麦芽根多糖的提取分离纯化及抗氧化研究
|
摘要
麦芽根是麦粒发芽后的须根,纤细而弯曲。麦芽根作为啤酒工业副产物约占大麦总投料的3%。麦芽根中含有氨基酸、蛋白质、碳水化合物、维生素和矿物质等多种元素和营养物质,目前,麦芽根在国内外主要作为饲料使用。本文以大麦麦芽根为原料,对其进行了基本营养成分分析,优化麦芽根多糖的提取工艺,并对其多糖进行分离纯化、纯度鉴定、理化性质、初级结构分析及生物活性等方面的研究。具体的研究内容及结果如下：
采用水提醇沉法提取麦芽根水溶性多糖,在单因素实验的基础上,通过正交试验研究了提取温度、提取时间及液料比对麦芽根多糖提取率的影响,从而确定了麦芽根多糖的最佳提取工艺参数：液料比40：1(V/W)、温度90℃、浸提3次、时间2h。
麦芽根水提取的粗多糖经脱蛋白、脱色、不同乙醇浓度沉淀后得到精制的麦芽根多糖BCP30、BCP50、BCP80。BCP80经过二乙氨基乙基(DEAE)纤维素柱层析,用0～1.0mol/L NaCl溶液洗脱,分别得到三个洗脱组分,浓缩、冷冻干燥后分别为白色粉末状的多糖BCPⅠ、BCPⅡ、BCPⅢ组分。BCPⅡ经Sephadex G-100柱层析得到两个对称峰BCPⅡa、BCPⅡb;BCPⅢ经Sephadex G-100层析分离得到一个组分为BCPⅢa。BCPⅡa、BCPⅡb和BCPⅢa经纸层析、醋酸纤维薄膜电泳和旋光法鉴定为均一组分。
麦芽根多糖纯品BCPⅡa、BCPⅡb、BCPⅢa为白色粉末状固体,可溶于水、稀酸碱,不溶于乙醇、丙酮等有机溶剂。苯酚-硫酸反应呈阳性,斐林试剂反应呈阴性,考马斯亮蓝染色反应呈阴性,碘—碘化钾反应呈阴性,Molish反应呈阳性,三氯化铁反应呈阴性。实验表明三种多糖是非还原性、不含多酚、无蛋白的多糖。经UV、IR和1H-NMR分析可知,三种多糖为β-糖苷键连接的吡喃环多糖。
采用水提旋蒸浓缩醇沉法提取并精制多糖,用50%和80%乙醇分段醇析分离得两种粗多糖BCP50和BCP80；用分光光度法研究了两种粗多糖在不同体系中对羟自由基、超氧自由基、DPPH自由基的清除作用、还原力以及用Rancimat油脂氧化仪测定其抗氧化活性。
上述的研究结果表明,分离纯化得到的麦芽根多糖为β-糖苷键吡喃环多糖。从麦芽根中在优化工艺条件下提取的水溶性多糖,具有一定的抗氧化活性,另外,研究麦芽根多糖的抗氧化活性为研究其免疫活性与抗肿瘤、抗突变、抗病毒等的研究提供理论基础。
Barley malt roots are fibrous roots of malt, slender and curved. Barley malt roots as a by-product of beer industry account for about 3% of total feed. Barley malt roots contain amino acids, proteins, carbohydrates, vitamins and minerals and other elements and nutrients. At present, Barley malt roots mainly used as feed all over the world. In this paper, barley malt roots as a raw material were systematic studied on the aspect of basic nutritional component, optimization of extraction of polysaccharides from malt roots, separation, purification, physical and chemical properties, primary structure and biological activity. The results are as follows:
Method of water extraction and alcohol precipitation was used for extracting water-soluble polysaccharide from malt roots. Orthogonal experiment based on the single factor experiments which were extraction temperature, extraction time and ratio of solid to liquid, on extraction rate of polysaccharides of malt roots. The optimum extraction parameters:liquid ratio 40:1 (V/W), temperature 90℃,extraction three times, time 2 hours.
The polysaccharides of malt roots BCP30, BCP50, BCP80were obtained by water extract, protein removal, decolorization, precipitation and refined with different concentration of ethanol. BCP80 was obtained after diethylamino ethyl (DEAE) cellulose column chromatography. Using 0 to 1.0 mol/L NaCl solution, elution, three fractions BCPⅠ,BCPⅡ,BCPⅢwere obtained. The three fractions were concentrated and freeze-dried, white powder were obtained. BCPⅡpurified by Sephadex G-100 column chromatography was obtained two symmetrical peaks BCPⅡa, BCPⅡb. BCPⅢpurified by Sephadex G-100 column chromatography was obtained a fraction named as BCPⅢa. By paper chromatography, cellulose acetate membrane electrophoresis and optical rotation, BCPⅡa, BCPⅡb and BCPⅢa were homogeneous fractions, respectively.
BCPⅡa, BCPⅡb, BCPⅢa are white powdery solid, soluble in water, dilute acid and alkali, insoluble in ethanol, acetone and other organic solvents. Phenol-sulfuric acid reaction was positive, negative Fehling reagent, Coomassie brilliant blue staining reaction was negative, iodine-potassium iodide reaction was negative, Molish positive for reaction, ferric chloride reaction was negative. Results show that the three fractions of polysaccharide are of non-reducing, non-polyphenol, protein-free polysaccharide. UV, IR and 1H-NMR analysis, the three fractions of polysaccharide areβ-glycosidic linkage of the pyran ring polysaccharide.
