Bitto is a Protected Designation of Origin raw milk cheese produced in a restricted Italian alpine area only during the summer transhumance. The indigenous microbial ecosystem of this artisanal cheese is considered a primary factor related to its typicality. The aim of the research was to investigate the dynamics of wild lactic acid bacteria (LAB) involved in Bitto production and to study the characteristics of LAB. A total of 210 LAB isolates from curd, whey and ripened cheese, were first molecularly analysed by means of randomly amplified polymorphic DNA (RAPD). After strain differentiation, LAB were identified at the species level using species-specific primers and 16S rRNA gene sequencing. Genotypic diversity and technological properties of major interest for cheese making (acidification ability, redox potential and caseinolytic activity) were also evaluated. The predominant species, in both curd and ripened cheese, was Enterococcus faecium , and there appeared a high degree of diversity in the genotypic and technological traits. By using 16S rRNA sequencing and RAPD-PCR as well as examining the phenotypic properties, the new isolates were shown to belong to a novel enterococcal species for which the name Enterococcus lactis has been proposed. Among the curd isolates, six bacteriocin producers were found belonging to E . faecium , Lactobacillus fermentum , Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus species.

Lysozyme is an antimicrobial enzyme that could be applied to counteract those bacterial species which, due to their own metabolic activity, possess notable beer spoilage ability and lead to loss of beer quality. Experiments were carried out to assess lysozyme potential to prevent the growth of beer spoilage bacteria, and to verify the effect of lysozyme on the microbiological stability and sensory characteristics of unpasteurized beer. Eight replicates, all from the same lot of Italian beer, were treated with 0 and 100 ppm lysozyme. Microbiological analyses were conducted bimonthly to investigate the presence of spoilage bacteria. Sensory analyses were performed to determine whether there were any significant differences in sensory impressions between beers produced with and without lysozyme. Lysozyme exerted a strong inhibitory action on the lactic acid bacteria (LAB) present in the beer and was very stable throughout the shelf life. Sensory tests revealed no unfavourable influence on beer flavour when using lysozyme. Indeed, the shelf life of beer with added lysozyme proved to be extended. Even as late as 1 month after the expiry date it still met with the panellists' approval. Lysozyme may be regarded as an effective agent for preventing microbiological contamination and prolonging the stability of unpasteurized beer.

Staphylococcus aureus is a known major cause of foodborne illnesses, and milk and dairy products are often contaminated by enterotoxigenic strains of this bacterium. In the present study, 122 S. aureus isolates collected from different dairy products were characterised by phenotypic properties, by the distribution of genes encoding staphylococcal enterotoxins (sea, sec, sed, seg, seh, sei, sej, and sel) and by randomly amplified polymorphic DNA PCR (RAPD-PCR). Moreover, strain resistance to vancomycin and methicillin (oxacillin) was studied. The differences in the RAPD-PCR profiles obtained with the primers M13 and AP4 revealed the presence of a great genetic heterogeneity among the different S. aureus strains. Using the primer AP4 and M13, eight groups were distinguished by RAPD-PCR cluster analysis, although, except in few cases, it was not possible to correlate the isolates of different animal species (cow or ovine) with the presence of se genes. None of the isolates showed resistance to vancomycin or methicillin.

The study concerns 130 Staphylococcus aureus strains isolated from different raw-milk dairy products (122 isolates) and human samples (eight isolates). Four different typing techniques were applied: biochemical profiles (Biolog GP), restriction fragment length polymorphism of coagulase gene (coaRFLP), random amplified polymorphic DNA (RAPD) and multilocus variable number tandem repeat analysis (MLVA). Moreover multiplex-PCR was used to study the distribution of genes encoding staphylococcal enterotoxins. The results of this study reveal marked genomic and phenotypic variability among the tested S. aureus. The considered techniques were all found useful for strain typing, but, based on discriminatory power as the key parameter of the typing system, MLVA and Biolog GP were found to be the most powerful techniques. The methods showed little concordance in terms of discerning the clusters of related strains.

