生物法生产低聚葡萄糖的研究
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摘要
本文旨在研究以生物法生产低聚葡萄糖,为低聚葡萄糖的工业化提供新的技术依据。
首先研究了肠膜明串珠菌(Leuconostoc mesenteroides)发酵产葡聚糖。文中依据肠膜明串珠菌的生长曲线和摇瓶发酵曲线,确定了肠膜明串珠菌对数生长期为26~36 h,稳定期为36~60 h;26 h之后发酵液中开始产生葡聚糖。通过摇床发酵实验确定了发酵液最佳配方:蔗糖100 g/L、蛋白胨5.0 g/L、CaCO_3 0.8 g/L、MgSO_4 0.02 g/L、(NH_4)_2SO_4 0.02 g/L;摇床发酵最佳条件:温度25℃、活化液接种量5 %、发酵液装液量100/250 mL、摇床转速150 r/min、发酵液起始pH值7.5。在此条件下,摇瓶和发酵罐发酵的蔗糖转化率分别为62.58 %和70.25 %。
第二研究了发酵液的脱蛋白、脱色及葡聚糖的提取和沉淀工艺。研究发现TCA法脱蛋白效果最好,以0.1 %TCA脱蛋白,蛋白的脱除率为81.5 %,葡聚糖的损失率为10.2 %;活性炭对发酵液的脱色效果较好,实验结果表明:10 g/L活性炭用于脱色发酵液时,脱色率为80.19 %,葡聚糖损失率为19.24 %;接着研究了NaCl、KCl、NaOH对葡聚糖提取的效果,结果表明:0.6 mol/L的KCl,60℃提取效果最好,葡聚糖得率为43.57 g/kg;最后考查了乙醇沉淀葡聚糖的效果,结果表明:60℃时,60 %乙醇终浓度沉淀葡聚糖效果较好,葡聚糖得率为41.28 g/kg;
第三研究了普鲁兰酶(Optimax EC3.2.1.41)酶解葡聚糖生产低聚葡萄糖。通过酶解实验确定酶解葡聚糖的最优条件为:pH 4.4、温度54℃、反应时间0.5 h、酶活浓度2000 ASPU/g,在此条件下,94.11 %的葡聚糖可以降解为低分子量的糖。
最后探讨了低聚葡萄糖的结构和性质。高效液相色谱表明酶解产物中低聚葡萄糖占74.55 %,比市面上销售的低聚异麦芽糖IMO500的活性成分高。红外光谱表明低聚葡萄糖含有α吡喃糖苷键。通过葡聚糖相对粘度的测定,发现低聚葡萄糖的粘度随着温度的增加而降低,随着溶液pH的增加而增加,pH在4.0~8.0的范围内粘度增加速度较快,低聚葡萄糖有着与其它低聚糖相当的热稳定性。通过低聚葡萄糖相对溶解度的测定,发现60℃低聚葡萄糖相对溶解度最大,达到82 %,冷冻干燥的产品溶解性最好,相对溶解度达到79~85 %。
The objective of this work focused on using biological method to produce a glucose oligosaccharide, which provided new technology for the industrialization of glucose oligosaccharide.
Firstly, dextran produced by Leuconostoc mesenteroides was stdutied. According to the growth curve of Leuconostoc mesenteroides and the time course of growth in shake flask, logarithmic growth phase of Leuconostoc mesenteroides was between 26 h and 36 h, and stationary phase was between 36 h and 60 h; after 26 h dextran was synthesised in fermentation broth. Fermentation experiments had been taken to determine the optimum fermentation liquid medium:sucrose 100 g/L, peptone 5.0 g/L, CaCO_3 0.8 g/L, MgSO_4 0.02 g/L, (NH_4)_2SO_4 0.02 g/L; the optimum fermentation conditions in the shaker: temperature is 25℃, the inocating rate of actitivation solution is 5 %, the culture medium of fermentation liquid is 100/250 mL, the rotation speed of shaking table is 150 r/min, the starting pH of fermentation liquid is pH 7.5. At optimum medium and optimum conditions, the conversion rate of sucrose in shake flask and in fermentor was 62.58 % and 70.25 %, respectively.