Rotary steam condensed by water extraction and alcohol precipitation of polysaccharide extracted and purified with 50% and 80% ethanol were two separate sub-analysis of alcohol polysaccharides BCP50 and BCP80,Spectrophotometry of the two polysaccharides in different systems on the hydroxyl radical, superoxide, DPPH radical scavenging, reducing power and lipid oxidation determined with the methord of Rancimat with witch antioxidant activity were measured.
The results show that the polysaccharides separated and purified from malt roots are beta glycosidic bond pyranoid ring polysaccharide. There exists antioxidant activity for malt extract extracted at optimization being water-soluble polysaccharide. And more, malt polysaccharide antioxidant activity offers the base for further studies of immune activity, antitumour, antimutation, antiviral. Therefore, the research on active, polysaccharides especially malt plays an important role and broad application prospect.
采用水提醇沉法提取麦芽根水溶性多糖,在单因素实验的基础上,通过正交试验研究了提取温度、提取时间及液料比对麦芽根多糖提取率的影响,从而确定了麦芽根多糖的最佳提取工艺参数：液料比40：1(V/W)、温度90℃、浸提3次、时间2h。
麦芽根水提取的粗多糖经脱蛋白、脱色、不同乙醇浓度沉淀后得到精制的麦芽根多糖BCP30、BCP50、BCP80。BCP80经过二乙氨基乙基(DEAE)纤维素柱层析,用0～1.0mol/L NaCl溶液洗脱,分别得到三个洗脱组分,浓缩、冷冻干燥后分别为白色粉末状的多糖BCPⅠ、BCPⅡ、BCPⅢ组分。BCPⅡ经Sephadex G-100柱层析得到两个对称峰BCPⅡa、BCPⅡb;BCPⅢ经Sephadex G-100层析分离得到一个组分为BCPⅢa。BCPⅡa、BCPⅡb和BCPⅢa经纸层析、醋酸纤维薄膜电泳和旋光法鉴定为均一组分。
麦芽根多糖纯品BCPⅡa、BCPⅡb、BCPⅢa为白色粉末状固体,可溶于水、稀酸碱,不溶于乙醇、丙酮等有机溶剂。苯酚-硫酸反应呈阳性,斐林试剂反应呈阴性,考马斯亮蓝染色反应呈阴性,碘—碘化钾反应呈阴性,Molish反应呈阳性,三氯化铁反应呈阴性。实验表明三种多糖是非还原性、不含多酚、无蛋白的多糖。经UV、IR和1H-NMR分析可知,三种多糖为β-糖苷键连接的吡喃环多糖。
采用水提旋蒸浓缩醇沉法提取并精制多糖,用50%和80%乙醇分段醇析分离得两种粗多糖BCP50和BCP80；用分光光度法研究了两种粗多糖在不同体系中对羟自由基、超氧自由基、DPPH自由基的清除作用、还原力以及用Rancimat油脂氧化仪测定其抗氧化活性。
上述的研究结果表明,分离纯化得到的麦芽根多糖为β-糖苷键吡喃环多糖。从麦芽根中在优化工艺条件下提取的水溶性多糖,具有一定的抗氧化活性,另外,研究麦芽根多糖的抗氧化活性为研究其免疫活性与抗肿瘤、抗突变、抗病毒等的研究提供理论基础。
Barley malt roots are fibrous roots of malt, slender and curved. Barley malt roots as a by-product of beer industry account for about 3% of total feed. Barley malt roots contain amino acids, proteins, carbohydrates, vitamins and minerals and other elements and nutrients. At present, Barley malt roots mainly used as feed all over the world. In this paper, barley malt roots as a raw material were systematic studied on the aspect of basic nutritional component, optimization of extraction of polysaccharides from malt roots, separation, purification, physical and chemical properties, primary structure and biological activity. The results are as follows:
Method of water extraction and alcohol precipitation was used for extracting water-soluble polysaccharide from malt roots. Orthogonal experiment based on the single factor experiments which were extraction temperature, extraction time and ratio of solid to liquid, on extraction rate of polysaccharides of malt roots. The optimum extraction parameters:liquid ratio 40:1 (V/W), temperature 90℃,extraction three times, time 2 hours.
The polysaccharides of malt roots BCP30, BCP50, BCP80were obtained by water extract, protein removal, decolorization, precipitation and refined with different concentration of ethanol. BCP80 was obtained after diethylamino ethyl (DEAE) cellulose column chromatography. Using 0 to 1.0 mol/L NaCl solution, elution, three fractions BCPⅠ,BCPⅡ,BCPⅢwere obtained. The three fractions were concentrated and freeze-dried, white powder were obtained. BCPⅡpurified by Sephadex G-100 column chromatography was obtained two symmetrical peaks BCPⅡa, BCPⅡb. BCPⅢpurified by Sephadex G-100 column chromatography was obtained a fraction named as BCPⅢa. By paper chromatography, cellulose acetate membrane electrophoresis and optical rotation, BCPⅡa, BCPⅡb and BCPⅢa were homogeneous fractions, respectively.
BCPⅡa, BCPⅡb, BCPⅢa are white powdery solid, soluble in water, dilute acid and alkali, insoluble in ethanol, acetone and other organic solvents. Phenol-sulfuric acid reaction was positive, negative Fehling reagent, Coomassie brilliant blue staining reaction was negative, iodine-potassium iodide reaction was negative, Molish positive for reaction, ferric chloride reaction was negative. Results show that the three fractions of polysaccharide are of non-reducing, non-polyphenol, protein-free polysaccharide. UV, IR and 1H-NMR analysis, the three fractions of polysaccharide areβ-glycosidic linkage of the pyran ring polysaccharide.
