• Carbohydrate catabolic flexibility in the mammalian intestinal commensal Lactobacillus ruminis revealed by fermentation studies aligned to genome annotations

      O'Donnell, Michelle M.; Forde, Brian M; Neville, B. Anne; Ross, R Paul; O'Toole, Paul W. (Biomed Central, 30/08/2011)
      Background: Lactobacillus ruminis is a poorly characterized member of the Lactobacillus salivarius clade that is part of the intestinal microbiota of pigs, humans and other mammals. Its variable abundance in human and animals may be linked to historical changes over time and geographical differences in dietary intake of complex carbohydrates. Results: In this study, we investigated the ability of nine L. ruminis strains of human and bovine origin to utilize fifty carbohydrates including simple sugars, oligosaccharides, and prebiotic polysaccharides. The growth patterns were compared with metabolic pathways predicted by annotation of a high quality draft genome sequence of ATCC 25644 (human isolate) and the complete genome of ATCC 27782 (bovine isolate). All of the strains tested utilized prebiotics including fructooligosaccharides (FOS), soybean-oligosaccharides (SOS) and 1,3:1,4-β-D-gluco-oligosaccharides to varying degrees. Six strains isolated from humans utilized FOS-enriched inulin, as well as FOS. In contrast, three strains isolated from cows grew poorly in FOS-supplemented medium. In general, carbohydrate utilisation patterns were strain-dependent and also varied depending on the degree of polymerisation or complexity of structure. Six putative operons were identified in the genome of the human isolate ATCC 25644 for the transport and utilisation of the prebiotics FOS, galacto-oligosaccharides (GOS), SOS, and 1,3:1,4-β-D-Gluco-oligosaccharides. One of these comprised a novel FOS utilisation operon with predicted capacity to degrade chicory-derived FOS. However, only three of these operons were identified in the ATCC 27782 genome that might account for the utilisation of only SOS and 1,3:1,4-β-D-Gluco-oligosaccharides. Conclusions: This study has provided definitive genome-based evidence to support the fermentation patterns of nine strains of Lactobacillus ruminis, and has linked it to gene distribution patterns in strains from different sources. Furthermore, the study has identified prebiotic carbohydrates with the potential to promote L. ruminis growth in vivo.
    • A case of bovine raw milk contamination with Listeria monocytogenes

      Hunt, Karen; Drummond, Niall; Murphy, Mary; Butler, Francis; Buckley, James F.; Jordan, Kieran; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine, Ireland; European Union (Biomed Central, 06/07/2012)
      During routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocytogenes was found to be a contaminant, at numbers < 100 cfu/ml. A strain with an indistinguishable pulsed-field gel electrophoresis pattern was isolated from the bulk milk two months later. Environmental swabs taken at the dairy environment were negative for the presence of L. monocytogenes, indicating a possible case of excretion of the L. monocytogenes directly into the milk. Milk samples were collected from the individual cows and analysed, resulting in the identification of L. monocytogenes excretion (at 280 cfu/ml) from one of the 4 mammary quarters of one dairy cow out of 180. When the infected cow was isolated from the herd, no L. monocytogenes was detected from the remaining herd. The pulsed-field gel electrophoresis pattern of the strain from the individual cow was indistinguishable from that originally isolated from the bulk milk. The infected cow did not show any clinical signs of disease, nor did the appearance of the milk have any physical abnormalities. Antibiotic treatment of the infected mammary quarter was found to be ineffective. This study shows that there can be risks associated with direct contamination of raw milk with L. monocytogenes.
    • Catabolic flexibility of mammalian-associated lactobacilli

