Production of volatile organic sulfur compounds (VOSCs) by basidiomycetous yeasts

Thirty-seven basidiomycetous yeasts belonging to 30 species of seven genera were grown on media containing l-cysteine or l-methionine as sole nitrogen sources with the objective of evaluating volatile organic sulfur compound (VOSC) production. The headspace of yeast cultures was analyzed by the solid-phase microextraction (SPME) sampling method, and volatile compounds were quantified and identified by GC-MS techniques. Ten strains assimilating L-methionine produced the following VOSCs: 3-(methylthio)-1-propanol, methanethiol, S-methyl thioacetate, dimethyl disulfide, dimethyl trisulfide, allyl methyl sulphide and 4,5-dihydro-3(2H)-thiophenone. Production was <1 mgl(-1) except for 3-(methylthio)-1-propanol of which between 40 and 400 mgl(-1) was synthesized. Higher alcohols (isobutyl alcohol, isoamyl alcohol and active amyl alcohol) and esters (ethyl acetate, ethyl propionate, n-propyl acetate, isobutyl acetate, n-propyl propionate, n-butyl acetate, isoamyl acetate, amyl acetate, isoamyl propionate, amyl propionate and 2-phenylmethyl acetate) were also sporadically produced. This is the first report of VOSCs production by basidiomycetous yeasts. Consequently, basidiomycetous yeasts may be considered an interesting new group of microbial VOSCs producers for the flavor industry.     

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.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.     

The Colorimetric Determination of Isobutyl and Isoamyl Alcohol in Fusel Oil Separately

The amounts of isobutyl and isoamyl alcohol which are the main constituents of fusel oil are determined fractionally. Isobutyl and isoamyl alcohol give different colors by treatment with p-dimethylaminobenzaldehyde in sulfuric acid. Using this color difference the amount of each of these alcohols in fusel oil is determined colorimetrically. These alcohols arise during alcoholic fermentation and are concentrated in the distillate by distillation, and affect the flavor of distilled spirits, such as brandy and whisky, i.e., butyl alcohol is buttery and amyl alcohol is aromatic. So determination of the amounts of these alcohols fractionally in these spirits furnishes a useful criterion for evaluating the character of these spirits.     

A study on volatile organic compounds (VOCs) produced by tropical ascomycetous yeasts

As a part of a program aiming at the selection of strains which might be of interest as sources of natural flavouring molecules, the production of volatile organic compounds (VOCs) by 98 ascomycetous yeast strains (representative of 40 species belonging to 12 genera) isolated from tropical environments was investigated. Volatiles produced were sampled by means of headspace solid-phase microextraction (SPME) and the compounds were analysed and identified by gas chromatography–mass spectroscopy (GC–MS). The VOCs produced were found to be alcohols (amyl alcohol and isoamyl alcohol), aldehydes (2-methyl-2-hexenal and 2-isopropyl-5-methyl-2-hexenal) and esters (ethyl isobutyrate, isobutyl acetate, isoamyl acetate, 2-methylbutyl acetate, ethyl isovalerate, isoamyl propionate and phenylmethyl acetate). Differences in VOC profiles were used to cluster the yeast strains into 25 VOC phenotypes. The different frequency of VOC phenotypes in three specific habitats was correlated to the divergent environmental conditions, possibly affecting the selection of specific yeasts. From a biotechnological viewpoint, this study reveals the potentiality of ascomycetous yeasts isolated from tropical environments as a promising source of VOCs relevant in food and fragrance industry. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enzymatic gallic acid esterification

Gallic acid esters of n-propyl and amyl alcohols have been produced by enzymatic synthesis in organic solvents using immobilized tannase. Studies indicate that maximum esterification of gallic acid occurs with amyl alcohol. The enzyme shows broad alcohol specificity. However, the enzyme exhibits absolute specificity for the acid portion of the ester. Studies were carried out on K(m), V(max), pH, and temperature optima.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that in the alcoholic fermentation of amino acids by yeast isobutyl alcohol is produced from alanine and n-propyl and active amyl alcohols are formed from α-amino-n-butyric acid or threonine contrary to the F. Ehrlich’s scheme. These results suggest the close relationship among the formation of these higher alcohols and biosynthesis of valine from alanine and biosynthesis of isoleucine from α-amino-n-butyric acid or threonine.

