| Chinese Sichuan sausage is a well-known traditional meat product with distinctive regional features in China that is adored by people for its unique flavor.Sichuan sausage manufactured from raw pork that is dried by cold air in a naturalistic environment,depending on microorganisms and endogenous enzymes from the raw material and auxiliary ingredients to generate a distinct flavour.Currently,studies have focused on microbial community structure,physicochemical characteristics,the identification of biogenic amines and amine-producing microorganisms,and others.There are few reports on the flavor formation of dominant strains of Sichuan sausages,in particular,comprehensive studies on the characteristic flavor substances of Sichuan sausages and the non-volatile metabolites associated with their development have not been studied.In this study,we used P.pentosaceus and S.carnosus as core microorganisms from Sichuan natural air-dried sausages to investigate the effects of inoculation alone and in combination on the physicochemical characteristics and metabolite profiles of Sichuan sausages and their correlation.A comprehensive analysis of non-volatile and volatile metabolites of sausages was further conducted to identify the characteristic flavor substances of sausages and their potential metabolic pathways regulated by non-volatile metabolites,providing new insights into the regulation of the characteristic flavor of sausages by microbial metabolism.The concrete research contents and conclusions are as follows:(1)The P.pentosaceus and S.carnosus were inoculated alone and mixed with prepared sausages,and the uninoculated sausages were used as a control group to study the changes in the physicochemical properties of the sausages during the preparation process.The results indicated that P.pentosaceus,S.carnosus alone and in mixture had no significant effect on sausage p H,Aw.The total viable counts(TVC)of sausage increased and then decreased in all three groups of inoculated strains,with consistently higher numbers than the control group.Peroxide value(POV)and Thiobarbituric acids(TBARS)were consistently elevated,and the mixed group always had the lowest levels,showing more prominent lipid primary and secondary antioxidant benefits.Chromaticity(L~*,a~*,and b~*)continued to decrease during sausage manufacturing,and mixed inoculation had the highest benefit in delaying the decline of sausage chromaticity.(2)Based on the HS-SPME/GC-MS technique,27 volatile compounds were detected in four groups of sausages,including 8 aldehydes,2 ketones,3 acids,7 alcohols,2 esters,1 furan,and 4 hydrocarbons,of which 7 were co-volatile compounds.The results of principal component analysis(PCA)and cluster analysis were consistent in demonstrating that the four sausage groups were clearly divided into cluster I(CT、PG)and cluster II(SG、MG),indicating significant differences in volatile compounds between the two clusters.A similar change in volatiles was observed in sausages inoculated with S.carnosus and P.pentosaceus alone,with a significant increase in the concentration of ketones,acids,and esters and a significant decrease in the concentration of aldehydes and alcohols;the concentration of important flavor compounds such as acids,esters,and ketones were all increased in the mix inoculated sausage.The orthogonal partial least squares discriminant analysis(OPLS-DA)model and random permutation test indicated that the results of the analysis of variance were accurate and reliable,which were used to further identify the 17 characteristic flavor substances.Five characteristic flavor substances such as vinyl hexanoate and 3-hydroxy-2-butanone were significantly elevated in the P.pentosaceus group;eight characteristic flavor substances such as 1-octen-3-ol and 3-methyl-1-butanol were significantly elevated in the S.carnosus group;and 13 compounds such as vinyl hexanoate,1-octen-3-ol,and 3-hydroxy-2-butanone were significantly elevated in the mixed inoculation group,which has a higher level.(3)UHPLC-MS was applied to characterize the non-volatile metabolite profiles of four groups of sausages,and further investigated the regulation pattern of sausage metabolites by individual and mixed inoculations of P.pentosaceus and S.carnosus.A total of 903 non-volatile metabolites were detected by LC-MS;these metabolites were classified under the Superclass classification hierarchy as follows:42.03%of lipids and lipid-like molecules,25.00%of organic acids and derivatives,11.41%of organoheterocyclic compounds,and others.Multivariate statistical analysis was applied to identify 35,57 and 57 differential metabolites from P.pentosaceus,S.carnosus and mixed group sausages,respectively,totaling 92differential metabolites.Based on KEGG pathway enrichment analysis,92 differential metabolites were enriched to a total of 32 metabolic pathways,with only arginine biosynthesis,purine metabolism,and glycerophospholipid metabolism pathways containing a total of 20metabolites having topological analysis results of-log10(p)>2 and pathway impact>0.1.Among the P.pentosaceus group,D-ribose 1-phosphate,inosine 5’-monophosphate(IMP),guanosine,adenine,inosine,and xanthine were absorbed and promoted the accumulation of N-acetyl-L-glutamic acid,L-citrulline,and L-arginine flavored amino acids.Among the S.carnosus group,PE(15:0/22:2(13Z,16Z))was converted to PC(16:0/18:1(11Z))via the Phosphatidylcholine(PC)biosynthesis pathway,while the expression of PC(P-18:0/0:0)and1,2-diacylglycerol-3-phosphate was significantly elevated.Among the S.carnosus group,differential metabolites in the arginine biosynthesis pathway were significantly increased,In particular,L-glutamic acid and L-arginine were 1.82 and 4.44 times elevated in flavor or flavor precursor amino acids.Meanwhile,PE(15:0/22:2(13,16))was completely converted to PC(16:0/18:1(11 Z)),acetylcholine,and an abundance of flavor precursor metabolism substrate was accumulated.(4)Applying PLSR and Pearson correlation to analyze the correlation between the physicochemical characteristics of sausages and the characteristic volatile compounds and the enriched non-volatile metabolites and the characteristic volatile compounds,respectively.Aiming to reveal the underlying association between physicochemical characteristics,non-volatile metabolites and characteristic flavor substances.The PLSR correlation analysis revealed that the 17 characteristic flavor compounds and physicochemical characteristics were mostly in the ellipse,indicating that there was a strongly correlated effect of sausage physicochemicals on the formation of characteristic flavor compounds.Pearson correlation analysis suggested that the 20 enriched non-volatile metabolites were significantly correlated with 17 characteristic flavor substances.(Z)-2-octen-1-ol,2-pentyl-furan,1-hexanol,4-methyl-1-pentanol were significantly and positively correlated with D-ribose 1-phosphate,inosine,IMP,PE(15:0/22:2(13Z,16Z));hexanal and pentanal were significantly and positively correlated with L-glutamine,xanthine,lecithin and acetylcholine;3-hydroxy-2-butanone,3-methyl-1butanol,octanal,and 2,3-butanediol were significantly and positively correlated with L-glutamine,PC(16:0/18:1(11Z)),PC(P-18:0/0:0),and N-acetyl-L-glutamate.The 20 non-volatile metabolites enriched play a determining role in the development and accumulation of volatile compounds characteristic of sausages. |
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