      O'Donnell, Michelle M.; O'Toole, Paul W.; Ross, R Paul (Biomed Central, 16/05/2013)
      Metabolic flexibility may be generally defined as “the capacity for the organism to adapt fuel oxidation to fuel availability”. The metabolic diversification strategies used by individual bacteria vary greatly from the use of novel or acquired enzymes to the use of plasmid-localised genes and transporters. In this review, we describe the ability of lactobacilli to utilise a variety of carbon sources from their current or new environments in order to grow and survive. The genus Lactobacillus now includes more than 150 species, many with adaptive capabilities, broad metabolic capacity and species/strain variance. They are therefore, an informative example of a cell factory capable of adapting to new niches with differing nutritional landscapes. Indeed, lactobacilli naturally colonise and grow in a wide variety of environmental niches which include the roots and foliage of plants, silage, various fermented foods and beverages, the human vagina and the mammalian gastrointestinal tract (GIT; including the mouth, stomach, small intestine and large intestine). Here we primarily describe the metabolic flexibility of some lactobacilli isolated from the mammalian gastrointestinal tract, and we also describe some of the food-associated species with a proven ability to adapt to the GIT. As examples this review concentrates on the following species - Lb. plantarum, Lb. acidophilus, Lb. ruminis, Lb. salivarius, Lb. reuteri and Lb. sakei, to highlight the diversity and inter-relationships between the catabolic nature of species within the genus.
    • Characterisation and application of fruit by-products as novel ingredients in gluten-free products

      O'Shea, Norah; Department of Agriculture, Food and the Marine, Ireland (2014-01)
      Literature has revealed that “waste” left from the processing of fruit can still contain a substantial quantity of macro and minor nutrients. The aim of this thesis was to ascertain the nutritional and structural properties and potential uses of two fruit by-products [apple pomace (Malus domestica Cv. “Karmijn de Sonnaville”) and orange pomace (Citrus sinensis L. Cv. “Valencia”)] in glutenfree bread and extruded snack formulations. The physicochemical and nutritional properties of the fruit by-products were initially studied. Apple pomace contained a high level of fibre and pectin. The isolated pectin was demonstrated to have a high level of methylation which developed viscous pastes. Orange pomace also had high levels of fibre and pectin, and it was an abundant source of minerals such as potassium and magnesium. Orange pomace had a poor gelling ability. The flour obtained after milling dried orange pomace was used in the formulation of gluten-free bread with the aid of a response surface design. Due to the fibrous properties of orange pomace flour, proofing and water addition were also studied. When added at levels greater than 6%, the loaf volume decreased. The number of cells per slice also decreased with increasing orange pomace addition. Inclusion of orange pomace at levels of up to 4% increased crumb softness. An optimised formulation and proofing time was derived using the optimisation tool; these consisted of 5.5% orange pomace, 94.6% water inclusion and with 49 minutes proofing. These optimised parameters doubled the total dietary fibre content of the bread compared to the original control. The pasting properties, rheology, microstructure and sensory characteristics of the optimised formulation (batter and bread) were investigated. Pasting results showed how orange pomace inclusions reduced the final viscosity of the batter, hence reducing the occurrence of starch gelatinisation. Rheological properties such as the storage modulus (G') and complex modulus (G*) increased in the orange pomace batter compared to the control batter. This demonstrates how the orange pomace as an ingredient improved the robustness of the formulation. Sensory panellists scored the orange pomace bread comparably to the control bread. Milled apple pomace was studied as a potential novel ingredient in an extruded snack. As extrusion requires the trialling of a number of extruder parameters, a response surface design was again used to develop an optimised snack. The parameters studied were apple pomace addition, die head temperature and screw speed. Screw speed had the most significant impact on extrudate characteristics. As screw speed increased the favourable extrudate characteristics such as radical expansion ratio, porosity and specific volume decreased. The inclusion of apple pomace had a negative effect on extrudate characteristics at levels greater than 8% addition. Including apple pomace reduced the hardness and increased the crispiness of the snack. Using the optimisation tool, the optimised and validated formulation and extrusion process contained the following parameters: 7.7% apple pomace, 150oC die head temperature and a screw speed of 69 rpm.
    • Characterization of functional properties of proteins from Ganxet beans (Phaseolus vulgaris L. var. Ganxet) isolated using an ultrasound-assisted methodology

