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.

A novel spectral fingerprint analysis to discriminate dry red wines.

A novel spectral fingerprint to discriminate different dry red wines was built using data visualization method. Twelve red wines with different vintages, cultivars and ageing methods from Changli and Shacheng were sampled. Nine fractions of each wine were collected with a reversed-phase C18 column, and then they were lyophilized. The residue of each fraction was resolved with synthetic wine of the same volume with the fraction sample. The transmittance spectra of wines and their fractions were recorded from 190 to 1100 nm. And the spectral data were visualized to show their visual differences directly. Mono-phenols in wine and fractions were analyzed by HPLC-DAD at wavelengths in the range where located the obvious differences of the spectral fingerprints. The results showed that the spectral differences of wine samples lied in the range of 190 to 600 nm. There were obvious differences in visual maps among wines with different vintages, mainly around 520 nm. The visualization differences among wines with distinct geographical origins lay in the F8 maps, and the differences from the aging methods almost cover the whole wavelength range visualized. However, wines from different grape cultivars had the similar visual characteristics. HPLC-DAD identified the possible monophenol groups for the spectral differences at 280, 313, 365 and 520 nm. It was concluded that the visualization of spectral data from 190 to 600 nm could be used to build red wine spectral fingerprint to distinguish dry red wines with different vintages, origins, and ageing methods.

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.

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.

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.

Phenolic matrix effect on aroma formation of terpenes during simulated wine fermentation - Part I: Phenolic acids.

In this study, fermentations were performed using simulated grape juice supplemented with terpene glycosides (TGs), extracted from Vitis vinifera L. Meili, and phenolic acids (gallic acid or p-coumaric acid). Free terpenes were detected using solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) every day during the fermentation, and the aromas of final wines were evaluated by panelists. Quantum mechanical (QM) calculations performed at density functional level of theory were used to uncover the structural and thermodynamic properties of the binding between phenolic acids and free terpenes (or TGs). Results showed that phenolic acids remarkably inhibited TG hydrolysis and free terpene volatilization, and affected wine aroma perception. QM calculations revealed that phenolic acids can bind to linalool and its glycoside spontaneously (ΔG < 0) via hydrogen bonding and dispersive forces. These findings indicate that the matrix effect of phenolic acids can effectively control the release and modulate the global feature of wine aromas.

Profiling terpene glycosides from ecolly, cabernet gernischet, and muscat hamburg grapes by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry.

Terpene glycosides (TGs) in the berry skins of Ecolly and Cabernet Gernischet (CG) grapes were profiled by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and compared to that of Muscat Hamburg (MH) grapes. The aroma glycosides in grape skins were extracted by methanol, purified using Amberlite XAD-2 resins, eluted in methanol/ethyl acetate, and analyzed by UPLC-Q-TOF-MS. MS/MS fragmentation and the TG database were used for identification, while semiquantification was achieved by the internal standard, octyl-ß-D-glucopyranoside. Seven, nine, and thirteen TGs were identified in Ecolly, CG, and MH, respectively, whereas only four TGs were detected in all the varieties. Compared to MH, linalool-3-O-α-L-arabinofuranosyl-ß-D-glucopyranoside was unique for Ecolly, and pyran linalool oxide-α-L-arabinofuranosyl-ß-D-glucopyranoside was unique for CG. Furthermore, the total concentrations of TGs were 7.32, 3.50, and 81.27 mg/kg in Ecolly, CG, and MH, respectively. Diendiol I-α-D-apiofuranosyl-ß-D-glucopyranoside, furanosyl-linalool oxide-7-O-α-L-arabinofuranosyl-ß-D-glucopyranoside, and nerol-l-O-α-D-apiofuranosyl-ß-D-glucopyranoside were most abundant in Ecolly, CG, and MH, respectively. PRACTICAL APPLICATION: Terpene glycosides (TGs) are important aroma precursors for wines and it is meaningful to reveal the profiles of TGs in grapes. By analyzing the differences of TGs among the Ecolly, Cabernet Gernischet (CG), and Muscat Hamburg grape varieties, it is expected to provide guidance for the study on the aroma characteristics and aroma enhancing brewing of Ecolly and CG grapes. The results may provide technical experience on aroma glycoside identification and their quantification in grapes and wines for future studies.

Increased glycosidase activities improved the production of wine varietal odorants in mixed fermentation of P. fermentans and high antagonistic S. cerevisiae.

