分离纯化低聚木糖中木二糖技术参数的研究
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摘要
木二糖是由两个木糖分子通过糖苷键结合而成，具有促双歧杆菌增殖、降血压、促进Ca吸收等作用，是一种非常重要的食品甜味剂和添加剂。本试验的目的是将超滤膜分离法和树脂色谱法两种纯化技术相结合，通过纯化工艺技术的优化，确定木二糖的生产工艺。本试验主包括三个方面：
1.超滤膜分离法初步纯化木二糖的技术参数研究。使用超滤膜对低聚木糖粗提液进行过滤操作，根据分离过程中的参数变化，确定分离过程所属模型。通过单因素试验确定最佳超滤膜分离参数为：料液温度为30℃，操作压力为0.15MPa，进样浓度浓度为2.52%的条件下，得到理论膜通量最大值为59.78L/m2·h。使用高效液相色谱法对滤出液测定，通过计算得出木二糖纯度达到45.0%±0.5%。
2.固定床色谱法进一步纯化木二糖的技术参数研究。超滤膜的滤出液中主要含有较低聚合度的木糖同系物，通过静态吸附试验筛选出分离木二糖的最佳树脂为Na树脂，进而通过动态吸附试验研究了不同流速、温度、乙醇浓度对木二糖在大孔树脂上的吸附与解吸性能的影响。结果表明：树脂最佳吸附条件为流速5BV/h，温度60℃；最佳解吸条件为洗脱液为10%乙醇溶液，洗脱流速为3BV/h。通过正交试验优化Na树脂吸附木二糖最佳的参数为：进样浓度20mg/mL、进样速率2.0mL/min、洗脱速率1.0mL/min。此条件下分离得到木二糖溶液经浓缩后总纯度达到91.66%，木二糖得率达到88.65。
3.木二糖结晶工艺参数的研究。本试验以木二糖结晶的量为指标，以Na树脂的洗脱液为原料，研究了不同溶剂、温度差、溶剂浓度和过饱和度对木二糖结晶的影响。通过旋转组合试验和使用响应面法分析，确定最佳的结晶条件为：结晶溶剂为90%乙醇；糖液浓度为90%；糖溶剂比为1:3；预冷起晶温度60℃；搅拌速度为80r/min。对晶体进行色谱和光谱分析可知：在本试验条件下得到木二糖总纯度达到91.05%，结晶率达到70.05%。
4.木二糖的鉴定与分析。使用薄层层析法和高效液相色谱法对超滤膜分离液和树脂色谱分离液进行定性和定量分析，超滤膜滤出液中主要含有木二糖和木糖，树脂分离得到的溶液中木二糖含量较高可达85.27%。使用显微熔点测定仪测定结晶体的熔点，对比前人得出的熔点数值可知：本试验结晶的木二糖晶体纯度高达90%。通过使用红外光谱仪对晶体的结构进行分析，可知结晶体是木二糖，纯度大于95%。
通过研究可以证实，从低聚木糖溶液中分离纯化木二糖的关键是树脂的配型正确，就能够很大程度上提高分离效率和得率。本试验选用Na树脂吸附木二糖，影响纯化的因素主要决定于洗脱剂的浓度是否能将木二糖顺利洗脱同时又不解吸其它杂糖。在后续结晶工艺中，树脂洗脱液作为母液结晶，可获得纯度超过90%的木二糖晶体，超过Bio Gel P-2凝胶获得的木二糖纯度81%，本试验的数据可以作为规模化生产的依据。
Xylobiose was integrated by glycosidic bond which have Growth-Promoting factors，hypotensive，penetrated Ca enhancement etl,is a all-important sweetening agent and additive.The purpose of thisexperiment study are that integrate ultrafiltrationmembrane process and resin chromatography,throughparameter optimization to determine manufacturing technique of Xybiose.This experiment contain threeaspects:
1.Technical parameters reseach of ultrafiltrationmembrane process separation and purificationXylobiose firstly.
To use ultrafiltration membrane to filter xylooligosaccharides crude extract,according to parametervariation separation process,on the basis of that to determin separation process belong to which model.Bysingle factor experiment the process parameters were optimized as following:feed solution temperature30℃,operating pressure of0.15Mpa,sample concentration was2.52%under the conditon can getActualmembrane flux53.2L/m2·h.
2.Technical parameters research of Xylobiose separation by resin chromatagraphy secondly.
