Mycotoxins In Wine Research Articles

A Portable Automated Microfluidic Platform for Point-of-Care Testing for Multiple Mycotoxins in Wine.

Food safety requires point-of-care testing (POCT) for mycotoxins, since their presence in wine significantly impacts the wine industry and poses a severe threat to human life. Traditional detection methods are usually limited to detecting one mycotoxin and cannot achieve high-throughput, automated, and rapid quantitative analysis of multiple mycotoxins in real samples. Here, we propose a portable automated microfluidic platform (PAMP) integrating a chemiluminescence (CL) imaging system and a microfluidic chip to realize POCT for multiple mycotoxins in real samples, simplifying complex manual operations, shortening the detection time, and improving the detection sensitivity. Specially, silicone films were used as substrates on microfluidic chips to incubate mycotoxin conjugations, and the streptavidin-biotin (SA-B) system and an indirect immunoassay were implemented on silicone films to improve the sensitivity of reaction results. Interestingly, these methods significantly improved detection results, resulting in sensitive detection of mycotoxins, including zearalenone (ZEA) ranging from 1 to 32 ng/mL, aflatoxin B1 (AFB1) ranging from 0.2 to 6.4 ng/mL, and ochratoxin A (OTA) ranging from 2 to 64 ng/mL. The recovery of samples reached 91.39-109.14%, which verified the reliability and practicability of the PAMP. This PAMP enables sensitive and rapid detection of multiple mycotoxins in markets or wineries that lack advanced laboratory facilities. Therefore, it is essential to develop a portable microfluidic platform for POCT to detect mycotoxins in real samples.

Determination of 10 mycotoxins in wine, baijiu, and huangjiu of the Chinese market by liquid chromatography tandem mass spectrometry and exposure estimation

In this study, liquid chromatography tandem mass spectrometry (LC–MS/MS) was employed to analyze the prevalence of 10 mycotoxins in 140 samples from the Chinese market, aiming to assess the exposure of Chinese individuals to these mycotoxins through the consumption of wine, baijiu, and huangjiu. Mycotoxins were detected in 98% of the samples, with fumonisins (FBs), deoxynivalenol (DON), and zearalenone (ZEN) exhibiting positive rates exceeding 50%. Regarding the exposure of the Chinese population to mycotoxins resulting from alcoholic beverage consumption, fruit wine intake made a relatively significant contribution to aflatoxin exposure, while baijiu showed a relatively significant contribution to ZEN exposure (1.84%). The analysis of the correlation between grape variety, wine region, and mycotoxin content demonstrated that FBs, ZEN, and DON were significantly influenced by grape variety and wine region. This research holds great significance in protecting human life and health, as well as in the production of safer alcoholic beverages.

Application of a Validated Method for the Identification and Quantification of Mycotoxins in Wines Using UPLC-MS/MS

The aim of the present study was to develop a rapid, simple and reliable method for the identification and quantification of six mycotoxins in wine using liquid chromatography with electrospray ionization tandem mass spectrometry. The analytical method was fully validated, and calibration curves were made with correlation coefficients >0.9970. A short analysis time and acceptable extraction efficiency were achieved by a direct extraction method of analytes (ochratoxin A, aflatoxin B1, B2, G1, G2 and Zearalenone) with acetonitrile. LOD values were from 0.03 to 0.27 μg kg−1, and LOQ values were from 0.08 to 0.81 μg kg−1, with recoveries at various values from 77 to 108%. The expanded uncertainty was 5–21% expressed at a coverage level of k = 2, at a confidence level of approximately 95%. The performance criteria of the method were fully met according to European legislation (EC) 401/2006. The method was successfully applied to wine samples from Cyprus. The method was simple, low cost, quick, accurate, and sensitive.

Mycotoxins in red wine: Occurrence and risk assessment

Mycological contamination of food products is a common problem in the food industry, associated with implications for consumer health. Red wine is regarded as an alcoholic beverage with health benefits, notably for the circulatory system. However, in spite of its health-promoting properties, it can also be a source of toxic substances. Wine is a documented source of ochratoxin A, which is one of the ubiquitous, highly toxic secondary metabolites produced by filamentous fungi. It is the only mycotoxin for which a regulatory maximum level in wine has been established. There are no legal regulations on the content of other mycotoxins in wine, as a product with an increased risk of mycological contamination, and so their levels are not monitored. The aim of this study was to determine the concentrations of ochratoxin A (OTA), deoxynivalenol (DON) as well as T-2 and HT-2 toxins in dry red wines. The study included 19 wines originating from Spain, France and Poland, from both conventional and organic viticulture. Mycotoxin levels were determined using immunoenzymatic assays (ELISA). The pH values were determined by potentiometric method. In all of the wines included in the study, mycotoxin levels exceeded the applicable limits. The levels of OTA and DON contamination were associated with the country of origin, and ochratoxin A was additionally linked to the farming method and certification status. The content of T-2 and HT-2 toxins was the only parameter to be negatively correlated with pH. In conclusion, wine may provide a source of many mycotoxins, not only OTA but also DON T-2 and HT-2 toxins, which was shown for the first time, and it appears further research is required to establish reference values for various mycotoxins in a wider range of foodstuffs, including wine.

