The Fermentation Technology Of Acid-resistant Thermostable α-amylase And Developing On Its Application

α-amylase is one of the most ancient enzymes in industrial application. It is widely used in food processing, alcohol and brewing, pharmaceutical, textile desizing, paper making, washing liquid, feed, oil exploration and other fields. The fermentation process of bacillus subtilis was optimized, and the hydrolysis saccharification process of the corn starch was also optimized, finally α-amylase was applied in the steamed bread.Through the single factor and orthogonal experiment, enzyme production process conditions were optimized. The results showed that the optimal medium composing of maltose 9%, corn starch 3%; tryptone 1.5%, urea 1.5%; manganese sulfate 0.5%; tween80 0.1%; culture volume(30 mL/250 mL). Under the condition of 37 oC, 200 r/min, it was cultivated 48 h, 72 h, 96 h respectively. Centrifuging the fermented liquid, then taking the clear liquid for the crude enzyme fluid, the acid and high temperature resistant α-amylase activity was measured under the condition of 90 oC, pH5.5. And the highest enzyme activity was improved from 43.1 U/mL to 89.6 U/mL.Taking corn starch for the substrate, through the single factor and orthogonal experiment, the two types of hydrolysis process were optimized.(1) The corn starch was hydrolyzed by the midrange thermal stable α-amylase and glucoamylase. The results showed that the optimum conditions were that: the additive amount of glucoamylase was 200 U/g, the additive amount of α-amylase was 25 U/g, the reaction temperature and time were 75 oC, 25 h respectively. The content of reducing sugar and total sugar were 256.1 mg/mL and 262.4 mg/mL respectively. The conversion rate was 85.4%.(2) Using the acid and high temperature resistant α-amylase and glucoamylase, corn starch should be liquefied before saccharification. The results showed that the optimum conditions were that the additive amount of glucoamylase was 300 U/g, the acid and high temperature resistant α-amylase was 400 U/g, the saccharification temperature was 75 oC, the reaction time was 15 h. Then the content of reducing sugar and total sugar were 290.5 mg/mL and 338 mg/mL respectively. The conversion rate was 96.8%. The two ways of hydrolyzing corn starch were both well, and the hydrolysis rate were both more than 85%. Midrange thermal stable α-amylase and glucoamylase hydrolyzing corn starch contemporary greatly simplified the production process. Another way of hydrolyzing corn starch greatly shortened the development time, and the hydrolysis rate was taller than the first way of hydrolyzing corn starch.Finally, taking yujiao 5 wheat as the experiment materials, then milling them, adding different amount of medium temperature of α-amylase, the acid and high temperature resistant α-amylase to the flour, through the falling number to determine the amount of enzyme. It was concluded that the amount of them were between 1 mg/kg and 5 mg/kg, and the falling number was about 200 s-300 s. So it was suitable for the production of steamed bread. Further more confirmed its optimum additive content respectively through the sensory evaluation scores and texture analyzer data. The results showed that: the additive content of medium temperature α-amylase, the acid and high temperature resistant α-amylase were 2 mg/kg, 3 mg/kg respectively; falling number were 282 s, 276 s respectively; then the hardness, chewiness and viscosity were the lowest, elasticity was moderate, sensory evaluation score was the highest.