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  • br Technical Note Fungal spoilage

    2023-01-31


    Technical Note Fungal spoilage of dairy products remains a major concern for dairy manufacturers despite the use of preventive and control approaches, including the use of chemical preservatives. Among methods that are increasingly used by industries for microbial contaminant control, biopreservation is gaining increasing attention due to strong societal (Gerez et al., 2013) and legislative (Fuselli et al., 2012; Stratford et al., 2013) demand for preservative-free products. Biopreservation can be defined as using natural or added microbiota or their antimicrobial compounds to preserve a food product (in terms of safety and quality) and possibly extend its shelf life (Stiles, 1996). Lactic Clindamycin HCl bacteria (LAB) are well known for their ability to exhibit antifungal activity (Schillinger and Villareal, 2010; Wulijideligen and Taku, 2011; Cheong et al., 2014) and protective cultures containing LAB, such as FreshQ (consisting of Lactobacillus paracasei and Lactobacillus rhamnosus strains; Chr. Hansen, Hørsholm, Denmark) or the HOLDBAC series (consisting of various lactobacilli and propionibacteria; Danisco/DuPont, Madison, WI), are currently used in dairy products for their antifungal properties (Varsha and Nampoothiri, 2016). Recently, Inglin et al. (2015) developed a high-throughput screening method to detect antimicrobial (including antifungal) activities using an agar-spot assay in 24-well plates. This method enabled screening of 2,000 assays per day. However, despite being a useful primary high-throughput screening method for developing protective cultures, this antifungal screening assay used de Man, Rogosa, and Sharpe (MRS) agar medium, which can strongly affect expression of antifungal properties and antifungal molecule activity. Indeed, as mentioned in several articles (Stiles et al., 2002; Delavenne et al., 2012; Le Lay et al., 2016), MRS contains acetate, which may reinforce LAB antifungal activity and artificially inflate the number of active isolates. However, assays applied to real food matrices (e.g., dairy product models such as yogurt) can rapidly become labor intensive, thereby reducing the number of isolates that can be screened to detect strains with antifungal activity (Lynch et al., 2014; Delavenne et al., 2015). In addition, these methods do not allow large-scale and rapid screening of antifungal cultures or compounds. In the present study, we present a high-throughput screening assay for antifungal activity in a cheese-mimicking matrix distributed in a 24-well plate. The proposed method allowed testing the antifungal activity of a large variety of microbial protective compounds: bacterial fermentation products, bacterial cultures used as adjunct cultures, and purified antifungal preservatives. To prepare the cheese-mimicking matrix, we used a standardized UF milk retentate (3.4×; 244 g/kg of DM, 65.8 g/kg total fat, 116.2 g/kg of total N, pH 6.53) prepared according to Hannon et al. (2006). Briefly, whole raw cow milk was heated to 50°C and skimmed using a cream separator (Westfalia, Handelsweg, the Netherlands). Skim milk was then microfiltered (0.8-µm Sterilox GP membrane, Pall Corp., Port Washington, NY) at 50°C using the pilot equipment GP7 (Brenet, Mamirolle, France) and cream was heat-treated for 2 min at 95°C followed by fat standardization (20 g/kg final concentration). The retentate was ultrafiltered at 0.02 µm at 50°C (T.I.A., Bollene, France) and sterilized sodium chloride (Sogebul, Sainte-Maure de Touraine, France) was added to reach 0.7% (wt/wt). The salted retentate was heat-treated for 2 min at 95°C (Microthermics, Raleigh, NC), distributed in 1-L sterile bottles, and kept at −20°C until use. Before plate preparation, 10 mL/L of a pH indicator (sterile solution of litmus 50 g/L), 106 cfu/L of commercial starter MA016 (Lactococcus lactis ssp. cremoris and L. lactis ssp. lactis, Elimeca, Thoissey, France), and 1.5 mL/L of 5× diluted and filtered (0.22 µm) rennet (Danisco, Dangé Saint-Romain, France) were added to the thawed retentate. After vigorous homogenization for 1 min, the retentate was distributed into 24-well plates (2 mL/well) and incubated for 1 h at 30°C and then for 3 d at 20°C, leading to the formation of a “mini-cheese” (pH 5) in each well. The exudate was then removed from the surface of each mini-cheese and plates were stored at 12°C until use.