      Lafarga, Tomas; Álvarez, Carlos; Bobo, Gloria; Aguilo-Aguayo, Ingrid; Generalitat de Catalunya; Juan de la Cierva contract award; Postdoctoral Senior Grant Ramon y Cajal; FJCI-2016-29541; RYC-2016-19949 (Elsevier, 2018-08-17)
      This study investigated different methods of extraction of protein from Ganxet beans (Phaseolus vulgaris L. var. Ganxet) and evaluated the functional properties of these valuable proteins. Overall, ultrasound processing (40 kHz, 250 W) resulted in higher yields and increased percentages of material solubilized and proteins recovered. The highest percentage of recovered protein was obtained after extraction using 0.4 M NaOH followed by ultrasound processing for 60 min and was calculated as 78.73 ± 4.88% (p < 0.05). Extraction using 0.4 M NaOH followed by sonication for 60 min resulted in the highest yield and percentage of solubilized material calculated as 37.98 ± 0.02 and 54.58 ± 0.19%, respectively (p < 0.05). The water- and oil-holding capacities of the Ganxet protein concentrate were calculated as 2.33 ± 0.12 and 2.69 ± 0.32 g of water or oil per g of protein concentrate, respectively. The highest emulsifying capacity was observed at pH 8.0 and was calculated as 69.4 ± 0.8%.
    • Characterization of Pro-Inflammatory Flagellin Proteins Produced by Lactobacillus ruminis and Related Motile Lactobacilli

      Neville, B. Anne; Forde, Brian M; Claesson, Marcus J.; Darby, Trevor; Coghlan, Avril; Nally, Kenneth; Ross, R Paul; O'Toole, Paul W.; Science Foundation Ireland; Irish Research Council for Science, Engineering and Technology; et al. (PLOS, 10/07/2012)
      Lactobacillus ruminis is one of at least twelve motile but poorly characterized species found in the genus Lactobacillus. Of these, only L. ruminis has been isolated from mammals, and this species may be considered as an autochthonous member of the gastrointestinal microbiota of humans, pigs and cows. Nine L. ruminis strains were investigated here to elucidate the biochemistry and genetics of Lactobacillus motility. Six strains isolated from humans were non-motile while three bovine isolates were motile. A complete set of flagellum biogenesis genes was annotated in the sequenced genomes of two strains, ATCC25644 (human isolate) and ATCC27782 (bovine isolate), but only the latter strain produced flagella. Comparison of the L. ruminis and L. mali DSM20444T motility loci showed that their genetic content and gene-order were broadly similar, although the L. mali motility locus was interrupted by an 11.8 Kb region encoding rhamnose utilization genes that is absent from the L. ruminis motility locus. Phylogenetic analysis of 39 motile bacteria indicated that Lactobacillus motility genes were most closely related to those of motile carnobacteria and enterococci. Transcriptome analysis revealed that motility genes were transcribed at a significantly higher level in motile L. ruminis ATCC27782 than in non-motile ATCC25644. Flagellin proteins were isolated from L. ruminis ATCC27782 and from three other Lactobacillus species, while recombinant flagellin of aflagellate L. ruminis ATCC25644 was expressed and purified from E. coli. These native and recombinant Lactobacillus flagellins, and also flagellate L. ruminis cells, triggered interleukin-8 production in cultured human intestinal epithelial cells in a manner suppressed by short interfering RNA directed against Toll-Like Receptor 5. This study provides genetic, transcriptomic, phylogenetic and immunological insights into the trait of flagellum-mediated motility in the lactobacilli.
    • CLA-producing adjunct cultures improve the nutritional value of sheep cheese fat