The ultrafiltrate from xylooligosaccharides crude extract after ultrafiltration membrane Na resin was aoptimal resin which separation and purification Xylobios and screening by static adsorption test.Studydifferent velocity of flow,temperature,ethanol concentration effect on the adsorption and desorption. Theresult show that the optimal adsorption condition was velocity of flow was5BV/h，temperature was60℃；The optimum desorption condition were that：eluate was10%ethanol，velocity of flow was3BV/h.Theconditions were optimized by orthogonal test.The optimum process parameters were determined by theconditions were optimized by orthogonal test was sample introduction concentration was20mg/mL,elutionrate2.0mL/min，washing out rate was1.0mL/min.The total purity quotient of Xylobiose up to91.66,yieldrate was86.55under this condition.
3.Technical parameters research of the technological parameter of Xylobiose crystallization.
This experiment take crystallize dose as index and eluate of Na resin as crude,study differentsolvent,difference in temperature,solvent strength and supersaturation effect on the Xylobiosecrystallizaion.By rotatory combination experiment and use response surface method to analyze,the bestcondition was determin:crystal-solvent was90%ethanol;the concentration of sugar solution was90%;thesugar and impregnant was1:3;quench temperature was60℃；mixing speed was80r/min.We can get theresults after chromatogram and spectroscopic analysis:the total purity quotient was91.05,the crystallizerate was70.05%.
4.The identify and analyze of Xylobiose.
To use thin layer chromatography and HPLC quantitative and qualitative analysis:filtrate main containXylobiose and xylose, the separating medium of Na resin contain85.27%Xylobiose.To survey and evalutemelting point and compared with the ancestors' numerical value can determine the purify of Xylobiosehigher.The molecular structure was analyzed by infrared spectrometer can certain that the crystallizationwas Xylobioseand its purify beyond95%.
The result can be confirmed through experiment, The key to purify Xybiose from Xylooliogosaccharides syrup is that resin type correctly, which can greatly improve the separationefficiency and yield rate. This experiment selects the Na resin adsorbed Xylobiose, the effects of purifiedkey factor mainly depends on the eluent concentration will be wabsorb Xylobiose and desorption of othermiscellaneous candy. In subsequent crystallization process, resin eluate as mother liquid crystal, can obtainpurity over90%Xylobiose crystal, it more than Bio Gel P-2gel resin obtained the purity, the test processcan be used as scale production.
1.超滤膜分离法初步纯化木二糖的技术参数研究。使用超滤膜对低聚木糖粗提液进行过滤操作，根据分离过程中的参数变化，确定分离过程所属模型。通过单因素试验确定最佳超滤膜分离参数为：料液温度为30℃，操作压力为0.15MPa，进样浓度浓度为2.52%的条件下，得到理论膜通量最大值为59.78L/m2·h。使用高效液相色谱法对滤出液测定，通过计算得出木二糖纯度达到45.0%±0.5%。
2.固定床色谱法进一步纯化木二糖的技术参数研究。超滤膜的滤出液中主要含有较低聚合度的木糖同系物，通过静态吸附试验筛选出分离木二糖的最佳树脂为Na树脂，进而通过动态吸附试验研究了不同流速、温度、乙醇浓度对木二糖在大孔树脂上的吸附与解吸性能的影响。结果表明：树脂最佳吸附条件为流速5BV/h，温度60℃；最佳解吸条件为洗脱液为10%乙醇溶液，洗脱流速为3BV/h。通过正交试验优化Na树脂吸附木二糖最佳的参数为：进样浓度20mg/mL、进样速率2.0mL/min、洗脱速率1.0mL/min。此条件下分离得到木二糖溶液经浓缩后总纯度达到91.66%，木二糖得率达到88.65。
3.木二糖结晶工艺参数的研究。本试验以木二糖结晶的量为指标，以Na树脂的洗脱液为原料，研究了不同溶剂、温度差、溶剂浓度和过饱和度对木二糖结晶的影响。通过旋转组合试验和使用响应面法分析，确定最佳的结晶条件为：结晶溶剂为90%乙醇；糖液浓度为90%；糖溶剂比为1:3；预冷起晶温度60℃；搅拌速度为80r/min。对晶体进行色谱和光谱分析可知：在本试验条件下得到木二糖总纯度达到91.05%，结晶率达到70.05%。
4.木二糖的鉴定与分析。使用薄层层析法和高效液相色谱法对超滤膜分离液和树脂色谱分离液进行定性和定量分析，超滤膜滤出液中主要含有木二糖和木糖，树脂分离得到的溶液中木二糖含量较高可达85.27%。使用显微熔点测定仪测定结晶体的熔点，对比前人得出的熔点数值可知：本试验结晶的木二糖晶体纯度高达90%。通过使用红外光谱仪对晶体的结构进行分析，可知结晶体是木二糖，纯度大于95%。
通过研究可以证实，从低聚木糖溶液中分离纯化木二糖的关键是树脂的配型正确，就能够很大程度上提高分离效率和得率。本试验选用Na树脂吸附木二糖，影响纯化的因素主要决定于洗脱剂的浓度是否能将木二糖顺利洗脱同时又不解吸其它杂糖。在后续结晶工艺中，树脂洗脱液作为母液结晶，可获得纯度超过90%的木二糖晶体，超过Bio Gel P-2凝胶获得的木二糖纯度81%，本试验的数据可以作为规模化生产的依据。
