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Every year, microbial contamination causes billions of tons of food wasted and millions of cases of illness. ε-Poly-l-lysine has potent, wide-spectrum inhibitory activity and is heat stable and biodegradable. It has been approved for food preservation by an increasing number of countries. ε-Poly-l-lysine is produced from soil bacteria of the genus Streptomyces, also producers of various antibiotic drugs and toxins and not considered to be a naturally occurring food component., ε-Poly-l-lysine is a potent antimicrobial produced through fermentation of Streptomyces and used in many Asian countries as a food preservative. It is synthesized and excreted by a special nonribosomal peptide synthetase (NRPS)-like enzyme called Pls. In this study, we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the Pls from Streptomyces in terms of domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus Pls promoter, we confirmed that its product is indeed ε-poly-l-lysine. A comprehensive sequence analysis suggested that Pls genes are widely spread among coryneform actinobacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain it in their genomes. This finding raises the possibility that ε-poly-l-lysine as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems. IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and millions of cases of illness. ε-Poly-l-lysine has potent, wide-spectrum inhibitory activity and is heat stable and biodegradable. It has been approved for food preservation by an increasing number of countries. ε-Poly-l-lysine is produced from soil bacteria of the genus Streptomyces, also producers of various antibiotic drugs and toxins and not considered to be a naturally occurring food component. The frequent finding of pls in cheese and skin bacteria suggests that ε-poly-l-lysine may naturally exist in cheese and on our skin, and ε-poly-l-lysine producers are not limited to filamentous actinobacteria.

Epsilon-polylysine (ε-PL) is an antimicrobial commercially produced by Streptomyces fermentation and widely used in Asian countries for food preservation. Here we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the ε-PL synthetase from Streptomyces in terms of enzymatic domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus ε-PL synthetase promoter, we confirmed that its product is indeed ε-PL. A comprehensive sequence analysis suggests that ε-PL synthetases are widely spread among coryneform bacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain Pls gene. This discovery raises the possibility that ε-PL as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems.IMPORTANCEEvery year, microbial contamination causes billions of tons of food wasted and millions of cases of foodborne illness. ε-PL is an excellent food preservative as it is potent, wide spectrum and is heat stable and biodegradable. It has not been accepted by all countries (e.g those in the EU) partially because it was not a natural composition of food but rather originated from the soil bacteria Streptomyces, a famous producer of various antibiotic drugs and toxins. The unexpected finding of ε-PL synthetases in cheese and skin bacteria suggests that ε-PL may naturally exist in cheese and on our skin.

A method for semi-preparative isolation of major resveratrol metabolites from human urine after oral intake of a trans-resveratrol-containing dietary supplement was developed. Pretreatment of the urine (6L) by using solid-phase extraction gave a brown oily residue (9.3g), which was separated using a combination of normal phase column chromatography and reversed-phase flash column chromatography resulting in fractions containing 1.1g crude trans-resveratrol-3-O-sulfate (M1), 86mg of a crude mixture of trans-resveratrol-3,5-O-disulfate (M2) and trans-resveratrol-3,4'-O-disulfate (M3), and 568mg of a crude mixture of trans-resveratrol-3-O-β-d-glucuronide (M4) and dihydroresveratrol-3-O-β-d-glucuronide (M5). Purification of the crude metabolites was performed by semi-preparative reversed-phase HPLC using a gradient of aqueous ammonium acetate (2.5mmol/L, pH 6.7)/acetonitrile for purification of M1, M2 and M3 or trifluoroacetic acid in water (pH 2.5)/acetonitrile for purification of M4 and M5. From a part of the crude metabolites (50-75mg), 47mg M1 (purity 98.7%), 14mg M2 (purity 96.1%), 10mg M3 (purity 96.3%), 38mg M4 (purity 98.2%) and 18mg M5 (purity 97.8%) were obtained. The structures of all isolated resveratrol metabolites were elucidated by spectroscopic and spectrometric methods such as 1D and 2D NMR, UV, and LC-MS. This method represents a novel approach to obtain resveratrol metabolites being the first method describing the direct isolation of pure resveratrol metabolites from urine samples in quantities sufficient for full chemical characterization and testing in vitro and in preclinical trials.

