Synthetic Grape Juice Medium Research Articles

Effect of interaction between Aspergillus carbonarius and non-ochratoxigenic grape-associated fungal isolates on growth and ochratoxin A production at different water activities and temperatures

The effect of water activity (0.90, 0.94, and 0.98 aw) and temperature (15, 20, and 25 °C) on the in vitro interactions between three ochratoxigenic strains of Aspergillus carbonarius (Ac-28, Ac-29, and Ac-33) and eleven non ochratoxigenic grape-associated fungal strains was assessed in this study. Fungal strains were allowed to grow in dual cultures on Synthetic Grape-juice Medium (SGM) for 15 days and fungal interactions were given a numerical score to obtain an Index of Dominance (ID) for each fungus. Results showed that in most cases A. carbonarius toxigenic strains were dominant against other fungal species. However, A. carbonarius presented mutual antagonism with A. section Nigri strains regardless of water activity (aw) and temperature. Moreover, interactions with Penicillium spinulosum and Cladosporium spp. at 15 °C, as well as Botrytis cinerea at 20 °C, showed that the antagonists were more competitive against A. carbonarius. In some cases, growth rates of A. carbonarius strains were either slightly stimulated or inhibited after interaction in dual cultures, depending on temperature, aw and competing species. Regarding OTA production, the presence of other species sometimes decreased the production or slightly enhanced it, depending on fungal competitor and environmental conditions. Overall, OTA production was higher at 15 °C/0.98 aw and 20 °C/0.98 aw for all target strains and at 20 °C/0.94 aw for Ac-33 strain only, but decreased at higher temperatures regardless of aw and interacting species.

The effect of nitrogen source on yeast metabolism and H2S formation

The impacts of aspartic acid and glutamine used as nitrogen supplements for alcoholic fermentations conducted by Saccharomyces were studied. Synthetic grape juice media and commercially prepared grape juices were supplemented with diammonium phosphate, aspartic acid, or glutamine to increase yeast-assimilable nitrogen concentrations to 250 mg N/L prior to fermentation. Two yeast strains, UCD522 and EC1118, were inoculated at 105 CFU/mL and fermentations were monitored for soluble solids, hydrogen sulfide (H2S), and residual amino acids. In general, unsupplemented media/juices fermented slower than supplemented ones, produced more H2S, and contained lower concentrations of amino acids after fermentation. Among the supplemented treatments, fermentation rates, H2S production, and amino acid utilization varied depending on the nitrogen source and yeast strain. Those fermentations supplemented with aspartic acid were generally slower and sometimes did not achieve dryness. In contrast, glutamine additions yielded fermentation rates and H2S production equivalent or better than other supplemented treatments. Based on these results, the use of glutamine appears promising as an alternative nitrogen supplement for wine production.

A pH control strategy for increased β-carotene production during batch fermentation by recombinant industrial wine yeast

The effects of different pH values, ranging from 4.0 to 7.0, on cell growth and β-carotene production by recombinant industrial wine yeast Saccharomyces cerevisiae T73-63 in a synthetic grape juice medium was investigated. Based on the kinetic analysis of the batch fermentation process, a two-stage pH control strategy was developed in which the pH was maintained at 7.0 for the first 24h and then shifted to 5.0 after 24h. Using this strategy, the highest β-carotene production (50.39mg/l) and the formation rate (1.40mg/l/h) were increased by 19.1% and 18.6%, respectively, compared to the maximum values of constant pH fermentation. The oxidative stress during β-carotene production was also determined in terms of the catalase (CAT) and superoxide dismutase (SOD) activities. Oxidative stress appears to be induced by the lowering of pH as indicated by the increase in activities of CAT and SOD due to pH shift from pH 7.0 to pH 5.0. Pre-treating cells with ascorbic acid (an antioxidative agent) reversed the improvement of β-carotene production while addition of H2O2 enhanced it. Considering that induction of oxidative stress is associated with increased β-carotene production, it was concluded that the enhancement of β-carotene production by the low-pH strategy involved the induction of oxidative stress.

