真姬菇子实体多糖的提取、纯化及抗氧化性研究
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摘要
真姬菇又名蟹味菇、玉蕈、鸿禧菇等,是一种很有开发价值和应用前景的珍稀食药用真菌,以其独特的营养价值和抗肿瘤、提高免疫力等药用功能而成为新近研究的热点。本文以真姬菇子实体为材料,对其多糖的提取、分离纯化、抗氧化活性进行了研究,取得以下主要结果:
1.以水为浸提液,通过单因素试验研究了浸提温度、浸提时间、液固比、乙醇用量和提取次数对真姬菇多糖得率的影响,并采用二次回归正交旋转组合设计试验对提取工艺进行优化。结果表明,多糖得率的主要影响因素及其主次顺序为浸提温度、浸提时间、液固比;在乙醇用量为2倍、提取次数为1次时,最佳提取工艺参数为浸提温度95℃、浸提时间2.84h、液固比11.6:1,多糖得率为4.47%。
2.经过对Sevag法、TCA法、胰蛋白酶法脱蛋白效果的比较,结果表明胰蛋白酶法效果较好。向多糖溶液中加入0.4倍多糖溶液体积的胰蛋白酶溶液(1mg/mL),保持pH6.0左右,在37℃±1℃水浴条件下酶解2h,蛋白质脱除率为87.69%,多糖保留率为89.04%。
3.透析后的真姬菇子实体多糖经DEAE-Sephadex A-25柱层析,用不同溶剂洗脱后分别得到灰白色、白色、淡黄色、灰白色4个多糖组分,经纸层析、Sephadex G-200凝胶柱层析、紫外光谱鉴定,均为单一组分。紫外光谱结果表明,4个多糖组分均不含核酸和蛋白质等杂质。
4.真姬菇粗多糖(HMⅠ)、脱蛋白粗多糖(HMⅡ)对O_2~-·有一定清除效果;对Fenton体系产生的·OH有较好的清除效果,比Vc稍低,且HMⅠ对·OH清除率较HMⅡ高;在DPPH·体系中,在浓度小于2.2mg/mL时,HMⅠ与HMⅡ清除率小于Vc,高于此浓度时大于Vc,但两者对DPPH·清除率基本没有差别;HMⅠ与HMⅡ均有一定的还原能力。HMⅠ、HMⅡ对·OH清除能力和对Fe~(3+)还原能力的IC_(50)值达到了极显著;对DPPH·清除能力的IC_(50)值达到了显著水平。HMⅠ抗氧化活性显著高于HMⅡ,亦即未脱蛋白多糖的抗氧化活性显著高于脱蛋白多糖。
本文创新点:
首次建立了真姬菇子实体多糖的热水提取工艺及模型,并通过脱蛋白、柱层析等建立了真姬菇子实体多糖的分离纯化工艺。
Hypsizigus marmoreus, also called Crab flavor mushroom, Hongxi mushroom, et al., is a kind of edible and medicinal mushroom. It is getting research hotspot recently for its distinctive nutrient and medicinal values on tumor and immunity.
The polysaccharides from Hypsizigus marmoreus fruit-body was extracted and purified, and its antioxidant properties were studied. All the results are showed below:
1. In order to optimize the technology for the extraction of polysaccharides from Hypsizigus marmoreus fruit-body, water was taken as extraction solvent and the effects of extraction temperature, extraction time, ratio of liquid to solid, ratio of ethanol to extract and extraction times on the yield of polysaccharides were investigated by the single factor experiments. Technology parameters were optimized by quadric regression orthogonal rotary design experiments. The results showed that the degree of the factors significantly influence on the yield of polysaccharides are extraction temperature, extraction time, ratio of liquid to solid, in a sequence. The optimum extraction conditions are as follows: volume of ethanol to extract for precipitation is 2 times, extraction times 1, extraction temperature 95℃, extraction time 2.84h, ratio of liquid to solid 11.6:1. The yield of polysaccharides can be up to 4.47% under the optimum extraction conditions.
