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DEVELOPMENT OF INNOVATIVE TECHNIQUES FOR STUDYING MICROBIAL POPULATIONSIN MILK AND DAIRY PRODUCTS
No full textABSTRACT
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
Changes in oxidation-reduction potential during milk fermentation by wild lactic acid bacteria
No full textOxidation-reduction potential (E h) is a fundamental physicochemical property of lactic acid bacteria that determines the microenvironment during the cheese manufacture and ripening. For this reason the E h is of growing interest in dairy research and the dairy industry. The objective of the study was to perform a comprehensive study on the reduction activity of wild lactic acid bacteria strains collected in different periods (from 1960 to 2012) from Italian dairy products. A total of 709 strains belonging to Lactococcus lactis, Enterococcus durans, E. faecium, E. faecalis and Streptococcus thermophilus species were studied for their reduction activity in milk. Kinetics of milk reduction were characterised by the minimum redox potential (Eh7) and time of reaching Eh7 (tmin), the maximum difference between two measures (Δmax) and the time at which these maximum differences occurred (t∗). Broad diversity in kinetic parameters was observed at both species and strain levels. E. faecalis and L. lactis resulted to be the most reducing species, while S. thermophilus was characterised by the lowest reducing power while the greatest heterogeneity was pointed out among E. durans and E. faecium strains. Considering the period of collection (1960-2012) we observed that the more recently isolated strains generally showed less reducing activity. This trend was particularly evident for the species E. durans, E. faecium and L. lactis while an opposite trend was observed in E. faecalis species. Data reported in this research provide new information for a deeper understanding of redox potential changes during milk fermentation due to bacterial growth. Gain knowledge of the redox potential of the LAB cultures could allow a better control and standardisation of cheesemaking process
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Bactericidal performance of nanostructured surfaces by fluorocarbon plasma
No full textThis study presents the characterization and antibacterial activity of nanostructured Si by plasma treatment method using a tetrafluoromethane (CF4) and hydrogen (H2) mixture. Nanostructured-Si is a synthetic nanomaterial that contains high aspect ratio nanoprotrusions on its surface, produced through a reactive-ion etching process. We have shown that the nanoprotrusions on the surfaces produce a mechanical bactericidal effect. Nanostructured-Si exhibited notable activity against three different microorganisms: Gram-negative (Escherichia coli), Gram-positive (Staphylococcus aureus) and spore-forming bacteria (Bacillus cereus) producing a > 5 log10 reduction after 24 h of incubation. Scanning electron microscopy was used to analysis the structure and morphology character of different surfaces evidencing the physical bactericidal activity of the Nanostructured-Si. These results provide excellent prospects for the development of a new generation of antibacterial surfaces
Mechanisms of clostridium tyrobutyricum removal through natural creaming of milk: a microscopy study
No full textClostridium tyrobutyricum is the main spoilage agent of late blowing defect (LBD) in Grana Padano and Parmigiano-Reggiano cheeses; LBD is characterized by openings and holes and is sometimes accompanied by cracks and an undesirable flavor. Even a very few spores remaining in the cheese curd may cause LBD; thus, it is essential to eradicate them during milk natural creaming. By this process, most of the bacteria, somatic cells, and spores rise to the top of the milk, together with the fat globules, and are removed with the cream. Previous studies suggested that milk immunoglobulins mediate the interactions between fat globules and bacteria that occur upon creaming but no direct evidence for this has been found. Moreover, other physical chemical interactions could be involved; for example, physical entrapment of spores among globule clusters. To maximize the efficiency of the natural creaming step in removing Cl. tyrobutyricum,, it is essential to understand the nature of spore globule interactions. With this aim, raw milk was contaminated with spores of Cl. tyrobutyricum before going to creaming overnight at 8 degrees C, after which spore and bacteria removal was >90%. The obtained cream was analyzed by light interference contrast and fluorescence microscopy and by transmission electron microscopy (TEM). Results showed that most of the vegetative cells and spores, which were stained with malachite green before addition to milk, adhered tightly to the surface of single fat globules, the membranes of which appeared heterogeneous when stained with the fluorescent dye DilC(18)(3)-DS. Using the same dye, we observed transient and persistent interactions among globules, with formation of clusters of different sizes and partial coalescence of adhering membranes. Transmission electron microscopy examination of replicates of freeze-fractured cream allowed us to observe tight adhesion of spores to fat globules. Ultrathin sections revealed that this adhesion is mediated by an amorphous, slightly electron-opaque material, sometimes granular in appearance. Bacteria also adhered to different fat globules, linking them together, which suggests that adhesion was strong enough to maintain a stable contact. Although we cannot exclude physical entrapment of bacteria among fat globule clusters, we show for the first time that most of the bacteria are adhered to fat globules by an electron-opaque material whose nature has yet to be determined. Immunoglobulins are certainly the best candidates for adhesion but other compounds may be involved
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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