Rotary steam condensed by water extraction and alcohol precipitation of polysaccharide extracted and purified with 50% and 80% ethanol were two separate sub-analysis of alcohol polysaccharides BCP50 and BCP80,Spectrophotometry of the two polysaccharides in different systems on the hydroxyl radical, superoxide, DPPH radical scavenging, reducing power and lipid oxidation determined with the methord of Rancimat with witch antioxidant activity were measured.
The results show that the polysaccharides separated and purified from malt roots are beta glycosidic bond pyranoid ring polysaccharide. There exists antioxidant activity for malt extract extracted at optimization being water-soluble polysaccharide. And more, malt polysaccharide antioxidant activity offers the base for further studies of immune activity, antitumour, antimutation, antiviral. Therefore, the research on active, polysaccharides especially malt plays an important role and broad application prospect.
引文
[1]Sher M.J.,et al. Nat. Cancer Inst[R],1976,4:82.
[2]Whister R.L.,Bushway A.,Singh P.R.,et.al. Noncytotoxic antitumor polysaccharides[J].Adv. Carbodydr. Chem. Biochen.1976,32,235.
[3]Viegechthart JFC, et al. Abstract of the 12th International Carbohydrate Symposium. Netherlands[J].Vonk Publishers.1984:566.
[4]Franz G.Polysaccharides in pharmacology. Current application and future concepts [J].Plant Med,1998,55(6):493-497.
[5]孟宪元,邢连宗.茜草多糖的提取与分析[J].北京中医,2005,24(1)：35-36.
[6]赵宇,李志富,任少红,等.海蒿子多糖的提取方法研究[J].泰山医学院学报,2004,25(5)：429-430.
[7]白英,秦全仁,贺银凤.麦胚多糖提取工艺的研究[J].粮油加工与食品机械,2004,12：67-68.
[8]董彩霞,黄建华,祝勇,等.酶解法提取香菇多糖的探讨[J].光谱实验室,2005,22(5)：947-950.
[9]念保义,王铮敏,黄河宁.超声提取、超滤分离香菇多糖工艺的研究[J].化学与生物工程,2004,21(4)：16-18.
[10]Hayashi Katsuhiko. Preparation method of acidic polysaccharide[P].J.P.:2001,2: 2001-2008.
[11]AkiraY, Hiroshi N. M.,Taka S.B., et al. Structure Determination of Polysaccha--rides in Aloe arborescensvar[J].Planta Med,1986,3:213-218.
[12]李小平.红枣多糖提取工艺研究及其生物功能初探[D].西安：陕西师范大学,2004,27-29
[13]韦巍,李雪华.多糖的研究进展.国外医学药学分册[M].2005,32(3)：179-184.
[14]邱宏端,林娟,宋智晶,等.植物多糖的提取及对双歧杆菌的增殖作用[J].农业工程学报,2002,18(2)：96-100.
[15]李宏燕,樊君.大枣多糖的水提醇沉工艺研究[J].宁夏工程技术,2005,4(3)：265-267.
[16]周峙苗,邹玲玲,李国兴.羊栖菜多糖饮料的试制[J].食品工业科技,2001,22(3)：60-62
[17]李战.三种紫球藻培养、胞外多糖提取及RAP分析[D].上海：上海师范大学,2004.
[18]李军生,何仁,伍时华,等.超声波处理对提高南瓜多糖提取率的影响[J].农业科技通讯,2003,9：33-33.
[19]孟江.大枣多糖的化学研究及大枣渣多糖提取纯化的工艺研究[D].郑州：河南中医学院,2003,43-48
[20]王谦,黄猛,赵兵,等.气升式反应器超声破碎海带提取硫酸酯多糖[J].生物工程学报,2001,(1)：58-61.
[21]王航宇,刘金荣,江发涛,等.新疆甘草多糖的超声提取及含量测定[J].基层中药杂志,2002,16(1)：7-8.
[22]赵兵,王玉春.循环气升式超声破碎鼠尾藻提取海藻多糖[J].中国海洋药物杂志,1999,72(4)：19.
[23]王白军,王国卫.白芥子多糖的微波提取与含量测定[J].海峡药学,2005,17(1)：42-44.
[24]刘青梅,杨性民,邓红霞,等.采用微波技术提取紫菜多糖的工艺研究[J].农业工程学报,2005,21(2)：153-156.
[25]江和源,蒋迎.茶叶多糖的微波辅助提取技术研究[J].食品科技,2003,10：17-19.
[26]张海容,韩伟珍.微波法与传统热水法提取香菇多糖的比较研究[J].食品研究与开发,2005,26(5)：68-71.
[27]王建华,汪建民,李林,等.枸杞多糖LBP2a的分离、纯化与结构特征[J].食品科学,2002,3(6)：44-48.
[28]蒋秋燕.张振华,乔旭光.大蒜中性多糖的分离纯化(Ⅱ)[J]中国食品学报,2006,6(2)：55-58.
[29]刘海青,刘春兰,杨万政.沙棘叶水溶性多糖的提取及分离纯化[J].海南大学学报自然科学版,2005,23(4)：327-329.
[30]朱丹,牛广财,孙希云,等.马齿苋多糖POPⅢa的分离纯化及其结构特征[J].激光生物学报,2006,15(6)：624-627.