A selected Pichia fermentans strain was simultaneously and sequentially inoculated in synthetic and real juice with S. cerevisiae strains of different antagonistic activities in a ratio 1:1 to observe the correlation between varietal odorants and glycosidase activities. Fermentations using pure S. cerevisiae strains were used for comparison. Yeast biomass and glycosidase activities were monitored, varietal odorants were detected using HS-SPME-GC/MS during fermentation. The final wine aroma attributes were analyzed by trained panelists. Results showed that co-inoculation with high antagonistic S. cerevisiae resulted in higher glycosidase activities than others. Pearson correlation analysis indicated that yeast biomass was positively related to glycosidase activities during fermentation. The increase in glycosidase activities was the main reason for the higher production of terpenes and C13-norisoprenoids, and for the lower C6 compound content, which lead to superior fruity and floral aromas in the final wine samples of the high antagonistic S. cerevisiae group.

Chemical profiles and aroma contribution of terpene compounds in Meili (Vitis vinifera L.) grape and wine.

The chemical profiles and aroma contribution of terpene compounds in Meili grapes and wine were analyzed. Bound terpene compounds were extracted using methanol, purified using Amberlite XAD-2 resin, concentrated in methanol/ethyl acetate, and enzymatically hydrolyzed to release aglycones. Free terpene compounds were identified using solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Wine aroma characteristics were quantified by a trained sensory panel. Seventeen terpene glycosides were quantified in grapes and wines as pentosyl-glucopyranoside, the content of which ranged from 804 to 836 µg/kg, and from 155 to 192 µg/L, respectively. Eight free terpenes were present in wines with their content ranging from 40.1 to 59.7 µg/L. Linalool was abundant both in bound and free terpenes, and mathematical regression revealed that terpenes, especially linalool (contribution efficient > 0.4), contributed heavily to Meili wine aroma. Finally, a molecular rearrangement scheme based on linalool was proposed in Meili grape and wine.

Evolution of volatile compounds treated with selected non-Saccharomyces extracellular extract during Pinot noir winemaking in monsoon climate.

The dynamic pattern of volatiles during Pinot Noir winemaking in monsoon climate with yeast extracellular extract (EE) treatment was analyzed. EE from selected Pichia fermentans and Rhodotorula mucilaginosa strains, and almond ß-glucosidase were added after 12-h alcohol fermentation, and the volatiles were determined every 24 h by GC-MS. After 6-month storage, wine aroma was evaluated instrumentally as well as by well-trained panelists. Results showed that enzyme treatments improved the contents of both varietal and fermentative volatiles. The levels of C6 compounds, terpenes, and higher alcohols increased constantly during alcohol fermentation, whereas acetates, short and medium chain fatty acid ethyl esters, phenylethyls, and fatty acids increased first, followed by gradual decrease. EE treatment retarded the decrease of fruity ester content in wine. Mathematical regression between wine aroma and volatiles showed that the relatively higher contents of acetates, ethyl esters, and C13-norisoprenoids in 6-month EE-treated wine were responsible for the improvement in floral aroma intensity.

Enhancing wine ester biosynthesis in mixed Hanseniaspora uvarum/Saccharomyces cerevisiae fermentation by nitrogen nutrient addition.

The dynamic changes of wine ester production during mixed fermentation with Hanseniaspora uvarum Yun268 and Saccharomyces cerevisiae F5 was investigated at different levels and timings of nitrogen nutrient addition. Nitrogen additions were performed by supplementing yeast assimilable nitrogen (YAN) into a synthetic grape must with defined composition. Ester precursors and extracellular metabolites involved in ester synthesis were analyzed throughout the fermentation. Results showed that nitrogen additions covering 50-200 mg/L YAN at the point of yeast inoculation slightly affected yeast competition and ester profiles. Interestingly, when YAN was supplemented in the mid-stage, the survival of H. uvarum Yun268 was enhanced, resulting in more than a 2-fold increase in the levels of higher alcohol acetates compared to that at the initial stage. Furthermore, carbon fluxes may be redistributed in the central pathway, which contributed to the production of medium-chain fatty acids and eventually triggered a 1.2-fold elevation in corresponding ethyl ester levels.

Increase of medium-chain fatty acid ethyl ester content in mixed H. uvarum/S. cerevisiae fermentation leads to wine fruity aroma enhancement.

Medium-chain fatty acid (MCFA) ethyl esters, as yeast secondary metabolites, significantly contribute to the fruity aroma of foods and beverages. To improve the MCFA ethyl ester content of wine, mixed fermentations with Hanseniaspora uvarum Yun268 and Saccharomyces cerevisiae were performed. Final volatiles were analyzed by gas solid phase microextraction-chromatography-mass spectrometry, and aroma characteristics were quantitated by sensory analysis. Results showed that mixed fermentation increased MCFA ethyl ester content by 37% in Cabernet Gernischt wine compared to that obtained by pure fermentation. Partial least-squares regression analysis further revealed that the improved MCFA ethyl esters specifically enhanced the temperate fruity aroma of wine. The enhancement of MCFA ethyl esters was attributed to the increased contents of MCFAs that could be induced by the presence of H. uvarum Yun268 in mixed fermentation. Meanwhile, the timing of yeast inoculations significantly affected the involving biomass of each strain and the dynamics of ethanol accumulation.

