Giorgio Giraffa

Functionality of enterococci in dairy products.

Enterococci have important implications in the dairy industry. They occur as nonstarter lactic acid bacteria (NSLAB) in a variety of cheeses, especially artisan cheeses produced in southern Europe from raw or pasteurised milk, and in natural milk or whey starter cultures. They play an acknowledged role in the development of sensory characteristics during ripening of many cheeses and have been also used as components of cheese starter cultures. The positive influence of enterococci on cheese seems due to specific biochemical traits such as lipolytic activity, citrate utilisation, and production of aromatic volatile compounds. Some enterococci of dairy origin have also been reported to produce bacteriocins (enterocins) inhibitory against food spoilage or pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Vibrio cholerae, Clostridium spp., and Bacillus spp. The technological application of enterocins, shown to be produced during cheese manufacture, led to propose enterococci as adjunct starter or protective cultures in cheeses. There is evidence that enterococci, either added as adjunct starters or present as nonstarter NSLAB, could find potential application in the processing of some fermented dairy products. Literature suggest that the complex biochemical and ecological phenomena explaining the technological functionality of the enterococci in dairy products, are still to be fully understood. Clearly, the clinical research on enterococci underlines also that the safety of dairy products containing enterococci is an issue that the industry must carefully address before proceeding to their application.

DNA-based, culture-independent strategies for evaluating microbial communities in food-associated ecosystems

Culture-independent molecular techniques are now available to study microbial ecosystems. They are opening interesting perspectives to problems related to composition and population dynamics of microbial communities in various environmental niches (e.g., soil, water) and foods. In fermented food products, estimates of true microbial diversity is often difficult chiefly on account of the inability to cultivate most of the viable bacteria. The increasing knowledge of gene sequences and the concomitant development of new culture-independent molecular techniques are providing new and effective tools to compare the diversity of microbial communities and to monitor population dynamics in minimally disturbed samples. In this review, recent advances in these techniques are reported. Possible applications to food-associated microbial ecosystems are emphasised.

Enterococci Isolated from Dairy Products: A Review of Risks and Potential Technological Use

Enterococci are ubiquitous bacteria which frequently occur in large numbers in dairy and other food products. Although they share a number of biotechnological traits (e.g., bacteriocin production, probiotic characteristics, usefulness in dairy technology), there is no consensus on whether enterococci pose a threat as foodborne pathogens, The potential pathogenicity of lactic acid bacteria (LAB), including enterococci, in human clinical infections and their association with endocarditis have recently become a matter of controversy, in spite of the fact that foods containing enterococci have a long history of safe use. This article provides literature data available on microbial ecology, biochemical properties, production of anti- Listeria bacteriocins, and potential pathogenic traits of enterococci isolated from different dairy products.

Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses

Ninety-eight Lactobacillus plantarum strains isolated from Italian and Argentinean cheeses were evaluated for probiotic potential. After a preliminary subtractive screening based on the presence of msa and bsh genes, 27 strains were characterized. In general, the selected strains showed high resistance to lysozyme, good adaptation to simulated gastric juice, and a moderate to low bile tolerance. The capacity to agglutinate yeast cells in a mannose-specific manner, as well as the cell surface hydrophobicity was found to be variable among strains. Very high β-galactosidase activity was shown by a considerable number of the tested strains, whereas variable prebiotic utilization ability was observed. Only tetracycline resistance was observed in two highly resistant strains which harbored the tetM gene, whereas none of the strains showed β-glucuronidase activity or was capable of inhibiting pathogens. Three strains (Lp790, Lp813, and Lp998) were tested by in vivo trials. A considerable heterogeneity was found among a number of L. plantarum strains screened in this study, leading to the design of multiple cultures to cooperatively link strains showing the widest range of useful traits. Among the selected strains, Lp790, Lp813, and Lp998 showed the best probiotic potential and would be promising candidates for inclusion as starter cultures for the manufacture of probiotic fermented foods.

An evaluation of chelex-based DNA purification protocols for the typing of lactic acid bacteria

An easy and rapid protocol to extract DNA to be used as template for polymerase chain reaction (PCR) fingerprinting experiments from cultivable lactic acid bacteria (LAB) is proposed. Different procedures for rapid extraction of DNA by chelex (iminodiacetid acid) ionic resin were compared. Factors affecting the quality and reproducibility of PCR fingerprinting profiles were also investigated. Two out of three chelex-based protocols allowed to obtain DNA samples which, after PCR amplification, provided electrophoretic patterns comparable with those obtained by classical lysozyme and phenol-chloroform DNA extraction. A good level of reproducibility and consistency of the InstaGene procedure was verified. The procedure is fast, practical, and the DNA is of quality similar to that obtained by phenol-chloroform extraction. Although applied to a little number of LAB strains, chelex-based protocols are potentially applicable to a vast array of organisms and/or biological materials.