In this report, we studied the formation of n-propyl alcohol and active amyl alcohol from α-amino-n-butyric acid using washed yeast cells.     

Carbon Sources for Polytomella caeca

SUMMARY. The colorless phytomonad, Polytomella caeca , has been grown in simple media with each of the following as the sole energy source: propionate, butyrate, valerate; butyl and amyl alcohols; DL-glyceraldehyde, and alpha-ketoglutaric acid. These compounds did not support growth in simple media: caproate, caprylic acid, isobutyrate, propyl, isopropyl and isobutyl alcohols. At present, C₅, seems to be the limit of length of the fatty acids that are suitable carbon and energy sources. Iso compounds were not utilized. The pH in the acid media increased during incubation; that in alcohol media decreased.     

Cider fermentation with three Williopsis saturnus yeast strains and volatile changes

Williopsis saturnus var. subsufficiens NCYC 2728, W. saturnus var. saturnus NCYC 22 and W. saturnus var. mrakii NCYC 500 were used to carry out cider fermentation to assess their impact on the volatile composition of cider. The changes of yeast cell population, °Brix and pH were similar among the three yeasts. Strain NCYC 500 grew best, with the highest cell population of 1.14 × 108 CFU ml−1, followed by strains NCYC 2728 and NCYC 22 (8 × 107 CFU ml−1 and 3.19 × 107 CFU ml−1 respectively). Esters were the most abundant volatiles produced, followed by alcohols. Among the esters, ethyl acetate, 2-phenylethyl acetate, isoamyl acetate, cis-3-hexenyl acetate and hexyl acetate were the major volatiles. The major alcohols were ethanol, isoamyl alcohol, 2-phenylethyl alcohol and isobutyl alcohol. The three Williopsis yeasts transformed volatile compounds during cider fermentation with significant variations in terms of volatile production and degradation. This study implied that fermentation with Williopsis yeasts could result in cider with a more complex yet fruity aroma.     

Green Synthesis of the Flavor Esters with a Marine Candida parapsilosis Esterase Expressed in Saccharomyces cerevisiae

Indian Journal of Microbiology - The green synthesis of the flavor esters, n-propyl acetate, isobutyl acetate and isoamyl acetate had the advantages over the chemical synthesis. The esterase from...     

Expression levels of the yeast alcohol acetyltransferase genes ATF1, Lg-ATF1, and ATF2 control the formation of a broad range of volatile esters

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Delta atf2Delta double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes.     

Growth of the salt-tolerant yeast Zygosaccharomyces rouxii in microtiter plates: effects of NaCl,pH and temperature on growth and fusel alcohol production from branched-chain amino acids

Zygosaccharomyces rouxii, a salt-tolerant yeast isolated from the soy sauce process, produces fusel alcohols (isoamyl alcohol, active amyl alcohol and isobutyl alcohol) from branched-chain amino acids (leucine, isoleucine and valine, respectively) via the Ehrlich pathway. Using a high-throughput screening approach in microtiter plates, we have studied the effects of pH, temperature and salt concentration on growth of Z. rouxii and formation of fusel alcohols from branched-chain amino acids. Application of minor variations in pH (range 3-7) and NaCl concentrations (range 0-20%) per microtiter plate well allowed a rapid and detailed evaluation of fermentation conditions for optimal growth and metabolite production. Conditions yielding the highest cell densities were not optimal for fusel alcohol production. Maximal fusel alcohol production occurred at low pH (3.0-4.0) and low NaCl concentrations (0-4%) at 25 degrees C. At pH 4.0-6.0 and 0-18% NaCl, considerable amounts of alpha-keto acids, the deaminated products from the branched-chain amino acids, accumulated extracellularly. The highest cell densities were obtained in plates incubated at 30 degrees C. The results obtained under various incubation conditions with (deep-well) microtiter plates were validated in Erlenmeyer shake-flask cultures.     

