响应面法优化琼胶的酶解条件

Optimization of Agar Enzymolysis Process by Response Surface Methodology

为了优化琼胶酶酶解琼胶制备琼胶寡糖的工艺条件,在单因素试验的基础上,运用软件Design-Expert8.0设计中心组合试验,以反应体系中还原糖生成量为评价指标,采用响应面分析法确定琼胶酶酶解的最优条件。优化结果:酶解温度40℃,p H 7.04,Na Cl添加量15 g/L,加酶量为体系的23.97%(14.4 U)、底物质量浓度11 g/L。在此最佳工艺条件下,还原糖质量浓度达1762.3 mg/L,比优化前提高了近3倍,同时琼胶的转化率达到30%。采用薄层色谱分析法优化前后酶解产物的降解情况,结果显示优化后的终产物基本只有新琼二糖,酶解反应比优化前更加完全,酶解效率更高。试验结果表明,采用基于中心组合设计的响应面分析方法能够较大地提高目标产物的产量,实现酶解工艺条件的优化。

In recent years, bioactivity studies have indicated that the agaro-oligosaccharides stemed from agar or agarose show a range of physiological activities. For agaro-oligosaccharide preparation, it is very important to select the suitable methods. On the one hand, though agaro-oligosaccharides can be prepared expediently via acid hydrolysis of agar, acid hydrolysis is still not suitable for large-scale production of high purity agaro-oligosaccharides, because the hydrolyzate is not uniformity and the hydrolysis reaction is not easily controllable. On the other hand, as the enzyme degrades polysaccharides with high specificity as well as proceeding smoothly under mild conditions, enzymatic hydrolysis of polysaccharides should be a prospective alternative to acidic hydrolysis. Hereby, this research is to exploit a simple enzymatic method of preparing agaro-oligosaccharides. Firstly, taking the amount of reducing sugar as an indicator to optimize agar enzymolysis condition by the single factor experiment, the influencing factors mainly includ enzyme dosage,substrate concentration, p H value, temperature and sodium chloride concentration, etc. Secondly, response surface methodology based on single factor experiments is employed to optimize the enzymatic hydrolysis condition of agar to obtain the maximum yield of reducing sugar and conversion of agar. Finally, the enzymatic hydrolysate before and after optimization is identified by thin-layer chromatography and it is shown that the terminal products mainly included neoagarobiose. The optimal processing condition of enzymatic hydrolysis is abtained as follows： p H value 7, temperature 40 ℃,substrate concentration 11 g/L, agarase dosage 15 U, sodium chloride concentration 15 g/L under stationary state. Under this optimal condition, the concentration of reducing sugar reached 1318.8 mg/L, meanwhile the conversion of agar came to 27.7%. After RSM optimization, the maximum of reducing sugar reach 1762.3 mg/L after 40 min of enzymolysis,which is almost 3 fold compared with the initial condition before optimization. Besides, the conversion of agar attained30%. The degree of enzymatic hydrolysis reaction is more complete than the degree before optimization, and the efficiency of enzymatic hydrolysis raise obviously. This study establishs feasible systems for the preparation of agaro-oligosaccharides and prepared for the isolation of various sizes of oligomers generated from agar. Surface response analysis based on CCD increased the target production and the best condition of enzymic hydrolysis is determined.