Research On Optimization Of Fermentation Conditions And Isolation Of Nitrite Reductase Produced By Lactobacillus Brevis

As the nitrogen cycle intermediate, nitrite is a potential carcinogen which can be widely found in nature and detected in many food, especially in vegetables. Nitrite is the product of nitrate reduction of bacteria; as a preservative and flavor enhancement, it can be utilized as the additives added to meat products. In recent years, excessive nitrite content of food, water, farming and the environment caused serious damage. People are working hard to find effective method to control or degrade nitrite. Lactobacillus is the human probiotic that can produce nitrite reductase. Enzymatic degradation of nitrite is a safe and effective new way.In this paper, we used a strain of Lactobacillus(Lactobacillus brevis) screened and identified in our laboratory. The strain can produce nitrite reductase. By optimizing the medium and fermentation conditions, enzyme production volume increased. We isolated the enzyme from the cell extract and studied NiRs enzymatic properties. In addition, the degradation of residual sodium nitrite in pickled vegetables and meat caused by nitrite reductase were studied. The main results are as follows: enzyme production capacity increased by optimization of nutrition, the enzyme concentration can reach 1827.4 U/mL which was 35.06% higher than the former enzyme concentration 1353.4 U/mL. After further optimization of its fermentation conditions we got the best fermentation condition:initial pH value of fermentation broth 6.42, inoculation 5.6%, callus 119μg/mL, culture under 30.55. On the base of twice optimization, nitrite reductase enzyme concentration reached 2139.2 U/mL, which was 58.06% higher than before,17.59% higher than the first optimization. Through separation and purification steps as salting, microfiltration, dialysis, DEAE-52 ion exchange chromatography and Sephadex G-150 gel chromatography, the specific activity of NiRs enzyme solution achieved 1334.06 U/mg that increased 5.14 times. Enzymatic study of the nitrite reductase show that temperature for enzyme reaction are 28-35℃, the optimum temperature is 30℃; Thermal stability of the enzyme is not good. So when temperature exceeds 40℃, enzyme activity loses 87.5%. Optimum pH of enzyme reaction is 5.5. Curve of pH's influence to the enzyme shows that the enzyme is an acidic enzyme. The Km value of NiRs is 120.52μg/ml. Metal ions'affect results on the enzyme show that:under low ion concentration, Al3+, Cu2+, Fe2+and Fe3+can increase the activity of enzyme; Ba2+, Ag+, Cd2+, Mg2+and Ca2+can inhibit the enzyme activity; Zn2+and K+don't affect on activity too much; under high ion concentration, Al3+, Cu2+, Fe2+, Fe3+and K+can increase enzyme activity; Zn2+, Ba2+, Ag+, Cd2+, Mg2+and Ca2+can inhibit the enzyme activity. The optimum concentration of Cu2+that promote activity is 5 mmol/L. The trace metal elements contained in NiRs are Cu and Fe whose concentrations were 552.4 mg/kg and 113.2 mg/kg. Molecular weight were measured as 41.83 kDa by SDS-PAGE. In the optimal reaction system, nitrite can be reduced under the national standards after 12 hours by adding enzyme to the food.