Biomedical subjects

Giorgio Giraffa

Publications and source records attributed to Giorgio Giraffa.

12 recordsLinked to original sources

PubMed2006

Characterization of spontaneous phage-resistant derivatives of Lactobacillus delbrueckii commercial strains.

A total of 44 spontaneous phage-resistant mutants were isolated from three commercial Lactobacillus delbrueckii strains by secondary culture and agar plate methods. Phenotypic characteristics related to their phage-resistance capacities, i.e. plaquing efficiency, phage-resistance stability, lysogeny and adsorption rates were determined. The morphological, biochemical (sugar fermentation patterns) and technological (acidifying and proteolytic activities and acidification kinetics) properties of mutants were also studied. Amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) was applied to confirm strain identity at the subspecies level. Random amplification of polymorphic DNA (RAPD-PCR) was used to determine genetic diversity among the isolates and their respective parent strains. The secondary culture method was the most useful for obtaining phage-resistant mutants. Phage resistance stability was a variable property among the isolates, but a high level of resistance was exhibited as quantified by the efficiency of plaquing. Furthermore, a total absence of spontaneous lysogeny was demonstrated. Adsorption rates were heterogeneously distributed among the three groups of mutants. All mutants isolated from two sensitive strains were similar to them with respect to technological properties. Two groups of mutants with distinctive technological properties were isolated from the other sensitive strain. PCR-ARDRA revealed that two out of three sensitive strains identified commercially as Lb. delbrueckii subsp. bulgaricus were actually Lb. delbrueckii subsp. lactis. Some of the phage-resistant mutants that were obtained might be used in culture rotation programs without regulatory restrictions when commercial strains become sensitive to phages present in industrial environments.

Adsorption↗

PubMed2006

Detection and identification of Lactobacillus delbrueckii subsp. lactis bacteriophages by PCR.

A sensitive PCR method amplifying an internal fragment of the major tail protein gene was developed to detect Lactobacillus delbrueckii subsp. lactis lytic bacteriophages in undefined, thermophilic whey starters used in Italy for production of Grana and Provolone cheeses. PCR was applied to several lytic Lb. delbrueckii subsp. lactis bacteriophages, which were highly diverse according to restriction analysis and phage host range. PCR detected the presence of phages in two out of 11 cultures, when applied to whey starters for Grana Padano cheese sampled from different cheese plants. The presence of actively growing phages in infected cultures was confirmed by traditional test. The PCR method proved to be useful to screen for the presence of Lb. delbrueckii subsp. lactis phages in thermophilic whey starters.

Animals↗

PubMed2006

Cultivability of Streptococcus thermophilus in Grana Padano cheese whey starters.

The application of a culture-independent approach, that of reverse transcriptase-length heterogeneity-PCR coupled with epifluorescence microscopy, allowed us to observe that Streptococcus thermophilus is metabolically active, but only partially cultivable in Grana Padano cheese whey starters. A short preincubation of the starters in sterile skimmed whey was followed by cultivation in sterile skimmed whey-enriched M17. This procedure restored the cultivability of S. thermophilus and enabled us to detect S. thermophilus at ranges (10(7)-10(8) CFU mL(-1)) which have rarely been reported in these cultures. The use of cheese whey as a cultivation-revitalization substrate can be useful to obtain an unbiased picture of the microbial composition of whey starters for Grana Padano cheese, thus avoiding an underestimation of S. thermophilus in these cultures.

Bacteriological Techniques↗

PubMed2006

Nonstarter lactobacilli isolated from soft and semihard Argentinean cheeses: genetic characterization and resistance to biological barriers.

