Formation Of Volatile Esters Research Articles

The influence of polyphenols on the hydrolysis and formation of volatile esters in wines during aging: An insight of kinetic equilibrium reaction

The evolution of volatile esters leads to changes in wine aroma during aging. In this study, polyphenol effect on ester equilibrium in wines was investigated through three aging experiments. Kinetic parameters of esters were calculated in four red wines. Results showed that the reaction rate constant (kobsd) was mainly determined by the molar concentration ratio of alcohols or acids to the corresponding esters. Phenolic matrix was more likely to influence the activation energy (Ea). Higher contents of total polyphenol led to the increase of Ea, resulting in the reactions less prone to happen but more susceptible to temperature changes. Combined with the practical wine aging and exogenous polyphenol addition experiments, the impact of polyphenol composition was revealed. Flavanols with higher polymerization degrees were found more beneficial for ester preservation than monomer flavanols or anthocyanins. This work could provide theoretical guidance in enhancing fruity aroma in wines via modulating phenolic matrix.

Continuous fed-batch strategy decreases acetic acid production and increases volatile ester formation in wines under high-gravity fermentation

High sugar fermentation elevates acetic acid levels in wines, which can be avoided by applying the continuous fed-batch strategy. In this study, yeast gene expressions and wine volatile compounds were evaluated by quantitative real-time PCR (RT-qPCR) and gas chromatograph mass spectrometry (GC-MS) in high-gravity (HG, 320 g/L sugars) fermentations with different batch strategies. The acetic acid concentration in continuous fed-batch fermentation wine was reduced by 51.69 %, compared with that in whole-batch fermentation wine. The acetyl-CoA synthase gene (ACS2) expression was up-regulated, whereas the glycerol-3-phosphate dehydrogenase gene (GPD1) expression was down-regulated on day 3 and day 7 during the continuous fed-batch fermentation. The volatile ester concentration in continuous fed-batch fermentation wine was 36.74 % higher than that in whole-batch fermentation wine. Overall, the continuous fed-batch strategy can modulate the expression of yeast genes involved in acetic acid metabolism and can increase volatile esters in wine under high sugar fermentation. Our findings provide a reference for the application of a continuous fed-batch strategy in high-sugar fermentation so as to improve the quality of the wine.

Integrated metabolome and transcriptome analysis of the regulatory network of volatile ester formation during fruit ripening in pear

'Nanguo' pear (Pyrus ussuriensis Maxim.) is a typical climacteric fruit with an attractive aroma after postharvest ripening. Esters are the key volatile compounds determining the typical aroma formation. However, the mechanism of aroma-related ester formation remains largely unknown. In this study, we performed transcriptome and metabolome analyses to reveal the changes of aroma-related compounds during pear ripening in the optimal taste period (OTP). During the pear ripening process, typical fatty acid-derived volatile organic compounds (VOCs) are transformed from aldehydes, alcohols, and ketones to esters, where ethyl hexanoate, hexyl acetate, and ethyl butanoate are the dominant esters in the OTP. Rich aroma-related esters in the OTP are associated with the accumulation of important precursors of aroma volatiles, including linoleic acid, α-linolenic acid, γ-linolenic acid, and oleic acid. Genes encoding key biosynthetic enzymes are associated with the altered levels of aroma-related esters. The candidate genes associated with the high levels of aroma-related esters in 'Nanguo' pears are PuFAD2, PuLOX2, PuLOX5, and PuAAT. Additionally, transcription factor (TF) genes such as PuWRKY24, PuIAA29, and PuTINY may play crucial roles in aroma formation during fruit ripening. Hence, we summarized the TFs that regulate VOC metabolism in different fruit species. The results provided a foundation for further research on aroma-related esters in 'Nanguo' pears and could help to elucidate the mechanisms regulating fruit quality improvement.

Transcriptional and epigenetic analysis reveals that NAC transcription factors regulate fruit flavor ester biosynthesis.

