Role of gas chromatography and olfactometry to understand the wine aroma: Achievements denoted by multidimensional analysis.

The human nose has been used as a detector in gas chromatography analysis to evaluate odoriferous compounds related to aroma and quality of wine. Several olfactometric techniques are available to access the description, intensity, and/or duration of the odor of each compound. Olfactometry can be associated with one-dimensional gas chromatography or multidimensional gas chromatography, including heart-cut gas chromatography and comprehensive two-dimensional gas chromatography. Multidimensional gas chromatography may help to resolve coeluted compounds and detect important trace components for the aroma. The identification of odor-active compounds may help to differentiate wines according to terroir, grapes cultivars used in winemaking or types of aging, understand the role of fungal infection of grapes for wine quality, find the best management practices in vineyard and vinification to obtain the greatest quality. In addition, when the instrumental techniques are combined with sensory analysis, even more accurate information may be obtained regarding the overall wine aroma. This review discloses the state of the art of olfactometric methods and the analytical techniques used to investigate odor-active compounds such as one-dimensional gas chromatography, multidimensional gas chromatography, and comprehensive two-dimensional gas chromatography. The advances in knowledge of wine aroma achieved with the use of these techniques in the target and profiling approaches were also discussed.

Colonization of Aspergillus carbonarius and accumulation of ochratoxin A in Vitis vinifera, Vitis labrusca, and hybrid grapes - research on the most promising alternatives for organic viticulture.

BACKGROUND: Aspergillus carbonarius has been identified as one of the main fungi that produce ochratoxin A (OTA) in grapes. This nephrotoxic mycotoxin has been legislated against in several countries and is a major concern for viticulture. Knowledge of resistance to, or susceptibility to, colonization by A. carbonarius may be useful in selecting the most promising cultivars for organic agriculture and could help in preventing fungal contamination in vineyards. This study aimed to evaluate the colonization potential and the capacity to produce OTA by A. carbonarius in Vitis vinifera, V. labrusca, and hybrid grapes. The correlation between OTA levels and grape berry characteristics was also analyzed. RESULTS: The OTA content was only strongly correlated with the thickness and hardness of the grape skins. The correlation between OTA levels and these parameters was negative (grapes with the least thickness and hardness had the highest OTA levels). Vitis vinifera grapes were more susceptible to A. carbonarius than V. labrusca and hybrid grapes at both 25 and 4 °C. Chardonnay (V. vinifera) grapes showed the highest levels of OTA, followed by Merlot, Cabernet Sauvignon, Tannat, and Moscato Branco. Italia grapes were the exceptions among V. vinifera cultivars, since they showed similar thickness, hardness, and fungal resistance as the V. labrusca and hybrid grapes. CONCLUSION: The highest resistance to A. carbonarius was observed in the following grapes: hybrids (BRS Lorena and BRS Violeta), V. labrusca (Isabel and Bordo), and V. vinifera (Italia). These cultivars can be prioritized in the implementation of organic viticulture. © 2020 Society of Chemical Industry.

Genomic analysis reveals genes that encode the synthesis of volatile compounds by a Bacillus velezensis-based biofungicide used in the treatment of grapes to control Aspergillus carbonarius.

Fungal control strategies based on the use of Bacillus have emerged in agriculture as eco-friendly alternatives to replace/reduce the use of synthetic pesticides. Bacillus sp. P1 was reported as a new promising strain for control of Aspergillus carbonarius, a known producer of ochratoxin A, categorized as possible human carcinogen with high nephrotoxic potential. Grape quality can be influenced by vineyard management practices, including the use of fungal control agents. The aim of this study was to evaluate, for the first time, the quality parameters of Chardonnay grapes exposed to an antifungal Bacillus-based strategy for control of A. carbonarius, supporting findings by genomic investigations. Furthermore, genomic tools were used to confirm that the strain P1 belongs to the non-pathogenic species Bacillus velezensis and also to certify its biosafety. The genome of B. velezensis P1 harbors genes that are putatively involved in the production of volatiles and hydrolytic enzymes, which are responsible for releasing the free form of aroma compounds. In addition to promote biocontrol of phytopathogenic fungi and ochratoxins, the treatment with B. velezensis P1 did not change the texture (hardness and firmness), color and pH of the grapes. Heat map and hierarchical clustering analysis (HCA) of volatiles evaluated by GC/MS revealed that Bacillus-treated grapes showed higher levels of compounds with a pleasant odor descriptions such as 3-hydroxy-2-butanone, 2,3-butanediol, 3-methyl-1-butanol, 3,4-dihydro-ß-ionone, ß-ionone, dihydroactinidiolide, linalool oxide, and ß-terpineol. The results of this study indicate that B. velezensis P1 presents desirable properties to be used as a biocontrol agent.

