| Liuyang douchi has a long history of more than 3,000 years.It is one of the typical representatives of Aspergillus-type douchi in China.Liuyang douchi has been widely used in food and Chinese patent medicine due to its low salt,delicious taste,unique flavor and functional characteristics.However,owing to low degree of industrialization,the coarse production mode and uncontrolled spontaneous fermentation during producing,it was easily polluted by microorganisms such as Aspergillus flavus.As a result,the quality of Liuyang douchi was unstable and the safety is not guaranteed.In previous study,small black beans were used and the dominant strains Aspergillus flavus 1(Aspergillus flavus7214,abbreviated as 7214 strains in this text,the registration numbers were CGMCC No.20734)and Aspergillus flavus 2(Aspergillus flavus7622,abbreviated as 7622 strains in this text,the registration number was CGMCC No.20735)which were isolated from traditional Liuyang douchi,the mixture of the two strains in equal proportion(77 strain)and the commercial Aspergillus oryzae were selected as starter culture,while the traditional natural fermentation mode was regards as a control.The differences in physicochemical indexs,volatile components and metabolic mechanism of Liuyang douchi fermented by different starter culture were explored.It could provide technical support for the stabilization and standardization of Liuyang douchi production.The main results were as follows:1.Fermentation characteristics of enhanced fermentation with different culturesThe changes of fungal colony count,bacterial colony count,protease activity,α-amylase activity,reducing sugar,total acid and amino acid nitrogen at different stages of intensified fermentation with different strains were compared.With the increase of fermentation time,the total number of bacterial and fungal colonies rising firstly and then decreasing.The total number of bacterial colonies reached the maximum at 2 d,and the total number of fungal colonies reached the maximum at 8 d,and then continued to decline.The activities of protease and α-amylase increased first and then decreased.The protease activity of 7214 strain sample reached the maximum of 319 U / m L,and the α-amylase activity of Aspergillus oryzae strain sample reached the maximum of 70 U / m L.However,reducing sugar showed a trend of first increase and then decrease,and the degradation of reducing sugar was the fastest in the samples from 7214 strain washing-qu to the end of pile-fermentation.The contents of total acid and amino acid nitrogen increased slowly at first and then increased rapidly.The contents of total acid and amino acid nitrogen in 7214 strain samples were up to 3.52 g/100 g and 1.47 g/100 g,respectively.2.The effect of enhanced fermentation of different strains on the flavor of Liuyang douchiThe differences of their sensory,smell,free amino acids,organic acids and volatile components by means of sensory evaluation,electronic nose,HPLC and GM-MS of traditional natural fermentation and enhanced fermentation douchi were further measured.The natural fermentation samples had higher scores in appearance while the samples enhanced fermented by strain 7214 had higher scores in aroma and mouthfeel,espcially in umami,sweetness and sourness in terms of taste.According to the response values of electronic nose,the differences of 5 samples were mainly reflected in nitrogen oxides,sulfides and organic sulfides.The sample enhanced fermented by 7622 strain enhanced fermentation was similar to the sample of natural fermentation,while the smell of the sample enhanced fermented by 7214 strain showed a notably difference.Except Aspergillus oryzae fermented douchi,the contents of total free amino acids in enhanced fermentation samples were higher than natural fermentated sample.Similarity,the content of organic acids in all enhanced fermentation samples were higher than that in natural fermentation samples,revealing that enhanced fermentation might produce more the precursor of flavor substance.Twenty kinds of volatile compunds were founded in all of douchi samples,which indicates a similar flavor characteristics of the enhanced fermentation and natural fermentation Liuyang douchi have.Benzaldehyde and phenacetaldehyde(the important flavor substances of douchi)were detected in all of 5 samples,but the levels of them in 7214 strain enhanced fermented sample was higher than other samples.At the same time,there were some components not found in other samples,such as ethyl linoleate(fruity),3-octanone(vanilla,creamy)and1-octene-3-ol(mushroom aroma)indicating that the flavoring substances of enhanced fermentation were more abundant.3.Metabolic Characteristics of Different Strains Enhanced Fermentation and Its Effect on Formation of Flavor Compounds in Liuyang DouchiCompared with natural samples,according to the composition analysis of metabolites(PLS-DA)and the cluster analysis of different metabolites.The number of up-regulated and down-regulated metabolites in each enhanced fermentation sample was between 1481 and2167,indicating that there were significant differences in the metabolic characteristics of different strains during fermentation.The fermentation metabolic characteristics of 7622 strains were similar to those of natural strains.The analysis of 231 differential metabolites metabolic pathways in different strain-enhanced fermentation samples can screen out 13 important metabolic pathways,among which the differences in amino acid biosynthesis and protein digestion and absorption are the largest.A total of 62 differential metabolites were identified from five different strains.The differential metabolites mainly included pyruvic acid,L-glutamic acid,L-lysine,L-arginine,L-glutamine,L-histidine and succinic acid.A total of 7 substances were found,which indicated that amino acid metabolism had an important influence on the flavor of Liuyang Douchi.Combined with correlation analysis,it was found that organic acid and amino acid metabolism were closely related to the formation of esters.For example,L-serine,N(6)-methylly lysine,cis-4-hydroxy-D-proline and other amino acid derivatives are positively correlated with the content of organic acids such as2-(methylamino)benzoic acid and erucic acid and the content of ethyl phenylacetate.In addition,amino acid metabolism and lipid metabolism were closely related to aldehyde formation,such as N-a-acetyl citrulline and fructose lysine were positively correlated with benzaldehyde and benzaldehyde.2-octaenal was positively correlated with inositol andα-dimorpholinic acid.Based on the above studies,it was found that the appearance of different inoculation intensified fermentation methods was similar to that of natural fermentation.The flavor substances of 7622 strain samples were similar to those of natural fermentation.The nutritional components and flavor of 7214 strain and 77 mixed strain samples were better than those of natural fermentation.7214 strain was more suitable for Liuyang Douchi intensified fermentation.Inoculation enhanced fermentation has great practical significance for stabilizing the flavor of Liuyang Douchi,which provides important technical support for traditional solid-state fermentation of traditional food flavor fidelity. |
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