Identification of Green Coffee Contaminated Microfungi of the Genus <i>Aspergillus</i> on the Basis of Polyphasic Approach

Abstract

The Aspergillus species are widespread in the environment, able to grow at high temperatures and minimal humidity, including in regions with a hot tropical climate. Some species have the potential to produce toxins. This causes the risk of contamination by fungi of the genus Aspergillus and the mycotoxins (MT) produced by them of plant materials and food products, which is possible at any stage of production, transportation and storage. In the volume of coffee imported to the Russian Federation, 85% is accounted for by raw materials (green coffee), for which the risks of mold damage remain at all stages preceding the roasting stage. It is relevant to study the species composition and toxinogenic properties of  Aspergillus spp., which contaminate food raw materials for the production of mass consumption foodstuffs. Contaminated products include coffee, which is one of the basic products of the consumer basket. Reliable data on species identification and toxigenic potential of Aspergillus spp. can be obtained only with an integrated approach based on polyphasic taxonomy. The purpose of this work is to study the species composition of fungi of the genus Aspergillus isolated from green coffee using an integrated approach based on polyphasic taxonomy. The species composition of fungi of the genus Aspergillus from the internal mycoflora of 16 samples of green coffee beans of  Arabica and Robusta. The species belonging of the isolated 34 singlspore isolates of Aspergillus spp. was determined by cultural and morphological methods and confirmed by molecular genetic analysis, i.e., RT-PCR with DNA markers of conservative sequences (ITS, CaM, β-tub), studied in vitro profile of produced secondary toxic metabolites. The dominance of species of the Niger section was established (A. niger, 90%, and  A. tubingensis, A. carbonarius); then, in decreasing order, the species of section Flavi followed (A. flavus, 100%), sections Circumdati (A. ochraceus, 40% and A. westerdijkiae, 60%). In section Fumigati there was one strain of A. fumigatus. Analysis of the profile of toxic metabolites by HPLC-MS/MS in the multi-detection mode showed the production of mycotoxins by the following species: A. niger – fumonisin B2 and ochratoxin A, A. flavus – aflatoxins B1 and B2 together with sterigmatocystin, A. westerdijkiae – ochratoxin A and penicillic acid, A. ochraceus – penicillic acid. Amounts of produced MT show a high toxinogenic potential of Aspergillus spp. Thus, 20 out of 34 strains produced significant amounts of dangerous, regulated mycotoxins: AFL B1, OTA, FB2. Non-toxinogenic isolates were represented by the species A. niger, A. carbonarius, A. tubingensis, A. flavus, and A. fumigatus. A study of the species composition and toxinogenic properties of green coffee contaminants of the genus Aspergillus using a polyphasic approach was carried out in Russia for the first time.