Carvacrol nanocapsules as a new antifungal strategy: Characterization and evaluation against fungi important for grape quality and to control the synthesis of ochratoxins.

Fungi are a problem for viticulture as they can lead to deterioration of grapes and mycotoxins production. Despite the widespread use of synthetic fungicides to control fungi, their impact on the agricultural ecosystem and human health demand safer and eco-friendly alternatives. This study aimed to produce, characterize and assess the antifungal activity of carvacrol loaded in nanocapsules of Eudragit® and chia mucilage as strategy for controlling Botrytis cinerea, Aspergillus flavus, Aspergillus carbonarius, and Aspergillus niger. Eudragit® and chia mucilage were suitable wall materials, as both favored the encapsulation of carvacrol into nanometric diameter particles. Fourier Transform Infrared Spectroscopy (FTIR) analysis suggested a successful incorporation of carvacrol into both nanocapsules, which was confirmed by presenting a good encapsulation efficiency and loading capacity. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) analyses revealed adequate thermal resistance. All fungi were sensible to carvacrol treatments and B. cinerea was the most sensitive compared to the Aspergillus species. Lower concentrations of encapsulated carvacrol than the unencapsulated form were required to inhibit fungi in the in vitro and grape assays. Additionally, lower levels of carvacrol (unencapsulated or encapsulated) were used to inhibit fungal growth and ochratoxin synthesis on undamaged grapes in comparison to those superficially damaged, highlighting the importance of management practices designed to preserve berry integrity during cultivation, storage or commercialization. When sublethal doses of carvacrol were used, the growth of A. niger and A. carbonarius was suppressed by at least 45 %, and ochratoxins were not found. The nanoencapsulation of carvacrol using Eudragit® and chia mucilage has proven to be an alternative to mitigate the problems with fungi and mycotoxins faced by the grape and wine sector.

Genome analysis of Pseudomonas strain 4B with broad antagonistic activity against toxigenic fungi.

Pseudomonas sp. 4B isolated from the effluent pond of a bovine abattoir was investigated as antifungal against toxigenic fungi. The complete genome of Pseudomonas 4B was sequenced using the Illumina MiSeq platform. Phylogenetic analysis and genome comparisons indicated that the strain belongs to the Pseudomonas aeruginosa group. In silico investigation revealed gene clusters associated with the biosynthesis of several antifungals, including pyocyanin, rhizomide, thanamycin, and pyochelin. This bacterium was investigated through antifungal assays, showing an inhibitory effect against all toxigenic fungi tested. Bacterial cells reduced the diameter of fungal colonies, colony growth rate, and sporulation of each indicator fungi in 10-day simultaneous growing tests. The co-incubation of bacterial suspension and fungal spores in yeast extract-sucrose broth for 48 h resulted in reduced spore germination. During simultaneous growth, decreased production of aflatoxin B1 and ochratoxin A by Aspergillus flavus and Aspergillus carbonarius, respectively, was observed. Genome analysis and in vitro studies showed the ability of P. aeruginosa 4B to reduce fungal growth parameters and mycotoxin levels, indicating the potential of this bacterium to control toxigenic fungi. The broad antifungal activity of this strain may represent a sustainable alternative for the exploration and subsequent use of its possible metabolites in order to control mycotoxin-producing fungi.

Flavoromic analysis of wines using gas chromatography, mass spectrometry and sensory techniques.

