• Association of bovine leptin polymorphisms with energy output and energy storage traits in progeny tested Holstein-Friesian dairy cattle sires

      Giblin, Linda; Butler, Stephen T.; Kearney, Breda M.; Waters, Sinead M.; Callanan, Michael J.; Berry, Donagh; Department of Agriculture, Food and the Marine, Ireland; Irish Dairy Levy Research Trust; Teagasc Walsh Fellowship Programme; RSF-06-0353; et al. (Biomed Central, 29/07/2010)
      Background: Leptin modulates appetite, energy expenditure and the reproductive axis by signalling via its receptor the status of body energy stores to the brain. The present study aimed to quantify the associations between 10 novel and known single nucleotide polymorphisms in genes coding for leptin and leptin receptor with performance traits in 848 Holstein-Friesian sires, estimated from performance of up to 43,117 daughter-parity records per sire. Results: All single nucleotide polymorphisms were segregating in this sample population and none deviated (P > 0.05) from Hardy-Weinberg equilibrium. Complete linkage disequilibrium existed between the novel polymorphism LEP-1609, and the previously identified polymorphisms LEP-1457 and LEP-580. LEP-2470 associated (P < 0.05) with milk protein concentration and calf perinatal mortality. It had a tendency to associate with milk yield (P < 0.1). The G allele of LEP-1238 was associated (P < 0.05) with reduced milk fat concentration, reduced milk protein concentration, longer gestation length and tended to associate (P < 0.1) with an increase in calving difficulty, calf perinatal mortality and somatic cells in the milk. LEP-963 exhibited an association (P < 0.05) with milk fat concentration, milk protein concentration, calving difficulty and gestation length. It also tended to associate with milk yield (P < 0.1). The R25C SNP associated (P < 0.05) with milk fat concentration, milk protein concentration, calving difficulty and length of gestation. The T allele of the Y7F SNP significantly associated with reduced angularity (P < 0.01) and reduced milk protein yield (P < 0.05). There was also a tendency (P < 0.1) for Y7F to associate with increased body condition score, reduced milk yield and shorter gestation (P < 0.1). A80V associated with reduced survival in the herd (P < 0.05). Conclusions Several leptin polymorphisms (LEP-2470, LEP-1238, LEP-963, Y7F and R25C) associated with the energetically expensive process of lactogenesis. Only SNP Y7F associated with energy storage. Associations were also observed between leptin polymorphisms and calving difficulty, gestation length and calf perinatal mortality. The lack of an association between the leptin variants investigated with calving interval in this large data set would question the potential importance of these leptin variants, or indeed leptin, in selection for improved fertility in the Holstein-Friesian dairy cow.
    • Bacteriocins: Novel Solutions to Age Old Spore-Related Problems?

      Egan, Kevin; Field, Des; Rea, Mary; Ross, R Paul; Hill, Colin; Cotter, Paul D.; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; DAFM 13/F/462; TIDA 14/TIDA/2286; et al. (Frontiers Media S. A., 08/04/2016)
      Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria, which have the ability to kill or inhibit other bacteria. Many bacteriocins are produced by food grade lactic acid bacteria (LAB). Indeed, the prototypic bacteriocin, nisin, is produced by Lactococcus lactis, and is licensed in over 50 countries. With consumers becoming more concerned about the levels of chemical preservatives present in food, bacteriocins offer an alternative, more natural approach, while ensuring both food safety and product shelf life. Bacteriocins also show additive/synergistic effects when used in combination with other treatments, such as heating, high pressure, organic compounds, and as part of food packaging. These features are particularly attractive from the perspective of controlling sporeforming bacteria. Bacterial spores are common contaminants of food products, and their outgrowth may cause food spoilage or food-borne illness. They are of particular concern to the food industry due to their thermal and chemical resistance in their dormant state. However, when spores germinate they lose the majority of their resistance traits, making them susceptible to a variety of food processing treatments. Bacteriocins represent one potential treatment as they may inhibit spores in the post-germination/outgrowth phase of the spore cycle. Spore eradication and control in food is critical, as they are able to spoil and in certain cases compromise the safety of food by producing dangerous toxins. Thus, understanding the mechanisms by which bacteriocins exert their sporostatic/sporicidal activity against bacterial spores will ultimately facilitate their optimal use in food. This review will focus on the use of bacteriocins alone, or in combination with other innovative processing methods to control spores in food, the current knowledge and gaps therein with regard to bacteriocin-spore interactions and discuss future research approaches to enable spores to be more effectively targeted by bacteriocins in food settings.
    • Bactofencin A, a New Type of Cationic Bacteriocin with Unusual Immunity

