Gallic acid improves color quality and stability of red wine via physico-chemical interaction and chemical transformation as revealed by thermodynamics, real wine dynamics and benchmark quantum mechanical calculations.

The aim of this study was to explore the copigmentation effect of gallic acid on red wine color and to dissect its mechanism at the molecular level. Three-dimensional studies, e.g., in model wine, in real wine and in silico, and multiple indicators, e.g., color, spectrum, thermodynamics and phenolic dynamics, were employed. The results showed that gallic acid significantly enhanced the color quality and stability of red wine. Physico-chemical interactions and chemical transformations should be the most likely mechanism, and physico-chemical interactions are also a prerequisite for chemical transformations. QM calculations of the physico-chemical interactions proved that the binding between gallic acid and malvidin-3-O-glucoside is a spontaneous exothermic reaction driven by hydrogen bonding and dispersion forces. The sugar moiety of malvidin-3-O-glucoside and the phenolic hydroxyl groups of gallic acid affect the formation of hydrogen bonds, while the dispersion interaction was related to the stacking of the molecular skeleton.

Intermolecular interactions between malvidin-3-O-glucoside and caffeic acid: Structural and thermodynamic characterization and its effect on real wine color quality.

The copigmentation effect between malvidin-3-O-glucoside and caffeic acid was comprehensive inquiry on the model wine solution, theoretical simulation and real wine. Thermodynamic parameters were determined by UV/Visible spectroscopy and Isothermal titration calorimetry (ITC). Theoretical data were obtained employing a dispersion-corrected density functional approach. The effects in real wines were investigated by adding the caffeic acid during different fermentation periods. Results shown that the copigmentation reaction between caffeic acid and malvidin-3-O-glucoside is a spontaneous exothermic reaction driven by hydrogen bonding and dispersions forces. Computations show that the polyhydroxyl sugar moiety and phenolic hydroxyl groups are the key active sites. The addition of caffeic acid in post-alcohol fermentation samples evidences an improving color characteristics in the wine.

Decoding Polysaccharides from Two Pichia Yeasts and Their Molecular Interaction with Wine Fruity Esters.

This study extensively characterized yeast polysaccharides (YPs) from Pichia fermentans (PF) and Pichia kluyveri (PK), with a specific focus on their structural attributes and their interaction with wine fruity esters in a model wine system. By finely tuning enzymatic reactions based on temperature, pH, and enzyme dosage, an optimal YP yield of 77.37% was achieved, with a specific mass ratio of cellulase, pectinase, and protease set at 3:5:2. There were four YP fractions (YPPF-W, YPPF-N, YPPK-W, and YPPK-N) isolated from the two yeasts. YPPF-N and YPPK-N were identified as glucans based on monosaccharide analysis and Fourier-transform infrared spectroscopy analysis. "Specific degradation-methylation-nuclear magnetic" elucidated YPPF-W's backbone structure as 1,3-linked α-l-Man and 1,6-linked α-d-Glc residues, while YPPK-W displayed a backbone structure of 1,3-linked α-Man residues, indicative of a mannoprotein nature. Isothermal titration calorimetry revealed spontaneous interactions between YPPK-W/YPPF-W and fruity esters across temperatures (25-45 °C), with the strongest interaction observed at 30 °C. However, distinct esters exhibited varying interactions with YPPK-W and YPPF-W, attributed to differences in molecular weights and hydrophobic characteristics. While shedding light on these intricate interactions, further experimental data is essential for a comprehensive understanding of yeast polysaccharides' or mannoproteins' impact on fruity esters. This research significantly contributes to advancing our knowledge of yeast polysaccharides' role in shaping the nuanced sensory attributes of wine.

Improving aromatic higher alcohol acetates in wines by co-fermentation of Pichia kluyveri and Saccharomyces cerevisiae: growth interaction and amino acid competition.

