Enzymes involved in polyunsaturated fatty acid saturation metabolism in lactic acid bacteria and its application for functional lipid synthesis

Polyunsaturated fatty acids and probiotic lactic acid bacteria are reported to be effective to prevent metabolic syndrome. The mechanism, however, was not clear yet. We revealed the polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a, which converted linoleic acid into conjugated linoleic acid (CLA)1). The enzyme system for this saturation metabolism was found to consist of four enzymes (hydratase2), dehydrogenase3), isomerase, enone reductase4)) and generate hydroxy fatty acids, oxo fatty acids, and conjugated fatty acids as intermediates. The homologous genes encoding these four enzymes were found in genome sequences of many gut microorganisms. Therefore, acting in concert, gut microbiota may mediate the unsaturated fatty acid saturation metabolism in gastrointestinal tract. Furthermore, we confirmed the existence of these fatty acids in host tissues depending on the existence of gut microbes using specific pathogen free (SPF) mouse and germ free mouse1). Successive analysis revealed health promoting activity of these hydroxy and oxo fatty acids, i.e., intestinal epithelial barrier protection5), anti-obesity6), and anti-diabetic activity7), etc. Therefore, we developed novel production system for these fatty acid metabolites using the enzymes from probiotic lactic acid bacteria8,9,10). 10-hydroxy-cis-12-octadecenoic acid (HYA), an initial intermediate of linoleic acid saturation, has immunomodulatory activity and ameliorates intestinal epithelial barrier impairment, etc5). HYA was found in foods such as cheese, bacon, milk and vegetable pickles, but at low level. We developed hydroxy fatty acid production process using fatty acid hydratase in probiotic lactic acid bacteria. HYA was produced from safflower oil rich in linoleic acid (approximately 75%) with high conversion rate of approximately 50% with Lactobacillus plantarum. We achieved the industrial scale production using 2,000 L fermenter and 500 L reactor. Other C18 Δ9 unsaturated fatty acids such as oleic acid, α-linolenic acid, and γ-linolenic acid were also converted to corresponding 10-hydroxy fatty acids. The various hydroxy fatty acids provided by this technology using fatty acid hydratase in probiotic lactic acid bacteria are promising as novel functional fatty acids. These studies could open a new application of the enzymes involved in polyunsaturated fatty acid saturation in lactic acid bacteria to novel functional lipid production. 1) S. Kishino, et al. : Proc. Natl. Acad. Sci. USA, 110, 17808 (2013). 2) M. Takeuchi, et al. : J. Biosci. Bioeng. 119, 636 (2015). 3) M. Takeuchi, et al. : J. Mol. Catal., B Enzym. 117, 7 (2015). 4) H. Feng, et al. : FEBS Journal, 282, 1526-1537 (2015). 5) J. Miyamoto, et al. : J. Biol. Chem., 290, 2902 (2015). 6) T. Nanthirudjanar, et al. : Lipids, 50, 1093-1102 (2015). 7) T. Goto, et al. : Biochem. Biophys. Res. Commun., 459, 597 (2015). 8) H. Sakurama, et al. : J. Lipid Res., 55, 1855 (2014). 9) M. Takeuchi, et al. : J. Appl. Microbiol., 120, 1282-1288 (2016). 10) M. Takeuchi, et al. : Biosci. Biotechnol. Biochem., 80, 2132-2137 (2016)

Effects of chocolate containing Leuconostoc mesenteroides strain NTM048 on immune function: a randomized, double-blind, placebo-controlled trial

Abstract Background Previous reports showed that oral administration of Leuconostoc mesenteroides strain NTM048 increases IgA levels and CD4+ T cell population in feces and mice, respectively, as revealed by flow cytometric analysis of splenocytes. This study aimed to evaluate the effect of chocolate supplemented with L. mesenteroides strain NTM048 (> 1.00 × 109 CFU/day, NTM048) on the immune parameters of healthy subjects, using a randomized, placebo-controlled, double-blinded study design. Methods Participants (mean age: 46.3 years) ingested 28 g of test food daily, at a time of their own choice, for 4 weeks. The immunological parameters of all participants were evaluated two times (pre- and post- ingestion). At the end of the study, various immunological parameters of the participants were measured and scoring of immunological vigor (SIV) was performed using a comprehensive algorithm. Results Ingestion of NTM048-supplemented chocolate significantly improved SIV in the NTM048 group (18.6 ± 1.6) compared to that in the placebo group (17.8 ± 2.0) after 4 weeks (p = 0.049). Several immunological parameters (CD8+T cells, CD8+CD28+ T cells, and memory T cells) were significantly elevated in the NTM048 group as compared to the placebo group (all p < 0.05). In addition, T cell proliferation index at post-ingestion significantly increased compared with that at pre-ingestion in the NTM048 (p = 0.017) and placebo groups (p = 0.037), although no differences were observed between the two groups. Conclusion Our results suggest that ingestion of chocolate supplemented with NTM048 is effective against the age-related decline in T cell-related immune functions. Trial registration UMIN Clinical Trials Registry UMIN000021989. Registered 19 April 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R00002532