Study On The Microbial Diversity And Metabolites During Panxian Ham Fermentation

Panxian ham,a well-known specialty in Guizhou Province,has been protected by national geographical indication since 2012.Panxian ham is made from traditional technology(spontaneous fermentation)with unique regional quality characteristics.With the development of the Panxian ham industry,the disadvantages of spontaneous fermentation have gradually emerged,such as long processing time,susceptibility to contamination,and quality fluctuations.Similar to other fermented foods,the microbial population of dry-cured ham is pivotal to taste and flavor formation.Therefore,the key to breaking through the bottleneck of Panxian ham enterprises and achieving standardized production is to reveal the relationship between the succession of microbial community structure and the formation of metabolites,and to screen starter which could be used in the production of Panxian ham.At the present study,Panxian ham at different stages(raw ham,post-salting,post-resting,and initial,middle,and final stages of ripening)was used as an experimental material to uncover the succession of microbial communities during spontaneous fermentation,and to analyze the dynamic changes of physicochemical characteristics and metabolites.This study aims to discover potential key microorganisms in Panxian ham through the correlation analysis between microorganisms and metabolites.The main results of this paper are as follows:(1)A total of 10 bacteria and 3 fungi at the phylum level were detected by high-throughput sequencing technology,Firmicutes(53.57%),Proteobacteria(40.36%),and Ascomycota(97.78%)were the dominant phyla.At the genus level,a total of 154 bacteria and 51 fungi genera were found,including 11 core bacteria and 6 core fungi with more than 1%average relative abundance,of which Staphylococcus(44.79%)was the dominant bacteria and Debaryomyces(41.03%)and Aspergillus(37.68%)were the dominant fungi.The result of?-diversity showed that the microbial richness and diversity of Panxian ham decreased significantly during spontaneous fermentation,but bacterial community was more complex than the fungal community.The?-diversity analysis showed that the succession direction of bacteria and fungi was generally the same,but the change of bacterial community structure was more intense.The results of PICRUSt indicated that metabolism,especially carbohydrate metabolism,global and overview maps,and amino acid metabolism,was the most important function of the bacterial community.In addition,the abundance of predicted functional genes involved in human diseases decreased significantly during Panxian ham fermentation,especially the gene sequences associated with cancer.(2)With the progress of spontaneous fermentation,the water content and water activity(a_w)continued to decrease,from 72.69%and 0.977 to 43.93%and 0.753,respectively.The Na Cl content and p H increased from 0 and 5.74 to 5.28%and 5.94,especially.The dynamic changes of these physicochemical characteristics not only help the formation of Panxian ham quality,but also inhibit the growth and reproduction of harmful microorganisms in Panxian ham.Intramuscular fat(IMF)content decreased significantly during fermentation,from 9.60%to 6.47%,but was higher than Celta ham(5.96%),indicating that Panxian ham may have better springiness.For color indicators,lightness(L*)and yellowness(b*)reduced significantly,while redness(a*)increased significantly during fermentation,which suggested that Panxian ham has a better color parameter.A total of 50 volatile flavor compounds,including 24 aldehydes,9 alcohols,7 ketones,7 acids,2hydrocarbons,and 1 pyridine,were detected from Panxian ham at different stages by HS-SPEM/GC-MS technology.The types and contents of flavor compounds increased during processing,from 25(218.50?g/kg)to 40(1044.49?g/kg).The PLSR regression model between sensory attributes and flavor compounds suggested that 3-methyl-butyraldehyde,dodecanal,hexadecanal,(Z)-9-octadecenal,and octadecanal were significantly related to the meaty,faint scent,and fats incense of Panxian ham.(3)GC-TOF-MS-based non-targeted metabolomics showed that continuous metabolic changes occurred during the entire fermentation process,with the most significant changes occurred in the initial stage of ripening.Based on OPLS-DA model and one-way analysis of variance(ANOVA),the variable importance in projection(VIP)values and p-value were calculated and thirty-one significantly different metabolites(SDMs)were identified as discriminant factor,including 15 amino acids,6 fatty acids,3 organic acids,2 sugars,2 polyols,2nucleic acids,and 1 other metabolite.Except for 2 sugars(glucose and fructose),the levels of all SDMs increased rapidly and reached the maximum at the initial stage of ripening.The accumulation of these metabolites was important for the taste and quality of Panxian ham.Pathway analysis suggested that these metabolites were involved in 30 pathways,of which“alanine,aspartate,and glutamate metabolism”,“glycine,serine,and threonine metabolism”,and“arginine and proline metabolism”were the most relevant metabolic pathways during Panxian ham fermentation.(4)Redundancy analysis(RDA)was used to study the correlation between physicochemical factors and microbial communities.The results showed that water content was the main environmental factor driving the bacterial community variation,while,p H and a_w were the major environmental factors driving the fungal community variation.In order to reveal potential key microorganisms in Panxian ham,the correlation network and multiple RDA were used to analyze the effect of microbial community on volatile flavor and non-volatile metabolites,respectively.According to the Pearson correlation coefficient(|r|>0.6)and p-value(p<0.05)of microorganisms and flavor compounds,Staphylococcus,Aspergillus,Debaryomyces,Pseudomonas,Brochothrix,Acinetobacter,and Chromohalobacter had significant contributions to the flavor quality of Panxian ham.Further,statistical RDA indicated that Staphylococcus,Debaryomyces,and Chromohalobacter promoted the production of amino and fatty acids;Cobetia and Aspergillus were associated with sugar metabolism;and Kushneria,Penicillium,and Yamadazyma were closely related with organic acids.Combining flavor contribution and metabolite correlation,four microorganisms were identified as the key microorganisms of Panxian ham,namely Staphylococcus,Aspergillus,Debaryomyces,and Chromohalobacter.