M17 agar

Viable counts of LAB, Enterobacteriaceae, coliforms, and fungi were determined in triplicate on the selective media for each species at storage days 1, 7, and 14 using the pour plate technique, as described by Mileriene et al. [37 (link)]. The whey cheese samples were mixed (1:10, w/v) with buffered peptone water (Liofilchem, Teramo, Italy) and submitted to 10 decimal serial dilutions. The LAB counts were enumerated on M17 agar (Oxoid, Basingstoke, Hampshire, UK) and incubated at 30 °C for 72 h [38 ]. The yeasts and molds were enumerated on potato dextrose agar (PDA) (Oxoid, UK), which was acidified with sterile lactic acid (pH 4.5) (Lab M Ltd., Heywood, UK) to inhibit bacteria growth after incubation at 25 °C for 120 h, followed by microscopic confirmation tests for each type of colony encountered [39 ]. The coliforms were enumerated on violet red bile agar (Liofilchem, Teramo, Italy) at 30 °C for 24 h [40 ]. The Enterobacteria were enumerated on violet red bile glucose agar (Liofilchem, Teramo, Italy) at 37 °C for 24 h [41 ].

A portion of 5.0 g grated cheese was aseptically weighed into a sterile stomacher bag and homogenized in 45 mL of 0.85% (w/v) sodium chloride solution (27810.295P; VWR Chemicals, Leuven, Belgium) for 1 min using a laboratory blender (MiniMix 100 P CC; Interscience, Saint-Nom-la-Bretèche, France), as described by Socaciu et al. [21 (link)]. Seven ten-fold serial dilutions (10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, and 10⁻⁸) were prepared from this stock solution (10⁻¹).
Lactobacilli and streptococci counts were determined in cheese according to the method described by the ISO 7889:2003|IDF 117:2003 standard [22 ] using MRS broth (110611; Merck KGaA, Darmstadt, Germany) and M17 agar (CM0785; Oxoid Ltd., Basingstoke, England), respectively. Enumeration of lactobacilli and streptococci colonies was performed after incubating inoculated plates at 37 °C for 48 h under anaerobic conditions. All samples were analyzed in triplicate, and results were reported as log cfu/g. In addition, the total lactic acid bacteria count (log cfu/g) was calculated as the sum of the lactobacilli and streptococci counts.

Microbiological analyses were performed according to Chaves et al. [23 (link)]. From samples of dried cocoa beans, the beans (from here they are beans without shell) and the shells were obtained by manual separation. Twenty grams of cocoa nibs and separate shells were homogenized in a Stomacher Lab-blender (Thomas Scientific, Swedesboro, NJ, USA) in 90 mL phosphate buffer solution (PBS, Biolife, Milan, Italy) sterile solution, pH 7.4. Decimal dilutions of the suspension were prepared in PBS, plated and incubated as follows: Enterobacteriaceae were counted and isolated in Violet Red Bile Glucose Agar (Oxoid, Basingstoke, UK) at 37 °C in anaerobiosis for 48 h; mesophilic aerobic bacteria in Plate Count Agar (PCA) at 30 °C for 48 h; thermophilic aerobic bacteria in PCA and incubated at 45 °C for 48 h; lactobacilli in De Man Rogose and Sharp (MRS) Broth (Oxoid, Basingstoke, UK) at 37 °C in anaerobiosis for 72 h; lactic streptococci in M17 agar (Oxoid, Basingstoke, UK) at 37 °C in anaerobiosis for 72 h; yeasts in Yeast Extract-Peptone-Dextrose (YPD) agar medium and Walerstein Laboratory (WL) medium agar (Biolife, Milan, Italy) at 25 °C for 48 h; moulds in DG18 Agar (Oxoid, Basingstoke, UK) and Czapec-Agar (Biolife, Milan, Italy) added with 150 ppm chloramphenicol (Sigma-Aldrich Italy, Milan, IT) for 5 days.

Samples of cheese were analyzed for protein [29 ], fat [30 ], moisture (oven drying at 102°C) [31 ] and salt [32 ]. The pH was measured by Foodtrode electrode (Hamilton, Bonaduz, Switzerland).
Microbiological analyses were carried out as described previously [33 ]. Twenty grams of sample were homogenized with 180 ml of sterile sodium citrate (2%, [wt/vol]) solution. Presumptive mesophilic lactobacilli and lactococci were enumerated onto MRS and M17 agar (Oxoid, Basingstoke, Hampshire, UK), respectively, under anaerobiosis at 30°C for 48 h. Presumptive thermophilic streptococci were enumerated onto M17 agar (Oxoid), under anaerobiosis at 42°C for 48 h. Enterococci were counted onto Slanetz & Barteley Agar (Oxoid) at 37°C for 48 h. Except for enterococci, the media for plating bacteria were supplemented with cycloheximide at 0.1 g/l.
The pH 4.6-insoluble and -soluble nitrogen fractions of the cheeses were analyzed by urea polyacrylamide gel electrophoresis (Urea-PAGE) and reverse phase high pressure liquid chromatography (RP-HPLC), as described by Andrews et al. [34 (link)], and Gobbetti et al. [35 ], respectively. Total and individual free amino acids (FAA) from the pH 4.6-soluble fraction were determined by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, UK), as described by Di Cagno et al. [36 (link)].

Twenty g of cheese was added to 180 ml of sterile sodium citrate solution (20 g/L) and homogenized for 3 min by a stomacher (BagMixer 400P, Interscience, Saint-Nom-la-Bretèche, France). Serial dilutions of the homogenized samples were done in a physiological solution (0.9% NaCl), and aliquots of each dilution were spread onto the surface of different selective agar media. The enumeration of Streptococcus thermophilus was done on M17 agar (Oxoid, Basingstoke, Hampshire, UK) (42 °C, 48 h), while Lactococci were counted on M17 agar after 48 h at 30 °C. Moreover, the presence of the biocontrol agent was assured by checking the bacterial colony morphology and through molecular identification of the colonies by sequencing the 16S rRNA region according De Angelis et al. [40 (link)]. Total coliforms and yeasts were detected on Violet Red Bile agar (VRBA, Oxoid, Basingstoke, Hampshire, UK) (37 °C, 24 h) and Yeast extract Peptone Dextrose agar (YPD, Oxoid, Basingstoke, Hampshire, UK) (25 °C, 48 h), respectively. The presence of pathogenic species such as Listeria monocytogenes, Salmonella enteritidis, and Escherichia coli was monitored during the refrigerated storage of cheeses according to the ISO methods 11290, 6579, and 16649, respectively. pH was analyzed in cheese samples diluted in distilled water at a ratio 1:1 by a pH-meter (PH BASIC 20, Crison, Hach Lange, Italy).