灰树花β-葡聚糖的高效提取及量化技术
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摘要
本文通过对灰树花β-葡聚糖的提取、测定、纯化和结构的研究,建立了荧光法测定1,3-β-葡聚糖的方法,并探讨了其测定影响因素;确定了灰树花β-葡聚糖高效提取的条件;分析了一种灰树花β-葡聚糖的结构。
1.研究表明可得然1,3-β-葡聚糖、酵母1,3/1,6-β-葡聚糖及灰树花中1,3-链葡聚糖与苯胺蓝的结合具有特异性;当以酵母1,3/1,6-β-葡聚糖为标准物时蛋白浓度在葡聚糖浓度的一半以下,蛋白对测定影响不大;当以可得然凝胶为标准物测量时,蛋白对测定影响不大,淀粉,纤维素和麦芽糊精添加会导致两种测定的荧光强度下降。
2.食用菌1,3-β-葡聚糖荧光法测定与鲎试剂法测定比较,结果表明荧光法简单可行。
3.通过响应面优化,得到灰树花多糖提取最佳条件为:提取功率137.6W;超声时间为16.9 min;酶解温度40℃,时间45 min,pH 6.0,加酶量1.5%;95℃提取两次,在此条件下,10 g灰树花多糖提取得理论值达到12.75%;灰树花β-葡聚糖提取最佳条件为:提取频率为148.3 W;时间15.6 min;温度为95℃,在此条件下,10 g灰树花多糖提取得理论值达到3.60%。
4.对热水提取粗β-葡聚糖进行离子交换层析、凝胶柱层析,纯化得到一种均一多糖GFWD,通过对其进行单糖组分分析,红外光谱分析及核磁共振分析,确定此多糖是一种1,3-β-葡聚糖。
本研究创新点在于:
1.建立了荧光法测定1,3-β-葡聚糖的方法,并研究了其影响因素。
2.以响应面法优化了灰树花β-葡聚糖的超声-酶法提取工艺。
3.测定了灰树花热水提、冷碱提取和热碱提取粗多糖中β-葡聚糖和1,3-β-葡聚糖的含量。
4.通过气相色谱、红外光谱和核磁共振解析了一种热水提取β-葡聚糖的结构。
Systematic studies on the extraction,quantificational,purification and structural characterization of Grifola frondosa polysaccharides and itsβ-glucan were carried out in the presented dissertation.Some new methods and points were developed on the basis of thesis of these studies.The results and creative points are described as follows:
1.The result showed curdlan,1,3/1,6-β-glucan of yeast and Grifola frondosa 1,3-β-glucan could bind to Aniline Blue specifically,when the content of the protein was the half of the content of 1,3/1,6-β-glucan,there was no effect on quantificational with the standard 1,3/1,6-β-glucan of yeast.The protein no effect on quantificational with the curdlan standard.Starch,the dextrin,glucose and cellulose no effect.
2.To contrast to detecting method about 1,3-β-glucan about Limulus factor G method and fluorescence method,the result show fluorescence method was simple and feasible.
3.By response surface methodology,the optimum polysaccharides conditions of ultrasonic and enzyme assisted extraction was that ultrasonic power was 137.6 W,ultrasonic time was 16.9 min;the optimumβ-glucan conditions was ultrasonic power was 148.3 W. ultrasonic time was 15.6 min.Hydrolyzed temperature was 40℃. hydrolyzed time was 45 min,pH was 6.0 and the quantity of papain was 1.5%of raw material,then extraction was twice under 95℃.By the conditions,the extraction rate of polysaccharides andβ-glucan were 12.75%and 3.60%.
4.Separated the crudeβ-glucan by anion exchange and gel chromatographic,then to analyze the homogeneous sugar and structure of aβ-glucan from Grifola frondosa by hot water through High Performance Liquid Chromatography(HPLC),Gas Chromatography (GC),Infrared spectroscopy(IR)and Nuclear Magnetic Resonance (NMR).
The innovations that originally studies are embodied in the following several points:
1.A simple detecting method about 1,3-β-glucan by fluorescence method established according to some scientific reports.
2.To found the optimum conditions of ultrasonic and enzyme assisted extraction by hot water.