Secondly,the deproteinization and decoloration of fermentation liquid; the extraction and the precipitation of dextran were studied. The method of TCA was the best to remove proteins, 0.1 % TCA was used for deproteinization, the rate of deproteinization reached 81.5 % while the rate of dextran losing was only 10.2 %. The decoloration effect is better by the method of active carbon, the rate of decoloration reached 80.19 % while the rate of dextran losing was 19.24 %.Then three extraction methods’effects of the dextran had been compared, NaCl, KCl, NaOH. The results showed that the method of 0.6 mol/L KCl was the best for extraction at 60℃, the yield rate of dextran is 43.57 g/kg. The effect of ethanol precipitation dextran was examined , the results showed that 60 % ethanol terminal concentration was more effective to deposite dextran at 60℃, the yield rate of dextran is 41.28 g/kg.
Thirdly, the production of glucose oligosaccharide by the pullulanase (Optimax EC 3.2.1.41) enzymolysising dextran was studied. Through the enzymolysis experiment, optimum conditions for enzymolysis dextran were: pH 4.4, the temperature 54℃, the reaction time 0.5 h, the enzyme concentration 2000 ASPU/g. The results showed that 94.11% dextran can be degradated to low molecular weight carbohydrate.
Finally, glucose oligosaccharide’structure and nature were preliminary discussed.The highly effective liquid chromatography (HPLC) indicated that glucose oligosaccharide was 74.55% in enzymatic hydrolysate, with higher active ingredient than Ismalto-oligosaccharides IMO500 in sales.The infrared chromatograph indicated that the glucose oligosaccharide included alpha pyranose glucoside linkage. Through the glucose oligosaccharide relative viscosity's determination, it was discovered that glucose oligosaccharide viscosity decreased while temperature increaseing; and increased while pH decreaseing; and in the pH 4~8 scope the viscosity increased faster; glucose oligosaccharide had the equivalent thermostability as well as other oligosaccharides. Through the glucose oligosaccharide relative solubility determination, it was discovered that the glucose oligosaccharide relative solubility was the biggest, reaching 82 % at 60℃; the solubility of product by the freeze-drying was the best, and the relative solubility reached 79~85 %.
首先研究了肠膜明串珠菌(Leuconostoc mesenteroides)发酵产葡聚糖。文中依据肠膜明串珠菌的生长曲线和摇瓶发酵曲线,确定了肠膜明串珠菌对数生长期为26~36 h,稳定期为36~60 h;26 h之后发酵液中开始产生葡聚糖。通过摇床发酵实验确定了发酵液最佳配方:蔗糖100 g/L、蛋白胨5.0 g/L、CaCO_3 0.8 g/L、MgSO_4 0.02 g/L、(NH_4)_2SO_4 0.02 g/L;摇床发酵最佳条件:温度25℃、活化液接种量5 %、发酵液装液量100/250 mL、摇床转速150 r/min、发酵液起始pH值7.5。在此条件下,摇瓶和发酵罐发酵的蔗糖转化率分别为62.58 %和70.25 %。
第二研究了发酵液的脱蛋白、脱色及葡聚糖的提取和沉淀工艺。研究发现TCA法脱蛋白效果最好,以0.1 %TCA脱蛋白,蛋白的脱除率为81.5 %,葡聚糖的损失率为10.2 %;活性炭对发酵液的脱色效果较好,实验结果表明:10 g/L活性炭用于脱色发酵液时,脱色率为80.19 %,葡聚糖损失率为19.24 %;接着研究了NaCl、KCl、NaOH对葡聚糖提取的效果,结果表明:0.6 mol/L的KCl,60℃提取效果最好,葡聚糖得率为43.57 g/kg;最后考查了乙醇沉淀葡聚糖的效果,结果表明:60℃时,60 %乙醇终浓度沉淀葡聚糖效果较好,葡聚糖得率为41.28 g/kg;
第三研究了普鲁兰酶(Optimax EC3.2.1.41)酶解葡聚糖生产低聚葡萄糖。通过酶解实验确定酶解葡聚糖的最优条件为:pH 4.4、温度54℃、反应时间0.5 h、酶活浓度2000 ASPU/g,在此条件下,94.11 %的葡聚糖可以降解为低分子量的糖。
最后探讨了低聚葡萄糖的结构和性质。高效液相色谱表明酶解产物中低聚葡萄糖占74.55 %,比市面上销售的低聚异麦芽糖IMO500的活性成分高。红外光谱表明低聚葡萄糖含有α吡喃糖苷键。通过葡聚糖相对粘度的测定,发现低聚葡萄糖的粘度随着温度的增加而降低,随着溶液pH的增加而增加,pH在4.0~8.0的范围内粘度增加速度较快,低聚葡萄糖有着与其它低聚糖相当的热稳定性。通过低聚葡萄糖相对溶解度的测定,发现60℃低聚葡萄糖相对溶解度最大,达到82 %,冷冻干燥的产品溶解性最好,相对溶解度达到79~85 %。
The objective of this work focused on using biological method to produce a glucose oligosaccharide, which provided new technology for the industrialization of glucose oligosaccharide.
