Eighty psychrotrophic bacterial strains, isolated from different northwest Italian bulk tank milks destined for Grana Padano cheese production, were identified by 16S rRNA gene amplification and partial sequence analysis of the rpoB gene. Pseudomonas spp. were the most commonly occurring contaminants, P. fluorescens being the predominant isolated species, along with Enterobacteriaceae, primarily Serratia marcescens. RAPD-PCR was used to study genetic variability and distinguish closely related strains; a high degree of genetic heterogeneity among the strains was highlighted. All the strains were characterized for their ability to produce proteases, lipases and lecithinases at different temperatures (7, 22, and 30 °C). Forty-one of the psychrotrophic strains were positive for all the enzymatic activities. The highest number of positive strains for all the incubation temperatures was found for lipolytic activity (59), followed by proteolytic (31) and lecithinase (28) activities, and the enzymatic traits varied among the Pseudomonas and Enterobacteriaceae strains. The proteolytic psychrotrophic strains were screened for the presence of the aprX gene, coding for a heat-resistant metalloprotease in Pseudomonas spp. The aprX gene was detected in 19 of 63 Pseudomonas strains, and was widespread in the P. fluorescens strains (14/19). PRATICAL APPLICATION: The study provides new data on the enzymatic activity of Gram-negative psychrotrophic bacteria, useful in developing strategies to control the proteo-lipolytic spoilage of raw and processed milk that causes gelation, off-flavors, and loss of sensory quality and shelf life.

BACKGROUND Anaerobic spore‐forming bacteria (ASFB) in milk derive from the farm environment, and the use of silages and management practices are the main responsible of milk ASFB contamination. The aim of this study was to evaluate the relationships between feeding, milking routine and cow hygiene and milk and Grana Padano cheese (produced with and without lysozyme) ASFB contamination. RESULTS The study involved 23 dairy farms. ASFB in corn silage were on average 2.34 ± 0.87 log10 MPN g−1. For grass, Italian ryegrass and alfalfa, ASFB (log10 MPN g−1) were numerically higher for silages (3.22) than hays (2.85). The use of corn silages of high quality (high lactic and acetic acids concentrations) decreased the milk ASFB contamination, whilst the use of herbage silages did not affect it. The presence (>40%) of cows with dirty udders increased the ASFB contamination of milk, while forestripping had a positive effect (−9% ASFB). Ripened Grana Padano had an ASFB count below the analytical limit; Clostridium tyrobutyricum DNA was found only in wheels produced without lysozyme, which also showed late blowing. CONCLUSION The factors increasing milk spore contamination were corn silage quality, cow udder hygiene and inadequate milking routine. Late blowing was present only in cheeses without lysozyme. © 2014 Society of Chemical Industry

Bovine mastitis caused by Prototheca is a serious and complex problem that accounts for high economic losses in the dairy industry. The main objective of this study was to identify and characterize at genetic level different Prototheca strains and provide the most complete data about protothecal antibiotic resistance. The study involves 46 isolates from Italian (13 strains) and Brazilian (33 strains) mastitic milk. These strains were identified by multiplex PCR and single strand conformation polymorphism analysis and characterized by randomly amplified polymorphic DNA (RAPD)-PCR. Moreover, biofilm production and antibiotic susceptibility were evaluated. Forty-two strains resulted as Prototheca zopfii genotype 2, whereas 4 isolates could belong to a potential new Prototheca species. The RAPD-PCR, performed with 3 primers (M13, OPA-4, and OPA-18), showed a notable heterogeneity among isolates and grouped the strains according to the species and geographical origin. Biofilm production was species-dependent and P. zopfii genotype 2 strains were classified as strong biofilm producers. In vitro antibiotic susceptibility tests indicated that Prototheca strains were susceptible to antibacterial drugs belonging to aminoglycosides group; the highest activity against Prototheca strains was observed in the case of colistin sulfate, gentamicin, and netilmicin (100% of susceptible strains). It is interesting to note that all the Italian P. zopfii genotype 2 strains showed lower minimum inhibitory concentration values than the Brazilian ones. Nisin showed more efficacy than lysozyme and potassium sorbate, inhibiting 31% of the strains. Results obtained in this study confirmed that RAPD-PCR is a rapid, inexpensive, and highly discriminating tool for Prototheca strains characterization and could give a good scientific contribution for better understanding the protothecal mastitis in dairy herd.

Sustainable exploitation of agro-industrial by-products has attracted great interest in cereal bran valorization. In this research, a polyphasic approach has been carried out to characterize maize bran at microbiological and chemical level during a sourdough like fermentation process, in order to enhance its technological and nutritional properties. Autochthonous microbiota was isolated at different refreshment steps and subjected to identification and molecular characterization. Fermentation was characterized by a rapid increase in lactic acid bacteria and yeasts, with a co-dominance, at the initial stage, of Weissella spp., Pediococcus spp. and Wickerhamomyces anomalus. At the end of the fermentation, a natural selection was produced, with the prevalence of Lactobacillus plantarum, Lactobacillus brevis and Kazachstania unispora. This is the first time that a specific association between LAB and yeasts is reported, during the maize bran fermentation process. Enzymatic activities related to this microbial consortium promoted a “destructuration” of the fiber fraction, an increase in soluble dietary fiber and a reduction of phytic acid content. Our data also evidenced a noticeable increment in ferulic acid. The results obtained indicate that fermentation processes represent an efficient biotechnological approach to increase nutritional and functional potential of maize bran. Moreover, the characterization of microbiota involved in natural fermentation process will allow the selection of specific biotypes, with appropriate metabolic and enzymatic activities, to conduct “tailored” fermentation processes and improve brans or whole-meal flours from both nutritional and technological points of view.