An investigation into novel bacteriocin producers isolated for shelf life extension of dairy products

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Sugrue, Ivan
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University College Cork
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This thesis investigates extension of the shelf life of dairy products and outlines the discovery and characterization of novel antimicrobials for application in food and health. The results demonstrate factors which affect the quality of yoghurt and extend the shelf life of pasteurised milk, and detail the identification of antimicrobial producing organisms and their characterization. Chapter 1 first describes food-borne pathogens associated with raw milk and the risks associated with its consumption followed by an overview of microbial by-products of dairy fermentation. Metabolites such as biogenic amines, bioactive peptides, and antimicrobial peptides known as bacteriocins which affect the product and the consumer are described. Chapter 2 outlines the production and techno-functional characterization of yoghurt produced using milk derived from different diets, perennial ryegrass (GRS) and total mixed ration (TMR). Using compositional, textural, microbial, and volatile analyses GRS yoghurts were found to contain significantly more protein, less lactose, altered colour and improved late shelf life texture characteristics. TMR yoghurts had increased quantities of volatile compounds associated with natural yoghurt. Chapter 3 describes the application of the well-studied bacteriocin and preservative, nisin A, to extend the shelf life of pasteurised milk. Using a range of concentrations in commercially produced pasteurised milk, nisin A impacted spoilage organisms at 1 - 10 µg ml-1 and completely prevented growth at 1 mg ml-1 as far as 49 days in cold storage. This study determines the efficacy of using a bacteriocin for shelf life extension of pasteurised milk. Chapter 4 details the identification of novel bacteriocin producing lactic acid bacteria using traditional screening methods. Of 823 isolates mainly from raw milk sources, seven strains were identified as putative bacteriocin producers. Using whole genome sequencing and predictive analysis four high quality genomes were generated, each of which contained gene clusters for bacteriocin production. Two closely related but separate Streptococcus sp. strains were found to encode a novel nisin variant, nisin I, detectable by mass spectrometry. A Lactococcus lactis isolate was found to encode a novel two component lantibiotic. One Streptococcus uberis isolate contained four operons for bacteriocin production, one of which encoded a novel two component lantibiotic. During the screen for bacteriocin producing isolates described in chapter 4, a strain of Actinomyces ruminicola (a species and genus without previous recorded bacteriocin production) was identified. Using mass spectrometry, N-terminal amino acid sequencing and comparative genomics, chapter 5 outlines the purification of a novel antimicrobial peptide, actifensin, which could then be identified within the sequenced genome. Using the gene encoding the structural peptide, 161 Actinomyces genomes were searched, finding 47 homologous genes displaying a remarkable level of sequence diversity. Actifensin peptides were found to bear similarity to conserved ubiquitous eukaryotic antimicrobial peptides, defensins. This study highlights conserved antimicrobial structures across kingdoms and describes a novel group of bacteriocins. In Chapter 6 an investigation was carried out into another Actinomyces sp. and other genera harbouring actifensin-like gene clusters. Synthesised actifensin was found to be less active than natural actifensin and lacked highly conserved disulphide bonds. Actinomyces oris CCUG 34286 containing seven afnA copies did not exhibit characteristic actifensin activity. As defensin-like structures are conserved throughout nature, other actifensin-like operons were sought using gene neighbouring analysis of hypothetical proteins within the actifensin operon. Gene clusters were detected across the phylum Actinobacteria bearing similarity to the actifensin operon, but traditional culturing and mass spectrometry did not detect actifensin homolog production. This study identified a range of encoded actifensin-like peptides outside the genus Actinomyces to be investigated for future applications. Overall, the results of this thesis detail quality properties of dairy products and expand upon the current knowledge of antimicrobial peptides.
Food fermentation , Dairy , Antimicrobial , Bacteriocins , Shelf life , Actifensin , Lactic acid bacteria
Sugrue, I. 2020. An investigation into novel bacteriocin producers isolated for shelf life extension of dairy products. PhD Thesis, University College Cork.