• Enrichment and Assessment of the Contributions of the Major Polyphenols to the Total Antioxidant Activity of Onion Extracts: A Fractionation by Flash Chromatography Approach

      Hossain, Mohammad Billal; Lebelle, Justine; Birsan, Rares; Rai, Dilip K.; Department of Agriculture, Food and the Marine; 06/NITAFRC/6 (MDPI, 2018-11-27)
      The present study extensively fractionated crude red onion extract in order to identify the polyphenols which contributed most in the total antioxidant capacity of the onion extract using a flash chromatography system. The flash separations produced 70 fractions which were tested for their total phenol content, total flavonoid content, and antioxidant capacities as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Out of these 70 fractions, four fractions which were representatives of the four major peaks of the flash chromatograms, were further analysed for their constituent polyphenols using liquid chromatography tandem mass spectrometry (LC-MS/MS). The main contributor of onion antioxidant capacity is quercetin glycoside followed by quercetin aglycone although quercetin aglycone had higher antioxidant capacity than its glycosidic counterparts. High abundance of quercetin glycosides such as quercetin-3,4′-diglucoside and quercetin-4′-glucoside had compensated for their relatively low antioxidant capacities. A Higher degree of glycosylation resulted in lower antioxidant capacity. The fractionation approach also contributed in enrichment of the onion antioxidant polyphenols. A >9 folds enrichment was possible by discarding the early fractions (fractions 1–15) which contained the main bulk of the extracts, predominantly sugars.
    • Enrichment and Assessment of the Contributions of the Major Polyphenols to the Total Antioxidant Activity of Onion Extracts: A Fractionation by Flash Chromatography Approach

      Hossain, Mohammad Billal; Lebelle, Justine; Birsan, Rares; Rai, Dilip K.; Department of Agriculture, Food and the Marine; FIRM 06/NITAFRC/6 (MDPI AG, 2018-11-27)
      The present study extensively fractionated crude red onion extract in order to identify the polyphenols which contributed most in the total antioxidant capacity of the onion extract using a flash chromatography system. The flash separations produced 70 fractions which were tested for their total phenol content, total flavonoid content, and antioxidant capacities as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Out of these 70 fractions, four fractions which were representatives of the four major peaks of the flash chromatograms, were further analysed for their constituent polyphenols using liquid chromatography tandem mass spectrometry (LC-MS/MS). The main contributor of onion antioxidant capacity is quercetin glycoside followed by quercetin aglycone although quercetin aglycone had higher antioxidant capacity than its glycosidic counterparts. High abundance of quercetin glycosides such as quercetin-3,4′-diglucoside and quercetin-4′-glucoside had compensated for their relatively low antioxidant capacities. A Higher degree of glycosylation resulted in lower antioxidant capacity. The fractionation approach also contributed in enrichment of the onion antioxidant polyphenols. A >9 folds enrichment was possible by discarding the early fractions (fractions 1–15) which contained the main bulk of the extracts, predominantly sugars.
    • Enrichment and Assessment of the Contributions of the Major Polyphenols to the Total Antioxidant Activity of Onion Extracts: A Fractionation by Flash Chromatography Approach

      Hossain, Mohammad Billal; Lebelle, Justine; Birsan, Rares; Rai, Dilip K.; Department of Agriculture, Food and the Marine; FIRM 06/NITAFRC/6 (MDPI AG, 2018-11-27)
      The present study extensively fractionated crude red onion extract in order to identify the polyphenols which contributed most in the total antioxidant capacity of the onion extract using a flash chromatography system. The flash separations produced 70 fractions which were tested for their total phenol content, total flavonoid content, and antioxidant capacities as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Out of these 70 fractions, four fractions which were representatives of the four major peaks of the flash chromatograms, were further analysed for their constituent polyphenols using liquid chromatography tandem mass spectrometry (LC-MS/MS). The main contributor of onion antioxidant capacity is quercetin glycoside followed by quercetin aglycone although quercetin aglycone had higher antioxidant capacity than its glycosidic counterparts. High abundance of quercetin glycosides such as quercetin-3,4′-diglucoside and quercetin-4′-glucoside had compensated for their relatively low antioxidant capacities. A Higher degree of glycosylation resulted in lower antioxidant capacity. The fractionation approach also contributed in enrichment of the onion antioxidant polyphenols. A >9 folds enrichment was possible by discarding the early fractions (fractions 1–15) which contained the main bulk of the extracts, predominantly sugars.
    • Evolution of gut microbiota composition from birth to 24 weeks in the INFANTMET Cohort

