Fungal spoilage is one of the causes of consequential losses in the dairy industry. In this context, the use of bioprotective cultures can be an alternative or a complementary approach to be considered. Lactic acid bacteria (LAB) and propionibacteria, as well as some fungal species, can exhibit antifungal activities with large differences in activity between strains. Therefore, it is necessary to develop high-throughput screening methods to test a large number of strains and find the most efficient ones. In the present study, we developed a miniaturized high-throughput screening technique to rapidly detect antifungal activities in a cheese-like model. This model, distributed in a 24-well plate, consisted of 5-fold concentrated whole milk ultrafiltration retentate (final fat concentration of 45%), rennet (0.03%) and inoculated with a mesophilic lactic commercial starter and a pH indicator. Each well of the plate could be considered as a miniature cheese of ~2 g. Potent antifungal isolates were cultured in two dairy media; (i) a 10%-reconstituted low heat skim milk supplemented with 45% anhydrous milk fat (LH) and (ii) a 6-fold concentrated milk ultrafiltration permeate sterilized by 0.22 μm filtration and complemented with 10 g/l yeast extract and a pH indicator (UF). After cultivation, cultures (100 µl) were deposited on the miniature cheese surfaces followed by inoculation in duplicate with 50 spores or cells of 4 different fungal targets (1 fungi/plate), e.g., Mucor racemosus, Galactomyces geotrichum, Penicillium commune and Yarrowia lipolytica, and incubation at 12°C for up to 15 days. We screened 505 bacterial isolates belonging to Lactobacillus, Lactococcus, Pediococcus, Leuconostoc and Propiobacterium genera and 198 fungal isolates belonging to 28 genera. This high-throughput screening for antifungal activity revealed that 52 and 216 bacteria, and, 53 and 89 fungi, inhibited at least one fungal target after cultivation in UF and LH, respectively. Among the 4 tested fungal targets, P. commune was the most frequently inhibited fungus while only few isolates were able to inhibit M. racemosus or Y. lipolytica. This method opens new possibilities to screen microorganisms for antifungal activities. These results also underline the importance of the culture and screening media used on the expression of antifungal activities by bacteria or fungi