Xylobiose was integrated by glycosidic bond which have Growth-Promoting factors，hypotensive，penetrated Ca enhancement etl,is a all-important sweetening agent and additive.The purpose of thisexperiment study are that integrate ultrafiltrationmembrane process and resin chromatography,throughparameter optimization to determine manufacturing technique of Xybiose.This experiment contain threeaspects:
1.Technical parameters reseach of ultrafiltrationmembrane process separation and purificationXylobiose firstly.
To use ultrafiltration membrane to filter xylooligosaccharides crude extract,according to parametervariation separation process,on the basis of that to determin separation process belong to which model.Bysingle factor experiment the process parameters were optimized as following:feed solution temperature30℃,operating pressure of0.15Mpa,sample concentration was2.52%under the conditon can getActualmembrane flux53.2L/m2·h.
2.Technical parameters research of Xylobiose separation by resin chromatagraphy secondly.
The ultrafiltrate from xylooligosaccharides crude extract after ultrafiltration membrane Na resin was aoptimal resin which separation and purification Xylobios and screening by static adsorption test.Studydifferent velocity of flow,temperature,ethanol concentration effect on the adsorption and desorption. Theresult show that the optimal adsorption condition was velocity of flow was5BV/h，temperature was60℃；The optimum desorption condition were that：eluate was10%ethanol，velocity of flow was3BV/h.Theconditions were optimized by orthogonal test.The optimum process parameters were determined by theconditions were optimized by orthogonal test was sample introduction concentration was20mg/mL,elutionrate2.0mL/min，washing out rate was1.0mL/min.The total purity quotient of Xylobiose up to91.66,yieldrate was86.55under this condition.
3.Technical parameters research of the technological parameter of Xylobiose crystallization.
This experiment take crystallize dose as index and eluate of Na resin as crude,study differentsolvent,difference in temperature,solvent strength and supersaturation effect on the Xylobiosecrystallizaion.By rotatory combination experiment and use response surface method to analyze,the bestcondition was determin:crystal-solvent was90%ethanol;the concentration of sugar solution was90%;thesugar and impregnant was1:3;quench temperature was60℃；mixing speed was80r/min.We can get theresults after chromatogram and spectroscopic analysis:the total purity quotient was91.05,the crystallizerate was70.05%.
4.The identify and analyze of Xylobiose.
To use thin layer chromatography and HPLC quantitative and qualitative analysis:filtrate main containXylobiose and xylose, the separating medium of Na resin contain85.27%Xylobiose.To survey and evalutemelting point and compared with the ancestors' numerical value can determine the purify of Xylobiosehigher.The molecular structure was analyzed by infrared spectrometer can certain that the crystallizationwas Xylobioseand its purify beyond95%.
The result can be confirmed through experiment, The key to purify Xybiose from Xylooliogosaccharides syrup is that resin type correctly, which can greatly improve the separationefficiency and yield rate. This experiment selects the Na resin adsorbed Xylobiose, the effects of purifiedkey factor mainly depends on the eluent concentration will be wabsorb Xylobiose and desorption of othermiscellaneous candy. In subsequent crystallization process, resin eluate as mother liquid crystal, can obtainpurity over90%Xylobiose crystal, it more than Bio Gel P-2gel resin obtained the purity, the test processcan be used as scale production.
引文
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