Ivan Sugrue

Spoilage of pasteurized milk is mainly caused by the presence of organisms that either survive pasteurization (psychrotolerant spore-formers) or re-contaminate milk in the processing environment (post-pasteurisation contaminants). Pasteurization of bovine milk by heat treating at 72°C for 15-30 seconds ensures milk quality without impairing its organoleptic and nutritional status and extends shelf life to 12-14 days at refrigeration temperatures. Nisin A is a class I bacteriocin known to inhibit gram positive bacteria and approved by the European Food and Safety Authority as a food preservative. It is commercially available as Nisaplin®, which contains a concentration of 2.5% w/w of nisin. This study examined the effect of Nisaplin® at different concentrations on spoilage of refrigerated commercial whole pasteurized milk over a period of 59 days. At a high concentration of 4 mgml-1, Nisaplin® reduced the total bacterial count below the limit of detection in the milk, and inhibition was visible for at least 14 days at 4°C. Previously isolated milk spoilage bacteria were identified using 16s rRNA gene sequencing and utilised as target indicators for bacteriocin production. Lactobacillus delbrueckii ssp. bulgaricus LMG6901, Microbacterium lacticum, and Pseudomonas aeruginosa were utilised as indicator strains in a screen of milk spoilage organisms for bacteriocin production. This resulted in identifying the putative bacteriocin producer Carnobacterium divergens, a lactic acid bacterium active against L. bulgaricus. The study concludes that Nisaplin® is effective in the reduction of microbial load and its effectiveness could be increased when combined with other preservative methods thus forming an extra hurdle in the milk.

Nisin A, the prototypical lantibiotic, is an antimicrobial peptide currently utilised as a food preservative, with potential for therapeutic applications. Here, we describe nisin E, a novel nisin variant produced by two Streptococcus equinus strains, APC4007 and APC4008, isolated from sheep milk. Shotgun whole genome sequencing and analysis revealed biosynthetic gene clusters similar to nisin U, with a unique rearrangement of the core peptide encoding gene within the cluster. The 3100.8 Da peptide by MALDI-TOF mass spectrometry, is 75% identical to nisin A, with 10 differences, including 2 deletions: Ser29 and Ile30, and 8 substitutions: Ile4Lys, Gly18Thr, Asn20Pro, Met21Ile, His27Gly, Val32Phe, Ser33Gly, and Lys34Asn. Nisin E producing strains inhibited species of Lactobacillus, Bacillus, and Clostridiodes and were immune to nisin U. Sequence alignment identified putative promoter sequences across the nisin producer genera, allowing for the prediction of genes in Streptococcus to be potentially regulated by nisin. S. equinus pangenome BLAST analyses detected 6 nisin E operons across 44 publicly available genomes. An additional 20 genomes contained a subset of nisin E transport/immunity and regulatory genes (nseFEGRK), without adjacent peptide production genes. These genes suggest that nisin E response mechanisms, distinct from the canonical nisin immunity and resistance operons, are widespread across the S. equinus species. The discovery of this new nisin variant and its immunity determinants in S. equinus suggests a central role for nisin in the competitive nature of the species.

The effects of feeding an 80% plant protein diet, with and without fish protein hydrolysate (FPH) supplementation, on the growth and gut health of Atlantic salmon were investigated. Fish were fed either (A) a control diet containing 35% fishmeal, (B) an 80% plant protein diet with 15% fishmeal, (C) an 80% plant protein diet with 5% fishmeal and 10% partly hydrolysed protein, or (D) an 80% plant protein diet with 5% fishmeal and 10% soluble protein hydrolysate. Fish on the 80% plant- 15% fishmeal diet were significantly smaller than fish in the other dietary groups. However, partly-hydrolysed protein supplementation allowed fish to grow as well as fish fed the control 35% fishmeal diet. Fish fed the FPH diets (diets C and D) had significantly higher levels of amino acids in their blood, including 48% and 27% more branched chain amino acids compared to fish on the 35% fishmeal diet, respectively. Plant protein significantly altered gut microbial composition, significantly decreasing α-diversity. Spirochaetes and the familiesMoritellaceae, Psychromonadaceae, HelicobacteraceaeandBacteroidaceaewere all found at significantly lower abundances in the groups fed 80% plant protein diets compared to the control fishmeal diet.

Bacteriocins (ribosomally produced antimicrobial peptides) are potential alternatives to current antimicrobials given the global challenge of antimicrobial resistance. We identified a novel bacteriocin from Actinomyces ruminicola with no previously characterized antimicrobial activity. Using publicly available genomic data, we found a highly conserved yet divergent family of previously unidentified homologous peptide sequences within the genus Actinomyces with striking similarity to eukaryotic defensins. These actifensins may provide a potent line of antimicrobial defense/offense, and the machinery to produce them could be used for the design of new antimicrobials given the degeneracy that exists naturally in their structure.

The Lactobacillus casei group (LCG), composed of the closely related Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus are some of the most widely researched and applied probiotic species of lactobacilli. The three species have been extensively studied, classified and reclassified due to their health promoting properties. Differentiation is often difficult by conventional phenotypic and genotypic methods and therefore new methods are being continually developed to distinguish the three closely related species. The group remain of interest as probiotics, and their use is widespread in industry. Much research has focused in recent years on their application for health promotion in treatment or prevention of a number of diseases and disorders. The LCG have the potential to be used prophylactically or therapeutically in diseases associated with a disturbance to the gut microbiota. The group have been extensively researched with regard to stress responses, which are crucial for their survival and therefore application as probiotics.

Microbial fermentation has been used historically for the preservation of foods, the health benefits of which have since come to light. Early dairy fermentations depended on the spontaneous activity of the indigenous microbiota of the milk. Modern fermentations rely on defined starter cultures with desirable characteristics to ensure consistency and commercial viability. The selection of defined starters depends on specific phenotypes that benefit the product by guaranteeing shelf life and ensuring safety, texture, and flavour. Lactic acid bacteria can produce a number of bioactive metabolites during fermentation, such as bacteriocins, biogenic amines, exopolysaccharides, and proteolytically released peptides, among others. Prebiotics are added to food fermentations to improve the performance of probiotics. It has also been found that prebiotics fermented in the gut can have benefits that go beyond helping probiotic growth. Studies are now looking at how the fermentation of prebiotics such as fructo-oligosaccharides can help in the prevention of diseases such as osteoporosis, obesity, and colorectal cancer. The potential to prevent or even treat disease through the fermentation of food is a medically and commercially attractive goal and is showing increasing promise. However, the stringent regulation of probiotics is beginning to detrimentally affect the field and limit their application.