      Renes, Erica; Gómez-Cortés, Pilar; de la Fuente, Miguel Ángel; Linares, Daniel M.; Tornadijo, M. Eugenia; Fresno, José M.; University of León; Juan de la Cierva research contract (Elsevier, 2018-09-10)
      The influence of the autochthonous CLA-producing Lactobacillus plantarum TAUL 1588 and Lactobacillus casei subsp. casei SS 1644 strains and the ripening time on the fatty acid (FA) content and sensory characteristics of sheep cheese were investigated. Three cheese types with different cultures and the control cheese were produced in duplicate and ripened for 8 months. 86 individual FA were determined by gas chromatography. Ripening time (2, 90, 180 and 240 days) did not have a significant effect (P > .05) on the FA content. However, the presence of both Lactobacillus CLA-producing strains led to a decrease of the saturated FA content and to 1.30, 1.19 and 1.27 times higher levels of vaccenic acid, CLA and omega-3, respectively, when compared to the control cheese. This combination allowed obtaining sheep milk cheeses with a healthier FA content, without appreciable changes on sensory characteristics. This work could be a promising approach to increase the bioactive fatty acid content of cheeses.
    • Commercial systems for ultra-rapid chilling of lamb

      Redmond, Grainne; McGeehin, Brian; Henchion, Maeve; Sheridan, James J.; Troy, Declan J.; Cowan, Cathal; Butler, Francis (Teagasc, 2001-08)
      The overall objective was to devise a rapid chilling system for the Irish lamb processing industry. The objective of the first trial was to assess the effect of ultra-rapid chilling in air at - 4ºC, -10ºC and -20ºC and subsequent ageing on the appearance and tenderness of lamb carcasses. The objective of the next trial was to investigate the effect of carcass splitting, which produces faster chilling rates and reduces skeletal constraint of muscles, on the tenderness of rapidly and conventionally chilled lamb. The next task was to compare the effects of immersion chilling and conventional air chilling on meat tenderness and evaporative weight loss in lamb carcasses. The next task was to assess the level of interest in industry. This required costings of the process and a survey of several lamb processors focusing on their perceptions of rapid chilling in general, its advantages and disadvantages, and the implications of adopting the new system. The final objective was to introduce the ultra-rapid chilling process to industry via a factory trial. Lambs were ultra-rapidly chilled and then exported to France for assessment.
    • 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 analysis of Salmonella susceptibility and tolerance to the biocide chlorhexidine identifies a complex cellular defense network

      Condell, Orla; Power, Karen A.; Handler, Kristian; Finn, Sarah; Sheridan, Aine; Sergeant, Kjell; Renaut, Jenny; Burgess, Catherine; Hinton, Jay C.D.; Nally, Jarlath E.; et al. (Frontiers Media SA, 2014-08-01)
      Chlorhexidine is one of the most widely used biocides in health and agricultural settings as well as in the modern food industry. It is a cationic biocide of the biguanide class. Details of its mechanism of action are largely unknown. The frequent use of chlorhexidine has been questioned recently, amidst concerns that an overuse of this compound may select for bacteria displaying an altered susceptibility to antimicrobials, including clinically important anti-bacterial agents. We generated a Salmonella enterica serovar Typhimurium isolate (ST24CHX) that exhibited a high-level tolerant phenotype to chlorhexidine, following several rounds of in vitro selection, using sub-lethal concentrations of the biocide. This mutant showed altered suceptibility to a panel of clinically important antimicrobial compounds. Here we describe a genomic, transcriptomic, proteomic, and phenotypic analysis of the chlorhexidine tolerant S. Typhimurium compared with its isogenic sensitive progenitor. Results from this study describe a chlorhexidine defense network that functions in both the reference chlorhexidine sensitive isolate and the tolerant mutant. The defense network involved multiple cell targets including those associated with the synthesis and modification of the cell wall, the SOS response, virulence, and a shift in cellular metabolism toward anoxic pathways, some of which were regulated by CreB and Fur. In addition, results indicated that chlorhexidine tolerance was associated with more extensive modifications of the same cellular processes involved in this proposed network, as well as a divergent defense response involving the up-regulation of additional targets such as the flagellar apparatus and an altered cellular phosphate metabolism. These data show that sub-lethal concentrations of chlorhexidine induce distinct changes in exposed Salmonella, and our findings provide insights into the mechanisms of action and tolerance to this biocidal agent.
    • Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation

