• Analysis of Health Benefits Conferred by Lactobacillus Species from Kefir

      Cotter, Paul D.; Slattery, Conor; O'Toole, Paul W.; Department of Agriculture, Food and Marine; Science Foundation Ireland; APC Microbiome Ireland; Vistamilk; Enterprise Ireland; European Union; 818368 (MDPI, 2019-06-01)
      Lactobacilli are among the most common microorganisms found in kefir; a traditional fermented milk beverage produced locally in many locations around the world. Kefir has been associated with a wide range of purported health benefits; such as antimicrobial activity; cholesterol metabolism; immunomodulation; anti-oxidative effects; anti-diabetic effects; anti-allergenic effects; and tumor suppression. This review critically examines and assesses these claimed benefits and mechanisms with regard to particular Lactobacillus species and/or strains that have been derived from kefir; as well as detailing further potential avenues for experimentation.
    • Aroma compound diacetyl suppresses glucagon-like peptide-1 production and secretion in STC-1 cells

      McCarthy, Triona; Bruen, Christine; O'Halloran, Fiona; Schellekens, Harriet; Kilcawley, Kieran; Cryan, John F.; Giblin, Linda; Teagasc Walsh Fellowship Programme; Enterprise Ireland; CC20080001 (Elsevier, 21/01/2017)
      Diacetyl is a volatile flavour compound that has a characteristic buttery aroma and is widely used in the flavour industry. The aroma of a food plays an important role in food palatability and thus intake. This study investigates the effect of diacetyl on the satiety hormone, glucagon-like peptide (GLP-1), using the enteroendocrine cell line, STC-1. Diacetyl decreased proglucagon mRNA and total GLP-1 from glucose stimulated STC-1 cells. This dampening effect on GLP-1 appears to be mediated by increasing intracellular cAMP levels, increasing synthesis of the G protein coupled receptor, GPR120, and its recruitment to the cell surface. Voltage gated Ca2+ channels, K+ATP channels and the α-gustducin taste pathway do not appear to be involved. These findings demonstrate that components contributing to food palatability suppress GLP-1. This ability of diacetyl to reduce satiety signals may contribute to overconsumption of some palatable foods.
    • Atypical Listeria innocua strains possess an intact LIPI-3

      Clayton, Evelyn M; Daly, Karen M.; Guinane, Caitriona M.; Hill, Colin; Cotter, Paul D.; Ross, R Paul; Enterprise Ireland; Science Foundation Ireland; 06/IN.1/B98; 10/IN.1/B3027 (Biomed Central, 08/03/2014)
      Background: Listeria monocytogenes is a food-borne pathogen which is the causative agent of listeriosis and can be divided into three evolutionary lineages I, II and III. While all strains possess the well established virulence factors associated with the Listeria pathogenicity island I (LIPI-1), lineage I strains also possess an additional pathogenicity island designated LIPI-3 which encodes listeriolysin S (LLS), a post-translationally modified cytolytic peptide. Up until now, this pathogenicity island has been identified exclusively in a subset of lineage I isolates of the pathogen Listeria monocytogenes. Results: In total 64 L. innocua strains were screened for the presence of LIPI-3. Here we report the identification of an intact LIPI-3 in 11 isolates of L. innocua and the remnants of the cluster in several others. Significantly, we can reveal that placing the L. innocua lls genes under the control of a constitutive promoter results in a haemolytic phenotype, confirming that the cluster is capable of encoding a functional haemolysin. Conclusions: Although the presence of the LIPI-3 gene cluster is confined to lineage I isolates of L. monocytogenes, a corresponding gene cluster or its remnants have been identified in many L. innocua strains.
    • Bile acids at the cross-roads of gut microbiome–host cardiometabolic interactions

      Ryan, Paul M; STANTON, CATHERINE; Caplice, Noel M; Science Foundation Ireland; Enterprise Ireland; SFI/12/RC/2273; CF/2013/3030A/B (Biomed Central, 28/12/2017)
      While basic and clinical research over the last several decades has recognized a number of modifiable risk factors associated with cardiometabolic disease progression, additional and alternative biological perspectives may offer novel targets for prevention and treatment of this disease set. There is mounting preclinical and emerging clinical evidence indicating that the mass of metabolically diverse microorganisms which inhabit the human gastrointestinal tract may be implicated in initiation and modulation of cardiovascular and metabolic disease outcomes. The following review will discuss this gut microbiome–host metabolism axis and address newly proposed bile-mediated signaling pathways through which dysregulation of this homeostatic axis may influence host cardiovascular risk. With a central focus on the major nuclear and membrane-bound bile acid receptor ligands, we aim to review the putative impact of microbial bile acid modification on several major phenotypes of metabolic syndrome, from obesity to heart failure. Finally, attempting to synthesize several separate but complementary hypotheses, we will review current directions in preclinical and clinical investigation in this evolving field.
    • Bovine milk oligosaccharides as anti-adhesives against the respiratory tract pathogen Streptococcus pneumoniae