Various sensory perceptions drive the quality and typicality of wines, with the volatile profile playing a fundamental role in the characteristics of odor, aroma and consequently flavor, which combines the smell (odor and aroma), taste, and trigeminal sensations. Efforts have been made in both the field of instrumental and sensory analysis to understand the relationship of volatile compounds with sensory attributes in omics approaches. Gas chromatography (monodimensional and two-dimensional (heartcutting and comprehensive)) associated with mass spectrometry (GC/MS, GC-GC/MS and GCxGC/MS) and chemometric tools have contributed to foodomics analyses, specifically those linked to metabolomics/volatilomics. These tools, along with the elucidation of sensory properties (sensomics), lead to advanced results in the field of flavoromics. They also help to define the best practices in both vineyard management and winemaking that enable the production of high-quality wines. The objective of this review is to report the challenges of determining the volatile profile of wines, pointing out the ways that can be followed in successful identification and quantification of volatile compounds. The state of the art of sensory evaluation methods is also addressed, providing information that helps in choosing the most appropriate sensory method to be conducted with chromatographic analysis to achieve more in-depth results in the field of flavoromics.

A Bacillus-based biofungicide agent prevents ochratoxins occurrence in grapes and impacts the volatile profile throughout the Chardonnay winemaking stages.

Bacillus-based biocontrol agents have emerged as a strategy to eliminate or reduce the use of synthetic fungicides that are detrimental to health and the environment. In vineyards, a special concern arises from the control of Aspergillus carbonarius, a fungus known for its potential to produce ochratoxins. Ochratoxin A (OTA) is the most toxic form among ochratoxins and its maximum limit in wine has been established in Europe and Brazil as 2 µg/kg. Wine quality, especially the volatile profile, may be influenced by the antifungal strategies, since fungicide residues are transferred from grapes to must during winemaking. The objective of this study was to evaluate, for the first time, the impact of a biocontrol strategy containing Bacillus velezensis P1 on the volatile profile and occurrence of ochratoxins when grapes infected with A. carbonarius were used in winemaking. The evaluation of ochratoxins was carried out by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS), and volatile compounds were analyzed using comprehensive two-dimensional gas chromatography coupled to quadrupole mass spectrometry (GC × GC/qMS). Six ochratoxins were identified in must prepared with Chardonnay grapes inoculated with A. carbonarius (ochratoxin α, ochratoxin ß, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide and OTA). Although winemaking causes a decrease in the levels of all forms of ochratoxins, the co-occurrence of these mycotoxins was verified in wine made with grapes containing A. carbonarius. B. velezensis P1 prevented the occurrence of ochratoxins in must, ensuring the safety of wines. Regarding the volatile profile, a predominant presence of terpenic compounds was verified in samples treated with B. velezensis when compared with those not treated with the biocontrol strategy, whereas the presence of A. carbonarius resulted in a higher concentration of volatile compounds with an odor described as fatty/waxy, possibly compromising wine quality. Therefore, B. velezensis P1 is a new biofungicide possibility to produce ochratoxin-free grapes and high-quality wines.

Adaptation of an olfactometric system in a GC-FID in combination with GCxGC/MS to evaluate odor-active compounds of wine.

A step-by-step approach to easily adapt and use a GC-FID as an olfactometer, as well as a detailed description of acquisition and interpretation of olfactometric data by the OSME (from the Greek word for odor, ὀσµÎ®) method. A Merlot wine was used to exemplifly this strategy and its volatiles were characterized, rendering 43 volatiles in 1D-GC/MS and 142 in GCxGC/MS. GC-O showed the presence of 24 odor-active compounds and GCxGC/MS indicated aditional 14 odor-active compounds, which were found as coelutions. Six compounds (isoamyl acetate, ethyl octanoate, ethyl decanoate, 3-methylthio-1-propanol, carvone, benzyl alcohol and nonanoic acid) were described in 1D-GC-O analyses as having distinct odors by the same and by different assessors. This fact indicated the presence of coeluting bands, which were resolved by GCxGC/MS. The adapted GC-O in combination with the use of GCxGC/MS may be a tool to more accurate investigation of the odor-active compounds of wine.

Aspergillus carbonarius-derived ochratoxins are inhibited by Amazonian Bacillus spp. used as a biocontrol agent in grapes.

