Flavor Metabolism Research Articles

Effect of enzymes addition on the fermentation of Chinese rice wine using defined fungal starter

A higher fermentation capability and quality of a rice wine was achieved, using mixed fungal (Rhizopus chinonsis R01, Asperigillus niger A20, Mucor pusillus M05 and Saccharomyces cerevisiae S10) starter fermentation of japonica rice supplemented with α-amylase (AM), glucoamylase (GAM) and acid protease (AP). Results showed that alcohol content, free amino acids (FAA) and volatile flavor substances concentrations were increased by 49.22%, 85.54%, and 25.71% compared with the control group respectively. To understand the role of enzymes during rice wine brewing, the effects of hydrolases on chemical and physical properties of cooking rice, on the yeast growth, enzyme productivity of molds were determined. Rice starch was hydrolyzed effectively by AM and GAM, the viscosity reduced significantly, the enzyme contact area expanded, which accelerated the further hydrolysis of japonica rice. Increase of hydrolysate promoted the fungi growth, while improved the ability of molds to produce enzymes. AP promoted the protease activity of M. pusillus M05 and A. niger A20, improved protein utilization, increased the content of FAA and formed higher level of flavor substances in rice wine. The results indicate that hydrolase addition can change the physic-chemical state of the mash to enhance microbial biotransformation and modify flavor metabolism.

Application of Wickerhamomyces anomalus in Simulated Solid-State Fermentation for Baijiu Production: Changes of Microbial Community Structure and Flavor Metabolism.

Wickerhamomyces anomalus is conducive to the synthesis of ester compounds in brewing the Chinese liquor Baijiu; esters are crucial for the quality of Baijiu. In this study, simulated solid-state fermentation for Baijiu production was used to explore whether artificial addition of W. anomalus could improve the flavor substance in Baijiu, and the underlying mechanisms. Two experimental groups were studied, in which W. anomalus Y3604 (Group A) and YF1503 (Group B) were added, respectively; in the control group (Group C), no W. anomalus was added. Adding strain Y3604 increased the content of esters in fermentation samples, especially ethyl acetate and ethyl caproate, and reduced the content of higher alcohols. Adding strain YF1503 had little effect on the ester content but decreased the content of higher alcohols. The diversity and abundance of prokaryotic genera in Group A and B samples were similar, but there were some differences compared with Group C. The correlations of genera in Group A or B samples were simple compared with group C. Although the predominant eukaryotic genera in the three groups were consistent, the abundance of each gene varied among groups. Based on our findings, bioaugmentation of Baijiu fermentation with W. anomalus will change the ethyl acetate content and cause changes in the levels of other flavor substances. We suggest that the changes in flavor substances caused by the addition of W. anomalus are mainly due to changes in the microbial community structure that result from the addition of W. anomalus.

Transformation of Microbial Negative Correlations into Positive Correlations by Saccharomyces cerevisiae Inoculation during Pomegranate Wine Fermentation.

The application of starter is a common practice to accelerate and steer the pomegranate wine fermentation process. However, the use of starter needs a better understanding of the effect of the interaction between the starter and native microorganisms during alcoholic fermentation. In this study, high-throughput sequencing combined with metabolite analysis was applied to analyze the effect of commercial Saccharomyces cerevisiae inoculation on the native fungal community interaction and metabolism during pomegranate wine fermentation. Results showed that there were diverse native fungi in pomegranate juice, including Hanseniaspora uvarum, Hanseniaspora valbyensis, S. cerevisiae, Pichia terricola, and Candida diversa Based on ecological network analysis, we found that S. cerevisiae inoculation transformed the negative correlations into positive correlations among the native fungal communities and decreased the Granger causalities between native yeasts and volatile organic compounds. This might lead to decreased contents of isobutanol, isoamylol, octanoic acid, decanoic acid, ethyl laurate, ethyl acetate, ethyl hexadecanoate, phenethyl acetate, and 2-phenylethanol during fermentation. This study combined correlation and causality analysis to gain a more integrated understanding of microbial interaction and the fermentation process. It provided a new strategy to predict certain behaviors between inoculated and selected microorganisms and those coming directly from the fruit.IMPORTANCE Microbial interactions play an important role in flavor metabolism during traditional food and beverage fermentation. However, we understand little about how selected starters influence interactions among native microorganisms. In this study, we found that S. cerevisiae inoculation changed the interactions and metabolisms of native fungal communities during pomegranate wine fermentation. This study not only suggests that starter inoculation should take into account the positive features of starters but also characterizes the microbial interactions established among the starters and the native communities. It may be helpful to select appropriate starter cultures for winemakers to design different styles of wine.

