Mrs agar medium

At each sampling time, physic-chemical analysis were carried out following the methodology described by Garrido-Fernandez et al. (1997) . The NaCl content (%), pH, and titratable acidity (expressed as g of lactic acid per 100 mL of brine) were determined in brine using an automatic titrator model Excellence (Mettler Toledo, Columbus, OH, United States). Samples of brines and fruits were also collected to determine LAB counts using specific MRS agar medium (Oxoid, Basingstoke, Hampshire, United Kingdom) according to methodology described by Benítez-Cabello et al. (2015) (link). Briefly, fruits (10 g) were washed twice with a 0.9% sterile NaCl solution for removing non- and low-adherent cells, pitted, weighed in sterile conditions, and transferred into a stomacher bag containing 25 mL of sterile saline solution. Then, the fruits were homogenized for 3 min at 300 rpm in a stomacher model Seward 400 (Seward, United Kingdom). Decimal dilutions of brines or the stomacher bag liquid were spread onto culture media using a spiral platemaker model easySpiral Dilute (Interscience, Saint Nom la Brétèche, France). The counts were determined using an automatic image analysis system model Scan4000 (Interscience, SaintNom la Brétèche, France) and expressed as log₁₀ CFU/mL (brine) or log₁₀ CFU/g (biofilm), respectively.

The social stomachs, the midguts and the beebread, in sterile saline solution were homogenized and serial decimal dilutions were obtained. Briefly, LAB were enumerated and isolated by plating serial decimal dilutions on MRS agar medium (Oxoid, Milan, Italy) adding 40 mg/L cycloheximide, on modified MRS containing 10% fructose. Plates were incubated for 48–72 h at 30 °C under anaerobic conditions using an anaerobic system (Anaerogen, Oxoid, Milan, Italy).
Representative numbers (10%) of colonies randomly picked from each assayed medium were purified by streaking on the suitable agar media. The colonies were randomly selected according to morphological differences (colony size and shape).
The pure isolates were tested for their Gram reaction, catalase activity and morphology. Gram-positive and catalase-negative were selected as presumptive LAB and were stored at − 80 °C in the corresponding liquid isolation medium, supplemented with 25% (v/v) of glycerol.

Presumptive LAB were enumerated using MRS agar medium (Oxoid, Basingstoke, Hampshire, United Kingdom) supplemented with cycloheximide (0.1 g/L). Plates were incubated at 30 °C for 48 h, under anaerobiosis (AnaeroGen and AnaeroJar, Oxoid). Cell densities of yeasts and molds were estimated on yeast peptone dextrose agar medium (YPDA, Sigma-Merck, Darmstadt, Germany) supplemented with chloramphenicol (0.1 g/L), through pour and spread plate enumeration, respectively, and incubated at 30 °C for 72 h. The attribution (yeast/mold) was performed by visual analysis of colony morphology. Total Enterobacteriaceae were determined on violet red bile glucose agar (VRBGA, Oxoid) at 37 °C for 24 h.

Samples of MBC were received on the day of production from four dairy factories located in different provinces of central and southern Italy (Latina-LT; Salerno-SA; Caserta-CE; Foggia-FG). We exclusively selected dairy plants with associated animal farming, which guarantees reproducible sources of milk and associated microbiota profiles for cheese production. Samples were stored at 4°C and processed within 12 h. Pooled or single samples of MBC were homogenized with a BagMixer400 (Interscience, France) in sodium citrate solution (2% w/v) at a concentration of 0.5 g/mL. In order to test the titer of mesophilic cultivable LAB, serial dilutions were made in Quarter Strength Ringer's solution and plated on MRS agar medium (Oxoid Ltd, Basingstoke, Hampshire, England), as previously reported [13 (link)]. Plates were incubated at 30°C for 48 h, under aerobic and anaerobic conditions (Anaerocult A, Merck, Germany).

A commercial pasteurized non-filtered elderberry juice was added with viable cells and CFE of L. casei. Viable cells of L. casei 2057, 2306, 2240, 2246, prepared as described previously, were inoculated into elderberry juice in order to reach 7 Log CFU/mL. The juice was fermented at 37°C for 48 h. Microbial counts before and after fermentation were performed in triplicate using MRS Agar medium (Oxoid, Italy) and incubating at 37°C for 48–72 h, under anaerobiosis. Elderberry juice was also added with CFE from the same strains, incubated at 37°C for 48 h and after this period the absence of cultivable cells was evaluated using MRS plate count agar. The eight experiments were carried out in triplicate and elderberry juice not added of L. casei was incubated at 37°C for 48 h and used as control. All the samples were maintained at −80°C until the analysis.