Bacillus spp. have been used as a biocontrol strategy to eliminate/reduce toxic fungicides in viticulture. Furthermore, the presence of fungi that are resistant to commonly used products is frequent, highlighting the need for new biocontrol strains. Aspergillus carbonarius can produce ochratoxins, including ochratoxin A (OTA), which has a regulatory maximum allowable limit for grape products. The purpose of this study was to assess the ability of four Amazonian strains of Bacillus (P1, P7, P11, and P45) to biocontrol A. carbonarius and various forms of ochratoxins in grapes. Berries treated with strain P1 presented no fungal colonies (100% reduction), while P7, P11 and P45 strains caused a reduction of 95, 95 and 61% on fungal counts, respectively. Six forms of ochratoxin were found in the grapes inoculated with A. carbonarius, including ochratoxin α, ochratoxin ß, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide, and OTA. Four of these ochratoxin forms (ochratoxin ß, ochratoxin α methyl-ester, ochratoxin α amide, N-formyl-ochratoxin α amide) are reported for the first time in grapes. These ochratoxins were identified using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS). All Bacillus strains inhibited the synthesis of OTA, which is the most toxic form of ochratoxin. No ochratoxin form was found when P1 and P7 were used. Although some forms of ochratoxin were detected in grapes treated with Bacillus spp. P11 and P45, the levels decreased by 97%. To our knowledge, this is the first report on the inhibition of Aspergillus carbonarius-derived ochratoxin by Bacillus species. P1 strain, identified as Bacillus velezensis, was found to be the most promising for completely inhibiting fungal growth and production of all ochratoxins.

Fate of enniatins in the Ale beer production stages analyzed by a validated method based on matrix-matched calibration and LC-QToF-MS.

Enniatins (ENA, ENA1, ENB and ENB1) are emerging mycotoxins reported in malt used for brewing. This study aimed to assess the fate of ENA, ENA1, ENB and ENB1 throughout Ale beer brewing using a validated method based on matrix-matched calibration (MMC) and liquid chromatography with quadrupole time-of-flight mass spectrometry detection. MMC avoided the inaccurate quantification of all enniatins, since the matrix effects that cause signal suppression have been overcome. The validated method displayed linearity (R2 > 0.99), recovery (>88%), repeatability and intermediate precision (RSD < 9.0%) in accordance with the different guidelines of method validation. The values of LOD (<0.1 µg kg-1) and LOQ (<0.5 µg kg-1) were sensitive enough to detect enniatins throughout brewing. In the Ale beer production stages, enniatins levels were significantly reduced, ensuring that beers were free of these mycotoxins. In contrast, enniatins were found in all by-products of beer production due to their low water solubility.

Patulin accumulation in apples during storage by penicillium expansum and penicillium griseofulvum strains.

A part of apples destined to juice production is generally of poor quality. Apples from cold storage or recently harvest (ground harvested or low quality apples) are stored under ambient conditions until they are processed. Since Penicillium expansum and P. griseofulvum are the principal fungal species isolated from stored apples in Brazil, the objective of this study was to investigate the ability of these strains to produce patulin in apples and report the consequences of this type of storage in loss of quality. The toxin was quantified using thin layer chromatography and charge-coupled device camera (TLC-CCD). The rate and quantities that P. expansum and P. griseofulvum can grow and produce patulin are highly dependent on the fungal strain and time. Lesion diameter resulted to be independent of the strain considered. The maximum period of time which apples were kept at cold storage (4 °C) without patulin accumulation was 27 days. When these apples were kept at 25 °C during 3 days, both factors lesion diameter and patulin production increased significantly. These results confirm that time in which apples are taken out from cold storage room before juice production is critical in order to prevent patulin accumulation.

QuEChERS-LC-QTOFMS for the simultaneous determination of legislated and emerging mycotoxins in malted barley and beer using matrix-matched calibration as a solution to the commercial unavailability of internal standards for some mycotoxins.

