雪莲果低聚果糖提取分离及分析研究
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摘要
本课题采用成本较低的雪莲果块茎为原料,利用水浴浸提技术提取低聚果糖,通过絮凝澄清、去蛋白、醇沉、树脂脱色等工艺对雪莲果低聚果糖提取液进行初步分离纯化,并对所得到的雪莲果低聚果糖粗品进行了定量和定性分析,具体如下:
第一部分研究了水浴浸提法提取雪莲果水溶性低聚果糖的提取工艺,利用抗坏血酸对雪莲果榨汁液进行护色,对护色剂的用量进行了分析,确定了护色剂的用量为与雪莲果的料液比1:3、浓度为0.3%时的护色效果最佳;对水浴浸提的温度、时间、料液比分别进行了单因素实验和正交试验,最佳提取工艺为:运用水浴浸提法,料液比1:15、提取时间为3h、提取温度为72℃,在优化后的条件下,FOS平均提取率为56.5%。
第二部分对雪莲果水溶性低聚果糖进行了初步分离纯化,运用阳离子聚丙烯酰胺凝胶(CPAM)的电性中和和架桥作用对雪莲果榨汁液进行絮凝纯化,考察了CPAM的添加量、絮凝pH值、絮凝时间、絮凝温度四个因素,通过正交试验优化后的工艺条件为:CPAM的添加量为0.08%、絮凝pH值为6、絮凝时间3h、絮凝温度52℃。再通过sevag法除去蛋白并对蛋白质进行定性定量检测,蛋白质浓度c与吸光度y之间的回归方程为:Y=0.041X+0.0043,R=0.998,去蛋白前紫外扫描在280nm处有吸收峰,然后醇沉多糖分离出低聚果糖得到初步分离纯化的低聚果糖粗品,计算粗品的百分含量为56.2%。
第三部分比较了D941、D318、DM-2、860021四种树脂对雪莲果FOS浸提液脱色效果的影响,选择了D318为最适树脂,通过静态吸附实验,确定了脱色时间为3h、脱色温度为52℃、树脂用量3.7%,pH值为5.2,运用优化后的条件,对雪莲果FOS提取液多次测定,T的平均值为90.4%。
第四部分介绍了雪莲果中低聚果糖的定量和定性分析方法,运用苯酚-硫酸法对雪莲果低聚果糖提取液总糖含量进行测定,绘制了总糖标准曲线:Y=0.0033X-0.0193,R=0.999,运用DNS比色法对提取液的还原糖含量进行测定,绘制了还原糖标准曲线为:Y=0.7787X-0.0564,R=0.999。得到雪莲果样品中FOS的含量为样品中总糖的含量减去还原糖的含量。利用硅胶板薄层层析法对糖类物质进行定性分析,用冰醋酸-氯仿-无水乙醇-水的展开系统能够较好的分开各种糖;同时运用高效液相色谱法检测出雪莲果样品中含有葡萄糖、果糖、蔗糖、蔗果三糖、蔗果四糖、蔗果五糖。
Yacon,a root crop rich in fructo-oligosaccharides(FOS),is the material of our research.We commit ourselves to the study of the extracting of water bath,flocculation,deproteinizing,depositing in alcohol and decoloring of FOS.Moreover,we also pay some attention to the quantitative analysis and qualitative analysis of FOS.The terms as follows:
In the first part,the extraction of the FOS from yacon with water bath system was studied.Ascorbic acid was utilized to the color protection of yacon mashed fruit,and analyze the quantity of color fixative,the best conditions is:the concentration of color fixative is 0.3% and the ratio of color fixative to yacon solution is 1:3.The single factor and orthogonal experiment was studied to the temperature of water bath,time and the ratio of solid to liquid.The optimum extraction condiions are that the temperature of extraction is 72℃,the ratio of solid to liquid is 1:15 and the time of extraction is 3h.Under the above conditions, the average extraction rate of yacon FOS is 56.5%.
In the second part,Preliminary separation and purification of the water-soluble FOS in yacon was researched.Floccular purification by CPAM was carried out depending on its charge-neutralization and bridging action, four factors of CPAM were tested ,there are the quantity of CPAM,pH of flocculation,time of flocculation,temperature of flocculation.The best conditions after orthogonal test are that the quantity is 0.08%,pH is 6,the time is 3h,the temperature is 52℃.Then deproteinizing was studied by sevag and the quantitative analysis and qualitative analysis of protein were undertaked,the regression equation of the concentration of protein(c) and absorbance(y) is Y=0.041X+0.0043,R=0.998.The absorption peak of protein can be seen at 280nm before deproteinizing.After that,the polysaccharose was deposited in alcohol and FOS was separated from solution,the FOS crude product was got and its percentage composition is 56.2%.
In the third part,Four types of resins were tested to determine the best one in decolorizing Yacon FOS,they are D941、D318、DM-2、860021.Four factors were ascertained in the static absorption experiments,which are that the time of decolorizing is 3h,temperature of decolorizing is 52℃,the quantity of resin is 3.7% and the pH is 5.2.In the optimum conditions,several yacon extracting solution were tseted,their one-off light transmittance in average is 90.4%.
In the last part,the quantitative analysis and qualitative analysis of FOS were undertaked.The total suger content in yacon were determined using phenol-H2SO4 method.The standard curve of total suger is:Y=0.0036X-0.0317,R=0.999.The reducing sugar content in yacon were determined using DNS colorimetry.The standard curve of reducing suger is:Y=0.7787X-0.0564,R=0.999.The total suger content minus the reducing sugar content equals the content of FOS in yacon.HPTLC by silica gel plate were used for the identification of suger components including fructose,glucose,sucrose,FOS in yacon.The mobile phase was acetic acid-chloroform-dried ethanol-water,which can separate kinds of sugar well,and all kinds of saccharides—glucose,sucrose, and FOS in yacon samples were detected by HPLC.
