Study of microbial diversity in raw milk and fresh curd used for Fontina cheese production by culture-independent methods

Abstract

The bacterial populations of raw milk employed for the production of Fontina cheese in alpine farms located in different valleys and altitudes (from 700 to 2246 m above sea level) were investigated by culture independent techniques. Total microbial DNA was isolated from milk and curd samples and used as template in Polymerase Chain Reaction (PCR) to study the hypervariable V3 region of the bacterial 16S rRNA gene and analyzed by Denaturing Gradient Gel Electrophoresis (DGGE). Representative bands of DGGE patterns were sequenced for identification purposes. The use of universal primer for PCR-DGGE allowed the description of the bacterial community, not only for the presence of lactic acid bacteria, but also for other adventitious species. DGGE profiles obtained from milk and fresh curd samples were generally different and typical for each farm, although some recurrent bands were observed. Cluster analysis of DGGE profiles did not show high similarity among samples and it was probably dependent on the different geographical areas of pastures. Some Lactic Acid Bacteria (LAB) recurred in many samples (Streptococcus thermophilus, Enterococcus faecium, Enterococcus faecalis, Lactococcus lactis, Leuconostoc lactis) indicating that alpine milk is a preferential niche for their colonization. The microbiota included not only mesophilic and thermoresistant LAB but also adventitious bacteria (Macrococcus caseolyticus, Rothia spp.) and psychrotrophic bacteria (Chryseobacterium spp., Pseudomonas spp.), that were found in almost all samples, but disappeared after the warming up at 47-48 degrees C of coagulated milk. Pantoea spp. was primarily found in curds and only with a low incidence in milk samples, indicating the environmental origin. Finally the sequencing data confirmed the presence of E. faecium, E. faecalis and S. thermophilus as major species present in the curd. These species were found also in raw milk, proving its importance as source of the typical fermenting microflora.