ABSTRACT Within the complex bacterial community of traditional raw milk cheeses, lactic acid bacteria (LAB), naturally present in the milk as adventitious contaminants, play the key role in the cheese manufacture and ripening process through their acidifying, proteolytic and flavouring activities. Moreover, LAB contribute considerably to the microbial safety, since they produce organic acids and bacteriocins that extend shelf-lives of raw milk products. Numerous LAB genera and species such as Lactobacillus, Lactococcus, Streptococcus, Leuconostoc and Enterococcus are involved in these metabolic activities. This natural microbial biodiversity can be considered a heritage which has to be protected and preserved, since alterations in the composition of the beneficial autochthonous microbiota is of critical importance in maintaining the peculiar features of the traditional raw milk products. On the contrary, LAB may have also negative traits, such as the ability to produce toxic biogenic amines, possession of transmittable antibiotic resistance genes, and the potential for expression of putative virulence factors. Dairy safety aspects should include careful examination of these harmful features. Therefore, the study of micro-organisms at species and strain level and the monitor of their dynamics in the microbial community throughout cheese manufacture and ripening is helpful to improve the quality and safety of the final product. Classical microbiological and molecular techniques have been widely used to investigate the dairy biodiversity and to achieve the characterization of bacterial species present in a complex microbial dairy ecosystem, highlighting the advantages and the limitations of both methods. The aims of PhD thesis were: I) to apply a polyphasic approach based on different phenotypic and molecular techniques for the characterization of the LAB isolated from dairy products collected throughout the technological process of an artisanal raw milk cheese, such as “Formagèla Valseriana”; II) to evaluate the application of total microbial DNA extracted directly from dairy products as template in PCR 16S-23S rRNA region spacer analysis (RSA-PCR) in order to achieve a rapid knowledge about their microbial communities, bypassing the previous step of isolation in pure culture; III) to investigate the influence of equipment (thermocycler) and reagents (Taq polymerase) on results obtained from RSA analysis; IV) to explore the functional and safety aspects of E. faecalis strains isolated from dairy products collected in different North Italy regions through the study of their biodiversity, bacteriocin, volatic organic compounds and biogenic amines production, virulence factors and antibiotic resistance. Firstly, evolution of bacterial groups during the cheese-making process of “Formagèla Valseriana” was revealed by viable counting of LAB and non-LAB micro-organisms. A total of 143 presumptive LAB strains from milk culture, curd and cheese samples were randomly selected from agar plates and subjected to morphological and phenotypic characterization (microscopic examination, Gram staining, catalase test, growth at 15 and 45 °C, CO2 production from glucose) and molecular identification. To investigate LAB biodiversity, the taxonomic position of the isolates was established through RSA-PCR combined with a species-specific PCR assay. Strains characterized by atypical classes of spacer were identified by partial 16S rRNA sequencing. Random amplified polymorphic DNA analysis (RAPD-PCR) was carried out to explore the genetic diversity of LAB isolates. In addition, this study was intended to evaluate the application of microbial DNA extracted directly from dairy products as template in RSA-PCR in order to achieve a rapid knowledge about their microbial communities, bypassing the previous step of isolation in pure culture. As expected in raw milk cheeses, LAB (i.e. lactobacilli, lactococci and enterococci) were quantitatively the dominant group for all samples. The combination of various molecular procedures allowed many species to be detected. In particular, milk culture showed a prevalence of Streptococcus thermophilus, whereas the most detected LAB in curd belonged to the species Lactococcus lactis ss lactis, Lc. garvieae and Enterococcus durans. E. durans was the species most frequently isolated from cheese samples, even if several species (E. faecalis, E. gilvus, E. italicus, Lb. paracasei ss paracasei, St. bovis, Lb. plantarum, Lb. coryniformis and Ln. mesenteroides), which are indicative of biological richness, were also present. RAPD-PCR revealed a high biodiversity at species level, but it also showed the presence of significant genetic variability among the isolates belonging to the same species. RSA, using as template DNA extracted directly from milk culture, did not prove to be a suitable method for discrimination of microbial species in a complex matrix, since when microbial community was very heterogeneous and rich, it was difficult discerning LAB species or genera. Actually, the band of S. thermophilus represents the only one that could be frequently identified. The 143 LAB strains were then screened for their inhibitory activity against Listeria monocytogenes and Staphylococcus aureus and for their acidifying activity. Seven and six strains, mainly belonging to the Enterococcus genus, exhibited antagonistic activity against L. monocytogenes and S. aureus, respectively. With regard to acidifying activity, the majority of isolates (88.8%) were found to be low acidifying isolates, causing a pH decrease lower than 1.5 pH units, whereas only 16 strains were classified as medium acidifying isolates and could be considered the fastest acidifying strains. Yet, after 24 h 74.1% of the isolates showed a medium acidifying ability and 9.1% were classified as high acidifying strains. As revealed by screening the microbial population of “Formagèla Valseriana”, the Enterococcus genus is one of the most prevalent within dairy microbial community. However, enterococci are ascribed the most controversial nature, presenting both