第一部分研究了水浴浸提法提取雪莲果水溶性低聚果糖的提取工艺,利用抗坏血酸对雪莲果榨汁液进行护色,对护色剂的用量进行了分析,确定了护色剂的用量为与雪莲果的料液比1:3、浓度为0.3%时的护色效果最佳;对水浴浸提的温度、时间、料液比分别进行了单因素实验和正交试验,最佳提取工艺为:运用水浴浸提法,料液比1:15、提取时间为3h、提取温度为72℃,在优化后的条件下,FOS平均提取率为56.5%。
第二部分对雪莲果水溶性低聚果糖进行了初步分离纯化,运用阳离子聚丙烯酰胺凝胶(CPAM)的电性中和和架桥作用对雪莲果榨汁液进行絮凝纯化,考察了CPAM的添加量、絮凝pH值、絮凝时间、絮凝温度四个因素,通过正交试验优化后的工艺条件为:CPAM的添加量为0.08%、絮凝pH值为6、絮凝时间3h、絮凝温度52℃。再通过sevag法除去蛋白并对蛋白质进行定性定量检测,蛋白质浓度c与吸光度y之间的回归方程为:Y=0.041X+0.0043,R=0.998,去蛋白前紫外扫描在280nm处有吸收峰,然后醇沉多糖分离出低聚果糖得到初步分离纯化的低聚果糖粗品,计算粗品的百分含量为56.2%。
第三部分比较了D941、D318、DM-2、860021四种树脂对雪莲果FOS浸提液脱色效果的影响,选择了D318为最适树脂,通过静态吸附实验,确定了脱色时间为3h、脱色温度为52℃、树脂用量3.7%,pH值为5.2,运用优化后的条件,对雪莲果FOS提取液多次测定,T的平均值为90.4%。
第四部分介绍了雪莲果中低聚果糖的定量和定性分析方法,运用苯酚-硫酸法对雪莲果低聚果糖提取液总糖含量进行测定,绘制了总糖标准曲线:Y=0.0033X-0.0193,R=0.999,运用DNS比色法对提取液的还原糖含量进行测定,绘制了还原糖标准曲线为:Y=0.7787X-0.0564,R=0.999。得到雪莲果样品中FOS的含量为样品中总糖的含量减去还原糖的含量。利用硅胶板薄层层析法对糖类物质进行定性分析,用冰醋酸-氯仿-无水乙醇-水的展开系统能够较好的分开各种糖;同时运用高效液相色谱法检测出雪莲果样品中含有葡萄糖、果糖、蔗糖、蔗果三糖、蔗果四糖、蔗果五糖。
Yacon,a root crop rich in fructo-oligosaccharides(FOS),is the material of our research.We commit ourselves to the study of the extracting of water bath,flocculation,deproteinizing,depositing in alcohol and decoloring of FOS.Moreover,we also pay some attention to the quantitative analysis and qualitative analysis of FOS.The terms as follows:
In the first part,the extraction of the FOS from yacon with water bath system was studied.Ascorbic acid was utilized to the color protection of yacon mashed fruit,and analyze the quantity of color fixative,the best conditions is:the concentration of color fixative is 0.3% and the ratio of color fixative to yacon solution is 1:3.The single factor and orthogonal experiment was studied to the temperature of water bath,time and the ratio of solid to liquid.The optimum extraction condiions are that the temperature of extraction is 72℃,the ratio of solid to liquid is 1:15 and the time of extraction is 3h.Under the above conditions, the average extraction rate of yacon FOS is 56.5%.
In the second part,Preliminary separation and purification of the water-soluble FOS in yacon was researched.Floccular purification by CPAM was carried out depending on its charge-neutralization and bridging action, four factors of CPAM were tested ,there are the quantity of CPAM,pH of flocculation,time of flocculation,temperature of flocculation.The best conditions after orthogonal test are that the quantity is 0.08%,pH is 6,the time is 3h,the temperature is 52℃.Then deproteinizing was studied by sevag and the quantitative analysis and qualitative analysis of protein were undertaked,the regression equation of the concentration of protein(c) and absorbance(y) is Y=0.041X+0.0043,R=0.998.The absorption peak of protein can be seen at 280nm before deproteinizing.After that,the polysaccharose was deposited in alcohol and FOS was separated from solution,the FOS crude product was got and its percentage composition is 56.2%.
In the third part,Four types of resins were tested to determine the best one in decolorizing Yacon FOS,they are D941、D318、DM-2、860021.Four factors were ascertained in the static absorption experiments,which are that the time of decolorizing is 3h,temperature of decolorizing is 52℃,the quantity of resin is 3.7% and the pH is 5.2.In the optimum conditions,several yacon extracting solution were tseted,their one-off light transmittance in average is 90.4%.
In the last part,the quantitative analysis and qualitative analysis of FOS were undertaked.The total suger content in yacon were determined using phenol-H2SO4 method.The standard curve of total suger is:Y=0.0036X-0.0317,R=0.999.The reducing sugar content in yacon were determined using DNS colorimetry.The standard curve of reducing suger is:Y=0.7787X-0.0564,R=0.999.The total suger content minus the reducing sugar content equals the content of FOS in yacon.HPTLC by silica gel plate were used for the identification of suger components including fructose,glucose,sucrose,FOS in yacon.The mobile phase was acetic acid-chloroform-dried ethanol-water,which can separate kinds of sugar well,and all kinds of saccharides—glucose,sucrose, and FOS in yacon samples were detected by HPLC.
引文
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