Optimization Of Chickpea Natto Fermentation Process And Purification Of Nattokinase

Natto is a traditional legume fermented food by inoculating Bacillus subtilis into cooked soybean and fermenting under certain conditions.It can dissolve thrombus,resist oxidation,resist tumor,prevent osteoporosis and so on.Nattokinase in natto has a good function of thrombolysis,which has become a research hotspot.In this study,four kinds of beans were selected as fermentation materials to optimize the solid fermentation process of natto.Meanwhile,the extraction and purification process of nattokinase was studied.In addition,the storage stability and storage life of fresh natto and nattokinase freeze-dried powder were explored through accelerated storage experiment.The main research contents and results are as follows:(1)Using nattokinase and natto sensory evaluation as indicators,the effects of bean species,cooking time,matrix moisture content,inoculation amount,fermentation temperature,fermentation time and post ripening time on natto fermentation were studied by single factor experiment and response surface experiment.The results showed that the content of nattokinase in chickpea fermentation nattokinase was the highest,reaching 986.3 IU/g,more higher than that in soybean.Chickpea natto optimum technological conditions for fermentation: quantity of 6%,fermentation temperature is 34 ?,the fermentation time of 47 h,the prediction model established nattokinase activity 978.2 IU/g,experiment the nattokinase activity 986.3 IU/g,with the theoretical prediction.The model can predict the natto fermentation process of chickpea.(2)Optimization of the purification process of nattokinase.Nattokinase was extracted and purified by centrifugation,salting out and Sephadex G-75 column chromatography,respectively.Salting out method,the optimal process for extraction of nattokinase: v ammonium sulfate: v thick enzyme fluid = 2:1,salting out stirring speed of 600 r/min,salting out temperature 40 ?,the extracting time 16 h,process under this extraction of nattokinase enzymes is 350.2 IU/mL.The optimal technique for purification of nattokinase by Sephadex G-75 column chromatography was as follows: the sample amount of nattokinase was 1 mL,the sample concentration was 0.1 g/mL,the amount of eluent was 150 mL,and the elution time was 500 min.The purified nattokinase activity reached 89032.6 IU/mL.(3)The storage stability of fresh natto and nattokinase freeze-dried powder was studied by accelerated storage experiment.Through accelerated storage test to estimate the fresh natto in 25 ?,4 ? storage,time needed for 10% loss of enzyme activity were 16.6 days and 1.28 years;The time required for enzyme activity loss of 50% was 0.26 years and 7.62 years,respectively.Nattokinase producing at 25 ?,4 ? storage,time needed for 10% loss of enzyme activity were 14.6 days and 0.47 years;The time required for enzyme activity loss of 50% was 0.26 years and 3.11 years,respectively.Through the research of this subject,a kind of bean suitable for natto fermentation chickpea was screened,and the optimal process of natto fermentation was defined,and the extraction and purification process of nattokinase was optimized.At the same time,the storage stability and storage life of fresh natto and nattokinase freeze-dried powder were determined by accelerated storage experiment.The completion of the project provides a theoretical basis for the large-scale production of natto and nattokinase.