2.2. Microbiological analysis
10 g of cheese (without rind) were placed into a sterile Stomacher
bag with 90 mL of sterile 2% (w/v) tri-sodium citrate solution
and blended for 5 min in a Stomacher (IUL instruments, Germany)
at room temperature. Serial dilutions were made using Ringers
solution and microorganisms were grown in different media
(Merck, Germany) and incubation conditions: Plate Count Agar
(PCA) for Mesophilic aerobic and Psychrotrophic, incubated at 30
and 16 C for 72 and 96e120 h, respectively; Violet-red Bile
Dextrose (VRBD) for Enterobacteriaceae, incubated at 37 C for 24 h;
Man, Rogosa and Sharpe (MRS) for Lactic Acid Bacteria (LAB)
incubated at 35 C for 72 h in an atmosphere with 5% CO2 (Merck
Anaerocult C); Manitol Salt Phenol-red Agar (MSA) for Micrococcaceae,
incubated at 35 C for 72 h.
In order to evaluate Listeria spp. levels, ALOA isolation medium
(Oxoid Ltd, UK) according to EN ISO 11290 (reference method) was
used. A validated analytical method (AFNOR n AES 10/03-09/00)
according to standard EN ISO 16140:2003 was followed. This could
allow a first identification of Listeria monocytogenes which in case of
presumptive detection was tried to be confirmed by means of the
“metabolic fingerprint” from discrete test reactions performed
within 96 well Micro-plate, using Biolog equipment (Biolog Inc.,
California, USA). After inoculation and incubation, the Micro-plate
is placed into the Micro-Station Reader for analysis. 95 different
carbon compounds including sugars, carboxylic acids, amino acids
and peptides for the microorganism identification were used to
provide discriminating biochemical characterizations. The unique
metabolic pattern generated by the organism is recorded and
compared with hundreds of identification profiles in a corresponding
Biolog Database.