Protective role of ascorbic acid in the decontamination of cow milk casein by gamma-irradiation.

PURPOSE: The aim of this work was to investigate the protective role of ascorbic acid on irradiation-induced modification of casein. MATERIALS AND METHODS: Casein stock solutions were irradiated with increasing doses 2-10 kGy using (60)Co Gamma rays at a dose rate D• = 136.73 Gy/min at room temperature. The total viable microorganism content of cow milk casein was evaluated by Plate Count Agar (PCA) incubation for 48 h at 37°C. Sodium dodecylsulfate gel electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption-Ionization Time-of-Flight mass spectrometry (MALDI-TOF-MS) analysis were used to evaluate the effect of gamma irradiation on casein integrity. RESULTS: Gamma irradiation reduced the bacterial contamination of casein solutions at a lower irradiation dose when performed in the presence of ascorbic acid. The irradiation treatment of casein in the absence of ascorbic acid with a dose of 4 kGy could reduce 99% of the original amount of bacterial colonies. However, in the presence of ascorbic acid the irradiation treatment of casein with a dose lower than 2 kGy could reduce 99% of the original amount of bacterial colonies which suggested that the irradiation dose lower than 2 kGy achieved almost the entire decontamination result. SDS-PAGE and MALDI-TOF-MS analysis showed that ascorbic acid protected cow milk casein from degradation and subsequent aggregation probably by scavenging oxygen and protein radicals produced by the irradiation. CONCLUSIONS: It is demonstrated that the combination of gamma irradiation and ascorbic acid produce additive effects, providing acceptable hygienic quality of cow milk casein and protects caseins against Reactive Oxygen Species (ROS) generated, during the irradiation process.

Effects of static magnetic fields on growth and membrane lipid composition of Salmonella typhimurium wild-type and dam mutant strains.

This study was carried out to explore the adaptive mechanisms of S. typhimurium particularly, the implication of the Dam methyltransferase in the remodelling of membrane lipid composition to overcome magnetic field stress. With this aim, we focused our analyses on the increase in viable numbers and membrane lipid modifications of S. typhimurium wild-type and dam mutant cells exposed for 10h to static magnetic fields (SMF; 200 mT). For the wild-type strain, exposure to SMF induced a significant decrease (p<0.05) of CFU at 6h, followed by an increase between 8 and 10h. Growth of the dam mutant was significantly affected (p<0.05) after 6h and no recovery was observed until 10h, highlighting a different behavior of SMF stressed wild-type and dam mutant strains. SMF significantly affected the phospholipid proportions in the two strains. The most affected were those of the acidic phospholipids, cardiolipins (CL). In the dam strain the phospholipid response to SMF followed a globally similar trend as in the wild-type with however lower effects, leading mainly to an unusual accumulation of CL. This would in part explain the different behavior of the wild-type and the dam strain. Results showed a significant increase of membrane cyclic fatty acids Cyc17 and Cyc19 in the wild-type strain but only the Cyc17 in the dam strain and a meaningful increase of the total unsaturated fatty acids (UFAs) to total saturated fatty acids (SFAs) ratios of the exposed cells compared to controls from 3 to 9h (p<0.05) for both strains. The net increase of the total UFAs to total SFAs ratios seemed to result mainly from the increase of (C18:1) proportion (p<0.05) and to a lower extent from that of (C16:1) (p<0.05). These modifications of cyclic and unsaturated fatty acid proportions constitute an adaptive response to SMF stress in S. typhimurium wild-type and dam mutants to maintain an optimum level of membrane fluidity under SMF.

Effects of dam and/or seqA mutations on the fatty acid and phospholipid membrane composition of Salmonella enterica serovar Typhimurium.

We examined the phospholipids (Phls) and the membrane fatty acid (FA) composition in Salmonella enterica serovar Typhimurium dam and/or seqA mutants. Phosphatidylglycerol, phosphatidylethanolamine (PE), and cardiolipin (CL) are the major Phls present in all the strains and accounted for greater than 95% of the total lipid phosphorus. Phosphatidic acid and phosphatidylserine are the minor ones. The seqA mutant showed a decrease in PE and an increase in CL and phosphatidylglycerol proportion compared with the wild-type strain. The same changes were observed with the seqA dam double mutant. However, the dam mutation caused an unusual accumulation of CL with a significant decrease in the PE content, compared with the isogenic wild-type strain. FA composition of the total lipids and the different fractions containing Phls have been determined. The major saturated FAs (SFAs) and unsaturated FAs (UFAs) found were C(14:0), C(16:0) and C(16:1w7), C(18:1w9), respectively. Cyclic FAs, cyc(17:0) and cyc(19:0), were also present in appreciable amounts. Moreover, dam and/or seqA mutations caused a decrease in UFA/SFA ratio and there was a progressive reduction in the content of C(16:1w7) and C(18:1w9), going through the order seqA, dam/seqA, and dam mutants. This decrease in UFA content was compensated for in all strains by an increase in the corresponding C(17-) and C(19-) cyclic FAs. So these UFAs were converted to their cyclopropane derivatives, which resulted in a low UFA/SFA ratio. SeqA and Dam proteins might regulate FA biosynthesis and Phls composition of Salmonella enterica serovar Typhimurium.

The effect of methylation on DNA replication in Salmonella enterica serovar typhimurium.

The DNA adenine methylase of Salmonella typhimurium methylates adenine at GATC sequences. Strains deficient in this methylase are not well transformed by methylated plasmids, but unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam(-) strains with fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. It will also be shown that plasmids isolated from dam(-) bacteria are hemimethylated by restriction enzyme digestion. These results may explain why newly formed daughter molecules are not substrates for immediate reinitiation of DNA replication in dam(-) bacteria.