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News

Research: Alternaria toxins in tomato products

23/09/2021 - Sophie Colvine

Elisabeth Varga and Doris Marko from the Department of Food Chemistry and Toxicology at the University of Vienna, discuss the issue of mycotoxins in tomato products.

Mycotoxins are secondary metabolites produced by various filamentous fungi (also commonly referred to as moulds). The toxic effects depend on the specific toxin as well as on the dose and both acute toxic (e.g. vomiting, nausea or even death) as well as chronic (e.g. carcinogenic or cytotoxic) effects are reported.

Since more than 30 years the estimation of the Food and Agriculture Organization (FAO) that 25% of the global food crops are contaminated with mycotoxins is highly cited, but the exact source is unknown. Last year a review was published assessing the validity of this statement (Eskola et al. 2020). The authors concluded that the FAO 25% estimate is true for mycotoxin occurrence above the set maximum levels in the European Union and by the Codex Alimentarius standards.

However, also due to the high sensitivity and low detection limits with the methods at hand nowadays, it seems that this figure underestimates the occurrence of mycotoxins above the detectable levels. According to this review approximately 60 to 80% of food crops are contaminated (Eskola et al. 2020). However, this number has to be set into perspective since contamination with these natural toxins cannot be completely avoided and not every occurrence is causing a health threat. In general, consumers should not be concerned about low level contamination since products are considered as safe. More than 100 countries have regulatory limits or guidance levels for mycotoxins in food and feed. In the European Union, Commission Regulation (EC) 1881/2006 and its amendments are setting maximum levels for some mycotoxins in certain food categories with a special focus on cereals and cereal-based foods.

In juices, currently only regulatory limits are set for ochratoxin A in grape juice and other grape products (2 μg/kg) and patulin in fruit juices, concentrated fruit juices and fruit nectars as well as cider and fermented apple drinks (50 μg/kg) and apple juice (10 μg/kg). While most people are familiar with the regulated mycotoxins, the vast majority of the several hundred mycotoxin and mycotoxin metabolites are only known to experts. Some of them have gained attention as ‘emerging’ mycotoxins and the group of Alternaria toxins belong to them.

Alternaria
Alternaria is a versatile and ubiquitous genus of fungi, and more than 250 Alternaria species are known so far. While some species are non-host specific (e.g. Alternaria alternata, Alternaria tenuissima), others are associated with specific hosts only (e.g. Alternaria carotiincultae on carrots or Alternaria solani on potatoes and tomatoes). As the fungi itself, also the produced toxins can be divided into host-specific (e.g. AAL toxins)and non-host specific (e.g. alternariol and its methyl ether, tenuazonic acid) toxins which can be further divided into several groups.

For more information you can read a recent review by Aichinger et al. (2021). Beside the mycotoxins produced by the fungi itself also the so called ‘modified’ Alternaria toxins should be considered. When the fungus infects the plant and produces the toxins, the plant tries to defend itself, modifies the toxins and stores them in the vacuoles. These modifications are in most cases either catalysed by glucosyltransferases resulting in glucosides of the respective analytes or sulphotransferases forming sulphate conjugates. Both metabolite groups have been described for Alternaria toxins before. These biotransformation processes eliminate the toxin from the plant metabolism but might still be of health concern when humans or animals are consuming them.

Toxicity
Since available data on the toxicity and dietary exposure of the emerging mycotoxins such as Alternaria toxins is scarce, the European Food Safety Authority (EFSA) asked the member states to gather more information (EFSA 2011). In their scientific opinion published in 2016 they concluded that the estimated mean chronic dietary exposures in some cases exceeded the toxicological threshold of concern (TCC) value (EFSA 2016). Especially the genotoxic Alternaria metabolites alternariol and its monomethyl ether are of concern in this regard. The highest mean exposure was observed for toddlers due to the unfavourable combination of high exposure and low body weight. The most important contributors for alternariol are fruit and fruit products, for alternariol monomethyl ether vegetable oil and for tenuazonic acid and tentoxin tomatoes and tomato products (EFSA 2016).

An important prerequisite for obtaining exposure data are reliable analytical methods to quantify these toxins in various food matrices. At the Department of Food Chemistry and Toxicology, Faculty of Chemistry, University of Vienna, a method for the determination of 17 Alternaria toxins in three different food matrices (tomato sauce, sunflower seed oil and wheat flour) was developed (Puntscher et al. 2018). Since especially for the modified Alternaria metabolites analytical standards are not commercially available, they were purified in house or synthesized by collaborators. After an homogenisation step, a representative sample (1.0 g) was weight-in and extracted with 5.0 mL methanol-water-acetic acid (79:20:1, v:v:v) for 60 min at room temperature on an overhead shaker. Thereafter, the samples were diluted 1+1 with methanol-water (10:90, v:v) and centrifuged for 15 min at 20 000 g at 4°C. A filtration step should be avoided since some Alternaria toxins get stuck on filter membranes like nylon and polyether sulphone (Aichinger et al. 2020). While this might be an interesting strategy to remove Alternaria toxins from fruit juices, it is not desired for analytical purposes.

The determination of the Alternaria toxins itself was performed by liquid chromatography coupled to tandem mass spectrometry (Puntscher et al. 2018). The fully validated method was among others used to determine Alternaria mycotoxins in 56 tomato sauce products obtained in local shops in Austria (n = 32) and Croatia (n = 8) or were ordered online (n = 12) (Puntscher et al. 2019). From the 17 Alternaria toxins included in the method, nine could be detected and quantified in the investigated tomato products.

Furthermore, in 80% of the samples at least one Alternaria toxin could be detected. This number fits very well to the above-mentioned estimation that 60 to 80% of the food crops are contaminated with mycotoxins. The most prevalent toxin in tomato products was tenuazonic acid with detectable levels in 71% of the samples and in concentrations up to almost 700 μg/kg (median 82 μg/kg). Beside alternariol (prevalence 30%) and its monomethyl ether (55%) also sulphate conjugates were detected in 9% (alternariol-3-sulphate) and 34% (alternariol monomethyl ether-3- sulphate) of the samples respectively. The level of contamination was in the low μg/kg range. The results of this study are very well in agreement with other studies investigating Alternaria metabolites in tomato products.

Conclusion
To obtain occurrence data of Alternaria toxins is of tremendous importance for a reliable risk assessment and especially tomatoes and tomato products should be in focus. We could contribute to the first step the provision of analytical methods for the determination of these mycotoxin class in different food matrices.

The next steps are the systematic assessment of the contamination levels and to obtain more toxicological data in vitro and in vivo. These are the prerequisites to propose monitoring guidelines for certain agricultural crops or to implement maximum contamination levels of Alternaria toxins in certain commodities.

Elizabeth Varga has presented this topic at the IFU Technical Webinars 2021. The webinar can be seen on-demand here.

Artice reprinted with permission from Fruit Juice Focus, September/October 2021 issue. The original article can be seen here

Source: Fruit Juice Focus

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