Synthetic Grape Medium Research Articles

Hydrogen sulfide production during early yeast fermentation correlates with volatile sulfur compound biogenesis but not thiol release.

Yeasts undergo intensive metabolic changes during the early stages of fermentation. Previous reports suggest the early production of hydrogen sulfide (H2S) is associated with the release of a range of volatile sulfur compounds (VSCs), as well as the production of varietal thiol compounds 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHA) from six-carbon precursors including (E)-hex-2-enal. In this study, we investigated the early H2S potential, VSCs/thiol output, and precursor metabolism of 11 commonly used laboratory and commercial Saccharomyces cerevisiae strains in chemically defined synthetic grape medium (SGM) within 12 hours after inoculation. Considerable variability in early H2S potential was observed among the strains surveyed. Chemical profiling suggested that early H2S production correlates with the production of dimethyl disulfide, 2-mercaptoethanol, and diethyl sulfide, but not with 3SH or 3SHA. All strains were capable of metabolizing (E)-hex-2-enal while the F15 strain showed significantly higher residue at 12 h. Early production of 3SH, but not 3SHA, can be detected in the presence of exogenous (E)-hex-2-enal and H2S. Therefore, the natural variability of early yeast H2S production contributes to the early output of selected VSCs, but the threshold of which is likely not high enough to contribute substantially to free varietal thiols in SGM.

Fermentation of Sauvignon blanc grape marc extract yields important wine aroma 3-sulfanylhexan-1-ol (3SH)

Grape marc, comprised of grape stems, seeds and skins, is an underutilised and potentially problematic waste material produced by the wine industry in often overwhelming quantities. This work aims to showcase this abundant waste material, for the first time, as a potentially valuable technological tool for improving wine quality through the production of the aroma compound 3-sulfanylhexan-1-ol (3SH) during fermentation.A scaled-up eco-friendly solid-liquid extraction method, using 100% water, to extract thiol precursor compounds 3-S-cysteinylhexan-1-ol (Cys-3SH) and 3-S-glutathionylhexan-1-ol (GSH-3SH) from Sauvignon blanc grape marc was undertaken. Quantitation of the vacuum-dried crude extract dissolved in model wine, using liquid chromatography tandem mass spectrometry (LC-MS/MS), showed that the extract contained 1.9 μg Cys-3SH and 11.46 μg GSH-3SH per gram of grape marc extract. Addition of low (14.6 g L−1) and high (58.3 g L−1) quantities of grape marc extract to synthetic grape medium inoculated with a commercial wine yeast strain, yielded appreciable concentrations of 3SH in finished wines (average of 216 ng L−1 for the low addition and 1244 ng L−1 for the high addition). Interestingly, the grape marc extract was found to accelerate the fermentation rate of the yeast, rather than cause inhibition or toxicity.

Addition of volatile sulfur compounds to yeast at the early stages of fermentation reveals distinct biological and chemical pathways for aroma formation

Volatile sulfur compounds (VSCs) greatly influence the sensory properties and quality of wine and arise via both biological and chemical mechanisms. VSCs formed can also act as precursors for further downstream VSCs, thus elucidating the pathways leading to their formation is paramount. Short-term additions of exogenous hydrogen sulfide (H2S), ethanethiol (EtSH), S-ethylthio acetate (ETA), methanethiol (MeSH) and S-methylthio acetate (MTA) were made to exponentially growing fermentations of synthetic grape medium. The VSC profiles produced from live yeast cells were compared with those from dead cells and no cells. Interestingly, this experiment allowed the identification of specific biochemical and/or chemical pathways; e.g. most of the conversion of H2S to EtSH, and the further step from EtSH to ETA, required the presence of live yeast cells, as did the conversion of MeSH to MTA. In contrast, the reaction from MTA to MeSH and ETA to EtSH was due primarily to chemical degradation. Ultimately, this research unravelled some of the complex interactions and interconversions between VSCs, pinpointing the key biochemical and chemical nodes. These pathways are highly interconnected and showcase the complexity of both the sulfur pathways in yeast and the reactive chemistry of sulfur-containing compounds.

The role of yeast ARO8, ARO9 and ARO10 genes in the biosynthesis of 3-(methylthio)-1-propanol from L-methionine during fermentation in synthetic grape medium.

