Expression of the catalase gene katA in starter culture Lactobacillus plantarum TISTR850 tolerates oxidative stress and reduces lipid oxidation in fermented meat product.

The catalase gene katA of Lactobacillus sakei SR911 was cloned and expressed in Escherichia coli UM2 and Lactobacillus plantarum TISTR850 under strong lactococcal promoter P59 in E. coli-lactococcus expression vector pIL1020. The L. plantarum TISTR850 is a catalase-deficient strain isolated from local fermented meat product. The recombinant L. plantarum TISTR850 was shown to decompose hydrogen peroxide, and catalase activity approximately three times higher that of natural catalase-producing strain L. sakei SR911. The recombinant protein was also detected by in situ activity staining of the catalase enzyme. The recombinant L. plantarum TISTR850 did not accumulate hydrogen peroxide under glucose-limited aerobic conditions and remained viable after 60 h of incubation. The recombinant and host strain L. plantarum TISTR850 were used as starter cultures in the fermented meat product, and lipid oxidation was monitored over a 7-day storage at 20 degrees C determined as thiobarbituric acid-reactive substances (TBARS) value. The lipid oxidation level in the fermented meat product seeded with the catalase genetically modified starter culture L. plantarum TISTR850 was significantly lower than that of the natural catalase-deficient strain.

Identification of deoxy-D-fructosyl phosphatidylethanolamine as a non-enzymic glycation product of phosphatidylethanolamine and its occurrence in human blood plasma and red blood cells.

The amino-carbonyl reaction (Maillard reaction), also known as glycation, of egg-yolk phosphatidylethanolamine (PE) was induced by incubating PE (50 mg/ml) with D-glucose (222 mM) in a methanol medium containing 2,6-ditert-butyl-p-cresol as an antioxidant at 37 degrees C for 4 days. The resultant product, glycated PE (gPE), was then isolated from the reaction mixture by two-step normal and reversed-phase high-performance liquid chromatography with UV diode array detection and was characterized as having a 1:1:1 elemental ratio of phosphorus:nitrogen:sugar. The Fourier transform-nuclear magnetic resonance spectrum and infrared absorbance spectrum indicate the isolated gPE to have been deoxy-D-fructosyl PE, which is an Amadori product of PE. The fast atom bombardmentmass spectrometric data for the glycation product of authentic dioleoyl PE (1,2-di-9-octadecenoyl-sn-glycero-3-phosphoethanolamine) show that the molecular weight of gPE corresponds to that of glucose-conjugated PE in the form of an Amadori product. This Amadori product formation was also confirmed in PE/phosphate buffer dispersions and in phosphatidylcholine-PE liposome/phosphate buffer suspensions in the presence of D-glucose at 37 degrees C. gPE was degraded by phospholipase A2, C and D. Freshly spiked blood plasma and red blood cells (RBC) from nomal human volunteers contained substantial levels of gPE, the concentration corresponding to at least 9 mol% of PE. Remarkable formation of gPE, up to 15-45 mol% of PE in human blood plasma and RBC, was further confirmed by prolonged incubation with 5-45 mM D-glucose. The gPE formation in RBC was found to be proportional to the glycated hemoglobin formation.

Phospholipid hydroperoxides and lipid peroxidation in liver and plasma of ODS rats supplemented with alpha-tocopherol and ascorbic acid.

Forty-five mutant male ODS rats, unable to synthesize ascorbic acid, were fed nine diets containing 5, 50 or 250 mg of vitamin E/kg diet and 150, 300 or 900 mg of vitamin C/kg diet for 21 days. The concentrations of vitamins C and E increased in liver and plasma in relation to the level of these vitamins in the diet. Vitamin C dietary supplementation increased the plasma vitamin E content at low levels of vitamin E intake, supporting the concept of an in vivo synergism between both antioxidant vitamins. Vitamin C, at the dietary levels studied, did not affect the lipid peroxidation. Vitamin E decreased liver and plasma endogenous levels of thiobarbituric acid-reactive substances and liver sensitivity to non-enzymatic lipid peroxidation. This was confirmed by a highly specific assay of lipid hydroperoxides using high performance liquid chromatography with chemiluminescence detection. The hepatic concentration of both phosphatidylcholine and phosphatidylethanolamine hydroperoxides decreased as the vitamin E content of the diet increased. The results show for the first time the capacity of vitamin E to protest against peroxidation of major phospholipids in vivo under basal unstressed conditions.

Pyrone hydroperoxide formation during the Maillard reaction and its implication in biological systems.

Hydroperoxide formation during Maillard reaction (amino-carbonyl reaction) was investigated using luminol-chemiluminescence-high performance liquid chromatography (CL-HPLC). From the equimolar reaction mixture of 1 M beta-alanine/D-glucose in phosphate buffer (pH 8.0) at 95 degrees C, two hydroperoxides and H2O2 were detected as chemiluminescent products in CL-HPLC, and the yields were proportional to the browning development. One of these hydroperoxides was isolated and identified as 3-hydroxy-5-hydroperoxy-2-methyl-5,6-dihydropyran-4-one (HMDP, pyrone hydroperoxide) by fast atom bombardment mass spectrometry. The HMDP formation was also confirmed in L-lysine/D-glucose and in bovine serum albumin/D-glucose with the physiological incubation at 37 degrees C for 4 days and 3 wk, respectively. Incubation at 37 degrees C of human plasma containing 5.5-25.0 mM of D-glucose for 60 h showed the glucose concentration-dependent formation of HMDP (10-35 microM of H2O2 equivalence). The HMDP was negative to thiobarbituric acid reaction and was degraded by peroxidases such as horseradish peroxidase, Athromyces ramosus peroxidase, heated cytochrome c, and microperoxidase. The results strongly suggested the formation of such hydroperoxide even in biological Maillard reaction termed as glycation, and implied its contribution in pathogenesis and oxidative lesions associated with hyperglycemia.