      Hill, Cian J; Lynch, Denise B; Murphy, Kiera; Ulaszewska, Marynka; Jeffery, Ian B; O'Shea, Carol A; Watkins, Claire; Dempsey, Eugene; Mattivi, Fulvio; Tuohy, Kieran; et al. (Biomed Central, 17/01/2017)
      Background The gut is the most extensively studied niche of the human microbiome. The aim of this study was to characterise the initial gut microbiota development of a cohort of breastfed infants (n = 192) from 1 to 24 weeks of age. Methods V4-V5 region 16S rRNA amplicon Illumina sequencing and, in parallel, bacteriological culture. The metabolomic profile of infant urine at 4 weeks of age was also examined by LC-MS. Results Full-term (FT), spontaneous vaginally delivered (SVD) infants’ microbiota remained stable at both phylum and genus levels during the 24-week period examined. FT Caesarean section (CS) infants displayed an increased faecal abundance of Firmicutes (p < 0.01) and lower abundance of Actinobacteria (p < 0.001) after the first week of life compared to FT-SVD infants. FT-CS infants gradually progressed to harbouring a microbiota closely resembling FT-SVD (which remained stable) by week 8 of life, which was maintained at week 24. The gut microbiota of preterm (PT) infants displayed a significantly greater abundance of Proteobacteria compared to FT infants (p < 0.001) at week 1. Metabolomic analysis of urine at week 4 indicated PT-CS infants have a functionally different metabolite profile than FT (both CS and SVD) infants. Co-inertia analysis showed co-variation between the urine metabolome and the faecal microbiota of the infants. Tryptophan and tyrosine metabolic pathways, as well as fatty acid and bile acid metabolism, were found to be affected by delivery mode and gestational age. Conclusions These findings confirm that mode of delivery and gestational age both have significant effects on early neonatal microbiota composition. There is also a significant difference between the metabolite profile of FT and PT infants. Prolonged breastfeeding was shown to have a significant effect on the microbiota composition of FT-CS infants at 24 weeks of age, but interestingly not on that of FT-SVD infants. Twins had more similar microbiota to one another than between two random infants, reflecting the influence of similarities in both host genetics and the environment on the microbiota.
    • Exploring the effects of pulsed electric field processing parameters on polyacetylene extraction from carrot slices

      Aguilo-Aguayo, Ingrid; Abreu, Corina; Hossain, Mohammad Billal; Altisent, Rosa; Brunton, Nigel; Viñas, Inmaculada; Rai, Dilip K.; Department of Agriculture, Food and the Marine; Generalitat of Catalonia; 06TNITAFRC6; et al. (MDPI, 2015-03-02)
      The effects of various pulsed electric field (PEF) parameters on the extraction of polyacetylenes from carrot slices were investigated. Optimised conditions with regard to electric field strength (1–4 kV/cm), number of pulses (100–1500), pulse frequency (10–200 Hz) and pulse width (10–30 μs) were identified using response surface methodology (RSM) to maximise the extraction of falcarinol (FaOH), falcarindiol (FaDOH) and falcarindiol-3-acetate (FaDOAc) from carrot slices. Data obtained from RSM and experiments fitted significantly (p < 0.0001) the proposed second-order response functions with high regression coefficients (R2) ranging from 0.82 to 0.75. Maximal FaOH (188%), FaDOH (164.9%) and FaDOAc (166.8%) levels relative to untreated samples were obtained from carrot slices after applying PEF treatments at 4 kV/cm with 100 number of pulses of 10 μs at 10 Hz. The predicted values from the developed quadratic polynomial equation were in close agreement with the actual experimental values with low average mean deviations (E%) ranging from 0.68% to 3.58%.
    • Fate of beta-glucan, polyphenols and lipophilic compounds in baked crackers fortified with different barley-milled fractions