3-(methylthio)-1-propanol (methionol), produced by yeast as an end-product of L-methionine (L-Met) catabolism, imparts off-odours reminiscent of cauliflower and potato to wine. Saccharomyces cerevisiae ARO genes, including transaminases Aro8p and Aro9p, and decarboxylase Aro10p, catalyse two key steps forming methionol via the Ehrlich pathway. We compared methionol concentrations in wines fermented by single Δaro8, Δaro9 and Δaro10 deletants in lab strain BY4743 versus wine strain Zymaflore F15, and F15 double- and triple-aro deletants versus single-aro deletants, using headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry.Deletion of two or more aro genes increased growth lag phase, with the greatest delay exhibited by F15 Δaro8 Δaro9. The single Δaro8 deletion decreased methionol by 44% in BY4743 and 92% in F15, while the Δaro9 deletion increased methionol by 46% in F15 but not BY4743. Single deletion of Δaro10 had no effect on methionol.Unexpectedly, F15 Δaro8 Δaro9 and F15 Δaro8 Δaro9 Δaro10 produced more methionol than F15 Δaro8. In the absence of Aro8p and Aro9p, other transaminases may compensate or an alternative pathway may convert methanethiol to methionol. Our results confirm that Ehrlich pathway genes differ greatly between lab and wine yeast strains, impacting downstream products such as methionol.

Antifungal and Antiochratoxigenic Activities of Essential Oils and Total Phenolic Extracts: A Comparative Study.

This study is intended to prevent ochratoxin A (OTA) production by Aspergillus carbonarius S402 using essential oils (EOs) and total phenolic compounds extracted from plants and herbs. The EOs used in this study are the following: bay leaves, cumin, fenugreek, melissa, mint, and sage. As for the phenolic compounds, they were extracted from bay leaves, cumin, fenugreek, melissa, mint, sage, anise, chamomile, fennel, rosemary, and thyme. The experiments were conducted on Synthetic Grape Medium (SGM) medium at 28 °C for 4 days. OTA was extracted from the medium with methanol and quantified using HPLC (High Performance Liquid Chromatography). Results showed that EOs had a greater impact than the total phenolic extracts on the OTA production. Reduction levels ranged between 25% (sage) and 80% (melissa) for the EOs at 5 µL mL−1, and 13% (thyme) and 69% (mint) for the phenolic extracts. Although they did not affect the growth of A. carbonarius, total phenolic extracts and EOs were capable of partially reducing OTA production. Reduction levels depended on the nature of the plants and the concentration of the EOs. Reducing OTA with natural extracts could be a solution to prevent OTA production without altering the fungal growth, thus preserving the natural microbial balance.

In vitro evaluation of essential oils against Aspergillus carbonarius isolates and their effects on Ochratoxin A related gene expression in synthetic grape medium

Clove, mandarin, cinnamon and lemongrass essential oils (EOs) were tested for their efficacy against 2 wild isolates (Ac29, Ac28) and a reference strain (5010) of Aspergillus carbonarius. Two different assays were performed on Malt Extract Agar (MEA) and Czapek Yeast Extract Agar (CYA) solid cultures, as well as on synthetic grape medium (SGM) liquid cultures. The impact of EOs on fungal growth and toxin production was measured after 7 days of incubation at 25 °C. Furthermore, the effect of clove, lemongrass and mandarin EOs on A. carbonarius OTA related genes AcOTAnrps, AcOTApks and laeA was investigated by relative expression using Real Time PCR on SGM cultures. Results showed that in solid cultures, complete fungal inhibition was obtained at 100–300 μl/l of EOs revealing a dose dependant effect of clove, cinnamon and lemongrass, whereas mandarin was proved to be less effective. EOs could not inhibit OTA production but only reduce it by 15–98% depending on the type and concentration of EO, even though in certain cases of low concentrations, EOs showed to enhance toxin production. In liquid cultures, a major effect on growth inhibition and OTA reduction was observed for all EOs. Regarding gene expression, down regulation of AcOTAnrps was observed in the majority of treatments that could be associated with toxin reduction. Moreover, AcOTApks exhibited some differential expression but laeA did not show any differences in the transcriptional profile. The above gene expression results may indicate a possible mode of action of EOs in OTA biosynthetic pathway.

Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA comprises a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described in A. carbonarius, which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the β-ketosynthase and acyl-transferase domains of the OTA PKSs that had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A quantitative RT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed, with AcOTApks reaching its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production.

Ecophysiological characterization of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger isolated from grapes in Spanish vineyards

The aim of this study was to evaluate the diversity of black aspergilli isolated from berries from different agroclimatic regions of Spain. Growth characterization (in terms of temperature and water activity requirements) of Aspergillus carbonarius, Aspergillus tubingensis and Aspergillus niger was carried out on synthetic grape medium. A. tubingensis and A. niger showed higher maximum temperatures for growth (>45°C versus 40–42°C), and lower minimum aw requirements (0.83aw versus 0.87aw) than A. carbonarius. No differences in growth boundaries due to their geographical origin were found within A. niger aggregate isolates. Conversely, A. carbonarius isolates from the hotter and drier region grew and produced OTA at lower aw than other isolates. However, little genetic diversity in A. carbonarius was observed for the microsatellites tested and the same sequence of β-tubulin gene was observed; therefore intraspecific variability did not correlate with the geographical origin of the isolates or with their ability to produce OTA. Climatic change prediction points to drier and hotter climatic scenarios where A. tubingensis and A. niger could be even more prevalent over A. carbonarius, since they are better adapted to extreme high temperature and drier conditions.