      Kelleher, Philip; Bottacini, Francesca; Mahony, Jennifer; Kilcawley, Kieran N; 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.
    • Comparative genomics of lactic acid bacteria reveals a niche-specific gene set

      O'Sullivan, Orla; O'Callaghan, John; Sangrador-Vegas, Amaia; McAuliffe, Olivia; Slattery, Lydia; Kaleta, Pawel; Callanan, Michael J.; Fitzgerald, Gerald F; Ross, R Paul; Beresford, Tom (Biomed Central, 05/03/2009)
      Background: The recently sequenced genome of Lactobacillus helveticus DPC4571 1 revealed a dairy organism with significant homology (75% of genes are homologous) to a probiotic bacteria Lb. acidophilus NCFM 2. This led us to hypothesise that a group of genes could be determined which could define an organism's niche. Results: Taking 11 fully sequenced lactic acid bacteria (LAB) as our target, (3 dairy LAB, 5 gut LAB and 3 multi-niche LAB), we demonstrated that the presence or absence of certain genes involved in sugar metabolism, the proteolytic system, and restriction modification enzymes were pivotal in suggesting the niche of a strain. We identified 9 niche specific genes, of which 6 are dairy specific and 3 are gut specific. The dairy specific genes identified in Lactobacillus helveticus DPC4571 were lhv_1161 and lhv_1171, encoding components of the proteolytic system, lhv_1031 lhv_1152, lhv_1978 and lhv_0028 encoding restriction endonuclease genes, while bile salt hydrolase genes lba_0892 and lba_1078, and the sugar metabolism gene lba_1689 from Lb. acidophilus NCFM were identified as gut specific genes. Conclusion: Comparative analysis revealed that if an organism had homologs to the dairy specific geneset, it probably came from a dairy environment, whilst if it had homologs to gut specific genes, it was highly likely to be of intestinal origin. We propose that this "barcode" of 9 genes will be a useful initial guide to researchers in the LAB field to indicate an organism's ability to occupy a specific niche.
    • Comparative Proteomic Profiling of Divergent Phenotypes for Water Holding Capacity across the Post Mortem Ageing Period in Porcine Muscle Exudate

      Di Luca, Alessio; Hamill, Ruth M; Mullen, Anne Maria; Slavov, Nikolai; Giuliano, Elia; Department of Agriculture, Food and the Marine, Ireland; 06RDNUIG470 (PLOS, 07/03/2016)
      Two dimensional Difference Gel Electrophoresis (2-D DIGE) and mass spectrometry were applied to investigate the changes in metabolic proteins that occur over a seven day (day 1, 3 and 7) post mortem ageing period in porcine centrifugal exudate from divergent meat quality phenotypes. The objectives of the research were to enhance our understanding of the phenotype (water holding capacity) and search for biomarkers of this economically significant pork quality attribute. Major changes in protein abundance across nine phenotype-by-time conditions were observed. Proteomic patterns were dominated by post mortem ageing timepoint. Using a machine learning algorithm (l1-regularized logistic regression), a model was derived with the ability to discriminate between high drip and low drip phenotypes using a subset of 25 proteins with an accuracy of 63%. Models discriminating between divergent phenotypes with accuracy of 72% and 73% were also derived comparing respectively, high drip plus intermediate phenotype (considered as one phenotype) versus low drip and comparing low drip plus intermediate phenotype (considered as one phenotype) versus high drip. In all comparisons, the general classes of discriminatory proteins identified include metabolic enzymes, stress response, transport and structural proteins. In this research we have enhanced our understanding of the protein related processes underpinning this phenotype and provided strong data to work toward development of protein biomarkers for water holding capacity.
    • Compared to casein, bovine lactoferrin reduces plasma leptin and corticosterone and affects hypothalamic gene expression without altering weight gain or fat mass in high fat diet fed C57/BL6J mice