Obesity, diabetes, hypertension, and hyperlipidemia constitute risk factors for morbidity and premature mortality. Based on animal and in vitro studies, resveratrol reverts these risk factors via stimulation of silent mating type information regulation 2 homolog 1 (SIRT1), but data in human subjects are scarce. The objective of this study was to examine the metabolic effects of high-dose resveratrol in obese human subjects. In a randomized, placebo-controlled, double-blinded, and parallel-group design, 24 obese but otherwise healthy men were randomly assigned to 4 weeks of resveratrol or placebo treatment. Extensive metabolic examinations including assessment of glucose turnover and insulin sensitivity (hyperinsulinemic euglycemic clamp) were performed before and after the treatment. Insulin sensitivity, the primary outcome measure, deteriorated insignificantly in both groups. Endogenous glucose production and the turnover and oxidation rates of glucose remained unchanged. Resveratrol supplementation also had no effect on blood pressure; resting energy expenditure; oxidation rates of lipid; ectopic or visceral fat content; or inflammatory and metabolic biomarkers. The lack of effect disagrees with persuasive data obtained from rodent models and raises doubt about the justification of resveratrol as a human nutritional supplement in metabolic disorders.

Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH) are increasing clinical problems for which effective treatments are required. The polyphenol resveratrol prevents the development of fatty liver disease in a number of experimental studies. We hypothesized that it could revert steatohepatitis, including hepatic inflammation and fibrosis, in an experimental NASH model. To induce hepatic steatohepatitis, a 65% fat, 2% cholesterol and 0.5% cholate (HFC) diet was fed to rats for 1 or 16 weeks, prior to treatment. Subsequently, the diet was supplemented with resveratrol (approx. 100mg/rat/day) to three intervention groups; week 2-4, 2-7 or 17-22. Treated animals were sacrificed at the end of each intervention period with appropriate control and HFC diet controls. Blood and liver were harvested for analysis. When commenced early, resveratrol treatment partially mitigated transaminase elevations, hepatic enlargement and TNFα induced protein-3 protein expression, but generally resveratrol treatment had no effect on elevated hepatic triglyceride levels, histological steatohepatitis or fibrosis. We observed a slight reduction in Collagen1α1 mRNA expression and no reduction in the mRNA expression of other markers of fibrosis, inflammation or steatosis (TGFβ, TNFα, α2-MG, or SREBP-1c). Resveratrol metabolites were detected in serum, including trans-resveratrol-3-O-sulphate/trans-resveratrol-4'-O-sulphate (mean concentration 7.9μg/ml). Contrary to the findings in experimental steatosis, resveratrol treatment had no consistent therapeutic effect in alleviating manifest experimental steatohepatitis.

Low-grade inflammation is seen with obesity and is suggested to be a mediator of insulin resistance. The eliciting factor of low-grade inflammation is unknown but increased permeability of gut bacteria-derived lipopolysaccharides (LPS) resulting in endotoxemia could be a candidate. Here we test the effect of LPS and the anti-inflammatory compound resveratrol on glucose homeostasis, insulin levels and inflammation. Mice were subcutaneously implanted with osmotic mini pumps infusing either low-dose LPS or saline for 28 days. Half of the mice were treated with resveratrol delivered through the diet. LPS caused increased inflammation of the liver and adipose tissue (epididymal and subcutaneous) together with enlarged spleens and increased number of leukocytes in the blood. Resveratrol specifically reduced the inflammatory status in epididymal fat (reduced expression of TNFa and Il1b, whereas the increased macrophage infiltration was unaltered) without affecting the other tissues investigated. By LC-MS, we were able to quantitate resveratrol metabolites in epididymal but not subcutaneous adipose tissue. LPS induced insulin resistance as the glucosestimulated insulin secretion during an oral glucose tolerance test was increased despite similar plasma glucose level resulting in an increase in the insulinogenic index (IGI; delta 0-15insulin/delta 0-15glucose) from 13.73 to 22.40 pmol/mmol (P < 0.001). This aberration in insulin and glucose homeostasis was normalized by resveratrol. In conclusion: Low-dose LPS enhanced the glucose-stimulated insulin secretion without affecting the blood glucose suggesting increased insulin resistance. Resveratrol restored LPS-induced alteration of the insulin secretion and demonstrated anti-inflammatory effects specifically in epididymal adipose tissue possibly due to preferential accumulation of resveratrol metabolites pointing towards a possible important involvement of this tissue for the effects on insulin resistance and insulin secretion.