BACKGROUND: Higher alcohol acetates (HAAs) are potent aroma-active esters that impart desirable fruity and floral aromas. However, the conversion of higher alcohol precursors into HAAs is extremely low in winemaking. To investigate the underlying yeast-yeast interaction on targeted improvement of aromatic HAAs, we evaluated fermentation activity, cell viability, amino acid consumption and HAA production when Pichia kluyveri and Saccharomyces cerevisiae were inoculated concurrently or sequentially. RESULTS: Pichia kluyveri PK-21 possessed the ability to survive and increased HAA level up to 5.2-fold in mixed fermentation. Such an increment may benefit from the efficient conversion of higher alcohol precursors into HAAs (>27-fold higher than S. cerevisiae). During mixed fermentation, the two yeasts exhibited crucial interactions regarding cell growth and amino acid competition. Saccharomyces cerevisiae dominated over the co-inoculated P. kluyveri by efficient uptake of amino acids and biomass production. However, this dominance decreased in sequential fermentation, where P. kluyveri growth increased due to the consumption of preferred amino acids prior to S. cerevisiae. Pearson correlation analysis indicated that phenylalanine and aspartic acid may act as positive amino acids in boosting P. kluyveri growth and HAA production. Laboratory-scale winemaking validated the fermentation performance of P. kluyveri in sequential inoculum, resulting in a balanced aroma profile with enhanced floral and tropical fruity characteristics in the final wines. CONCLUSION: This study proposes a microbial, non-genetically engineered approach for targeted increase of HAA production in winemaking and the findings provide new insights into yeast-yeast interactions. © 2024 Society of Chemical Industry.

Asymmetric Paired Electrocatalysis: Enantioselective Olefin-Sulfonylimine Coupling.

Asymmetric electrocatalysis offers exciting new strategies for the synthesis of chiral molecules through novel reaction pathways. However, simultaneous activation of reactants on both electrodes via asymmetric paired electrolysis, which is more energy efficient and economic than single half-electrode synthesis, remains a formidable challenge. Herein, an asymmetric olefin-sulfonylimine coupling via paired electrocatalysis is presented for the first time. In this protocol, Co-catalyzed hydrogen atom transfer on the anode and Ni-catalyzed sulfonylimine reduction on the cathode were seamlessly cross-coupled. The new catalytic system enables the formation of chiral amine products bearing a tetrasubstituted carbon stereocenter with a high enantioselectivity (up to 96% ee).

Identification and validation of seed dormancy loci and candidate genes and construction of regulatory networks by WGCNA in maize introgression lines.

KEY MESSAGE: Seventeen PHS-QTLs and candidate genes were obtained, including eleven major loci, three under multiple environments and two with co-localization by the other mapping methods; The functions of three candidate genes were validated using mutants; nine target proteins and five networks were filtered by joint analysis of GWAS and WGCNA. Seed dormancy (SD) and pre-harvest sprouting (PHS) affect yield, as well as grain and hybrid quality in seed production. Therefore, identification of genetic and regulatory pathways underlying PHS and SD is key to gene function analysis, allelic variation mining and genetic improvement. In this study, 78,360 SNPs by SLAF-seq of 230 maize chromosome segment introgression lines (ILs), PHS under five environments were used to conduct GWAS (genome wide association study) (a threshold of 1/n), and seventeen unreported PHS QTLs were obtained, including eleven QTLs with PVE > 10% and three QTLs under multiple environments. Two QTL loci were co-located between the other two genetic mapping methods. Using differential gene expression analyses at two stages of grain development, gene functional analysis of Arabidopsis mutants, and gene functional analysis in the QTL region, seventeen PHS QTL-linked candidate genes were identified, and their five molecular regulatory networks constructed. Based on the Arabidopsis T-DNA mutations, three candidate genes were shown to regulate for SD and PHS. Meanwhile, using RNA-seq of grain development, the weighted correlation network analysis (WGCNA) was performed, deducing five regulatory pathways and target genes that regulate PHS and SD. Based on the conjoint analysis of GWAS and WGCNA, four pathways, nine target proteins and target genes were revealed, most of which regulate cell wall metabolism, cell proliferation and seed dehydration tolerance. This has important theoretical and practical significance for elucidating the genetic basis of maize PHS and SD, as well as mining of genetic resources and genetic improvement of traits.

Synergy Effect between Fruity Esters and Potential Odorants on the Aroma of Hutai-8 Rose Wine Revealed by Threshold, S-Curve, and σ-τ Plot Methods.