      O'Shea, Eileen F.; O'Connor, Paula M.; O'Sullivan, Orla; Cotter, Paul D.; Ross, R Paul; Hill, Colin; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; 04R; 07/CE/B1368 (American Society for Microbiology, 29/10/2013)
      Bacteriocin production is an important probiotic trait of intestinal bacteria. In this study, we identify a new type of bacteriocin, bactofencin A, produced by a porcine intestinal isolate Lactobacillus salivarius DPC6502, and assess its potency against pathogenic species including Staphylococcus aureus and Listeria monocytogenes. Genome sequencing of the bacteriocin producer revealed bfnA, which encodes the mature and highly basic (pI 10.59), 22-amino-acid defensin-like peptide. Matrixassisted laser desorption ionization–time of flight (MALDI-TOF) mass spectral analysis determined that bactofencin A has a molecular mass of 2,782 Da and contains two cysteine residues that form an intramolecular disulfide bond. Although an ABC transporter and transport accessory protein were also present within the bacteriocin gene cluster, a classical bacteriocin immunity gene was not detected. Interestingly, a dltB homologue was identified downstream of bfnA. DltB is usually encoded within the dlt operon of many Gram-positive bacteria. It is responsible for D-alanylation of teichoic acids in the cell wall and has previously been associated with bacterial resistance to cationic antimicrobial peptides. Heterologous expression of this gene conferred bactofencin A-specific immunity on sensitive strains of L. salivarius and S. aureus (although not L. monocytogenes), establishing its role in bacteriocin immunity. An analysis of the distribution of bfnA revealed that it was present in four additional isolates derived from porcine origin and absent from five human isolates, suggesting that its distribution is host specific. Given its novelty, we anticipate that bactofencin A represents the prototype of a new class of bacteriocins characterized as being cationic, with a DltB homologue providing a cognate immunity function.
    • Comparative Analysis of Pyrosequencing and a Phylogenetic Microarray for Exploring Microbial Community Structures in the Human Distal Intestine

      Claesson, Marcus J.; O'Sullivan, Orla; Wang, Qiong; Nikkila, Janne; Marchesi, Julian R.; Smidt, Hauka; de Vos, Willem M.; Ross, R Paul; O'Toole, Paul W.; Department of Agriculture, Food and the Marine, Ireland; et al. (PLOS, 20/08/2009)
      Background: Variations in the composition of the human intestinal microbiota are linked to diverse health conditions. Highthroughput molecular technologies have recently elucidated microbial community structure at much higher resolution than was previously possible. Here we compare two such methods, pyrosequencing and a phylogenetic array, and evaluate classifications based on two variable 16S rRNA gene regions. Methods and Findings: Over 1.75 million amplicon sequences were generated from the V4 and V6 regions of 16S rRNA genes in bacterial DNA extracted from four fecal samples of elderly individuals. The phylotype richness, for individual samples, was 1,400–1,800 for V4 reads and 12,500 for V6 reads, and 5,200 unique phylotypes when combining V4 reads from all samples. The RDP-classifier was more efficient for the V4 than for the far less conserved and shorter V6 region, but differences in community structure also affected efficiency. Even when analyzing only 20% of the reads, the majority of the microbial diversity was captured in two samples tested. DNA from the four samples was hybridized against the Human Intestinal Tract (HIT) Chip, a phylogenetic microarray for community profiling. Comparison of clustering of genus counts from pyrosequencing and HITChip data revealed highly similar profiles. Furthermore, correlations of sequence abundance and hybridization signal intensities were very high for lower-order ranks, but lower at family-level, which was probably due to ambiguous taxonomic groupings. Conclusions: The RDP-classifier consistently assigned most V4 sequences from human intestinal samples down to genuslevel with good accuracy and speed. This is the deepest sequencing of single gastrointestinal samples reported to date, but microbial richness levels have still not leveled out. A majority of these diversities can also be captured with five times lower sampling-depth. HITChip hybridizations and resulting community profiles correlate well with pyrosequencing-based compositions, especially for lower-order ranks, indicating high robustness of both approaches. However, incompatible grouping schemes make exact comparison difficult.
    • Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation

      Kelleher, Philip; Bottacini, Francesca; Mahony, Jennifer; Kilcawley, Kieran; van Sinderen, Douwe; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; 10/RD/TMFRC/704; 13/IA/1953; 14/TIDA/2287; et al. (Biomed Central, 29/03/2017)
      Background Lactococcus lactis is among the most widely studied lactic acid bacterial species due to its long history of safe use and economic importance to the dairy industry, where it is exploited as a starter culture in cheese production. Results In the current study, we report on the complete sequencing of 16 L. lactis subsp. lactis and L. lactis subsp. cremoris genomes. The chromosomal features of these 16 L. lactis strains in conjunction with 14 completely sequenced, publicly available lactococcal chromosomes were assessed with particular emphasis on discerning the L. lactis subspecies division, evolution and niche adaptation. The deduced pan-genome of L. lactis was found to be closed, indicating that the representative data sets employed for this analysis are sufficient to fully describe the genetic diversity of the taxon. Conclusions Niche adaptation appears to play a significant role in governing the genetic content of each L. lactis subspecies, while (differential) genome decay and redundancy in the dairy niche is also highlighted.
    • Comparative Genomic Analysis Reveals a Diverse Repertoire of Genes Involved in Prokaryote-Eukaryote Interactions within the Pseudovibrio Genus

      Romano, Stefano; Fernandez-Guerra, Antonio; Reen, F. Jerry; Glockner, Frank O.; Crowley, Susan P.; O'Sullivan, Orla; Cotter, Paul D.; Adams, Claire; Dobson, Alan D. W.; O'Gara, Fergal; et al. (Frontiers Media S. A., 30/03/2016)
      Strains of the Pseudovibrio genus have been detected worldwide, mainly as part of bacterial communities associated with marine invertebrates, particularly sponges. This recurrent association has been considered as an indication of a symbiotic relationship between these microbes and their host. Until recently, the availability of only two genomes, belonging to closely related strains, has limited the knowledge on the genomic and physiological features of the genus to a single phylogenetic lineage. Here we present 10 newly sequenced genomes of Pseudovibrio strains isolated from marine sponges from the west coast of Ireland, and including the other two publicly available genomes we performed an extensive comparative genomic analysis. Homogeneity was apparent in terms of both the orthologous genes and the metabolic features shared amongst the 12 strains. At the genomic level, a key physiological difference observed amongst the isolates was the presence only in strain P. axinellae AD2 of genes encoding proteins involved in assimilatory nitrate reduction, which was then proved experimentally. We then focused on studying those systems known to be involved in the interactions with eukaryotic and prokaryotic cells. This analysis revealed that the genus harbors a large diversity of toxin-like proteins, secretion systems and their potential effectors. Their distribution in the genus was not always consistent with the phylogenetic relationship of the strains. Finally, our analyses identified new genomic islands encoding potential toxin-immunity systems, previously unknown in the genus. Our analyses shed new light on the Pseudovibrio genus, indicating a large diversity of both metabolic features and systems for interacting with the host. The diversity in both distribution and abundance of these systems amongst the strains underlines how metabolically and phylogenetically similar bacteria may use different strategies to interact with the host and find a niche within its microbiota. Our data suggest the presence of a sponge-specific lineage of Pseudovibrio. The reduction in genome size and the loss of some systems potentially used to successfully enter the host, leads to the hypothesis that P. axinellae strain AD2 may be a lineage that presents an ancient association with the host and that may be vertically transmitted to the progeny.
    • Detection and Enumeration of Spore-Forming Bacteria in Powdered Dairy Products

      McHugh, Aoife; Feehily, Conor; Hill, Colin; Cotter, Paul D.; Department of Agriculture, Food and the Marine, Ireland (Frontiers, 31/01/2017)
      With the abolition of milk quotas in the European Union in 2015, several member states including Ireland, Luxembourg, and Belgium have seen year on year bi-monthly milk deliveries to dairies increase by up to 35%. Milk production has also increased outside of Europe in the past number of years. Unsurprisingly, there has been a corresponding increased focus on the production of dried milk products for improved shelf life. These powders are used in a wide variety of products, including confectionery, infant formula, sports dietary supplements and supplements for health recovery. To ensure quality and safety standards in the dairy sector, strict controls are in place with respect to the acceptable quantity and species of microorganisms present in these products. A particular emphasis on spore-forming bacteria is necessary due to their inherent ability to survive extreme processing conditions. Traditional microbiological detection methods used in industry have limitations in terms of time, efficiency, accuracy, and sensitivity. The following review will explore the common spore-forming bacterial contaminants of milk powders, will review the guidelines with respect to the acceptable limits of these microorganisms and will provide an insight into recent advances in methods for detecting these microbes. The various advantages and limitations with respect to the application of these diagnostics approaches for dairy food will be provided. It is anticipated that the optimization and application of these methods in appropriate ways can ensure that the enhanced pressures associated with increased production will not result in any lessening of safety and quality standards.
    • Effect of pre-treatment on the generation of dipeptidyl peptidase-IV- and prolyl endopeptidase-inhibitory hydrolysates from bovine lung

