甘薯蔓多糖特性与分子硫酸化修饰的研究
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摘要
甘薯(Ipomoea batatas(L.)Lam.),属旋花科多年生植物,是福建省特有的经济作物之一,具有丰富的营养价值以及药用功效。本项目拟采用福建金山763甘薯的副产物—其叶、茎、蔓(统称甘薯蔓)为原料,利用超声波辅助提取甘薯蔓多糖;对其进行脱色、分离纯化;分析探讨甘薯蔓多糖的分子特性;研究甘薯蔓多糖分子硫酸化修饰工艺,并考察硫酸化修饰对甘薯蔓多糖抗氧化性能的影响,主要研究结果如下:
(1)采用超声波技术与热水浸提相结合辅助提取甘薯蔓多糖,探讨了料液比、提取温度、超声波功率、超声波处理—水提时间、提取液pH对甘薯蔓多糖提取的影响效果。采用L16(45)正交试验设计试验获得的甘薯蔓多糖提取的最佳工艺为:料液比l:50,超声波功率500W。浸提温度75℃,浸提时间90min。以此优化方案,在pH为近中性条件下,提取甘薯蔓多糖2次,甘薯蔓多糖平均提取率为3.86%。
(2)采用H_2O_2脱色法对甘薯蔓多糖进行脱色,通过[L_9(3~3)]正交试验,以脱色后甘薯蔓粗多糖含量、·OH清除率为衡量指标,获得甘薯蔓多糖H_2O_2脱色最佳工艺为:在添加5‰(w/v)EDTA作为护色剂,调节脱色液pH值8.8的辅助条件下,H_2O_2使用量为甘薯蔓多糖溶液的1.0%(v/v)、脱色温度50℃、脱色时间1h。在上述条件下脱色后甘薯蔓粗多糖的保存率为70.3%,其?OH清除率为原甘薯蔓粗多糖的85.4%。
(3)经SephadexG-75凝胶色谱分离、纯化的甘薯蔓多糖为单一级分,冷冻干燥后为浅褐色絮状粉末,易溶于水;在100℃以内,以及常规食品加工pH范围内,稳定性较好。甘薯蔓多糖比旋光度「a」20D = +78o (0.071,H2_O),特性粘度[η]= 10.421;其与硫酸—苯酚反应呈棕褐色,与蒽酮反应呈暗绿色;与I2-KI、FeC13、硫酸一咔唑、斐林试剂均呈阴性;与考马斯亮蓝反应呈阳性;经HPGPC与紫外光谱进行分析,鉴定甘薯蔓多糖为单一级分,并结合有蛋白质的多糖络合物,其中蛋白质含量为35.4%;确定其分子量为1824736D。
通过对甘薯蔓多糖红外光谱指纹区的特征吸收解析表明:甘薯蔓多糖含有羧基,是一种中偏酸性多糖;甘薯蔓多糖在954.81cm-1处有吸收峰,属β-D-葡萄吡喃糖,同时结合1H-NMR谱图特征证明该糖链几乎由β-构型糖苷键连接。
(4)采用氨基磺酸-甲酰胺法对甘薯蔓多糖进行硫酸化修饰,通过四元二次通用旋转组合试验设计方案,建立的甘薯蔓多糖硫酸化修饰的动力学方程为: Y=1.41000+0.02750X_1+0.02500X_2+0.01250X_3+0.01083X_4-0.09521X_1~2-0.08146X_2~2-0.08646X_3~2-0.08646X_4~2 -0.03500X_1X_2-0.01500X_3X_4
通过该方程获得的甘薯蔓多糖硫酸化修饰最佳工艺参数组合为:当各影响因素均处于0水平时,即氨基磺酸用量30mg、甲酰胺用量8mL、酯化时间3h、酯化温度90℃,可使甘薯蔓多糖获得理想的硫酸取代度,DS为1.42;甘薯蔓多糖硫酸衍生物的清除·OH自由基的能力与其DS成正相关性,当其DS为1.42时,其清除·OH-自由基的能力最大,SA值81.29%,是天然甘薯蔓多糖的3倍。
Sweet potato (Ipomoea batatas(L.)Lam.),is a special economical resource in Fujian,which has rich nutrition and special health value.Ultrasonic-assisted technology,decoloration technology,purified technology,sulfate modification and the anti-oxidantive effect of sulfated derivatives of sweet potato vines polysaccharide (PSPV) were studied.Therefore,basic data for development of biological activity and the appliaction of Biopharmaceutical of PSPV were offered.
(1) In this study,combined with traditional hot-water extraction,we have disscussed the main affect factors involved in the ultrasonic-assisted extraction procession of the PSPV.The four factors,solid-liquid ratio、extraction temperature、extraction time and uhrasonic power,which were affected the extraction procession were optimized by L16(45) orthogonal test.The obtained results indicated that the optimized parameters were as follows:the solid-liquid ratio 1:50,ultrasonic power 500W and extraction temperature at 75℃for 90 minutes.At this optimization scheme,the method was repeated for twice under neutral pH and the extraction ratio of PSPV was about 3.86%.
