• 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.
    • Large-scale genome-wide analysis links lactic acid bacteria from food with the gut microbiome

      Pasolli, Edoardo; De Filippis, Francesca; Mauriello, Italia E.; Cumbo, Fabio; Walsh, Aaron M.; Leech, John; Cotter, Paul D.; Segata, Nicola; Ercolini, Danilo; European Union; et al. (Springer Science and Business Media LLC, 2020-05-25)
      Lactic acid bacteria (LAB) are fundamental in the production of fermented foods and several strains are regarded as probiotics. Large quantities of live LAB are consumed within fermented foods, but it is not yet known to what extent the LAB we ingest become members of the gut microbiome. By analysis of 9445 metagenomes from human samples, we demonstrate that the prevalence and abundance of LAB species in stool samples is generally low and linked to age, lifestyle, and geography, with Streptococcus thermophilus and Lactococcus lactis being most prevalent. Moreover, we identify genome-based differences between food and gut microbes by considering 666 metagenome-assembled genomes (MAGs) newly reconstructed from fermented food microbiomes along with 154,723 human MAGs and 193,078 reference genomes. Our large-scale genome-wide analysis demonstrates that closely related LAB strains occur in both food and gut environments and provides unprecedented evidence that fermented foods can be indeed regarded as a possible source of LAB for the gut microbiome.
    • Meta-analysis of cheese microbiomes highlights contributions to multiple aspects of quality

      Walsh, Aaron M.; Macori, Guerrino; Kilcawley, Kieran N.; Cotter, Paul D.; Science Foundation Ireland; European Commission; Department of Agriculture, Food and Marine; SFI/12/RC/2273P1; SFI/12/RC/2273P2; 818368; et al. (Springer Science and Business Media LLC, 2020-08-13)
      A detailed understanding of the cheese microbiome is key to the optimization of flavour, appearance, quality and safety. Accordingly, we conducted a high-resolution meta-analysis of cheese microbiomes and corresponding volatilomes. Using 77 new samples from 55 artisanal cheeses from 27 Irish producers combined with 107 publicly available cheese metagenomes, we recovered 328 metagenome-assembled genomes, including 47 putative new species that could influence taste or colour through the secretion of volatiles or biosynthesis of pigments. Additionally, from a subset of samples, we found that differences in the abundances of strains corresponded with levels of volatiles. Genes encoding bacteriocins and other antimicrobials, such as pseudoalterin, were common, potentially contributing to the control of undesirable microorganisms. Although antibiotic-resistance genes were detected, evidence suggested they are not of major concern with respect to dissemination to other microbiomes. Phages, a potential cause of fermentation failure, were abundant and evidence for phage-mediated gene transfer was detected. The anti-phage defence mechanism CRISPR was widespread and analysis thereof, and of anti-CRISPR proteins, revealed a complex interaction between phages and bacteria. Overall, our results provide new and substantial technological and ecological insights into the cheese microbiome that can be applied to further improve cheese production.
    • Microbial Succession and Flavor Production in the Fermented Dairy Beverage Kefir

      Walsh, Aaron M.; Crispie, Fiona; Kilcawley, Kieran; O'Sullivan, Orla; O'Sullivan, Maurice G.; Claesson, Marcus J.; Cotter, Paul D.; Science Foundation Ireland; SFI/12/RC/2273; SFI/11/PI/1137; et al. (2018-11-05)
      Kefir is a putatively health-promoting dairy beverage that is produced when a kefir grain, consisting of a consortium of microorganisms, is added to milk to initiate a natural fermentation. Here, a detailed analysis was carried out to determine how the microbial population, gene content, and flavor of three kefirs from distinct geographic locations change over the course of 24-h fermentations. Metagenomic sequencing revealed that Lactobacillus kefiranofaciens was the dominant bacterial species in kefir during early stages of fermentations but that Leuconostoc mesenteroides became more prevalent in later stages. This pattern is consistent with an observation that genes involved in aromatic amino acid biosynthesis were absent from L. kefiranofaciens but were present in L. mesenteroides. Additionally, these shifts in the microbial community structure, and associated pathways, corresponded to changes in the levels of volatile compounds. Specifically, Acetobacter spp. correlated with acetic acid; Lactobacillus spp. correlated with carboxylic acids, esters and ketones; Leuconostoc spp. correlated with acetic acid and 2,3-butanedione; and Saccharomyces spp. correlated with esters. The correlation data suggest a causal relationship between microbial taxa and flavor that is supported by observations that addition of L. kefiranofaciens NCFB 2797 increased the levels of esters and ketones whereas addition of L. mesenteroides 213M0 increased the levels of acetic acid and 2,3-butanedione. Finally, we detected genes associated with probiotic functionalities in the kefir microbiome. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein and can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods. IMPORTANCE Traditional fermented foods represent relatively low-complexity microbial environments that can be used as model microbial communities to understand how microbes interact in natural environments. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein. In the process, the link between individual species, and associated pathways, with flavor compounds is revealed and several genes that could be responsible for the purported gut health-associated benefits of consuming kefir are identified. Ultimately, in addition to providing an important fundamental insight into microbial interactions, this information can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods.
    • A pilot study demonstrating the altered gut microbiota functionality in stable adults with Cystic Fibrosis

      Fouhy, Fiona; Ronan, N. J.; O'Sullivan, Orla; McCarthy, Y.; Walsh, Aaron M.; Murphy, D.M.; Daly, M.; Flanagan, E. T.; Fleming, C.; McCarthy, M.; et al. (Nature Publishing Group, 2017-07-27)
      Cystic Fibrosis (CF) and its treatment result in an altered gut microbiota composition compared to non-CF controls. However, the impact of this on gut microbiota functionality has not been extensively characterised. Our aim was to conduct a proof-of-principle study to investigate if measurable changes in gut microbiota functionality occur in adult CF patients compared to controls. Metagenomic DNA was extracted from faecal samples from six CF patients and six non-CF controls and shotgun metagenomic sequencing was performed on the MiSeq platform. Metabolomic analysis using gas chromatography-mass spectrometry was conducted on faecal water. The gut microbiota of the CF group was significantly different compared to the non-CF controls, with significantly increased Firmicutes and decreased Bacteroidetes. Functionality was altered, with higher pathway abundances and gene families involved in lipid (e.g. PWY 6284 unsaturated fatty acid biosynthesis (p = 0.016)) and xenobiotic metabolism (e.g. PWY-5430 meta-cleavage pathway of aromatic compounds (p = 0.004)) in CF patients compared to the controls. Significant differences in metabolites occurred between the two groups. This proof-of-principle study demonstrates that measurable changes in gut microbiota functionality occur in CF patients compared to controls. Larger studies are thus needed to interrogate this further.