      Lafarga, Tomas; Hayes, Maria; Department of Agriculture, Food and the Marine, Ireland; Teagasc Walsh Fellowship Programme; 11/F/043 (Teagasc (Agriculture and Food Development Authority), Ireland, 25/05/2017)
      The aim of this work was to study the effect of two different pre-treatments, high temperature (100 °C, 5 min) and high pressure (600 MPa, 3 min), on the potential of the enzymes papain, collagenase and Alcalase® to generate bioactive hydrolysates containing dipeptidyl peptidase-IV- (DPP-IV; EC 3.4.14.5) and prolyl endopeptidase- (PEP; EC 3.4.21.26) inhibitory peptides from bovine lung. Both pre-treatments resulted in an increase in the degree of hydrolysis over a 24 h period (P < 0.001) and significantly increased the DPP-IV- and PEP-inhibitory activities of the generated hydrolysates (P < 0.001). Generated hydrolysates included an Alcalase hydrolysate of pressure-treated bovine lung, which was the most active, and showed DPP-IV and PEP half-maximal inhibitory concentration (IC50) values of 1.43 ± 0.06 and 3.62 ± 0.07 mg/ mL, respectively. The major peptides contained in this hydrolysate were determined by liquid chromatography-tandem mass spectrometry, and results demonstrated that bovine lung is a good substrate for the release of bioactive peptides when proper pre-treatment and enzymatic treatment are applied.
    • Encapsulation of a Lactic Acid Bacteria Cell-Free Extract in Liposomes and Use in Cheddar Cheese Ripening

      Nongonierma, Alice B.; Abrlova, Magdalena; Kilcawley, Kieran; Department of Agriculture, Food and the Marine, Ireland (MDPI AG., Basel, Switzerland, 13/03/2013)
      A concentrated form of cell free extract (CFE) derived from attenuated Lactococcus lactis supsb. lactis 303 CFE was encapsulated in liposomes prepared from two different proliposome preparations (Prolipo Duo and Prolipo S) using microfluidization. Entrapment efficiencies of 19.7 % (Prolipo S) and 14.0 % (Prolipo Duo) were achieved and the preparations mixed in the ratio 4 (Prolipo Duo):1 (Prolipo S). Cheddar cheese trials were undertaken evaluating the performance of CFE entrapped in liposomes, empty liposomes and free CFE in comparison to a control cheese without any CFE or liposomes. Identical volumes of liposome and amounts of CFE were used in triplicate trials. The inclusion of liposomes did not adversely impact on cheese composition water activity, or microbiology. Entrapment of CFE in liposomes reduced loss of CFE to the whey. No significant differences were evident in proteolysis or expressed PepX activity during ripening in comparison to the cheeses containing free CFE, empty liposomes or the control, as the liposomes did not degrade during ripening. This result highlights the potential of liposomes to minimize losses of encapsulated enzymes into the whey during cheese production but also highlights the need to optimize the hydrophobicity, zeta potential, size and composition of the liposomes to maximize their use as vectors for enzyme addition in cheese to augment ripening.
    • Future Protein Supply and Demand: Strategies and Factors Influencing a Sustainable Equilibrium

      Henchion, Maeve; Hayes, Maria; Mullen, Anne Maria; Fenelon, Mark; Tiwari, Brijesh K; Department of Agriculture, Food and the Marine, Ireland; 11/F/043 (MDPI, 20/07/2017)
      A growing global population, combined with factors such as changing socio-demographics, will place increased pressure on the world’s resources to provide not only more but also different types of food. Increased demand for animal-based protein in particular is expected to have a negative environmental impact, generating greenhouse gas emissions, requiring more water and more land. Addressing this “perfect storm” will necessitate more sustainable production of existing sources of protein as well as alternative sources for direct human consumption. This paper outlines some potential demand scenarios and provides an overview of selected existing and novel protein sources in terms of their potential to sustainably deliver protein for the future, considering drivers and challenges relating to nutritional, environmental, and technological and market/consumer domains. It concludes that different factors influence the potential of existing and novel sources. Existing protein sources are primarily hindered by their negative environmental impacts with some concerns around health. However, they offer social and economic benefits, and have a high level of consumer acceptance. Furthermore, recent research emphasizes the role of livestock as part of the solution to greenhouse gas emissions, and indicates that animal-based protein has an important role as part of a sustainable diet and as a contributor to food security. Novel proteins require the development of new value chains, and attention to issues such as production costs, food safety, scalability and consumer acceptance. Furthermore, positive environmental impacts cannot be assumed with novel protein sources and care must be taken to ensure that comparisons between novel and existing protein sources are valid. Greater alignment of political forces, and the involvement of wider stakeholders in a governance role, as well as development/commercialization role, is required to address both sources of protein and ensure food security.
    • Future Protein Supply and Demand: Strategies and Factors Influencing a Sustainable Equilibrium