INTERACTIVE EFFECTS OF SELECTED NUTRIENTS AND FERMENTATION TEMPERATURE ON H2S PRODUCTION BY WINE STRAINS OF SACCHAROMYCES

ABSTRACTMetabolic interactions between yeast assimilable nitrogen (YAN), biotin, pantothenic acid, and fermentation temperature that affect H2S production by wine yeast during alcoholic fermentation were examined. Strains of Saccharomyces cerevisiae (UCD 522 and EC1118) were inoculated into a synthetic grape juice medium with H2S evolution monitored under fermentative conditions. While a number of interactions affected the evolution of H2S, YAN as a factor by itself was found to be not significant (P > 0.05) for both yeasts examined. Maximal cumulative H2S production for strain UCD 522 occurred in media fermented at 30C with 60 mg/L YAN, 10 µg/L biotin, and 50 µg/L pantothenic acid while minimum production was observed with 250 mg/L YAN and 250 µg/L pantothenate. Similarly, strain EC1118 produced the most H2S at 30C, but with 250 mg/L YAN, 0.5 µg/L biotin, and 50 µg/L pantothenic acid and the least in media that contained 250 mg/L YAN and 250 µg/L pantothenic acid.PRACTICAL APPLICATIONS“Reduced” off‐odors of wines, primarily associated with sulfur‐containing molecules such as H2S, continue to be a difficulty facing winemakers worldwide. One strategy for wineries to limit these problems is to add yeast nutrients prior to fermentation, most commonly, nitrogen‐containing compounds such as diammonium phosphate. However, nitrogen deficiency is not always the sole cause for these problems. Rather, the current research suggests the need to consider factors other than nitrogen including availability of biotin and pantothenic acid as well as fermentation temperature in order to minimize these off‐odors.

Survey of Hydrogen Sulfide Production in Wine Strains ofSaccharomyces cerevisiae

The level of variation in hydrogen sulfide formation as a function of yeast genetic background was evaluated for 170 strains, comprised of 169 commercial strains and one native isolate, using six different media: BiGGY agar (assess basal levels of sulfite reductase), synthetic grape juice media with differing nitrogen and vitamin content, and Syrah grape juice. Statistical and cluster analysis placed the strains into 15 clusters, ranging from 1 to 43 members. Sulfide production was not well correlated with the nitrogen content of the medium. In general, sulfide formation was highest in the Syrah juice, in both total number of strains producing the compound and in level of production. There was no correlation between colony color on BiGGY agar and level of production of H₂S in any of the liquid media. Strains producing high levels of H₂S in Syrah also tended to produce H₂S in at least one of the synthetic media. Many moderate to low level producers of H₂S in Syrah produced trace to undetectable levels of H₂S in synthetic media. Thus, synthetic media can be used to identify strains with the potential to produce high levels of H₂S during fermentation. However, a low level of sulfide production in synthetic juices does not indicate low level of production in juice, and strain evaluation in actual juices is still necessary.

Validation of a predictive model for the growth of Botrytis cinerea and Penicillium expansum on grape berries

The objective of this study was to develop and to validate a model for predicting the combined effect of temperature and a w on the radial growth rate, μ, of Botrytis cinerea and Penicillium expansum on grape berries. The proposed strategy was based on the gamma-concept developed previously [Zwietering, M.H., Wijtzes, T., de Wit, J.C., van't Riet, K. 1992. A decision support system for prediction of the microbial spoilage in foods. Journal of Food Protection. 12, 973–979]: μ = μ opt.γ(T).γ( a w ), where the gamma functions were cardinal models with inflection (CMI), μ opt the radial growth rate on grape berries. Firstly, the cardinal temperatures and a w 's were estimated independently from experiments carried out on Potato Dextrose Agar. Secondly, the gamma concept was validated i/ on a synthetic grape juice medium (SGJ) and ii/ on a grape juice agar (GJA). Accuracy and bias factors were closer to 1 with the latter analogue, thus suggesting that GJA should be preferred to SGJ. Thirdly, an experimental protocol taken into account the isotropic nature of fungal growth was developed for estimating μ opt on grape berries. This study demonstrated that CMI's can be validated on agri-food products over a wide range of temperature and a w using the described methodology.

