Wine Contamination Research Articles

Non-Invasive Tools to Detect Smoke Contamination in Grapevine Canopies, Berries and Wine: A Remote Sensing and Machine Learning Modeling Approach.

Bushfires are becoming more frequent and intensive due to changing climate. Those that occur close to vineyards can cause smoke contamination of grapevines and grapes, which can affect wines, producing smoke-taint. At present, there are no available practical in-field tools available for detection of smoke contamination or taint in berries. This research proposes a non-invasive/in-field detection system for smoke contamination in grapevine canopies based on predictable changes in stomatal conductance patterns based on infrared thermal image analysis and machine learning modeling based on pattern recognition. A second model was also proposed to quantify levels of smoke-taint related compounds as targets in berries and wines using near-infrared spectroscopy (NIR) as inputs for machine learning fitting modeling. Results showed that the pattern recognition model to detect smoke contamination from canopies had 96% accuracy. The second model to predict smoke taint compounds in berries and wine fit the NIR data with a correlation coefficient (R) of 0.97 and with no indication of overfitting. These methods can offer grape growers quick, affordable, accurate, non-destructive in-field screening tools to assist in vineyard management practices to minimize smoke taint in wines with in-field applications using smartphones and unmanned aerial systems (UAS).

Potentially Smoke Tainted Red Wines: Volatile Phenols and Aroma Attributes

Malodourous compounds, including volatile phenols (VPs) are frequently found at concentrations below their odour thresholds in wine, and may therefore be considered to present no threat to wine quality. Most investigations into smoke taint quantify compounds by chemical/analytical means, or investigate sensory effects of supra- and peri-threshold contamination in model wine. In this project, twelve wines (submitted by the South African industry as potentially smoke tainted) were screened for VPs using GC-MS, and characterized using descriptive analysis (DA) by a sensory panel highly trained in smoke taint evaluation. Results were compared statistically to elucidate relationships between chemical and sensory characteristics. It was demonstrated, using the combined dataset that concentration and composition of VPs in the wines correlated well with certain sensory attributes. Guaiacol was present in most samples at peri- or suprathreshold levels, but was not correlated with taint unless in combination with other phenols, in which case it was associated with ‘smoky’, ‘ashy’ and ‘herbaceous’ attributes. Wines with supra-threshold levels of VPs showed negative attributes (‘chemical / plastic’, ‘tar / BR’ and ‘medicinal / Elastoplast™’). In some cases, sensory effects (‘earthy / dusty / potato skin’, ‘mouldy / musty’ and ‘cooked vegetables (veg.)’) could not be attributed to supra-threshold VP contamination, and therefore seemed to be due to combinations of VPs at subthreshold levels. Associations between negative attributes and historical bushfire events prior to harvest were found for a number of the wines. This study emphasizes the importance of understanding effects of VPs on wine aroma, and escalating awareness and sensitivity to these issues in the wine industry.

Quantification of toxic metals during different winemaking stages

Heavy metals in beverages can constitute serious problems to human health. Consumption of wine may contribute to the daily dietary intake of pollutants, especially of toxic (heavy) metals. These compounds are also known as priority pollutants due to their potential toxic effects, if concentrations are not kept under allowable limits. Many characteristics such as: quality, origin, aroma and health safety of wine are influenced by environmental and anthropogenic factors. Hence, the contamination of wine by priority pollutants may occur at different stages of vine-growing, due to the application of agricultural chemicals, or at different stage of winemaking and ageing, because of the extended contact of wine with winemaking equipment materials (aluminium, brass, glass, stainless steel and wood, etc.), or chemicals used for cleaning and sanitation. The aim of this study is to identify and quantify the heavy metal ions from red wines. A particular focus was attributed to Zn and Cd from destemming-pressing-filtration-bottling stages in Fetească neagră grape variety from Cotnari vine growing region of Romania. Results indicated that heavy metals were linked to diverse Zn and Cd sources and complexes during the winemaking processes. Concentration of Zn and Cd were generally higher in must than in wine, although heavy metals concentrations were lower than the limits recommended by the International Organization of Vine and Wine for human health safety.

Bottle capsules as a barrier against airborne 2,4,6-trichloroanisole

The possibility of 2,4,6-trichloroanisole (TCA) migration through synthetic stoppers and into wine from highly contaminated air was shown by several authors. However, those experiments were usually conducted without bottle capsules, which are a common part of wine packaging. In the current study, we demonstrated that the presence of capsules (without open holes) above synthetic stoppers can reduce wine contamination by airborne d5-TCA by about 10 times or more. Generally, metallic capsules revealed better barrier properties than polyvinyl chloride counterparts. Application of EVOH film on the external surface of the polyvinyl chloride capsules usually resulted in a lower level of wine contamination. Additionally it was demonstrated, that relatively short exposure (3 months) of the bottles to highly contaminated air could cause a considerable absorption of d5-TCA by synthetic stoppers, which can subsequently lead to wine contamination after 12 months.

