马铃薯淀粉全酶法制备磷酸寡糖的研究
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摘要
磷酸寡糖(Phosphoryl Oligosaccharides,POs)是从马铃薯淀粉水解产物中分离得到的分子中带有磷酸酯键(C-O-P),且聚合度在3～6之间的麦芽寡糖的混合物。其具有抑制磷酸钙形成、抗龋齿、抗淀粉老化等多种特性,是应用安全、广泛的新型功能性低聚糖。磷酸寡糖的研究始于上世纪90年代,目前日本已有磷酸寡糖产品面市。在国内磷酸寡糖的研究还基本属于空白,自主研发制备磷酸寡糖,具有重要的现实意义。
本文以马铃薯淀粉为原料制备低DE值的麦芽低聚糖浆,再通过柱层析的方法从麦芽低聚糖糖浆中分离制备了磷酸寡糖,并对其进行了定性分析。为进一步探讨磷酸寡糖的制备工艺,早日实现磷酸寡糖工业化生产和广泛应用奠定了一定的理论基础和实施依据。
本文通过喷射液化的方法制备了低DE值的麦芽低聚糖浆。工艺条件为:用水将淀粉乳浓度调至7°Bé,加入209,0000U耐高温α淀粉酶,于105℃的条件下喷射液化4～8min,再于层流罐内95℃条件下保温30min。灭酶后进入糖化工艺,加入1075,7900U糖化酶,于60℃条件下反应60min,升温灭酶。通过高效液相对制备的麦芽低聚糖糖浆进行组分分析,发现样品中包括葡萄糖及麦芽2～7糖,其中以麦芽二糖、三糖、四糖、五糖居多,说明该工艺制备的麦芽低聚糖符合分离制备磷酸寡糖的要求。
本文考察了两种弱碱性阴离子交换树脂HJ-30和HJ-31对磷酸寡糖的洗脱收率。实验结果表明,在一定的洗脱条件下HJ-30和HJ-31对磷酸寡糖的洗脱收率分别为87.3%和74.1%,前者更适于磷酸寡糖的分离制备。以HJ-30为层析柱(柱3cm×60cm)填料,考察了洗脱液浓度和洗脱速度对分离磷酸寡糖的影响,得到了分离磷酸寡糖的最佳工艺条件:去离子水洗脱中性糖后,以0.5mol/L的NaCl为洗脱液,1.2ml/min的速度,洗脱磷酸寡糖。
对制备的磷酸寡糖样品进行红外光谱结构分析,图谱分析结果表明样品为结合有磷酯键P-O-C的糖类物质。同时采用薄层色谱法(Thin Layer Chromatography,TLC)对制备的磷酸寡糖进行定性分析,建立了磷酸寡糖的TLC条件,通过优化展开剂和显色剂的条件,确定了磷酸寡糖的TLC最佳展开剂为正丁醇:乙酸:水(3:1:1),显色剂为苯胺-二苯胺-磷酸。此外,本文还采用高效阴离子交换色谱(High-PerformanceAnion-Exchange Chromatography,HPAEC)对POs进行了组分分析。结果表明本文制备得到的磷酸寡糖是分子中结合有磷酸基团的葡萄糖和聚合度在2～7之间的麦芽寡糖的混合物。TLC与HPAEC分析结果均与文献报道一致。
Phosphoryl Oligosaccharides,newly produced malto oligosaccharides(DP3～7) having C-O-P groups in their molecules,are isolated from potato starch hydrolysate.It is a new type of functional oligosaccharides with many functional properties.For example,it could inhibit the formation of calcium phosphate,prevent caries and suppress the retrogradation of starch.Researches on POs started from 90's of the last century.At present, products can be found in the markets of Japan,mainly to Phosphoryl Oligosaccharides of calcium(Pos-Ca).Until recently,there have been little researches on POs in China.In order to develop this new kind of oligosaccharide and to make full use of raw material of potato starch,the experiments on POs have been initiated and the objective of this dissertation is to prepare Pos from potato starch by enzymatic hydrolysis.