Nonstarter lactic acid bacteria isolated from Argentinean cheeses were identified and characterized by focusing on their resistance to biological barriers, along with other physiological features of potential interest, in the search for future probiotic organisms. Lactobacilli were enumerated and isolated from semihard and soft cheeses made with multistrain Streptococcus thermophilus starters. Lactobacilli counts in 1-week-old cheeses were between 10(5) and 10(7) CFU/g and then reached 10(7) CFU/ g in all 1-month samples, while streptococci were always above 10(9) CFU/g. A total number of 22 lactobacilli isolates were retained, identified, and characterized by in vitro tests. Species identity was determined by carbohydrate metabolism and species-specific PCR assays. Genetic diversity was explored by random amplified polymorphic DNA (RAPD) PCR analysis. The Lactobacillus strains were assigned to the species L. casei, L. plantarum, L. rhamnosus, L. curvatus, L. fermentum, and L. perolens. All the strains studied tolerated 25 ppm of lysozyme, and most of them showed resistance to 0.3% bile. After incubation in gastric solution (pH 2.0), counts decreased by several log units, ranging from 3.2 to 7.0. The strains were able to grow in the presence of bile salts, but only three isolates were capable of deconjugation. The nonstarter lactobacilli that were assayed fermented the prebiotic substrates (especially lactulose and inulin). Some strains showed high cell hydrophobicity and beta-galactosidase activity, as well as inhibitory activity against pathogenic bacteria. It was concluded that most of the lactobacilli isolated in this study demonstrated resistance to biological barriers and physiological characteristics compatible with probiotic properties, which make them suitable for further research in in vivo studies aimed at identifying new probiotic organisms.

Argentina↗

PubMed2005

Application of reverse transcriptase PCR-based T-RFLP to perform semi-quantitative analysis of metabolically active bacteria in dairy fermentations.

A method consisting of reverse transcriptase (RT)-PCR amplification of 16S rRNA from the total microbial community, coupled with T-RFLP, was optimized for semi-quantitative characterization of the metabolically active population in defined strain cultures of Lactococcus lactis ssp. lactis and Leuconostoc citreum, two mesophilic lactic acid bacteria (LAB) species routinely used in cheese manufacture. The set of PCR primers selected efficiently amplified the 16S rRNA from both bacterial species. The digestion of the PCR products with DdeI yielded different terminal restriction fragments (T-RFs) for each species. Nevertheless, additional T-RFs due to formation of chimeric molecules and pseudo-T-RFs derived from partly single-stranded 16S rRNA amplicons were observed in both species, although in minor amounts. Twenty PCR cycles were determined as the optimum to minimize the presence of artifactual fragments and to avoid underestimation of populations due to the saturation effect on DNA quantification caused by a PCR product excess. T-RFLP analysis showed a good repeatability when applied to mixed dairy cultures. Dynamics of two defined mixed starters consisting of a L. lactis ssp. lactis strain and a L. citreum strain were studied by this method and results compared to those obtained by a culture-dependent technique. The data indicated the suitability of T-RFLP to perform semi-quantitative analyses of microbial populations. Some slight differences could be explained by the presence of metabolically active cells that could not be detected by colony counting. RT-PCR-based T-RFLP can be an alternative to classical methods in order to study dynamics of metabolically active populations in relatively simple microbial ecosystems, such as defined dairy starter cultures.

Animals↗

PubMed2005

Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases.

About a thousand lactic acid bacteria (LAB) isolated from dairy products, especially cheeses, were identified and typed by species-specific PCR and RAPD-PCR, respectively. RAPD-PCR profiles, which were obtained by using the M13 sequence as a primer, allowed us to implement a large database of different fingerprints, which were analysed by BioNumerics software. Cluster analysis of the combined RAPD-PCR fingerprinting profiles enabled us to implement a library, which is a collection of library units, which in turn is a selection of representative database entries. A library unit, in this case, can be considered to be a definable taxon. The strains belonged to 11 main RAPD-PCR fingerprinting library units identified as Lactobacillus casei/paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus and Lactococcus lactis. The possibility to routinely identify newly typed, bacterial isolates by consulting the library of the software was valued. The proposed method could be suggested to refine previous strain identifications, eliminate redundancy and dispose of a technologically useful LAB strain collection. The same approach could also be applied to identify LAB strains isolated from other food ecosystems.

Animals↗

PubMed2005

Heterogeneity of putative surface layer proteins in Lactobacillus helveticus.

