【作者】 孙俊良；

【导师】 李新华；

【作者基本信息】 沈阳农业大学 ， 食品科学与工程， 2009， 博士

【摘要】 α-淀粉酶是一种重要的工业用酶,广泛应用于食品、纺织和造纸等行业。本课题采用酶法制备不同DE值糊精,研究了酶法生产糊精的工艺,并以不同DE值糊精作为碳源,研究了枯草芽孢杆菌的产酶特性,用二次回归旋转设计优化发酵培养基和培养条件,研究了α-淀粉酶的酶学性质,确定了深层发酵工艺和发酵动力学模型,并进行了中试放大研究。最后探讨了不同DE值糊精中有效成分诱导枯草芽孢杆菌的产酶机理。主要研究结果如下:1.中温淀粉酶和高温淀粉酶联用制备糊精可以有效地缩短酶解反应时间,减少酶的用量,提高效率和降低成本。正交试验结果表明,温度对淀粉水解产品的DE值影响最大,反应时间的影响最小。制备糊精较为适宜的水解条件为温度90℃,时间10min,中温淀粉酶用量4 U/g淀粉,高温淀粉酶用量7U/g淀粉。在此基础上适当调整酶解时间或加酶量,可以获得不同DE值的糊精产品。2.淀粉乳酶解前用超声波处理,然后酶解制备糊精,可获得分子分布更为均匀的糊精样品。超声波作用条件为:超声功率50W,超声全程时间范围可选择5-15min,超声波工作时间选择2-5s范围,间隔时间可选择20s以上。在此基础上调整加酶量或酶解时间,可获得不同DE值的糊精样品。超声波处理后淀粉的粘度降低;超声波处理会侵蚀淀粉颗粒表面,造成不同程度的破裂;高强度的超声波降解支链淀粉的作用增强,超声波处理制备的糊精分子分布更为均匀,大分子比例减少,分支化度较小,线性分子数量增多。3.糊精的性质:糊精与原淀粉相比粒度均变小。DE值22以下的糊精具有结晶结构,保持A型特征峰,峰强随DE值的增大逐渐减弱。说明水解首先发生在无定形区,其次才是结晶区。不同DE值糊精的表观粘度随DE值的增大而逐渐降低;热重分析（TG）分析表明,糊精的热稳定性较原淀粉高;DE值较大的糊精的透光率较高。Sephadex G50凝胶过滤层析结果表明,DE值较大的糊精线性较强。HPLC分析结果表明双酶法所得糊精分子量分布比较均匀,七糖以下组分中含有较大比例的单糖、二糖、三糖和五糖。4.不同DE值糊精作为碳源对B.subtilis ZJF-1A5产α-淀粉酶的产酶特性:糊精的DE值控制在16-22,B.subtilis ZJF-1A5的接种量控制在3%-4%,产酶量及酶活较高。发酵工艺单因素条件为:最佳条件温度为37℃,初始pH为5.0,摇床转速为210 r/min,培养时间为54 h。通过二次回归正交旋转设计,所得回归方程为:Y=151.6+14.7X₁-7.8 X₂+26.9X₃-19.7 X₁²-4.5 X₂²+6.8 X₁X₃。相应得到优化的工艺参数为:培养温度为37℃,发酵培养基pH为5,时间为58 h,α-淀粉酶活力达到理论最大值206.5 U/mL。经检验该工艺参数可靠。在发酵摇床试验中,以DE值为16-22的糊精为碳源时,和以淀粉和葡萄糖为碳源时相比,酶活分别提高了80.2%和219.3%。经5L级全自动发酵罐中试验证,利用DE值为16-22的糊精为碳源,酶活可达到469.8±27.7 U/mL,与淀粉作为碳源相比,酶活力提高了41.9%。5.菌株ZJF-1A5分泌的主要α-淀粉酶的最适反应温度在60℃,最适pH值是6.0,其活性受酸性变化影响不大,在pH值4左右,仍能保持较高的酶活水平,因此该酶为酸性淀粉酶,具有较高的工业应用价值。菌株ZJF-1A5分泌的主要α-淀粉酶能够被Mg²⁺、Ca²⁺、Na⁺、Al³⁺激活,且Al³⁺对ZJF-1A5分泌的主要α-淀粉酶的激活能力最强,目前尚未见其他关于Al³⁺激活α-淀粉酶的报道。6.一定DE值的糊精诱导B.subtilis ZJF-1A5产α-淀粉酶的量增加。糊精中主要的诱导成分是糊精中的麦芽低聚糖,通过基本培养基发酵,添加不同聚合度的麦芽低聚糖,实验结果表明,麦芽五糖的诱导活性最强,证实麦芽低聚糖在糊精诱导B.subtilis ZJF-1A5产α-淀粉酶量升高过程中起到重要作用。更多还原

