Panreac

Samples of fresh milk (FM) at arrival to the cheese factory, acidified milk (AM) before renneting, acidified curd (AC) before kneading, and fresh cheese (CH) after salting were collected from two artisanal raw-milk cheese factories (Tulancingo Valley, Mexico) on three working days. FM and AM (250 ml), AC (500 g), and CH (500 g) samples were transported into sterile screw-capped flasks or sterile containers at 4ºC to the laboratory and analyzed within 4 h after sampling. Representative portions (10 ml: FM or AM; 10 g: AC or CH) were homogenized with 90 ml of buffered peptone water (peptone 0.1%, NaCl 0.85%) using a Stomacher blender (Seward). Decimal dilutions were prepared and pour plated using the two-layer method in M17 agar (Oxoid), and in Man, Rogosa, and Sharpe agar (MRS; Oxoid) previously acidified (pH 5.5) with lactic acid (Panreac); the plates were incubated at 30ºC for 48-72 h. Four colonies were randomly selected from FM, AM and AC, and three colonies from CH, reaching a total of 180 isolates (90 per medium). Isolates were recovered in Tryptone Soy Broth (TSB; Bacto) with 0.5% (w/v) of yeast extract (YE; Difco) (TSB-YE) at 37°C for 24 h. Aliquots (1 ml) were centrifuged (12,000 rpm, 3 min) in Eppendorf tubes; the supernatants were discarded and the pellets were suspended in 1 ml of MRS broth with 50% (v/v) of glycerol (Acofarma) and stored at -40 ºC.