The parameters that affect the supercritical extraction OF 2,4,6-trichloroanisol from cork

The contamination of wine by 2,4,6-trichloroanisol (TCA) derived from the cork stopper is a huge problem for the wine industry. This work shows the results of TCA extraction using supercritical CO2. Cork granules (6% moisture) were placed forming a fixed bed within a high-pressure vessel. The CO2 was made to continuously flow over this. The TCA removal was compared at different operating conditions (pressure, temperature, flow rate or extraction time). TCA removal was not possible in dried cork. Density had a positive impact in TCA removal due to the solvent capacity increase. An increase in the supercritical fluid temperature over 60 °C impaired the TCA extraction. High residence times benefited the extraction. At high CO2 density of 585 kg m−3, TCA elimination below the detection level was achieved in 8 min residence time. This required relatively low solvent to cork mass ratios (43 kg CO2 kg−1).

Wine Contamination with Ochratoxins: A Review

Ochratoxin A (OTA) is the main mycotoxin occurring in wine. This review article is focused on the distribution of this toxin and its producing-fungi in grape berries, as well as on the fate of OTA during winemaking procedures. Due to its toxic properties, OTA levels in wine are regulated in different countries; therefore, it is necessary to apply control and detoxification methods that are also discussed in this revision.

Adhesion properties and surface hydrophobicity of Pichia manshurica strains isolated from organic wines

This study focuses on the determination of microorganisms present in spoiled bottled wines of a Montepulciano d’Abruzzo organic wine and establish the source of this contamination. The yeasts belonging to Pichia manshurica species were genetically characterized by PFGE, and tested for their resistance to potassium metabisulphite and some cleaning agents determining the minimum inhibitory concentrations (MIC) and biofilms formation in YPD and pasteurized must. PFGE analysis revealed the presence of 2 karyotypes with 3 or 4 bands. The most effective detergent was the alkaline powder one, while the less active was the peracetic acid based sanitizing agent. MIC values for potassium metabisulphite ranged from 100 to 200 ppm. Regarding adhesion properties all strains showed a certain degree of hydrophobicity, as revealed by the affinity for hexadecane and decane, and were able to form biofilm on polystyrene plates: sessile cells reached values of 7.85 log cfu/mL in YPD and 6.95 log cfu/mL in must. All strains formed MAT structures and an air-liquid biofilm. This paper shows that organic wine can be subjected to contamination driven by not very resistant strains. Moreover, membrane filters used for wine sterilization act as a vector of wine contamination.

A new method for the detection of early contamination of red wine by Brettanomyces bruxellensis using Pseudomonas putida 4-ethylphenol methylene hydroxylase (4-EPMH)

Brettanomyces/Dekkera bruxellensis is a cause of major concern for the winemaking industry worldwide. If a slight presence of this spoilage yeast in red wine adds a Brett character, a strong contamination has irreversible and detrimental effects on the organoleptic qualities due to the production of volatile phenols such as 4-ethylphenol. Time is a key factor in the treatment of B. bruxellensis contaminations. Nowadays, the diagnostic and quantification resources available are time consuming and too expensive, making them either inadequate or inaccessible to most of the winemakers. This study was focused on a new, easy to use, inexpensive method that could allow winemakers to directly detect B. bruxellensis contamination in red wine at an early stage, hence, reducing wine spoilage. In this work, the ability of Pseudomonas putida 4-ethylphenol methylene hydroxylase was tested in order to catabolize the 4-ethylphenol and to elaborate an enzymatic assay with the purpose of detecting early contaminations by B. bruxellensis in red wine. We have developed a colorimetric enzymatic assay, based on the redox state of the 4-ethylphenol methylene hydroxylase co-factor, cytochrome C, that can detect and quantify low concentrations of 4-ethylphenol. The range of concentrations detected is well below the level detectable by the human nose. Combined to an enrichment step, this method allows the detection of B. bruxellensis at an initial concentration of less than 10 cells per ml.

