Alternaria toxins in wheat from the Autonomous Province of Vojvodina,Serbia: a preliminary survey

Although Fusarium species remain a main source of mycotoxin contamination of wheat, in recent years, due to the evident climatic changes, other mycotoxigenic fungi have been recognised as important wheat contaminants. Alternaria species, especially A. alternata, have been found as contaminants of wheat as well as wheat-based products. Under favourable conditions A. alternata very often produce alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA) and others Alternaria toxins. The aim of the present study was to examine the presence of three Alternaria toxins (AOH, AME and TeA) in wheat samples harvested during three years (2011–13). To this end, 92 samples were collected during wheat harvesting from different growing regions of the Autonomous Province of Vojvodina, which represents the most important wheat-growing area in Serbia. The presence of Alternaria toxins was analysed by HPLC with electrospray ionisation triple quadrupole mass spectrometry (LC-ESI-MS/MS). Among all the analysed wheat samples, 63 (68.5%) were contaminated with TeA, 11 (12.0%) with AOH and 6 (6.5%) with AME. Furthermore, the maximum and mean toxin concentrations were 2676 and 92.4 µg kg^?1, 48.9 and 18.6 µg kg^?1, and 70.2 and 39.0 µg kg^?1 for TeA, AOH and AME, respectively. Co-occurrence of three Alternaria toxins in wheat samples was detected in six samples; a combination of two toxins was found in two samples; and 64 samples contained one toxin. The results showed that among 92 analysed wheat samples, only 20 (21.7%) samples were without Alternaria toxins. The presence of Alternaria toxins was also investigated in terms of weather conditions recorded during the period of investigation, as well as with the sampling region. This study represents the first preliminary report of the natural occurrence of Alternaria toxins in wheat (Triticum aestivum) from Serbia.     

Alternaria toxins in Argentinean wheat,bran, and flour

Alternaria species have been reported to infect a wide variety of vegetables, fruits, and cereal crops. Wheat is one of the most consumed cereal worldwide. A sensitive HPLC-DAD methodology was applied to quantify alternariol (AOH), alternariol methyl ether (AME) and tenuazonic acid (TeA) in 65 samples of whole wheat, bran, and flour. The extraction methodology allowed extracting the three toxins simultaneously. Limits of detection in wheat were 3.4, 4.5, and 0.5 µg kg^?1 for AOH, AME and TeA, respectively. For bran, these data were 3.1, 4.5, and 12 µg kg^?1 and for flour 50, 70, and 14 µg kg^?1, respectively. The studied recoveries were higher than 70% and RSD was below 10%. Wheat and bran samples showed low AOH and AME contamination compared to TeA. The averages levels found for TeA in wheat, bran and flour were 19,190, 16,760, and 7360 µg kg^?1, respectively.     

Effects of gamma radiation on the growth of Alternaria alternata and on the production of alternariol and alternariol monomethyl ether in sunflower seeds

The objective of the present study was to evaluate the effects of different gamma radiation doses on the growth of Alternaria alternata and on the production of toxins alternariol (AOH), and alternariol monomethyl ether (AME) in sunflower seed samples. After irradiation with 2, 5 and 7 kGy, the spore mass was resuspended in sterile distilled water and the suspension was inoculated into sunflower seeds. The number of colony-forming units per gram (CFU/g) was determined after culture on Dichloran Rose Bengal Chloramphenicol and Dichloran Chloramphenicol Malt Extract Agar. The presence of AOH and AME was investigated by liquid chromatography coupled to mass spectrometry. The radiation doses used resulted in a reduction of the number of A. alternata CFU/g and of AOH and AME levels when compared to the nonirradiated control group. Maximum reduction of the fungus (98.5%) and toxins (99.9%) was observed at a dose of 7 and 5 kGy, respectively. Under the present conditions, gamma radiation was found to be an alternative for the control of A. alternata and, consequently, of AOH and AME production in sunflower seeds.     