      McManus, Bettina; Korpela, Riitta; O'Connor, Paula M.; Schellekens, Harriet; Cryan, John F.; Cotter, Paul D.; Nilaweera, Kanishka N. (Biomed Central, 08/12/2015)
      Background Several studies in both humans and rodents have examined the use of lactoferrin as a dietary solution to weight gain and visceral fat accretion and have shown promising results in the short term (up to 7 weeks). This study examined the effects of giving lactoferrin over a longer period of time. Methods For 13 weeks, male C57/BL6J mice were given a diet containing 10 % kJ fat and 20 % kJ casein (LFD) or a diet with 45 % kJ fat and either 20 % kJ casein (HFD) or 20 % kJ lactoferrin (HFD + Lac). Physiological, metabolic, and biochemical parameters were investigated. Gene expression was investigated by Real-Time PCR and microarray. All data was assessed using t-test, ANOVA or ANCOVA. Gene Set Enrichment Analysis was used to interpret microarray data and assess the impact on gene sets with common biological roles. Results By the end of the trial, HFD + Lac fed mice did not alter energy balance, body composition, bodyweight, or weight gain when compared to the HFD group. Notably, there were no changes in subcutaneous or epididymal adipose leptin mRNA levels between high fat diet groups, however plasma leptin was significantly reduced in the HFD + Lac compared to HFD group (P < 0.05) suggesting reduced leptin secretion. Global microarray analysis of the hypothalamus indicate an overall reduction in gene sets associated with feeding behaviour (P < 0.01) and an up-regulation of gene sets associated with retinol metabolism in the HFD + Lac group compared to the HFD group (P < 0.01). Genes in the latter catergory have been shown to impact on the hypothalamic-pituitary-adrenal axis. Notably, plasma corticosterone levels in the HFD + Lac group were reduced compared to the HFD fed mice (P < 0.05). Conclusions The data suggests that prolonged feeding of full-length dietary lactoferrin, as part of a high fat diet, does not have a beneficial impact on weight gain when compared to casein. However, its impact on leptin secretion and accompanying changes in hypothalamic gene expression may underlie how this dietary protein alters plasma corticosterone. The lactoferrin fed mouse model could be used to identify leptin and corticosterone regulated genes in the hypothalamus without the confounding effects of body weight change.
    • Comparing Apples and Oranges?: Next Generation Sequencing and Its Impact on Microbiome Analysis

      Clooney, Adam G; Fouhy, Fiona; Sleator, Roy D.; O'Driscoll, Aisling; Stanton, Catherine; Cotter, Paul D.; Claesson, Marcus J.; Science Foundation Ireland; European Union; SFI/12/RC/2273; et al. (PLOS, 05/02/2016)
      Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number ofmethodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance inmicrobiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced withMiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.
    • Comparison of antioxidant activities of bovine whey proteins before and after simulated gastrointestinal digestion

      Corrochano, Alberto R.; Sariçay, Yunus; Arranz, Elena; Kelly, Philip M.; Buckin, Vitaly; Giblin, Linda; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 13 F 354-WheyGSH (Elsevier, 2018-10-24)
      Oxidative stress caused by free radicals has been implicated in several human disorders. Dietary antioxidants can help the body to counteract those reactive species and reduce oxidative stress. Antioxidant activity is one of the multiple health-promoting attributes assigned to bovine whey products. The present study investigated whether this activity was retained during upper gut transit using a static simulated in vitro gastrointestinal digestion (SGID) model. The capacity to scavenge free radicals and reduce ferric ion of whey protein isolate (WPI), individual whey proteins, and hydrolysates pre- and post-SGID were measured and compared using various antioxidant assays. In addition, the free AA released from individual protein fractions in physiological gut conditions were characterized. Our results indicated that the antioxidant activity of WPI after exposure to the harsh conditions of the upper gut significantly increased compared with intact WPI. From an antioxidant bioactivity viewpoint, this exposure negates the need for prior hydrolysis of WPI. The whey protein α-lactalbumin showed the highest antioxidant properties post-SGID (oxygen radical absorbance capacity = 1,825.94 ± 50.21 μmol of Trolox equivalents/g of powder) of the 4 major whey proteins tested with the release of the highest amount of the antioxidant AA tryptophan, 6.955 μmol of tryptophan/g of protein. Therefore, α-lactalbumin should be the preferred whey protein in food formulations to boost antioxidant defenses.
    • Comparison of methods for the identification and sub-typing of O157 and non-O157 Escherichia coli serotypes and their integration into a polyphasic taxonomy approach