The combination of QuEChERS-LC-QTOFMS and matrix-matched calibration (MMC) to simultaneously determinate legislated and emerging mycotoxins in malt and beer was evaluated for the first time. The method performance was satisfactory displaying suitable linearity (R2 >0.99) and recovery (71-102%). The lowest values (in µg kg-1) of LOD (0.01) and LOQ (0.05) were found for enniatins, while the highest LOD (15) and LOQ (50) were reported for fumonisin B1. Precision and sensitivity (RSD <10%) were in accordance with the different guidelines of method validation. MMC was important to avoid inaccurate quantification of all mycotoxins due to signal enhancement or suppression. Another advantage was the enhanced throughput, requiring 1.2 min of analysis per analyte. The detection of legislated (aflatoxins, ochratoxin A, deoxynivalenol, fumonisin B1, zearalanone, T-2 and HT-2 toxin) and emerging mycotoxins (enniatins, beauvericin, moniliformin and sterigmatocystin) allowed verifying compliance with legislation and generating data to support the establishment of limits for emerging mycotoxins.

Role of partial dehydration in a naturally ventilated room on the mycobiota, ochratoxins, volatile profile and phenolic composition of Merlot grapes intended for wine production.

Dehydration of grapes has been used in various regions of the world to produce special wines, aiming to add value to oenological products. Post-harvest dehydration in rooms may be carried out regardless of weather conditions, without the additional cost of a specific infrastructure, in addition to the benefits of protecting the grapes from damages and environmental pollution. The objective of this study was to verify, for the first time, the impact of the dehydration in a naturally ventilated room on the quality of Merlot grapes. Physicochemical characteristics, mycobiota, occurrence of mycotoxins, volatile profile and phenolic composition of grapes were monitored on 7th, 14th and 21st days of dehydration (weight loss of 10, 20 and 27%, respectively). A decrease in aw (6%), pH (4%), and berry hardness (58%), along with an increase in total soluble solid content (15%) were observed during dehydration. The presence of Pestalotiopsis clavispora, Neopestalotiopsis clavispora, Colletotrichum siamense and Alternaria porri was favored during the dehydration process, while a decrease in the occurrence of Aspergillus niger and Phanerochaete sp. was verified. A. niger isolates showed no potential to produce forms of ochratoxins. These toxins were also not found in the grape samples. Regarding the volatile profile, 1-hexanal, 2-hexenal, and 1-octanal gave rise to the corresponding alcohols during dehydration, such as 1-hexanol, 2-hexen-1-ol, and 1-octanol. Acids (hexanoic, decanoic, and 3-hexenoic) resulted in the respective ethyl esters (hexanoate, decanoate, and ethyl 3-hexenoate) during dehydration. Terpenes as limonene, myrcene, and geraniol decreased throughout dehydration, while their biotransformation products (α-terpineol, 6-methyl-5-hepten-2-one, and linalool, respectively) had an increase in concentration. The phenolic content oscillated during dehydration, with an emphasis on increased levels of four hydroxybenzoic acids (ethyl gallate, p-hydroxybenzoic acid, gallic acid-hexose, and gallic acid), two hydroxycinnamic acids (caffeic acid and caftaric acid), two flavonols (kaempeferol galactoside and quercetin) and two anthocyanins (peonidin 3-O-hexoside and delphinidin 3-O-hexoside). Grapes of satisfactory quality were produced by dehydration in a naturally ventilated room. Even small wine producers can be encouraged to implement this procedure for the diversification of oenological products, as it has no costs related to the implementation of chambers/tunnels.

Volatile profile and aroma potential of tropical Syrah wines elaborated in different maturation and maceration times using comprehensive two-dimensional gas chromatography and olfactometry.

The influence of different combinations of Syrah grape maturation degree (19, 21 and 23 °Brix) and maceration times (10, 20 and 30 days) on the volatile profile and aroma potential was evaluated for the first time through different chromatographic platforms (GC × GC/TOFMS, GC-O-OSME, GC-FID and GC/MS). GC × GC/TOFMS analyses resulted in 145 identified compounds and among these 29 were determined to be the most important for wine differentiation. The aroma compounds allowed the discrimination of Syrah wines made with grapes macerated for a shorter time (ten days) due to the higher levels of volatile compounds. The evaluation of these wines through GC-O-OSME together with GC-FID, MS resulted in the designation of 19 °Brix as the most appropriate grape maturation degree to obtain a greater number of volatiles with pleasant odor and higher intensity and persistence. GC × GC/TOFMS allowed five and six co-elutions to be resolved, involving, respectively, ten and twelve important wine compounds.

