Biocontrol and mechanisms of yeasts on ochratoxin A – producing fungi in coffee berries

Abstract

Yeasts can be applied in different biotechnological processes in industry, food production, or in the field as the biocontrol of toxigenic fungi. Fungi produce substances such as ochratoxin A (OTA), which is toxic to humans and animals. Biocontrol can be applied in the post-harvest stage using volatile organic compound (VOC) yeasts, as they do not leave residues in food. This study aimed to evaluate the possible biocontrol mechanisms of 32 isolates of Candida, Meyerozyma, Pichia, Wickerhamomyces, Rhodotorula and Saccharomyces. In vitro tests evaluated were killer toxin production; VOC production and mycelial growth of Aspergillus carbonarius CCDCA 10608 and A. ochraceus CCDCA 10612 in co-cultivation with yeasts; fungistatic or fungicidal effect; β-1,3-glucanase production; production of antagonistic substances obtained from cell-free extracts. In vivo tests on dried red coffee (Coffea arabica) Catuaí fruits were carried out by inoculating S. cerevisiae CCMA strains (0159; 1302) and evaluating the biocontrol against CCDCA isolates (10608; 10612). The presence of OTA in the husk and coffee bean of the initial and final time was evaluated by enzyme -linked immunosorbent assay (ELISA). The formation of a biofilm and the interaction of yeast and fungus, on the surface of coffee fruits, were analyzed by scanning electron microscopy (SEM). The dynamic behavior of the inoculation of S. cerevisiae CCMA1302 and A. carbonarius during the fermentation process was evaluated by the real time PCR technique. Yeasts had more than one biocotrole mechanism. S. cerevisiae CCMA (0159; 1299; 1302) showed production of VOC, killer toxin and β-1,3-glucanase. In the killer test, eleven isolated were killer positive. In relation to the production of VOCs, eight compounds had an inhibitory effect on A. ochraceus CCDCA10612 and seven on A. carbonarius CCDCA10608. There was no spore production by A. carbonarius in co-cultivation with S. cerevisiae CCMA 1306 and mycelial growth was inhibited in the rate to 65%. A. ochraceus showed inhibition of mycelial growth in the presence of S. cerevisiae strains CCMA 1313 (69.83%) and CCMA 1306 (79.88%). S. cerevisiae CCMA 1302 was the best producer of β-1,3-glucanase in 120 hours. In the tests using the cell-free supernatant no antagonistic action was observed and therefore the fungi developed normally. The yeasts evaluated presented fungistatic action. In coffee, OTA was detected in 17 (24.28%) of the analyzed samples at concentrations ranging from 0.04 to 10.11 μg / kg. The formation of biofilms by the yeasts strains (0159; 1302) in the coffee husk was observed by scanning electron microscopy, these were antagonists in the development and production of OTA due to the competition for space and nutrients. There was a decrease in the population of A. carbonarius in the presence of yeast CCMA 1302, at the initial fermentation time (6.2 log spores / g) and at the end (5.7 log spores / g). Thus, positive results were obtained both in in vitro and in vivo tests. Most strains of S. cerevisiae showed the best in vitro test results. S. cerevisiae CCMA 1302 is a candidate for biocontrol agent of ochratoxigenic fungi in coffee fruits.