[31]苏彦珍,孙红月,胡奇芬.不同工艺路线制备生脉口服液的比较[J].中国药学杂志,1999,24(2)：93-95.
[32]郑宗坤,许贤华,陈志行,等.超滤提取香菇多糖的研究[J].中国生化药物杂志,2000,21(2)：73-75.
[33]白英.麦胚多糖的分离纯化及组成分析[J].食品科技,2005,4：86-87.
[34]魏传晚,曾和平,王晓娟,等.多糖及其研究进展简述[J].广东化工,2004,1：36-40.
[35]袁静明.胶层析法及其应用[M].科学出版社,1975.
[36]HAkan Bjorndal, Bengt Lindberg, Sigfrid Svensson. Mass spectrometry of partially methylated alditol acetates[J].Carbohydr Research,1967,5(4):433-440.
[37]江咏,李晓玺,李琳,等.多糖类亲和分离材料的研究进展[J].现代化工,2006,26(8)：19-24.
[38]高小荣,刘培勋.多糖结构分析研究进展[J].天津药学,2003,15(6)：67-70
[39]Xin D.,Kelvin K. C.,Hei W. L.,et.al. Fingerprint porfiling of acid hydrolyzates of polysaccharides extracted from fruiting bodies and spores of LingZhi by high-performance thin-layer chromatography[J].Journal of Chromatography A, 2003,1018(1):85-95.
[40]Xiaoming Q., Ryo Y, Koichi A.,et al. Structural features of arabinogalactan-proteins from the fruit of Lycium chinense Mill[J].Carbohydrate Research,2001,333:79-85.
[41]Zhaojing W., Dianhui L., Zhongyan L.Structure of polysaccharides from the fruiting body of Hericium erinaceus Pers[J].Carbohydrate Polymers,2004,57(3): 241-247.
[42]Christelle L., Jean-marc N., Bernard K.,et al. Hydrolysis of wheat bran and straw by an endoxylanase:Production and structural Characterization of cinnamoyl--oligosaccharides[J].Carbohdrate Research,1999,319(1-4):102-111.
[43]Siddhartha N.,Thomas W. E, John M. Swiader. Analysis of Mono-and Polysaccharides in Creeping Bentgrass Turf Using Near Infrared Reflectance Spectroscopy [J].Crop Science,2005,45(1):266-273.
[44]张丽萍,张翼伸.金顶侧耳多糖及其化学修饰产物水溶液构象的圆二色谱法测定[J].生物化学杂志,1994,10(5)：6-33.
[45]Robin E.B.L, Wei L., Delphi C.,et al. Rapid structural characterization of the arabinogalactan and lipoarabinomannan in live my-cobacterial cells using 2D and 3D HR-MAS NMR:structural changes in the arabinan due to ethambutol treatment and gene mutationare observed[J].Glycobiology,2005,5(2):139-151.
[46]Hakan Y, Andrej W., Goran W..Structural studies of the O-polysaccharide from the Escherichia coli O77 lipopolysaccharide[J].Carbohydrate Research,2001,333(2): 179-183.
[47]胡亚男,王义明,罗国安.质谱在糖分析中的应用[J].药学学报,2000,35(5)：397-400.
[48]田媛,张尊建.糖类结构的核磁共振波谱及质谱分析[J].药学进展,2003,27(2)：87-90.
[49]王宏钧，张惠苓,卢葛章.二维核磁共振谱解析及其应用[J].光谱实验室,1994,04：3-82.
[50]王顺春,方积年.香菇多糖硫酸化衍生物的制备及其结构分析[J].生物化学与生物物理学报,1999,31(5)：594-597.
[51]鲍幸峰,方积年.原子力显微镜在生物大分子结构中的应用进展[J].分析化学,2000,28(10)：1300-1307.
[52]孙润广,张静.甘草多糖螺旋结构的原子力显微镜研究[J].化学学报,2006,24：2467-2472.
[53]梁忠岩,倪秀珍.中草药多糖的应用研究[J].长春师范学院学报,2001,5：38-41.
[54]孔庆胜,王彦英.南瓜多糖的分离、纯化及其降血脂作用[J].中国生化药物杂志,2000,21(3)：130-132.
[55]刘青,李新芳.南沙参多糖对受照射小鼠遗传损伤的拮抗作用[J].中药药理与临床,2001,17(6)：21-22.
[56]王惠燕.党参多糖对小鼠细胞免疫的调节作用[J].中国药理学通讯,1989,5(6)：276-278.
[57]齐德生,于炎湖.黑木耳多糖抗肿瘤作用的实验研究[J].华中农业大学学报,1994,13(2)：160-163.
[58]孟运莲,蔡丽华,吴慧芬,等.化学修饰的茯苓多糖抗肿瘤效应的免疫组织化学观察[J].武汉大学学报(医学版),2007,1(28)：67-69.
[59]徐文清,高文远,沈秀.银耳孢子多糖抑制肿瘤及放射增效作用的研究[J].中国生化药物杂志,2006,6(27)：351-353.
[60]陈地龙,刘永刚,王亚平,等.人参多糖对K562细胞增殖抑制及诱导分化的实验研究[J].第三军医大学学报,2005,6(27)：517-520.
[61]陈西广，刘万顺,刘晨光.几丁质的研究与进展[J].生物工程进展,1997,17(3)：5-9.
[62]黄建设,龙丽娟,张儡.海洋天然产物及其生理活性的研究进展[J].海洋通报,2001,4：13-21.
[63]张艺,杨明.日研究中药多糖的新进展[J].中医药信息杂志,1997,4(5)：39-40.
