Selection And Fermentation Optimization Of Lactococcus Lactis With High Productivity Diacetyl And Related Enzyme Study

Diacetyl is one of the most effective favour substances in fermented dairy. As a secondary metabolite, diacetyl is synthetized during later phase of fermentation. The yield of diacetyl produced by lactococcus lactis is low, most of lactococcus lactis synthetize acetoin instead of deacetyl. The experiment gain one strain of lactococcus lactis with high yield of diacetyl from the lab. After fermentation optimization process the yield of diacetyl rocketed. Many enzymes influence the yield of diacetyl in anabolic path way,so the experiment studies activities of related enzymes and contains isolation and purification of the key enzyme,a-acetolactate decarboxylase(ALDC).The diacetyl yield of Lactococcus selected form the lab reaches10.96mg/L,16S rDNA identification result proves the strain is Lactococcus lactis.Response surface method was used to enhance the yield of diacetyl and optimized the fermentation process as:mass concentration of skim milk is10%,inoculum concentration is2%, initial pH of fermentation medium is6.5,fermentation temperature is37℃,liquid medium volume reaches100ml,static cultivation.The diacetylyield is enhanced to22.38mg/L approximately.Four related enzymes of diacetyl were studied, the result approves diacetyl reductase is effected significantly by temperature, the temperature rise above40℃the activity descend evidently, while the pH value has no evident influence on diacetyl reductase activity. The activity of citrate lyase is stable when the temperature changes, optimum temperature is35℃.The activity could be influenced evident-ly if the pH value has vatiation, the optimum pH value is5.5, while the pH value is above6.5,the citrate lyase descends fiercely. Acetolactate synthase can afford the change of temperature and pH value, the optimum pH value is5.5. The optimum temperature of ALDC activity is40℃,when the temperature is above, the activity reduces evidently.The optimum pH value of ALDC is6.5, change of pH value partly influence the activity of ALDC.In order to obtain Gel pure ALDC,such methods as ammonium sulfate precipitation, gel chromatography and gel electrophoresis are used.After calculation the mocular weight of ALDC is73.12KDa. Enzyme activity indicates that Fe+、 Zn2+、Mg2+activate ALDC intense, Ba2+、Ca2+activate ALDC while Cu2+restrain the activity apparently. There branched chain amino acids have significant influence on ALDC activity.IR was used to detect ALDC, it approves that usions ammonia I strip absorption peak is on1661cm-1while there is no usions mammonia II strip absorption peak, the usions ammonia Ⅲstrip absorption peak appears on1300cm-1.