Ochratoxin A presence in Cabernet Sauvignon wine changes antioxidant activity in vitro and oxidative stress markers in vivo.

Ochratoxin A (OTA) is a mycotoxin found in grape products and oxidative stress has been reported as an important mechanism involved in its toxicity, classified as possible carcinogenic to humans. Conversely, phenolics are known bioactive compounds in grapes and display great antioxidant properties. However, the biological effects of the concomitant presence of phenolic compounds and OTA remains unclear. The aim of this study was to evaluate, for the first time, the effect of OTA presence in Cabernet Sauvignon wine on antioxidant activity in vitro and on oxidative stress markers in vivo. In addition, the phenolic composition of wine was evaluated by LC-DAD-MS/MS. In vitro assays were based on spectrophotometric methods, while in vivo assays were performed evaluating oxidative stress markers in the nematode Caenorhabditis elegans, an alternative model to animal testing. A total of 23 phenolic compounds were identified in the Cabernet sauvignon red wine, including the anthocyanins delphinidin-3-O-glicoside and malvidin-3-O-glicoside, the flavonol quercetin-3-O-glucuronide and the phenolic acids caffeic, verbascoside and caftaric. Trans-resveratrol and trans-piceid were the only stilbenes found in the samples. OTA presence in the red wine was accompanied by reduction in GSH content and increase in hydroxyl radical generation in vitro. The presence of OTA in wine also increased lipoperoxidation and induced overexpression of the antioxidant enzymes superoxide dismutase and catalase in vivo. This study demonstrates that OTA presence in red wine can reduce its antioxidant potential in vitro and induces oxidative stress in vivo, without affecting the phenolic compounds levels in the samples. Thus, this work provides insights into the negative effects of the presence of OTA in wine, not only by its known toxicity, but also by prejudicing the antioxidant potential of wine. It is important to be aware of these effects when developing a complete description of OTA toxicity in humans.

Effect of Aspergillus carbonarius on ochratoxin a levels, volatile profile and antioxidant activity of the grapes and respective wines.

Aspergillus carbonarius can produce a possibly carcinogenic mycotoxin named ochratoxin A (OTA). The metabolism of this fungus can also impact grape and wine quality as it influences the volatile and phenolic profiles, which are related to aroma and antioxidant activity, respectively. The objective of this study was to evaluate the effect of A. carbonarius on OTA levels and for the first time on volatile profile and antioxidant activity of grapes and their respective wines. Cabernet Sauvignon (CS, red) grapes presented higher susceptibility to A. carbonarius than Moscato Italico (MI, white) grapes and OTA levels in their respective musts were in accordance with this same trend. However, vinification of red grapes resulted in 67% reduction of OTA, while the reduction observed with white wines was 45%. The presence of acids (hexanoic, octanoic, nonanoic and decanoic, fatty odor) was found to be an indicative of the fungus incidence in grapes. These acids were precursors of esters that might impart negative aroma (methyl nonanoate and isoamyl octanoate, fatty odor) or provide desirable fruity characteristics (ethyl hexanoate, ethyl octanoate and methyl octanoate) for wine. In addition, terpenes were detected only in wines produced with grapes (CS and MI) inoculated with A. carbonarius. The presence of A. carbonarius increased the antioxidant activity of CS grapes. For MI grapes and both wines (CS and MI) no differences were verified in the antioxidant activity of the samples affected or not affected by this fungus. Although A. carbonarius occurrence has shown no influence on the antioxidant activity of wines, it produced OTA and has negatively influenced the wine odor profile, due to the production of some volatiles that impart a deleterious effect on wine aroma.

Carbonyl compounds in different stages of vinification and exposure risk assessment through Merlot wine consumption.