      Gangopadhyay, Nirupama; O'Shea, Norah; Brunton, Nigel P.; Gallagher, Eimear; Harrison, Sabine M.; Rai, Dilip K.; Department of Agriculture, Food and the Marine; FIRM 11/SF/317 (Elsevier BV, 2019-07-18)
      Four types of crackers were prepared, whereby wheat flour was substituted with different percentages of barley flour and bran. These formulations were compared to a 100% wheat flour (control) cracker with respect to β-glucan, polyphenols and lipophilic bioactives. Incorporation of barley fractions enriched the β-glucan, and phenolic content, as well as in vitro antioxidant capacities of the crackers. However, some polyphenols including procyanidin C and ferulic acid could not be detected in the crackers owing to the probable degradation of these compounds during baking. The β-glucan, flavanols (catechin and procyanidin B), as well as fatty acids and sterols were least affected; while the α-tocotrienols showed degradation following the baking process. Overall, barley fractions can serve as valued ingredients for enhancing the health-salutary components of fortified crackers or the products thereof.
    • Food for thought: The role of nutrition in the microbiota-gut–brain axis

      Oriach, Clara Seira; Robertson, Ruairi C.; STANTON, CATHERINE; Cryan, John F.; Dinan, Timothy G.; Science Foundation Ireland; Health Research Board of Ireland; Sea Change Strategy, NutraMara programme; Department of Agriculture, Food and the Marine; SFI/12/RC/2273; et al. (Elsevier, 2016-01-21)
      Recent research has provided strong evidence for the role of the commensal gut microbiota in brain function and behaviour. Many potential pathways are involved in this bidirectional communication between the gut microbiota and the brain such as immune mechanisms, the vagus nerve and microbial neurometabolite production. Dysbiosis of gut microbial function has been associated with behavioural and neurophysical deficits, therefore research focused on developing novel therapeutic strategies to treat psychiatric disorders by targeting the gut microbiota is rapidly growing. Numerous factors can influence the gut microbiota composition such as health status, mode of birth delivery and genetics, but diet is considered among the most crucial factors impacting on the human gut microbiota from infancy to old age. Thus, dietary interventions may have the potential to modulate psychiatric symptoms associated with gut–brain axis dysfunction. Further clinical and in vivo studies are needed to better understand the mechanisms underlying the link between nutrition, gut microbiota and control of behaviour and mental health.
    • Food for thought: The role of nutrition in the microbiota-gut–brain axis

      Oriach, Clara Seira; Robertson, Ruairi C; STANTON, CATHERINE; Cryan, John F.; Dinan, Timothy G.; Science Foundation Ireland; Health Research Board of Ireland; Sea Change Strategy NutraMara programme; SMART FOOD project; Department of Agriculture, Food and the Marine; et al. (Elsevier, 2016-01-21)
      Recent research has provided strong evidence for the role of the commensal gut microbiota in brain function and behaviour. Many potential pathways are involved in this bidirectional communication between the gut microbiota and the brain such as immune mechanisms, the vagus nerve and microbial neurometabolite production. Dysbiosis of gut microbial function has been associated with behavioural and neurophysical deficits, therefore research focused on developing novel therapeutic strategies to treat psychiatric disorders by targeting the gut microbiota is rapidly growing. Numerous factors can influence the gut microbiota composition such as health status, mode of birth delivery and genetics, but diet is considered among the most crucial factors impacting on the human gut microbiota from infancy to old age. Thus, dietary interventions may have the potential to modulate psychiatric symptoms associated with gut–brain axis dysfunction. Further clinical and in vivo studies are needed to better understand the mechanisms underlying the link between nutrition, gut microbiota and control of behaviour and mental health.
    • Generation of Bioactive Hydrolysates and Peptides from Bovine Hemoglobin with In Vitro Renin, Angiotensin-I-Converting Enzyme and Dipeptidyl Peptidase-IV Inhibitory Activities

      Lafarga, Tomas; Rai, Dilip K.; O'Connor, Paula M.; Hayes, Maria; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; 11/F/043 (Wiley, 02/03/2016)
      Bovine hemoglobin was selected for use in the generation of bioactive hydrolysates with potential for use as functional food ingredients for prevention of disorders such as hypertension, obesity and diabetes. Bovine hemoglobin was isolated and hydrolyzed with papain, which was selected using in silico analysis. The generated hydrolysate was enriched by ultrafiltration and further purified by high performance liquid chromatography. A number of peptides were identified using de novo peptide sequencing and these peptides were chemically synthesized to confirm their bioactivity in vitro. Three multifunctional peptides with both, ACE-I and renin-inhibitory properties and one peptide with ACE-I-inhibiting properties were identified. These included the di-peptide HR with ACE-I and renin IC50 values of 0.19 and 7.09 mM, respectively. The generated papain hydrolysate of bovine hemoglobin not only inhibited the enzymes ACE-I and renin but also the enzyme DPP-IV, which has been linked to type-2 diabetes.
    • Genome Sequence of Geobacillus stearothermophilus DSM 458, an Antimicrobial-Producing Thermophyllic Bacterium, Isolated from a Sugar Beet Factory