Effect of temperature and water activity on growth and ochratoxin A production boundaries of twoAspergillus carbonariusisolates on a simulated grape juice medium

To develop and validate a logistic regression model to predict the growth and ochratoxin A (OTA) production boundaries of two Aspergillus carbonarius isolates on a synthetic grape juice medium as a function of temperature and water activity (a(w)). A full factorial design was followed between the factors considered. The a(w) levels assayed were 0.850, 0.880, 0.900, 0.920, 0.940, 0.960, 0.980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40 degrees C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3.4-4.1% false-positives and 0.7-1.4% false-negatives. No growth was observed at 10 degrees C and 40 degrees C for all a(w) levels assayed, with the exception of 0.980 a(w)/40 degrees C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0.7-1.4% accounting for false-positives and 2.0-2.7% false-negatives. No OTA production was detected at 10 degrees C or 40 degrees C regardless of a(w), and at 0.850 a(w) at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15-20 degrees C) as opposed to the growth boundary that shifted to higher temperature levels (25-30 degrees C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false-positive and three false-negative predictions out of 68 growth cases and 13 false-positive predictions out of 45 OTA production cases. The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a(w). The proposed modelling approach helps the understanding of fungal-food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.

Mycelial growth and ochratoxin A production byAspergillussectionNigrion simulated grape medium in modified atmospheres

To evaluate the impact of modified atmosphere packaging on in vitro growth of Aspergillus carbonarius and Aspergillus niger, and possible effects on ochratoxin A (OTA) biosynthesis. Ochratoxigenic isolates belonging to the species A. carbonarius and A. niger were grown on a synthetic grapejuice medium (SNM) and packaged in combinations of controlled O(2) (1% and 5%) and CO(2) levels (0% and 15%), and in air as a control. Colony diameters were recorded every 3 days up to 21 days, and OTA was analysed after 7, 14 and 21 days. The greatest reductions in mycelial growth rate were observed at 1% O(2) followed by 1% O(2)/15% CO(2), whereas 5% O(2) stimulated the growth of all isolates. OTA production by A. carbonarius and A. niger isolates was minimized at 1% O(2)/15% CO(2) and 1% O(2), respectively, after 7 days of incubation. Maximal OTA accumulation after 7 days was observed for all isolates in the control pack and at 5% O(2). Of the atmospheres tested, only 1% O(2) combined with 15% CO(2) consistently reduced fungal growth and OTA synthesis by A. carbonarius and A. niger. Storage under modified atmospheres is unlikely to be suitable as the sole method for OTA minimization and grape preservation; other inhibitory factors are necessary.

Impact of Water Activity and Temperature on Growth and Ochratoxin A Production of Two Aspergillus carbonarius Isolates from Wine Grapes in Greece

The aim of this study was to determine the effects of water activity (aw; 0.85 to 0.98) and temperature (10 to 40°C) on the radial growth rate and ochratoxin A (OA) production of two Aspergillus carbonarius isolates in vitro. The isolates were obtained from wine grapes cultivated in Greece, and the trial was conducted with a synthetic grape juice medium similar in composition to grapes between veraison (beginning of color change) and ripeness. Fungal growth and OA production data were collected for 55 days. Response surface curves and cardinal values for aw and temperature were obtained using multiple regression analysis. The lag phase lasted from less than 1 to 10 days. Both isolates grew optimally at 30 to 35°C and 0.96 aw, but maximum OA production occurred under suboptimal growth conditions (15 to 20°C and 0.93 to 0.96 aw). Growth also was observed at 0.85 aw and 25°C, however at this same aw the fungus failed to produce mycelium at any other temperatures tested. The isolates produced OA at 15 to 30°C and 0.90 to 0.98 aw. Maximum OA production was detected after 25 days of incubation at 20°C and 0.96 aw and was 3.14 and 2.67 μgg−1, respectively, for the two strains. The isolated strains used in this study were more xerotolerant than others from the Mediterranean basin. These data will allow producers to identify and thus monitor critical environmental conditions effectively in wine grapes. These data also increase the knowledge base concerning the ability of A. carbonarius to grow and produce toxin under different ecological conditions and can contribute to the development of secondary models for the prediction and risk assessment of OA in wine production.