Involvement of branched-chain amino acid aminotransferases in the production of fusel alcohols during fermentation in yeast

Organoleptic compounds produced by yeast during the fermentation of wort have a great impact on beer smell and taste. Among them, fusel alcohols are the major abundant volatile compounds. The availability of Saccharomyces cerevisiae mutants in which the genes coding for the two branched-chain amino acid aminotransferases have been deleted offers the possibility of further defining the role of these enzymes in the formation of higher alcohols. Comparing the production profiles of different strains, it is clear that they are not all influenced in the same way by branched-chain amino acid aminotransferase mutations. First of all, as propanol is synthesised from alpha-ketobutyrate, the first metabolic intermediate in the anabolic pathway of isoleucine, neither the eca39 nor eca40 mutations have any effect on the production of this higher alcohol. On the other hand, it can be concluded that the eca40 mutation has a drastic effect on the production of isobutanol. To a certain extent, the same conclusion can be made for the production of active amyl alcohol and isoamyl alcohol, although the results suggest that another route could lead to the formation of these two higher alcohols.     

Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Δ atf2Δ double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes.     

Fermentative capability and aroma compound production by yeast strains isolated from Agave tequilana Weber juice

Five yeast strains isolated from agave juice were studied for their fermentative and aromatic capacity. The experiments were performed using agave juice supplemented with ammonium sulphate, as is commonly done in tequila distilleries. Three strains classified as Saccharomyces cerevisiae showed high biomass and ethanol production, as well as higher ethanol tolerance than those classified as Kloeckera africana and Kloeckera apiculata, which showed scarce growth. The results suggest that Kloeckera strains were affected by nutritional limitation and/or toxic compounds present in agave juice. Agave juice analyses showed a lower amino acid content than those reported in grape juice. S. cerevisiae strains produced predominantly amyl and isoamyl alcohols, n-propanol, 2-phenyl ethanol, succinic acid, glycerol, methanol, isoamyl acetate, ethyl hexanoate, acetaldehyde and isobutanol, whereas Kloeckera strains showed a high production of acetic acid, 2-phenyl ethyl acetate and ethyl acetate. The methanol concentration was significantly different among the yeasts studied. The diversity between three S. cerevisiae strains were higher for the aromatic profile than for genetic level and kinetic parameter. On the other hand, the diversity of Kloeckera yeasts were lower than Saccharomyces yeasts even when belonging to two different species.     

Identification of volatile flavour compounds obtained in culture of Kluyveromyces marxianus

When Kluyveromyces marxianus was cultivated on a defined medium, flavour volatile compounds accumulated in the broth. Besides superior alcohols and aldehydes, acids and fruit esters could be analyzed by gas chromatography and coupled gas chromatography-mass spectrometry. The predominant components are isoamyl alcohol, 2-phenylethyl alcohol and isobutyric acid with 180 mg/L, 400 mg/L and 290 mg/L broth, respectively.     

Electroantennogram responses of Nilaparvata lugens (Homoptera: Delphacidae) to plant volatile compounds

Electroantennogram (EAG) recordings were made from the plaque organ receptors of the rice brown plant hopper, Nilaparvata lugens (St?l). The receptors of the distal plaque organ of both male and female hoppers responded to the air from above chopped rice plants, and those of females gave a larger response. Female hoppers, tested with 27 plant volatiles, gave dose-related responses to 16 of the compounds, including a range of green leaf volatiles. The aliphatic aldehydes hexanal and (E)-2-hexenal elicited a larger response than a range of green leaf alcohols. Of the remaining compounds, acetophenone, benzaldehyde, ethyl and methyl benzoate, and amyl and isoamyl acetate were among the most effective in eliciting responses. Receptor recovery times showed some variation between compounds. A comparison of the dose-related response of 16 compounds in paraffin oil and in pentane showed that considerably larger responses were recorded when the highly volatile solvent pentane was used.     

Regulation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genetic engineering on the production of acetate esters and higher alcohols during Chinese Baijiu fermentation

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.     

Influence of the physiological state of the inoculum on fermentation of musts from Pedro Ximénez grapes by Saccharomyces cerevisiae

Two inocula in different physiological states, namely in the exponential growth phase and in the declining phase were prepared from a strain of Saccharomyces cerevisiae. With these inocula were fermented musts from grapes of the Pedro Ximénez variety, sterilized by filtration. Cell growth and the activity of the enzymes alcohol dehydrogenase and aldehyde dehydrogenase [NADP+ and NAD(P)+] were found to vary with the state of the inoculum. This was reflected in the specific rate of production and, in some instances, in the final concentration of acetaldehyde, acetic acid, ethanol, isoamyl alcohols, phenethyl alcohol and their esters in the wine.