Firstly, dextran produced by Leuconostoc mesenteroides was stdutied. According to the growth curve of Leuconostoc mesenteroides and the time course of growth in shake flask, logarithmic growth phase of Leuconostoc mesenteroides was between 26 h and 36 h, and stationary phase was between 36 h and 60 h; after 26 h dextran was synthesised in fermentation broth. Fermentation experiments had been taken to determine the optimum fermentation liquid medium:sucrose 100 g/L, peptone 5.0 g/L, CaCO_3 0.8 g/L, MgSO_4 0.02 g/L, (NH_4)_2SO_4 0.02 g/L; the optimum fermentation conditions in the shaker: temperature is 25℃, the inocating rate of actitivation solution is 5 %, the culture medium of fermentation liquid is 100/250 mL, the rotation speed of shaking table is 150 r/min, the starting pH of fermentation liquid is pH 7.5. At optimum medium and optimum conditions, the conversion rate of sucrose in shake flask and in fermentor was 62.58 % and 70.25 %, respectively.
Secondly,the deproteinization and decoloration of fermentation liquid; the extraction and the precipitation of dextran were studied. The method of TCA was the best to remove proteins, 0.1 % TCA was used for deproteinization, the rate of deproteinization reached 81.5 % while the rate of dextran losing was only 10.2 %. The decoloration effect is better by the method of active carbon, the rate of decoloration reached 80.19 % while the rate of dextran losing was 19.24 %.Then three extraction methods’effects of the dextran had been compared, NaCl, KCl, NaOH. The results showed that the method of 0.6 mol/L KCl was the best for extraction at 60℃, the yield rate of dextran is 43.57 g/kg. The effect of ethanol precipitation dextran was examined , the results showed that 60 % ethanol terminal concentration was more effective to deposite dextran at 60℃, the yield rate of dextran is 41.28 g/kg.
Thirdly, the production of glucose oligosaccharide by the pullulanase (Optimax EC 3.2.1.41) enzymolysising dextran was studied. Through the enzymolysis experiment, optimum conditions for enzymolysis dextran were: pH 4.4, the temperature 54℃, the reaction time 0.5 h, the enzyme concentration 2000 ASPU/g. The results showed that 94.11% dextran can be degradated to low molecular weight carbohydrate.
Finally, glucose oligosaccharide’structure and nature were preliminary discussed.The highly effective liquid chromatography (HPLC) indicated that glucose oligosaccharide was 74.55% in enzymatic hydrolysate, with higher active ingredient than Ismalto-oligosaccharides IMO500 in sales.The infrared chromatograph indicated that the glucose oligosaccharide included alpha pyranose glucoside linkage. Through the glucose oligosaccharide relative viscosity's determination, it was discovered that glucose oligosaccharide viscosity decreased while temperature increaseing; and increased while pH decreaseing; and in the pH 4~8 scope the viscosity increased faster; glucose oligosaccharide had the equivalent thermostability as well as other oligosaccharides. Through the glucose oligosaccharide relative solubility determination, it was discovered that the glucose oligosaccharide relative solubility was the biggest, reaching 82 % at 60℃; the solubility of product by the freeze-drying was the best, and the relative solubility reached 79~85 %.
引文
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