[64]R. S.BHATTY.Hull-less barley bran:a potential new product from an old grain[J]. Cereal Food world,1995,40(11):819-912.
[65]赵启美,何佳,李西波.微生物转化麦芽根为蛋白饲料的研究[J].中国酿造,2001,03：23-24.
[66]段作营,刘军昌,于瑞嵩,等.麦芽根中5’-磷酸二酯酶的提取及制备[J].食品与发酵工业,2002,28(6)：5-8.
[67]王肇慈.粮油食品品质分析(第二版)[M].北京：中国轻工业出版社,2000.
[68]Eric W., Roger A., Per A.. Ioslation and chemical characterization of water soluble mixed-linked β-glucans and arabionxylans in oat milling fraction[J]. Carbohydrate Polymer.1993,20:15-123.
[69]Michel Dubois, K.A. Gilles, J.K. Hamilton, et.al. Colorimetric methord for determination of sugar and related substances[J]. Analytical Chemistry, 1956,28(3):350-356.
[70]张惟杰.糖类复合物生化研究技术[M],浙江大学出版社,1994,16-17.
[71]肖桂武,曾和平.龙葵多糖的分离、纯化和鉴定(Ⅱ)[J].中草药,2000,31：162-164.
[72]汪东风,谢晓凤,严峻,等.茶多糖等有效成分综合提取研究[J].中草药,1998,29：739-740.
[73]Marion M. Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254.
[74]Fukuda K. Uematsu T. Hamada A., et.al The polysaccharide from Lampteromyces japonicus[J].Chem.Pharm. Bull(Tokyo),1975,23(9):1955-9.
[75]徐大伦,欧昌荣,黄晓春,等.浒苔多糖脱蛋白方法的研究[J].水产科学,2005,24(5)：26-27.
[76]付学鹏.植物多糖脱色技术的研究[J].食品研究与开发,2007,28(11)：166-169
[77]向东,王国政,赖凤英,等.活性炭对南瓜粗多糖液的脱色研究[J].河南科学,2004,22(6)：780-783.
[78]黄文书,杨海燕,李焕荣,等.枸杞多糖的脱色工艺[J].食品研究与开发,2008,29(3)：95-98.
[79]夏玮,吕庆,张文清,等.大孔吸附树脂脱色桑叶多糖的研究[J].食品与发酵工业,2007,33(2)：141-144.
[80]张文雄,梁宏,秦海错,等.螺旋藻酸性杂多糖的分类纯化和分析[J].中草药,2006,3(15)：326-328.
[81]张惟杰.多糖复合物生化技术[M].上海：上海科技出版社.1987,65-68,110,209-212.
[82]汉丽萍,梁宗岩,张丽萍,等.高山红景天多糖RSA的分离纯化和组成分析[J].中国药学杂志,2003,37(6)：418-421.
[83]韩亚珊.食品化学实验指导[M].北京：北京农业大学出版社,1992：16-19.
[84]王卫国,吴强,胡保坤,等.几种测定灰树花多糖中蛋白质含量方法的比较研究[J].中国食用菌,2003,22(1)：27-30.
[85]王爱军,王风山,王友联,等.低浓度蛋白质含量测定方法的比较[J].中国生化药物杂志,2003,24(2)：8-80.
[86]张部昌,吴东儒.天门冬多糖的分离纯化及其部分理化性质[J].安徽大学学报(自然科学版),1994,18(2)：88-94.
[87]Parker F.S.. Application of infrared, Raman and resonance Raman spectroscopy in biochemistry[J].Journal of Molecular Structure,1985,128(4):366.
[88]Sekkal M., Huvenne J.P.,Legrand P., et al..Direct Structural identification of polysaccharides from red algae by FTIR microspectrometry(Ⅰ):localization of agar in gracilatia Verrucosa ections[J].Mikrochim Acta.,1993,112:110.
[89]Wang Z.,Fang J.N..Application of nuclear magnetic resonance spectroscope in the structural determination of polysacchadde[J].Chinese Journal Analytical Chemistry, 2000,28:240-247.
[90]黄芳,蒙义文.活性多糖的研究进展[J].天然产物研究与开发,1999,11(5)：90-98.
[91]王庭欣,朱惠民,周明河.植物多糖抑瘤活性的研究进展[J].癌变·畸变·突变,2000,12(2)：118-120.
[92]Jianhong Yang, Yumin Du,Ronghua Huang, et al. Chemical modification and antitumour activity of Chinese lacquer polysaccharide from lac tree Rhus vemicifera[J].Carbohydrate Polymers,2005,59(1):101-107.
[93]李志孝,黄成钢,蔡育军,等.天门冬多糖的化学结构及体外抗氧化活性[J].药学学报,2000,35(5)：358-362.
[94]张振明,葛斌,许爱霞,等.女贞子多糖的抗衰老作用[J].中国药理学与毒理学杂志,2006,20(2)：108-111.
[95]周林珠,杨祥良,周井炎,等.多糖抗氧化作用研究进展[J].中国生化药物杂志,2002,23(4)：210-212.
[96]A Kumaran,R Joel karunakaran. Antioxidant and free radical scavenging activity of an aqueous extract of Coleus aromaticus[J].Food Chemistry,1997,(1):109-114.
[97]王雅,赵萍,赵坤,等.沙米绿原酸提取工艺优化及抗氧化性能研究[J]食品与发酵工业,2007,(10)：131-134.
[98]赵萍,王雅,张轶,等.葵花籽粕乙醇提取物的抗氧化活性的研究[J].食品科学,2007,(10)：219-222.