Coagulase gene restriction fragment length polymorphism (RFLP), six-locus variable-number tandem-repeat analysis patterns (MLVA) and detection of enterotoxin genes (se) (sea,sec,sed,seg,seh,sei,sejandsel) were used to determine the phylogenetic relationship among isolates ofStaphylococcus aureusisolated from dairy products from different regions of Italy. A total of 25Staph. aureuswere subtyped into 16 coagulase genotypes by RFLP, and MLVA revealed marked genomic variability. Furthermore, 17 of the isolates harboured at least one toxin gene, with the predominance ofsea,sedandsejamong cow isolates andsec-selamong the goat and sheep strains. Combined RFLP, MLVA polymorphism andsegenes were found to be useful techniques for discriminating several genetic variants inStaph. aureusisolates.

Milk and dairy products are frequently contaminated with enterotoxigenic Staphylococcus aureus, which is often involved in staphylococcal food poisoning. The distribution of genes encoding staphylococcal enterotoxins (SE) in S. aureus isolated from bovine, goat, sheep and buffalo milk and dairy products was verified by the presence of the corresponding SE production. A total of 112 strains of S. aureus were tested for SE production by immuno-enzymatic (SEA-SEE) and reversed passive latex agglutination (SEA-SED) methods, while multiplex-PCR was applied for SE genes (sea, sec, sed, seg, seh, sei, sej and sel). Of the total strains studied, 67% were detected to have some SE genes (se), but only 52% produced a detectable amount of the classic antigenic SE types. The bovine isolates frequently had enterotoxin SEA, SED and sej, while SEC and sel predominated in the goat and sheep strains. The results demonstrated (i) marked enterotoxigenic S. aureus strain variations, in accordance with strain origin and (ii) the two methods resulted in different information but concurred on the risk of foodstuff infection by S. aureus.

AIMS:To verify to what degree reducing capacity is a characterizing parameter of a species, and of the strains themselves within a given species, of lactic acid bacteria.METHODS AND RESULTS:Eighty-eight strains belonging to 10 species of lactic acid bacteria (LAB) isolated from traditional Italian cheeses were studied for their reduction activity: Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Streptococcus thermophilus, Lactococcus lactis ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus and Pediococcus pentosaceus. It was observed that the lactococci reached minimum redox potential before the lactobacilli. The reduction rate of Enterococcus spp. and L. lactis ssp. lactis was higher than that of the streptococci and Lactobacillus spp. All the P. pentosaceus strains had poor reduction activity compared with the other species.CONCLUSIONS:The evolution of the redox potential in milk over a time span of 24 h has been found to be a parameter that characterizes a species: the different courses corresponding to the species in question are clearly evident, and interesting differences can also be noted within the same species.SIGNIFICANCE AND IMPACT OF THE STUDY:The reduction aptitude of strains might be used to select and adapt appropriate strains for use as starters for dairy products.

In the present study, 68 enterococcal isolates collected from dairy products in north–west Italy were characterised by randomly amplified polymorphic DNA-PCR (RAPD-PCR) and by their technologically relevant biochemical properties. After the comparison of RAPD-PCR profiles with the profiles of the enterococcal type strains, isolates were assigned to the species Enterococcus faecalis (35 isolates), E. faecium (27 isolates) and E. durans (6 isolates). Phenotypic identification based on carbohydrate fermentation profile galleries gave different results from RAPD-PCR in 17.6% of the cases (10 E. faecium and 2 E. durans isolates). With respect to biochemical activities, the majority of the strains displayed weak acidification activity in milk. High values for redox activity were detected for 80% of E. faecalis isolates, while E. faecium provided the most lipolytic species. Proteolytic activity was generally low, except for 10 E. faecalis isolates that displayed values ⩾0.5mg tyrosine 5mL-1 of milk. None of the isolates carried vanA or vanB (vancomycin resistance) genetic determinants.

A multiplex PCR for the simultaneous detection of Staphylococcus aureus 23S rRNA, the coagulase and thermonuclease genes as well as the enterotoxin genes sea, sec, sed, seg, seh, sei, sej, sel was developed. The method was used to determine the presence of enterotoxigenic types for 93 S. aureus strains isolated from milk and dairy products. The data obtained by mPCR resulted comparable to those obtained by immunoassay methods. In addition, the mPCR assays also amplified some se genes, whose toxins are undetectable by immunoassay. Multiplex amplification can be obtained starting from 1 pg of DNA, showing the excellent specificity and high sensitivity of the assay.

