Research On Low Purine, Low MK-7Content And High Nattokinase Activity Product

Nattokinase products have won a good reputation in the market for its strongthrombolytic capacity, long half-life in body (8hours) and excellent oral effect. However,some nattokinase products on sell contained high contents of vitamin K2(MK-7) and purine,which had negative effects on thrombolytic and people with gout. In this paper, thedetermination methods of nattokinase activity, purine and MK-7were established. To obtaina product with low contents of purine, MK-7and high nattokinase activity, the bacterialisolation and fermentation conditions were researched. The results can be concluded asfollows.The determination of purine and MK-7in fermentation broth had been established byHPLC. The optimal conditions for purine detection were: column, Hitachi La ChromC18(4.6×250mm,5μm); mobile phase, methanol:tetrabutyl ammonium hydroxide:acetic acid:water（10:1.485:1.485:987.03, v/v/v/v）; mobile velocity of flow was0.7mL/min; columntemperature was25℃and the UV detection wavelength was254nm. The optimal conditionsfor MK-7detection were: column, Aglient SB-C18（4.6×250mm,5μm); mobile phase,methanol: dichloromethane（9:1，v/v）; mobile velocity of flow was1.0mL/min; columntemperature was40℃and the UV detection wavelength was270nm.A bacterium with low contents of purine, MK-7and high enzyme activity was isolatedfrom14kinds of natto products in the market. The isolation bacterium was named Y-2andidentified as Bacillus by16s RNA method. The analysis of free purine and nucleosides inbroth demonstrated that free purine bases were the dominant material (98.41%in total purinematerials). Additionally, analysis of total purine content in fermentation broth revealed thatthe TP content was84.99mg/L after fermentation which was higher than blank group (41.33mg/L). It meant that purine material in fermentation broth were not only from rural material(soybean) but also from microbial metabolism.In order to obtain high enzyme activity with low purine content, several fermentationfactors were optimized. From the single factor experiment, some factors which could beconcluded as N/C, fermentation temperature, fermentation time and pH had significantinfluence on nattokinase activity and purine content. And the nattokinase activity revealednegtive relationship with the purine content. From the orthogonal experiment, the finaloptimum conditions were defined as nitrogen source was whole peas power; carbon sourcewas lactose; N/C was1:1, pH for broth was7.0, inoculum was2%, fermentation temperaturewas37℃, fermentation time was36h. The corresponding enzyme activity was2835.50IU/mL which was significantly higher than products on sel（l1327.42IU/mL~1573.21IU/mL）.While the concentrations of total purine and MK-7were29.36mg/L and231.60μg/100g respectively, which was significantly lower than products on sell（total purine,41.74mg/L~65.32mg/L; MK-7,436.54μg/100g~512.14μg/100g）.Accelerated storage experiments were selected to research the nattokinase productsstability. The results revealed that nattokinase activity would lose10%in22.7d for25℃andin307.21d for4℃. The verification experiment for25℃was researched which revealed thatenzyme activity projected survival rate was only1.39%~7.62%higher than the actualdetection of survival which could meet the calculation demand.