DEVELOPMENT OF INNOVATIVE TECHNIQUES FOR STUDYING MICROBIAL POPULATIONSIN MILK AND DAIRY PRODUCTS
Tesi di Dottorato
Data di Pubblicazione:
2010
Citazione:
DEVELOPMENT OF INNOVATIVE TECHNIQUES FOR STUDYING MICROBIAL POPULATIONSIN MILK AND DAIRY PRODUCTS / T. Silvetti ; tutor: Milena Brasca, Roberta Lodi. Universita' degli Studi di Milano, 2010 Dec 17. 23. ciclo, Anno Accademico 2010.
Abstract:
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 knowled
Tipologia IRIS:
Tesi di dottorato
Elenco autori:
T. Silvetti
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