Bioengineering strategies to improve functional qualities of nisin

The abuse of antibiotics and the emergence of multi-drug resistant bacterial strains have created the need to explore alternative methods of controlling microbial pathogens. The bacteriocin family of antimicrobial peptides has been proposed as one such alternative to classic antibiotics. Nisin A belongs to the subgroup of bacteriocins called the lantibiotics, which contain several unusual amino acids as a consequence of enzyme-mediated post-translational modifications. As nisin is produced by generally regarded as safe (GRAS) microorganisms, it could potentially be applied in a clinical setting. However, as lantibiotics are naturally produced in such small quantities, this can hinder their industrial potential. In order to overcome this, several approaches can be utilised. For example, given the gene encoded nature of lantibiotics, genetic engineering approaches can be implemented in order to yield variants with enhanced properties. Here, the use of mutagenesis-based strategies was employed to obtain a derivative of nisin with enhanced bioactivity in vitro. Investigations with purified peptide highlighted the enhanced specific activity of this variant, nisin M21V, against food-borne Listeria monocytogenes strains. Furthermore, this specific enhanced bioactivity was evident in a mouse model of listeriosis. Reductions in bioluminescence and microbial counts in organs from infected mice were observed following treatment with nisin M21V compared to that of wild-type nisin A. Peptide bioengineering approaches were also implemented to obtain additional novel derivatives of nisin. The generation of “S5X” and “S33X” banks (representing a change of natural serines at positions 5 and 33 to all possible alternative residues) by a combination of site-saturation and site-directed mutagenesis led to the identification of several derivatives exhibiting improved stability. This allowed the rational design of variants with enhanced stability compared to that of wild type nisin. Another means of tackling issues associated with lantibiotic yield is to combine lantibiotics with other antimicrobials. This could circumvent the need for enhanced production while also reducing concentrations of the peptide antimicrobials. We observed that combinations of nisin variants and low levels of plant essential oils (thymol, carvacrol, trans-cinnamaldehyde) significantly controlled Gram negative foodborne pathogens in in vitro assays compared to nisin A-essential oil combinations. This enhanced control was also evident in model food systems. Nisin variants used in conjunction with carvacrol significantly reduced numbers of E. coli O157:H7 in apple juice while a commercial nisin preparation used in combination with citric acid significantly controlled C. sakazakii in infant milk formula. It is noteworthy that while nisin is generally associated with Gram positive targets, upon combination with plant essential oils the spectrum of inhibition was broadened to Gram negative targets.

The application of next generation sequencing to profile microbe related cheese quality defects

High throughput next generation sequencing, together with advanced molecular methods, has considerably enhanced the field of food microbiology. By overcoming biases associated with culture dependant approaches, it has become possible to achieve novel insights into the nature of food-borne microbial communities. In this thesis, several different sequencing-based approaches were applied with a view to better understanding microbe associated quality defects in cheese. Initially, a literature review provides an overview of microbe-associated cheese quality defects as well as molecular methods for profiling complex microbial communities. Following this, 16S rRNA sequencing revealed temporal and spatial differences in microbial composition due to the time during the production day that specific commercial cheeses were manufactured. A novel Ion PGM sequencing approach, focusing on decarboxylase genes rather than 16S rRNA genes, was then successfully employed to profile the biogenic amine producing cohort of a series of artisanal cheeses. Investigations into the phenomenon of cheese pinking formed the basis of a joint 16S rRNA and whole genome shotgun sequencing approach, leading to the identification of Thermus species and, more specifically, the pathway involved in production of lycopene, a red coloured carotenoid. Finally, using a more traditional approach, the effect of addition of a facultatively heterofermentative Lactobacillus (Lactobacillus casei) to a Swiss-type cheese, in which starter activity was compromised, was investigated from the perspective of its ability to promote gas defects and irregular eye formation. X-ray computed tomography was used to visualise, using a non-destructive method, the consequences of the undesirable gas formation that resulted. Ultimately this thesis has demonstrated that the application of molecular techniques, such as next generation sequencing, can provide a detailed insight into defect-causing microbial populations present and thereby may underpin approaches to optimise the quality and consistency of a wide variety of cheeses.