Food spoilage is a major issue for the food industry, leading to food waste, substantial economic losses for manufacturers and consumers, and a negative impact on brand names. Among causes, fungal contamination can be encountered at various stages of the food chain (e.g., post-harvest, during processing or storage). Fungal development leads to food sensory defects varying from visual deterioration to noticeable odor, flavor, or texture changes but can also have negative health impacts via mycotoxin production by some molds. In order to avoid microbial spoilage and thus extend product shelf life, different treatments—including fungicides and chemical preservatives—are used. In parallel, public authorities encourage the food industry to limit the use of these chemical compounds and develop natural methods for food preservation. This is accompanied by a strong societal demand for ‘clean label’ food products, as consumers are looking for more natural, less severely processed and safer products. In this context, microbial agents corresponding to bioprotective cultures, fermentates, culture-free supernatant or purified molecules, exhibiting antifungal activities represent a growing interest as an alternative to chemical preservation. This review presents the main fungal spoilers encountered in food products, the antifungal microorganisms tested for food bioprotection, and their mechanisms of action. A focus is made in particular on the recent in situ studies and the constraints associated with the use of antifungal microbial agents for food biopreservation.

Consumer's demand for naturally preserved food products is growing and the use of bioprotective cultures is an alternative to chemical preservatives or a complementary tool to hurdle technologies to avoid or delay fungal spoilage of dairy products. To develop antifungal cultures for the dairy product biopreservation, experiments were conducted both in vitro and in situ. Firstly, the antifungal activity of 32 strains of lactic acid bacteria (LAB) and propionibacteria was screened alone, and then on combinations based on 5 selected lactobacilli strains. This screening was performed in yogurt and cheese models against four major spoilage fungi previously isolated from contaminated dairy products (Penicillium commune, Mucor racemosus, Galactomyces geotrichum, and Yarrowia lipolytica). Selected combinations were then tested as adjunct cultures in sour cream and semi-hard cheeses produced at a pilot scale to evaluate their antifungal activity during challenge tests against selected fungal targets (P. commune, M. racemosus, and Rhodotorula mucilaginosa) and shelf life tests; and their impact on product organoleptic properties. The screening step allowed selecting two binary combinations, A1 and A3 composed of Lactobacillus plantarum L244 and either Lactobacillus harbinensis L172 or Lactobacillus rhamnosus CIRM-BIA1113, respectively. In situ assays showed that the A1 combination delayed the growth of P. commune, M. racemosus and R. mucilaginosa for 2–24 days on sour cream depending of the antifungal culture inoculum, without effect on organoleptic properties at low inoculum (10⁶ colony-forming units (CFU)/mL). Moreover, the A1 and A3 combinations also delayed the growth of P. commune in semi-hard cheese for 1–6 days and 1 day, respectively. Antifungal cultures neither impacted the growth of starter cultures in both sour cream and cheese nor the products' pH, although post acidification was observed in sour cream supplemented with these combinations at the highest concentrations (2.10⁷ CFU/mL). The combination of both in vitro and in situ screening assays allowed developing 2 antifungal combinations exhibiting significant antifungal activity and providing future prospects for use as bioprotective cultures in dairy products.

In this study, we developed a high-throughput antifungal activity screening method using a cheese-mimicking matrix distributed in 24-well plates. This method allowed rapid screening of a large variety of antifungal agent candidates: bacterial fermented ingredients, bacterial isolates, and preservatives. Using the proposed method, we characterized the antifungal activity of 44 lactic acid bacteria (LAB) fermented milk-based ingredients and 23 LAB isolates used as protective cultures against 4 fungal targets (Mucor racemosus, Penicillium commune, Galactomyces geotrichum, and Yarrowia lipolytica). We also used this method to determine the minimum inhibitory concentration of a preservative, natamycin, against 9 fungal targets. The results underlined the strain-dependency of LAB antifungal activity, the strong effect of fermentation substrate on this activity, and the effect of the screening medium on natamycin minimum inhibitory concentration. Our method could achieved a screening rate of 1,600 assays per week and can be implemented to evaluate antifungal activity of microorganisms, fermentation products, or purified compounds compatible with dairy technology.