Plant-based alternatives to animal-derived products have gained significant prominence in recent years, with the market for these products experiencing continuous growth. In response to this trend, we developed an ultra-high-performance liquid chromatography method, coupled with tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous quantification of 15 mycotoxins from Fusarium (deoxynivalenol (DON), DON-3-glucoside (D3G), 3-acetyl-DON, 15-acetyl-DON, HT-2 toxin, T-2 toxin), Alternaria (alternariol, alternariolmonomethylether (AME), tenuazonic acid (TeA)), and Aspergillus species (aflatoxin (AF) B1, B2, G1, G2, sterigmatocystin (STC), ochratoxin A) in plant-based meat, cheese, and fish substitutes. Sample extraction was performed using a QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach, followed by dispersive solid phase extraction (dSPE) for clean-up. Quantification was achieved through stable isotope dilution analysis (SIDA). The method demonstrated limits of detection (LOD) ranging from 0.01µg/kg (AFB2 and STC) to 14.1µg/kg (D3G) and limits of quantification (LOQ) between 0.02µg/kg (STC) and 41.9µg/kg (D3G). This method was successfully applied to quantify mycotoxins in 32 plant-based alternative products, revealing contamination predominantly with Alternaria toxins such as AME and TeA. Seitan-based products were identified as having the highest levels of contamination. In total, 27 out of 32 analyzed products contained at least one of the target mycotoxins. With the obtained data, we performed a risk assessment and revealed a potential risk, especially for toddlers, but also for individuals of all ages, due to the presence of Alternaria toxins. However, none of the detected concentrations exceeded the established regulatory limits or recommended values, as plant-based alternatives are not specifically included in the relevant food safety regulations.

The rise of difficult-to-treat fungal infections necessitates novel therapeutic strategies. In this study, endophytic fungi were isolated from Acalypha hispida leaves and molecularly identified as Penicillium oxalicum via 18S rRNA sequencing. LC-MS/MS analysis of the fungal extract revealed major bioactive compounds, including linoleic acid, sinapinic acid, alternariol monomethyl ether, ellagic acid, and kaurenic acid. Oily-core poly (ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) nanocapsules (PEGylated PLGA NCs) were developed to encapsulate the fungal extract, improving stability and bioavailability. The PEGylated PLGA NCs exhibited controlled particle size, positive surface charge, and spherical morphology. In vitro, the PEGylated PLGA NCs demonstrated antifungal activity against Candida albicans with inhibition zones of 10-14mm. In vivo, treatment significantly improved histological features of the kidney, liver, and spleen, and reduced tumor necrosis factor-alpha and cyclooxygenase-2 expression. In silico studies further confirmed the potential of the major compounds of the fungal extract to inhibit C. albicans aspartic proteinases SAP4-6. These findings suggest that PEGylated PLGA NCs loaded with P. oxalicum extract represent a promising antifungal therapeutic strategy.

Toxic metabolites of Alternaria spp. are considered as “emerging mycotoxins” and have not been yet regulated by legislation in many countries. Alternaria species can grow and produce these mycotoxins in various products including fruits and fruit juices. In the present study, UV-C treatment (3971.09 J m−2, 1–8 h) was used for the first time to degrade Alternaria toxins [tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME)] in apple juice. The effect of the treatment on quality characteristics of apple juice was also evaluated. UV-C treatment was very effective in degrading toxins. AME was the most affected toxin (decreased by half) after a treatment for 1 h. Degradation rates of 90.52, 77.01 and 75.92% were observed in the levels of TeA, AOH and AME, respectively, at the end of treatment for 8 h. Soluble solid content, pH and titratable acidity values did not change significantly after UV-C treatment, however turbidity values increased by 92% at the end of treatment. One of the major effects of UV-C treatment was on the color of the apple juice. Transmittance value at 440 nm increased while a* and b* values decreased significantly (p < 0.05) which shows that UV-C had a deleterious effect on color pigments of the juice. Significant decreases were observed also in phenolic content and antioxidant activity of the UV-C treated juice. These results demonstrate that UV-C is quite effective in degrading Alternaria toxins in apple juice but has detrimental effects on some quality characteristics, especially for prolonged treatment periods.