      Henchion, Maeve; Hayes, Maria; Mullen, Anne Maria; Fenelon, Mark; Tiwari, Brijesh K; Department of Agriculture, Food and the Marine, Ireland (MDPI, 20/07/2017)
      A growing global population, combined with factors such as changing socio-demographics, will place increased pressure on the world’s resources to provide not only more but also different types of food. Increased demand for animal-based protein in particular is expected to have a negative environmental impact, generating greenhouse gas emissions, requiring more water and more land. Addressing this “perfect storm” will necessitate more sustainable production of existing sources of protein as well as alternative sources for direct human consumption. This paper outlines some potential demand scenarios and provides an overview of selected existing and novel protein sources in terms of their potential to sustainably deliver protein for the future, considering drivers and challenges relating to nutritional, environmental, and technological and market/consumer domains. It concludes that different factors influence the potential of existing and novel sources. Existing protein sources are primarily hindered by their negative environmental impacts with some concerns around health. However, they offer social and economic benefits, and have a high level of consumer acceptance. Furthermore, recent research emphasizes the role of livestock as part of the solution to greenhouse gas emissions, and indicates that animal-based protein has an important role as part of a sustainable diet and as a contributor to food security. Novel proteins require the development of new value chains, and attention to issues such as production costs, food safety, scalability and consumer acceptance. Furthermore, positive environmental impacts cannot be assumed with novel protein sources and care must be taken to ensure that comparisons between novel and existing protein sources are valid. Greater alignment of political forces, and the involvement of wider stakeholders in a governance role, as well as development/commercialization role, is required to address both sources of protein and ensure food security
    • Gene-trait matching across the Bifidobacterium longum pan-genome reveals considerable diversity in carbohydrate catabolism among human infant strains

      Arboleya, Silvia; Bottacini, Francesca; O'Connell-Motherway, Mary; Ryan, C. Anthony; Ross, R Paul; van Sinderen, Douwe; STANTON, CATHERINE; Science Foundation Ireland; Department of Agriculture, Food and the Marine, Ireland; SFI/12/RC/2273; et al. (Biomed Central, 08/01/2018)
      Background Bifidobacterium longum is a common member of the human gut microbiota and is frequently present at high numbers in the gut microbiota of humans throughout life, thus indicative of a close symbiotic host-microbe relationship. Different mechanisms may be responsible for the high competitiveness of this taxon in its human host to allow stable establishment in the complex and dynamic intestinal microbiota environment. The objective of this study was to assess the genetic and metabolic diversity in a set of 20 B. longum strains, most of which had previously been isolated from infants, by performing whole genome sequencing and comparative analysis, and to analyse their carbohydrate utilization abilities using a gene-trait matching approach. Results We analysed their pan-genome and their phylogenetic relatedness. All strains clustered in the B. longum ssp. longum phylogenetic subgroup, except for one individual strain which was found to cluster in the B. longum ssp. suis phylogenetic group. The examined strains exhibit genomic diversity, while they also varied in their sugar utilization profiles. This allowed us to perform a gene-trait matching exercise enabling the identification of five gene clusters involved in the utilization of xylo-oligosaccharides, arabinan, arabinoxylan, galactan and fucosyllactose, the latter of which is an abundant human milk oligosaccharide (HMO). Conclusions The results showed high diversity in terms of genes and predicted glycosyl-hydrolases, as well as the ability to metabolize a large range of sugars. Moreover, we corroborate the capability of B. longum ssp. longum to metabolise HMOs. Ultimately, their intraspecific genomic diversity and the ability to consume a wide assortment of carbohydrates, ranging from plant-derived carbohydrates to HMOs, may provide an explanation for the competitive advantage and persistence of B. longum in the human gut microbiome.
    • Gene-trait matching across the Bifidobacterium longum pan-genome reveals considerable diversity in carbohydrate catabolism among human infant strains