To analyze the contribution of fruity esters on wine aroma perception, the interaction levels between 12 esters and key odorants of Hutai-8 rose wine were investigated using threshold, S-curve, and σ-τ plot methods, and the aroma enhancement performance of esters was verified by using addition experiments. Results indicated that esters enhance the sweet, floral, and fruity traits of citronellol, ß-damascenone, and nerolidol, especially at subthreshold levels. Meanwhile, esters increased the floral and fruity characteristics of key fermentative odorants mainly by additive effects, with acetate esters possessing a better synergy ability. In contrast, the synergy levels between binary esters were less influenced by the concentration but more by the compound structure and aroma. Additionally, moderately subjoining the type and content of esters in wine proved that their synergy effects improved the sweet trait and decreased the sour fruit trait. This finding characterized that the contribution of esters to the wine aroma was obtained by the combined synergy of odorants at a suitable concentration.

Unlocking the Nucleophilicity of Strong Alkyl C-H Bonds via Cu/Cr Catalysis.

Direct functionalization of inert C-H bonds is one of the most attractive yet challenging strategies for constructing molecules in organic chemistry. Herein, we disclose an unprecedented and Earth abundant Cu/Cr catalytic system in which unreactive alkyl C-H bonds are transformed into nucleophilic alkyl-Cr(III) species at room temperature, enabling carbonyl addition reactions with strong alkyl C-H bonds. Various aryl alkyl alcohols are furnished under mild reaction conditions even on a gram scale. Moreover, this new radical-to-polar crossover approach is further applied to the 1,1-difunctionalization of aldehydes with alkanes and different nucleophiles. Mechanistic investigations reveal that the aldehyde not only acts as a reactant but also serves as a photosensitizer to recycle the Cu and Cr catalysts.

Molecular Rearrangement of Four Typical Grape Free Terpenes in the Wine Environment.

In order to analyze the molecular rearrangement of terpenes in wine during aging, the changes in linalool, α-terpineol, nerol, and geraniol in model wine were investigated in the dark at low temperature for 90 days. Headspace-gas chromatograph-mass spectrometer/olfactometry was used for qualitative and relative quantitation of terpenes. Quantum mechanical calculation was used to analyze the Gibbs free energy. The results showed that nerol was converted into d-limonene, terpinolene, linalool, and α-terpineol. Geraniol was converted into ß-ocimene, terpinolene, and linalool. Linalool was converted into terpinolene. The conversion rate of nerol to terpinolene was the highest with 5.94%. α-Terpineol was not converted spontaneously into other terpenes due to its lowest Gibbs free energy, indicating that the cyclization and isomerization could occur spontaneously through an exotherm reaction. However, the dehydroxylation of linalool, nerol, and geraniol required an energy source.

Fate of carotenoids in yeasts: synthesis and cleavage.

Carotenoids and their cleavage products (norisoprenoids) have excellent functional properties with diverse applications in foods, medicaments, cosmetics, etc. Carotenoids can be oxidatively cleaved through nonspecific reactions or by carotenoid cleavage oxygenases (CCOs), the product of which could further modify food flavor. This review provides comprehensive information on both carotenoid synthesis and cleavage processes with emphasis on enzyme characterization and biosynthetic pathway optimization. The use of interdisciplinary approaches of bioengineering and computer-aided experimental technology for key enzyme modification and systematic pathway design is beneficial to monitor metabolic pathways and assess pathway bottlenecks, which could efficiently lead to accumulation of carotenoids in microorganisms. The identification of CCOs spatial structures isolated from different species has made a significant contribution to the current state of knowledge. Current trends in carotenoid-related flavor modification are also discussed. In particular, we propose the carotenoid-synthesizing yeast Rhodotorula spp. for the production of food bioactive compounds. Understanding the behavior underlying the formation of norisoprenoids from carotenoids using interdisciplinary approaches may point toward other areas of investigation that could lead to better exploiting the potential use of autochthonous yeast in flavor enhancement.

Isolation, Characterization, and Compositional Analysis of Polysaccharides from Pinot Noir Wines: An Exploratory Study.