Impact of pantothenic acid addition on H 2S production by Saccharomyces under fermentative conditions

The impact of addition of pantothenic acid at different times during alcoholic fermentation by two different strains of Saccharomyces under fermentative conditions was studied. Two strains of Saccharomyces cerevisiae, EC1118 and UCD522, were inoculated into synthetic grape juice media containing either 50 or 250 μg/l pantothenic acid. To those media containing 50 μg/l pantothenic acid, an additional 200 μg/l of the vitamin was added either 48 or 96 h after yeast inoculation. While yeast viabilities and fermentation rates were similar for all treatments, H 2S production varied depending on yeast strain and time of addition of the vitamin. In general, H 2S production was significantly higher for UCD522 compared to EC1118. Furthermore, adding pantothenic acid to deficient musts containing 50 μg/l resulted in a cessation of H 2S production within 24 h of addition. Based on these findings, pantothenic acid can be added before or during alcoholic fermentation as a means to reduce problems such as the formation of H 2S.

Effect of temperature and water activity on growth and ochratoxin A production by Australian Aspergillus carbonarius and A. niger isolates on a simulated grape juice medium

The effect of water activity (0.92, 0.95, 0.965 and 0.98) and temperature (15 °C, 25 °C, 30 °C and 35 °C) on growth rate and ochratoxin A (OA) production by five strains of Aspergillus carbonarius and two strains of A. niger isolated from Australian vineyards was characterised on a synthetic grape juice medium. Maximum growth for A. carbonarius occurred at ca 0.965 a w and 30 °C, and for A. niger, at ca 0.98 a w and 35 °C. The optimum temperature for OA production was 15 °C and little was produced above 25 °C. The optimum a w for toxin production was 0.95–0.98 for A. carbonarius and 0.95 for A. niger. Toxin was produced in young colonies after and, typically, did not continue to accumulate the entire surface area of the plate was colonised. Rather, the amount decreased as colonies aged. Trends for growth and OA production were similar among Australian isolates and those from European grapes, as reported in the literature.

Biodegradation of ochratoxin A byAspergillussection Nigri species isolated from French grapes: a potential means of ochratoxin A decontamination in grape juices and musts

Ochratoxin A (OTA) is a very dangerous mycotoxin, the presence of which is often reported in different foods, as well as in beverages such as grapes, grape juices and wines. Detoxifying these products is therefore of prime importance in protecting consumer health, and biological approaches have been the most promising methods. In this report, 40 isolates representing the black apergilli species Aspergillus carbonarius, A. niger aggregate and A. japonicus, isolated on French grapes, were assessed for OTA degradation capacities in CZAPEK yeast extract broth (CYB) and in a synthetic grape juice medium (SGM) contaminated with OTA at 2 mg L(-1) (5 microM). It was clearly observed that in both media these fungi had the ability to degrade OTA to OTalpha (ochratoxinalpha). However, there were differences between the media used and species tested during OTA degradation. In SGM and CYB, 77% and 45% of the isolates, respectively were able to degrade more than 80% of the OTA. Despite a better growth on SGM, specific OTA degradation was higher on CYB for most of the isolates. Kinetic studies carried out on SGM with three black Aspergillus isolates all showed different OTA degradation rates. After 9 days of incubation, OTalpha had decreased, whereas an unknown compound appeared. A. niger could be the first interesting species for OTA detoxification processes, followed by A. japonicus.