In this thesis,maltooligosaccharides with a low dextrose equivalent(DE)were prepared from potato starch,and then Pos were separated from it by column layer analysis. The qualitative analysis of Pos was also presented.This Study established theoretical and practical basis for the further research in preparation technology,and also benefited the early realization of its application and industrial production.
A jet liquefaction process was adopted to prepare maltose oligosaccharides with a low dextrose equivalent.The following conditions of the process are obtained:Potato starch with the concentration of 7°Bé,thermostableα-amylase with the amount of 167,2000U,jet liquefaction temperature 105℃,liquefaction with the temperature and time of 95℃and 30min respectively.And then glucoamytase with the amount of 1075,7900U was added to the syrup and maintained for 60min at 60℃.In final,amylase was deactivated for 10min at 100℃.The composition of maltooligosaccharides was analyzed by HPLC.The result showed that glucose and maltose oligosaccharides(mainly to DP2, DP3,DP4,DP5)were included in the sample we prepared.
Weakly basic anion-exchange resin HJ-30 and HJ-31 were Surveyed according to the yield for Pos.The results demonstrated that:the Pos yields of HJ-30 and HJ-31 were 87.3%and 74.1%respectively,so the former was obviously better than the latter.Using HJ-30 as the column packing,influence of the solvent concentration and the ratio of elution on the separation of Pos was investigated.The result indicated that the optimum conditions to prepare Pos were as follows:after eluting neutral oligosaccharides with de-ionized waters,Pos was obtained with 0.5mol/L NaCl and the eluting rate was 1.2ml/min.
Structural analysis of POs was done by infrared chromatography.The results showed that thereare phosphate ester bonds in the sample.TLC and HPAEC were also empolyed in the qualitative analysis of POs.For TLC,the optimum solvent(butyl alcohol:acetic acid: water=3:1:1)and Visualization reagent(aniline-Diphenylamine-phosphorous acid)were presented by optimizing the condition of solvent and Visualization reagent.The results demonstrated that there were four carbohydrate components(DP3～7)in the sample of Pos at least.For HPAEC,the results demonstrated that at least one or more kinds of oligosaccharides(included G1～G7)in sample of Pos.
本文以马铃薯淀粉为原料制备低DE值的麦芽低聚糖浆,再通过柱层析的方法从麦芽低聚糖糖浆中分离制备了磷酸寡糖,并对其进行了定性分析。为进一步探讨磷酸寡糖的制备工艺,早日实现磷酸寡糖工业化生产和广泛应用奠定了一定的理论基础和实施依据。
本文通过喷射液化的方法制备了低DE值的麦芽低聚糖浆。工艺条件为:用水将淀粉乳浓度调至7°Bé,加入209,0000U耐高温α淀粉酶,于105℃的条件下喷射液化4～8min,再于层流罐内95℃条件下保温30min。灭酶后进入糖化工艺,加入1075,7900U糖化酶,于60℃条件下反应60min,升温灭酶。通过高效液相对制备的麦芽低聚糖糖浆进行组分分析,发现样品中包括葡萄糖及麦芽2～7糖,其中以麦芽二糖、三糖、四糖、五糖居多,说明该工艺制备的麦芽低聚糖符合分离制备磷酸寡糖的要求。
本文考察了两种弱碱性阴离子交换树脂HJ-30和HJ-31对磷酸寡糖的洗脱收率。实验结果表明,在一定的洗脱条件下HJ-30和HJ-31对磷酸寡糖的洗脱收率分别为87.3%和74.1%,前者更适于磷酸寡糖的分离制备。以HJ-30为层析柱(柱3cm×60cm)填料,考察了洗脱液浓度和洗脱速度对分离磷酸寡糖的影响,得到了分离磷酸寡糖的最佳工艺条件:去离子水洗脱中性糖后,以0.5mol/L的NaCl为洗脱液,1.2ml/min的速度,洗脱磷酸寡糖。
对制备的磷酸寡糖样品进行红外光谱结构分析,图谱分析结果表明样品为结合有磷酯键P-O-C的糖类物质。同时采用薄层色谱法(Thin Layer Chromatography,TLC)对制备的磷酸寡糖进行定性分析,建立了磷酸寡糖的TLC条件,通过优化展开剂和显色剂的条件,确定了磷酸寡糖的TLC最佳展开剂为正丁醇:乙酸:水(3:1:1),显色剂为苯胺-二苯胺-磷酸。此外,本文还采用高效阴离子交换色谱(High-PerformanceAnion-Exchange Chromatography,HPAEC)对POs进行了组分分析。结果表明本文制备得到的磷酸寡糖是分子中结合有磷酸基团的葡萄糖和聚合度在2～7之间的麦芽寡糖的混合物。TLC与HPAEC分析结果均与文献报道一致。