Inhibition of A. carbonarius growth and reduction of ochratoxin A by bacteria and yeast composites of technological importance in culture media and beverages

Five composites of yeast and six of bacterial isolates from fermented products were studied, in order to assess their ability to inhibit Aspergillus carbonarius growth and reduce OTA concentration in culture media and beverages. The antagonistic effect of the above composites against A. carbonarius growth was studied in synthetic grape medium of pH 3.5 and a(w) 0.98, 0.95, 0.92 after incubation at 25°C. Different combinations of initial inocula of bacteria or yeast composites and fungi were used (10(2)cfu/mL vs 10(5)spores/mL; 10(5)cfu/mL vs 10(2)spores/mL; and 10(5)cfu/mL vs 10(5)spores/mL). Regarding the OTA reduction experiment, 10(3) and 10(7)cfu/mL of the bacteria and yeast composites were inoculated in liquid media of different pH (3.0, 4.0, 5.0, and 6.1 or 6.5) and initial OTA concentration (50 and 100μg/L) and incubated at 30°C. Moreover, grape juice, red wine, and beer were supplemented with 100μg/L of OTA and inoculated with composites of 16 yeasts (16YM) and 29 bacterial (29BM) strains (10(7)cfu/mL) to estimate the kinetics of OTA reduction at 25°C for 5days. Fungal inhibition and OTA reduction were calculated in comparison to control samples. None of the bacterial composites inhibited A. carbonarius growth. The high inoculum of yeast composites (10(5) cfu/mL) showed more efficient fungal inhibition compared to cell density of 10(2) cfu/mL. All yeast composites showed higher OTA reduction (up to 65%) compared to bacteria (2-25%), at all studied assays. The maximum OTA reduction was obtained at pH 3.0 by almost all yeast composites. For all studied beverages the decrease in OTA concentration was higher by yeasts (16YM) compared to bacteria (29BM). The highest OTA reduction was observed in grape juice (ca 32%) followed by wine (ca 22%), and beer (ca 12%). The present findings may assist in the control of A. carbonarius growth and OTA production in fermented foodstuffs by the use of proper strains of technological importance.

Characterisation of a pks gene which is expressed during ochratoxin A production by Aspergillus carbonarius

Aspergillus carbonarius is considered the main fungus responsible for ochratoxin A (OTA) contamination in grapes. OTA is a potent nephrotoxin and a possible human carcinogen with a polyketide derived structure. Fungal polyketide synthases (PKSs) have recently been demonstrated to be involved in OTA biosynthesis in both Penicillium and Aspergillus species. We report here on the identification and characterisation of part of a novel polyketide synthase gene, ACpks from A. carbonarius. The sequence appears to encode conserved ketosynthase and acyl transferase domains, which are characteristic of previously characterised PKS enzymes. Expression of the ACpks gene is differentially regulated, with transcription being observed when the fungus was grown on synthetic grape medium and on OTA permissive medium (MM) whereas no transcription was detected when the fungus was grown on OTA restrictive medium (YES). ACpks expression was also observed when A. carbonarius was grown at low pH, with concomitant increases in OTA production. This correlation between ACpks gene expression and OTA production suggests the likely involvement for the product of this gene in ochratoxin A biosynthesis in the fungus. From a preliminary screening of Aspergillus isolates with ACpks specific primers, ACpks gene homologues appear to be present in A. sclerotioniger and A. ibericus, two species of section Nigri which are closely related to A. carbonarius.

Aspergillus carbonarius growth and ochratoxin A production on a synthetic grape medium in relation to environmental factors

The effects of water activity (0.90-0.99 a(w)), temperature (15-37 degrees C), and their interaction on growth and ochratoxin A (OTA) production by eight isolates of Aspergillus carbonarius were investigated on synthetic nutrient medium (SNM) with composition similar to grapes. Growth data were modelled by an multiple linear regression and response surface models were obtained. Aspergillus carbonarius grew much faster at 30 degrees C than at the other temperature levels tested and its growth rate increased with increasing a(w), maximum growth rate being between 0.95 and 0.99 a(w). In general, isolates grew faster at 35-37 degrees C than at 20 degrees C, although no significant differences were found between these temperatures. OTA accumulation was also favoured by high a(w) levels, and although it was observed in the whole range of temperatures, maximum amounts were detected at 20 degrees C. No OTA was found at the most unfavourable growth conditions. Optimum a(w) level for growth seems to correspond with optimum for OTA production, meanwhile the most propitious temperature for the toxin production was below the best one for growth. Prediction of A. carbonarius growth would allow estimating their presence and therefore, the OTA production, as it was found that conditions for the toxin production were more limited than those permitting growth.