      Arboleya, Silvia; Bottacini, Francesca; O'Connell-Motherway, Mary; Ryan, C. Anthony; Ross, R Paul; van Sinderen, Douwe; STANTON, CATHERINE; Science Foundation Ireland; Department of Agriculture, Food and the Marine, Ireland; SFI/12/RC/2273; et al. (Biomed Central, 08/01/2018)
      Background Bifidobacterium longum is a common member of the human gut microbiota and is frequently present at high numbers in the gut microbiota of humans throughout life, thus indicative of a close symbiotic host-microbe relationship. Different mechanisms may be responsible for the high competitiveness of this taxon in its human host to allow stable establishment in the complex and dynamic intestinal microbiota environment. The objective of this study was to assess the genetic and metabolic diversity in a set of 20 B. longum strains, most of which had previously been isolated from infants, by performing whole genome sequencing and comparative analysis, and to analyse their carbohydrate utilization abilities using a gene-trait matching approach. Results We analysed their pan-genome and their phylogenetic relatedness. All strains clustered in the B. longum ssp. longum phylogenetic subgroup, except for one individual strain which was found to cluster in the B. longum ssp. suis phylogenetic group. The examined strains exhibit genomic diversity, while they also varied in their sugar utilization profiles. This allowed us to perform a gene-trait matching exercise enabling the identification of five gene clusters involved in the utilization of xylo-oligosaccharides, arabinan, arabinoxylan, galactan and fucosyllactose, the latter of which is an abundant human milk oligosaccharide (HMO). Conclusions The results showed high diversity in terms of genes and predicted glycosyl-hydrolases, as well as the ability to metabolize a large range of sugars. Moreover, we corroborate the capability of B. longum ssp. longum to metabolise HMOs. Ultimately, their intraspecific genomic diversity and the ability to consume a wide assortment of carbohydrates, ranging from plant-derived carbohydrates to HMOs, may provide an explanation for the competitive advantage and persistence of B. longum in the human gut microbiome.
    • Gene-trait matching across the Bifidobacterium longum pan-genome reveals considerable diversity in carbohydrate catabolism among human infant strains

      Arboleya, Silvia; Bottacini, Francesca; O'Connell-Motherway, Mary; Ryan, C. Anthony; Ross, R Paul; van Sinderen, Douwe; STANTON, CATHERINE; Science Foundation Ireland; Department of Agriculture, Food and the Marine, Ireland; SFI/12/RC/2273; et al. (Biomed Central, 08/01/2018)
      Background Bifidobacterium longum is a common member of the human gut microbiota and is frequently present at high numbers in the gut microbiota of humans throughout life, thus indicative of a close symbiotic host-microbe relationship. Different mechanisms may be responsible for the high competitiveness of this taxon in its human host to allow stable establishment in the complex and dynamic intestinal microbiota environment. The objective of this study was to assess the genetic and metabolic diversity in a set of 20 B. longum strains, most of which had previously been isolated from infants, by performing whole genome sequencing and comparative analysis, and to analyse their carbohydrate utilization abilities using a gene-trait matching approach. Results We analysed their pan-genome and their phylogenetic relatedness. All strains clustered in the B. longum ssp. longum phylogenetic subgroup, except for one individual strain which was found to cluster in the B. longum ssp. suis phylogenetic group. The examined strains exhibit genomic diversity, while they also varied in their sugar utilization profiles. This allowed us to perform a gene-trait matching exercise enabling the identification of five gene clusters involved in the utilization of xylo-oligosaccharides, arabinan, arabinoxylan, galactan and fucosyllactose, the latter of which is an abundant human milk oligosaccharide (HMO). Conclusions The results showed high diversity in terms of genes and predicted glycosyl-hydrolases, as well as the ability to metabolize a large range of sugars. Moreover, we corroborate the capability of B. longum ssp. longum to metabolise HMOs. Ultimately, their intraspecific genomic diversity and the ability to consume a wide assortment of carbohydrates, ranging from plant-derived carbohydrates to HMOs, may provide an explanation for the competitive advantage and persistence of B. longum in the human gut microbiome.
    • Invited review: Whey proteins as antioxidants and promoters of cellular antioxidant pathways

      Corrochano, Alberto R.; Buckin, Vitaly; Kelly, Philip; Giblin, Linda; Department of Agriculture, Food and the Marine, Ireland; Teagasc Walsh Fellowship Programme; 13 F 454; 13 F 454-WheyGSH (Elsevier for American Dairy Science Association, 28/03/2018)
      Oxidative stress contributes to cell injury and aggravates several chronic diseases. Dietary antioxidants help the body to fight against free radicals and, therefore, avoid or reduce oxidative stress. Recently, proteins from milk whey liquid have been described as antioxidants. This review summarizes the evidence that whey products exhibit radical scavenging activity and reducing power. It examines the processing and treatment attempts to increase the antioxidant bioactivity and identifies 1 enzyme, subtilisin, which consistently produces the most potent whey fractions. The review compares whey from different milk sources and puts whey proteins in the context of other known food antioxidants. However, for efficacy, the antioxidant activity of whey proteins must not only survive processing, but also upper gut transit and arrival in the bloodstream, if whey products are to promote antioxidant levels in target organs. Studies reveal that direct cell exposure to whey samples increases intracellular antioxidants such as glutathione. However, the physiological relevance of these in vitro assays is questionable, and evidence is conflicting from dietary intervention trials, with both rats and humans, that whey products can boost cellular antioxidant biomarkers.
    • Mesophilic sporeformers identified in whey powder by using shotgun metagenomic sequencing