The objective of this research was to estimate for the first time the transformations that the free form of some target carbonyl compounds may undergo during winemaking and assess the exposure risk to these compounds through the consumption of the Merlot commercial wines under study. Acrolein and furfural were found in grapes and the respective wines, although levels were observed to decline throughout the winemaking process. Formaldehyde was found in all stages of wine production in levels lower than the limit of quantification of the method and ethyl carbamate was not found in samples. Acetaldehyde seems to be a precursor of acetoin and 2,3-butanediol, since the levels of this aldehyde decreased along winemaking and the formation of the ester and alcohol was verified. Furfural levels decreased, while the occurrence of furan-containing compounds increased during winemaking. The formation of acetaldehyde during alcoholic fermentation and the potential environmental contamination of grapes with acrolein and furfural are considered as the critical points related to the presence of toxic carbonyl compounds in the wine. Acrolein was found in the samples under study in sufficient quantities to present risk to human health, while other potentially toxic carbonyl compounds did not result in risk. This study indicated for the first time the presence of acrolein in grapes suggesting that environmental pollution can play an important role in the levels of this aldehyde detected in wines. Reduction of the emission of this aldehyde to the environment may be achieved by replacing wood burning by another heat source in fireplaces or wood stones, and abandoning the practice of burning garbage and vegetation.

Influence of ripeness and maceration of the grapes on levels of furan and carbonyl compounds in wine - Simultaneous quantitative determination and assessment of the exposure risk to these compounds.

The validated method based on the use of headspace solid phase microextraction (HS-SPME) coupled with the comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC×GC/TOFMS) proved to be appropriate for this first simultaneous quantitative determination of six toxic compounds (formaldehyde, acetaldehyde, ethyl carbamate, furan, furfural and acrolein) found in wines. Acetaldehyde and acrolein coeluted with other wine compounds, which indicated that difficulties could arise if only one-dimensional gas chromatography was used for the determination of these compounds. The advancement of the ripeness degree and increasing the grape maceration time seems to result in higher concentrations of toxic compounds. The exposure to furan, acrolein and ethyl carbamate through wine consumption may pose risks to consumer health, since calculated MOE values were lower than 10,000.

Exposure risk assessment to ochratoxin A through consumption of juice and wine considering the effect of steam extraction time and vinification stages.

The goals of this study were (i) to verify the effect of steam extraction used in juice production and the stages of vinification on the ochratoxin A (OTA) levels found in grapes naturally contaminated, and (ii) evaluate the risk of exposure to this toxin when the daily consumption of juice and wine is followed to prevent cardiovascular disease. OTA-producing fungi were isolated from Cabernet Sauvignon, Moscato Itálico and Concord grapes harvested from the same vineyard and intended to produce red wine, white wine and juice, respectively. The highest levels of this toxin were found in the Concord grapes used for juice production. Although greater reduction in OTA levels occurred during juice production (73%) compared to winemaking (66 and 44%, for red and white, respectively), the estimated OTA exposure through juice was higher than the tolerable intake established for this toxin by JECFA. The risk associated with juice consumption, rather than wine, can be explained by (i) higher OTA levels found in Concord must than those of Cabernet and Moscato, indicating that Concord grapes appear to be more susceptible to OTA production by toxigenic fungi; and (ii) the daily recommended juice consumption is higher than those proposed to red wine.

Inhibition of mycotoxin-producing fungi by Bacillus strains isolated from fish intestines.

Bacillus strains isolated from the aquatic environment of the Brazilian Amazon region were tested for their activity against mycotoxigenic fungi. All tested bacteria showed antifungal activity, inhibiting at least 7 indicator fungi. Four Bacillus strains showing promising antifungal results were subsequently evaluated for their activity in reducing mycelial growth rate, sporulation, spore germination percentage, and mycotoxin production. Bacillus sp. P1 and Bacillus sp. P11 had a remarkable antifungal effect on toxigenic fungi. Washed bacterial cell suspension of strains P1 and P11 (107CFU/ml) reduced by >70% the fungal colony diameters, including a complete inhibition of ochratoxin A (OTA) producing Aspergillus spp. Significant reduction of growth rate, sporulation and spore germination were also observed. The bacteria influenced the production of mycotoxins, causing a reduction around 99 and 97% in AFB1 and OTA concentration, respectively. Chromatographic analysis revealed the presence of lipopeptides (iturin A and surfactin isomers) in butanol extracts of cell-free supernatants and cell pellets of strains P1 and P11. Furthermore, antifungal activity of these extracts was confirmed against A. flavus A12 and A. carbonarius ITAL293, producers of AFB1 and OTA, respectively. These bacterial strains could be promising biocontrol agents against toxigenic fungi.