Research On Preparation Of Immobilized Acid Protease And Its Effect On Accelerating Maturity Of New Vinegar

The Purpose of this research is to accelerate maturity of new vinegar by immobilized acid protease.Through the screening of four kinds of common natural polymer gels, we find the most suitable carrier and method that acid protease was fixed with the method of adsorption by which sodium alginate(SA) and No.2 gel were mixed as carrier material. Based on single factor experiments and orthogonal test to determine the immobilization conditions, the enzymatic properties of immobilized acid protease was also investigated. Optimum conditions which the immobilized enzyme accelerating aging were obtained through single factor experiments and response surface optimization test, then analyzed the effect of immobilized acid protease on the composition of vinegar. The main results were as follows:1. The immobilized optimum conditions for the SA-xanthan gum was obtained:The total concentration of gel was 1.6g/100ml, the ratios of SA to xanthan gum was 4:1, concentrations of calcium chloride was 4%, immobilization time was 2.5h, absorption time was 2.5h, pH of the enzyme was 3.5, the amount of enzyme was 1650U/g.2. The enzymatic properties of the immobilized enzyme were investigated. Under the optimum immobilized conditions, the activity yield was 74.5%, optimum reaction temperature was 45℃, half-life was 17.39 h in vinegar, the thermal stability is better than that of free enzyme.3. The optimum reaction conditions of immobilized enzyme accelerating aging were investigated. According to single factor test, three factors which are more significant for the aging of vinegar were selected, and they are the catalysis temperature, reaction time and adding amount of immobilized enzyme. With the amino acid nitrogen as the evaluation index, according to Box-Beknhen central composite experimental design and response surface analysis, regression equation was derived using statistical software Design-Expert.V8.0.6, obtained the regression equation:Y=0.28+0.0021X1+0.002625X+0.003025X3+0.0016X1X2-0.0012X1X3-0.00205X2X3-0.003755X12-0.002955X22-0.001705X32.Through statistical analysis, this regression equation can make a better fit to the test, and it can be used to determine the best conditions for accelerating aging by immobilized acid protease. Optimum conditions were obtained by the optimization: reaction temperature 46.2℃, reaction time 8.59h, the immobilized enzyme dosage was 9.94 g. When the aging was completed, the amino acid nitrogen in vinegar can reach 0.283g/100ml, increased 0.0291g/100ml.4.17 kinds of amino acids were detected by amino acid analyzer in the vinegar, Sweet amino acids accounted for 48.05% of total amino acids, the bitter taste of amino acids accounted for 37.66% of total amino acids,flavor amino acids accounted for 14.28% of total amino acids. Sweet amino acids, bitter amino acids and flavor amino acids were increased dealing by the immobilized enzyme in vinegar. Evaluated by sensory the taste was soft slightly and stimulus was weakened, its body was transparent.