现代提取分离技术在食药用菌多糖分离纯化中的应用

详细综述了超声波法,酶解法,超滤法,透析法,色谱法等现代提取分离技术在食药用菌多糖分离纯化中的应用,简单阐述了各种技术的优缺点,并对其应用前景作了简要探讨。     

灰树花多糖的提取及抗氧化活性

采用单因素和正交试验法对热水浸提、超声波辅助和微波辅助提取灰树花多糖的工艺进行研究,分别获得了3种方法的最佳条件,并发现微波辅助法最佳,其最优条件为:微波功率800 W、微波时间3 min,浸提2h,多糖得率为13.05％,比热水浸提法提高了51.22％.利用提取的灰树花多糖进行抗氧化性研究,发现灰树花多糖对O2-·和·OH都具有一定的清除作用,且多糖浓度与其对O2-·的清除作用存在量效关系,但不如·OH明显.     

微波辅助提取灰树花多糖工艺研究

采用提取时间、微波功率、液料比的单因素试验和正交试验法优化微波辅助提取灰树花多糖条件.结果表明,以净多糖得率为指标,影响微波辅助提取灰树花多糖的主次因素为:提取时间＞微波功率＞液料比,并且提取时间和微波功率的影响达到了极显著水平.灰树花多糖最佳提取工艺条件为:提取时间为10 min,微波功率为80%(全功率为800 W),液料比为25∶1.创立了一种用苯酚-硫酸法测定多糖时排除蛋白质干扰的方法.     

灰树花蛋白聚糖的提取、分离及氨基酸组成分析

目的:研究灰树花(Grifola frondosa)蛋白聚糖的提取与分离方法及氨基酸组成.方法:采用碱提法提取蛋白聚糖,QSepharose FF离子交换层析进行纯化,SDS - PAGE电泳分离蛋白聚糖及结合蛋白,高效液相色谱法鉴定蛋白聚糖的纯度及分子量,并测定18种氨基酸的含量.结果:分离出蛋白聚糖GFP Ⅰ、GFPⅡ和GFPⅢ,糖含量分别为:43.69％、40.88％、28.33％,蛋白含量分别为:40.42％、34.65％、51.19％.其中GFPⅢ为均一成分,纯度93.61％,重均相对分子量(Mr) 150kDa,GFPⅢ结合蛋白分子量约为33.0kDa,为单一谱带.氨基酸组成分析结果显示:GFPⅢ的氨基酸总和达到66.60g/100g,其中8种必需氨基酸总量为26.14g/100g,4种呈味氨基酸总量为25.43g/100g,Asp、Glu、Leu、Ala和Lys含量较高.结论:分离出的蛋白聚糖为结构均一成分,纯度较高,可用于质量研究和生物学研究.     

夏枯草多糖的提取、分离与纯化技术研究

对夏枯草多糖的分离纯化方法作了探讨,通过正交试验,筛选出最佳工艺条件。采用纸层析、琼脂糖凝胶电泳和紫外分光光度法对多糖纯度进行鉴定,红外光谱法对多糖分子结构进行分析,通过分级沉淀和气相色谱仪分析了多糖的单糖组成。     

黑木耳多糖的提取与纯化

黑木耳干子实体经复合酶解结合热水浸提法提取,超过滤,鞣酸法去蛋白,DEAE-52和Sephacryl S-400分子筛柱层析纯化得到精美糖(AAP1)。其含糖量为89．5％。经薄板层析得到单一斑点,经琼脂糖凝胶电泳得到单一区带,表明为分子大小均一的单一组分多糖。     

鬼臼多糖的分离纯化及其抗肿瘤活性

鬼臼经乙醚脱脂后用热水抽提,用蛋白酶法和Sevag法相结合除去蛋白质,乙醇分级沉淀,经DEAE－Sepharose和Superdex G-75或Sephacryl S-200HR柱层析纯化得到两种多糖组分EPS－A和EPS－B。经分析该两种多糖均为单一组分,用分子筛层析法测定了EPS－A和EPS－B的分子量分别为15kD和175kD。纸层析和气相层析分析得知,EPS－B含有木糖,阿拉伯糖,鼠李糖,葡萄糖,甘露糖和半乳糖,其摩尔比为0．37：2．20：0．57：9．83：1．0：3．42,而EPS－A中仅含葡萄糖,体内试验结果表明,EPS－B多糖对小鼠腹水肝癌细胞生长有一定的抑制作用。     

