Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation

Contamination with filamentous fungi during cocoa bean fermentation and drying reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop anti-fungal lactic acid bacteria (LAB)-yeast co-cultures by selecting anti-fungal strains best adapted to the cocoa bean fermentation process from 362 LAB and 384 yeast strains isolated from cocoa bean post-harvest processes. The applied multiphasic screening approach included anti-fungal activity tests in vitro and in vivo and assessment of the carbon metabolism and stress tolerance of the anti-fungal strains in cocoa pulp simulation medium. The anti-fungal strains, Lactobacillus fermentum M017, Lactobacillus fermentum 223, Hanseniaspora opuntiae H17, and Saccharomyces cerevisiae H290, were selected based on their high inhibition capacity and their well-adapted metabolism. Up to seven filamentous fungal strains of the genera Aspergillus, Penicillium, and Gibberella were inhibited by 63 and 75% of the maximal inhibition zone by M017 and 223, respectively, and by 25 and 31% by the strains H17 and H290, respectively. Both Lb. fermentum strains converted the medium's glucose, fructose, and citric acid into 20.4–23.0 g/l of mannitol, 3.9–6.2 g/l acetic acid, and 8.6–10.3 g/l lactic acid, whereas the two yeast strains metabolized glucose and fructose to produce 7.4–18.4 g/l of ethanol. The Lb. fermentum strains were further characterized as ethanol, acetic acid, and temperature tolerant and both yeast strains as ethanol and lactic acid tolerant. Finally, the anti-fungal in vivo assays revealed that the two Lb. fermentum strains completely inhibited growth of the citrinin-producing strain, P. citrinum S005, and the potentially fumonisin-producing strain, G. moniliformis S003, on the surface of cocoa beans. All four selected anti-fungal strains, i.e. Lb. fermentum M017, Lb. fermentum 223, H. opuntiae H17, and Sacc. cerevisiae H290, inhibited at 51–95% growth of aflatoxin-producer A. flavus S075 as single cultures while preventing its growth by 100% when combined into four co-cultures, each composed of a Lb. fermentum strain and one of the two yeast strains. As a conclusion, these four LAB-yeast co-cultures are recommended for future applications to limit the growth of filamentous fungi and the concomitant mycotoxin production during the fermentation of cocoa beans