Flavor-associated volatile chemicals make major contributions to consumers' perception of fruits. Although great progress has been made in establishing the metabolic pathways associated with volatile synthesis, much less is known about the regulation of those pathways. Knowledge of how those pathways are regulated would greatly facilitate efforts to improve flavor. Volatile esters are major contributors to fruity flavor notes in many species, providing a good model to investigate the regulation of volatile synthesis pathways. Here we initiated a study of peach (Prunus persica L. Batsch) fruits, and identified that the alcohol acyltransferase PpAAT1 contributes to ester formation. We next identified the transcription factor (TF) PpNAC1 as an activator of PpAAT1 expression and ester production. These conclusions were based on in vivo and in vitro experiments and validated by correlation in a panel of 30 different peach cultivars. Based on homology between PpNAC1 and the tomato (Solanum lycopersicum) TF NONRIPENING (NOR), we identified a parallel regulatory pathway in tomato. Overexpression of PpNAC1 enhances ripening in a nor mutant and restores synthesis of volatile esters in tomato fruits. Furthermore, in the NOR-deficient mutant tomatoes generated by CRISPR/Cas9, lower transcript levels of SlAAT1 were detected. The apple (Malus domestica) homolog MdNAC5 also stimulates MdAAT1 expression via binding to this gene's promoter. In addition to transcriptional control, epigenetic analysis showed that increased expression of NACs and AATs is associated with removal of the repressive mark H3K27me3 during fruit ripening. Our results support a conserved molecular mechanism in which NAC TFs activate ripening-related AAT expression, which in turn catalyzes volatile ester formation in multiple fruit species.

Functional divergence of principal alcohol o-acyltransferase for biosynthesis of volatile acetate esters among tomato wild species (Solanum Sect. Lycopersicon)

Volatile esters are the chemicals that have multiple physiological functions including plant defense responses and reproduction. From a human perspective, the esters largely contribute to the fruity aroma of freshy fruits. Composition of volatile esters show a significant diversity among the wild tomato species (Solanum sect. Lycopersicon). To address the basis for this divergence, here we conducted functional analysis of a gene encoding major alcohol o-acyltransferase (AAT1) that catalyzes volatile ester formation. Although AAT1 transcripts were highly expressed in the ripe fruits of all the wild species examined, their enzymatic properties significantly differed due to amino acid sequence variations. Notably, AAT1s from S. pennellii showed the highest ability to produce acetate esters whereas AAT1s from S. neorickii, S. chmielewskii and S. habrochaites had the lowest activities. Further, screenings using domain-swapped or point-mutated AAT1s allowed us to identify Met/Thr352 as one of the critical residues related to the transferase activity with acetyl-CoA. This finding is potentially applied to aroma engineering in which a site-directed mutagenesis at this position in alcohol o-acyltransferases could enable to manipulate volatile ester levels in ripe fruits.

Peach Carboxylesterase PpCXE1 Is Associated with Catabolism of Volatile Esters.

Peach fruit volatile acetate esters impact consumer sensory preference and contribute to defense against biotic stresses. Previous studies showed that alcohol acyltransferase (AAT) family PpAAT1 is correlated with volatile ester formation in peach fruits. However, fruits also contain carboxylesterase (CXE) enzymes that hydrolyze esters. The functions of this family with regard to volatile ester content has not been explored. Here, we observed that content of acetate ester was negatively correlated with expression of PpCXE1. Recombinant PpCXE1 protein exhibited hydrolytic activity toward acetate esters present in peach fruit. Kinetic analysis showed that PpCXE1 showed the highest catalytic activity toward E-2-hexenyl acetate. Subcellular localization demonstrated that PpCXE1 is present in the cytoplasm. Transient expression in peach fruit and stable overexpression in tomato fruit resulted in significant reduction of volatile esters in vivo. Taken together, the results indicate that PpCXE1 expression is associated with catabolism of volatile acetate esters in peach fruit.

Monitoring of the production of flavour compounds by analysis of the gene transcription involved in higher alcohol and ester formation by the brewer's yeastSaccharomyces pastorianususing a multiplex RT-qPCR assay

Lager yeast produces a broad range of aroma-active substances during fermentation, of which higher alcohols and volatile esters are vital for the complex flavour of beer. Lager yeast contains several genes that encode the proteins responsible for flavour production. Investigation of gene expression in the hybrid Saccharomyces pastorianus is meaningful as it combines genes from both S. cerevisiae (Sc-type) and S. bayanus (Sb-type). In this study, we developed a GeXP analyser-based multiplex polymerase chain reaction assay to simultaneously assess the transcription of BAP2, BAT1, BAT2, ATF1, ATF2, EEB1, EHT1 and IAH1, involved in higher alcohol and volatile ester biosynthesis, within the S. pastorianus genome from both sub-genomes. GeXP proved to be an effective method of performing a gene expression study by providing a continuous profile of the expression of multiplex genes. This offers the possibility of using this type of data to monitor and control fermentations. Profiles of higher alcohol and volatile ester formation, and the transcription of specific genes, indicated that the synthesis of flavour compounds appears to correlate, at least in part, to the transcription of certain genes. As well as the differential expression of various genes, the differential expression of orthologous genes was also observed during fermentation, suggesting that the Sc-type and Sb-type genes may have different functions. Our results will further the understanding and manipulation of the phenotype of commercially important lager yeasts. Copyright © 2014 The Institute of Brewing & Distilling

