| Rice-acid,as the traditional"sour food culture crystallization"of Guizhou Miao and Dong nationalities,is highly favored by consumers.The compound seasoning made of rice-acid(white sour soup)and red sour soup which is widely used in cooking sour soup fish and other flavor foods.However,the current rice-acid fermentation still stays in the traditional process,and there are few studies on the extraction of acid-producing and flavor-enhancing strains and the optimization of the fermentation system,resulting in flavor deterioration and even spoilage.Therefore,our project aims to improve the flavor quality of traditional fermented rice-acid,analyze the diversity of the rice-acid fermentation flora and the relationship of flavor quality,screen excellent strains that produce acid and aroma,build a rice-acid intensified fermentation model.Taking characteristic flavor substances as the interesting point,combined with omics technology to analyze the biological regulation in the intensified rice-acid.To reveal the flavor formation mechanism of the two-strains synergistic fermentation of rice-acid from the gene level and protein level.It is expected to provide scientific basis for the development of fermented rice-acid,mining of functional strains,improvement of flavor quality,etc,it has guiding significance for the industrial application of high-quality rice-acid.The main conclusions are as follows:(1)Analysis of microbial community of fermented rice-acid and its correlations with characteristic flavorThe 62 bacterial genera in 10 rice-acid samples by high-throughput sequencing.Lactobacillus had the highest relative abundance(up to 94%),followed by Acetobacter and Prevotella.Meanwhile,there was little difference in abundance of 57fungal genera,mainly including Naumovia,Pichia,Emericella,Candida,Paecilomyces,Kluyveromyces,Malassezia,Saccharomyces and Rhodotorula;The microorganisms which were closely correlated with key organic acids in rice-acid included Lactobacillus,Acetobacter,Pichia,and Kluyveromyces,etc.The microorganisms which were correlated with VFCs included Acetobacter,Prevotella,Kluyveromyces and Saccharomyces,etc.;In particular,Lactobacillus,Pichia,Malassezia,Clavispora and Rhizopus were significantly positively correlated with lactic acid in rice-acid.Kluyveromyces,Saccharomyces and Emericella were significantly positively correlated with ethanol and ethyl acetate.(2)Screening of acid and aroma high-producing lactic acid bacteria and yeasts in rice-acid and construction of rice-acid intensified fermentation modelUsing characteristic flavor as an indicator,we selected 86 lactic acid bacteria and33 yeasts to determine that the high acid-producing strain was L.paracasei H4-11 and the strong aroma-producing strain was K.marxianus L1-1.The rice-acid intensified fermentation model was:the content of selenium rice flour was 8.0%,the temperature was 30?,the inoculation concentration of L.paracasei H4-11 and K.marxianus L1-1 were 7.80×10~7 CFU/m L and 4.95×10~5 CFU/m L,respectively.The fermentation time was 96 h.During the fermentation period,the L-lactic acid content increased significantly(P<0.05),and the content of L-lactic acid on Day 3 of the two-strain fermentation(H-group)reached 22.08±0.42 g/kg,which was higher than that of the L-group and L-group;OAV values showed potential flavor compounds which mainly included 13 kinds of ethyl acetate,ethyl propionate,isobutyl acetate,ethanol,etc.The ethyl acetate concentration of Y-group(rice-acid inoculated with K.marxianus L1-1)and H-group reached 241.37±6.20 mg/L and 193.83±6.94 mg/L on Day 3,respectively.The results showed that the content of characteristic flavor components of intensified fermented rice-acid was significantly higher than that of naturally fermented rice-acid(P<0.05).(3)The regulation mechanism of taste substance and L-lactic acid high-producing of rice-acid inoculated with L.paracasei H4-11Comparing the fermented rice-acid on Day 3 to Day 1,L.paracasei H4-11 is involved in ribosomal transcription,ABC transporters,and purine metabolism related genes and protein expression levels all show certain differences.There were mainly 5up-regulated genes and proteins related to amino acid metabolism,which could be closely related to the formation of rice-acid flavor substances.While there were down-regulated proteins related to the metabolism of glyoxylic acid and dicarboxylate metabolism.There were mainly 3 down-regulated proteins related to tryptophan metabolism,which could be related to the decrease of astringency