Coupling SPE on-line pre-enrichment with HPLC and MS/MS for the sensitive detection of multiple allergens in wine

Mass spectrometry (MS) represents an essential tool in proteomics studies, in the last years also exploited for monitoring allergens contamination in food products. Milk and egg are renowned allergens often used as fining agents to promote clarification of wines, therefore any residual amount in the end-products could represent a menace for allergic individuals. In view of this, it is of paramount importance to have at disposal sensitive analytical methods able to detect traces of milk and egg allergens in food. In this work we describe the upgrade and the optimization of an analytical workflow based on the use of a pre-enrichment column coupled with HPLC separation and MS/MS detection for the selective and sensitive detection of milk and egg allergens in white wine. Two different sample pre-treatments based on the use of mass cut-off filters or size exclusion cartridges were evaluated and compared, before tryptic digestion and LC-SRM-MS/MS analysis of the resulting peptides mixture. The devised UF based method coupled with peptide on-line pre-enrichment enabled to reach the lowest LODs down at 0.036 μg/mL and 0.05 μg/mL for egg and milk allergens respectively, proving to be the most sensitive strategy for monitoring allergens contamination in wine.

A Rapid Assay to Detect Toxigenic Penicillium spp. Contamination in Wine and Musts.

Wine and fermenting musts are grape products widely consumed worldwide. Since the presence of mycotoxin-producing fungi may greatly compromise their quality characteristics and safety, there is an increasing need for relatively rapid “user friendly” quantitative assays to detect fungal contamination both in grapes delivered to wineries and in final products. Although other fungi are most frequently involved in grape deterioration, secondary infections by Penicillium spp. are quite common, especially in cool areas with high humidity and in wines obtained by partially dried grapes. In this work, a single-tube nested real-time PCR approach—successfully applied to hazelnut and peanut allergen detection—was tested for the first time to trace Penicillium spp. in musts and wines. The method consisted of two sets of primers specifically designed to target the β-tubulin gene, to be simultaneously applied with the aim of lowering the detection limit of conventional real-time PCR. The assay was able to detect up to 1 fg of Penicillium DNA. As confirmation, patulin content of representative samples was determined. Most of analyzed wines/musts returned contaminated results at >50 ppb and a 76% accordance with molecular assay was observed. Although further large-scale trials are needed, these results encourage the use of the newly developed method in the pre-screening of fresh and processed grapes for the presence of Penicillium DNA before the evaluation of related toxins.

Occurrence of ethyl carbamate in three types of Chinese wines and its possible reasons.

A total of 75 wines including 30 white wines, 31 red wines, and 14 sparkling wines were obtained from several regions in China (Sinkiang, Tonghua, Huailai, Yantai, Changli, Shanxi, Gansu, and Ningxia). Ethyl carbamate (EC) was detected by gas chromatography mass spectrometry. The EC concentration ranged from less than 1.16 to 38.56 μg/L, and the concentrations in 17 wines exceeded the U.S. limit for table wines (15 μg/L). The concentrations of EC increased in the order of white, red, and sparkling wines with the corresponding mean concentrations of 6.12, 9.22, and 14.03 μg/L. The relationship between EC concentration and wine type suggested that EC concentrations in wines might be affected by vinification patterns, most likely due to the difference between EC precursors in different vinification processes. This work provides a novel clue for EC contamination in different wines.

Diversity of Mycotoxin-Producing Black Aspergilli in Canadian Vineyards.

Several Aspergillus species produce ochratoxin A (OTA) and/or fumonisins on wine and table grapes. The relevant species and their mycotoxins have been investigated in a number of wine-producing regions around the world; however, similar data have not been reported for Canadian vineyards. A multiyear survey of black Aspergilli in Niagara, ON, vineyards was conducted to determine the diversity of species present and to assess the risk of OTA and fumonisin contamination of wine grapes from this region. From 2012 to 2014, 253 black Aspergilli were isolated from soil samples and the fruits of 10 varieties of grapes. The isolates were identified by DNA sequencing: Aspergillus welwitschiae (43%), Aspergillus uvarum (32%), Aspergillus brasiliensis (11%), Aspergillus tubingensis (9%), and Aspergillus niger (4%). Aspergillus carbonarius, the primary OTA producer on grapes in other parts of the world, was isolated only once, and this is the first report for it in Canada. All 10 A. niger strains produced fumonisins, but, in contrast, only 26% of the 109 A. welwitschiae isolates were producers, and no strains of either species produced OTA. Grape samples were analyzed for OTA and fumonisins from sites where strains with mycotoxigenic potential were isolated. Fumonisin B2 (FB2) was detected in 7 of 22 (32%) of these grape samples in the 1-15 ppb range, but no OTA was detected. Additionally, the recently reported nonaminated fumonisins were detected in 3 of 22 grape samples. These results suggest that fumonisin-producing Aspergilli can occur in Ontario vineyards but, at present, the risk of contamination of grapes appears low. The risk of OTA contamination in Niagara wine is also low because of the low prevalence of A. carbonarius.