Wine aroma response to different participation of selected Hanseniaspora uvarum in mixed fermentation with Saccharomyces cerevisiae.

Wine aroma response to a selected Hanseniaspora uvarum Yun268 strain was investigated using different inoculation strategies with commercial Saccharomyces cerevisiae yeast, namely, simultaneous fermentation (SiF), sequential fermentation (SeF), S. cerevisiae fermentation treated with extracellular extract of H. uvarum (EE), and pure S. cerevisiae fermentation (PF). Contributive volatiles in the perception of enhanced aroma traits were uncovered by partial least-squares regression. Results showed that controlled inoculation resulted into different amounts of H. uvarum Yun268, which distinctively affected the chemical and sensory profiles of wines. The concentration of aromatic compounds could be increased by H. uvarum Yun268 yeasts via high levels of ß-glucosidase activity and fatty acids. Terpenes, C13-norisoprenoids, acetate esters, ethyl esters, and fatty acids served as the impact volatiles that contributed to the enhanced aroma traits. SiF specifically increased the contents of C13-norisoprenoids, terpenes, and ethyl esters, while EE enhanced varietal volatile content rather than those of fermentative ones. However, excessive H. uvarum Yun268 in sequential inoculation elevated the concentrations of acetate esters and volatile phenols, triggering nail polish odor in Cabernet Sauvignon red wines.

Aroma modulation of Cabernet Gernischt dry red wine by optimal enzyme treatment strategy in winemaking.

Cabernet Gernischt (CG) is a famous Chinese wine grape cultivar, the red wine of which is known for its green trait, especially when produced from grapes cultivated in regions with monsoon climate. To modify CG wine aroma, three enzyme preparations (H. uvarum extracellular enzyme, AR2000, and pectinase) were introduced in different winemaking stages with Saccharomyces cerevisiae. Free and bound aroma compounds in young wines were detected using headspace solid-phase micro-extraction and gas chromatography-mass spectrometry, and aroma characteristics were quantified by trained panelists. Results showed that simultaneous inoculation of enzymes and yeasts improved wine aroma. Partial least-squares regression revealed that the green trait was due mainly to varietal compounds, especially C6 compounds, and could be partly weakened by fermentative compounds. Moreover, H. uvarum enzyme treatments enriched the acid fruit note of CG wine by enhancing the synergistic effect of varietal volatiles and certain fermentative compounds, such as esters and phenylethyls.

Aroma Glycosides in Grapes and Wine.

The major aroma components in grapes and wine include free volatile compounds and glycosidic nonvolatile compounds. The latter group of compounds is more than 10 times abundant of the former, and constitutes a big aroma reserve in grapes and wine. This review summarizes the research results obtained recently for the identification of aroma glycosides in grapes and wine, including grape glycoside structures, differences in aroma glycosides among grape varieties, hydrolysis mechanisms, and the factors that influence them. It also presents the analytical techniques used to identify the glycosidic aroma precursors. The operational strategies, challenges, and improvements of each step encountered in the analysis of glycosidic aroma precursors are described. This review intends to provide a convenient reference for researchers interested in the methods used for the determination of the aroma glucosides composition and the recognition of their chemical structures.

Evaluation of aroma enhancement for "Ecolly" dry white wines by mixed inoculation of selected Rhodotorula mucilaginosa and Saccharomyces cerevisiae.

To improve the aroma profile of Ecolly dry white wine, the simultaneous and sequential inoculations of selected Rhodotorula mucilaginosa and Saccharomyces cerevisiae were performed in wine making of this work. The two yeasts were mixed in various ratios for making the mixed inoculum. The amount of volatiles and aroma characteristics were determined the following year. Mixed fermentation improved both the varietal and fermentative aroma compound composition, especially that of (Z)-3-hexene-1-ol, nerol oxide, certain acetates and ethyls group compounds. Citrus, sweet fruit, acid fruit, berry, and floral aroma traits were enhanced by mixed fermentation; however, an animal note was introduced upon using higher amounts of R. mucilaginosa. Aroma traits were regressed with volatiles as observed by the partial least-square regression method. Analysis of correlation coefficients revealed that the aroma traits were the multiple interactions of volatile compounds, with the fermentative volatiles having more impact on aroma than varietal compounds.