      Ryan, Joseph Thomas; Slattery, Helen; Hickey, Rita M.; Marotta, Mariarosaria; Enterprise Ireland; CC20080001 (Elsevier, 2018-02-20)
      Streptococcus pneumoniae is a Gram-positive pathogen, which is regularly found in the upper respiratory tract of healthy individuals. Increased numbers of S. pneumoniae have been observed colonising the upper respiratory tract of children affected by respiratory tract infections. Galβ1-4GlcNAcβ1-3Gal has been previously identified as one of the receptors involved in the adherence and translocation of S. pneumoniae. As this structure is similar to the milk oligosaccharide lacto-N-neoTetraose, many studies have investigated if free milk oligosaccharides can inhibit the adhesion of S. pneumoniae to epithelial cells of the respiratory tract. Here, we demonstrate that bovine oligosaccharides, which were extracted from demineralised whey, using a combination of membrane filtration and chromatography, were capable of reducing S. pneumoniae adhesion to pharynx and lung cells in vitro when tested at physiological concentrations. This study strengthens the potential use of bovine derived milk oligosaccharides as functional ingredients to reduce the incidence of infectious diseases.
    • Delivery of β-carotene to the in vitro intestinal barrier using nanoemulsions with lecithin or sodium caseinate as emulsifiers

      Gasa-Falcon, Ariadna; Arranz, Elena; Odriozola-Serrano, Isabel; Martín-Belloso, Olga; Giblin, Linda; Ministerio de Economía y Competitividad; Enterprise Ireland; European Union; Science Foundation Ireland; Agencia de Gestio d’Ajuts Universitaris I de Recerca; et al. (Elsevier BV, 2021-01)
      To increase the intestinal delivery of dietary β-carotene, there is a need to develop nanostructured food systems to encapsulate this fat soluble bioactive. The aim of this study was to evaluate the bioacessibility and bioavailability across the intestinal barrier of β-carotene-enriched nanoemulsions stabilised with two emulsifiers (lecithin or sodium caseinate) by coupling an in vitro gastrointestinal digestion with two in vitro cell culture models (Caco-2 or co-culture of Caco-2/HT29-MTX). Nanoemulsions stabilised with lecithin had significantly higher β-carotene in the gastrointestinal digested micellar fraction, lower β-carotene in the Caco-2 (and Caco-2/HT29-MTX) apical compartment and significantly higher β-carotene in Caco-2 cellular content compared to β-carotene-enriched nanoemulsions stabilised with sodium caseinate. Finally, to assess anti-inflammatory activity of digested nanoemulsions, lipopolysaccharide stimulated macrophages were exposed to Caco- 2 basolateral samples with levels of TNF-α and IL-β, subsequently quantified. A TNF-α response from stimulated THP-1 macrophages was elicited by basolateral samples, regardless the emulsifier used to formulate nanoemulsions. This study demonstrated that β-carotene permeability is influenced by the food derived emulsifier used for stabilising nanoemulsions, indicating that composition may be a critical factor for β-carotene delivery.
    • The Effect of Dietary Supplementation with Spent Cider Yeast on the Swine Distal Gut Microbiome