Monitoring of 49 Pesticides and 17 Mycotoxins in Wine by QuEChERS and UHPLC–MS/MS Analysis

An effective method for the determination of 49 pesticide residues and 17 mycotoxins in wine by a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method and ultrahigh-performance liquid chromatography-tandem mass spectrometry was developed. The target compounds were extracted with 1% (v/v) formic acid-acetonitrile, and no cleanup steps were required. The extracts were separated on a C18 chromatographic column (2.1 mm × 50 mm, 1.7 µm) with acetonitrile and water with 0.2% formic acid solution and ammonium acetate (10 mM) as the mobile phases under gradient elution at a flow rate of 0.2 mL/min. The determination was conducted using electrospray ionization in positive ion mode with multiple reaction monitoring. The analytes were quantified by comparison with matrix-matched standard solutions. The good linearities were obtained in the range of 0.05 to 500.0 µg/kg, and the correlation coefficients were all greater than 0.9935. The average recoveries of the 66 target compounds ranged from 69% to 119%, and the RSDs were in the range of 1% to 10%. The limits of detection were in the range of 0.05 to 20.0 µg/kg. The method was proved to be rapid, selective, sensitive, and stable, and it has been applied to analysis of 64 wine samples.

Simultaneous determination of pesticides and mycotoxins in wine by direct injection and liquid chromatography-tandem mass spectrometry analysis

A simple and rapid method for simultaneous determination of pesticides and mycotoxins in red wine is presented. Sample preparation approach, called direct injection, consists of a sequence of only three steps: centrifugation, dilution and filtration. The analysis of extracts were performed by UPLC-MS/MS for determination of pesticides and mycotoxins. The method was assessed for linearity, limits of detection and quantification, matrix effects, selectivity, accuracy and precision. For recovery experiments, mycotoxins were divided in two groups according to their sensitivity in the UPLC-MS/MS system. More than 80% of the mycotoxins were reliably quantified at the lowest spike level studied (1 μg kg−1 for group 1 and 50 μg kg−1 for group 2). From the 185 evaluated pesticides, 144 showed acceptable results at 10 μg kg−1, the lowest spiked level. Matrix effects were, in most of the cases, negative, and that was observed for both pesticides and mycotoxins.

Rapid assessment of mycotoxins in wine by on-line SPE-UHPLC-FLD

According to the latest statistics, grapes are one of the largest fruit crops worldwide. In this regard, it is important to consider all factors influencing quality of grapes and wine. In the last years, scientist focused on the study of mycotoxins that can influence the quality of wine. It is considered that toxins produced by moulds, causing significant economic losses, affect approximately one quarter of the world grape production. If the selective sorting of infected grapes is not done adequately, wine will present a major risk to consumers, mycotoxins being considered by the “International Agency for Cancer Research” a carcinogenic compound. The main mycotoxins monitored in this study come from Aspergillus sp., and are represented by aflatoxins B1, B2, G1, G2 and ochratoxin A. This study purpose is to develop a faster method for the analysis of mycotoxins, in order to increase rapidity and efficiency for the evaluation of the degree of infestation in wine. The purposed method is using an on-line large volume injection coupled to pre-concentration of sample (SPE) which is directly transfer to the ultra-high-pressure liquid chromatography (UHPLC) column for separation and the detection by means of the fluorescence detector (FLD). As the maximum tolerated level for mycotoxins in wines is 2 ppm, this method is able to detect under this limits of quantification with RSD below 2%.

