Bioengineering of nisin to enhance functionality against dairy pathogens

The bacteriocin class of antimicrobial peptides have emerged as a viable alternative to at least partially fill the void created by the end of the golden age of antibiotic discovery. Along with this potential use in a clinical setting, bacteriocins also play an important role as bio-preservatives in the food industry. This thesis focuses on a specific bacteriocin group, the lantibiotics (Lanthionine-containing antibiotics). Their numerous methods of appliance in a food setting and how their gene-encoded nature can be modified to improve on overall bioactivity and functionality are explored here. The use of a lantibiotic (lacticin 3147) producing starter culture to control the Crohn’s disease-linked pathogen Mycobacterium paratuberculosis was assessed in a raw milk cheese. Although lacticin 3147 production did not effectively control the pathogen, the study provided an impetus to employ a variety of PCR-based mutagenesis techniques with a view to the creation of enhanced lantibiotic derivatives. Through the use of these techniques, a number of enhanced derivatives were generated from the ‘hinge’ region of the nisin peptide. Furthermore, a derivative in which the three hinge amino acids were replaced with three alanines represents the first enhanced derivative of nisin to have been designed through a rational process. This derivative also formed the backbone for the creation of an active, trypsin resistant, variant. Through the employment of further mutagenesis methods a derivative was created with potential use as an oral anti-bacterial in the future. Finally a number of lead nisin derivatives were investigated to assess their anti- Streptococcus agalactiae ability, a mastitis associated pathogen. Also a system was developed to facilitate the large scale production of these candidates, or other nisin derivatives, from dairy substrates.

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.

An assessment of the influence of the industry distribution chain on the oxygen levels in commercial modified atmosphere packaged cheddar cheese using non-destructive oxygen sensor technology

The establishment and control of oxygen levels in packs of oxygen-sensitive food products such as cheese is imperative in order to maintain product quality over a determined shelf life. Oxygen sensors quantify oxygen concentrations within packaging using a reversible optical measurement process, and this non-destructive nature ensures the entire supply chain can be monitored and can assist in pinpointing negative issues pertaining to product packaging. This study was carried out in a commercial cheese packaging plant and involved the insertion of 768 sensors into 384 flow-wrapped cheese packs (two sensors per pack) that were flushed with 100% carbon dioxide prior to sealing. The cheese blocks were randomly assigned to two different storage groups to assess the effects of package quality, packaging process efficiency, and handling and distribution on package containment. Results demonstrated that oxygen levels increased in both experimental groups examined over the 30-day assessment period. The group subjected to a simulated industrial distribution route and handling procedures of commercial retailed cheese exhibited the highest level of oxygen detected on every day examined and experienced the highest rate of package failure. The study concluded that fluctuating storage conditions, product movement associated with distribution activities, and the possible presence of cheese-derived contaminants such as calcium lactate crystals were chief contributors to package failure.

Effect of galactose metabolising and non-metabolising strains of Streptococcus thermophilus as a starter culture adjunct on the properties of Cheddar cheese made with low or high pH at whey drainage

Cheddar cheese was made using control culture (Lactococcus lactis subsp. lactis), or with control culture plus a galactose-metabolising (Gal+) or galactose-non-metabolising (Gal-) Streptococcus thermophilus adjunct; for each culture type, the pH at whey drainage was either low (pH 6.15) or high (pH 6.45). Sc. thermophilus affected the levels of residual lactose and galactose, and the volatile compound profile and sensory properties of the mature cheese (270 d) to an extent dependent on the drain pH and phenotype (Gal+ or Gal-). For all culture systems, reducing drain pH resulted in lower levels of moisture and lactic acid, a higher concentration of free amino acids, and higher firmness. The results indicate that Sc. thermophilus may be used to diversify the sensory properties of Cheddar cheese, for example from a fruity buttery odour and creamy flavour to a more acid taste, rancid odour, and a sweaty cheese flavour at high drain pH.

Autoinducer-2 plays a crucial role in gut colonization and probiotic functionality of Bifidobacterium breve UCC2003

In the present study we show that luxS of Bifidobacterium breve UCC2003 is involved in the production of the interspecies signaling molecule autoinducer-2 (AI-2), and that this gene is essential for gastrointestinal colonization of a murine host, while it is also involved in providing protection against Salmonella infection in Caenorhabditis elegans. We demonstrate that a B. breve luxS-insertion mutant is significantly more susceptible to iron chelators than the WT strain and that this sensitivity can be partially reverted in the presence of the AI-2 precursor DPD. Furthermore, we show that several genes of an iron starvation-induced gene cluster, which are downregulated in the luxS-insertion mutant and which encodes a presumed iron-uptake system, are transcriptionally upregulated under in vivo conditions. Mutation of two genes of this cluster in B. breve UCC2003 renders the derived mutant strains sensitive to iron chelators while deficient in their ability to confer gut pathogen protection to Salmonella-infected nematodes. Since a functional luxS gene is present in all tested members of the genus Bifidobacterium, we conclude that bifidobacteria operate a LuxS-mediated system for gut colonization and pathogen protection that is correlated with iron acquisition.