芦荟多糖的分离纯化及性质研究

采用水提醇沉法提取芦荟多糖,经DEAE-C32柱层析分离,Sephades G-100进一步纯化,得AⅠ、AⅡ和AⅢ三种芦荟多糖。Sephadex G-100凝胶色谱表明,AⅠ组分为均一组分,其分子量约为3.8×10~4。借助气相色谱技术,研究了芦荟粗多糖和AⅠ组分的单糖组成。另外,红外光谱鉴定芦荟多糖主要为吡喃多糖。     

千斤拔多糖的提取纯化及其结构鉴定

利用正交试验考察千斤拔多糖的提取工艺,并比较脱蛋白方法中的Sevag法和三氯乙酸法的纯化效果,总糖含量测定采用苯酚-硫酸法,蛋白质含量测定采用考马斯亮蓝法;采用DEAE-52纤维柱法来分离多糖,并运用HPLC色谱来分析千斤拔多糖中的单糖成分。结果表明:经正交试验得出千斤拔多糖的最佳提取条件为时间2.5 h,料液比为1∶30,温度80℃,其多糖得率为8.558%。对比两种脱蛋白的方法,Sevage法萃取3次时蛋白的脱除效果最好。经DEAE-52纤维柱来分离多糖共分得7个组分。经HPLC色谱鉴定出有葡萄糖,甘露糖和阿拉伯糖,主要单糖成分为葡萄糖。     

白芨粗多糖提取方法的比较研究

以白芨（Bletilla Striata(Thunb.)Reichb.f.）块茎为原料,运用多种理化方法,提取白芨粗多糖,经红外吸收光谱、紫外吸收光谱的比较和鉴定确诊提取物中有多糖特征吸收,测定粗多糖中总糖、还原糖、蛋白质等组成物质的含量,以多糖纯度和提取率为主要指标,对提取方法予以比较。结果表明热水处理和室温下超声处理为较理想的提取方法。     

Content of Extractive Substances and Polysaccharides in Fruit Bodies of Grifola frondosa Cultivated on Lignocellulose Substrates Depending on Extraction Methods

Russian Journal of Bioorganic Chemistry - The work is devoted to a comparative analysis of the quantitative content of extractives and water-soluble polysaccharides isolated from the fruit bodies...     

Purification and characterization of an aminopeptidase from the edible basidiomycete Grifola frondosa

An aminopeptidase was purified 178-fold from an extract of Grifola frondosa by ammonium sulfate precipitation and a series of column chromatographies on phenyl-Toyopearl, Sephadex G-25, and Mono-Q. The molecular mass of the enzyme was estimated to be 27 kDa and 30 kDa by gel filtration and SDS-PAGE, respectively. The enzyme had an optimum pH of 8.5 and was stable between pH 6.0 and pH 10.5, and it also had a high level of heat stability. The enzyme was inactivated by EDTA and o-phenanthroline, and it was also strongly inhibited by bestatin, but no inhibitory effect of DFP was observed. The enzyme preferentially hydrolyzed peptides containing hydrophobic residues in the N-terminal position.     

Purification and Characterization of a Trehalose Synthase from the Basidiomycete Grifola frondosa