It has been reported that polysaccharides in wine can interact with tannins and other wine components and modify the sensory properties of the wine. Unfortunately, the contribution of polysaccharides to wine quality is poorly understood, mainly due to their complicated structure and varied composition. In addition, the composition and molecular structure of polysaccharides in different wines can vary greatly. In this study, the polysaccharides were isolated from pinot noir wine, then separated into high-molecular-weight (PNWP-H) and low-molecular-weight (PNWP-L) fractions using membrane-based ultrafiltration. Each polysaccharide fraction was further studied using size exclusion chromatography, UV-Vis, FT-IR, matrix-assisted laser desorption/ionization-high-resolution mass spectrometry, and gas chromatography-mass spectrometry (GC-MS). The results showed that PNWP-L and PNWP-H had different chemical properties and compositions. The FT-IR analysis showed that PNWPs were acidic polysaccharides with α- and ß-type glycosidic linkages. PNWP-L and PNWP-H had different α- and ß-type glycosidic linkage structures. FT-IR showed stronger antisymmetric and symmetric stretching vibrations of carboxylate anions of uronic acids in PNWP-L, suggesting more uronic acid in PNWP-L. The size exclusion chromatography results showed that over 72% of the PNWP-H fraction had molecular sizes from 25 kDa to 670 kDa. Only a small percentage of smaller molecular polysaccharides was found in the PNWP-H fraction. In comparison, all of the polysaccharides in the PNWP-L fraction were below 25 KDa, with a majority distributed approximately 6 kDa (95.1%). GC-MS sugar composition analysis showed that PNWP-L was mainly composed of galacturonic acid, rhamnose, galactose, and arabinose, while PNWP-H was mainly composed of mannose, arabinose, and galactose. The molecular size distribution and sugar composition analysis suggested that the PNWP-L primarily consisted of rhamnogalacturonans and polysaccharides rich in arabinose and galactose (PRAG). In comparison, PNWP-H were mostly mannoproteins and polysaccharides rich in arabinose and galactose (PRAG). Further research is needed to understand the impacts of these fractions on wine organoleptic properties.

Effects of inoculation timing and mixed fermentation with Pichia fermentans on Oenococcus oeni viability, fermentation duration and aroma production during wine malolactic fermentation.

There has been a growing interest in developing co-inoculum of Oenococcus oeni and Saccharomyces cerevisiae/non-Saccharomyces for simultaneous malolactic fermentation (MLF) and alcoholic fermentation (AF) of wines. This study sought to elucidate the effects of two crucial factors (inoculation timing and paired yeast) on the fermentation performance and aroma production of Merlot wine. O. oeni used for MLF was concurrently or sequentially inoculated with two yeast cultures (i.e., single S. cerevisiae and mixed S. cerevisiae /Pichia fermentans H5Y-28) used for AF. Inoculation timing determined the overall vinification duration, and conditioned the production of principle higher alcohols, terpene and O. oeni-mediated volatiles. In contrast, paired yeast improved O. oeni viability, and showed significant effect on aromatic esters and volatile fatty acids. Possibly due to lower ethanol stress, co-inoculum allowed O. oeni to initiate MLF during AF, resulting in 45% reduction of total fermentation time. Meanwhile, O. oeni growth was stimulated by P. fermentans, with 1.7-fold of the maximum population higher than that in co-fermentation without P. fermentans. Such stimulation of O. oeni growth also occurred in sequential fermentation where P. fermentans had been replaced by S. cerevisiae. Only in sequential inoculum, P. fermentans induced high levels of 3-methylbutyl acetate, ethyl 3-methylbutanoate, ethyl hexanoate and ethyl octanoate, which may result in enhanced fresh fruity trait of wines. These findings suggested a positive effect of P. fermentans H5Y-28 on O. oeni and MLF. This work provides an alternative approach to improve wine MLF and aroma outcomes using friendly non-Saccharomyces yeast with appropriate inoculation strategy.

Oxidation-reduction potential affects medium-chain fatty acid ethyl ester production during wine alcohol fermentation.

The medium-chain fatty acid ethyl esters (MCFAEEs) are a group of important aroma compounds generated during wine production. Wine alcohol fermentation involves several redox processes, which are affected by the oxidation-reduction potential (ORP). However, the mechanism via which ORP regulates MCFAEE production remains unclear. To investigate the effect of ORP on MCFAEE production, wine alcohol fermentation was performed using Saccharomyces cerevisiae under different ORPs. The results demonstrated that the ORPs studied (except for 90 mV) did not significantly affect cell growth, sugar consumption, and ethanol production, while the MCFAEE concentration in the simulated wines can be manipulated by ORP operation. MCFAEE levels increased till 96 h, and then decreased. The maximum MCFAEE level of 1222.97 µg/L was obtained after 96 h at 0 mV, which was 45.32% higher than that of the control. During the increase, higher relative expression of ACC1, FAS1, FAA2 and EEB1, elevated external citric acid flux, and moderate intracellular NADP+/NADPH ratio were observed at 0 mV compared to that at other ORPs. During the decrease, lowest relative expression of POX1 was detected at 0 mV. We showed for the first time the relationship between ORP operation and MCFAEE production in winemaking, which will improve the aroma quality of wine.

