Application Research Of Natto Solid-state Fermentation And Nattokinase Separation And Purification

Thrombosis and embolism serious threat to human health,morbidity and mortality rate ranks first in all kinds of diseases.Natto kinase as a new generation of thrombolytic drugs has a strong fibrinolytic activity,low cost,good safety,the advantages of long duration,so the natto kinase is of great significance to promote the development of thrombolytic drugs.This paper studies the natto solid-state fermentation process,compares three methods of drying of natto,optimized the purification natto kinase ultrafiltration method,and studied its stability,has provided the preparation of natto and natto kinase of processing and utilization of new ideas.To obtain high-quality natto,test ordinary soybeans as raw material,solid and optimized natto fermentation process.With content of natto kinase and mucus production rate as index,The effects of initial water content,inoculum amount,fermentation temperature,fermentation time of nattokinase content and yield of mucus.According to the results of single factor experiment.To optimize the orthogonal rotation combination design technology of solid fermentation of natto,optimal parameters of solid natto fermentation process for the initial moisture content of 51%.Inoculation quantity was 0.15%,fermentation temperature is 43 ?,fermentation time is 24 h,under the conditions of nattokinase content was 0.076 mg/mL,mucus yield of 17.60%.The raw material of solid state fermentation of natto,Comparison of three kinds of drying methods on the influence of natto kinase activity,analysis nutrients and amino acid composition of dride nano powder.The results show that the vacuum freeze-drying at 10-15h,nattokinase activity loss rate of less than 17%;dried with hot air at 50-60 ? drying conditions 16-32h nattokinase activity loss rate of 28-43%control the moisture content within 10%;when cold drying at 24-28 ? dried 48-72h nattokinase activity loss rate in 46-51%,control the moisture content 12-8%;Fully consider a hot air drying and drying of the product is more suitable for natto.The ash content of the dried sodium soy flour was 5.77%,a fat content of 24.24%,38.13%protein content,reducing sugar content was 6.27%,the total amino acid content of 32.08g/100g,essential amino acid content of 11.12 g/100g.Test saturation of 20%and 60%saturation of ammonium sulfate fractional salting Preparation of crude enzyme solution to the crude enzyme solution as raw material by response surface analysis separation of ultrafiltration purification of nattokinase process optimization.In nattokinase vitality index,the effects of operating pressure,feed temperature,feed solution pH influence on the vitality of nattokinase.The single factor results,ultrafiltration separation and purification of nattokinase to optimize response surface design to determine the ultrafiltration separation and purification of nattokinase optimum operating pressure 0.2MPa,feed temperature 17 ?,solid-liquid pH 7.2,under the conditions of nattokinase activity was 981.98U/mL.Ultrafiltration separation and purification to obtain pure nattokinase enzyme specific activity was 6546.53U/mg,the purification fold of 5.99 times,the recovery was 66.5%.Pure enzyme by SDS-PAGE electrophoresis verified as pure electrophoresis,a molecular weight of 28KD.Nattokinase should be stored at low temperatures,to stand at 37 ? 6h inactivation is not significantly higher than that at 55 ? enzyme inactivated Rapidly.When the pH of 6-12,activity of nattokinase enzyme was stable,it should be kept under alkaline conditions.Mg2+,Ca2+ has a weak activation to nattokinase;Ba2+,K+,Na+ little impact,Zn2+,Cu2+,Fe2+,Fe3+ have different degrees of inhibition to nattokinase.Glycerin,gelatin,sodium alginate,chitosan,skimmed milk powder on the thermal stability of nattokinase contributes soluble starch is not obvious,CMC of nattokinase thermal stability to some extent.Isolated and purified by ultrafiltration nattokinase powder is white and smooth,and yield is 1.7%.