A trehalose synthase (TSase) that catalyzes the synthesis of trehalose from d-glucose and α-d-glucose 1-phosphate (α-d-glucose 1-P) was detected in a basidiomycete, Grifola frondosa. TSase was purified 106-fold to homogeneity with 36% recovery by ammonium sulfate precipitation and several steps of column chromatography. The native enzyme appears to be a dimer since it has apparent molecular masses of 120 kDa, as determined by gel filtration column chromatography, and 60 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although TSase catalyzed the phosphorolysis of trehalose to d-glucose and α-d-glucose 1-P, in addition to the synthesis of trehalose from the two substrates, the TSase equilibrium strongly favors trehalose synthesis. The optimum temperatures for phosphorolysis and synthesis of trehalose were 32.5 to 35°C and 35 to 37.5°C, respectively. The optimum pHs for these reactions were 6.5 and 6.5 to 6.8, respectively. The substrate specificity of TSase was very strict: among eight disaccharides examined, only trehalose was phosphorolyzed, and only α-d-glucose 1-P served as a donor substrate with d-glucose as the acceptor in trehalose synthesis. Two efficient enzymatic systems for the synthesis of trehalose from sucrose were identified. In system I, the α-d-glucose 1-P liberated by 1.05 U of sucrose phosphorylase was linked with d-glucose by 1.05 U of TSase, generating trehalose at the initial synthesis rate of 18 mmol/h in a final yield of 90 mol% under optimum conditions (300 mM each sucrose and glucose, 20 mM inorganic phosphate, 37.5°C, and pH 6.5). In system II, we added 1.05 U of glucose isomerase and 20 mM MgSO₄ to the reaction mixture of system I to convert fructose, a by-product of the sucrose phosphorylase reaction, into glucose. This system generated trehalose at the synthesis rate of 4.5 mmol/h in the same final yield.Trehalose (1-α-d-glucopyranosyl-α-d-glucopyranoside) is a nonreducing disaccharide with an α,α-1,1 glycosidic linkage and is widely distributed in plants, insects, fungi, yeast, and bacteria (7). Due to the absence of reducing ends in trehalose, it is highly resistant to heat, pH, and Maillard’s reaction (24). In trehalose-producing organisms, this compound may serve as an energy reserve, a buffer against stresses such as desiccation and freezing, and a protein stabilizer (5, 7, 26, 31, 32). If trehalose can be produced economically, then it has potential commercial applications as a sweetener, a food stabilizer, and an additive in cosmetics and pharmaceuticals (6, 25). Recently, trehalose production through fermentation of yeast (17) and Corynebacterium (30), enzymatic processes from starch (18, 34) and maltose (19, 22, 23, 33), and extraction from transformed plants (10) has been reported.Our approach to trehalose production is to use an enzymatic process to produce trehalose from sucrose, one of the least expensive sugars. Since sucrose is efficiently converted to α-d-glucose 1-phosphate (α-d-glucose 1-P) and fructose by sucrose phosphorylase (SPase), we screened microorganisms for an enzyme that converts α-d-glucose 1-P to trehalose on the assumption that the combination of the putative trehalose synthase (TSase) and SPase would convert sucrose into trehalose. Although similar enzyme activities have been reported in the basidiomycete Flammulina velutipes (11) and in the yeast Pichia fermentans (27), these enzymes have not been well characterized.Our objectives were (i) to screen microorganisms, primarily fungi, for TSase activity; (ii) to purify and characterize the TSase; (iii) to identify the enzymatic process by which trehalose is produced from sucrose; and (iv) to identify an enzymatic process for production of trehalose from sucrose in which the fructose component is also converted to trehalose.     

灰树花多糖的分子修饰及其活性研究进展

灰树花是一种高蛋白低脂肪的食品,具有免疫调节、抗氧化和抗肿瘤等生物活性。其中,多糖是灰树花的主要活性成分之一。对多糖进行分子修饰是提高它原有的生物活性或增加新活性的重要途径之一。综述了灰树花多糖的分子修饰方法,以及修饰后灰树花多糖的生物活性及其构效关系的研究进展,以期为灰树花多糖的深入研究和开发利用提供参考。     

Purification and characterization of a novel prolyl aminopeptidase from Maitake (Grifola frondosa)

We have found a novel prolyl aminopeptidase in Grifola frondosa. The enzyme was purified by DEAE-Sepharose CL-6B, Butyl-Toyopearl, Sephacryl S-100, and Mono-Q column chromatographies. The purified enzyme exists as a dimer and gives high activity toward L-proline-p-nitroanilide. The enzyme was strongly inhibited by p-chloromercuribenzoic acid and iodoacetic acid and markedly inhibited by phenylmethylsulfonyl fluoride and arphamenin A.     

红果参多糖的提取纯化及抗氧化活性研究

利用超声波辅助水提醇沉法提取并纯化红果参多糖.以抗坏血酸(VC)作参照物,考察了红果参多糖对羟基自由基、超氧阴离子自由基、亚硝基的清除效果及对油脂的抗氧化性能.结果表明:红果参多糖能很好的清除羟基自由基和超氧阴离子自由基,其清除能力与多糖浓度呈正相关性,对油脂的氧化有明显的抑制作用,对亚硝基的清除效果不明显.