径向色谱技术分离黑木耳抗氧化活性多糖的研究
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摘要
黑木耳是我国珍贵的药食两用胶质真菌,具有极其丰富的营养价值和药用价值,其生物活性的有效成分主要为黑木耳多糖。据大量研究表明,黑木耳多糖具有调节免疫功能、降血脂、抗衰老、抗氧化、抗肿瘤、抗凝血、抗血栓等药理生物活性。径向色谱技术是复杂样品快速分离、制备最有效的工具之一。其采用了径向流动技术,样品和流动相是从柱的圆周围流向柱圆心或从圆心流向圆周围,具有速度快、压降低、操作方便、便于线性放大等优点,解决了传统的轴向色谱技术所存在的问题,在分离纯化生物大分子方面具有很大的优势。为了更有效地分离纯化黑木耳多糖,得到高抗氧化活性和高纯度的黑木耳多糖,因此本课题对应用径向色谱技术分离纯化黑木耳多糖进行了研究。
通过黑木耳多糖脱色方法的比较实验,得出树脂吸附法是一种较理想的黑木耳粗多糖脱色方法,既能取得良好的脱色效果,又不会造成多糖的大量损失。多糖保留率为68.6%,脱蛋白质率为55.8%。树脂脱色工艺为：AB-8大孔树脂中加入质量分数5.0%的多糖在pH 4.5、40℃下,以100 r／min的转速恒温振荡2小时后过滤。
确定了DEAE树脂对黑木耳多糖吸附的最佳pH值为7.0,多糖吸附量为5.69 mg/mL；在pH值保持不变的情况下,树脂对黑木耳多糖的吸附量随初始浓度的增加而增大,当多糖浓度大于2.5 mg/mL时,多糖吸附量的增幅的趋势变缓。通过动态解吸实验,确定了最佳的洗脱液浓度范围为0.1-0.5 mol／L NaCl。
使用柱规格(i.d.=2.3 cm,h=50 cm)的DEAE轴向色谱柱对黑木耳多糖进行分离,得到两个色谱峰,分别命名为AAPⅠ和AAPⅡ。采用DPPH法和H2O2/Fe2+体系测定了AAPⅠ和AAPⅡ的抗氧化活性,得出AAPⅡ纯度较高、具有较强的抗氧化活性。
通过单因素实验的综合分析可以得出,确定了采用Toyopearl DEAE-650 M 500 mL径向色谱柱分离黑木耳抗氧化活性多糖AAPⅡ的最适条件为：上样体积200 mL、上样量2000 mg、上样液和洗脱液pH 7.0、洗脱流速45 mL／min、操作温度20℃。线性放大径向色谱柱,黑木耳多糖的上样量也可提高到512 g,分离可得116.8 g的AAPⅡ样品,达到近公斤级的制备规模。
对径向色谱技术与轴向色技术分离黑木耳多糖进行了比较,得出：径向色谱柱压降低,可以进行大流量操作,分离速度比轴向色谱柱大大提高；且径向色谱技术更易于线性放大,可用于大规模的生产分离和纯化。
以上研究结果可以为径向色谱技术分离纯化黑木耳多糖的规模化生产提供技术支持,也能为其它多糖的分离纯化提供科学依据。
Auricularia auricula is the precious medicinal and edible gelatinous fungi, and extremely rich in nutritional and medicinal value. Polysaccharides in Auricularia auricula are potential biological ingredients. According to a large number of studies, polysaccharides in Auricularia auricula possess bioactivities in regulating immunity, reducing blood lipids, anti-aging, anti-oxidation, anti-tumor, anti-coagulation and anti-thrombotic. Radial chromatography is one of the most effective instruments of rapid separation and preparation of complex samples. Because it adopts the radial flow technology, samples and mobile phase flow from the surrounding of the column to the center of a circle or from the center of a circle to the surrounding, with rapid speed, low pressure, easy to operate, easy to linear amplification, etc., which solves the problems of the traditional axial chromatographic technique, and has a great advantage of the separation and purification of biological macromolecules. In order to more effectively purify polysaccharides in Auricularia auricula and get higher antioxidant activity and higher purity polysaccharides in Auricularia auricula, this paper studied on the application of radial chromatography on the separation and purification of polysaccharides in Auricularia auricula.
Decolorization of polysaccharides in Auricularia auricula was studied. The results showed that the resin adsorption was an excellent decolorating method. After decolorization, polysaccharides retention rate was 68.6%, protein removal rate was 55.8%.
The effects of adsorption and desorption of polysaccharides in Auricularia auricula on DEAE anion-exchange resin were studied based on experiments of static adsorption, dynamic adsorption and desorption. The results showed that the concentration and pH value of the sample solution were the key factors of the resin exchange capacity, and the concentration of the eluent influenced the elution curve. The optimum sample concentration was 2.5 mg/mL, the optimum pH was 7.0. Under the optimum condition adsorption capacity of the polysaccharides was 5.69 mg/mL. The concentration of eluent from 0.1 mol/L to 0.5 mol/L gave a good separation result.
AAPII, a polysaccharide component with a higher antioxidant activity from a separation by Toyopearl DEAE-650 M, was selected as the target product of polysaccharides in Auricularia auricula of radial column chromatography with Toyopearl DEAE-650 M.
Through comprehensive analysis of single factor experiments, the optimum operating conditions were obtained as fllow:sample volume, 200 mL; sample amount,2000 mg; pH value,7.0; flow rate of sample load and elution,45 mL/min; and temperature,20℃.