      McHugh, Aoife; Feehily, Conor; Tobin, John; Fenelon, Mark; Hill, Colin; Cotter, Paul D.; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; 14/F/883; 11/P1/1137 (American Society for Microbiology, 01/10/2018)
      Spoilage and pathogenic spore-forming bacteria are a major cause of concern for producers of dairy products. Traditional agar-based detection methods employed by the dairy industry have limitations with respect to their sensitivity and specificity. The aim of this study was to identify low-abundance sporeformers in samples of a powdered dairy product, whey powder, produced monthly over 1 year, using novel culture-independent shotgun metagenomics-based approaches. Although mesophilic sporeformers were the main target of this study, in one instance thermophilic sporeformers were also targeted using this culture-independent approach. For comparative purposes, mesophilic and thermophilic sporeformers were also tested for within the same sample using culture-based approaches. Ultimately, the approaches taken highlighted differences in the taxa identified due to treatment and isolation methods. Despite this, low levels of transient, mesophilic, and in some cases potentially pathogenic sporeformers were consistently detected in powder samples. Although the specific sporeformers changed from one month to the next, it was apparent that 3 groups of mesophilic sporeformers, namely, Bacillus cereus, Bacillus licheniformis/Bacillus paralicheniformis, and a third, more heterogeneous group containing Brevibacillus brevis, dominated across the 12 samples. Total thermophilic sporeformer taxonomy was considerably different from mesophilic taxonomy, as well as from the culturable thermophilic taxonomy, in the one sample analyzed by all four approaches. Ultimately, through the application of shotgun metagenomic sequencing to dairy powders, the potential for this technology to facilitate the detection of undesirable bacteria present in these food ingredients is highlighted.
    • Oral Delivery of Nisin in Resistant Starch Based Matrices Alters the Gut Microbiota in Mice

      Gough, Ronan; Cabrera-Rubio, Raul; O'Connor, Paula M.; Crispie, Fiona; Miao, Song; Hill, Colin; Ross, R Paul; Cotter, Paul D.; Nilaweera, Kanishka; Rea, Mary; et al. (Frontiers, 15/06/2018)
      There is a growing recognition of the role the gastrointestinal microbiota plays in health and disease. Ingested antimicrobial proteins and peptides have the potential to alter the gastrointestinal microbiota; particularly if protected from digestion. Nisin is an antimicrobial peptide that is used as a food preservative. This study examined the ability of nisin to affect the murine microbiota when fed to mice in two different starch based matrices; a starch dough comprising raw starch granules and a starch gel comprising starch that was gelatinized and retrograded. The effects of the two starch matrices by themselves on the microbiota were also examined. Following 16S rRNA compositional sequencing, beta diversity analysis highlighted a significant difference (p = 0.001, n = 10) in the murine microbiota between the four diet groups. The differences between the two nisin containing diets were mainly attributable to differences in the nisin release from the starch matrices while the differences between the carriers were mainly attributable to the type of resistant starch they possessed. Indeed, the differences in the relative abundance of several genera in the mice consuming the starch dough and starch gel diets, in particular Akkermansia, the relative abundance of which was 0.5 and 11.9%, respectively (p = 0.0002, n = 10), points to the potential value of resistance starch as a modulator of beneficial gut microbes. Intact nisin and nisin digestion products (in particular nisin fragment 22–31) were detected in the feces and the nisin was biologically active. However, despite a three-fold greater consumption of nisin in the group fed the nisin in starch dough diet, twice as much nisin was detected in the feces of the group which consumed the nisin in starch gel diet. In addition, the relative abundance of three times as many genera from the lower gastrointestinal tract (GIT) were significantly different (p < 0.001, n = 10) to the control for the group fed the nisin in starch gel diet, implying that the starch gel afforded a degree of protection from digestion to the nisin entrapped within it.
    • Partitioning of starter bacteria and added exogenous enzyme activities between curd and whey during Cheddar cheese manufacture