【Abstract】 α-amylase as an important industrial enzyme was widely used in food industries, textile technology, paper manufacturing and so on. In this study, the method preparing dextrin with different DE value by combination of common and thermalα-amylase was investigated. Dextrin with different DE value was used as carbon source and its effect on enzyme production was studied. Medium and conditions of fermentation were optimized using quadratic regression revolution design. The properties ofα-amylase produced by B.subtilis ZJF-1A5 were studied. The batch process and dynamics model were studied and medium-scale up was also investigated. The inducing mechanism of dextrin with different DE value on production ofα-amylase by B.subtilis ZJF-1A5 was discussed. The main results were as following:1. By successive use of double-enzyme method （medium-temperature and high-temperatureα-amylases）, the method preparing dextrin was obtained, which could reduce the time of enzymatic hydrolysis and addition of enzyme. According to the result of single-factor experiment, an orthogonal test was designed and carried out, the optimum condition was determined as follows: reaction temperature 90℃, hydrolysis time 20 min, addition of medium-temperatureα-amylase 4 U/g starch and addition of high-temperatureα-amylase 7 U /g starch. On the basis of above conditions, dextrin with different DE value could be obtained by adjusting the hydrolysis time or enzyme addition.2. Starch was treated by ultrasonic and then hydrolyzed by the method of medium-temperature and thermalα-amylases, the molecular distribution of the obtained dextrin was more even. The ultrasonic conditions were as following: ultrasonic power 50W, total stimulation time 5-15min, ultrasonic stimulation time 2-5s and ultrasonic intermissive time 20s. On the basis of above conditions, dextrin with different DE value could be obtained by adjusting the hydrolysis time or enzyme addition. It was considered by integrated analysis that the surfaces of starch granules were subjected to erode and the pasting viscosity of starch decreased. The molecular weight distributions of dextrin by enzyme method with ultrasonic assistance were more even. The proportion of large molecules in the product reduced while the amount of linear molecules increased.3. The particle of the dextrin changed significantly compared with the starch. The obtained dextrin subjected to enzyme hydrolysis showed the narrowest size distribution. The crystal type of A -type of dextrin with DE value below 22 had been partly preserved and possessed crystal structure and the peak intensity was gradually weakened with the increase of DE value. This result revealed that amorphous areas composed of amylopetin （in coral regions of starch particles） were hydrolyzed firstly in the process of the enzyme hydrolysis. Apparent viscosity of dextrin with different value decreased with the increase of DE value. TG analysis showed that the thermal stability of the dextrin was higher than that of the raw starch. Transmittances of dextrin with large DE value were higher than that with low DE value. Gel filtration chromatography by Sephadex G50 showed the amount of linear molecules of dextrin with larger DE value increased. HPLC analysis showed molecular weight distributions of dextrin by medium-temperature and high-temperatureα-amylases method were more even and contained large proportion of glucose, maltose, maltotriose and maltopentaose in the components of oligosaccharide the polymerization degree （DP） was below seven.4. Yield and activity ofα-amylase were relatively high when the DE value of dextrin was controlled in the range of 16-22 and inoculation amount of B.subtilis ZJF-1A5 was 3%-4%. Single factor conditions of fermentation process parameters were temperature 37℃, pH 5.0, rotation speed 210 r/min and the culture time 54h.Regression equation was obtained by quadratic orthogonal rotation and the equation was as following: Y = 151.6 + 14.7 X₁ - 7.8 X₂+ 26.9 X₃- 19.7 X₁²- 4.5 X₂²+ 6.8 X₁X₃. The optimum parameters were determined as follows: temperature 37℃, pH 5.0 and the culture time 58 h. Theoretical maximum ofα-amylase activity reached 206.5 U/mL. And the product has a good application prospect.Compared with starch and glucose, dextrins with DE value 16-22 were used as carbon source in shaking-bottle incubating test, the activities ofα-amylase increased by 80.2% and 219.3%, respectively.The pilot test in 5L automatic fermentor showed that theα-amylase activity reached 469.8±27.7 U/mL when dextrins with DE value 16-22 were used as carbon sources. Compared with starch used as carbon sources, theα-amylase activity increased by 41.9%.5. The properties of a-amylase by B.subtilis ZJF-1A5 were studied. Its optimum temperature was 60℃. Its optimum pH of was 6.0 andα-amylase activity changed little at pH 4.0. So the enzyme was a kind of acidα-amylase and has perspective application in industry.The result showed that Mg²⁺, Ca²⁺, Na⁺ and Al³⁺ were the activators ofα-amylase produced by B.subtilis ZJF-1A5 and activation ability of Al³⁺ was the strongest. At present there was no report about that Al³⁺ activated the activity ofα-amylase.6. The yield ofα-amylase by B.subtilis ZJF-1A5 increased by using dextrin with a certain DE value range as carbon source. The effective induced component in dextrins might be oligosaccharide. By ordinary fermention with oligosaccharide as carbon source, the results showed that inducing activity of maltopentaose was the strongest. It further confirmed that dextrins played important roles during the process of production ofα-amylase by B.subtilis ZJF-1A5.更多还原