      Prieto-Calvo, M.A.; Omer, M.K.; Alveseike, O.; Lopez, M.; Alvarez-Ordonez, Avelino; Prieto, Maria Luz; Research Council of Norway; INIA, Spain; Foundation for Levy on Foods; Norwegian Research Fees Fund for Agricultural Goods; et al. (Teagasc (Agriculture and Food Development Authority), Ireland, 30/12/2016)
      Phenotypic, chemotaxonomic and genotypic data from 12 strains of Escherichia coli were collected, including carbon source utilisation profiles, ribotypes, sequencing data of the 16S–23S rRNA internal transcribed region (ITS) and Fourier transform-infrared (FT-IR) spectroscopic profiles. The objectives were to compare several identification systems for E. coli and to develop and test a polyphasic taxonomic approach using the four methodologies combined for the sub-typing of O157 and non-O157 E. coli. The nucleotide sequences of the 16S–23S rRNA ITS regions were amplified by polymerase chain reaction (PCR), sequenced and compared with reference data available at the GenBank database using the Basic Local Alignment Search Tool (BLAST) . Additional information comprising the utilisation of carbon sources, riboprint profiles and FT-IR spectra was also collected. The capacity of the methods for the identification and typing of E. coli to species and subspecies levels was evaluated. Data were transformed and integrated to present polyphasic hierarchical clusters and relationships. The study reports the use of an integrated scheme comprising phenotypic, chemotaxonomic and genotypic information (carbon source profile, sequencing of the 16S–23S rRNA ITS, ribotyping and FT-IR spectroscopy) for a more precise characterisation and identification of E. coli. The results showed that identification of E. coli strains by each individual method was limited mainly by the extension and quality of reference databases. On the contrary, the polyphasic approach, whereby heterogeneous taxonomic data were combined and weighted, improved the identification results, gave more consistency to the final clustering and provided additional information on the taxonomic structure and phenotypic behaviour of strains, as shown by the close clustering of strains with similar stress resistance patterns.
    • Comparison of the nutritional composition of experimental fermented milk:wheat bulgur blends and commercially available kishk and tarhana products

      O'Callaghan, Yvonne C.; Shevade, Ashwini V.; Guinee, Timothy P.; O'Connor, Tom P.; O'Brien, Nora M.; Department of Agriculture, Food and the Marine; 14/F/805 (Elsevier, 2018-11-05)
      Dried, fermented blends of dairy products and cereals, such as kishk and tarhana, are foodstuffs traditionally consumed in many regions as they possess good nutritional qualities and extended storage stability. This study examined the nutritional composition of kishk or tarhana type products and compared with experimental blends of fermented milk and wheat bulgur containing 60–80% milk. The blends with higher milk contents had levels of protein (18.9%) and fat (5.8%) at the concentrations specified in fortified blended foods as outlined by the World Food Program. Higher milk contents were also associated with higher contents of calcium (323.2 mg/100 g), phosphorus (335.3 mg/100 g), vitamin A (486.7 µg/100 g) and α-tocopherol (174.5 µg/100 g). The nutritional content of the experimental fermented milk:wheat bulgur blends compared favourably with that of the commercial samples. These blends may be suitable as base products, to be fortified with micronutrients, for the development of fortified blended foods (FBFs) for humanitarian distribution.
    • The competitiveness of the Irish food processing industry

      Pitts, Eamonn; O'Connell, Larry; McCarthy, B. (Teagasc, 2001-07)
      Ways of measuring industrial competitiveness are discussed and an analysis of the competitiveness of the food sector as a whole and of three sub-sectors are presented. The techniques employed were Revealed Comparative Advantage and the Porter Diamond.