Adjustment of impact odorants in Hutai-8 rose wine by co-fermentation of Pichia fermentans and Saccharomyces cerevisiae.

Hutai-8 grape (Vitis vinifera × Vitis labrusca) was fermented into rose wine with Saccharomyces cerevisiae to characterize the key aroma compounds by sensomics approach, and fermented with selected Pichia fermentans and S. cerevisiae for aroma enhancement. In total, 82 odorants were detected in single-fermentation Hutai-8 rose wine, 22 aroma active compounds, mainly varietal odorants and fruity esters, were further quantitated for aroma reconstitution tests, and successfully mimicked sweet, floral, and fruity traits. Omission tests excluded the importance of isobutyl acetate, ethyl butyrate, 1-nonanol for aroma development. Furthermore, the successfully recombined co-fermentation Hutai-8 rose wine revealed 21 aroma active compounds identified from 79 odorants. The complexity of wine fruity/floral characteristics was improved through increased levels of inactive odorants and decreased levels of off-odorants. Moreover, rose wine made by co-inoculation had greater color stability compared to single-fermentation. This work provides insights into the understanding of mechanisms underlying modulation of co-fermentation for improved wine quality.

Localization, purification, and characterization of a novel ß-glucosidase from Hanseniaspora uvarum Yun268.

ß-Glucosidase is a key enzyme that hydrolyzes nonvolatile glycosylated precursors of aroma compounds and enhances the organoleptic quality of wines. In this study, a novel ß-glucosidase from Hanseniaspora uvarum Yun268 was localized, purified, and characterized. Results indicated that ß-glucosidase activity was mainly distributed within the cells. After purification via ammonium sulfate precipitation combined with chromatography, ß-glucosidase specific activity increased 8.36 times, and the activity recovery was 56.90%. The enzyme had a molecular mass of 74.22 kDa. It has a Michaelis constant (Km ) of 0.65 mmol/L, and a maximum velocity (Vmax ) of 5.1 nmol/min under optimum conditions; and Km of 0.94 mmol/L, and Vmax of 2.8 nmol/min under typical winemaking conditions. It exhibited the highest activity at 50°C and pH 5.0 and was stable at a temperature range of 20-80°C and pH range of 3.0-8.0. The enzyme has good tolerance to Fe3+ , especially maintaining 93.68% of its activity with 10 mmol/L of Fe3+ . Ethanol (<20%) and glucose (<150 g/L) inhibited its activity only slightly. Therefore, ß-glucosidase from H. uvarum Yun268 has excellent biochemical properties and a good application potential in winemaking. PRACTICAL APPLICATION: Winemaking is a biotechnological process in which exogenous ß-glucosidase is used to overcome the deficiency of endogenous ß-glucosidase activity in grapes. By localizing, purifying, and characterizing of ß-glucosidase from Hanseniaspora uvarum Yun268, it is expected to reveal its physical and chemical characteristics to evaluate its oenological properties in winemaking. The results may provide the basis for promoting the release of varietal aroma and improving wine sensory quality in the wine industry.

The implication of phenolic acid matrix effect on the volatility of ethyl acetate in alcohol-free wine model: Investigations with experimental and theoretical methods.

Hydroxycinnamic acids and ethyl acetate were assessed in simulated alcohol-free wine solutions to explore the effect of phenolic acids on the aroma volatility of esters. The results showed that the phenolic acids could inhibit the volatilization of ethyl acetate, and the extent of inhibition was influenced by the concentration and structure of the phenolic compounds. The ultraviolet absorption spectra of the phenolic acids and ethyl acetate confirmed the interaction between the two compounds. The thermodynamic parameters of the interaction implied a spontaneous exothermic interaction, driven primarily by hydrophobic effects. Meanwhile, the results of the fluorescence-quenching analysis indicated electron transfer between the reactants. The quantum chemical investigations revealed negative and positive charge density distributions in the structures of ethyl acetate and the phenolic acids, respectively. These results will provide some data reference and theoretical support for further research on the effects of phenolic acid matrix on other structural esters.

The effects of cell-cell contact between Pichia kluyveri and Saccharomyces cerevisiae on amino acids and volatiles in mixed culture alcoholic fermentations.

