Analysis Of The Microbiome Based On Metatranscriptomic Technology And Its Effects On The Flavor During Noni Fruit Fermentation

Noni fruit is rich in nutrients and has attracted much attention because of its many active substances.But when the fruit ripens,the puree becomes soft and juicy,with a bitter taste and a strong unpleasant odor that puts off most consumers.Microbial fermentation plays an important role in flavor and quality of products.However,due to the high acidity and antibacterial activity of Noni juice,the structure and metabolic function of active microbiome in natural fermentation process are still unclear.In this study,metatranscriptomic and metabonomic technologies were used to explore the metabolic functions of active microbiome and their effects on the flavor of products,so as to provide a scientific basis for product quality control.1.The metatranscriptomic sequencing library of microbiome in naturally fermented noni juice(GNF)was constructed and sequenced with RNA-Seq high-throughput sequencing technology.The assembled and de-redundant sequences were annotated to analyze the microbial structure during GNF fermentation.The results are as follows:Acetobacter contributed the most to GNF fermentation,and the relative abundance of the transcripts increased from 0.9% to 84% at the 3rd day of fermentation.Acetobacter sp.and Gruconobacter sp.appeared in succession within 30 days of fermentation,with the sum of the relative abundance of their transcripts exceeding 90%.The fermentation species after30 days were Acetobacter aceti,Gluconobacter sp.and Gluconacetobacter sp.,and the average relative abundences of the transcripts were 56.2%,25.1% and 13.8%,respectively.2.Functional annotation and analysis of unigenes were carried out by comparison with the KEGG database,and 7301 KEGG Orthology(KO)were obtained,which could be annotated to 370 metabolic pathways.According to principal components analysis(PCA)and non-metric multidimensional scaling(NMDS)of KOs,GNF was divided into three fermentation stages: phase 1(day 1-9),phase 2(day 10-30)and phase 3(day 31-60).The microbial activities of GNF fermentation are mainly related to carbohydrate metabolism,energy metabolism and amino acid metabolism.At the end of phase 2,the fermentation was basically completed.A complete but modified variant citric acid cycle involved by core acetic acid bacteria was highly expressed throughout the fermentation process.This pathway was the most important carbohydrate metabolism and contributes to energy production through depleting cytoplasmic acetate.Oxidative phosphorylation is the major energy source of fermented microbiome.Fermented microorganisms of GNF also participated in diverse amino acids and fatty acids synthesis and metabolism.In addition,antibiotic secondary metabolites were produced by microbiome during GNF fermentation.3.Physical and chemical indexes of GNF were analyzed,and HS-SPME-GC-MS combined with aroma activity(OAV)analysis was used to evaluate the contribution of volatiles to the overall flavor.Then GC-MS was used to quantify the odor substances.The results are as follows:In essence,GNF is a fruit vinegar with unique organic acids.During fermentation,the acidity increased while the sugar decreased,and it stabilizes at phase 2.The main organic acids are citric acid,malic acid,succinic acid and pyruvic acid.A total of 68 volatiles were identified in GNF,mainly esters,acids and alcohols.24 odor activity substances were screened out by OAV > 1 to constitute the characteristic flavor of GNF.The concentration of hexanoic acid,butanoic acid and octanoic acid were588,361,18 mg/L respectively,which were the sources of unpleasant flavor of GNF.Their concentrations decreased significantly with the fermentation and decreased to 148,193 and11 mg/L at phase 3.4.Three strains that could grow well in Noni juice were screened from different fermentation stages of GNF and identified as Acetobacter by molecular biology,but their16 S r RNA had less than 97% identities with Acetobacter species.Therefore,the genome of the isolated strains was further sequenced to construct the gene framework.After ANI identification of the genome,the three isolated strains were determined to be homologous,which was a potential new species of Acetobacter.5.Acetobacter N2 was inoculated to ferment noni puree to investigate the effect of acetic acid bacteria inoculation and fermentation on flavor and quality of products.The main organic acids of JAF were citric acid and succinic acid,while the concentration of pyruvic acid,oxalic acid,malic acid and lactic acid decreased significantly,and the content of acetic acid was not statistically significant.The flavor and taste of JAF were improved,which not only ensured the stability of the product,but also did not affect the contents of total phenols,total flavonoids,active substances such as deacetylasperulosidic acid and asperulosidic acid.Compared with JAF,the acidity and sugar content of JEAF were increased,which improved the flavor of the juice.In addition,enzyme treatment increased the contents of total phenols and deacetylasperulosidic acid in JEAF.At the same time,the fermentation time was shortened,and the fermentation was basically completed on the 4th day.In general,microbial function during natural fermentation of noni fruit was revealed by metatranscriptomic sequencing.Odor compounds were identified by characteristic flavor analysis.The influence of core strains screened from GNF on flavor and quality of products was studied,so as to contribute to product optimization and utilization of microbial resources.