Research advance in regulation of fruit quality characteristics by microRNAs

Abstract MicroRNAs (miRNAs) are short (19–24 nucleotides in length) noncoding RNAs that have a profound effect on gene expression. By completely or almost perfectly base-pairing with their individual target mRNAs they cause mRNA cleavage or repression of translation. As important regulators, miRNAs plays an important role in the regulation of fruit quality. Extensive studies have been reported in fruits, however current studies are mostly focused on the identification of miRNAs and the prediction and validation of target genes. This review summarizes research progress on the role of miRNAs in regulating fruit ripening and senescence and quality characteristics, such as coloration, flavor metabolism, and texture for providing information for future research.

Effects of fortification of Daqu with various yeasts on microbial community structure and flavor metabolism.

Fortification of Daqu with isolated functional strains can influence the metabolic activity of the microbial community, and thus alter the flavors of the Baijiu produced with Daqu as a fermentation starter. Here, we analyzed the microbial community dynamics of, and volatile compound production by, Daqu fortified respectively with three high-yield ethyl caproate-producing yeasts (Saccharomyces cerevisiae Y7#09, Hyphopichia burtonii F12507 and Clavispora lusitaniae YX3307), or with a mixture of these three strains, during the fermentation of Baijiu. The microbial community was investigated using Illumina HiSeq technology. Three bacterial genera (Bacillus, Lactobacillus and Enterobacter) and four fungal genera (Pichia, Clavispora, Saccharomyces and Saccharomycopsis) were dominant in the microbial communities. The volatile compounds were examined by gas chromatography-mass spectrometry. Forty-one flavor compounds were detected in all samples, including seven alcohols, 26 esters and four aldehydes. In particular, an increase in ethyl caproate content was associated with Daqu fortified with S. cerevisiae Y7#09, C. lusitaniae YX3307, or the mixed inoculum. The ester content of these fortified Daqu was higher in the later stage of the fermentation than that in unfortified Daqu, or in Daqu fortified with H. burtonii F12507. Our results show that fortification of Daqu with aroma-producing yeast strains influenced the microbial community composition in the Daqu and affected its metabolic activity. Overall, this study reveals the features of fortified Daqu microbial communities in different phases and improves understanding of the relationships between aroma-producing yeast and the metabolic activity of microbial communities in Baijiu production.

Bioturbation effect of fortified Daqu on microbial community and flavor metabolite in Chinese strong-flavor liquor brewing microecosystem.

Traditional spontaneous fermentation in microecosystem with microbial successions and environmental variables results in inconsistent quality of fermented foods. We therefore propose the directional bioturbation for microbiota regulation and metabolites production in food fermentation. Here, we revealed the bioturbation effect of fortified Daqu on microbial community based on taxonomic composition, co-occurrence network, and metabolic potential, using Chinese strong-flavor liquor fermentation as a microecosystem. According to principal coordinate analysis, microbial communities were obviously influenced by the bioturbation of fortified Daqu. More specifically, bioturbation increased the abundances of Caproiciproducens, Clostridium, Aspergillus, Candida, Methanobacterium, and Methanosarcina, while decreased that of Lactobacillus. Meanwhile, higher abundances of most genes that encoding enzymes involved in interspecies hydrogen transfer between hexanoic acid bacteria and methanogens were observed in the bioturbated ecosystem by PICRUSt approach. Additionally, co-occurrence analysis showed that bioturbation increased the diversity and complexity of interspecies interactions in microecosystem, which contributed to higher production of flavor metabolites such as hexanoic acid, ethyl hexanoate, and hexyl hexanoate. These results indicated that the bioturbation of fortified Daqu is feasible for flavor metabolism by interspecies interactions of functional microbiota in liquor fermentation. Taken together, it is of great importance for regulating Chinese liquor and even other foods fermentation by bioturbation.

A genome-scale metabolic network of the aroma bacterium Leuconostoc mesenteroides subsp. cremoris.

Leuconostoc mesenteroides subsp. cremoris is an obligate heterolactic fermentative lactic acid bacterium that is mostly used in industrial dairy fermentations. The phosphoketolase pathway (PKP) is a unique feature of the obligate heterolactic fermentation, which leads to the production of lactate, ethanol, and/or acetate, and the final product profile of PKP highly depends on the energetics and redox state of the organism. Another characteristic of the L. mesenteroides subsp. cremoris is the production of aroma compounds in dairy fermentation, such as in cheese production, through the utilization of citrate. Considering its importance in dairy fermentation, a detailed metabolic characterization of the organism is necessary for its more efficient use in the industry. To this aim, a genome-scale metabolic model of dairy-origin L. mesenteroides subsp. cremoris ATCC 19254 (iLM.c559) was reconstructed to explain the energetics and redox state mechanisms of the organism in full detail. The model includes 559 genes governing 1088 reactions between 1129 metabolites, and the reactions cover citrate utilization and citrate-related flavor metabolism. The model was validated by simulating co-metabolism of glucose and citrate and comparing the in silico results to our experimental results. Model simulations further showed that, in co-metabolism of citrate and glucose, no flavor compounds were produced when citrate could stimulate the formation of biomass. Significant amounts of flavor metabolites (e.g., diacetyl and acetoin) were only produced when citrate could not enhance growth, which suggests that flavor formation only occurs under carbon and ATP excess. The effects of aerobic conditions and different carbon sources on product profiles and growth were also investigated using the reconstructed model. The analyses provided further insights for the growth stimulation and flavor formation mechanisms of the organism.

