Tea contamination by mycotoxins and azole-resistant mycobiota - The need of a One Health approach to tackle exposures.

Despite tea beneficial health effects, there is a substantial risk of tea contamination by harmful pathogens and mycotoxins. A total of 40 tea samples (17 green (raw) tea; 13 black (fermented) tea; 10 herbal infusions or white tea) were purchased from different markets located in Lisbon district during 2020. All products were directly available to consumers either in bulk (13) and or in individual packages (27). Bacterial analysis was performed by inoculating 150 µL of samples extracts in tryptic soy agar (TSA) supplemented with 0.2 % nystatin medium for mesophilic bacteria, and in Violet Red bile agar (VRBA) medium for coliforms (Gram-negative bacteria). Fungal research was performed by spreading 150 µL of samples in malt extract agar (MEA) supplemented with 0.05 % chloramphenicol and in dichloran-glycerol agar (DG18) media. The molecular detection of the Aspergillus sections Fumigati, Nidulantes, Circumdati and Flavi was carried out by Real Time PCR (qPCR). Detection of mycotoxins was performed using high performance liquid chromatograph (HPLC) with a mass spectrometry detector. Azole resistance screening was achieved following the EUCAST guidelines. The highest counts of total bacteria (TSA) were obtained in green raw tea (81.6 %), while for coliform counts (VRBA) were found in samples from black raw tea (96.2 %). The highest fungal counts were obtained in green raw tea (87.7 % MEA; 69.6 % DG18). Aspergillus sp. was the most prevalent genus in all samples on MEA (54.3 %) and on DG18 (56.2 %). In the raw tea 23 of the samples (57.5 %) presented contamination by one to five mycotoxins in the same sample. One Aspergillus section Fumigati isolate from green tea beverage recovered form itraconazole-Sabouraud dextrose agar (SDA) medium, presented itraconazole and posaconazole E-test MICs above MIC90 values. Our findings open further discussion regarding the One-Health approach and the necessary investment in researching biological hazards and azole-resistance associated with the production and consumption of tea (in particular green tea).

Dietary Supplements Based on Red Yeast Rice-A Source of Citrinin?

Citrinin (CIT) is secondary metabolite of filamentous molds. This mycotoxin has nephrotoxic, hepatotoxic, embryocidal, and fetotoxic properties. It is also produced by several species of the three genera Penicillium spp., Aspergillus spp., and Monascus spp., which are used to make red yeast rice (RYR). The material for this study consisted of 15 dietary supplements containing an extract of fermented red rice, available on the Polish market. Samples were extracted using a MeOH-H2O mixture, cleaned-up with an immunoaffinity CitriTest HPLC column, and quantified by HPLC-FLD. None of the analyzed samples contained CIT above the established limit of detection (LOD). Studies on the presence of toxic metabolites in red yeast rice show the importance of regulating this product and of clear information on the label regarding the standardized amounts of monacolin.

Drinking Green Tea: Despite the Risks Due to Mycotoxins, Is It Possible to Increase the Associated Health Benefits?

Tea has been consumed for thousands of years. Despite the different varieties, particular emphasis has been placed on green tea (GT), considering the associated health benefits following its regular consumption, some of which are due to its polyphenol constituents, such as epigallocatechin-3-gallate (EGCG). Tea is not prone to the growth of microorganisms, except fungus, when proper storage, handling, and packing conditions are compromised. Consequently, mycotoxins, secondary metabolites of fungi, could contaminate tea samples, affecting human health. In the present study, we aimed to assess the balance between risks (due to mycotoxins and high levels of EGCG) and benefits (due to moderate intake of EGCG) associated with the consumption of GT. For this, 20 GT samples (10 in bulk and 10 in bags) available in different markets in Lisbon were analyzed through a LC-MS/MS method, evaluating 38 different mycotoxins. Six samples revealed detectable values of the considered toxins. Current levels of mycotoxins and EGCG intake were not associated with health concerns. Scenarios considering an increasing consumption of GT in Portugal showed that drinking up to seven cups of GT per day should maximize the associated health benefits. The present study contributes to the future establishment of GT consumption recommendations in Portugal.

Ochratoxin A and citrinin in green coffee and dietary supplements with green coffee extract.

The aim of this study was to determine the degree of mold contamination and mycotoxin levels in commercially available green coffee products and dietary supplements with green coffee extract. The study included 34 samples from green coffee products: raw beans (n = 16), ground coffee (n = 15) and instant coffee (n = 3), as well as 22 samples from dietary supplements in form of capsules (n = 19), tablets (n = 2) and sachets (n = 1). Total mold count was determined with spread-plate method. Anamorphic mold were identified based on their microscopic morphology and the type of sporulation. Concentrations of mycotoxins, ochratoxin A and citrinin, were quantified by means of HPLC-fluorescence detection. Molds, typically Aspergillus spp. and Penicillium spp., were found in 94% of green coffee beans, 100% of ground and instant coffee samples, and 55% of dietary supplement samples. None of the samples contained detectable levels of citrinin. Ochratoxin A (0.4 ng/g) was detected in only one sample of raw green coffee beans, but in up to 40% and 67% of ground and instant coffee samples, respectively. Mean concentrations of ochratoxin A in ground and instant coffee samples were 3.28 ng/g and 4.09 ng/g, respectively, and maximum concentrations amounted to 6.65 ng/g and 7.44 ng/g, respectively. Ochratoxin A (mean concentration 9.60 ng/g, maximum level 31.4 ng/g) was also detected in up to 58% of the supplement capsules, but in none of tablets and sachets.

Advantageous Extraction, Cleanup, and UHPLC-MS/MS Detection of Patulin Mycotoxin in Dietary Supplements and Herbal Blends Containing Hawberry from Crataegus spp.

Patulin (PAT) is a highly genotoxic mycotoxin still found as the common contaminant of various kinds of spoiled fruits and related commodities which are often endorsed as the health-enhancing products. Thus, a fast and convenient liquid-solid extraction followed by a solid-phase cleanup with the MycoSep®228 AflaPat multifunctional column was used for the highly efficient isolation of PAT with an average recovery of 112.7% from commercial dietary supplements and herbal blends formulated with dried hawberry. Analysis of the PAT content was carried out using gradient elution with a Synergi Polar C18 column (150 × 2 mm, 4 µm) and UHPLC system equipped with a mass spectrometer. PAT was detected in all (n=14) commercial single-component dietary supplements formulated with dried hawberry belonging to Crataegus monogyna and/or Crataegus laevigata. Similarly, PAT was detected in 67% of the studied multicomponent commercial herbal blends (n=6) that contained-in addition to hawberry-different amounts of apple, chokeberry, elderberry, hibiscus, or mallow. Moreover, the PAT content was determined in the hawberry collected from the mature wild hawthorn trees belonging to three botanical species, Crataegus monogyna Jacq., Crataegus laevigata (Poiret) DC, and Crataegus rhipidophylla Gand, growing in the recreational forest areas and in the law-protected state national forest park in Poland. In conclusion, to prevent PAT accumulation and reduce the health risk of consumers in globalizing markets, the implementation of improved cultivation/processing practices of hawthorn trees and hawberry as well as increased analytical control related to the presence of PAT in dietary supplements and herbal blends formulated with fresh, dried, or frozen hawberry should be urgently recommended.