Pesticide residues screening in wine by mass spectrometry

Recently, a study (from PAN Europe) covered 40 bottles of wine – 34 conventional and six organic ones – purchased inside the EU. According to the results, the 34 bottles of conventional wine together contained 148 pesticide residues. All 34 bottles contained from one to ten pesticides, bringing the average per bottle to more than four. Of the six bottles of organic wine tested, one sample contained a low concentration of a possibly carcinogenic pesticide. According to PAN Europe, the “contamination of wines is a direct result of over-reliance on pesticides in grape production”. This study, between others, to prove the importance of develop methods sensivity and confident for pesticide detection in wine. A multi-residue method was developed for the determination ca of 250 pesticide residues in wine using Quechers extraction, gas chromatography-tandem mass spectrometry (GC-MS-MS) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). The method was validated with the evaluation of follow parameters: Linearity, Precision, Accuracy, Matrix effect, Limit of detection and Limit of Quantification. The method was approved and was able to quantify pesticide residues in more than 60 samples of wine.

Fungicide multiresidue monitoring in international wines by immunoassays

Azoxystrobin, boscalid, cyprodinil, fenhexamid, and pyrimethanil are new generation fungicides extensively employed in order to combat diseases affecting vineyards worldwide. Owing to their physico-chemical characteristics, residues of these compounds on grapes are transferred to must and wine. In this study, a survey of the occurrence of these fungicides in international wines was carried out by using rapid antibody-based assays. Results are discussed as a function of wine type and sample geographical origin. 44.4% of the samples contained at least one of the targets (>10μgL−1). Fungicide residue occurrences were 22.4%, 19.2%, 18.8%, 6.8%, and 1.2% for pyrimethanil, boscalid, fenhexamid, cyprodinil, and azoxystrobin, respectively, while residue contents higher than 100μgL−1 were found in 8.4% of the samples. This study shows that contamination of commercial wines with pesticides is an issue of worldwide relevance with potential implications for consumer health and international trade.

Impact of processed earwigs and their faeces on the aroma and taste of 'Chasselas' and 'Pinot Noir' wines

The abundance of the European earwig Forficula auricularia L. (Dermaptera, Forficulidae) in European vineyards increased considerably over the last few years. Although earwigs are omnivorous predators that prey on viticultural pests such as grape moths, they are also known to erode berries and to transfer fungal spores. Moreover, they are suspected to affect the human perception of wines both directly by their processing with the grapes and indirectly by the contamination of grape clusters with their faeces. In this study we artificially contaminated grapes with F. auricularia adults and/or their faeces and determined the impact on aroma and taste of white 'Chasselas' and red 'Pinot noir' wines. Whereas the addition of five living adults/kg grapes affected the olfactory sensation of 'Chasselas' wines only marginally, 0.6 gram of earwig faeces/kg grapes had a strong effect on colour, aroma and the general appreciation of 'Chasselas' wines. Faeces-contaminated wines were less fruity and less floral, the aroma was described as faecal and they were judged to be of lower quality. The contamination of 'Pinot noir' grapes with four different densities of living earwig adults (e.g. 0, 5, 10 and 20 individuals/kg grapes) showed that only wines contaminated with more than 10 earwigs/kg grapes smelled and tasted significantly different than the uncontaminated control wine. Earwig-contaminated 'Pinot noir' wines were judged to be of lower quality. The descriptors “animal”, “reductive”, “vegetal”, “acidic”, “bitter” and “tannic” characterised their sensory perception. In conclusion, our results show that there is a real risk of wine contamination by F. auricularia . In particular, earwig faeces and earwig adults at densities above a threshold of 5 to 10 individuals/kg grapes have the potential to reduce the quality of wines. The evolution of earwig populations in vineyards should therefore be monitored carefully in order to anticipate problems during vinification.

Determination of trichloroanisole and trichlorophenol in wineries’ ambient air by passive sampling and thermal desorption–gas chromatography coupled to tandem mass spectrometry

The present paper describes the calibration of selected passive samplers used in the quantitation of trichlorophenol and trichloroanisole in wineries’ ambient air, by calculating the corresponding sampling rates. The method is based on passive sampling with sorbent tubes and involves thermal desorption–gas chromatography–triple quadrupole mass spectrometry analysis. Three commercially available sorbents were tested using sampling cartridges with a radial design instead of axial ones. The best results were found for Tenax TA™. Sampling rates (R-values) for the selected sorbents were determined. Passive sampling was also used for accurately determining the amount of compounds present in the air. Adequate correlation coefficients between the mass of the target analytes and exposure time were obtained. The proposed validated method is a useful tool for the early detection of trichloroanisole and its precursor trichlorophenol in wineries’ ambient air while avoiding contamination of wine or winery facilities.