Grana Padano cheese whey starters: microbial composition and strain distribution.

The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and lactobacilli were quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT-LH-PCR), a culture-independent technique successfully applied to study whey starter ecosystems. The dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identified by M13-PCR fingerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190 strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%). This result was in good agreement with the qualitative whey starter composition observed by RT-LH-PCR. M13-PCR fingerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy plant-specific rather than correlated with the different production areas.

Bacteriocin-Like Inhibitors of Streptococcus lactis Against Listeria monocytogenes

Culture extracts containing inhibitory substances produced by seven Streptococcus lactis strains and different concentrations of nisin were tested on six strains of Listeria monocytogenes . The antimicrobial effect was revealed by the presence of zones of inhibition after spotting of 10 μl of neutralized, filter sterilized, and catalase treated culture extracts onto agar lawns of L. monocytogenes . Culture extracts demonstrated a bactericidal action without cell lysis of pathogenic organisms. Furthermore, a wide sensitivity of inhibitor substances to proteolytic enzymes was observed. Thus the antimicrobial compounds could be defined as bacteriocin-like substances.

Molecular Diversity within Lactobacillus helveticus as Revealed by Genotypic Characterization

ABSTRACT Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium that is used in the manufacture of Swiss type and long-ripened Italian cheeses, such as Emmental, Grana, and Provolone cheeses. Substantial differences in several technologically important characteristics are found among L. helveticus strains isolated from natural dairy starter cultures. In the present study we investigated the genotypic diversity of 74 strains isolated from different dairy cultures used for manufacturing Grana and Provolone cheeses and six collection strains. A restriction fragment length polymorphism analysis of both total genomic DNA and the 16S rRNA gene (ribotyping) was used as genotypic fingerprinting. A multivariate statistical analysis of the data enabled us to identify significant genotypic heterogeneity inL. helveticus. We found that genotypic fingerprinting could be used to distinguish strains; in particular, it was possible to associate the presence of specific strain genotypes with dairy ecosystem sources (e.g., Grana or Provolone cheese). Our data contribute to the description of microbial heterogeneity in L. helveticus and provide a more solid basis for understanding the functional and ecological significance of the presence of differentL. helveticus biotypes in natural dairy starter cultures.

Genotypic and phenotypic diversity of Lactobacillus delbrueckii subsp. lactis strains of dairy origin

Eighty-nine strains of Lactobacillus delbrueckii subsp. lactis isolated from Italian hard and semi-hard cheeses and artisan starter cultures were characterised by phenotypic and genotypic methods. Phenotypic diversity was evaluated by studying biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR and pulsed field gel electrophoresis (PFGE). Phenotypic characterisation indicated a wide variability of the acidifying activity within Lact. delbrueckii subsp. lactis. Although the data was variable, it allowed us to evidence groups of strains with different acidifying properties, especially in terms of acidification intensity. Concerning peptidase activity, Lact. delbrueckii subsp. lactis showed a homogeneously high x-prolil-dipeptidil-aminopeptidase activity and a considerable but more heterogeneous lysil-aminopeptidase activity. The increased resolution obtained by the use of two molecular typing techniques, i.e. RAPD-PCR and PFGE, allowed to widen the level of strain heterogeneity. Technological and ecological pressures are determinant in selecting Lact. delbrueckii subsp. lactis sub-populations which are more functional to the different cheese technologies.

Inhibition of Listeria innocua in Milk by Bacteriocin-Producing Enterococcus faecium 7C5

Enterococcus faecium 7C5 produces a bacteriocin active against Listeria monocytogenes and Listeria innocua . In co-cultures of the strain 7C5 with a thermophilic starter, which was composed of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus , the acidifying activity of the latter was not affected. In contrast, the rate of bacteriocin production was lower when compared with the pure culture of strain 7C5. In co-cultures of L. innocua CNRZ LIN 11 with the thermophilic starter or with strain 7C5, a bacteriostatic effect on Listeria growth was observed. In the co-culture of L. innocua with both strain 7C5 and the thermophilic starter, a complete listerial inhibition occurred. The combined inhibitory effect of the pH decrease and bacteriocin production, which were shown to be synergistic, was demonstrated both at 37°C and under temperature conditions reproducing the first 24 h of a soft-cheese technology. This data substantially supported the potential of using bacteriocin-producing strains as a culture adjunct to inhibit Listeria during cheese manufacturing.