[99]王建华,张民,甘璐,等.构杞多糖的抗羟基自由基氧化作用[J].食品科学,2001,(1)：11-12.
[100]Smirnoff N., Q. Cumbes. Hyroxyl radicals cavenging activity of compatible solutes[J].Photochemistry,1989,28(4):1051-1056.
[101]魏文树,谭建权.云芝多糖对活性氧清除机制的增强作用[J].中国药学杂志,1997,4(23)：199-201.
[2]Whister R.L.,Bushway A.,Singh P.R.,et.al. Noncytotoxic antitumor polysaccharides[J].Adv. Carbodydr. Chem. Biochen.1976,32,235.
[3]Viegechthart JFC, et al. Abstract of the 12th International Carbohydrate Symposium. Netherlands[J].Vonk Publishers.1984:566.
[4]Franz G.Polysaccharides in pharmacology. Current application and future concepts [J].Plant Med,1998,55(6):493-497.
[5]孟宪元,邢连宗.茜草多糖的提取与分析[J].北京中医,2005,24(1)：35-36.
[6]赵宇,李志富,任少红,等.海蒿子多糖的提取方法研究[J].泰山医学院学报,2004,25(5)：429-430.
[7]白英,秦全仁,贺银凤.麦胚多糖提取工艺的研究[J].粮油加工与食品机械,2004,12：67-68.
[8]董彩霞,黄建华,祝勇,等.酶解法提取香菇多糖的探讨[J].光谱实验室,2005,22(5)：947-950.
[9]念保义,王铮敏,黄河宁.超声提取、超滤分离香菇多糖工艺的研究[J].化学与生物工程,2004,21(4)：16-18.
[10]Hayashi Katsuhiko. Preparation method of acidic polysaccharide[P].J.P.:2001,2: 2001-2008.
[11]AkiraY, Hiroshi N. M.,Taka S.B., et al. Structure Determination of Polysaccha--rides in Aloe arborescensvar[J].Planta Med,1986,3:213-218.
[12]李小平.红枣多糖提取工艺研究及其生物功能初探[D].西安：陕西师范大学,2004,27-29
[13]韦巍,李雪华.多糖的研究进展.国外医学药学分册[M].2005,32(3)：179-184.
[14]邱宏端,林娟,宋智晶,等.植物多糖的提取及对双歧杆菌的增殖作用[J].农业工程学报,2002,18(2)：96-100.
[15]李宏燕,樊君.大枣多糖的水提醇沉工艺研究[J].宁夏工程技术,2005,4(3)：265-267.
[16]周峙苗,邹玲玲,李国兴.羊栖菜多糖饮料的试制[J].食品工业科技,2001,22(3)：60-62
[17]李战.三种紫球藻培养、胞外多糖提取及RAP分析[D].上海：上海师范大学,2004.
[18]李军生,何仁,伍时华,等.超声波处理对提高南瓜多糖提取率的影响[J].农业科技通讯,2003,9：33-33.
[19]孟江.大枣多糖的化学研究及大枣渣多糖提取纯化的工艺研究[D].郑州：河南中医学院,2003,43-48
[20]王谦,黄猛,赵兵,等.气升式反应器超声破碎海带提取硫酸酯多糖[J].生物工程学报,2001,(1)：58-61.
[21]王航宇,刘金荣,江发涛,等.新疆甘草多糖的超声提取及含量测定[J].基层中药杂志,2002,16(1)：7-8.
[22]赵兵,王玉春.循环气升式超声破碎鼠尾藻提取海藻多糖[J].中国海洋药物杂志,1999,72(4)：19.
[23]王白军,王国卫.白芥子多糖的微波提取与含量测定[J].海峡药学,2005,17(1)：42-44.
[24]刘青梅,杨性民,邓红霞,等.采用微波技术提取紫菜多糖的工艺研究[J].农业工程学报,2005,21(2)：153-156.
[25]江和源,蒋迎.茶叶多糖的微波辅助提取技术研究[J].食品科技,2003,10：17-19.
[26]张海容,韩伟珍.微波法与传统热水法提取香菇多糖的比较研究[J].食品研究与开发,2005,26(5)：68-71.
[27]王建华,汪建民,李林,等.枸杞多糖LBP2a的分离、纯化与结构特征[J].食品科学,2002,3(6)：44-48.
[28]蒋秋燕.张振华,乔旭光.大蒜中性多糖的分离纯化(Ⅱ)[J]中国食品学报,2006,6(2)：55-58.
[29]刘海青,刘春兰,杨万政.沙棘叶水溶性多糖的提取及分离纯化[J].海南大学学报自然科学版,2005,23(4)：327-329.
[30]朱丹,牛广财,孙希云,等.马齿苋多糖POPⅢa的分离纯化及其结构特征[J].激光生物学报,2006,15(6)：624-627.
[31]苏彦珍,孙红月,胡奇芬.不同工艺路线制备生脉口服液的比较[J].中国药学杂志,1999,24(2)：93-95.
[32]郑宗坤,许贤华,陈志行,等.超滤提取香菇多糖的研究[J].中国生化药物杂志,2000,21(2)：73-75.
[33]白英.麦胚多糖的分离纯化及组成分析[J].食品科技,2005,4：86-87.
[34]魏传晚,曾和平,王晓娟,等.多糖及其研究进展简述[J].广东化工,2004,1：36-40.
[35]袁静明.胶层析法及其应用[M].科学出版社,1975.
