A Preliminary Study On Macroproteomics And Metabolomics Of The Raw Milk During Refrigerated Storage

During the refrigeration of raw milk,exogenous enzyme secreted by the microorganisms convert the nutrients in milk into small molecule metabolites such as amino acids and fatty acids,making the nutrient composition change,and eventually leads to spoilage of the raw milk.The raw milk was refrigerated at 4℃ for 6 days.The macroproteomics and non-target metabolomics techniques were used to study the changes of the microbial protein and metabolites in raw milk which was refrigerated for 0 day,1 day,3 days,4 days and 6 days.The results are as follows:（1）Lable free technology was used for macroproteomics research.A total of 341 microbial proteins were identified during the refrigeration of raw milk,and most of them appeared in 3 days refrigeration.PCA analysis showed that the microbial proteins in the samples changed significantly at the end of refrigeration stage.Throughout the refrigeration,lots of proteins’function were cell wall/membrane/envelope biogenesis and transcription.The number of proteins involved in the metabolic pathways of two-component system and legionellosis were all greater than 6 at each time point.The relative abundance of proteins involved in amino sugar and nucleotide sugar metabolism was higher and always greater than 20%during the process.The mainly microbial proteins were produced by Pseudomonas,Clostridium,Lactococcus,Acinetobacter,Mycobacterium,and Lactobacillus.The relative abundance of proteins from Pseudomonas was the highest,which was always greater than 38%in the stage.With the extension of refrigeration time,the number of differentially expressed proteins increased.Among the pathways that the differentially expressed proteins involved in,aminoacyl-tRNA biosynthesis pathway changed significantly after 1 day.After 3 days,plant-pathogen interaction pathway and necroptosis pathway changed significantly.After 4 days,plant-pathogen interaction,quorum sensing,glycolysis/gluconeogenesis,carbon fixation in photosynthetic organisms and HIF-1 signaling pathway changed significantly.After 6 days,quorum sensing pathway and necroptosis pathway changed significantly.（2）The differently expressed metabolites were identificated by LC-MS.Compared with 0 d,the number of significantly differently expressed metabolites gradually increased with the extension of refrigeration time.The number of significantly differently expressed metabolites for 1 d/0 d,3 d/0 d,4 d/0 d and 6 d/0 d were 8,24,25,and 30 in positive ion mode.In negative ion mode,they were 12,28,35,32.Among them,the number increased most when refrigerated for 1 day to 3 days,indicating the composition of raw milk changed drastically within 3 days.Compared with 0 day,the expression of 6 amino acid metabolites increased significantly and continuously after 4 days and later,and the expression of 13 fatty acids and their derivatives continued to decrease for 3 days and later.Enrichment analysis of differential metabolite metabolic pathways found that with the extension of refrigeration time,the metabolic pathways related to lipid metabolism,amino acid metabolism,carbohydrate metabolism,and bacterial infections changed significantly.（3）Integrating proteomics and metabolomics for correlation analysis,the results show that the differentially expressed protein from microorganisms and metabolites did not involve the same pathway of 1 d/0 d,and as the time of refrigeration extended,the common metabolic pathway number increased.So the influence of microbial protein on metabolites in raw milk might increased The correlations between differentially expressed proteins from microorganisms and metabolites showed that most of the differentially expressed proteins were positively correlated,while the correlations between differentially expressed metabolites and differentially expressed proteins with differentially expressed metabolites were more complicated.Correlation analysis showed the proteins as WP₀26727825.1,CDA85602.1,WP₀94167221.1,WP₁03230885.1,and WP₀87679563.1 which had a strong correlation with most differentially expressed metabolites,might be the key microbial proteins for raw milk spoilage.