Improving flavor metabolism of Saccharomyces cerevisiae by mixed culture with Wickerhamomyces anomalus for Chinese Baijiu making

Yeasts are the most important microorganisms in the fermentation of Chinese Baijiu and the interaction of these yeasts could impact the quality of Baijiu. In this study, we initially characterized the Baijiu yeasts and evaluated their fermentation potential. Wickerhamomyces anomalus GZ3 and Saccharomyces cerevisiae G20 were found to generate high yields of ethyl acetate (2.76g/L) and 2-phenylethanol, respectively. Results also indicated that the use of W.anomalus along with S.cerevisiae increased volatile compounds production, the maximum ethyl acetate production was observed in S.cerevisiae and W.anomalus at 106:106 ratio and have increased by 33 % compared with single culture of W.anomalus. Besides, there was a significant increase of 2-phenylethanol (3.29g/L) production in single culture of S.cerevisiae with the addition of l-phenylalanine. However, the conversion of l-phenylalanine in mixed culture had significant impact on the yeasts interaction and end flavor of Baijiu. Thus, the present study provided new insights into yeasts interactions in Baijiu fermentation and the effect of some primary metabolites on the end flavor and Baijiu quality.

Identification of Novel Alleles Conferring Superior Production of Rose Flavor Phenylethyl Acetate Using Polygenic Analysis in Yeast.

ABSTRACTFlavor compound metabolism is one of the last areas in metabolism where multiple genes encoding biosynthetic enzymes are still unknown. A major challenge is the involvement of side activities of enzymes having their main function in other areas of metabolism. We have applied pooled-segregant whole-genome sequence analysis to identify novel Saccharomyces cerevisiae genes affecting production of phenylethyl acetate (2-PEAc). This is a desirable flavor compound of major importance in alcoholic beverages imparting rose- and honey-like aromas, with production of high 2-PEAc levels considered a superior trait. Four quantitative trait loci (QTLs) responsible for high 2-PEAc production were identified, with two loci each showing linkage to the genomes of the BTC.1D and ER18 parents. The first two loci were investigated further. The causative genes were identified by reciprocal allele swapping into both parents using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. The superior allele of the first major causative gene, FAS2, was dominant and contained two unique single nucleotide polymorphisms (SNPs) responsible for high 2-PEAc production that were not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation. Exchange of both superior alleles in the ER18 parent strain increased 2-PEAc production 70%, nearly to the same level as in the best superior segregant. Our results show that polygenic analysis combined with CRISPR/Cas9-mediated allele exchange is a powerful tool for identification of genes encoding missing metabolic enzymes and for development of industrial yeast strains generating novel flavor profiles in alcoholic beverages.

Juice volatile composition differences between Valencia orange and its mutant Rohde Red Valencia are associated with carotenoid profile differences

A targeted approach using HS-SPME-GC–MS was performed to investigate volatile compounds of ordinary Valencia (VAL) and its more deeply colored mutant Rohde Red Valencia orange (RRV) at different developmental stages. Fifty-six volatile components classified into six chemical groups were quantified. The individual volatile compounds in each group were significantly influenced by the genotype and harvest date. Cluster analysis suggested two principal clusters. Cluster I included fruits of VAL and RRV harvested in July, September, and November and was characterized by the 2-ethyl-1-hexanol acetate and linalool chemotype; cluster II included fruits harvested in January and March, with the valencene/β-myrcene chemotype. Principal component analysis confirmed that fruits of both cultivars harvested at different dates possessed a unique aroma active profile, especially, the odor-active volatile-norisoprenoids degraded from C40 carotenoid were significantly higher in RRV than in VAL at full maturity. These results could provide information for further study of the flavor metabolism pathways in the two closely related cultivars.

