Optimization Of α-Dextranase Produced By Fermentation Of Chaetomium Gracile

Dextran is a kind of viscous polysaccharide produced by microorganism in process of cane sugaring. Its existence can bring great harm to the production of white sugar. Main methods to eliminate dextran include physical, chemical and biological methods at present, but these methods have their own defects can’t be solved, more or less. The most safe and effective method to eliminate dextran is to directly add a-dextranase to liquid glucose in recent dextran removal method. However, this method is restricted by the defect that a-dextranase prepared by current technology is in low enzyme activity, high cost and difficulty to embody industrial application. Therefore, it becomes very critical to produce a type of efficient, low-cost and environment-friendly a-dextranase. Chaetomium gracile is a safe bacterial strain for food and it can produce and prepare a-dextranase in fermentation process, and it has great potential application in research. Due to the stronger self-flocculence of Chaetomium gracile, the enzyme activity of a-a-dextranase produced by fermentation of Chaetomium gracile is lower, which greatly restricts the application of this type of enzyme in food and medicine.This paper aims to optimize fermentation technology of Chaetomium gracile to improve enzyme activity and production, and build up the foundation for industrialized production. Main research contents of this topic include:1. Adopt optimization strategy of fermentation and improve enzyme production efficiency.This topic is to optimize fermentation culture and process of Chaetomium gracile then perform single factor experiment and orthogonal experiment in terms of addition of calcium carbonate, fermentation temperature, rotation speed, optimized choice of nitrogen source and mass proportion of nitrogen source and carbon source. The result indicated that nitrogen source was corn steep liquor replacing yeast extract, and the mass proportion of carbon and nitrogen source was 1:1, and fermentation temperature was 29℃, and fermentation rotation speed 210 r/min was the optimum process condition when enzyme activity was 172.8 U/mL increasing by 68.1%.2. Introduce ultrasonic technique to the fermentation process and improve enzyme production efficiency.Introduce ultrasonic technique to the fermentation process of Chaetomium gracile and study the influence of different ultrasonic powers, times and frequencies on fermentation through single factor experiment. The result of research had found that low-frequency ultrasonic field could significantly improve the enzyme activity and production of α-dextranase. Experiments showed that with the minimum ultrasonic treatment conditions:ultrasonic power 300 W, ultrasonic time 20 S/20 min and ultrasonic frequency 20 KHZ, the enzyme activity increased from 102.9 U/mL to 193.4 U/mL, increasing by 87.9%.3. Adopt two-step induction method and two-step fermentation thallus transferring method, improve enzyme production efficiency.Firstly, the study was concentrated on variation of dry cell weight and enzyme production from Chaetomium gracile when glucose and dextran served as substrate., the result of research showed that when glucose was treated as substrate, no enzyme was produced; when dextran served as substrate, the strain could produceα-dextranase, however, the growth of thallus was slow; Based on experimental result, so setting induction fermentation technology that glucose was used for cell growth in early period and then adding dextran to produce a-dextranase in later period. The optimum induction concentration was 1% and the optimum beginning induction time was 20 h according to enzyme activity measurement result, enzyme activity was 152.4 U/mL and increased by 49.3%, fermentation period was shorten by 4 h.Besides, the study designed two-step fermentation thallus transferring method to improve the enzyme production efficiency, namely cultivating thallus with glucose fermentation, and then collecting and transferring to fresh dextran fermentation medium to produce enzyme. Measured the change of enzyme activity and made growth curve. The result indicates that the thallus transferring method has significant effect. The enzyme activity at this time is 200.1 U/mL, 96% higher than control group.4. Reduce bacteria self-flocculation and improve enzyme production efficiency.To resolve problem of self-flocculation, adding appropriate amount of glass bead into fermentation culture to improve bacteria dispersion. This topic studies the influence of adding different glass bead diameter, addition quantity and time on cell morphology and production of a-dextranase in experiment. Experiment result indicated that enzyme activity was 145.8 U/mL when fermentation period was 12 h, adding glass beads which density was 10 glass beads/25 mL with diameter 5mm into 25 mL culture, increasing by 42.2% compared with the situation without glass beads.Integrated all methods of improving a-dextranase production from Chaetomium gracile, to verify the effect of process optimization. Results show that enzyme activity reached to 349.5 U/mL, increased by 241.0% compared with the control group.