Occurrence of emerging mycotoxins in cereals and cereal-based products from the Korean market using LC-MS/MS

The present study aimed to investigate the occurrence of emerging mycotoxins in cereals (n = 61) and cereal-based products (n = 36) collected from Korean market. First of all, using the quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction method, and ultrahigh-pressure liquid chromatography (UPLC) with triple quadruple tandem mass spectrometry (MS/MS), we developed a simple and fast method for quantitative determination of eight emerging mycotoxins including alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), beauvericin (BEA) and enniatins (ENs; ENA, ENA1, ENB and ENB1). The developed analytical method was validated in parameters of linearity, precision and accuracy. For UPLC-MS/MS analysis, the recoveries of emerging mycotoxins from spiked samples at three concentration levels ranged from 82.7% to 108.8% with RSDs between 0.4% and 14.7%. Analytical methods were applied to determine the contamination of mycotoxins in cereal and cereal-based product samples. Sixty-three of the total 97 samples were contaminated with at least one emerging mycotoxin. The maximum number of emerging mycotoxins observed in a single sample was six out of eight analytes. The highest level of contamination was detected in cereal at 70.9 μg/kg for alternariol monomethyl ether (AME). However, currently there is no international standard for emerging mycotoxins in food. Accordingly, it is necessary to establish a database of emerging mycotoxins contamination through continuous monitoring.     

Oxidative metabolism of the mycotoxins alternariol and alternariol-9-methyl ether in precision-cut rat liver slices in vitro

Scope: Monohydroxylation of alternariol (AOH) and alternariol‐9‐methyl ether (AME) has previously been reported as a prominent metabolic route under cell‐free conditions. This pathway gives rise to several catechol metabolites and may therefore be of toxicological relevance. Methods and results: To clarify whether hydroxylation of AOH and AME occurs under in vivo‐like conditions in the presence of conjugation reactions, the metabolism of the Alternaria toxins has now been studied in precision‐cut rat liver slices. Four catechol metabolites of AOH and two of AME, together with several of their O‐methylation products, as catalyzed by catechol‐O‐methyl transferase, were clearly identified after incubation of the liver slices with AOH and AME. These metabolites were predominantly present as conjugates with glucuronic acid and/or sulfate. In preliminary studies with bile duct‐cannulated male rats dosed with AOH by gavage, the four monohydroxylated metabolites of AOH could also be demonstrated in the bile either as catechols or as O‐methyl ethers. Conclusion: These experiments clearly show that AOH and AME undergo catechol formation in vivo and warrant closer examination of the toxicological significance of this metabolic pathway.     

PRODUCTION OF ALTERNARIOL AND ALTERNARIOL METHYL ETHER BY ALTERNARIA SPP.

Five isolates of Alternaria alternata, five isolates of A. tenuis, one isolate of A. hemicota and three isolates of unidentified Alternaria spp. were examined for their production of alternariol methyl ether (AME) and alternariol (AOH) in complex liquid media. The data were compared with the production of these two toxins on solid substrate rice cultures. A new high-performance liquid chromatography method with minimum detection levels for AME and AOH of 20 ng and 40 ng, respectively, was used to quantitate the toxins. All isolates produced AME and AOH, but the amount varied considerably with the isolates and media tested. Of the three different liquid media tested, the highest quantity of AME and AOH was produced in a modified Czapek-Dox broth. The highest yield of toxins was obtained in the solid rice cultures. As much as 125 mg of AME and 16 mg of AOH were produced by one A. tenuis isolate on 300 g of rice after 14 days of incubation at 25°C in darkness.     