Determination of Alternaria mycotoxins in wine and juice using ionic liquid modified countercurrent chromatography as a pretreatment method followed by high-performance liquid chromatography

Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA) are some of the main Alternaria mycotoxins that can be found as contaminants in food materials. The objective of this study was to develop a pretreatment method with countercurrent chromatography (CCC) for enrichment and cleanup of trace Alternaria mycotoxins in food samples prior to high-performance liquid chromatography (HPLC) analysis. An Analytical CCC instrument with a column volume 22.5mL was used, and a two-phase solvent system composed of ethyl acetate and water modified with 6% [HOOMIM][Cl] in mass to volume ratio was selected. Under the optimized CCC operation conditions, trace amounts of AOH, AME, and TeA in large volume of liquid sample were efficiently extracted and enriched in the stationary phase, and then eluted out just by reversing the stationary phase as mobile phase in the opposite flowing direction tail-to-head. The enrichment and elution strategies are unique and can be fulfilled online with high enrichment factors (87–114) and high recoveries (81.14–110.94%). The method has been successively applied to the determination of Alternaria mycotoxins in real apple juice and wine samples with the limits of detection (LOD) in the range of 0.03–0.14μgL−1. Totally 12 wine samples and 15 apple juice samples from the local market were analyzed. The detection rate of AOH and AME in both kinds of the samples were more than 50%, while TeA was found in relatively high level of 1.75–49.61μgL−1 in some of the apple juice samples. The proposed method is simple, rapid, and sensitive and could also be used for the analysis and monitoring of Alternaria mycotoxin in other food samples.

Development, optimization and validation of a multimethod for the determination of 36 mycotoxins in wines by liquid chromatography–tandem mass spectrometry

A fast and efficient multimethod for the determination of 36 mycotoxins in wine, using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS), was developed, optimized, validated and implemented in routine analysis. A simplified, quick extraction was performed with acetonitrile, derived from the QuEChERS (quick, easy, cheap, effective, rugged and safe) approach, which was traditionally developed for pesticides analysis. This study aimed at a single extraction and chromatographic separation for 36 mycotoxins.Optimization tests were performed to find the proper ratio of wine: water and extraction solvent and the need for an additional buffering step with ammonium formate/formic acid and a dispersive SPE cleanup with various sorbents. The dSPE steps did not show significant improvement in analysis results, therefore, it was not applied in the final method to be validated. The mycotoxins were separated and detected on a UPLC–MS/MS system, used in the ESI positive ionization mode. The various mycotoxins were divided in three different concentration level groups, according to their sensitivity in UPLC–MS/MS. The validation was performed by analyzing recovery samples at three different spike levels with six replicates (n=6) at each level. Linearity (r2) of calibration curves, accuracy (recovery %), instrument limits of detection and method limits of quantification (LOD and LOQ), precision (RSD%) and matrix effects (%) were determined for each individual mycotoxin. From the 36 mycotoxins analyzed by UPLC–MS/MS (ESI+), 35 showed average recoveries in the range 70–120%, and 86% of these with a RSD≤20% at the lowest spike level (for Group I, II and III, respectively, 1, 50 and 10µgkg−1). The higher spike levels showed even better results. Only nivalenol could not be quantified at any concentration level. The method LOQ for 86% of the mycotoxins studied was the lowest spike level tested. The matrix effect observed was low for most mycotoxins analyzed and had no significant influence on the analytical results obtained. The developed procedure was applied successfully in routine analysis in a survey of wine samples originating from different countries.

Rapid Method for the Determination of Multiple Mycotoxins in Wines and Beers by LC-MS/MS Using a Stable Isotope Dilution Assay

A "dilute and shoot" method for the liquid chromatography-tandem mass spectrometric (LC-MS/MS) determination of multiple mycotoxins (aflatoxins B1, B2, G1, G2, ochratoxin A (OTA), fumonisins (F) B1 and B2, zearalenone, deoxynivalenol, T-2 toxin, and HT-2 toxin) in wines and beers has been developed and validated. Separation was accomplished using ultrahigh-performance liquid chromatography (UHPLC) with <10 min analysis time. Mycotoxins were detected by dynamic multiple reaction monitoring (MRM) in positive electrospray ionization mode. Due to matrix effects, (13)C-uniformly labeled mycotoxins were added to the sample extracts prior to LC-MS/MS analysis. With external calibration, recoveries were 18-148% for white wines, 15-118% for red wines, and 20-125% for beers, at three spiking levels. The (13)C-labeled internal standards compensated for matrix effects effectively, with overall recoveries of 94-112% for white wines, 80-137% for red wines, and 61-131% for beers, with greater recoveries for FB1 and FB2, at three spiking levels. The relative standard deviation was <20% for all analytes in the wines and beers. This method was applied to a USDA-funded nationwide survey of domestic and imported wines and beers for the determination of OTA and extended to include other mycotoxins.