The seperation of polysaccharides in Auricularia auricula were compared bewteen radial and axial chromatographic technique. The results indicated that radial chromatography column had lower pressure with large flow operation, and the seperation efficiency improved markedly, much faster than axial chromatography column. Radial chromatographic technique was much easier to linear amplification, which can be used for the separation and purification of large-scale production.
The above research results will be able to provide technical support for the large-scale purification of polysaccharide AAPⅡfrom Auricularia auricula with radial chromatography, and also provide scientific basis for other polysaccharides.
通过黑木耳多糖脱色方法的比较实验,得出树脂吸附法是一种较理想的黑木耳粗多糖脱色方法,既能取得良好的脱色效果,又不会造成多糖的大量损失。多糖保留率为68.6%,脱蛋白质率为55.8%。树脂脱色工艺为：AB-8大孔树脂中加入质量分数5.0%的多糖在pH 4.5、40℃下,以100 r／min的转速恒温振荡2小时后过滤。
确定了DEAE树脂对黑木耳多糖吸附的最佳pH值为7.0,多糖吸附量为5.69 mg/mL；在pH值保持不变的情况下,树脂对黑木耳多糖的吸附量随初始浓度的增加而增大,当多糖浓度大于2.5 mg/mL时,多糖吸附量的增幅的趋势变缓。通过动态解吸实验,确定了最佳的洗脱液浓度范围为0.1-0.5 mol／L NaCl。
使用柱规格(i.d.=2.3 cm,h=50 cm)的DEAE轴向色谱柱对黑木耳多糖进行分离,得到两个色谱峰,分别命名为AAPⅠ和AAPⅡ。采用DPPH法和H2O2/Fe2+体系测定了AAPⅠ和AAPⅡ的抗氧化活性,得出AAPⅡ纯度较高、具有较强的抗氧化活性。
通过单因素实验的综合分析可以得出,确定了采用Toyopearl DEAE-650 M 500 mL径向色谱柱分离黑木耳抗氧化活性多糖AAPⅡ的最适条件为：上样体积200 mL、上样量2000 mg、上样液和洗脱液pH 7.0、洗脱流速45 mL／min、操作温度20℃。线性放大径向色谱柱,黑木耳多糖的上样量也可提高到512 g,分离可得116.8 g的AAPⅡ样品,达到近公斤级的制备规模。
对径向色谱技术与轴向色技术分离黑木耳多糖进行了比较,得出：径向色谱柱压降低,可以进行大流量操作,分离速度比轴向色谱柱大大提高；且径向色谱技术更易于线性放大,可用于大规模的生产分离和纯化。
以上研究结果可以为径向色谱技术分离纯化黑木耳多糖的规模化生产提供技术支持,也能为其它多糖的分离纯化提供科学依据。
Auricularia auricula is the precious medicinal and edible gelatinous fungi, and extremely rich in nutritional and medicinal value. Polysaccharides in Auricularia auricula are potential biological ingredients. According to a large number of studies, polysaccharides in Auricularia auricula possess bioactivities in regulating immunity, reducing blood lipids, anti-aging, anti-oxidation, anti-tumor, anti-coagulation and anti-thrombotic. Radial chromatography is one of the most effective instruments of rapid separation and preparation of complex samples. Because it adopts the radial flow technology, samples and mobile phase flow from the surrounding of the column to the center of a circle or from the center of a circle to the surrounding, with rapid speed, low pressure, easy to operate, easy to linear amplification, etc., which solves the problems of the traditional axial chromatographic technique, and has a great advantage of the separation and purification of biological macromolecules. In order to more effectively purify polysaccharides in Auricularia auricula and get higher antioxidant activity and higher purity polysaccharides in Auricularia auricula, this paper studied on the application of radial chromatography on the separation and purification of polysaccharides in Auricularia auricula.
Decolorization of polysaccharides in Auricularia auricula was studied. The results showed that the resin adsorption was an excellent decolorating method. After decolorization, polysaccharides retention rate was 68.6%, protein removal rate was 55.8%.
The effects of adsorption and desorption of polysaccharides in Auricularia auricula on DEAE anion-exchange resin were studied based on experiments of static adsorption, dynamic adsorption and desorption. The results showed that the concentration and pH value of the sample solution were the key factors of the resin exchange capacity, and the concentration of the eluent influenced the elution curve. The optimum sample concentration was 2.5 mg/mL, the optimum pH was 7.0. Under the optimum condition adsorption capacity of the polysaccharides was 5.69 mg/mL. The concentration of eluent from 0.1 mol/L to 0.5 mol/L gave a good separation result.
AAPII, a polysaccharide component with a higher antioxidant activity from a separation by Toyopearl DEAE-650 M, was selected as the target product of polysaccharides in Auricularia auricula of radial column chromatography with Toyopearl DEAE-650 M.
Through comprehensive analysis of single factor experiments, the optimum operating conditions were obtained as fllow:sample volume, 200 mL; sample amount,2000 mg; pH value,7.0; flow rate of sample load and elution,45 mL/min; and temperature,20℃.
The seperation of polysaccharides in Auricularia auricula were compared bewteen radial and axial chromatographic technique. The results indicated that radial chromatography column had lower pressure with large flow operation, and the seperation efficiency improved markedly, much faster than axial chromatography column. Radial chromatographic technique was much easier to linear amplification, which can be used for the separation and purification of large-scale production.
The above research results will be able to provide technical support for the large-scale purification of polysaccharide AAPⅡfrom Auricularia auricula with radial chromatography, and also provide scientific basis for other polysaccharides.
引文
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