quality of Sinonovacula constricta stored under three different temperatures(-1.5,10 and 20 °C)were analyzed by sensory,physicochemical and microbiological methods,and the results showed that the storage temperature of-1.5 °C was best for keeping Sinonovacula constricta alive.The major quality indices that reflect the freshness of Sinonovacula constricta were sensory score,TVB-N value,K' value,texture(hardness,springiness and adhesiveness)and total viable count,and the pH value was a reference index.2.The results showed that the initial bacterial flora in fresh Sinonovacula constricta was complicated,which was mainly composed of Arcobacter spp.(16.2%),Bacteroidales(8.4%)and Psychrilyobacter spp.(7.2%).At the end of keeping-alive period,Pseudomonas spp.became the dominant spoilage bacteria,which accounted for 26.2%.The other dominant bacteria were presented as Vibrionaceae,Psychromonas spp.,Arcobacter spp.and Psychrilyobacter spp.,with the proportion of 17.8%,14.4%,13%and 11.8%,respectively.The growth dynamics of Pseudomonas spp.at low temperatures(-1.5?15.0 ?)could be described by the modified Gompertz model.The determination coefficient(R2)exceeded 0.98 and the bias factor and accuracy factor indicated that the model was established within statistical acceptable ranges.The square-root model for temperature dependence of both the specific growth rate and lag-phase duration showed better linear effectiveness,with the determination coefficients(R2)of 0.963 and 0.970,respectively.The established Gompertz model can be used for predicting the growth of Pseudomonas spp.in the range of-1.5?15.0 ?.3.A total of 27 differentially expressed proteins in Sinonovacula constricta stored under controlled freezing-point storage,including 4 up-regulated and 23 down-regulated,were identified by using the iTRAQ-based 2D LC-MS/MS technology.Gene Ontology(GO)analyses indicated that these differentially expressed proteins were mostly related to energy metabolism process,stress response,DNA duplication and protein synthesis.Pathways enrichment analyses of the differential expressed proteins were performed by using the KEGG database,and the pathways that were probably related to cold tolerance of Sinonovacula constricta were screened.Results showed that the differentially expressed proteins were significantly enriched in MAPK signaling pathway,oocyte meiosis pathway and long-term potentiation.The MAPK signaling pathway,oxidative phosphorylation,TCA cycle,calcium signaling pathway,starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism and carbon metabolism were probably related to cold tolerance of Sinonovacula constricta.Particularly,the MAPK signaling pathway was the most important,the proteins involved in this pathway included ribosomal protein S6 kinase 2 alpha,HSP 70,calcineurin B subunit and two uncharacterized proteins.