http://hdl.handle.net/10468/246 2017-10-29T23:51:19Z http://hdl.handle.net/10468/4774 Simulated gastrointestinal digestion of nisin and interaction between nisin and bile Gough, Ronan; O'Connor, Paula M.; Rea, Mary C.; Gómez-Sala, Beatriz; Miao, Song; Hill, Colin; Brodkorb, André Nisin, an antimicrobial peptide showing activity against many Gram positive bacteria, is widely used as a food preservative. The simulated gastrointestinal digestion of nisin (variant A) was studied using the in vitro INFOGEST digestion method. Following oral, gastric and small intestinal digestion, there was no intact nisin in the system and the nisin was primarily digested by pancreatin. After digestion, six nisin fragments (1–11, 1–12, 1–20, 1–21, 1–29 and 1–32) were identified by reversed phase high performance liquid chromatography and mass spectroscopy and four of these nisin fragments (1–20, 1–21, 1–29 and 1–32) demonstrated low antibacterial activity against Lactococcus lactis HP in agar diffusion activity assays. Additionally, it was observed that bile salts form a complex with nisin. This was examined by atomic force microscopy, turbidity and dynamic light scattering, which showed that this interaction resulted in significantly larger bile salt micelles. The presence of bile salts at physiological levels significantly altered the relative amounts of the nisin fragments 1–12, 1–20 and 1–29 produced during an in vitro digestion. This study highlights the importance of including bile in simulated digestions of antimicrobial peptides in order to obtain a more accurate simulation of the in vivo digestion products and their activity. 2017-08-14T00:00:00Z http://hdl.handle.net/10468/4188 Strain-level metagenomic analysis of the fermented dairy beverage nunu highlights potential food safety risks Walsh, Aaron M.; Crispie, Fiona; Daari, Kareem; O'Sullivan, Orla; Martin, Jennifer C.; Arthur, Cornelius T.; Claesson, Marcus J.; Scott, Karen P.; Cotter, Paul D. The rapid detection of pathogenic strains in food products is essential for the prevention of disease outbreaks. It has already been demonstrated that whole metagenome shotgun sequencing can be used to detect pathogens in food but, until recently, strain-level detection of pathogens has relied on whole metagenome assembly, which is a computationally demanding process. Here, we demonstrate that three short read alignment-based methods, MetaMLST, PanPhlAn, and StrainPhlAn, can accurately, and rapidly, identify pathogenic strains in spinach metagenomes which were intentionally spiked with Shiga toxin-producing Escherichia coli in a previous study. Subsequently, we employ the methods, in combination with other metagenomics approaches, to assess the safety of nunu, a traditional Ghanaian fermented milk product which is produced by the spontaneous fermentation of raw cow milk. We show that nunu samples are frequently contaminated with bacteria associated with the bovine gut, and worryingly, we detect putatively pathogenic E. coli and Klebsiella pneumoniae strains in a subset of nunu samples. Ultimately, our work establishes that short read alignment-based bioinformatics approaches are suitable food safety tools, and we describe a real-life example of their utilisation. Importance: Foodborne pathogens are responsible for millions of illnesses, annually. Here, we demonstrate that short read alignment-based bioinformatics tools can accurately, and rapidly, detect pathogenic strains in food products from shotgun metagenomics data. The methods used here are considerably faster than both traditional culturing methods and alternative bioinformatics approaches that rely on metagenome assembly, and thus they can potentially be used for more high-throughput food safety testing. Overall, our results suggest that whole metagenome sequencing can be used as a practical food safety tool to prevent diseases or link outbreaks to specific food products. 2017-06-16T00:00:00Z http://hdl.handle.net/10468/4189 Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli Mesa-Pereira, Beatriz; O'Connor, Paula M.; Rea, Mary C.; Cotter, Paul D.; Hill, Colin; Ross, R. Paul The bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli TunerTM (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinations of these genes, revealed that the structural and the transporter genes alone are sufficient to permit heterologous production and secretion in this host. Even though the accessory protein, usually associated with optimal disulfide bond formation, was not required for bacteriocin synthesis, its presence did result in greater pediocin PA-1 production. The simplicity of the system and the fact that the associated bacteriocins could be recovered from the extracellular medium provides an opportunity to facilitate protein engineering and the overproduction of biologically-active bacteriocins at industrial scale. Additionally, this system could enable the characterization of new bacteriocin operons where genetic tools are not available for the native producers. 2017-06-08T00:00:00Z http://hdl.handle.net/10468/4080 When regulation challenges innovation: the case of the genus Lactobacillus Salvetti, Elisa; O'Toole, Paul W. The majority of probiotic bacteria belong to the genus Lactobacillus which includes a large number of safe species integral to fermented food production. In the European Union the conversion of ensuing data into successful claims that are compliant with regulatory requirements has proved difficult. Furthermore, the study of lactobacilli has been challenging because of their phenotypic and genomic diversity. Here issues pertaining to the marketing authorization of novel foods and probiotics are outlined, taking Lactobacillus genus as reference. We highlight the drawbacks regarding the taxonomic characterization and the safety assessment of these bacteria and the validation of their beneficial mechanisms. 2017-06-01T00:00:00Z