Validation of analytical method for α‐dicarbonyl compounds using gas chromatography–nitrogen phosphorous detector and their levels in alcoholic beverages

This study aimed to establish an analytical method for α‐dicarbonyl compounds (α‐DCs) including glyoxal, methylglyoxal and diacetyl, to determine the content of α‐DCs in 101 various alcoholic beverages using gas chromatography–nitrogen phosphorous detector (GC‐NPD) and to perform exposure assessment. The limit of detection and limit of quantification for α‐DCs were 0.05–0.22 and 0.15–0.70 μg g^?1, respectively. The accuracy and precision were validated in five matrices. The raspberry fruit wine had the highest value at 139.74 μg g^?1 total α‐DCs. The lowest α‐DC concentration among the beverages was detected in rice wine (Makgeolli) at 1.59 μg g^?1. The levels of α‐DCs in various samples were detected as follows: 1.59–56.68 μg g^?1 in rice wine (Makgeolli), 2.73–16.77 μg g^?1 in beer, 8.22–139.74 μg g^?1 in fruit wine and 8.17–91.56 μg g^?1 in rice wine (Cheongju). The estimated daily intake of α‐DCs in the intake‐only group and population group was calculated as 4.22–97.94 μg kg^?1 bw day^?1 and 0.28–7.13 μg kg^?1 bw day^?1, respectively.     

Novel oxidative in vitro metabolites of the mycotoxins alternariol and alternariol methyl ether

The Alternaria toxins alternariol (AOH; 3,7,9-trihydroxy-1-methyl-6H-benzo[c]chromen-6-one) and alternariol methyl ether (AME, 3,7-dihydroxy-9-methoxy-1-methyl-6H-benzo[c]chromen-6-one) are common contaminants of food and feed, but their oxidative metabolism in mammals is as yet unknown. We have therefore incubated AME and AOH with microsomes from rat, human, and porcine liver and analyzed the microsomal metabolites with HPLC and GC-MS/MS. Seven oxidative metabolites of AME and five of AOH were detected. Their chemical structures were derived from their mass spectra using deuterated trimethylsilyl (TMS) derivatives, and from the information obtained from enzymatic methylation. Several of the metabolites were identified by comparison with synthetic reference compounds. AME as well as AOH were monohydroxylated at each of the four possible aromatic carbon atoms and also at the methyl group. In addition, AME was demethylated to AOH and dihydroxylated to a small extent. As the four metabolites arising through aromatic hydroxylation of AME and AOH are either catechols or hydroquinones, the oxidative metabolism of these mycotoxins may be of toxicological significance.     

Mycotoxin production by Alternaria strains isolated from Argentinean wheat

The toxigenic potential of Alternaria strains isolated from Argentinean wheat was investigated. A total of 123 strains were assayed for the production of tenuazonic acid (TA), alternariol (AOH) and alternariol monomethyl ether (AME). All but one of the isolates were able to produce at least one of the three mycotoxins. TA was produced by 72% of the strains (1-14782 mg/kg), AOH by 87% (4-622 mg/kg) and AME by 91% (7-2625 mg/kg). The average level of TA detected for all strains (1757 mg/kg) was higher than the average level of both alternariols (162 mg/kg for AOH and 620 mg/kg for AME). TA was the toxin produced at the highest concentration but in lower frequency. Most of the strains were able to synthesize more than one toxin: 74 isolates (60%) were positive for all three toxins, 30 (24%) for both AOH and AME, 5 (4%) for both TA and AME, and 2 (2%) for TA and AOH. The widespread occurrence of Alternaria in wheat and its ability to produce mycotoxins suggests the possible occurrence of its toxins in wheat naturally infected with this fungus.     

Rapid Quantification Method of Three <Emphasis Type="Italic">Alternaria</Emphasis> Mycotoxins in Strawberries

Validation of simple and rapid method for three Alternaria mycotoxins determination including alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) in strawberries is described. The extraction procedure was based on a simple liquid–liquid extraction with ethyl acetate, which provided the highest recoveries and the lowest matrix effect. Analytes were determinated by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The obtained relative recoveries were higher than 63 % for the studied mycotoxins in strawberries at the limit of quantification (LQ). Good linearity (r ²?>?0.993) and quantification limits (3–14 ng/g) were obtained. Repeatability, expressed as relative standard deviation, was always lower than 6 %, whereas interday precision was lower than 13 % for the developed method. The method was applied to analyse 24 strawberry samples commercialized in markets of the Valencia city (Spain). Analysed samples were only contaminated with AOH and AME.     