(2) Under the EDTA subsidiary conditions , H_2O_2 was used as the decolorant,which was carried on the decoloration to the PSPV.Through the [L_9(3~3)] orthogonal test,we took the content of PSPV,the hydroxy free radical percentage clearance as the weight target.The result indicated that the best technological parameter combination was:Under the condition that the content of EDTA was 5‰of the polysaccharide dissolution fluid and the pH of solution was 8.8,the amount of H_2O_2 was 1.5% of the polysaccharide dissolution fluid,the temperature was 50℃,the time was 1h,and the pH value is 8.8.Under this condition,the survival rate of PSPV was 70.3% and its hydroxy free radical percentage clearance was 85.4% of original PSPV after the decolorization.
(3) The crude PSPV was removed proteins by Sevag’s methods,and was isolated and purified by SephadexG-75.Light brown powder State objects was obtained after freeze-drying and was soluble in water.The PSPV was thermal stability at 0℃~100℃among the food processing pH value.The specific rotatory power of PSPV [α]_D~20=+78°(0.071,H2O)and inherent viscosity [η]= 10.421.The results of color reaction indicated:Reacting with phenol-sulfuric acid reagent,anthrone reagent ,PSPV turned into blown and blind green respectively.However,it turned out to be colorless while reacting with I -KIFeCl2 3 carbazole- sulfuric acid reagent and fehling reagent.It was positive while reacting wih Coomassie BrilliantG-250 reagentthe.The pure PSPV was identified by HPGPC and UV and was a bioactive glycoprotein which contained protein 35.4%.HPLC showed that PSPV had a molecular weight of 1824736,the polydispersity of 1.027394.
According to the analysis of IR fingerprint characteristics absorption,the result showed that the PSPV was an acidic polysaccharide containing a carboxyl.In addition, the results of 954.81cm-1 department absorption peak and 1H-NMR showed that the configuration of polysaccharide wasβ– glycosidic.
(4) The method of using sulfamic acid as the agent and formamide as solvent was adopted to for sulfamic modification on the polysaccharides from sweet potato vines (PSPV) in this paper..Basing on the rotating perpendicular combination experiments, the dynamic equation of sulfamic modification on PSPV was established,the dynamic equation: Y=1.41000+0.02750X_1+0.02500X_2+0.01250X_3+0.01083X_4-0.09521X_1~2-0.08146X_2~2-0.08646X_3~2-0.08646X_4~2 -0.03500X_1X_2-0.01500X_3X_4
The result indicated that the optimum technological parameters of sulfamic modification on PSPV were to add the amount of sulfamic acid 30mg;formamide volume 8 mL;and esterification time 3 hours;esterification temperature 90℃;the DS of the derivatives on PSPV was 1.42.Moreover,the scavenging ability of sulfated derivatives of PSPV on·OH was positively related with the degree of substitution (DS), and which antioxidant activity was the largest,the SA was 81.29%,treble than the natural PSPV,when the DS was 1.42.
(1)采用超声波技术与热水浸提相结合辅助提取甘薯蔓多糖,探讨了料液比、提取温度、超声波功率、超声波处理—水提时间、提取液pH对甘薯蔓多糖提取的影响效果。采用L16(45)正交试验设计试验获得的甘薯蔓多糖提取的最佳工艺为:料液比l:50,超声波功率500W。浸提温度75℃,浸提时间90min。以此优化方案,在pH为近中性条件下,提取甘薯蔓多糖2次,甘薯蔓多糖平均提取率为3.86%。
(2)采用H_2O_2脱色法对甘薯蔓多糖进行脱色,通过[L_9(3~3)]正交试验,以脱色后甘薯蔓粗多糖含量、·OH清除率为衡量指标,获得甘薯蔓多糖H_2O_2脱色最佳工艺为:在添加5‰(w/v)EDTA作为护色剂,调节脱色液pH值8.8的辅助条件下,H_2O_2使用量为甘薯蔓多糖溶液的1.0%(v/v)、脱色温度50℃、脱色时间1h。在上述条件下脱色后甘薯蔓粗多糖的保存率为70.3%,其?OH清除率为原甘薯蔓粗多糖的85.4%。
(3)经SephadexG-75凝胶色谱分离、纯化的甘薯蔓多糖为单一级分,冷冻干燥后为浅褐色絮状粉末,易溶于水;在100℃以内,以及常规食品加工pH范围内,稳定性较好。甘薯蔓多糖比旋光度「a」20D = +78o (0.071,H2_O),特性粘度[η]= 10.421;其与硫酸—苯酚反应呈棕褐色,与蒽酮反应呈暗绿色;与I2-KI、FeC13、硫酸一咔唑、斐林试剂均呈阴性;与考马斯亮蓝反应呈阳性;经HPGPC与紫外光谱进行分析,鉴定甘薯蔓多糖为单一级分,并结合有蛋白质的多糖络合物,其中蛋白质含量为35.4%;确定其分子量为1824736D。
通过对甘薯蔓多糖红外光谱指纹区的特征吸收解析表明:甘薯蔓多糖含有羧基,是一种中偏酸性多糖;甘薯蔓多糖在954.81cm-1处有吸收峰,属β-D-葡萄吡喃糖,同时结合1H-NMR谱图特征证明该糖链几乎由β-构型糖苷键连接。
(4)采用氨基磺酸-甲酰胺法对甘薯蔓多糖进行硫酸化修饰,通过四元二次通用旋转组合试验设计方案,建立的甘薯蔓多糖硫酸化修饰的动力学方程为: Y=1.41000+0.02750X_1+0.02500X_2+0.01250X_3+0.01083X_4-0.09521X_1~2-0.08146X_2~2-0.08646X_3~2-0.08646X_4~2 -0.03500X_1X_2-0.01500X_3X_4
通过该方程获得的甘薯蔓多糖硫酸化修饰最佳工艺参数组合为:当各影响因素均处于0水平时,即氨基磺酸用量30mg、甲酰胺用量8mL、酯化时间3h、酯化温度90℃,可使甘薯蔓多糖获得理想的硫酸取代度,DS为1.42;甘薯蔓多糖硫酸衍生物的清除·OH自由基的能力与其DS成正相关性,当其DS为1.42时,其清除·OH-自由基的能力最大,SA值81.29%,是天然甘薯蔓多糖的3倍。