Identification, mycotoxigenic ability and biosynthesis genes of Alternaria spp. from apples in China.

Sulfitation by Aspergillus oryzae PC2 unveils a novel detoxification pathway for alternariol monomethyl ether: Experimental and computational study

Natural occurrence of Alternaria and Fusarium and their mycotoxins in malting barley grains from Argentina.

Emerging Alternaria mycotoxins tenuazonic acid (TeA), alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) and tentoxin (TEN) were detected in samples of spices and herbs. A total of 92 samples purchased in Italian markets were analyzed by using a UPLC-MS/MS method. TeA was the predominant mycotoxin with the highest percentage of positive samples (53%), followed by AME, AOH and TEN with overall means of 801.5, 2.4, 6.2 and 10.8 µg/kg, respectively. All samples were negative to ALT. The spices with higher levels of mycotoxins were flax seeds, paprika, red chili and licorice; regarding herbs, they were basil, sage and oregano. While TeA was found to be the most abundant mycotoxin equally in Italian and Lebanese samples, AOH and AME showed significantly lower levels in Italian samples, as Lebanese samples contained concentrations up to 14 times higher. Considering the mean levels of TeA in samples, the daily consumption of >8.7 g of flax seeds, >9.3 g of paprika and >5.8 g of red chili will exceed the threshold of toxicological concern (TTC) of TeA, which is 1500 ng/kg bw for a day. The high percentage of positive samples and the high levels of some mycotoxins observed demonstrate the susceptibility of spices and herbs to Alternaria mycotoxin contamination. These results provide an overview of emerging Alternaria mycotoxins in spices and herbs marketed in Italy and represent a valuable starting point to assess human exposure and support future studies aimed at establishing safe limits to protect human health.

Mycotoxins are toxic secondary metabolites produced by fungi, frequently present in food and representing significant health hazards. Exposure occurs through the consumption of contaminated foods or animal-derived products from livestock fed with contaminated feed. This study evaluated internal exposure to twelve mycotoxins in 492 first-morning urine samples from adults, aged 18–65 years, in the Valencian Community, Spain. Samples were analysed using a “dilute-and-shoot” approach followed by UHPLC-MS/MS. Aflatoxins (AFs) were the most frequently detected, with a geometric mean (GM) of 1.17 ng/mL and a 95th percentile (P95) of 6.04 ng/mL. Alternariol (AOH), present in 63% of samples, showed high concentrations (GM: 0.98 ng/mL; P95: 4.74 ng/mL). Emerging mycotoxins such as alternariol monomethyl ether (AME), citrinin (CIT), and sterigmatocystin (STER) were also considered due to their potential health impacts. Exposure levels correlated with variables including sex, age, annual income, smoking status, and recent consumption of meat and cereals. Probable daily intakes (PDIs) were estimated from urinary concentrations to support risk assessment. Hazard Quotients (HQs), Margins of Exposure (MOEs), the Hazard Index (HI) and the total Margin of Exposure (MOET) were calculated to evaluate the risk associated with mycotoxin exposure. Findings suggest that potential health risks cannot be excluded.