Aroma compounds and characteristics of noble-rot wines of Chardonnay grapes artificially botrytized in the vineyard.

Aroma characteristics and their impact volatile components of noble-rot wines elaborated from artificial botrytized Chardonnay grapes, obtained by spraying Botrytis cinerea suspension in Yuquan vineyard, Ningxia, China, were explored in this work. Dry white wine made from normal-harvested grapes and sweet wine produced from delay-harvested grapes were compared. Wine aromas were analysed by trained sensory panelists, and aroma compounds were determined by SPME-GC-MS. Results indicated that esters, fatty acids, thiols, lactones, volatile phenols and 2-nonanone increased markedly in noble-rot wines. In addition to typical aromas of noble-rot wines, artificial noble-rot wines were found to contain significant cream and dry apricot attributes. Partial Least-Squares Regression models of aroma characteristics against aroma components revealed that non-fermentative odorants were the primary contributor to dry apricot attribute, especially, thiols, C13-norisoprenoids, lactones, terpenols and phenolic acid derivatives, while cream attribute was dependent on both fermentative and non-fermentative volatile components.

Potential of Glycosidase from Non-Saccharomyces Isolates for Enhancement of Wine Aroma.

The aim of this work was to rapidly screen indigenous yeasts with high levels of ß-glucosidase activity and assess the potential of glycosidase extracts for aroma enhancement in winemaking. A semiquantitative colorimetric assay was applied using 96-well plates to screen yeasts from 3 different regions of China. Isolates with high ß-glucosidase activity were confirmed by the commonly used pNP assay. Among 493 non-Saccharomyces isolates belonging to 8 generas, 3 isolates were selected for their high levels of ß-glucosidase activity and were identified as Hanseniaspora uvarum, Pichia membranifaciens, and Rhodotorula mucilaginosa by sequence analysis of the 26S rDNA D1/D2 domain. ß-Glucosidase in the glycosidase extract from H. uvarum strain showed the highest activity in winemaking conditions among the selected isolates. For aroma enhancement in winemaking, the glycosidase extract from H. uvarum strain exhibited catalytic specificity for aromatic glycosides of C13 -norisoprenoids and some terpenes, enhancing fresh floral, sweet, berry, and nutty aroma characteristics in wine.

Modulating the formation of Meili wine aroma by prefermentative freezing process.

The influence of a prefermentative freezing process on changes of aromatic characteristics and volatile compounds in Meili wines was studied to optimize freezing parameters and reveal the mathematical relationship between aromatic characteristics and volatile compounds. The wines obtained were characterized by sensory evaluation and stir bar sorptive extraction (SBSE) followed by a thermal desorption-gas chromatography-mass spectrometry analysis. A total of 28 aromatic descriptors from 6 categories of wine aroma terminology were identified by judging with high "modified frequency (MF%)". In addition, 19 varietal aroma compounds and 36 fermentation aroma compounds were quantitated, followed by the determination of odor activity values (OAVs). On the basis of the data obtained, principal component analysis (PCA) was used to find the relationship between characteristic aroma terms and different freezing conditions, and then partial least-squares regression (PLSR) was proposed to establish the mathematical relationship between the resulting terms and impact odorants. Natural thawing treatment on frozen must resulted in higher aroma quality with higher extraction of varietal aroma compounds. Lower frozen maceration temperature contributed to higher esters and organic acids. Impact aroma compounds were related to models for floral, sweet fruit, temperate fruit, and vegetal, whereas the model of rose and strawberry contained only varietal volatile compounds, and temperate fruit could be regressed by impact fermentation aroma compounds.

Impact of cover crops in vineyard on the aroma compounds of Vitis vinifera L. cv Cabernet Sauvignon wine.

This study compared the influence of different cover crops with clean tillage on wine aroma compounds of 5-year-old Cabernet Sauvignon vines. White clover, alfalfa, and tall fescue were used in the vineyard and compared with clean tillage. Aroma compounds of wine were analysed by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC/MS). Forty-seven volatile compounds were identified and quantified. Wines made from grapes grown with various cover crops had higher levels of aroma compounds. Ethyl acetate, isoamyl acetate, ethyl octanoate, ethyl hexanoate, phenylethyl acetate, isoamyl alcohol, linalool, citronellol, ß-damascenone, α-ionone, and 5-amyl-dihydro-2(3H)-furan were the impact odorants of sample wines. Wines from cover crop also had higher contents of these impact odorants than the control. For different cover crops, alfalfa sward yielded the highest levels, followed by the tall fescue treatment. According to the data analysis of aroma compounds and sensory assess, permanent cover crop may have the potential to improve wine quality.