臭氧处理对交链孢菌生长及其毒素积累的抑制作用

交链孢菌（Alternaria spp.）易侵染农作物,引起农产品病害,而且能够代谢产生交链孢毒素,包括细交链孢酮酸（tenuazonic acid,TeA）、交链孢酚（alternariol,AOH）、交链孢酚单甲醚（alternariol monomethyl ether,AME）等,严重影响人体健康。因此,亟需高效、安全的方法用以防控交链孢菌及其毒素积累。本实验研究了臭氧处理对体外互隔交链孢（A. alternata）生长及其产毒能力的影响。结果表明,臭氧处理组的菌落直径显著低于对照组（P＜0.05）,且臭氧处理可显著抑制互隔交链孢产生TeA、AOH、AME这3 种交链孢毒素;利用扫描电子显微镜观察臭氧处理后互隔交链孢的微观形态,发现孢子和菌丝发生了凹陷、褶皱、断裂等异常现象;臭氧处理后的交链孢菌对番茄果实的致病力明显减弱,同时交链孢菌的产毒能力明显降低,20 mg/L臭氧处理条件下TeA、AOH、AME含量比对照组分别减少了36.1%、89.9%、93.2%。此外,臭氧对TeA、AOH、AME具有降解作用,降解率随着臭氧质量浓度的增加和处理时间的延长而显著提高,TeA经过20 mg/L臭氧处理30 min即可被完全降解,AOH及AME经20 mg/L臭氧处理120 min后降解率可达90%以上。综上,臭氧处理可以作为农产品及其制品中互隔交链孢及其毒素污染的防治手段。     

Rapid determination of Alternaria mycotoxins in tomato samples by pressurised liquid extraction coupled to liquid chromatography with fluorescence detection

ABSTRACT

A sensitive and reliable method using pressurised liquid extraction (PLE) followed by molecularly imprinted solid phase extraction (MISPE) and high performance liquid chromatography with fluorescence detection (HPLC–FLD) has been developed for the analysis of alternariol (AOH) and alternariol monomethyl ether (AME) in tomato samples. Influence of several extraction parameters that affect PLE efficiency were evaluated for the simultaneous extraction of both mycotoxins in the selected samples. AOH and AME were optimally extracted using MeOH/water (25:75, v/v) at 70°C as solvent, a pressure of 1000 psi and a single extraction cycle. The resulting PLE extracts were pre-concentrated by molecularly imprinted solid phase extraction (MISPE) cartridges followed of analysis by HPLC with fluorescence detection (λ_exc = 258, λ_em = 440 nm). The proposed method was applied to the analysis of AOH and AME in fortified tomato samples (20–72 µg· kg–1) with recoveries of 84–97% (RSD < 8%, n = 6) for AOH and 67–91% (RSD < 13%, n = 6) for AME. The detection limit for AOH and AME were 7 and 15 µg· kg^–1, respectively. The ensuing PLE–MISPE–HPLC–FLD method was validated for the analysis of both mycotoxins in tomato samples in accordance with European Commission Decision 2002/657/EC.     

Determination of 4‐ethylcatechol in a Merlot wine using sensory evaluation and the electronic tongue

The objective of this study was to determine the influence of 4‐ethylcatechol (4‐EC) on the sensory profile of Brettanomyces‐contaminated Merlot wine and evaluate electronic tongue discrimination. Using sensory evaluation panels, the consumer detection threshold (DT) and the consumer rejection threshold (CRT) of 4‐EC were determined in Merlot containing 493, 714, 1035 and 1500 μg L^?1 4‐EC. The DT of 4‐EC in Merlot was 823 μg L^?1, while the CRT was 1323 μg L^?1. The electronic tongue discriminated index (DI = 82%) among the samples, with hierarchical clustering showing a clear distinction between the control sample and the spiked samples. The lowest concentration distinguished by the electronic tongue was 493 μg L^?1, a lower value than the sensory threshold determined. These findings suggest that for the detection of 4‐EC in Merlot, the e‐tongue may be more sensitive than some consumers and the e‐tongue may be a suitable methodology for detection of subthreshold concentrations of chemical compounds in wine.     