This study aimed to assess the prevalence of four Alternaria toxins (alternariol [AOH], alternariol monomethyl ether [AME], tenuazonic acid [TeA], and tentoxin [TEN]) in various foods and assess the risk of Alternaria-toxin exposure in Zhejiang Province, China. A total of 325 samples were collected in this study, and at least one type of Alternaria toxin was detected in 53.85% of the samples. Wheat flour had a high detection rate of 97.41%, and TeA was the most prevalent compound in terms of concentration and detection rate. Assessment of Alternaria toxins using the threshold of toxicological concern (TTC) method showed that the majority of the population had a low exposure risk. Population-wide dietary exposure assessment suggested a potential health risk for some residents with 95th percentile (P95) assessment values 0.0038, 0.0128, and 0.0047 µg/kg b.w. for AOH from wheat flour and AOH and AME from Coix rice, respectively, exceeding the TTC value of 0.0025 µg/kg b.w. Probabilistic assessment showed that the mean exposure of children aged ≤6 years to AOH via wheat flour for P92 and of those aged 7–12 years for P93 were both 0.0025 µg/kg b.w. Exposures to TeA and TEN were within the acceptable limits (below the TTC value of 1.5 µg/kg b.w.). Age-group probabilistic and point assessments indicated that children aged ≤6 and 7–12 years are at higher exposure risk. This study provides a useful reference for developing limiting values and legislation for Alternaria toxins in food.

Insights into Alternaria in apple fruit causing mouldy core, external infection and mycotoxin production under retail and storage conditions.

Sensitive determination of Alternaria toxins in cereals and cereal-based foods using online MAX SPE column cleanup coupled with liquid chromatography-tandem mass spectrometry.

Concerns about mycotoxin contamination by Alternaria spp. in tomato-based products emphasize the need for understanding the effect of the environment on their production. In the current study, we focused on three species frequently associated with tomato (A. alternata, A. solani, and A. tenuissima) by evaluating the effects of different temperatures (5 to 40 °C) and substrata (PDA and V8) on mycelial growth and the production of mycotoxins (alternariol, alternariol monomethyl ether, and tenuazonic acid). Both biological processes were supported between 5 and 35 °C, with optimal temperatures between 20 and 30 °C, depending on the species. Temperature and its interaction with species significantly (p < 0.05) affected both processes. However, the species factor alone was not significant (p > 0.05), indicating that environmental conditions affect Alternaria spp. growth and mycotoxin production more than the species itself does. Mathematical equations were developed to describe the effect of temperature on mycelial growth, as well as on the production of AOH, AME, and TeA, for each Alternaria species. High concordance (CCC ≥ 0.807) between observed and predicted data and low levels of residual error (RMSE ≤ 0.147) indicated the high goodness of fit of the developed equations, which may be used for the development of models to predict Alternaria contamination both in field and during post-harvest storage.

In order to understand the current situation of mycotoxin contamination and risk assessment of dietary exposure of coix seed (Coix lacryma-jobi) sold in Gansu Province, China, a total of 67 coix seed samples were collected from the local markets and supermarkets in 14 prefectures and cities in Gansu Province. Sixteen mycotoxins were determined by isotope dilution liquid chromatography-tandem mass spectrometry. The results showed that 37.3% of coix seed was contaminated by mycotoxins. Among them, fumonisin B1 (FB1), fumonisin B2(FB2), fumonisin B3 (FB3) and zearalenone (ZEN) had the highest prevalence in coix seed, 10.5%, 11.9%, 17.9% and 10.5%, respectively. Coix seed was susceptible to mixed contamination of various mycotoxins, which was often detected in the forms of an FB1, FB2, and FB3 combination (35.7%) and an ZEN-FBs combination (21.5%). In this study, the exposure assessment was evaluated separately according to the use of coix seed for food or clinical use. It was found that the EDI values of ZEN, FB1, FB2, FB3, AME (alternariol monomethyl ether), TeA (tenuazonic acid), and TEN (tentoxin) calculated from the intake of coix seed for both medicinal and edible purposes were less than their TDIs, indicating that the health risk of exposure to the above toxins caused by the consumption of coix seed was within an acceptable range.