This study used a double-compartment fermenter to assess yeast growth, fermentation activity, and aroma production in response to cell-cell contact during mixed culture fermentation of Pinot noir grape must with Pichia kluyveri and Saccharomyces cerevisiae. Furthermore, amino acids were analyzed in order to study yeast interactions and possible reasons for aroma modulation as a response to cell-cell contact. Our results show that cell-cell contact between the two yeasts decreased cell viability of each yeast during mixed culture fermentation, and that it increased acetate and ethyl ester production and decreased varietal volatile levels. Moreover, it increased the consumption of glutamic acid and the biosynthesis of some specific amino acids related to cell growth, mainly histidine, glycine and proline, while suppressing the production of higher alcohols through the Ehrlich pathway. These results may contribute to an improved understanding, and thus control, of aroma production in mixed culture wine fermentations.

Engineering Rhodosporidium toruloides for limonene production.

BACKGROUND: Limonene is a widely used monoterpene in the production of food, pharmaceuticals, biofuels, etc. The objective of this work was to engineer Rhodosporidium toruloides as a cell factory for the production of limonene. RESULTS: By overexpressing the limonene synthase (LS), neryl pyrophosphate synthase (NPPS)/geranyl pyrophosphate synthase and the native hydroxy-methyl-glutaryl-CoA reductase (HMGR), we established a baseline for limonene production based on the mevalonate route in Rhodosporidium toruloides. To further enhance the limonene titer, the acetoacetyl-CoA thiolase/HMGR (EfMvaE) and mevalonate synthase (EfMvaS) from Enterococcus faecalis, the mevalonate kinase from Methanosarcina mazei (MmMK) and the chimeric enzyme NPPS-LS were introduced in the carotenogenesis-deficient strain. The resulting strains produced a maximum limonene titer of 393.5 mg/L. CONCLUSION: In this study, we successfully engineered the carotenogenesis yeast R. toruloides to produce limonene. This is the first report on engineering R. toruloides toward limonene production based on NPP and the fusion protein SltNPPS-CltLS. The results demonstrated that R. toruloides is viable for limonene production, which would provide insights into microbial production of valuable monoterpenes.

Genome-wide identification, characterization, and expression analysis of the monovalent cation-proton antiporter superfamily in maize, and functional analysis of its role in salt tolerance.

Na+, K+ and pH homeostasis are important for plant life and they are controlled by the monovalent cation proton antiporter (CPA) superfamily. The roles of ZmCPAs in salt tolerance are not fully elucidated. In this study, we identified 35 ZmCPAs comprising 13 Na+/H+ exchangers (ZmNHXs), 16 cation/H+ exchanger (ZmCHXs), and 6 K+ efflux antiporters (ZmKEAs). All ZmCPAs have transmembrane domains and most of them were localized to plasma membrane or tonoplast. ZmCHXs were specifically highly expressed in anthers, while ZmNHXs and ZmKEAs showed high expression in various tissues. ZmNHX5 and ZmKEA2 were up-regulated in maize seedlings under both NaCl and KCl stresses. Yeast complementation experiments revealed the roles of ZmNHX5, ZmKEA2 in NaCl tolerance. Analysis of the maize mutants further validated the salt tolerance functions of ZmNHX5 and ZmKEA2. Our study highlights comprehensive information of ZmCPAs and provides new gene targets for salt tolerance maize breeding.

Fine tuning of medium chain fatty acids levels increases fruity ester production during alcoholic fermentation.

Pichia fermentans Z9Y-3 and its intracellular enzymes were inoculated along with S. cerevisiae in synthetic grape must to modulate fruity ester production. The levels of ester-related enzymes, ester precursors, and fruity esters were monitored every 24 h during fermentation. Results showed that the levels of ethyl acetate, acetate higher alcohol esters (AHEs), short chain fatty acid ethyl esters (SFEs), and medium chain fatty acid ethyl esters (MFEs) were significantly enhanced in mixed fermentation. Pearson correlation analysis further revealed that higher alcohols and fatty acids played a more important role in fruity ester production than enzymes; Particularly, the correlation coefficient between fatty acids and MFEs was 0.940. In addition, supplementation of medium chain fatty acids (7.2 mg/L) at the metaphase of single S. cerevisiae fermentation improved ethyl acetate, AHE, SFE, and MFE production by 42.56%, 21.00%, 61.33%, and 90.04%, respectively, although the high level of ethyl acetate might result in off-flavors.