A total of 70 enterococcal strains isolated from traditional Italian raw milk cheeses were identified by API 20 Strep and Biolog GP. All the identifications were confirmed by species-specific PCR. Four species of genus Enterococcus were found: E. faecalis, E. faecium, E. durans and E. hirae. E. faecalis and E. faecium were the dominant enterococcal species isolated in artisanal cheeses. According to the most representative species and the origin of isolation, 21 strains ( 11 E. faecalis and 10 E. faecium) were selected for the conductimetric analysis. The influence of pH (5.0; 5.5; 6.0 and 6.5) and temperature ( 25 degrees C and 37 degrees C) on the metabolism and the development of enterococci was evaluated by determination of Generation Time (GT), Detection Time (DT) and the maximum conductance value (Delta mu S) of the curve during a period of 48 hours. Conductance trials showed that both species were able to adapt to adverse cultural conditions ( low values of pH and temperature) during cheese-making and ripening. E. faecalis appeared to be less sensitive on the whole. In the worst conditions ( pH 5.0, 25 degrees C), after an adaptation phase ( about 10 h), these microorganisms showed GT values almost double those at 37 degrees C. The same behavior can occur in cheese, where enterococci can reach numbers of up to 10(5) - 10(8) cfu center dot g(-1) during ripening. This could explain the fact that enterococci ( in particular, E. faecalis and E. faecium) represent the typical and important microflora in raw milk cheese.

Temperature (T), pH and NaCl concentration are the parameters that control the rate of propionic acid bacteria (PAB) growth. The indirect conductimetric technique was employed and a medium containing yeast extract, Na acetate, L (+)-cysteine chloride and Na lactate was formulated to amplify the electric signal. Sixteen PAB strains isolated from milk for Grana cheesemaking were tested in different combinations of 4 parameters (temperature, pH, NaCl and species); data were expressed as percentage of growth delay compared to standard conditions (pH 6.1, 30 degrees C, no NaCl added). Decreasing temperature caused the most significant growth delay (131% at 22 degrees C and 438% at 15 degrees C), while PAB growth with decreasing pH and increasing NaCl concentration was less affected in the most restrictive conditions (236% at pH 5.2 and 222% at 2.5% NaCl respectively). A slight stimulating effect was observed at 30 degrees C and low NaCl content (from 193% at 0% down to 187% at 0.5%). The double combinations T x pH and T x NaCl further increased delay values up to 489% (T x pH) and to 482% (T x NaCl) in the most restrictive conditions, and temperature was always the most important factor; the stimulating effect due to NaCl was amplified at 30 degrees C for all concentrations and at 22 degrees C at 0.5%. A significant difference was found in the behaviour of the 4 tested species: P. thoeni was the most inhibited, while P. freudenreichii and P. acidipropionici underwent the lowest growth reduction. Strictly controlled temperature (under 22 degrees C), curd acidification (under pH 5.4) and brine salt concentration are the cheesemaking steps identified as the critical points for containing PAB growth; the reliability of the proposed method suggests further individualization of the most suitable factor levels to contain the late blowing defect.

This work aimed to improve the knowledge and the control of interactions between lactic acid bacteria (LAB) and propionibacteria (PAB) in order to control the propionic acid fermentation in European hard cheeses, and therefore their quality. Among more than 50 couples of strains LAB/PAB used in cheese making, some pairs of strains were chosen in function of quality results on cheeses (sensorial analysis). A whey model system and an alternative conductimetric method, with good reproducibility were used to study interactions. The lipolytic activity of PAB strains varied by a factor 4 to 5. The aroma of ripened Emmental cheese was linked to the level and the type of lipolysis in the cheese, correlated with lipolytic activity of PAB. The main pathways of the utilisation of lactate by PAB, studied by 13C RMN, were useful to understand the balance between final products: acetic, propionic acid, CO2.