Alternaria species are important fungal pathogens occurring worldwide in wheat, causing both productive and qualitative losses, and posing a toxicological risk to human health due to the production of their mycotoxins in kernels. This study aimed to investigate the occurrence of Alternaria species and their mycotoxins in 48 wheat grain samples collected from the northeast to the southeast of Algeria. Seventy-two representative Alternaria strains were molecularly analyzed using a multi-locus sequence approach and evaluated for their capability to produce mycotoxins under in vitro conditions. Alternaria alternata, representing 42% of the strains, was the dominant species, followed to a lesser extent by species included in the Infectoriae section (26%). In addition, three species not previously reported in Algerian wheat, A. eureka, A. consortialis and A. tellustris, were identified, accounting for 5% of the total strains. Mycotoxin analyses showed high contamination of grains with alternariol monomethyl ether, alternariol and tenuazonic acid, occurring in 75, 69 and 35% of the samples, respectively. Moreover, 41 out of 48 samples showed the co-occurrence of multiple Alternaria mycotoxins. This study provides, for the first, time a clear picture of the occurrence and the distribution of Alternaria species on wheat in Algeria. Finally, the extensive monitoring activities carried out revealed the great biodiversity of Alternaria species able to colonize wheat grains. Moreover, findings on mycotoxin contamination raise concerns about the significant mycotoxigenic risk in Algerian wheat, emphasizing the need for strict monitoring and regulatory measures on Alternaria mycotoxins in food and feed.

Introduction and methodsThe distribution of tenuazonic acid (TeA), alternariol (AOH), alternariol monomethyl ether (AME), and tentoxin (TEN) between the pulp and peel was determined in different tomato varieties after artificial inoculation with three Alternaria species (Alternaria alternata, Alternaria tenuissima, and Alternaria solani) and incubation for 3 weeks. The role of heat treatments, similar to pasteurization, in their stability was also investigated.Results and discussionUnlike AME that was never detected, TeA, AOH, and TEN were determined at different levels in the pulp and peel. Specifically, AOH remained mainly in the peel, where the inoculation was carried out, while TeA and TEN migrated into the pulp and were also found in the discarded liquid accumulated during the incubation period. Heat treatments reduced TeA, AOH, and TEN to varying degrees. In particular, the TeA level was slightly reduced after treatment both at 100°C (approximately 10%) and 121°C (approximately 20%), while a reduction of approximately 30% was achieved after the double heat treatment (treatment at 100°C followed by treatment at 121°C). AOH was found to be less stable to heat treatments, showing a reduction of around 50% after treatment at 100°C and up to 80% after double heating treatments. TEN was reduced by approximately 50% only after the combined treatment of 100°C + 121°C.

Sweet pepper (Capsicum annuum) is a highly nutritious and economically important vegetable grown worldwide. Black mold, caused by mycotoxin-producing Alternaria spp., is a common postharvest disease during cold storage and transport, leading to significant produce losses. A better understanding of the infection process is essential for improving disease control. This study examined Alternaria alternata isolates infecting green (mature unripe) and red (ripe) pepper fruit. Findings indicate that black mold can infect fruit at both ripening stages, with differences in symptom progression, growth rates, and sporulation. Disease development was influenced by fruit ripeness in a temperature-dependent manner. At 7°C, lesion size and sporulation were similar on green and red fruit, but at 22°C, lesions were significantly larger on red fruit (P < 0.05). Microscopic studies revealed comparable conidial germination on both fruit stages; however, appressoria formation was less frequent on green fruit early in infection. Fungal penetration into the pericarp occurred 8 hours post-inoculation through cuticle wounds, with hyphae growing intercellularly among pericarp walls. By 24 hours post-inoculation, cell contents were disorganized, and cell walls had dissolved. In red fruit, vascular bundles were destroyed, whereas in green fruit, they remained intact. At 22°C, high levels of the mycotoxins altenuene, alternariol, and alternariol monomethyl ether were detected in both green and red infected fruit. The susceptibility of mature green fruit to black mold highlights the need for effective field treatments to prevent pathogen establishment and reduce postharvest disease.

Monte Carlo simulation-based risk assessment of human exposure to multi-mycotoxins in Platycladi semen.