Generic sample treatment method for simultaneous determination of multiclass pesticides and mycotoxins in wines by liquid chromatography–mass spectrometry

In this work, a generic sample treatment method for simultaneous determination of multiclass pesticides and mycotoxins in wines is presented. The proposed method is based on solid-phase extraction (SPE) using polymeric-type SPE cartridges. To evaluate the proposed sample treatment, a liquid chromatography electrospray time-of-flight mass spectrometry method was used for testing 60 selected representative multiclass pesticides and 9 mycotoxins. Two different polymeric sorbents were evaluated, with hydrophilic–lipophilic-balanced (HLB) polymer cartridges being selected (Oasis™ HLB) as the most suitable for the present study. The identification and confirmation of the compounds was based on retention time and accurate mass measurements of the protonated molecules ([M+H]+). Limits of detection were below 1μgL−1 for the 87% of the studied compounds. With the selected 4:1 preconcentration factor, 70% of the target compounds showed relatively low matrix effects, corresponding to signal suppressions lower than 30%. Recovery studies (n=10) were carried out at two concentration levels, 2.5μgL−1 and 25μgL−1, obtaining mean recovery rates between 70 and 120% for the 90% of studied analytes. The relative standard deviation (RSD%) values of the entire procedure were below 15% in most cases (97% of the studied analytes). The proposed method was successfully applied to 24 red wine samples produced in different regions of Spain. The concentration levels of the target compounds found in the studied samples were in compliance with the current regulations. Aflatoxin B2 and metalaxyl were the most detected compounds (75% and 50% of the studied samples, respectively).

Sensing of Mycotoxin Producing Fungi in the Processing of Grains

Due to the growing demand for rapid, sensitive and inexpensive detection of contaminations in food and feed, both in the field and after harvest, non-invasive and non-destructive spectroscopic methods are reviewed for the direct identification of fungi and mycotoxins on grain and in grain products. Different model samples containing defined contaminations of fungi and/or mycotoxins as well as real-world samples were investigated using spectroscopic methods (steady-state and time-resolved fluorescence spectroscopy, ultraviolet/visible/near infrared (NIR) spectroscopy, two-photon-induced fluorescence (TPIF)). To evaluate the capability of TPIF for on-line and in-line mycotoxin analysis in real-world samples, fundamental parameters of different mycotoxins, such as two-photon cross-sections, were determined in beer and wine. A comparison showed that the disturbance due to background emission originating from matrix constituents is significantly reduced under two-photon excitation conditions, and TPIF can be applied for a qualitative and quantitative analysis of mycotoxins in wine and beer. The combination of data obtained from different spectroscopic methods (such as optimised excitation and emission wavelengths, fluorescence decay times and fluorescence quantum efficiency on the one hand side and NIR spectroscopy on the other side) is promising for the qualitative as well as quantitative identification of fungi and mycotoxins. Moreover, NIR reflection spectra yield additional information on ingredients, moisture content and the presence (or absence) of fungi in the sample. The spectroscopic techniques are complemented by chemometric tools to extract the desired chemical information, e.g. with respect to the presence of contaminations.

Mycotoxins in wine with special attention on ochratoxin A

Wine quality is a complex, multi - layered conception consisting of numerous factors such as sensory characteristics, chemical composition, legislation, market - consumer, with hygienic - toxicological factor being of special importance due to growing demands for health safe foods. This paper shows the results of studies carried out up till now concerning the mycotoxins in wine (Aflatoxins, Trichothecens, Patulin), with special attention paid to ochratoxin A, most frequently present in grapes, must and wine, and to the influence of certain technological operations and processes during wine making. Due to its high toxicity, the presence of ochratoxin A has been limited to 2 ?g/l by EU EG regulation 123/2005.

Mycotoxin production from fungi isolated from grapes.

In order to assess the potential for producing mycotoxins, fungi were isolated from wine producing grapes. The isolates were identified and Penicillium expansum, the most well recognized mycotoxin producer, was analysed for mycotoxin production by TLC. Many of the strains produced patulin and/or citrinin, often depending on whether they were grown on a grape or yeast extract sucrose media. Citrinin was produced by all strains grown in the yeast extract sucrose medium, but only one strain (from 51) was able to produce this compound in grape juice medium. Patulin was produced in the yeast extract medium by 20 strains and in grape juice medium by 33 strains. The presence of mycotoxins in wine producing grapes is discussed. Grapes contamination with patulin seems not to contribute to wine contamination, and no ochratoxin producing fungi was identified.