and bitterness during the fermentation of rice-acid;The differentially expressed genes and proteins in amino sugar and nucleotide sugar metabolism,starch and sucrose metabolism were organic acids which provides energy to L-lactic acid formation;The 4 genes in glycolysis pathway were up-regulated,of which LSEI?2549 was inseparable from the formation of L-lactic acid;Pyruvate metabolism was annotated 7 up-regulated genes and 10 down-regulated genes,especially LSEI?2156 gene was significantly up-regulated;There were mainly 1 up-regulated gene and 6 down-regulated genes in TCA cycle,which could be related to the precursor substances formation of L-lactic acid.Using ELISA and PRM technology,4 key metabolic enzymes and 14 proteins related to taste substances and L-lactic acid were verified respectively by L.paracasei H4-11,which were consistent with the quantitative results of 4D LFQ.(4)The formation mechanism of ethyl acetate high-producing of rice-acid inoculated with K.marxianus L1-1Comparing the fermented rice-acid on Day 3 to Day 1,in K.marxianus L1-1,there were mainly 3 up-regulated genes and proteins related to starch and sucrose metabolism,amino sugar and nucleotide sugar metabolism,which could provide energy for the formation of acids,alcohols and esters;There were mainly 7up-regulated genes and proteins related to glycolysis/gluconeogenesis and pyruvate metabolism and 1 down-regulated ALD protein,which could play an important role in the formation of ethyl acetate-based esters;In TCA cycle and pyruvate metabolism,7main up-regulated differentially expressed genes and proteins and 1 down-regulated DLD1 protein played an important role in organic acid formation.Using ELISA and PRM technology,the 5 key metabolic enzymes and 15 proteins related to ethyl acetate and organic acid of K.marxianus L1-1 were verified,respectively,which were consistent with the quantitative results of 4D LFQ.(5)The flavor enhancement mechanism of intensified rice-acid inoculated with L.paracasei H4-11 and K.marxianus L1-1Comparing the fermented rice-acid on Day 3 to Day 1,the combined analysis results of RNA-seq and 4D LFQ proteomics technology showed that the up-regulated KEGG pathways were annotated by differentially expressed genes and proteins in L.paracasei H4-11 mainly included starch and sucrose metabolism,pyruvate metabolism,amino sugar and nucleotide sugar metabolism,and glycolysis/gluconeogenesis.While the up-regulated KEGG pathways were annotated by differentially expressed genes and proteins in K.marxianus L1-1 mainly included glycolysis/gluconeogenesis,TCA cycle,pyruvate metabolism,and pathways related to antioxidant capacity;Compared with the rice-acid inoculated with single starter,the synergistically intensified rice-acid had a richer characteristic flavor.The organic acid(taste substance)was mainly L-lactic acid,and the volatile flavor component was mainly ethyl acetate.Meanwhile,the signal intensity of astringency and bitterness were reduced,and the concentration of volatile sulfur compounds was reduced.On Day 3 of intensified rice-acid,in L.paracasei H4-11,there were 2 key up-regulated genes and 7 key down-regulated genes,and 10 key up-regulated proteins and 8 key down-regulated proteins were enriched and expressed in 7 KEGG pathways including amino sugar and nucleotide sugar metabolism.In K.marxianus L1-1,there were 7key up-regulated genes and 2 key down-regulated genes,the 6 key up-regulated proteins and 23 key down-regulated proteins were enriched and expressed in 7 KEGG pathways including starch and sucrose metabolism;The flavor formation metabolic pathway of the two-strain cooperative fermentation system showed that in L.paracasei H4-11,L-lactate dehydrogenase,phosphoglucomutase,acetate kinase,alcohol dehydrogenase and acetyl-Co A were up-regulated,D-lactate dehydrogenase and N-Acetyltransferase were down-regulated.In K.marxianus L1-1,Acetyl-Co A,acetaldehyde dehydrogenase,acyl-coenzyme A,N-acetyltransferase and L-lactate dehydrogenase were up-regulated,Hexokinase,alcohol dehydrogenase and alcohol O-acetyltransferase,etc.were down-regulated.Which synergistically promote the formation of characteristic flavor(mainly L-lactic acid and ethyl acetate)of intensified rice-acid;Using ELISA and PRM technology,it was verified that L.paracasei H4-11 and K.marxianus L1-1 were associated with 5 key metabolic enzymes and 27 proteins related to the characteristic flavor of rice-acid,respectively,which were consistent with the quantitative results of 4D LFQ. |
PDF Full Text Request