Search for the contamination source of butyltin compounds in wine: agglomerated cork stoppers

A possible butyltin contamination source in wine was studied in this paper. Agglomerated cork stoppers, which were produced in Portugal, Spain, and Italy, used in wine bottles were examined. The domestic cork products, cork granules, and mucus used for cork products were also analyzed. The levels of mono- and dibutyltin compounds in corks were found in the range from <0.0024 to 3.3 and from <0.0029 to 6.7 microg of Sn/g, respectively. A low level of tributyltin contamination was also found in 2 of 31 tested samples. The presence of butyltin compounds in agglomerated cork stoppers was confirmed by GC-MS. Experimental results indicated that all overseas agglomerated cork stoppers studied contained mono- and/or dibutyltins. Butyltins were not detected in cork granules, mucus, most of the natural cork stoppers, and domestic agglomerated cork products. The concentrations of mono- and dibutyltins increased with the time in a 30-day experiment, showing that butyltin compounds can leach from agglomerated cork to the wine. When the butyltin concentrations in wine samples were compared with their levels in the corresponding agglomerated cork stoppers, a correlation was found. The potential harm of such food contamination was evaluated by the toxic research of butyltin compounds using Daphnia sp. as the experimental model.     

Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition

Alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) are Alternaria mycotoxins produced by the most common post-harvest pathogens of fruits. The production of these metabolites depends on several environmental factors, mainly temperature, water activity, pH and the technological treatments that have been applied to the product. In this study, the occurrence of AOH, AME and TEN was evaluated in strawberries samples stored at different temperatures ranges (at 22 ± 2 or 6 ± 2°C) and different periods (up to 1 month) simulating the current practice of consumer’s storage conditions. Sample extraction was performed using a liquid–liquid extraction method prior to LC-MS/MS analysis. AOH was the most prevalent mycotoxins with a 42% at strawberries stored at (22 ± 2)°C and 37% stored at (6 ± 2)°C. The highest AOH levels were found in samples conserved at (22 ± 2)°C ranging between 26 and 752 ng g^–1. AME levels ranged between 11 and 137 ng g^–1, which were found mainly in stored samples at (6 ± 2)°C for more than 28 days. None sample presented levels of TEN in either of the studied conditions.     

Migration of phenolic compounds from different cork stoppers to wine model solutions: antioxidant and biological relevance

Considering the enological interest of cork, this study aimed to identify and quantify the phenolic compounds able to migrate from different classes (natural cork stoppers “Flor” and “Third” quality and microagglomerate cork stopper) of cork stoppers into bottled wine model solutions. Another aim was to evaluate some antioxidant and biological features of cork phenolics that migrated into the wine model solutions. The main phenolic acids and aldehydes detected were as follows: gallic and protocatechuic acid detected both around 3.5 mg/L and vanillin and protocatechuic aldehyde detected around 2.5 and 1.5 mg/L after 27 months of bottling, respectively. Trace amounts of more complex polyphenols, namely hydrolysable tannins (castalagin/vescalagin and mongolicain A/B), were also detected. Two antioxidant features of the wine model solutions bottled with different wine cork stoppers were studied, namely the antiradical capacity and the reducing capacity, being the natural cork stoppers the ones with the higher activities. The intestinal absorption of the compounds in each wine model solution after 27 months in bottled was evaluated. The simpler phenolic compounds were able to cross Caco-2 cell model. The antiproliferative activity of the same wine model solutions was also evaluated against gastric and breast cancer cells. All samples were active against the two cell lines, which highlight the possible health outcomes of wine sealed with cork stoppers.     

