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Title: Actinomycete diversity of European smear-ripened cheeses
Author: Bora, Nagamani
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2011
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Regional cheeses in Europe, including red smear cheeses, are part of the cultural heritage of their source region and an important, traditional industry. However, the incidence of Listeria, including Listeria monocytogenes, has been relatively high, and despite extensive upgrading of hygiene in the production of these cheeses across Europe it has not decreased. The anti-listerial activity of the complex microbial surface flora has been seen as protective in their production, and the use of defined microbial inocula, or the manipulation of the surface microflora, as tools in the fight against contamination by Listeria. However, the microbial flora, bacteria and yeast, especially bacteria, on different red smear cheeses have been poorly defined and identified. The surface microflora needs to be properly identified to enable the selection of strains which generate the colour, aroma and organoleptic qualities of specific cheeses and to screen for anti-listerial activity – identifying the bacterial flora was the subject of this study. In this project 1424 isolates from 5 European red smear cheeses were dereplicated by rep-PCR and representatives of the rep-PCR clusters sent for comparison with reference strains at the Belgium Coordinated Collections of Microorganisms (BCCM) Laboratorium voor Microbiologie, Universiteit Gent (LMG) culture collection, Ghent. The 16S rRNA genes of unidentified strains were sequenced and novel strains subjected to a full polyphasic taxonomic identification. Four novel species were described Agrococcus casei, Mycetocola reblochoni, Brevibacterium superficiens and Leucobacter lepicola. The remaining diversity of isolates, as identified using molecular fingerprints (rep-PCR, this thesis, and Box PCR, Ghent) and 16S rDNA sequencing from 3 stages of ripening for each of 5 cheeses gave a comparative overview of the bacteria and their variation, across cheeses and with stage of ripening. Although many of the strains recovered were identified to known species, the molecular characterisation showed extensive sub-specific variation. This was exemplified by the isolation of Brevibacterium linens. Although a commercial strain of B. linens was typically inoculated with the initial smear wash and B. linens was isolated from many cheese surface samples, the strains isolated were not the same as the strains used for inoculation. Common species of Brevibacterium, Corynebacterium and Microbacterium were isolated from multiple cheeses. vi Following the introduction of molecular ecological techniques it has become clear that isolation techniques give a biased view of community composition (Tabacchioni et al., 2000), often missing dominant members of the community and isolating rapid growing but numerically insignificant strains. Therefore denaturing gradient gel electrophoresis (DGGE) was applied to DNA extracted and amplified from 15 duplicate samples of the surface microflora, from 3 stages of ripening and from 5 cheeses. Extensive optimisations of procedures for the DGGE method in terms of DNA extraction procedures to the use of GC clamped primers were developed on Tilsit. The DNA extraction method was optimised and a nested PCR approach, using actinobacterial specific primers for the 16S gene, was utilised to generate DGGE profiles for all five smear cheeses at early, middle and late ripening stages. Selected bands were excised and sequenced. A combined approach of cloning with DGGE, with Tilsit and Livarot cheeses, gave similar results showing the robustness of the method. The molecular analysis revealed different complex patterns of bands for each cheese, and changes with ripening stage. However, part of the complexity was probably due to single strand DNA leading to multiple sequences present in single bands and multiple bands from the same sequence. Excision of bands, re-amplification and re-analysis generated multiple single sequence bands that could be sequenced. Most of the species revealed by molecular analysis using DGGE and sequencing were similar to recognised species of Brevibacterium, Corynebacterium and Microbacterium though not always assigned to the same species as the isolates from the same cheese sample. Some unexpected species were detected such as Citrococcus and Propionibacterium. The project provided a comprehensive overview of the actinobacterial flora present on five red smear cheeses and provides the source information for the future study of anti-listerial properties and to develop defined inocula. However, the example of Brevibacterium linens and the extensive sub-specific variation detected may indicate that commercially developed inocula may still be outcompeted by natural flora.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available