Isolation of mycotoxins producing black aspergilli in herbal teas available on the Swiss market

Abstract

Black aspergilli are among the predominant fungal contaminants in herbal teas. Despite the ability of some species in this group to produce the mycotoxins ochratoxin A (OTA) and fumonisins no study had been done to investigate in detail their presence in this commodity. In the present work conventional herbal teas available on the Swiss market were investigated for black aspergilli contamination. Black aspergilli were found in 16 of 22 samples, ranging from 10 to 3500 colony forming units per gram of herbal tea. Recovered isolates were identified to the species level by calmodulin sequencing. The most frequently isolated species were Aspergillus niger, Aspergillus acidus, Aspergillus awamori and Aspergillus tubingensis. Aspergillus carbonarius, the most important OTA-producing black Aspergillus, could not be recovered. A. niger and A. awamori isolates were tested for their ability to produce fumonisins and OTA in vitro. Fumonisins were produced by 76% of A. niger and 37% of A. awamori isolates. 7% of A. niger and none A. awamori isolates could produce OTA. In total, 12 of 22 samples were found to be contaminated with black aspergilli able to produce major mycotoxins. Our results indicate that mycotoxins producing black aspergilli are widespread fungal contaminants of herbal teas. Therefore, their presence as well as the occurrence of their mycotoxins should be further investigated to assess health risks linked with the consumption of this commodity.

Introduction

Members of the Aspergillus section Nigri, also known as black aspergilli, represent one of the most important source of mycotoxins contamination of foods and feeds. Major mycotoxins produced by this group of filamentous fungi are ochratoxin A (OTA) and fumonisins of the B series, in particular FB₂ (Nielsen, Mogensen, Johansen, Larsen, & Frisvad, 2009). OTA is a potent nephrotoxic and carcinogenic toxin produced by different species belonging to the genus Aspergillus and Penicillium (Bennett & Klich, 2003). Black aspergilli able to produce OTA are Aspergillus carbonarius, Aspergillus sclerotioniger, and a low percentage of Aspergillus niger and Aspergillus awamori strains (Frisvad et al., 2011, Varga et al., 2011). Fumonisins are carcinogenic mycotoxins originally described in the genus Fusarium (Scott, 2011). Fumonisins production in the A. niger species had been reported few years ago following the discovery of a putative fumonisins biosynthesis gene cluster in the A. niger genome (Frisvad et al., 2007, Pel et al., 2007). Recent studies showed that a high percentage (>50%) of A. niger and A. awamori isolated from coffee, grapes and raisins are able to synthetize fumonisins (Logrieco et al., 2009, Noonim et al., 2009, Varga et al., 2010).

Since only few species inside the section Nigri can produce OTA and fumonisins, a correct identification of black aspergilli isolated from commodities is necessary to evaluate the subsequent risk of mycotoxins contamination (Nielsen et al., 2009). The Aspergillus section Nigri comprises 26 species that can be distinguished without ambiguity using calmodulin sequencing (Varga et al., 2011). In fact, species recognition based on morphology alone is very difficult, in particular for those species closely related to A. niger, which are included in the so-called A. niger aggregate (Samson et al., 2007). This group comprises some of the most common species of black aspergilli, including A. niger, A. awamori, Aspergillus tubingensis and Aspergillus acidus (Nielsen et al., 2009, Perrone et al., 2011). Of these morphological indistinguishable species, only A. niger and its sibling A. awamori are known to produce major mycotoxins (Frisvad et al., 2011, Perrone et al., 2011).

Herbal teas, defined as extracts made with plant materials which do not contain Camellia sinensis, have been shown to be prone to mycotoxin contaminations, including fumonisins and OTA (Martins et al., 2001, Santos et al., 2009). Different studies done in the past to assess the microbiological quality of herbal teas reported black aspergilli, and in particular A. niger, as one of the most frequently isolated group of filamentous fungi (Bugno et al., 2006, Halt, 1998, Tournas and Katsoudas, 2008). However, species determination of molds collected in these works was based only on morphological observations. This implies that black aspergilli classified as A. niger could also belong to the other atoxigenic species of the A. niger aggregate. Moreover, since fumonisins production by A. niger became apparent only recently, these studies were mainly focused on the presence of aflatoxin-producing Aspergillus flavus/Aspergillus parasiticus isolates, and black aspergilli strains were not tested for mycotoxins production. A more focused investigation on black aspergilli in herbal teas is therefore needed to better evaluate their potential role in mycotoxins contamination of this commodity.

The aim of this work is the isolation and characterization of black aspergilli from conventional herbal teas available on the Swiss market. Species identity of recovered aspergilli was determined using calmodulin sequencing and potentially toxigenic isolates were tested for mycotoxins production in vitro.