Dairy associations for the targeted control of opportunistic Candida.

Antifungal and antibacterial activities of twenty-six combinations of lactic acid bacteria, propionibacteria, acetic acid bacteria and dairy yeasts inoculated in whey and milk were investigated. Associations including acetic acid bacteria were shown to suppress growth of the opportunistic yeast Candida albicans in well-diffusion assays. The protective effect of milk fermented with the two most promising consortia was confirmed in Caco-2 cell culture infected with C. albicans. Indeed, these fermented milks, after heat-treatment or not, suppressed lactate dehydrogenase release after 48 h while significant increase in LDH release was observed in the positive control (C. albicans alone) and with fermented milk obtained using commercial yogurt starter cultures. The analysis of volatile compounds in the cell-free supernatant using solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) showed accumulation of significant amount of acetic acid by the consortium composed of Lactobacillus delbrueckii 5, Lactobacillus gallinarum 1, Lentilactobacillus parabuchneri 3, Lacticaseibacillus paracasei 33-4, Acetobacter syzygii 2 and Kluyveromyces marxianus 19, which corresponded to the zone of partial inhibition of C. albicans growth during well-diffusion assays. Interestingly, another part of anti-Candida activity, yielding small and transparent inhibition zones, was linked with the consortium cell fraction. This study showed a correlation between anti-Candida activity and the presence of acetic acid bacteria in dairy associations as well as a significant effect of two dairy associations against C. albicans in a Caco-2 cell model. These two associations may be promising consortia for developing functional dairy products with antagonistic action against candidiasis agents.

Antifungal activity of fermented dairy ingredients: Identification of antifungal compounds.

Fungi are commonly identified as the cause for dairy food spoilage. This can lead to substantial economic losses for the dairy industry as well as consumer dissatisfaction. In this context, biopreservation of fermented dairy products using lactic acid bacteria, propionibacteria and fungi capable of producing a large range of antifungal metabolites is of major interest. In a previous study, extensive screening was performed in vitro and in situ to select 3 dairy fermentates (derived from Acidipropionibacterium jensenii CIRM-BIA1774, Lactobacillus rhamnosus CIRM-BIA1952 and Mucor lanceolatus UBOCC-A-109193, respectively) with antifungal activity. The aim of the present study was to determine the main compounds responsible for this antifungal activity. Fifty-six known antifungal compounds as well as volatiles were targeted using different analytical methods (conventional LC and GC, GC-MS, LC-QToF). The most abundant antifungal compounds in P. jensenii-, L. rhamnosus- and M. lanceolatus-derived fermentates corresponded to propionic and acetic acids, lactic and acetic acids, and butyric acid, respectively. Many other antifungal compounds (organic acids, free fatty acids, volatile compounds) were identified but at lower levels. In addition, an untargeted approach using nano LC-MS/MS identified a 9-amino acid peptide derived from αs2-casein in the L. rhamnosus-derived fermentate. This peptide inhibited M. racemosus and R. mucilaginosa in vitro. This study provides new insights on the molecules involved in antifungal activities of food-grade microorganism fermentates which could be used as antifungal ingredients in the dairy industry.

Identification and quantification of natural compounds produced by antifungal bioprotective cultures in dairy products.

In the context of growing consumer demand for clean label foods, antifungal cultures offer alternatives to chemical preservatives for the reduction of food fungal spoilage. Selected binary combinations of lactobacilli strains were recently successfully used to inhibit Penicillium commune and Mucor racemosus in four dairy products. Our aim was to identify the compounds most likely involved in their antifungal activity. Four chromatographic methods, targeting 56 antifungal compounds as well as volatiles, were combined. Overall, 53 antifungal compounds were detected, of which 33 were in significantly higher amounts in at least one product inoculated with an antifungal culture compared to the controls. They were present at concentrations below their MIC and thus could act in synergy. Among them, the most commonly identified were acetic, hydroxyphenyllactic, phenyllactic, 3-phenylpropanoic, 3-(4-hydroxyphenyl)propanoic and 5-oxopyrrolidine-2-carboxylic acids, diacetyl, acetoin, and an unidentified volatile. This extensive study contributes to improve the knowledge about the action mode of antifungal lactobacilli.

Selection of Algerian lactic acid bacteria for use as antifungal bioprotective cultures and application in dairy and bakery products.

In the context of a demand for "preservative-free" food products, biopreservation appears as a promising alternative to either replace or reduce the use of chemical preservatives. The purpose of this study was to evaluate the antifungal activity of a collection of lactic acid bacteria (n = 194), and then to evaluate the applicability and efficacy of selected ones used as bioprotective cultures against mold spoilers in dairy and bakery products. First, lactic acid bacteria were isolated from various Algerian raw milk samples and Amoredj, a traditional fermented product. Secondly, in vitro screening tests against Mucor racemosus UBOCC-A-109155, Penicillium commune UBOCC-A-116003, Yarrowia lipolytica UBOCC-A-216006, Aspergillus tubingensis AN, Aspergillus flavus T5 and Paecilomyces formosus AT allowed for the selection of 3 active strains, namely Lactobacillus plantarum CH1, Lactobacillus paracasei B20 and Leuconostoc mesenteroides L1. In situ tests were then performed to validate their activity in actual products (sour cream and sourdough bread) challenged with fungal spoilers. These tests showed that antifungal LAB could slow the fungal target growth and could be candidates of interest for industrial applications. Finally, organic acids and various antifungal compounds produced in sour cream and sourdough bread by the selected LAB, and thus potentially supporting the observed antifungal activity, were identified and quantified by HPLC and LC-QTOF.

Antifungal Activity of Lactic Acid Bacteria Combinations in Dairy Mimicking Models and Their Potential as Bioprotective Cultures in Pilot Scale Applications.

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.

Antifungal Microbial Agents for Food Biopreservation-A Review.

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.