The S-layer-encoding genes of 21 Lactobacillus helveticus strains were characterized. Phylogenetic analysis based on the identified S-layer genes revealed two main clusters, one which includes a sequence similar to that of the slpH1 gene of L. helveticus CNRZ 892 and a second cluster which includes genes similar to that of prtY. These results were further confirmed by Southern blot hybridization. This study demonstrates S-layer gene variability in the species L. helveticus.

Amino Acid Sequence↗

PubMed2004

Monitoring of the bacterial composition of dairy starter cultures by RAPD-PCR.

Randomly amplified polymorphic DNA (RAPD)-PCR was used to verify the species composition of commercial dairy starters and to detect possible shifts in strain composition of these cultures. After RAPD-PCR analysis, not all the strains isolated in the years 2001 and 2002 fell within the same dendrogram cluster of the strains isolated in the year 2000 and used as reference strains. Changes in composition of the microbial population and/or voluntary immission of new biotypes with respect to the original strain formulation had occurred in the starters. The microbial composition of modern dairy starters represents a key point because the complex relationships among microorganisms can easily be altered. Little variations in the microbial composition could have unexpected effects on cheese quality.

Bacteria↗

PubMed2004

Studying the dynamics of microbial populations during food fermentation.

The dynamics of growth, survival and biochemical activity of microorganisms in food are the result of stress reactions in response to the changing of the physical and chemical conditions into the food microenvironment, the ability to colonise the food matrix and to growth into a spatial heterogeneity, and the in situ cell-to-cell ecological interactions which often happen in a solid phase. In food, ecological approaches to study the evolution of microbial flora would be useful to comprehend better the microbiological processes involved in food processing and ripening, to improve microbiological safety by monitoring in situ pathogenic bacteria, and to evaluate the effective compositions of the microbial populations. This paper gives a general overview of biotechnological approaches to study microbial populations in food fermentation.

Bacteria↗

PubMed2003

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.

Animals↗

PubMed2002

A polyphasic approach to highlight genotypic and phenotypic diversities of Lactobacillus helveticus strains isolated from dairy starter cultures and cheeses.

In the present work, 67 strains of Lactobacillus helveticus isolated from whey starter cultures and cheeses were identified and grouped by genotypic and phenotypic methods. Strains were identified by sugar fermentation pattern, by cell-wall protein profile, and by probe hybridisation. Phenotypic diversity was evaluated by a chemometric model taking into account biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR, which provided stran-specific patterns and revealed the occurrence of different strains. The RAPD-PCR profiles were clustered according to their similarities: the groups obtained, together with the cell-wall protein profiling and the chemometric information, could be sometimes correlated with the type of cheese and/or dairy niches used as sources of strains. A computerised analysis of genotypic and phenotypic information could be successfully applied for rapid and reliable differentiation and characterisation of Lb. helveticus isolates occurring in different dairy products.

Animals↗

PubMed2002

Enterococci from foods.

Enterococci have recently emerged as nosocomial pathogens. Their ubiquitous nature determines their frequent finding in foods as contaminants. In addition, the notable resistance of enterococci to adverse environmental conditions explains their ability to colonise different ecological niches and their spreading within the food chain through contaminated animals and foods. Enterococci can also contaminate finished products, such as fermented foods and, for this reason, their presence in many foods (such as cheeses and fermented sausages) can only be limited but not completely eliminated using traditional processing technologies. Enterococci are low grade pathogens but their intrinsic resistance to many antibiotics and their acquisition of resistance to the few antibiotics available for treatment in clinical therapy, such as the glycopeptides, have led to difficulties and a search for new drugs and therapeutic options. Enterococci can cause food intoxication through production of biogenic amines and can be a reservoir for worrisome opportunistic infections and for virulence traits. Clearly, there is no consensus on the acceptance of their presence in foodstuffs and their role as primary pathogens is still a question mark. In this review, the following topics will be covered: (i) emergence of the enterococci as human pathogens due to the presence of virulence factors such as the production of adhesins and aggregation substances, or the production of biogenic amines in fermented foods; (ii) their presence in foods; (iii) their involvement in food-borne illnesses; (iv) the presence, selection and spreading of antibiotic-resistant enterococci as opportunistic pathogens in foods, with particular emphasis on vancomycin-resistant enterococci.

Biogenic Amines↗