RESUMEN

Lytic bacteriophages (phages) offer a great potential as biocontrol agents for spoilage Clostridium tyrobutyricum, responsible for butyric acid fermentation in semi-hard and hard ripened cheeses, resulting in late gas blowing defect. With this aim, we have isolated, identified and characterized new lytic phages of C. tyrobutyricum, and have evaluated their efficacy to control cheese late blowing by adding them to manufacture milk. Silage, soil, milk and cheese from dairy farms were screened for anti-clostridial phages, obtaining 96 isolates active against C. tyrobutyricum. According to host range, source and plaque morphology, we obtained 20 phage profiles, 8 of them (represented by phages FA3, FA21, FA29, FA52, FA58, FA67, FA70 and FA88) showing a wider host range and high quality lysis, which were further characterized. Selected isolates showed a non-contractile tail, belonging to the Siphoviridae family, and were grouped into 3 restriction profiles. Viable phages were detected after storage in sodium-magnesium buffer (SM buffer), skim milk and acidified skim milk (pH 5) for 7 d at 4 °C, 12 °C and 37 °C, although a decline in infectivity was observed in some cases. Good phage survival was also detected during semi-hard cheese manufacture and ripening (60 d), and cheese lactococci counts, pH, dry matter values, and volatile compounds were not affected by phage addition. In semi-hard cheese, phage FA67 impaired the early germination of C. tyrobutyricum spores and caused a significant decrease in clostridial vegetative cells counts at 14 d of ripening, delaying by 2 weeks the consumption of lactic acid, formation of butyric acid and appearance of late blowing symptoms, compared to the spoilt control cheese without the phage. This is the first report on the application of phage to control C. tyrobutyricum in cheese.

Asunto(s)

RESUMEN

This study was carried out to assess the survival of Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic Escherichia coli (aEPEC) during the traditional manufacturing and ripening of Spanish hard cheese from raw cow's milk. Milk samples were spiked with up to 3.1-3.5 log cfu/mL of one strain of STEC (O140:H32 serotype) and one of aEPEC (serotype O25:H2). The first steps of cheesemaking allow for a STEC and aEPEC increase of more than 1 log cfu/mL (up to 4.74 log cfu/g and 4.55 log cfu/g, respectively). After cheese pressing, a steady reduction of both populations was observed, with the STEC strain being more sensitive. The studied pathogenic E. coli populations decreased by 1.32 log cfu/g in STEC and 0.59 log cfu/g in aEPEC in cheese ripened during a minimum period of 60 d. Therefore, a moderate contamination by these diarrhoeagenic E. coli pathotypes, in particular, with aEPEC, on cheese manufactured from raw milk may not be totally controlled through the cheesemaking process and during a maturation of 90 d. These findings remark the importance of improvement in bacteriological quality of raw milk and cross-contamination prevention with diarrhoeagenic E. coli in the dairy industry.