The Preliminary Study On The Oxidative Stress Resistant Mechanism Of A Lactococcus Lactis Recombinant Strain

Lactococcus lactis has already acquired the status of generally recognized as safe (GRAS), and when incorporated into food products, it is frequently exposed to different stress conditions, such as those induced by high temperature, salt, acid, and oxygen conditions. Much effort has been made to overcome these stress problems in lactic acid bacteria, and this has involved the application of Lactococcus lactis in different fields.Preliminary studies have found that Lactococcus lactis had acquired higher antioxidant ability when expressing mtg (from Streptoverticillium mobaraense) in a nisin-inducible expression system. Based on the above, firstly, in this study the oxidative stress resistance of the two strains was compared to confirm the differences between the two strains in anti-oxidant stress ability. Then, the mechanism of the oxidative stress resistance was primarily studied.The result has showed as follows:The biomass of the recombinant strain under aerobic growth is increased visibly. The biomass of the recombinant strain at stationary phase is2.782times that of the control strain. And the recombinant strain exhibits a secondary growth in it late stationary phase. Also, the recombinant stain showed an exceptional ability on the carbon source utilization under aerobic conditions. The OD600of the recombinant strain grown in glucose-free M17medium reached2.825, while the control strain performed just about no growth in the same condition. In a word, the recombinant strain was good at coping with the oxygen conditions.Using the16s rRNA sequence analysis and random amplified polymorphic diagnosis (RAPD), it was confirmed the recombinant strain and the control strain were Lactococcus lactis ssp. cremoris.The survival of the recombinant strain is3.165times that of the control strain under the the stress condition of50mmol/L H2O2for10min, and39.171times when exposed for20min. Under the stress condition of150mmol/L H2O2for15min, the survival of the recombinant strain is1456.4times that of the control strain. The superoxide anion scavenging ability of the recombinant strain is10.574times that of the control strain. Moreover, the hydroxyl radical scavenging capacity of the recombinant strain is2.679times that of the control strain. These results suggested that the antioxidant capacity of the recombinant strain was significantly improved. Oxidative stress resistance mechanisms of the recombinant strain were premilinarily studied. The experiments found that the protoplast of the recombinant strain showed the same antioxidant capacity as the intact control strain cell. But when compared the capacity of the antioxidant capacity in their protoplast cell, the protoplast of the recombinant strain still showed a slight advantage. So all the results suggests that the oxidative stress resistance of the recombinant strain not only derived from its thickened cell wall, but also related to the metabolic regulation within its cytoplastWhen the MTG activity (expressed by the recombinant strain) was inhibited partially, the antioxidant capacity of the recombinant strain was decreased significantly. The superoxide anion scavenging capacity of the recombinant strain (shaking culture) is16.129times that of the control strain. But in present of3mmol/L cystamine (to inhibit the intracellular MTG activity), the superoxide anion scavenging capacity of the recombinant strain is induced to9.230times tthat of the control strain. While under static culture conditions, the superoxide anion scavenging capacity of the recombinant strain is dropped from10.878to1.801times that of the control strain. As to the hydroxyl radical scavenging ability, the recombinant strain (shaking culture) is4.279times that of the control strain. But the multiple drop to3.312times in present of3mmol/L cystamine. And under static culture conditions, the ratio changed from4.618to1.161. So the antioxidant s capacity of the recombinant strain is closely associated to the intracellular activity of MTG.When the plasmid contained in the recombinant strain and the control strain were eliminated, the oxygen free radical scavenging ability of the two bacteria are basically the same. This phenomenon implied that the high antioxidant capacity of the recombinant strain probably stemed from the recombinant plasmid.