Volatile Bio-ester Production from Orange Pulp-Containing Medium Using Saccharomyces cerevisiae

The dynamics of orange pulp (OP) as a fermentation feedstock for the production of volatile esters of “fruity” aroma by using a commercial wine yeast strain (Saccharomyces cerevisiae var. cerevisiae) was investigated. To achieve this goal, the kinetic behaviour of yeast (cell growth, substrate assimilation and volatile ester formation) was studied in OP complemented with nutritive medium under two different aeration conditions. The results were compared with those obtained from conventional processes in nutritive medium containing glucose as a sole carbon source. The results obtained revealed that the yeast cells were able to grow in the OP-containing medium after a 12-h lag period, probably due to the inhibitory effect of limonene. OP was found to stimulate the de novo synthesis of isoamyl acetate, phenylethyl acetate and ethyl esters (hexanoate, octanoate, decanoate and dodecanoate) by S. cerevisiae. This was strongly evident in the case of limited oxygen supply. Based on the above findings, OP appears to be a promising choice for bioflavour production by yeast.

Hydrolysis and formation of volatile esters in New Zealand Sauvignon blanc wine

The evolution of the varietal thiol 3-mercaptohexanol acetate (3MHA) and other key aroma compounds has been monitored in New Zealand Sauvignon blanc wines stored for 1year at three different temperatures (5, 10 and 18°C). The main processes that occurred in the Sauvignon blanc wines during bottle ageing were hydrolysis of 3MHA and other acetate esters, hydrolysis of ethyl esters of fatty acids, and the formation of ethyl esters of branched acids. The kinetic parameters of ester hydrolysis, including reaction rate constants and activation energies, were determined, which allow prediction of future wine composition based upon storage temperature and time. It was found that 3MHA had the highest reaction rate constant, meaning that this compound is the most unstable, particularly at higher storage temperatures, and that it disappeared very fast during wine storage.

Antisense suppression of alcohol acetyltransferase gene in ripening melon fruit alters volatile composition

Volatile esters are characteristic aroma components of melon (Cucumis melo L.) fruit. Alcohol acetyltransferase (AAT) (EC2.3.1.84) catalyzes the reactions of aliphatic and aromatic alcohols to their esters in the presence of acyl CoA in fruit and flowers. In this study, melon plants were transformed with a gene construct containing a melon AAT cDNA (AAT2) in an antisense orientation, driven by the constructive cauliflower mosaic virus 35S promoter, by ovary-injection transformation method. The transformed plants displayed a range of decreased AAT activities in the ripening fruit. The relative content of volatile esters was reduced, and the average relative content in the transgenic fruit was 78% of the wild-type fruit. However, the relative contents of aldehydes and alcohols increased; the average relative contents were 3.2 times and 2.4 times of those in the wild-type fruit, respectively. Apart from the difference in the volatile compounds, no other phenotypic alterations were noted, including other quality indices, such as the sugar, vitamin C and carotenoid contents. Our study indicates that AAT plays the key role in volatile ester formation in melon fruit.

Unexpected events in sulfated zirconia catalyst during glycerol-to-acrolein conversion

The aim of the work was the analysis of the phenomena occurring during glycerol dehydration in the presence of sulfated zirconia used as solid acid catalysts; the attention was focused on catalyst deactivation. A close correlation between the sulfate content and catalytic behavior was found. In addition to the accumulation of carbon residues on the catalyst surface, there were other phenomena contributing to catalyst deactivation: (i) the self-reduction of sulfuric into sulfurous groups – an event which occurred, however, only in samples with the higher S content under anaerobic conditions – and (ii) the leaching of S from catalysts, due to the hydrolysis of sulfate groups and the formation of volatile esters. These findings are of general interest in relation to both the transformation of glycerol into acrolein by means of gas-phase dehydration and the use of sulfated zirconia catalysts for high-temperature, acid-catalyzed reactions.