      Upadrasta, Aditya; O'Sulivan, Lisa; O'Sullivan, Orla; Sexton, Noel; Lawlor, Peadar G; Hill, Colin; Fitzgerald, Gerald F; STANTON, CATHERINE; Ross, R Paul; Enterprise Ireland; et al. (PLOS, 09/10/2013)
      Background: There is an increasing need for alternatives to antibiotics for promoting animal health, given the increasing problems associated with antibiotic resistance. In this regard, we evaluated spent cider yeast as a potential probiotic for modifying the gut microbiota in weanling pigs using pyrosequencing of 16S rRNA gene libraries. Methodology and Principal Findings: Piglets aged 24–26 days were assigned to one of two study groups; control (n = 12) and treatment (n = 12). The control animals were fed with a basal diet and the treatment animals were fed with basal diet in combination with cider yeast supplement (500 ml cider yeast containing ,7.6 log CFU/ml) for 21 days. Faecal samples were collected for 16s rRNA gene compositional analysis. 16S rRNA compositional sequencing analysis of the faecal samples collected from day 0 and day 21 revealed marked differences in microbial diversity at both the phylum and genus levels between the control and treatment groups. This analysis confirmed that levels of Salmonella and Escherichia were significantly decreased in the treatment group, compared with the control (P,0.001). This data suggest a positive influence of dietary supplementation with live cider yeast on the microbial diversity of the pig distal gut. Conclusions/Significance: The effect of dietary cider yeast on porcine gut microbial communities was characterized for the first time using 16S rRNA gene compositional sequencing. Dietary cider yeast can potentially alter the gut microbiota, however such changes depend on their endogenous microbiota that causes a divergence in relative response to that given diet.
    • The effect of ovine milk fermentation on the antithrombotic properties of polar lipids

      Lordan, Ronan; Walsh, Aaron M.; Crispie, Fiona; Finnegan, Laura; Cotter, Paul D.; Zabetakis, Ioannis; Enterprise Ireland; Department of Biological Sciences, University of Limerick, Ireland; IP-2016-0488Y (Elsevier, 2019-01-24)
      The effect of fermentation on the antithrombotic properties of polar lipids in ovine milk has been assessed through the production of yoghurts. The total lipids (TL), total neutral lipids (TNL), and total polar lipids (TPL) were extracted. The fatty acid profiles of all yoghurt polar lipids were analysed by GC-MS. The levels of MUFA increased in the fatty acids of the polar lipids, but there was a reduction in PUFA as milk was fermented to yoghurt. The bioactivity of each lipid extract was assessed against platelet-activating factor (PAF) induced platelet aggregation. All yoghurt polar lipids exhibited potent antithrombotic activities with IC50 values ranging from 45 to 77 µg. Shotgun metagenomics determined the species-level microbial composition and functional potential of the yoghurts. Yoghurts containing L. acidophilus seem to correlate with greater bioactivity. Several phospholipid biosynthetic genes have been identified in the most antithrombotic yoghurts. This study has demonstrated that fermentation enhances the antithrombotic properties of yoghurt polar lipids against PAF.
    • Host Specific Diversity in Lactobacillus johnsonii as Evidenced by a Major Chromosomal Inversion and Phage Resistance Mechanisms

      Guinane, Caitriona M.; Kent, Robert M.; Norberg, Sarah; Hill, Colin; Fitzgerald, Gerald F; STANTON, CATHERINE; Ross, R Paul; Enterprise Ireland; CFTD/07/116 (PLOS, 20/04/2011)
      Genetic diversity and genomic rearrangements are a driving force in bacterial evolution and niche adaptation. We sequenced and annotated the genome of Lactobacillus johnsonii DPC6026, a strain isolated from the porcine intestinal tract. Although the genome of DPC6026 is similar in size (1.97mbp) and GC content (34.8%) to the sequenced human isolate L. johnsonii NCC 533, a large symmetrical inversion of approximately 750 kb differentiated the two strains. Comparative analysis among 12 other strains of L. johnsonii including 8 porcine, 3 human and 1 poultry isolate indicated that the genome architecture found in DPC6026 is more common within the species than that of NCC 533. Furthermore a number of unique features were annotated in DPC6026, some of which are likely to have been acquired by horizontal gene transfer (HGT) and contribute to protection against phage infection. A putative type III restriction-modification system was identified, as were novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) elements. Interestingly, these particular elements are not widely distributed among L. johnsonii strains. Taken together these data suggest intra-species genomic rearrangements and significant genetic diversity within the L. johnsonii species and indicate towards a host-specific divergence of L. johnsonii strains with respect to genome inversion and phage exposure.
    • Identification of short peptide sequences in the nanofiltration permeate of a bioactive whey protein hydrolysate