3.Determinated of Grifola frondosa 1,3-β-glucan by hot water,cold alkali and hot alkali.
4.To analyze structure of aβ-glucan from Grifola frondosa by hot water through GC,IR,NMR.
1.研究表明可得然1,3-β-葡聚糖、酵母1,3/1,6-β-葡聚糖及灰树花中1,3-链葡聚糖与苯胺蓝的结合具有特异性;当以酵母1,3/1,6-β-葡聚糖为标准物时蛋白浓度在葡聚糖浓度的一半以下,蛋白对测定影响不大;当以可得然凝胶为标准物测量时,蛋白对测定影响不大,淀粉,纤维素和麦芽糊精添加会导致两种测定的荧光强度下降。
2.食用菌1,3-β-葡聚糖荧光法测定与鲎试剂法测定比较,结果表明荧光法简单可行。
3.通过响应面优化,得到灰树花多糖提取最佳条件为:提取功率137.6W;超声时间为16.9 min;酶解温度40℃,时间45 min,pH 6.0,加酶量1.5%;95℃提取两次,在此条件下,10 g灰树花多糖提取得理论值达到12.75%;灰树花β-葡聚糖提取最佳条件为:提取频率为148.3 W;时间15.6 min;温度为95℃,在此条件下,10 g灰树花多糖提取得理论值达到3.60%。
4.对热水提取粗β-葡聚糖进行离子交换层析、凝胶柱层析,纯化得到一种均一多糖GFWD,通过对其进行单糖组分分析,红外光谱分析及核磁共振分析,确定此多糖是一种1,3-β-葡聚糖。
本研究创新点在于:
1.建立了荧光法测定1,3-β-葡聚糖的方法,并研究了其影响因素。
2.以响应面法优化了灰树花β-葡聚糖的超声-酶法提取工艺。
3.测定了灰树花热水提、冷碱提取和热碱提取粗多糖中β-葡聚糖和1,3-β-葡聚糖的含量。
4.通过气相色谱、红外光谱和核磁共振解析了一种热水提取β-葡聚糖的结构。
Systematic studies on the extraction,quantificational,purification and structural characterization of Grifola frondosa polysaccharides and itsβ-glucan were carried out in the presented dissertation.Some new methods and points were developed on the basis of thesis of these studies.The results and creative points are described as follows:
1.The result showed curdlan,1,3/1,6-β-glucan of yeast and Grifola frondosa 1,3-β-glucan could bind to Aniline Blue specifically,when the content of the protein was the half of the content of 1,3/1,6-β-glucan,there was no effect on quantificational with the standard 1,3/1,6-β-glucan of yeast.The protein no effect on quantificational with the curdlan standard.Starch,the dextrin,glucose and cellulose no effect.
2.To contrast to detecting method about 1,3-β-glucan about Limulus factor G method and fluorescence method,the result show fluorescence method was simple and feasible.
3.By response surface methodology,the optimum polysaccharides conditions of ultrasonic and enzyme assisted extraction was that ultrasonic power was 137.6 W,ultrasonic time was 16.9 min;the optimumβ-glucan conditions was ultrasonic power was 148.3 W. ultrasonic time was 15.6 min.Hydrolyzed temperature was 40℃. hydrolyzed time was 45 min,pH was 6.0 and the quantity of papain was 1.5%of raw material,then extraction was twice under 95℃.By the conditions,the extraction rate of polysaccharides andβ-glucan were 12.75%and 3.60%.
4.Separated the crudeβ-glucan by anion exchange and gel chromatographic,then to analyze the homogeneous sugar and structure of aβ-glucan from Grifola frondosa by hot water through High Performance Liquid Chromatography(HPLC),Gas Chromatography (GC),Infrared spectroscopy(IR)and Nuclear Magnetic Resonance (NMR).
The innovations that originally studies are embodied in the following several points:
1.A simple detecting method about 1,3-β-glucan by fluorescence method established according to some scientific reports.
2.To found the optimum conditions of ultrasonic and enzyme assisted extraction by hot water.
3.Determinated of Grifola frondosa 1,3-β-glucan by hot water,cold alkali and hot alkali.
4.To analyze structure of aβ-glucan from Grifola frondosa by hot water through GC,IR,NMR.
引文
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