Volatile Ester Suppression and Recovery following 1-Methylcyclopropene Application to Apple Fruit

The effect of 1-methylcyclopropene (1-MCP) on biosynthesis of volatiles and fruit ripening in apple (Malus ×domestica Borkh.) was investigated using `Golden Delicious', `Jonagold', and `Redchief Delicious' fruit. Application of 1-MCP to `Golden Delicious' at the preclimacteric stage effectively inhibited ripening as determined by decreased expression of genes for 1-amino-1-cyclopropane carboxylic acid (ACC) oxidase (ACO), and ACC synthase, ACO protein content, climacteric ethylene production, respiration, and volatile ester biosynthesis. Exogenous ethylene applied after 1-MCP treatment did not induce ethylene production, respiration, or volatile production. Activity for alcohol acyltransferase, which catalyzes the final step in ester formation, was demonstrable for 1-MCP-treated fruit, indicating no strict limitation on ester formation is imposed by this enzyme and that ester formation in 1-MCP-treated apple fruit is at least partially limited by reduced substrate synthesis. Once volatile ester formation had been suppressed by 1-MCP, the recovery of volatile synthesis required ≈3 weeks for `Jonagold' and 4 weeks for `Delicious' when held in air at 22 °C. For the first 2 months of storage at 0 °C in air, `Jonagold' and `Delicious' required ≈3 weeks holding at 22 °C for volatile biosynthesis to initiate; after 5 months in refrigerated storage, volatile formation was evident at the time of removal from cold storage. For `Jonagold' fruit held in controlled atmosphere (CA) storage for 2, 5, and 7 months at 0 °C, at least 3 weeks holding at 22 °C were required for volatile formation to begin to recover. The maximal amount of volatile formation was reduced 50% by 1-MCP relative to nontreated control fruit. CA storage had a similar impact on maximal volatile formation. The marketing of 1-MCP-treated fruit soon after treatment might result in the delivery of fruit to the consumer with little likelihood of recovery of volatile ester formation prior to consumption.

Functional characterization of enzymes forming volatile esters from strawberry and banana.

Volatile esters are flavor components of the majority of fruits. The last step in their biosynthesis is catalyzed by alcohol acyltransferases (AATs), which link alcohols to acyl moieties. Full-length cDNAs putatively encoding AATs were isolated from fruit of wild strawberry (Fragaria vesca) and banana (Musa sapientum) and compared to the previously isolated SAAT gene from the cultivated strawberry (Fragaria x ananassa). The potential role of these enzymes in fruit flavor formation was assessed. To this end, recombinant enzymes were produced in Escherichia coli, and their activities were analyzed for a variety of alcohol and acyl-CoA substrates. When the results of these activity assays were compared to a phylogenetic analysis of the various members of the acyltransferase family, it was clear that substrate preference could not be predicted on the basis of sequence similarity. In addition, the substrate preference of recombinant enzymes was not necessarily reflected in the representation of esters in the corresponding fruit volatile profiles. This suggests that the specific profile of a given fruit species is to a significant extent determined by the supply of precursors. To study the in planta activity of an alcohol acyltransferase and to assess the potential for metabolic engineering of ester production, we generated transgenic petunia (Petunia hybrida) plants overexpressing the SAAT gene. While the expression of SAAT and the activity of the corresponding enzyme were readily detected in transgenic plants, the volatile profile was found to be unaltered. Feeding of isoamyl alcohol to explants of transgenic lines resulted in the emission of the corresponding acetyl ester. This confirmed that the availability of alcohol substrates is an important parameter to consider when engineering volatile ester formation in plants.

Aroma Production in Climacteric Fruit: A Comparison of Tomato, Apple, and Banana

Volatile aroma compounds produced by apple, banana, and tomato are produced throughout development, however, those associated with ripening and edible quality are dependent upon ethylene action. In apple and banana, characteristic aroma is, in large part, dependent upon the formation of volatile esters. In tomato, many of the characteristic aromas are dependent upon tissue disruption and result from aldehydes and alcohols following lipid degradation. For apple and banana, the enzyme alcohol acyl-CoA transferase (AAT, EC 2.3.1.84) is the enzyme responsible for the final reaction in the pathway for ester formation and catalyzes the union of an alcohol and the CoA derivative of fatty acids. In both tissues, AAT gene expression was detected prior to the onset of ester production. In apple, AAT expression was found to be closely tied with the onset of autocatalytic ethylene synthesis. In banana, ethylene synthesis peaked and began to decline well before ester synthesis began. However, the expression of AAT increased as ester production increased for both tissues. Tomato fruit, like apple and banana, produced characteristic aromas following the onset of the ethylene climacteric, suggesting changes in the activity of various components of the lipoxygenase pathway. In all three tissue types, there are continuous, significant shifts in the aroma profile as fruit ripen age, suggesting shifts in specific metabolic pathways associated with precursor synthesis or degradation.

Volatile ester formation in roses. Identification of an acetyl-coenzyme A. Geraniol/Citronellol acetyltransferase in developing rose petals.