A high-performance liquid chromatography tandem mass spectrometry method has been developed and validated to quantify alternariol, alternariol monomethyl ether, tenuazonic acid, altenuene, altertoxin I, and tentoxin, and implemented to better understand the risks associated with Alternaria contamination of tomatoes and fate of the toxins during processing. This method has been developed for routine use, by reducing the cost, duration, and complexity of manipulations. Limits of quantification were below EU recommendations 2022/553, reaching 1.2–3.7 µg kg−1 for alternariol and alternariol monomethyl ether and 9.4–18.4 µg kg−1 for tenuazonic acid depending on the matrix. Apparent recovery ranged between 85 and 103%, and intraday repeatability was <15%. Different Alternaria strains isolated from tomatoes were assessed for their toxin production profiles, and the impact of processing operations on Alternaria toxins naturally occurring in tomatoes was evaluated on a pilot scale. Tenuazonic acid was the predominant toxin produced by Alternaria strains and contaminating tomatoes. Processing operations did not reduce toxin accumulation, which demonstrates its thermostability. Additionally, tomato skin and seeds residues, which are reused for different applications, was 2.6 times more contaminated than tomato pulp.

The mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME), produced by Alternaria spp., are common contaminants of food and feed and are a potential threat to animal and human health. To date, the most prominent data gaps for their comprehensive risk assessment concern information on their in vivo absorption, distribution, metabolism and excretion (ADME) and toxicokinetic behavior. The aim of this study was to determine the absolute oral bioavailability, quantitative toxicokinetic characteristics and biotransformation of AOH and AME in vivo in pigs, using crossover trials with intravenous and oral administration of a single dose of both mycotoxins at 2 mg/kg b.w. Plasma profiles of the mycotoxins and phase I and II metabolites were studied in the vena jugularis using UPLC-MS/MS and LC-HRMS methods. Furthermore, plasma from the vena portae was analyzed to study presystemic biotransformation. Urine was collected to determine the urinary excretion and metabolite profiles. Results reveal a low absolute oral bioavailability of AOH (15%) and AME (9%), caused by a low absorption and/or extensive first-pass biotransformation in the liver to mainly phase II, and to a lesser extent phase I metabolites. Quantitative toxicokinetic modeling of the IV data showed a high total body clearance for both AOH and AME (12.9 and 16.8 L/(h*kg b.w.), respectively), a high volume of distribution (4.97 and 5.15 L/kg b.w., respectively) and a short elimination half-life of 0.16 and 0.21 h, respectively. These findings may contribute to the risk assessment of AOH and AME and to the development of candidate biomarkers of exposure in biomonitoring studies since pigs are considered a suitable animal model to extrapolate to humans.

The aim of this study was to investigate the effects of extrusion processing parameters-moisture content (M = 20 and 24%), feeding rate (FR = 20 and 25 kg/h), and screw speed (SS = 300, 390 and 480 RPM), on the content of deoxynivalenol (DON), 15-Acetyl Deoxynivalenol (15-AcDON), 3-Acetyl Deoxynivalenol (3-AcDON), HT-2 Toxin (HT-2), tentoxin (TEN) and alternariol monomethyl ether (AME), using a pilot single-screw extruder in whole-grain triticale flour. The temperature at the end plate of the extruder ranged between 97.6 and 141 °C, the absolute pressure was from 0.10 to 0.42 MPa, the mean retention time of material in the barrel was between 16 and 35 s, and the specific energy consumption was from 91.5 to 186.6 Wh/kg. According to the standard score, the optimum parameters for the reduction of the content of analysed mycotoxins were M = 24 g/100 g, FR = 25 kg/h, SS = 480 RPM, with a reduction of 3.80, 60.7, 61.5, 86.5, 47.7, and 55.9% for DON, 3-AcDON, 15-AcDON, HT-2, TEN, and AME, respectively. Under these conditions, the bulk density, pellet hardness, water absorption index, and water solubility index of the pellet were 0.352 g/mL, 13.7 kg, 8.96 g/g, and 14.9 g/100 g, respectively.