BackgroundThe aim of this study was to determine the catalytic activity and physiological role of myosin-cross-reactive antigen (MCRA) from Bifidobacterium breve NCIMB 702258. MCRA from B. breve NCIMB 702258 was cloned, sequenced and expressed in heterologous hosts (Lactococcus and Corynebacterium) and the recombinant proteins assessed for enzymatic activity against fatty acid substrates.ResultsMCRA catalysed the conversion of palmitoleic, oleic and linoleic acids to the corresponding 10-hydroxy fatty acids, but shorter chain fatty acids were not used as substrates, while the presence of trans-double bonds and double bonds beyond the position C12 abolished hydratase activity. The hydroxy fatty acids produced were not metabolised further. We also found that heterologous Lactococcus and Corynebacterium expressing MCRA accumulated increasing amounts of 10-HOA and 10-HOE in the culture medium. Furthermore, the heterologous cultures exhibited less sensitivity to heat and solvent stresses compared to corresponding controls.ConclusionsMCRA protein in B. breve can be classified as a FAD-containing double bond hydratase, within the carbon-oxygen lyase family, which may be catalysing the first step in conjugated linoleic acid (CLA) production, and this protein has an additional function in bacterial stress protection.

This study was designed to isolate different strains of the genus Bifidobacterium from the fecal material of neonates and to assess their ability to produce the cis-9, trans-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be Bifidobacterium species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml ؊1 by using gas-liquid chromatography. The most efficient producers belonged to the species Bifidobacterium breve, of which two different strains converted 29 and 27% of the free linoleic acid to the cis-9, trans-11 isomer per microgram of dry cells, respectively. In addition, a strain of Bifidobacterium bifidum showed a conversion rate of 18%/g dry cells. The ability of some Bifidobacterium strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.

We have previously demonstrated that oral administration of a metabolically active Bifidobacterium breve strain, with ability to form cis-9, trans-11 conjugated linoleic acid (CLA), resulted in modulation of the fatty acid composition of the host, including significantly elevated concentrations of c9, t11 CLA and omega-3 (n-3) fatty acids in liver and adipose tissue. In this study, we investigated whether a recombinant lactobacillus expressing linoleic acid isomerase (responsible for production of t10, c12 CLA) from Propionibacterium acnes (PAI) could influence the fatty acid composition of different tissues in a mouse model. Linoleic-acid-supplemented diets (2 %, w/w) were fed in combination with either a recombinant t10, c12 CLA-producing Lactobacillus paracasei NFBC 338 (Lb338), or an isogenic (vector-containing) control strain, to BALB/c mice for 8 weeks. A third group of mice received linoleic acid alone (2 %, w/w). Tissue fatty acid composition was assessed by GLC at the end of the trial. Ingestion of the strain expressing linoleic acid isomerase was associated with a 4-fold increase (P,0.001) in t10, c12 CLA in adipose tissues of the mice when compared with mice that received the isogenic non-CLA-producing strain. The livers of the mice that received the recombinant CLA-producing Lb338 also contained a 2.5-fold (albeit not significantly) higher concentration of t10, c12 CLA, compared to the control group. These data demonstrate that a single gene (encoding linoleic acid isomerase) expressed in an intestinal microbe can influence the fatty acid composition of host fat. INTRODUCTIONEvidence is emerging to support the concept that the enteric microbiota can exert profound effects on human health and disease, involving complex host-bacteria interactions that are as yet poorly understood. The gut microbiota is important to the host with regard to metabolic functions, providing nutrients and conferring an ability to resist bacterial infections (Marchesi & Shanahan, 2007;Wilks, 2007), as well as playing a dominant role in the education of the intestinal mucosal immune responses. Furthermore, the enteric microbiota has been shown to exert effects on disease processes outside the gut, including an impact on obesity and non-alcoholic fatty liver disease (Dumas et al., 2006;Marchesi & Shanahan, 2007).Conjugated linoleic acid (CLA) is a beneficial bacterial metabolite formed via microbial isomerization of linoleic acid. CLA is a collective term describing different isomers of linoleic acid with conjugated double bonds, the cis-9, trans-11 (c9, t11) CLA isomer being the most abundant form and the t10, c12 CLA isomer accounting for~1 % of total milk fat CLA (Jensen, 2002). It has been reported that t10, c12 CLA is the most potent isomer in terms of potential to prevent cell proliferation and induce apoptosis in cancer cells (Cho et al., 2005(Cho et al., , 2006Kim et al., 2002a; Lee et al., 2006b; Ochoa et al., 2004). t10, c12 CLA is also associated with decreased body fat and increased lean body mass in various animal...

The linoleic acid isomerase enzyme from Propionibacterium acnes responsible for bioconversion of linoleic acid to trans-10, cis-12 conjugated linoleic acid (t10, c12 CLA) was cloned and overexpressed in Lactococcus lactis and Escherichia coli, resulting in between 30 and 50 % conversion rates of the substrate linoleic acid to t10, c12 CLA. The anti-proliferative activities of the fatty acids produced following isomerization of linoleic acid by L. lactis and E. coli were assessed using the human SW480 colon cancer cell line. Fatty acids generated from both L. lactis and E. coli contained a mixture of linoleic acid and t10, c12 CLA at a ratio of ∼1.35 : 1. Following 5 days of incubation of SW480 cells with 5–20 μg ml−1 (17.8–71.3 μM) of the t10, c12 CLA, there was a significant (P<0.001) reduction in growth of the SW480 cancer cells compared with the linoleic acid control. Cell viability after treatment with the highest concentration (20 μg ml−1) of the t10, c12 CLA was reduced to 7.9 % (L. lactis CLA) and 19.6 % (E. coli CLA), compared with 95.4 % (control linoleic acid) and 31.7 % (pure t10, c12 CLA). In conclusion, this is believed to represent the first report in which recombinant strains are capable of producing CLA with an anti-proliferative potential.