      Le Maux, Solene; Nongonierma, Alice B.; Murray, Brian A.; Kelly, Philip; Fitzgerald, Richard J.; Enterprise Ireland; TC2013-0001 (Elsevier, 16/09/2015)
      Short peptides in food protein hydrolysates are of significant interest as they may be highly bioactive whilst also being bioavailable. A dipeptidyl peptidase IV (DPP-IV) inhibitory whey protein hydrolysate (WPH) was fractionated using nanofiltration (NF) with a 200 Da MWCO membrane. The DPP-IV half maximal inhibitory concentration of the NF permeate (IC50 = 0.66 ± 0.08 mg protein equivalent mL− 1) was significantly more potent (P > 0.05) than that of the starting WPH (IC50 = 0.94 ± 0.24 mg protein equivalent mL− 1) and associated retentate (IC50 = 0.82 ± 0.13 mg protein equivalent mL− 1). This confirmed the contribution of short peptides within the NF permeate to the overall DPP-IV inhibitory activity. An hydrophilic interaction liquid chromatography (HILIC-) and reverse-phase (RP-) liquid chromatography tandem mass spectrometry (LC–MS/MS) strategy, based on two retention time models, allowed detection of eight free amino acids and eight di- to tetrapeptides in the NF permeate. The potential sequences of the peptides within the NF permeate were then ranked on the basis of their highest probability of occurrence. A confirmatory study with synthetic peptides showed that valine–alanine (VA), valine–leucine (VL), tryptophan–leucine (WL) and tryptophan–isoleucine (WI) displayed DPP-IV IC50 values < 170 μM. The NF and LC–MS strategies employed herein represent a new approach for the targeted identification of short peptides within bioactive food protein hydrolysates.
    • In vivo activity of Nisin A and Nisin V against Listeria monocytogenes in mice

      Campion, Alicia; Casey, Patrick G.; Field, Des; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Programme for Research in Third-Level Institutions; Irish Research Council for Science, Engineering and Technology; Enterprise Ireland; Science Foundation Ireland (Biomed Central, 01/02/2013)
      Background: Lantibiotics are post-translationally modified antimicrobial peptides, of which nisin A is the most extensively studied example. Bioengineering of nisin A has resulted in the generation of derivatives with increased in vitro potency against Gram-positive bacteria. Of these, nisin V (containing a Met21Val change) is noteworthy by virtue of exhibiting enhanced antimicrobial efficacy against a wide range of clinical and food-borne pathogens, including Listeria monocytogenes. However, this increased potency has not been tested in vivo. Results: Here we address this issue by assessing the ability of nisin A and nisin V to control a bioluminescent strain of Listeria monocytogenes EGDe in a murine infection model. More specifically, Balb/c mice were infected via the intraperitoneal route at a dose of 1 × 105 cfu/animal and subsequently treated intraperitoneally with either nisin V, nisin A or a PBS control. Bioimaging of the mice was carried out on day 3 of the trial. Animals were then sacrificed and levels of infection were quantified in the liver and spleen. Conclusion: This analysis revealed that nisin V was more effective than Nisin A with respect to controlling infection and therefore merits further investigation with a view to potential chemotherapeutic applications.
    • Microbiome and metabolome modifying effects of several cardiovascular disease interventions in apo-E−/− mice

      Ryan, Paul M; London, Lis E E; Bjorndahl, Trent C; Mandal, Rupasri; Murphy, Kiera; Fitzgerald, Gerald F; Shanahan, Fergus; Ross, R Paul; Wishart, David S; Caplice, Noel M; et al. (Biomed Central, 13/03/2017)
      Background There is strong evidence indicating that gut microbiota have the potential to modify, or be modified by the drugs and nutritional interventions that we rely upon. This study aims to characterize the compositional and functional effects of several nutritional, neutraceutical, and pharmaceutical cardiovascular disease interventions on the gut microbiome, through metagenomic and metabolomic approaches. Apolipoprotein-E-deficient mice were fed for 24 weeks either high-fat/cholesterol diet alone (control, HFC) or high-fat/cholesterol in conjunction with one of three dietary interventions, as follows: plant sterol ester (PSE), oat β-glucan (OBG) and bile salt hydrolase-active Lactobacillus reuteri APC 2587 (BSH), or the drug atorvastatin (STAT). The gut microbiome composition was then investigated, in addition to the host fecal and serum metabolome. Results We observed major shifts in the composition of the gut microbiome of PSE mice, while OBG and BSH mice displayed more modest fluctuations, and STAT showed relatively few alterations. Interestingly, these compositional effects imparted by PSE were coupled with an increase in acetate and reduction in isovalerate (p < 0.05), while OBG promoted n-butyrate synthesis (p < 0.01). In addition, PSE significantly dampened the microbial production of the proatherogenic precursor compound, trimethylamine (p < 0.05), attenuated cholesterol accumulation, and nearly abolished atherogenesis in the model (p < 0.05). However, PSE supplementation produced the heaviest mice with the greatest degree of adiposity (p < 0.05). Finally, PSE, OBG, and STAT all appeared to have considerable impact on the host serum metabolome, including alterations in several acylcarnitines previously associated with a state of metabolic dysfunction (p < 0.05). Conclusions We observed functional alterations in microbial and host-derived metabolites, which may have important implications for systemic metabolic health, suggesting that cardiovascular disease interventions may have a significant impact on the microbiome composition and functionality. This study indicates that the gut microbiome-modifying effects of novel therapeutics should be considered, in addition to the direct host effects.
    • Physiological Gut Oxygenation Alters GLP‐1 Secretion from the Enteroendocrine Cell Line STC‐1