2. The removal of protein in crude Hypsizigus marmoreus fruit-body polysaccharides was studied by three methods including Sevag method, TCA method and trypsin method. The research showed that the trypsin method was optimum. The deproteinizaton rate was 87.69%, the retain rate of polysaccharides was 89.04% under the conditions that ratio of polysaccharides to 1mg/mL trypsin is 1:0.4, pH6.0 and water bath temperature is 37℃±1℃for 2h.
3. There were offwhite, white, straw yellow and offwhite polysaccharides component were obtained by the DEAE-Sephadex A-25 column chromatography using four different solvent, from Hypsizigus marmoreus fruit-body polysaccharides which was deproteinized, decolored and dialyzed. The four components were homogeneous, which were identified by paper chromatography, Sephadex G-200 gel column chromatography and UV spectrum. The UV spectrum indicated the four polysaccharide components do not contain nucleic acid, protein and other impurities.
4. The antioxidative properties of polysaccharide extracts from fruitbodies of Hypsizigus marmoreus were studied and compared with Vc using superoxide radical system, hydroxyl radical system, 1,1-diphenyl-2-picrylhydrazyl radical system and reducing power. The results showed that HMⅠ(non-deproteinization Hypsizigus marmoreus polysaccharides) and HMⅡ(deproteinization Hypsizigus marmoreus polysaccharides) had different scavenging activity in these radical systems. Their scavenging activity to superoxide radical were not abvious, but to hydroxyl radical had a better scavenging effectiveness(both lower than Vc) and HMⅠwas higher than HMⅡ. With regard to the scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals, both HMⅠand HMⅡwere less than Vc when the concentration lower than 2.2mg/mL and more than Vc when the concentration higher than 2.2mg/mL, but they had not significant difference. Their reducing power were similarly equal and less than Vc. IC_(50) value of HMⅠand HMⅡwere terribly significant on hydroxyl radical scavenging capability and reducing power, respectively, and significant on 1,1-diphenyl-2-picrylhydrazyl radicals. In a whole, the antioxidative activity of HMⅠwas significantly higher than HMⅡ.
The creative points are as follows:
Hot water extract technology and model of polysaccharides from Hysizigus marmoreus fruit-body were built. Isolation and purification technology of polysaccharides were established by deproteinization, DEAE-Sephadex A-25 column chromatography, et al.
1.以水为浸提液,通过单因素试验研究了浸提温度、浸提时间、液固比、乙醇用量和提取次数对真姬菇多糖得率的影响,并采用二次回归正交旋转组合设计试验对提取工艺进行优化。结果表明,多糖得率的主要影响因素及其主次顺序为浸提温度、浸提时间、液固比;在乙醇用量为2倍、提取次数为1次时,最佳提取工艺参数为浸提温度95℃、浸提时间2.84h、液固比11.6:1,多糖得率为4.47%。
2.经过对Sevag法、TCA法、胰蛋白酶法脱蛋白效果的比较,结果表明胰蛋白酶法效果较好。向多糖溶液中加入0.4倍多糖溶液体积的胰蛋白酶溶液(1mg/mL),保持pH6.0左右,在37℃±1℃水浴条件下酶解2h,蛋白质脱除率为87.69%,多糖保留率为89.04%。
3.透析后的真姬菇子实体多糖经DEAE-Sephadex A-25柱层析,用不同溶剂洗脱后分别得到灰白色、白色、淡黄色、灰白色4个多糖组分,经纸层析、Sephadex G-200凝胶柱层析、紫外光谱鉴定,均为单一组分。紫外光谱结果表明,4个多糖组分均不含核酸和蛋白质等杂质。
4.真姬菇粗多糖(HMⅠ)、脱蛋白粗多糖(HMⅡ)对O_2~-·有一定清除效果;对Fenton体系产生的·OH有较好的清除效果,比Vc稍低,且HMⅠ对·OH清除率较HMⅡ高;在DPPH·体系中,在浓度小于2.2mg/mL时,HMⅠ与HMⅡ清除率小于Vc,高于此浓度时大于Vc,但两者对DPPH·清除率基本没有差别;HMⅠ与HMⅡ均有一定的还原能力。HMⅠ、HMⅡ对·OH清除能力和对Fe~(3+)还原能力的IC_(50)值达到了极显著;对DPPH·清除能力的IC_(50)值达到了显著水平。HMⅠ抗氧化活性显著高于HMⅡ,亦即未脱蛋白多糖的抗氧化活性显著高于脱蛋白多糖。
本文创新点:
首次建立了真姬菇子实体多糖的热水提取工艺及模型,并通过脱蛋白、柱层析等建立了真姬菇子实体多糖的分离纯化工艺。
Hypsizigus marmoreus, also called Crab flavor mushroom, Hongxi mushroom, et al., is a kind of edible and medicinal mushroom. It is getting research hotspot recently for its distinctive nutrient and medicinal values on tumor and immunity.
