Inulin Preparation And Flexible Chitosan Immobilized Inulinase Hydrolysis Of Inulin

This paper studies on the characterization of the Polyphenol Oxidase (PPO) of Helianthus tuberosus L, the pretreatment of the unsalted Helianthus tuberosus L, the extraction of inulin by the technique of Microwave-Assisted Extraction, the refinement of inulin, the separation and purification of inulin by ultrafiltration membrane technique, the flexible immobilized inulinase on the Chitosan Support, the characterization of immobilized inulinase and the continuous hydrolysis of inulin by immobilized inulinase.The Polyphenol Oxidase (PPO) of Helianthus tuberosus L. was extracted by 0.1mol/L lemon acid buffer, and the characterization of PPO was researched. Experimental results showed that the optimal pH and temperature were 7.4 and 20℃respectively, and the Km value and Vm value with catechol substrate were 11.11 mmol/L and 0.536 OD₄₁₅/min respectively, and the PPO would be inactive in 2 min when the water tempreture was 90℃. Ascorbic acid and Hydrogen sodium of sulphurous acid were strong inhibitors, the effective concentrations of inhibitor were 0.40 g/L and 0.520g/L respectively.The pretreatment of the unsalted Helianthus tuberosus L. was achieved by the uniform design, and the optimization process was obtained as follows: the unsalted Jerusalem Anichoke is scalded for 10 min under the temperature of 100℃, then desiccated for 7 hours under the temperature of 50℃.The inulin from Helianthus tuberosus L. was extracted effectively with the technology of Microwave-Assisted Extraction, and the optimization process of Microwave-Assisted Extraction for inulin was attained as follows: 1:18 as the proportion of Helianthus tuberosus L. to water, 400w as the microwave power, 270s as the microwave radiation time, 95℃as the extracted temperature, and 40 min as the extracted time. Under this optimal condition, the rate of extraction was 99%.The crude solution of inulin was refined through the treatment of the method of lime-phosphoric acid, anion exchange resin and cation exchange resin, and the prepared solution of inulin was obtained. The content of inulin from the prepared solution was decreased from 3.44g/100mL to 2.94 g/100mL, the transmittance was rised from 0.6 to 96.1, the remanent content of inulin was 85.46%, and the inulin solution was purified partially.Through the treatment of the prepared solution of inulin with ultrafiltration membrane technique, the molecular weight distribution of inulin was obtained as follows: the rate of molecular weigh more than 10KDa was 25.3%, between 5KDa and 10KDa was 10.7%, between 3KDa and 5KDa was 11.2%, less than 3KDa was 31.6%, respectively. Overall recovery of inulin was 78.8%. The purity of inulin that was deposited by ethanol and dried after the treatment of 3KDa ultrafiltration membrane was 76.74%, which was more than that of the inland inulin, and only less 10% than that of the import inulin.With the method of the flexible immobilized inulinase on the Chitosan Support, the immobilized inulinase was gained. The conditions of immobilization as follows: the oretical oxidation degree of DAS was 1, enzyme amount was 0.5 mL, immobilized temperature was 15℃, pH was 5.8 which was maked up by phosphoric acid buffer solution, immobilized time was 24 hours. Under that optimal conditions, the recovery of immobilized enzyme activity was more than 95%.The Optimal reaction temperature of the immobilized inulinase was 60℃, the Optimal reaction pH was 4.6, and the Km value, Vm value and I/S was 1.95μmol/mL, 2.12μmol/min and 1.04 respectively. The immobilized inulinase have good temperature stability and pH stability.In the experiment of the continuous hydrolysis of inulin by immobilized inulinase, hydrolysis rate of substrate was decreased gradually under the same concentration of substrate as the flow rate was increased, but the constant volume rate of production was increased gradually. Hydrolysis rate of substrate was decreased gradually under the same flow rate as the concentration of substrate was increased, but the constant volume rate of production was increased gradually. In consideration of the solubility of inulin and the production efficiency, the concentration of inulin should be 10%. The operational stability is the main indicator of evaluating the use value of the immobilized enzyme. Through computation, the attenuation coefficient and the half life of immobilized enzyme was 0.01521 and 45d respectively.