链格孢霉菌侵染番茄产毒机制

为研究链格孢霉侵染番茄后的产毒机制,本实验将链格孢霉孢子悬液接种于蕃茄上,并分别置于4 ℃和25 ℃下培养,15 d内进行3 次取样。样品经高分辨质谱检测后,使用MZmine、Xcalibur软件对检测结果进行分析,原质谱数据集经Mzmine软件处理后导入全球天然产物社会分子网络平台,采用基于特征的分子网络（feature-based molecular networking,FBMN）建立真菌毒素及代谢物分子网络,实现对链格孢毒素代谢通路的可视化分析。结果表明,接种后番茄在4 ℃下未产生任何毒素,25 ℃下链格孢酚（alternariol,AOH）、链格孢甲基醚（alternariol monomethyl ether,AME）、细链格孢菌酮酸（tenuazonic acid,TeA）、腾毒素（tentoxin,TEN）在5、10、15 d时均被检出,链格孢霉烯（altenuene,ALT）直至15 d时才被检出,3 次取样中TeA含量均最高。利用MZmine软件的碎片诊断过滤功能和Xcalibur软件精确分子质量技术验证了AOH和AME硫酸盐结合的隐蔽型真菌毒素的存在。FBMN分析结果表明3 种毒素（AOH、AME、TeA）有明显的代谢通路,代谢产物的种类较多,而未在番茄中检测到TEN和ALT代谢产物。本研究阐明了链格孢霉毒素在番茄中的产生及代谢机制,可为番茄中链格孢毒素的有效控制及番茄的贮藏保鲜提供理论支持。     

Effect of autochthonous starter cultures on the volatile flavour compounds of Chinese traditional fermented fish (Suan yu)

Effect of autochthonous starter cultures on the volatile flavour compounds of Chinese traditional fermented fish was studied. Lactobacillus plantarum 120, Staphylococcus xylosus 135 and Saccharomyces cerevisiae 31, isolated from Suan yu, were selected as starter cultures. Volatiles were extracted by headspace solid‐phase microextraction (SPME) and analysed by gas chromatography–mass spectrometry technology (GC‐MS). Esters and alcohols were the main components of volatiles, accounting for over 50 percentage points in all samples. The highest content of esters (3034.54 μg kg^?1) was observed in S1 inoculated with L. plantarum 120, while the highest content of alcohols (2164.53 μg kg^?1) and ketones (379.98 μg kg^?1) was detected in S3 inoculated with S. cerevisiae 31. The content of acids and aldehydes was lower in inoculated samples. Principal component analysis revealed that the volatile composition was primarily influenced by the nature of the starter cultures. L. plantarum 120 and S. xylosus 135 could accelerate fermentation.     

Production of alternaria mycotoxins by Alternaria alternata isolated from weather-damaged wheat

The production of Alternaria mycotoxins by Alternaria alternata isolated from Chinese weathered wheat kernels were first investigated on polished rice and durum wheat grains. These mycotoxins included alternariol (AOH) and its monomethyl ether (AME), altenuene (ALT), altertoxin I (ATX-I), and tenuazonic acid (TA). Of 25 isolates tested, all were AOH and AME producers, 21 (84%) coproduced ALT and ATX-I, and 8 (32%) produced TA in rice culture. TA was the most abundant toxin produced at a level ranging from 1,369 to 3,563 mg/kg. Much smaller amounts of AOH, AME, ALT, and ATX-I were present with average concentrations of 54, 40, 44, and 8 mg/kg, respectively. There were linear correlations between the level of AOH and AME (r = 0.846), alternariols (AOH plus AME) and ALT (r = 0.785), and ATX-I and TA (r = 0.553). Polished rice medium seems to support a bit more production of Alternaria metabolites than wheat but with an insignificant difference in concentrations (P > 0.05). A study of the time-course of toxin production by A. alternata isolates indicated that AOH production began faster than any other toxins monitored, and ALT production exhibited a progressive increase throughout the experiment. TA producers might reveal their considerably higher ability to produce toxin in the field despite their low frequency.