[36]HAkan Bjorndal, Bengt Lindberg, Sigfrid Svensson. Mass spectrometry of partially methylated alditol acetates[J].Carbohydr Research,1967,5(4):433-440.
[37]江咏,李晓玺,李琳,等.多糖类亲和分离材料的研究进展[J].现代化工,2006,26(8)：19-24.
[38]高小荣,刘培勋.多糖结构分析研究进展[J].天津药学,2003,15(6)：67-70
[39]Xin D.,Kelvin K. C.,Hei W. L.,et.al. Fingerprint porfiling of acid hydrolyzates of polysaccharides extracted from fruiting bodies and spores of LingZhi by high-performance thin-layer chromatography[J].Journal of Chromatography A, 2003,1018(1):85-95.
[40]Xiaoming Q., Ryo Y, Koichi A.,et al. Structural features of arabinogalactan-proteins from the fruit of Lycium chinense Mill[J].Carbohydrate Research,2001,333:79-85.
[41]Zhaojing W., Dianhui L., Zhongyan L.Structure of polysaccharides from the fruiting body of Hericium erinaceus Pers[J].Carbohydrate Polymers,2004,57(3): 241-247.
[42]Christelle L., Jean-marc N., Bernard K.,et al. Hydrolysis of wheat bran and straw by an endoxylanase:Production and structural Characterization of cinnamoyl--oligosaccharides[J].Carbohdrate Research,1999,319(1-4):102-111.
[43]Siddhartha N.,Thomas W. E, John M. Swiader. Analysis of Mono-and Polysaccharides in Creeping Bentgrass Turf Using Near Infrared Reflectance Spectroscopy [J].Crop Science,2005,45(1):266-273.
[44]张丽萍,张翼伸.金顶侧耳多糖及其化学修饰产物水溶液构象的圆二色谱法测定[J].生物化学杂志,1994,10(5)：6-33.
[45]Robin E.B.L, Wei L., Delphi C.,et al. Rapid structural characterization of the arabinogalactan and lipoarabinomannan in live my-cobacterial cells using 2D and 3D HR-MAS NMR:structural changes in the arabinan due to ethambutol treatment and gene mutationare observed[J].Glycobiology,2005,5(2):139-151.
[46]Hakan Y, Andrej W., Goran W..Structural studies of the O-polysaccharide from the Escherichia coli O77 lipopolysaccharide[J].Carbohydrate Research,2001,333(2): 179-183.
[47]胡亚男,王义明,罗国安.质谱在糖分析中的应用[J].药学学报,2000,35(5)：397-400.
[48]田媛,张尊建.糖类结构的核磁共振波谱及质谱分析[J].药学进展,2003,27(2)：87-90.
[49]王宏钧，张惠苓,卢葛章.二维核磁共振谱解析及其应用[J].光谱实验室,1994,04：3-82.
[50]王顺春,方积年.香菇多糖硫酸化衍生物的制备及其结构分析[J].生物化学与生物物理学报,1999,31(5)：594-597.
[51]鲍幸峰,方积年.原子力显微镜在生物大分子结构中的应用进展[J].分析化学,2000,28(10)：1300-1307.
[52]孙润广,张静.甘草多糖螺旋结构的原子力显微镜研究[J].化学学报,2006,24：2467-2472.
[53]梁忠岩,倪秀珍.中草药多糖的应用研究[J].长春师范学院学报,2001,5：38-41.
[54]孔庆胜,王彦英.南瓜多糖的分离、纯化及其降血脂作用[J].中国生化药物杂志,2000,21(3)：130-132.
[55]刘青,李新芳.南沙参多糖对受照射小鼠遗传损伤的拮抗作用[J].中药药理与临床,2001,17(6)：21-22.
[56]王惠燕.党参多糖对小鼠细胞免疫的调节作用[J].中国药理学通讯,1989,5(6)：276-278.
[57]齐德生,于炎湖.黑木耳多糖抗肿瘤作用的实验研究[J].华中农业大学学报,1994,13(2)：160-163.
[58]孟运莲,蔡丽华,吴慧芬,等.化学修饰的茯苓多糖抗肿瘤效应的免疫组织化学观察[J].武汉大学学报(医学版),2007,1(28)：67-69.
[59]徐文清,高文远,沈秀.银耳孢子多糖抑制肿瘤及放射增效作用的研究[J].中国生化药物杂志,2006,6(27)：351-353.
[60]陈地龙,刘永刚,王亚平,等.人参多糖对K562细胞增殖抑制及诱导分化的实验研究[J].第三军医大学学报,2005,6(27)：517-520.
[61]陈西广，刘万顺,刘晨光.几丁质的研究与进展[J].生物工程进展,1997,17(3)：5-9.
[62]黄建设,龙丽娟,张儡.海洋天然产物及其生理活性的研究进展[J].海洋通报,2001,4：13-21.
[63]张艺,杨明.日研究中药多糖的新进展[J].中医药信息杂志,1997,4(5)：39-40.
[64]R. S.BHATTY.Hull-less barley bran:a potential new product from an old grain[J]. Cereal Food world,1995,40(11):819-912.
[65]赵启美,何佳,李西波.微生物转化麦芽根为蛋白饲料的研究[J].中国酿造,2001,03：23-24.
[66]段作营,刘军昌,于瑞嵩,等.麦芽根中5’-磷酸二酯酶的提取及制备[J].食品与发酵工业,2002,28(6)：5-8.
[67]王肇慈.粮油食品品质分析(第二版)[M].北京：中国轻工业出版社,2000.