      Egan, Kevin; Kelleher, Philip; Field, Des; Rea, Mary; Ross, R Paul; Cotter, Paul D.; Hill, Colin; Department of Agriculture, Food and the Marine; Science Foundation Ireland; DAFM 13/F/462; et al. (American Society for Microbiology, 2017-10-26)
      This paper reports the full genome sequence of the antimicrobial-producing bacterium Geobacillus stearothermophilus DSM 458, isolated in a sugar beet factory in Austria. In silico analysis reveals the presence of a number of novel bacteriocin biosynthetic genes.
    • Genome Sequence of Geobacillus stearothermophilus DSM 458, an Antimicrobial-Producing Thermophilic Bacterium, Isolated from a Sugar Beet Factory

      Egan, Kevin; Kelleher, Philip; Field, Des; Rea, Mary; Ross, R Paul; Cotter, Paul D.; Hill, Colin; Department of Agriculture, Food and the Marine; Science Foundation Ireland; 13/F/462; et al. (American Society for Microbiology, 2017-10-26)
      This paper reports the full genome sequence of the antimicrobial-producing bacterium Geobacillus stearothermophilus DSM 458, isolated in a sugar beet factory in Austria. In silico analysis reveals the presence of a number of novel bacteriocin biosynthetic genes.
    • Gut microbes from the phylogenetically diverse genus Eubacterium and their various contributions to gut health

      Mukherjee, Arghya; Lordan, Cathy; Ross, R. Paul; Cotter, Paul D.; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship; Science Foundation Ireland; EU; 15/F/635; 2017047; et al. (Taylor & Francis, 2020-08-23)
      Over the last two decades our understanding of the gut microbiota and its contribution to health and disease has been transformed. Among a new ‘generation’ of potentially beneficial microbes to have been recognized are members of the genus Eubacterium, who form a part of the core human gut microbiome. The genus consists of phylogenetically, and quite frequently phenotypically, diverse species, making Eubacterium a taxonomically unique and challenging genus. Several members of the genus produce butyrate, which plays a critical role in energy homeostasis, colonic motility, immunomodulation and suppression of inflammation in the gut. Eubacterium spp. also carry out bile acid and cholesterol transformations in the gut, thereby contributing to their homeostasis. Gut dysbiosis and a consequently modified representation of Eubacterium spp. in the gut, have been linked with various human disease states. This review provides an overview of Eubacterium species from a phylogenetic perspective, describes how they alter with diet and age and summarizes its association with the human gut and various health conditions.
    • The Gut Microbiota Composition in Dichorionic Triplet Sets Suggests a Role for Host Genetic Factors

      Murphy, Kiera; O'Shea, Carol A; Ryan, C. Anthony; Dempsey, Eugene; O'Toole, Paul W.; STANTON, CATHERINE; Ross, R Paul; Department of Agriculture, Food and the Marine; Science Foundation Ireland; Teagasc Walsh Fellowship Programme; et al. (PLoS, 14/04/2015)
      Monozygotic and dizygotic twin studies investigating the relative roles of host genetics and environmental factors in shaping gut microbiota composition have produced conflicting results. In this study, we investigated the gut microbiota composition of a healthy dichorionic triplet set. The dichorionic triplet set contained a pair of monozygotic twins and a fraternal sibling, with similar pre- and post-natal environmental conditions including feeding regime. V4 16S rRNA and rpoB amplicon pyrosequencing was employed to investigate microbiota composition, and the species and strain diversity of the culturable bifidobacterial population was also examined. At month 1, the monozygotic pair shared a similar microbiota distinct to the fraternal sibling. By month 12 however, the profile was more uniform between the three infants. Principal coordinate analysis (PCoA) of the microbiota composition revealed strong clustering of the monozygotic pair at month 1 and a separation of the fraternal infant. At months 2 and 3 the phylogenetic distance between the monozygotic pair and the fraternal sibling has greatly reduced and by month 12 the monozygotic pair no longer clustered separately from the fraternal infant. Pulse field gel electrophoresis (PFGE) analysis of the bifidobacterial population revealed a lack of strain diversity, with identical strains identified in all three infants at month 1 and 12. The microbiota of two antibiotic-treated dichorionic triplet sets was also investigated. Not surprisingly, in both triplet sets early life antibiotic administration appeared to be a major determinant of microbiota composition at month 1, irrespective of zygosity. By month 12, early antibiotic administration appeared to no longer exert such a strong influence on gut microbiota composition. We hypothesize that initially host genetics play a significant role in the composition of an individual’s gut microbiota, unless an antibiotic intervention is given, but by month 12 environmental factors are the major determinant.
    • High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