The polysaccharides from Hypsizigus marmoreus fruit-body was extracted and purified, and its antioxidant properties were studied. All the results are showed below:
1. In order to optimize the technology for the extraction of polysaccharides from Hypsizigus marmoreus fruit-body, water was taken as extraction solvent and the effects of extraction temperature, extraction time, ratio of liquid to solid, ratio of ethanol to extract and extraction times on the yield of polysaccharides were investigated by the single factor experiments. Technology parameters were optimized by quadric regression orthogonal rotary design experiments. The results showed that the degree of the factors significantly influence on the yield of polysaccharides are extraction temperature, extraction time, ratio of liquid to solid, in a sequence. The optimum extraction conditions are as follows: volume of ethanol to extract for precipitation is 2 times, extraction times 1, extraction temperature 95℃, extraction time 2.84h, ratio of liquid to solid 11.6:1. The yield of polysaccharides can be up to 4.47% under the optimum extraction conditions.
2. The removal of protein in crude Hypsizigus marmoreus fruit-body polysaccharides was studied by three methods including Sevag method, TCA method and trypsin method. The research showed that the trypsin method was optimum. The deproteinizaton rate was 87.69%, the retain rate of polysaccharides was 89.04% under the conditions that ratio of polysaccharides to 1mg/mL trypsin is 1:0.4, pH6.0 and water bath temperature is 37℃±1℃for 2h.
3. There were offwhite, white, straw yellow and offwhite polysaccharides component were obtained by the DEAE-Sephadex A-25 column chromatography using four different solvent, from Hypsizigus marmoreus fruit-body polysaccharides which was deproteinized, decolored and dialyzed. The four components were homogeneous, which were identified by paper chromatography, Sephadex G-200 gel column chromatography and UV spectrum. The UV spectrum indicated the four polysaccharide components do not contain nucleic acid, protein and other impurities.
4. The antioxidative properties of polysaccharide extracts from fruitbodies of Hypsizigus marmoreus were studied and compared with Vc using superoxide radical system, hydroxyl radical system, 1,1-diphenyl-2-picrylhydrazyl radical system and reducing power. The results showed that HMⅠ(non-deproteinization Hypsizigus marmoreus polysaccharides) and HMⅡ(deproteinization Hypsizigus marmoreus polysaccharides) had different scavenging activity in these radical systems. Their scavenging activity to superoxide radical were not abvious, but to hydroxyl radical had a better scavenging effectiveness(both lower than Vc) and HMⅠwas higher than HMⅡ. With regard to the scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals, both HMⅠand HMⅡwere less than Vc when the concentration lower than 2.2mg/mL and more than Vc when the concentration higher than 2.2mg/mL, but they had not significant difference. Their reducing power were similarly equal and less than Vc. IC_(50) value of HMⅠand HMⅡwere terribly significant on hydroxyl radical scavenging capability and reducing power, respectively, and significant on 1,1-diphenyl-2-picrylhydrazyl radicals. In a whole, the antioxidative activity of HMⅠwas significantly higher than HMⅡ.
The creative points are as follows:
Hot water extract technology and model of polysaccharides from Hysizigus marmoreus fruit-body were built. Isolation and purification technology of polysaccharides were established by deproteinization, DEAE-Sephadex A-25 column chromatography, et al.
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