beneficial and negative features. They represent important nosocomial pathogens, but are also recognized to produce bacteriocin with antibacterial activity and to enhance flavour and aroma development in cheese. For this reason, 40 Enterococcus faecalis strains isolated from raw milk products (milk, curd and cheese) collected throughout the technological process of various North-west Italian traditional cheeses (Aosta Valley, Piedmont and Lombardy) over a 13 year period (1997-2009) were examined. The genetic biodiversity of E. faecalis isolates was assessed through RAPD-PCR and repetitive extragenic palindromic PCR (rep-PCR). Phenotypic and molecular protocols were applied in order to investigate the incidence of antibiotic resistance and virulence factors among strains. They were examined for their antimicrobial activity against 18 foodborne spoilage and pathogenic bacteria. Investigation was also made to identify their bacteriocinogenic potential by searching for bacteriocin-encoding genes (enterocin A, enterocin B, bacteriocin 31, enterocin P, enterocin Q, enterocin L50A-B, mundticin KS, enterocin CRL35, enterocin AS-48, and enterocin 1071A-B). In addition, they were inoculated in milk and submitted to head-space solid-phase-micro- extraction gas chromatography-mass spectrometry analysis in order to study their capability for production of volatile organic compounds (VOCs). Resistance to streptomycin (50%), quinupristin-dalfopristin (32.5%), tetracycline (25%), rifampicin (7.5%), chloramphenicol (5%) and erythromycin (2.5%) were evidenced. Ampicillin, ciprofloxacin, levofloxacin, mupirocin, nitrofurantoin, penicillin G and vancomycin were revealed effective antimicrobials against all the strains considered. Tetracycline resistance was also confirmed by detection of tet(M), tet(K), tet(L), tet(S) and transmissibility of resistance by integrase gene (int) of the Tn916/Tn1545 family of transposons. The high incidence of tet(M) gene, found in 90% of the isolates, doesn’t correlate with their phenotypic expression, thus highlighting the presence of silent genes. The phenotypic assay of haemolytic activity showed positive results (β-haemolysis) only in 2 strains. Among the 7 virulence determinants tested, only gel, asa1, esp and efaA genes were harboured. None other gene encoding for either different virulence factors (cylA, hyl, ace), or amino-acid decarbolylase activity (hdc, tdc, odc) was detected. Our results suggest that dairy enterococci don’t represent the major potential reservoir for the spread of the investigated detrimental traits in contrast to other strains of different origin. Considering their antimicrobial activity, all the E. faecalis tested were active against at least 1 of the 18 indicator strains. Remarkable inhibitory effects against harmful or detrimental microbial species, including Bacillus cereus (44.7% of strains), Escherichia coli (17.5%), L. monocytogenes (15%), St. aureus (2.6%), and Clostridium sporogenes (21.1%), was detected. Moderate antagonism towards LAB (65.8% of strains), especially Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus helveticus, was found, too. However, 31.6% of E. faecalis strains were reported as ideal preservatives. Of the 10 enterocin structural genes explored, only gene for enterocin AS-48 was identified, suggesting that the antimicrobial activity of the phenotypically positive isolates should necessarily be due to another enterogenic or non-enterogenic compound. A significant genetic variability was pointed out, but it was no possible to link the enterocin phenotypes and genotypes with the strain origin. No relation with the geographical origin or the period in which the strains were isolated was also found with regard to the VOCs production and very different enzymatic activities among strains was detected. The major volatile compounds produced were: ethanol, diacetyl, acetoin, acetic and benzoic acids. The results of the present study suggested that a polyphasic approach, combining different phenotypical and genotypical methods, may represent an essential tool to obtain a more effective, accurate and exhaustive investigation of the microbial typing. Finally, even if RSA-PCR was proven to be a rapid, reliable and cost-effective alternative to other PCR methods for the microbial identification and typing, the possibility to compare RSA-PCR results obtained with different thermal cyclers and Taq DNA polymerases has not been investigated effectively. Four models of thermal cyclers of 3 different commercial brands and 4 brands of Taq DNA polymerase were evaluated for their effects on the reproducibility of the RSA-PCR. Five reference LAB strains (St. thermophilus, Lb. helveticus, Lb. delbrueckii subsp. lactis, Lb. delbrueckii subsp. bulgaricus and Lb. fermentum) and a DNA mixture, obtained combining together the DNAs extracted from each reference strain in the same ratio, were used in the experiment. A wide variety of RSA-PCR profiles in terms of band number and size were observed in relation to thermal cycler and Taq DNA polymerase tested, highlighting the relevant influence of these two variables on RSA-PCR reproducibility. It is therefore recommended to choose, within each laboratory, appropriate operating procedures in accordance with one’s equipment and reagents.
DEVELOPMENT OF INNOVATIVE TECHNIQUES FOR STUDYING MICROBIAL POPULATIONSIN MILK AND DAIRY PRODUCTS / T. Silvetti ; tutor: Milena Brasca, Roberta Lodi. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2010 Dec 17. ((23. ciclo, Anno Accademico 2010.
|Titolo:||DEVELOPMENT OF INNOVATIVE TECHNIQUES FOR STUDYING MICROBIAL POPULATIONSIN MILK AND DAIRY PRODUCTS|
|Tutor esterno:||BRASCA, MILENA|
|Data di pubblicazione:||17-dic-2010|
|Settore Scientifico Disciplinare:||Settore AGR/16 - Microbiologia Agraria|
|Citazione:||DEVELOPMENT OF INNOVATIVE TECHNIQUES FOR STUDYING MICROBIAL POPULATIONSIN MILK AND DAIRY PRODUCTS / T. Silvetti ; tutor: Milena Brasca, Roberta Lodi. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2010 Dec 17. ((23. ciclo, Anno Accademico 2010.|
|Appare nelle tipologie:||Tesi di dottorato|