Analysis of grapes and the first stages of the vinification process in wine contamination with Brettanomyces bruxellensis

Brettanomyces bruxellensis is a major cause of wine spoilage due to the production of ethyl phenols, and it has become a major worldwide oenological concern in recent years. The most critical factor in volatile phenol production is the presence of microorganisms responsible for biosynthesis. In this work, carried out during three consecutive harvests, grapes and the first step in grape processing (stemming–crushing) have been evaluated as the origin of wine contamination by these spoilage yeasts. Results showed that there was nil or minimal presence of Brettanomyces yeasts in grapes and on the stemmer, in levels that the method was not able to detect. This shows that the main contamination of wines by this microorganism occurs in later stages of the vinification process and/or during storage. The contamination of many wines with Brettanomyces, either via the fruit or from the winery environment, during the early stages of vinification and before the start of aging, was confirmed by analyzing 100 recently made red wines, in which this yeast was detected in a high percentage of wines (27 %). However, the level of Brettanomyces yeasts found in the samples was low, with values which would not be sufficient to cause organoleptic defects. Consequently, this study confirms that many wines are still tainted by Brettanomyces when the winemaking phase comes to an end. It was also found that wines with problems during fermentation had a higher level of Brettanomyces. This is why it is essential to ensure strict controls during fermentation processes and conservation to prevent wine becoming spoiled or tainted.

Contamination of wines and spirits by phthalates: types of contaminants present, contamination sources and means of prevention

This research determines the concentrations of various phthalates in French wines and grape spirits marketed in Europe or intended for export. Dibutyl phthalate (DBP), diethylhexyl phthalate (DEHP) and butyl benzyl phthalate (BBP) were the most frequently detected compounds in the wines analysed. While only 15% of the samples examined contained quantifiable concentrations (> 0.010 mg kg–1) of DEHP and BBP, 59% of the wines contained significant quantities of DBP, with a median value as high as 0.0587 mg kg–1. Only 17% of the samples did not contain any detectable quantity of at least one of the phthalates and 19% contained only non-quantifiable traces. In the spirits analysed, DBP (median = 0.105 mg kg–1) and DEHP (median = 0.353 mg kg–1) were the substances measured at the highest concentrations, as well as the most frequently detected (90% of samples). BBP was present in 40% of the samples at an average concentration of 0.026 mg kg–1. Di-isobutyl phthalate (DiBP), which is not permitted in contact with food, was found in 25% of the spirits tested. According to the specific migration limits (SML) for materials in contact with food, slightly more than 11% of the wines analysed were non-compliant, as they exceeded the SML for DBP (0.3 mg kg–1); just under 4% were close to the SML for DEHP. Concerning spirits, 19% of the samples analysed were considered non-compliant to the SML for DBP and nearly 7% were close to the SML for DEHP. The aged grape spirits analysed were often excessively contaminated with DiBP, which is not permitted to be used in contact with food (> 0.01 mg kg–1). A study of various materials frequently present in wineries revealed that a relatively large number of polymers sometimes contained high concentrations of phthalates. However, the epoxy resin coatings used on vats represented the major source of contamination.

Factors affecting distribution and abundance of Aspergillus section Nigri in vineyard soils from grapevine growing regions of Argentina

Aspergillus species belonging to section Nigri are the main fungi responsible for ochratoxin (OTA) contamination in grapes and wine. These species live as saprophytes in the superficial layer of the vineyard soil. We evaluated the biodiversity of potentially ochratoxigenic strains of Aspergillus section Nigri isolated from vineyard soils from different grapevine growing regions of Argentina. The isolates were characterized by classical and molecular methods. A multiple correspondence analysis was performed to identify the overall correlation of the Aspergillus group distribution with environmental conditions, geographical characteristics and vineyard practices. Aspergillus niger aggregate was the prevalent group (71%) and A. carbonarius made up only 2%. Species discrimination by species-specific primers showed that in A. niger aggregate 89% were A. tubingensis; 97% of the uniseriate were A. japonicus/A. aculeatus. Isolates belonging to these groups were unable to produce OTA. Our results clearly demonstrate a strong association between presence of A. carbonarius, high average temperatures and drip irrigation. Precipitation levels appear as a secondary factor, and altitude, vineyard age, predominant species, grape variety or total fungal count showed no association with A. carbonarius. We demonstrated a low prevalence of ochratoxigenic species in vineyard soil from the grape-growing regions of Argentina.

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.