      Doolan, I. A.; Nongonierma, Alice B.; Kilcawley, Kieran; Wilkinson, M.G.; Department of Agriculture, Food and the Marine, Ireland; 04/R&D/C/238 (Elsevier, 26/07/2013)
      Partitioning of starter bacteria and enzyme activities was investigated at different stages of Cheddar cheese manufacture using three exogenous commercial enzyme preparations added to milk or at salting. The enzyme preparations used were: Accelase AM317, Accelase AHC50, Accelerzyme CPG. Flow cytometric analysis indicated that AHC50 or AM317 consisted of permeabilised or dead cells and contained a range of enzyme activities. The CPG preparation contained only carboxypeptidase activity. Approximately 90% of starter bacteria cells partitioned with the curd at whey drainage. However, key enzyme activities partitioned with the bulk whey in the range of 22%–90%. An increased level of enzyme partitioning with the curd was observed for AHC50 which was added at salting, indicating that the mode of addition influenced partitioning. These findings suggest that further scope exists to optimise both bacterial and exogenous enzyme incorporation into cheese curd to accelerate ripening.
    • Potential of cultivar and crop management to affect phytochemical content in winter-grown sprouting broccoli (Brassica oleracea L. var. italica)

      Reilly, Kim; Valverde, Juan; Finn, Leo; Rai, Dilip K.; Brunton, Nigel; Sorenson, Jens C; Sorenson, Hilmer; Gaffney, Michael; Department of Agriculture, Food and the Marine, Ireland; 06/NITAFRC6 (Wiley, 08/07/2013)
      BACKGROUND: Variety and crop management strategies affect the content of bioactive compounds (phenolics, flavonoids and glucosinolates) in green broccoli (calabrese) types, which are cultivated during summer and autumn in temperate European climates. Sprouting broccoli types are morphologically distinct and are grown over the winter season and harvested until early spring. Thus they show considerable potential for development as an import substitution crop for growers and consumers during the ‘hungry gap’ of early spring. The present study investigated the effect of variety and management practices on phytochemical content in a range of sprouting broccoli varieties. RESULTS: Yields were significantly higher in white sprouting broccoli varieties. Levels of phenolics and flavonoids were in the range 81.6-270.4 and 16.9–104.8 mg 100g -1 FW respectively depending on year and cultivar, and were highest in varieties TZ 5052, TZ 5055, Red Admiral and Improved White Sprouting. In-row spacing did not affect flavonoid content. Phenolic and flavonoid content generally increased with increasing floret maturity and levels were high in edible portions of the crop. Crop wastes (leaf and flower) contained 145.9-239.3 and 21.5–116.6 mg 100g -1 FW total phenolics and flavonoids respectively depending on cultivar, tissue and year. Climatic factors had a significant effect on phenolic and flavonoid content. Levels of total and some individual glucosinolates were higher in sprouting broccoli than in the green broccoli variety Ironman. CONCLUSION: Levels of total phenolics, flavonoids and glucosinolates are higher in sprouting than green broccoli types. Sprouting broccoli represents an excellent source of dietary bioactive compounds.
    • Production of bioactive substances by intestinal bacteria as a basis for explaining probiotic mechanisms: Bacteriocins and conjugated linoleic acid

      O'Shea, Eileen F.; Cotter, Paul D.; STANTON, CATHERINE; Ross, R Paul; Hill, Colin; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; 04R & DC; 07/CE/B1368 (Elsevier Science B.V., 16/01/2012)
      The mechanisms by which intestinal bacteria achieve their associated health benefits can be complex and multifaceted. In this respect, the diverse microbial composition of the human gastrointestinal tract (GIT) provides an almost unlimited potential source of bioactive substances (pharmabiotics) which can directly or indirectly affect human health. Bacteriocins and fatty acids are just two examples of pharmabiotic substances which may contribute to probiotic functionality within the mammalian GIT. Bacteriocin production is believed to confer producing strains with a competitive advantage within complex microbial environments as a consequence of their associated antimicrobial activity. This has the potential to enable the establishment and prevalence of producing strains as well as directly inhibiting pathogens within the GIT. Consequently, these antimicrobial peptides and the associated intestinal producing strains may be exploited to beneficially influence microbial populations. Intestinal bacteria are also known to produce a diverse array of health-promoting fatty acids. Indeed, certain strains of intestinal bifidobacteria have been shown to produce conjugated linoleic acid (CLA), a fatty acid which has been associated with a variety of systemic health-promoting effects. Recently, the ability to modulate the fatty acid composition of the liver and adipose tissue of the host upon oral administration of CLA-producing bifidobacteria and lactobacilli was demonstrated in a murine model. Importantly, this implies a potential therapeutic role for probiotics in the treatment of certain metabolic and immunoinflammatory disorders. Such examples serve to highlight the potential contribution of pharmabiotic production to probiotic functionality in relation to human health maintenance.