Standardised methodology for testing malolactic bacteria and wine yeast compatibility

Metabolic interaction of wine yeast and malolactic (ML) bacteria may stimulate or inhibit malolactic fermentation (MLF). To study this interaction in the absence of extrinsic grape-derived and processing factors, a standardised methodology using a chemically defined medium was developed to reproducibly characterise the intrinsic metabolic interaction between any wine yeast and bacteria pair. The methodology can be adapted to study the modifying effects of extrinsic factors on yeast-bacteria interaction. The methodology involves measuring the growth characteristics of the chosen ML bacterium in the wine produced by the yeast of interest under standardised conditions. The methodology was applied to four Saccharomyces cerevisiae wine yeasts which have been characterised in terms of growth response of an Oenococcus oeni strain. Three yeasts gave compatible interactions, in which the level of bacterial growth stimulation was variable; the fourth yeast inhibited bacterial growth and produced a high concentration of sulfite. To test for the possibility that the bacterial growth response was inhibited by the depletion of nutrients by the fermentation yeast, supplementation of the wines with essential nutrients gave a strain-dependent growth response. A treatment designed to remove or inactivate yeast-derived inhibitory factors was also investigated. This treatment partly alleviated bacterial inhibition caused by the sulfite-producing yeast strain. To verify that the standardised methodology relates to grape juice and wine, the synthetic grape juice medium was replaced with a Chardonnay juice of comparable composition prepared from microbiologically sound grapes; similar results were obtained, suggesting that the grape-derived extrinsic factors in this case did not appreciably modify the intrinsic yeast-bacteria metabolic interaction.

Survey of Hydrogen Sulphide Production by Wine Yeasts

Twenty-one strains of commercial wine yeasts and 17 non-Saccharomyces species of different provenance were surveyed for their ability to produce hydrogen sulphide in synthetic grape juice medium indicator agar with different nitrogen sources, as well as in natural grape juice. Bacto Biggy agar, a commercially available bismuth-containing agar, was used to compare our results with others previously reported in the literature. Under identical physiological conditions, the strains used in this study displayed similar growth patterns but varied in colony color intensity in all media, suggesting significant differences in sulphite reductase activity. Sulphite reductase activity was absent for only one strain of Saccharomyces cerevisiae. All other strains produced an off-odor to different extents, depending significantly (P <0.05) on medium composition. Within the same species of some non-Saccharomyces yeasts, strain variation existed as it did for Saccharomyces. In natural musts, strains fell into three major groups: (i) nonproducers, (ii) must-composition-dependent producers, and (iii) invariable producers. In synthetic media, the formation of sulphide by strains of S. cerevisiae results from the reduction of sulphate. Therefore, this rapid screening methodology promises to be a very useful tool for winemakers for determining the risk of hydrogen sulphide formation by a given yeast strain in a specific grape juice.

Functional genomic analysis of a commercial wine strain of Saccharomyces cerevisiae under differing nitrogen conditions.

DNA microarray analysis was used to profile gene expression in a commercial isolate of Saccharomyces cerevisiae grown in a synthetic grape juice medium under conditions mimicking a natural environment for yeast: High-sugar and variable nitrogen conditions. The high nitrogen condition displayed elevated levels of expression of genes involved in biosynthesis of macromolecular precursors across the time course as compared to low-nitrogen. In contrast, expression of genes involved in translation and oxidative carbon metabolism were increased in the low-nitrogen condition, suggesting that respiration is more nitrogen-conserving than fermentation. Several genes under glucose repression control were induced in low-nitrogen in spite of very high (17%) external glucose concentrations, but there was no general relief of glucose repression. Expression of many stress response genes was elevated in stationary phase. Some of these genes were expressed regardless of the nitrogen concentration while others were found at higher levels only under high nitrogen conditions. A few genes, FSP2, RGS2, AQY1, YFL030W, were expressed more strongly with nitrogen limitation as compared to other conditions.