Sweet potato (Ipomoea batatas(L.)Lam.),is a special economical resource in Fujian,which has rich nutrition and special health value.Ultrasonic-assisted technology,decoloration technology,purified technology,sulfate modification and the anti-oxidantive effect of sulfated derivatives of sweet potato vines polysaccharide (PSPV) were studied.Therefore,basic data for development of biological activity and the appliaction of Biopharmaceutical of PSPV were offered.
(1) In this study,combined with traditional hot-water extraction,we have disscussed the main affect factors involved in the ultrasonic-assisted extraction procession of the PSPV.The four factors,solid-liquid ratio、extraction temperature、extraction time and uhrasonic power,which were affected the extraction procession were optimized by L16(45) orthogonal test.The obtained results indicated that the optimized parameters were as follows:the solid-liquid ratio 1:50,ultrasonic power 500W and extraction temperature at 75℃for 90 minutes.At this optimization scheme,the method was repeated for twice under neutral pH and the extraction ratio of PSPV was about 3.86%.
(2) Under the EDTA subsidiary conditions , H_2O_2 was used as the decolorant,which was carried on the decoloration to the PSPV.Through the [L_9(3~3)] orthogonal test,we took the content of PSPV,the hydroxy free radical percentage clearance as the weight target.The result indicated that the best technological parameter combination was:Under the condition that the content of EDTA was 5‰of the polysaccharide dissolution fluid and the pH of solution was 8.8,the amount of H_2O_2 was 1.5% of the polysaccharide dissolution fluid,the temperature was 50℃,the time was 1h,and the pH value is 8.8.Under this condition,the survival rate of PSPV was 70.3% and its hydroxy free radical percentage clearance was 85.4% of original PSPV after the decolorization.
(3) The crude PSPV was removed proteins by Sevag’s methods,and was isolated and purified by SephadexG-75.Light brown powder State objects was obtained after freeze-drying and was soluble in water.The PSPV was thermal stability at 0℃~100℃among the food processing pH value.The specific rotatory power of PSPV [α]_D~20=+78°(0.071,H2O)and inherent viscosity [η]= 10.421.The results of color reaction indicated:Reacting with phenol-sulfuric acid reagent,anthrone reagent ,PSPV turned into blown and blind green respectively.However,it turned out to be colorless while reacting with I -KIFeCl2 3 carbazole- sulfuric acid reagent and fehling reagent.It was positive while reacting wih Coomassie BrilliantG-250 reagentthe.The pure PSPV was identified by HPGPC and UV and was a bioactive glycoprotein which contained protein 35.4%.HPLC showed that PSPV had a molecular weight of 1824736,the polydispersity of 1.027394.
According to the analysis of IR fingerprint characteristics absorption,the result showed that the PSPV was an acidic polysaccharide containing a carboxyl.In addition, the results of 954.81cm-1 department absorption peak and 1H-NMR showed that the configuration of polysaccharide wasβ– glycosidic.
(4) The method of using sulfamic acid as the agent and formamide as solvent was adopted to for sulfamic modification on the polysaccharides from sweet potato vines (PSPV) in this paper..Basing on the rotating perpendicular combination experiments, the dynamic equation of sulfamic modification on PSPV was established,the dynamic equation: Y=1.41000+0.02750X_1+0.02500X_2+0.01250X_3+0.01083X_4-0.09521X_1~2-0.08146X_2~2-0.08646X_3~2-0.08646X_4~2 -0.03500X_1X_2-0.01500X_3X_4
The result indicated that the optimum technological parameters of sulfamic modification on PSPV were to add the amount of sulfamic acid 30mg;formamide volume 8 mL;and esterification time 3 hours;esterification temperature 90℃;the DS of the derivatives on PSPV was 1.42.Moreover,the scavenging ability of sulfated derivatives of PSPV on·OH was positively related with the degree of substitution (DS), and which antioxidant activity was the largest,the SA was 81.29%,treble than the natural PSPV,when the DS was 1.42.
引文
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