The aroma of roses (Rosa hybrida) is due to more than 400 volatile compounds including terpenes, esters, and phenolic derivatives. 2-Phenylethyl acetate, cis-3-hexenyl acetate, geranyl acetate, and citronellyl acetate were identified as the main volatile esters emitted by the flowers of the scented rose var. "Fragrant Cloud." Cell-free extracts of petals acetylated several alcohols, utilizing acetyl-coenzyme A, to produce the corresponding acetate esters. Screening for genes similar to known plant alcohol acetyltransferases in a rose expressed sequence tag database yielded a cDNA (RhAAT1) encoding a protein with high similarity to several members of the BAHD family of acyltransferases. This cDNA was functionally expressed in Escherichia coli, and its gene product displayed acetyl-coenzyme A:geraniol acetyltransferase enzymatic activity in vitro. The RhAAT1 protein accepted other alcohols such as citronellol and 1-octanol as substrates, but 2-phenylethyl alcohol and cis-3-hexen-1-ol were poor substrates, suggesting that additional acetyltransferases are present in rose petals. The RhAAT1 protein is a polypeptide of 458 amino acids, with a calculated molecular mass of 51.8 kD, pI of 5.45, and is active as a monomer. The RhAAT1 gene was expressed exclusively in floral tissue with maximum transcript levels occurring at stage 4 of flower development, where scent emission is at its peak.

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

FACTORS THAT INFLUENCE VOLATILE ESTER BIOSYNTHESIS IN ‘DELICIOUS’ APPLES

We studied the influence of the intensity of light reaching fruit surfaces, cultivar, and maturity on amounts of the principal aroma and flavor volatiles (acetate esters) emitted from 'Delicious' apple fruits, and the relationship between anthocyanin accumulation and acetate ester emission. Light treatments of full sun (FS), 53% FS, 19% FS, and <1% FS were created by season-long bagging of 'Starkrimson' and 'Topred' fruit with shade cloth. Anthocyanin levels decreased with decreased light exposure, with 'Topred' fruits generally accumulating more anthocyanin than 'Starkrimson'. Contents of all flavonoid classes were also reduced as light level decreased. Volatile ester emissions were strongly affected by light exposure, with maxima occurring below 100% FS. Ester emission from 'Starkrimson' fruit at 179 days after full bloom (DAFB) was roughly twice that from 'Topred' in both years. The primary ester of both strains at all light exposures was 2-methyl butyl acetate, accounting for 50 to 75% of total ester content, with butyl and hexyl acetates as secondary components. Fruit position on the canopy also affected color and aroma/flavor of fruit. Western and southern aspect fruit generally emitted more esters than northern and eastern aspect fruit; anthocyanin accumulation was maximal in fruit from northern and eastern aspects. Differences in ester, anthocyanin, and flavonoid content due to light exposure were as seen in the main study. These data suggest that anthocyanin accumulation and volatile ester formation increase together with increasing light exposure, but at higher light exposures, continued increases in anthocyanin production come at the expense of acetate ester biosynthesis. For these strains, this happened between 53% FS and FS. Results suggest that 'Delicious' apples managed for maximum color have less volatile content and less potential for volatile development.

Partial purification and some properties of alcohol acyltransferase from strawberry fruits

The enzyme system concerning volatile ester formation in strawberry Fragaria ananassa×Duchessne var. Chandler was studied. Protein with alcohol acyltransferase activity was purified about 29-fold from Chandler strawberry fruits by ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The enzyme activity had a pH optimum of 8.0 and an optimum temperature of 35 o C. The apparent M r estimated by gel filtration was 70 000. The enzyme was tested for its preference in using different acyl-CoAs and alcohols. Maximum activity was obtained using acetyl-CoA and hexyl alcohol as substrates. A clear correlation was observed between substrate preference and volatile esters present in strawberry var. Chandler

Studies on flavor development in strawberries. I. Formation of volatile esters in strawberries.

Aliphatic alcohols including methyl, ethyl, isopropyl, npropyl, isobutyl, nbutyl, isoamyl, namyl and hexyl alcohol were converted to their acetate, propionate, nbutyrate, isovalerate and caproate esters during the incubation with strawberry fruit tissue. Formate, isobutyrate and n-valerate esters were formed when alcohols were incubated together with these fatty acids and strawberry.Seventy esters were formed from various combinations of alcohols and acids by means of incubation with strawberry.No ester formation was observed when strawberry was homogenized.

FORMATION OF ESTERS FROM LOWER FATTY ACIDS BY VARIOUS YEAST SPECIES

A study has been made of the total formation of volatile esters and of the formation of ethyl esters from butyric acid, valeric acid, isocaproic acid and caproic acid in fermentation with a number of yeast species of various genera. The distribution between the esters studied was essentially the same for all the yeast species tested although the quantitative results differed considerably. There was no correlation between the total formation of volatile esters and the formation of esters from acids added to the medium.