Phosphoryl Oligosaccharides,newly produced malto oligosaccharides(DP3～7) having C-O-P groups in their molecules,are isolated from potato starch hydrolysate.It is a new type of functional oligosaccharides with many functional properties.For example,it could inhibit the formation of calcium phosphate,prevent caries and suppress the retrogradation of starch.Researches on POs started from 90's of the last century.At present, products can be found in the markets of Japan,mainly to Phosphoryl Oligosaccharides of calcium(Pos-Ca).Until recently,there have been little researches on POs in China.In order to develop this new kind of oligosaccharide and to make full use of raw material of potato starch,the experiments on POs have been initiated and the objective of this dissertation is to prepare Pos from potato starch by enzymatic hydrolysis.
In this thesis,maltooligosaccharides with a low dextrose equivalent(DE)were prepared from potato starch,and then Pos were separated from it by column layer analysis. The qualitative analysis of Pos was also presented.This Study established theoretical and practical basis for the further research in preparation technology,and also benefited the early realization of its application and industrial production.
A jet liquefaction process was adopted to prepare maltose oligosaccharides with a low dextrose equivalent.The following conditions of the process are obtained:Potato starch with the concentration of 7°Bé,thermostableα-amylase with the amount of 167,2000U,jet liquefaction temperature 105℃,liquefaction with the temperature and time of 95℃and 30min respectively.And then glucoamytase with the amount of 1075,7900U was added to the syrup and maintained for 60min at 60℃.In final,amylase was deactivated for 10min at 100℃.The composition of maltooligosaccharides was analyzed by HPLC.The result showed that glucose and maltose oligosaccharides(mainly to DP2, DP3,DP4,DP5)were included in the sample we prepared.
Weakly basic anion-exchange resin HJ-30 and HJ-31 were Surveyed according to the yield for Pos.The results demonstrated that:the Pos yields of HJ-30 and HJ-31 were 87.3%and 74.1%respectively,so the former was obviously better than the latter.Using HJ-30 as the column packing,influence of the solvent concentration and the ratio of elution on the separation of Pos was investigated.The result indicated that the optimum conditions to prepare Pos were as follows:after eluting neutral oligosaccharides with de-ionized waters,Pos was obtained with 0.5mol/L NaCl and the eluting rate was 1.2ml/min.
Structural analysis of POs was done by infrared chromatography.The results showed that thereare phosphate ester bonds in the sample.TLC and HPAEC were also empolyed in the qualitative analysis of POs.For TLC,the optimum solvent(butyl alcohol:acetic acid: water=3:1:1)and Visualization reagent(aniline-Diphenylamine-phosphorous acid)were presented by optimizing the condition of solvent and Visualization reagent.The results demonstrated that there were four carbohydrate components(DP3～7)in the sample of Pos at least.For HPAEC,the results demonstrated that at least one or more kinds of oligosaccharides(included G1～G7)in sample of Pos.
引文
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[4]倪红,杨艳燕,阎达中.寡糖的开发现状及其应用研究进展[J].湖北大学学报,2003,6,25(2):148-151
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