      O'Sullivan, Daniel; Fallico, Vincenzo; O'Sullivan, Orla; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Giblin, Linda; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; 2012205 (Biomed Central, 17/11/2015)
      Background: The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results: Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions: In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.
    • Impact of direct and indirect heating systems in broiler units on environmental conditions and flock performance

      Smith, Shaun; Meade, Joseph; Gibbons, James; McGill, Kevina; Bolton, Declan; Whyte, Paul; Department of Agriculture, Food and the Marine; 11SF328 (Elsevier BV, 2016-11-11)
      This study compared the impact of three indirect heating systems to direct gas flame heaters on a selection of flock performance and environmental indicators in commercial broiler units. No statistically significant differences (P≥0.05) were found in flock mortality rates, bird weight, water consumption, stress response, carbon dioxide, ammonia, temperature, relative humidity, litter quality, within-flock Campylobacter levels or mean Campylobacter counts when flock data from any of the three indirect heating systems were compared to flocks reared in houses with direct heating systems. Differences in litter quality were observed between upper and lower litter layers in all houses, regardless of heating type, which may have implications for bird health and welfare. Carbon dioxide concentrations in houses with direct heating systems were significantly higher than those in houses with indirect heating systems during the first 10 days of bird life (P≤0.05). This was due to the increased use of heating systems during this period of the flock cycle. Differences in CO2 concentrations had no effect on flock performance, possibly due to the fact that concentrations did not exceed known safe levels. A statistically significant increase in stress response was observed in birds as a result of partial depopulation (thinning) within houses, irrespective of heating system type used (P≤0.05). Stress associated with thinning may have consequences for bird welfare and food safety. In conclusion, the results of our study suggest that indirect heating systems do not appear to negatively impact on flock performance, stress response, within-flock Campylobacter levels or mean Campylobacter counts and do not appear to significantly alter environmental conditions within broiler houses when compared to houses equipped with direct heating systems. Indirect systems are a viable alternative for heating broiler houses in terms of flock performance, bird welfare and food safety.
    • Impact of pulsed electric field pre-treatment on nutritional and polyphenolic contents and bioactivities of light and dark brewer's spent grains

      Kumari, Bibha; Tiwari, Brijesh K; Walsh, Des; Griffin, Tomás; Islam, Nahidul; Lyng, James G.; Brunton, Nigel; Rai, Dilip K.; Department of Agriculture, Food and the Marine; European Union; et al. (Elsevier, 2019-04-30)
      Pulsed electric field (PEF) pre-treatment, at 2.8 kV/cm with 3000 pulses of 20 μs pulse-width, was applied on brewer's spent grains (BSG) followed by aqueous extraction at 55 °C, 220 rpm for 16 h. PEF pre-treatment showed significantly increased yields (p < 0.05) of carbohydrate, protein, starch and reducing sugar in extracts from dark BSG compared to untreated samples. Light BSG extracts had significantly higher (p < 0.05) levels of free d-glucose and total free amino acids (18.5–33.3 and 21–25 mg/g dry weight extract (Dwe)), compared to dark extracts (5 and 1.2 mg/g Dwe respectively). Dark BSG extracts showed significantly higher (p < 0.05) total phenolics (3.97–4.88 mg GAE/g Dwe) compared to light BSG extracts (0.83–1.40 mg GAE/g Dwe). Furthermore, PEF treated light BSG showed higher antimicrobial activity with minimum inhibition concentration (MIC) of 50 and 25 mg/mL against Salmonella typhimurium and Listeria monocytogenes, respectively compared to the untreated extracts (>50 mg/mL) with lowest MIC value of 1.56 mg/mL against Staphylococcus aureus. All the BSG extracts induced the release of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and chemokines (IL-8, MCP-1 and MIP-1α) confirming immunomodulatory activity.
    • Inhibition of L. monocytogenes Biofilm Formation by the Amidase Domain of the Phage vB_LmoS_293 Endolysin