Regulating yeast flavor metabolism by controlling saccharification reaction rate in simultaneous saccharification and fermentation of Chinese Maotai-flavor liquor

Maotai-flavor liquor is produced by simultaneous saccharification and fermentation (SSF), in which filamentous fungi produce hydrolases to degrade the starch into fermentable sugar. Saccharomyces cerevisiae simultaneously transforms the sugars to ethanol and flavor compounds. The saccharification rate plays an important role in regulating the liquor yield and flavor profile. This work investigated the effect of saccharification rate on fermentation by regulating the inoculation ratio (1:0.1, 1:0.5, 1:1, 1:5, 1:10) of S. cerevisiae and Aspergillus oryzae, the main saccharification agent. We found no significant difference in reducing sugar content among the mixed cultures with different ratios. This indicated a balance of the saccharification rate and the sugar consumption rate, in which the former was controlled by the interaction between A. oryzae and S. cerevisiae, and the latter controlled the metabolism of the two species. The ethanol yield was the highest in ratios of 1:0.5, 1:1, and 1:5, while the total production of flavor compounds was the highest for the ratio of 1:0.5, which was mainly attributed to the vigorous metabolism of S. cerevisiae. The inoculum ratio of 1:10 produced the second highest content of flavor compounds in which a large number of alcohols and esters were derived from the vigorous metabolism of A. oryzae. This indicated that the saccharification rate significantly influenced the flavor metabolism. This study improves understanding of the interaction and cooperation between A. oryzae and S. cerevisiae in co-culture fermentation for Chinese liquor making.

HEAT-INDUCED CHANGES IN VOLATILES OF MILK AND EFFECTS OF THERMAL PROCESSING ON MICROBIAL METABOLISM OF YOGURT

Flavor and texture of yogurt are highly associated with the consumer acceptability. The volatiles in the headspace of heated milk and yogurt were analyzed to assess the effects of preheat treatment on microbial metabolism. Seventeen and 25 volatiles were found in fresh raw milk and reconstituted milk using Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry, while 23 and 31 volatiles were investigated in their yogurt products, respectively. The levels of aldehydes, methyl ketones, alkanes, olefinic aldehydes and ketenes increased in liquid milk with increasing the intensity of heat treatments. Meanwhile, their levels in respective yogurt showed similar changes. 2-Pentanone, 2,3-butanedione and 2,3-pentanedione that were metabolized by starter culture organisms decreased due to increasing the intensity of heat treatments, suggesting that these heat treatments can inhibit the flavor metabolism of organisms. By analyzing the texture profile of yogurt, hardness, adhesiveness and gumminess increased with increasing the intensity of heat treatments; however, little difference was observed between the raw milk samples and the reconstituted milk samples. PRACTICAL APPLICATIONS This study demonstrated the differences in flavor impact compounds and textural properties of yogurt due to the preheat treatment and microbial metabolism. Results explain the source of volatile compounds in yogurt processing. Information on the changes in flavor as affected by heating processes would help in understanding their suitability in yogurt production. The research on flavor metabolism would shed light on the final application of some approaches to improve yogurt such as modification of milk medium, screening of the flavor-producing bacteria and the processing control of flavor production. Also, the finding could be used in the production of flavoring agent.

A candidate gene association study on muscat flavor in grapevine (Vitis vinifera L.)

BackgroundThe sweet, floral flavor typical of Muscat varieties (Muscats), due to high levels of monoterpenoids (geraniol, linalool and nerol), is highly distinct and has been greatly appreciated both in table grapes and in wine since ancient times. Muscat flavor determination in grape (Vitis vinifera L.) has up to now been studied by evaluating monoterpenoid levels through QTL analysis. These studies have revealed co-localization of 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS) with the major QTL positioned on chromosome 5.ResultsWe resequenced VvDXS in an ad hoc association population of 148 grape varieties, which included muscat-flavored, aromatic and neutral accessions as well as muscat-like aromatic mutants and non-aromatic offsprings of Muscats. Gene nucleotide diversity and intragenic linkage disequilibrium (LD) were evaluated. Structured association analysis revealed three SNPs in moderate LD to be significantly associated with muscat-flavored varieties. We identified a putative causal SNP responsible for a predicted non-neutral substitution and we discuss its possible implications for flavor metabolism. Network analysis revealed a major star-shaped cluster of reconstructed haplotypes unique to muscat-flavored varieties. Moreover, muscat-like aromatic mutants displayed unique non-synonymous mutations near the mutated site of Muscat genotypes.ConclusionsThis study is a crucial step forward in understanding the genetic regulation of muscat flavor in grapevine and it also sheds light on the domestication history of Muscats. VvDXS appears to be a possible human-selected locus in grapevine domestication and post-domestication. The putative causal SNP identified in Muscat varieties as well as the unique mutations identifying the muscat-like aromatic mutants under study may be immediately applied in marker-assisted breeding programs aimed at enhancing fragrance and aroma complexity respectively in table grape and wine cultivars.