Study On Sufu Paste Flavor Ripening And Mechanism Based On Amino Acid Metabolism

Sufu,douchi,soy sauce and bean sauce are known as four traditional fermented soybean appetizers in China.As a key factor in the evaluation of Sufu quality,characteristics flavor determines the consumer acceptance.However,due to the traditional production process,the flavor post-ripening takes a lot of time and the quality is difficult to guarantee.In order to solve these problems,the volatile flavor components（VFCs）and microbiota characteristics of eight famous Sufu samples were analyzed.Subsequently,the flavor associated strains were selected on the basis of the the interactions between characteristic VFCs and dominant microbiota.According to amino acid metabolism,the strains with high enzyme activities were chosen in terms of transaminase,protease and peptidase,and were applied to accelerate the ripening of Sufu paste.Based on amino acid metabolism,the main biochemical pathways of the flavor formation were analyzed by metagenome technology combined with bioinformatics analysis.The results will provide a scientific basis for shortening Sufu post-ripening and promoting the improvement of flavor quality of traditional fermented soybean appetizers.The main results are given below:（1）The results of physicochemical and taste characteristics of eight famous Sufu samples indicated that the content ofγ-aminobutyric acid（GABA）in bacteria-fermented Sufu（KS）was 57.95 mg/g DM,which was approximately 8 times higher than that of naturally-fermented Sufu（SS）and mould-fermented Sufu（WS）.The essential amino acid（EAA）contents ranged from 15.23 to 49.57 mg/g DM,and the patterns were comparable to those of eggs and milk proteins.According to the characteristics of amino acids,MSG-like and bitter were the major amino acid groups,followed by sweet.Additionally,potential tasty peptide profiles were typed by the segment of molecular weight（MW）<300 Da,ranging from 79.22%to 95.24%.Furthermore,the complex taste impression based on the electronic tongue showed that the bitterness of each Sufu was similar,while the umami,astringency and acid were the highest in bacteria-fermented Sufu（KS）.（2）Among the total 202 bacteria and 125 fungi species identified in the eight famous Sufu samples,16 bacteria and 7 fungi species were revealed as dominant community members by Illumina Miseq high-throughput sequencing technology.The VFC-microbiota correlation was characterized by Spearman’s rank correlation coefficients（ρ）,and Pichia fermentans,Kodamaea ohmeri,Saccharomyces rouxii,Lactococcus lactis subsp.and Sphingobacterium sp.were selected as the potential flavor associated strains.（3）According to the subordinate function values,the mixed starter,composed by Pichia fermentans,Kodamaea ohmeri and Lactococcus lactis subsp.was prepared with the indexs of protease,peptidase and transaminase,and applied to accelerate Sufu paste post-ripening.The optimal parameters were as follows:fermentation time15 d,fermentation temperature 28℃,addition amount of starter 0.10%at the ratio of2:1:1（Pichia fermentans:Kodamaea ohmeri:Lactococcus lactis subsp.）,addition amount ofα-ketoglutarate 0.4%and addition amount of salt 5%.Under the optimal condition,the total acid and amino acid nitrogen content were 0.14 g/100g and 1.35g/100g respectively,which were better than Sufu standard（AAN≥1.30 g/100g;TA≤0.35 g/100g）.The nutritional and functional increased significantly（p<0.05）,in which GABA increased from 0.02 mg/g DM（tofu）to 30.96 mg/g DM（Sufu paste）,and VB₂ reached up to 4.97 mg/g DM,about 8 times higher than that before fermentation.After post-ripening of 15 d,a total of 85 VFCs were tentatively identified,and tended to be stable.Among them,58 VFCs contributed to the flavor quality of Sufu paste,in which 13 VFCs were recognized as the main characteristic VFCs,including 2-methylbutyl acetate,2-amylfuran,2-methylbutanal,hexanal,octanal,heptanal,styrene,ethyl caproate,2-pentanone,ethyl butyrate,（E）-anethole,2,3-pentanedione and benzeneacetaldehyde.（4）According to the KEGG metabolic pathway annotation,amino acid metabolism（41.3%）was the highest metabolic pathways,followed by aromatic amino acids metabolism（21.9%）and sulphuric amino acids metabolism（6.8%）,up to 70%.Lactococcus lactis subsp.and Brevibacterium lines were the main species involved in aromatic amino acids metabolism.Pichia fermentans,Actinomucor elegans and Sphingobacterium sp.were the main species involved in branched-chain amino acids metabolism.Pichia fermentans,Saccharomyces rouxii and Lactobacillus fermentum were the main species involved in sulphuric amino acids metabolism.（5）Through metagenome combined with bioinformatics analysis,phenylalanine,leucine,isoleucine,tyrosine,valine and aspartic acid were the main amino acids involved in flavor formation during Sufu paste fermentation.The corresponding related metabolic pathways were mainly transamination,decarboxylation,dehydrogenation,oxidation and so on.The associated enzymes included transaminase,dehydrogenase,decarboxylase,oxidase and lyase,in which transaminases（EC:2.6.1.1,EC:2.6.1.5,EC:2.6.1.6,EC:2.6.1.42,EC:2.6.1.57 and EC:2.6.1.58）were the key enzymes.The results illustrated the formation pathways of VFCs such as benzeneacetaldehyde,styrene,benzeneethanol,2-methylbutanal,3-methylbutanal,2-methylbutanol,3-methylbutanol,2-methylbutyric acid and 2-methylbutyl acetate.