      Pennone, Vincenzo; Sanz-Gaitero, Marta; O'Connor, Paula; Coffey, Aidan; Jordan, Kieran; van Raaij, Mark J; McAuliffe, Olivia; Spanish Ministry of Science; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; et al. (MDPI, 2019-08-06)
      Listeria monocytogenes is a ubiquitous Gram-positive bacterium that is a major concern for food business operators because of its pathogenicity and ability to form biofilms in food production environments. Bacteriophages (phages) have been evaluated as biocontrol agents for L. monocytogenes in a number of studies and, indeed, certain phages have been approved for use as anti-listerial agents in food processing environments (ListShield and PhageGuard Listex). Endolysins are proteins produced by phages in the host cell. They cleave the peptidoglycan cell wall, thus allowing release of progeny phage into the environment. In this study, the amidase domain of the phage vB_LmoS_293 endolysin (293-amidase) was cloned and expressed in Escherichia. coli(E. coli). Muralytic activity at different concentrations, pH and temperature values, lytic spectrum and activity against biofilms was determined for the purified 293-amidase protein. The results showed activity on autoclaved cells at three different temperatures (20 °C, 37 °C and 50 °C), with a wider specificity (L. monocytogenes 473 and 3099, a serotype 4b and serogroup 1/2b-3b-7, respectively) compared to the phage itself, which targets only L. monocytogenes serotypes 4b and 4e. The protein also inhibits biofilm formation on abiotic surfaces. These results show the potential of using recombinant antimicrobial proteins against pathogens in the food production environment.
    • Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives

      Healy, Brian; Field, Des; O'Connor, Paula M.; Hill, Colin; Cotter, Paul D.; Ross, R Paul; Department of Agriculture, Food and the Marine; Science Foundation Ireland; 08/RD/C/691; 10/IN.1/B3027 (PLoS, 11/11/2013)
      Nisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.
    • 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; Teagasc Walsh Fellowship Programme; 13 F 454-WheyGSH (Elsevier, 2018-03-28)
      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.
    • Lactoferrin affects the adherence and invasion of Streptococcus dysgalactiae spp. dysgalactiae in mammary epithelial cells

      O'Halloran, Fiona; Beecher, Christine; Chaurin, Valerie; Sweeney, Torres; Giblin, Linda; Department of Agriculture, Food and the Marine; Irish Dairy Levy Research Trust; Teagasc Walsh Fellowship Programme; 06RDTMFRC437; 06RDTMFRC445 (Elsevier, 24/03/2016)
      Streptococcus dysgalactiae ssp. dysgalactiae is an important causative agent of bovine mastitis worldwide. Lactoferrin is an innate immune protein that is associated with many functions including immunomodulatory, antiproliferative, and antimicrobial properties. This study aimed to investigate the interactions between lactoferrin and a clinical bovine mastitis isolate, Strep. dysgalactiae ssp. dysgalactiae DPC5345. Initially a deliberate in vivo bovine intramammary challenge was performed with Strep. dysgalactiae DPC5345. Results demonstrated a significant difference in lactoferrin mRNA levels in milk cells between the control and infused quarters 7 h postinfusion. Milk lactoferrin levels in the Strep. dysgalactiae DPC5345 infused quarters were significantly increased compared with control quarters at 48 h postinfusion. In vitro studies demonstrated that lactoferrin had a bacteriostatic effect on the growth of Strep. dysgalactiae DPC5345 and significantly decreased the ability of the bacteria to internalize into HC-11 mammary epithelial cells. Confocal microscopy images of HC-11 cells exposed to Strep. dysgalactiae and lactoferrin further supported this effect by demonstrating reduced invasion of bacteria to HC-11 cells. The combined data suggest that a bovine immune response to Strep. dysgalactiae infection includes a significant increase in lactoferrin expression in vivo, and based on in vitro data, lactoferrin limits mammary cell invasion of this pathogen by binding to the bacteria and preventing its adherence.