EPS 생산 및 Helicobacter pylori urease에 억제효과가 있는 김치유산균 발효공정 최적화

Abstract

The pro-biotic lactic acid bacteria used to produce EPS (Exo-polysaccharide) and inhibited Helicobacter pylori urease. Many thesis didn't report optimization of fermentation process using MRS broth components. Some thesis used industrial broth, optimized the culture conditions for lactic acid bacteria isolated from Kimchi. In this research tried to optimize MRS broth components for improving production and decreasing the broth cost at laboratory. We used two finds of lactic acid bacteria isolated from Kimchi. The one is producing EPS(Exo-polysaccharide) with lactic acid bacteria isolated from Kimchi. We were already optimized culture conditions using MRS broth components. The optimal broth components were peptone 1 g/L, yeast extract 0.5 g/L, ammonium citrate 2 g/L, sodium acetate 1 g/L, dipotassium phosphate 1 g/L and optimal temperature and pH for growth and EPS production in batch cultivation were found to be 30℃ and 6.5, respectively. There was 100rpm agitation and the optimum aeration was between the 1.0~2.0 vvm. The suitable sucrose concentration was 30 g/L for both lactic acid bacteria growth and EPS production. Using these conditions the maximum cell mass and EPS production were 5.12 g/L and 0.5 g/L in the batch cultivation. Using these conditions we started fed batch cultivation. The feeding medium was condensed by 10times, and we supplied substrate by 1 mL/min after 12hr. The cell growth and exo-polysaccharide production have been improved as cultivation time goes by. We increase the agitation speed between 100~150 rpm, because of decreasing oxygen concentration in a fermenter. These conditions were fit in cell growth and exo-polysaccharide production. The maximum cell growth was 13.6 g/L and exo-polysaccharide was 7.21 g/L. For considering substrate decreases, we used DO stat method. At the second research we have been studied inhibition of Helicobacter pylori urease of lactic acid bacteria isolated from Kimchi. Using batch reactor and micro plate reader, we have found out optimal conditions for second lactic acid bacteria. In the batch reactor system, the optimum aeration rate was 1vvm at the initial pH 6.0 and 37℃. For the agitation speed, we have used stational cultivation. The maximum cell mass was 5.16 g/L. With these conditions we have developed fed batch fermentation. Using DO stat control method, we were supplied substrate by 5 mL/min after 16hr, and condensed medium by 10times. There was no agitation speed, just controlled aeration between 1~2vvm. The maximum cell growth was 10.9 g/L. At the third research we have been found the Lactobacillus strains isolated from fermented Kimchi produced H. pylori urease inhibitor which is not known by far. The inhibition types of enzymes kinetics was competitive inhibition with substrate urea and the inhibition constant was estimated from experimental data. The enzyme kinetic parameters of urease were 6.743 x 10-3 mM/min for Vm and 0.257 mMfor Km. With the increase of protein concentration in culture broth, the enzyme reaction was decreased. The change of Km was proportional to inhibitor concentration and we determined that the enzyme kinetics follows competitive inhibition and its inhibition constant Ki is 7.57 mg/l. The H. pylori urease inhibitor production with Lactobacillus fermentation was 46 mg/l and its production was greatly improved with fed batch fermentation.