[68]Eric W., Roger A., Per A.. Ioslation and chemical characterization of water soluble mixed-linked β-glucans and arabionxylans in oat milling fraction[J]. Carbohydrate Polymer.1993,20:15-123.
[69]Michel Dubois, K.A. Gilles, J.K. Hamilton, et.al. Colorimetric methord for determination of sugar and related substances[J]. Analytical Chemistry, 1956,28(3):350-356.
[70]张惟杰.糖类复合物生化研究技术[M],浙江大学出版社,1994,16-17.
[71]肖桂武,曾和平.龙葵多糖的分离、纯化和鉴定(Ⅱ)[J].中草药,2000,31：162-164.
[72]汪东风,谢晓凤,严峻,等.茶多糖等有效成分综合提取研究[J].中草药,1998,29：739-740.
[73]Marion M. Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Analytical Biochemistry,1976,72:248-254.
[74]Fukuda K. Uematsu T. Hamada A., et.al The polysaccharide from Lampteromyces japonicus[J].Chem.Pharm. Bull(Tokyo),1975,23(9):1955-9.
[75]徐大伦,欧昌荣,黄晓春,等.浒苔多糖脱蛋白方法的研究[J].水产科学,2005,24(5)：26-27.
[76]付学鹏.植物多糖脱色技术的研究[J].食品研究与开发,2007,28(11)：166-169
[77]向东,王国政,赖凤英,等.活性炭对南瓜粗多糖液的脱色研究[J].河南科学,2004,22(6)：780-783.
[78]黄文书,杨海燕,李焕荣,等.枸杞多糖的脱色工艺[J].食品研究与开发,2008,29(3)：95-98.
[79]夏玮,吕庆,张文清,等.大孔吸附树脂脱色桑叶多糖的研究[J].食品与发酵工业,2007,33(2)：141-144.
[80]张文雄,梁宏,秦海错,等.螺旋藻酸性杂多糖的分类纯化和分析[J].中草药,2006,3(15)：326-328.
[81]张惟杰.多糖复合物生化技术[M].上海：上海科技出版社.1987,65-68,110,209-212.
[82]汉丽萍,梁宗岩,张丽萍,等.高山红景天多糖RSA的分离纯化和组成分析[J].中国药学杂志,2003,37(6)：418-421.
[83]韩亚珊.食品化学实验指导[M].北京：北京农业大学出版社,1992：16-19.
[84]王卫国,吴强,胡保坤,等.几种测定灰树花多糖中蛋白质含量方法的比较研究[J].中国食用菌,2003,22(1)：27-30.
[85]王爱军,王风山,王友联,等.低浓度蛋白质含量测定方法的比较[J].中国生化药物杂志,2003,24(2)：8-80.
[86]张部昌,吴东儒.天门冬多糖的分离纯化及其部分理化性质[J].安徽大学学报(自然科学版),1994,18(2)：88-94.
[87]Parker F.S.. Application of infrared, Raman and resonance Raman spectroscopy in biochemistry[J].Journal of Molecular Structure,1985,128(4):366.
[88]Sekkal M., Huvenne J.P.,Legrand P., et al..Direct Structural identification of polysaccharides from red algae by FTIR microspectrometry(Ⅰ):localization of agar in gracilatia Verrucosa ections[J].Mikrochim Acta.,1993,112:110.
[89]Wang Z.,Fang J.N..Application of nuclear magnetic resonance spectroscope in the structural determination of polysacchadde[J].Chinese Journal Analytical Chemistry, 2000,28:240-247.
[90]黄芳,蒙义文.活性多糖的研究进展[J].天然产物研究与开发,1999,11(5)：90-98.
[91]王庭欣,朱惠民,周明河.植物多糖抑瘤活性的研究进展[J].癌变·畸变·突变,2000,12(2)：118-120.
[92]Jianhong Yang, Yumin Du,Ronghua Huang, et al. Chemical modification and antitumour activity of Chinese lacquer polysaccharide from lac tree Rhus vemicifera[J].Carbohydrate Polymers,2005,59(1):101-107.
[93]李志孝,黄成钢,蔡育军,等.天门冬多糖的化学结构及体外抗氧化活性[J].药学学报,2000,35(5)：358-362.
[94]张振明,葛斌,许爱霞,等.女贞子多糖的抗衰老作用[J].中国药理学与毒理学杂志,2006,20(2)：108-111.
[95]周林珠,杨祥良,周井炎,等.多糖抗氧化作用研究进展[J].中国生化药物杂志,2002,23(4)：210-212.
[96]A Kumaran,R Joel karunakaran. Antioxidant and free radical scavenging activity of an aqueous extract of Coleus aromaticus[J].Food Chemistry,1997,(1):109-114.
[97]王雅,赵萍,赵坤,等.沙米绿原酸提取工艺优化及抗氧化性能研究[J]食品与发酵工业,2007,(10)：131-134.
[98]赵萍,王雅,张轶,等.葵花籽粕乙醇提取物的抗氧化活性的研究[J].食品科学,2007,(10)：219-222.
[99]王建华,张民,甘璐,等.构杞多糖的抗羟基自由基氧化作用[J].食品科学,2001,(1)：11-12.
[100]Smirnoff N., Q. Cumbes. Hyroxyl radicals cavenging activity of compatible solutes[J].Photochemistry,1989,28(4):1051-1056.
[101]魏文树,谭建权.云芝多糖对活性氧清除机制的增强作用[J].中国药学杂志,1997,4(23)：199-201.