      Kondrashina, Alina; Papkovsky, Dmitri; Giblin, Linda; Enterprise Ireland; TC20130001 (Wiley, 29/09/2017)
      1 Scope Enteroendocrine cell lines are routinely assayed in simple buffers at ≈20% oxygen to screen foods for bioactives that boost satiety hormone levels. However, in vivo, enteroendocrine cells are exposed to different phases of food digestion and function at low oxygen concentration, ranging from 7.5% in the stomach to 0.5% in the colon–rectal junction. 2 Methods and results The objective of this study is to investigate the effect of physiologically relevant O2 concentrations of the gut on the production and secretion of the satiety hormone, glucagon‐like peptide 1 (GLP‐1), from the murine enteroendocrine cell line, secretin tumor cell line (STC‐1), in response to dairy macronutrients as they transit the gut. GLP‐1 exocytosis from STC‐1 cells is influenced by both oxygen concentration and by individual macronutrients. At low oxygen, STC‐1 cell viability is significantly improved for all macronutrient stimulations and cyclic adenosine monophosphate levels are dampened. GLP‐1 secretion from STC‐1 cells is influenced by both the phase of yogurt digestion and corresponding O2 concentration. Atmospheric oxygen at 4.5% combined with upper gastric digesta, which simulates ileum conditions, yields the highest GLP‐1 response. 3 Conclusion This demonstrates the importance of considering physiological oxygen levels and food digestion along gastrointestinal tract for reliable in vitro analysis of gut hormone secretion.
    • A Review on the Applications of Next Generation Sequencing Technologies as Applied to Food-Related Microbiome Studies

      Cao, Yu; Fanning, Seamus; Proos, Sinead; Jordan, Kieran; Srikumar, Shabarinath; Department of Agriculture, Food and the Marine; Enterprise Ireland; 13/F/423; IP 2015 0380 (Frontiers, 2017-09-21)
      The development of next generation sequencing (NGS) techniques has enabled researchers to study and understand the world of microorganisms from broader and deeper perspectives. The contemporary advances in DNA sequencing technologies have not only enabled finer characterization of bacterial genomes but also provided deeper taxonomic identification of complex microbiomes which in its genomic essence is the combined genetic material of the microorganisms inhabiting an environment, whether the environment be a particular body econiche (e.g., human intestinal contents) or a food manufacturing facility econiche (e.g., floor drain). To date, 16S rDNA sequencing, metagenomics and metatranscriptomics are the three basic sequencing strategies used in the taxonomic identification and characterization of food-related microbiomes. These sequencing strategies have used different NGS platforms for DNA and RNA sequence identification. Traditionally, 16S rDNA sequencing has played a key role in understanding the taxonomic composition of a food-related microbiome. Recently, metagenomic approaches have resulted in improved understanding of a microbiome by providing a species-level/strain-level characterization. Further, metatranscriptomic approaches have contributed to the functional characterization of the complex interactions between different microbial communities within a single microbiome. Many studies have highlighted the use of NGS techniques in investigating the microbiome of fermented foods. However, the utilization of NGS techniques in studying the microbiome of non-fermented foods are limited. This review provides a brief overview of the advances in DNA sequencing chemistries as the technology progressed from first, next and third generations and highlights how NGS provided a deeper understanding of food-related microbiomes with special focus on non-fermented foods.
    • Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

      Molloy, Evelyn M.; Field, Des; O'Connor, Paula M.; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Irish Research Council for Science, Engineering and Technology; Enterprise Ireland; Science Foundation Ireland (PLOS, 11/03/2013)
      It is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.