Temperature and duration of heating of sunflower oil affect ruminal biohydrogenation of linoleic acid in vitro

Sunflower oil heated at 110 or 150°C for 1, 3, or 6 h was incubated with ruminal content in order to investigate the effects of temperature and duration of heating of oil on the ruminal biohydrogenation of linoleic acid in vitro. When increased, these 2 parameters acted together to decrease the disappearance of linoleic acid in the media by inhibiting the isomerization of linoleic acid, which led to a decrease in conjugated linoleic acids and trans-C18:1 production. Nevertheless, trans-10 isomer production increased with heating temperature, suggesting an activation of Δ9-isomerization, whereas trans-11 isomer production decreased, traducing an inhibition of Δ12-isomerization. The amount of peroxides generated during heating was correlated with the proportions of biohydrogenation intermediates so that they might explain, at least in part, the observed effects. The effects of heating temperature and duration on ruminal bacteria community was assessed using capillary electrophoresis single-strand conformation polymorphism. Ruminal bacterial population significantly differed according to heating temperature, but was not affected by heating duration. Heating of fat affected ruminal biohydrogenation, at least in part because of oxidative products generated during heating, by altering enzymatic reactions and bacterial population

Identification and Characterization of Three Novel Lipases Belonging to Families II and V from Anaerovibrio lipolyticus 5ST

Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14)

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78 % following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-014-6355-6) contains supplementary material, which is available to authorized users

Buwchitin:A Ruminal Peptide with Antimicrobial Potential against <i>Enterococcus faecalis</i>

Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis. Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC3(5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalispublishersversionPeer reviewe

The rumen microbiome:An underexplored resource for novel antimicrobial discovery

Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic applicationpublishersversionPeer reviewe

Wheat aleurone polyphenols increase plasma eicosapentaenoic acid in rats

International audienceMethods: These studies were designed to assess whether wheat polyphenols (mainly ferulic acid [FA]) increased the very-long-chain omega-3 fatty acids (VLC n-3) [eicosapentaenoic acid (EPA) and docosahex-aenoic acid (DHA)] in rats. Wheat aleurone (WA) was used as a dietary source of wheat polyphenols. Two experiments were performed; in the first one, the rats were fed WA or control pellets (CP) in presence of linseed oil (LO) to provide alpha-linolenic acid (ALA), the precursor of VLC n-3. In the second one, the rats were fed WA or CP in presence of control oil (CO) without ALA. The concentrations of phenolic acid metabolites in urine were also investigated. Results: The urinary concentration of conjugated FA increased with WA ingestion (p B0.05). Plasma EPA increased by 25% (p B0.05) with WA in the CO group but not in the LO group. In contrast, there was no effect of WA on plasma DHA and omega-6 fatty acids (n-6). Finally, both n-3 and n-6 in the liver remained unchanged by the WA. Conclusion: These results suggest that WA consumption has a significant effect on EPA in plasma without affecting n-6. Subsequent studies are required to examine whether these effects may explain partly the health benefits associated with whole wheat consumption

Wheat aleurone polyphenols increase plasma eicosapentaenoic acid in rats

Methods: These studies were designed to assess whether wheat polyphenols (mainly ferulic acid [FA]) increased the very-long-chain omega-3 fatty acids (VLC n-3) [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] in rats. Wheat aleurone (WA) was used as a dietary source of wheat polyphenols. Two experiments were performed; in the first one, the rats were fed WA or control pellets (CP) in presence of linseed oil (LO) to provide alpha-linolenic acid (ALA), the precursor of VLC n-3. In the second one, the rats were fed WA or CP in presence of control oil (CO) without ALA. The concentrations of phenolic acid metabolites in urine were also investigated. Results: The urinary concentration of conjugated FA increased with WA ingestion (pB0.05). Plasma EPA increased by 25% (pB0.05) with WA in the CO group but not in the LO group. In contrast, there was no effect of WA on plasma DHA and omega-6 fatty acids (n-6). Finally, both n-3 and n-6 in the liver remained unchanged by the WA. Conclusion: These results suggest that WA consumption has a significant effect on EPA in plasma without affecting n-6. Subsequent studies are required to examine whether these effects may explain partly the health benefits associated with whole wheat consumption