雪莲果中糖类成分分析及低聚果糖制备工艺路线初探

【作者】 武莹；

【导师】 马长华；

【作者基本信息】 北京中医药大学 ， 中医学， 2008， 硕士

【摘要】 雪莲果(Smallanthus sonchifolius [Poepp. & Endl.] H. Robinson),又名亚贡(Yacon),为菊科(Compositae or Asteraceae)植物家族的一员,原产于南美洲安第斯山,是生活在安第斯山高山地区的土著印弟安人传统的块茎食品。雪莲果干物质(Dry matter,DM)的主要成分为低聚果糖,在南美洲,常被作为糖尿病病人、各种消化功能或肾功能紊乱病人的保健食品。近年来,雪莲果在我国云南省引种成功。本研究以雪莲果为研究对象,借助高效薄层层析(HPTLC)技术和液相色谱-质谱(LC- MS)联用技术对我国云南产雪莲果中糖类成分进行定性检测,运用苯酚硫酸法和高效液相-示差折光法(HPLC-RI)对雪莲果中的糖类成分进行了定量分析,运用上述建立的方法对雪莲果中糖类成分的提取和初步纯化工艺进行了考查,并制备得到了雪莲果低聚果糖粗品。第一部分分别采用薄层层析法和液质联用技术对雪莲果中的主要成分——糖类成分,包括果糖、葡萄糖、蔗糖、蔗果低聚糖(GF2-GF9)进行了定性鉴别。其中,薄层层析采用高效硅胶薄层板、冰醋酸:氯仿:无水乙醇:水=9:11:11:2(v/v)为展开剂、苯胺-二苯胺-磷酸显色剂;液质联用定性分析采用Agilent LC– MSD Trap XCT Plus液相色谱-紫外-离子阱质谱联用仪,ΜltimateTMXB-NH2(5μm,4.6mm×250mm)分析柱,流动相为乙腈:水=60:40(v/v),柱温40℃。结果显示,目标待测物均得到了较好的分离。第二部分建立了苯酚-硫酸比色法和高效液相-示差折光法测定雪莲果中糖类成分含量的分析方法。苯酚-硫酸比色法是测定各种糖类成分含量的简单、有效的方法。低聚糖在浓硫酸作用下,先水解成单糖,并迅速脱水生成糖醛衍生物,与苯酚反应生成橙黄色溶液,此溶液在适当波长处(490nm)有特征吸收,实验中得到苯酚-硫酸法在0.04mg～0.24mg线性关系良好。高效液相色谱法采用ΜltimateTMXB-NH2(5μm,4.6mm×250mm)分析柱,Waters R401型示差折光检测器,流动相乙腈:水=85:15(v/v)和乙腈:水=70:30(v/v),柱温35℃,等度洗脱,分别测定了单糖(果糖,葡萄糖)和低聚糖(蔗糖,蔗果三糖,蔗果四糖,蔗果五糖),得到了六种糖的回归方程,果糖在0.9~16.2mg/mL,葡萄糖在0.6~10.8mg/mL,蔗糖在0.6mg/mL~ 8mg/mL,蔗果三糖在0.45~6mg/mL,蔗果四糖在0.45~6mg/mL,蔗果五糖在0.27~ 3.6mg/mL分别呈良好的线性关系。实验还对流动相的有机相比例和柱温两大影响因素做了考查。最后一部分对雪莲果中干物质占鲜重百分比进行了测定,运用前述已建立的方法,以总糖回收率和澄清度为指标考查了壳聚糖絮凝法对雪莲果提取液澄清作用,将实验所得到的最优条件制备的雪莲果提取澄清液进行高效液相进样分析,分别计算了果糖、葡萄糖、蔗糖、蔗果三糖、蔗果四糖、蔗果五糖的转移率,并经过絮凝、冻干得到了GF2~GF4含量>20%的低聚果糖粗品。更多还原

【Abstract】 Yacon (Smallanthus sonchifolius [Poepp. & Endl.] H. Robinson) is a member of the Asteraceae and is an under- exploited native tuber crop which constitutes part of the diet of Indian tribes living in the high mountain areas of the Andes.Yacon stores oligofructans (fructo-,oligo-saccharides, FOS) as its main component of dry matter (DM) and is commonly consumed by people suffering from diabetes or from various digestive or renal disorders. Recently,yacon has been successfully introduced in Yun Nan Province.In this reserch,suger components were identified by high performance thin layer chromatography(HPTLC) and liquid chromatography-mass spectrometry (LC-MS). The suger components in yacon were determined using phenol-H2SO4 method and detected by UV detector.High performance liquid chromatography- differential refractive index detector(HPLC-RID) was used for the assay of suger components in yacon.At last a pragmatic method had been established to produce coarse yacon oligofructans via several procedures.In the first part,HPTLC and LC-MS were used for the identification of eleven suger components including fructose,glucose,sucrose,GF2~GF9 in yacon.We used high performance silica gel plate,the mobile phase was acetic acid-chloroform- dried ethanol-water(9:11:11:2,v/v), used amino benzene-phenylene diamine- phosphoric acid as chromogenic agent; Agilent LC - MSD Trap XCT Plus system and the mobile phase acetonitrile-10mM aqueous ammonium acetate(60:40,v/v) was used, Chromatography was performed at a constant temperature of 40℃on a UltimateTMXB-NH2 column for LC-MS detection.The result indicated that most suger components were well separated.In the second part, the suger components in yacon were determined using phenol-H2SO4 method.Oligosaccharides are hydrolyzed in H2SO4 into mono- saccharides,then quickly dehydrate and produce an orange-yellow color under the interaction with phenol,this solution has an especial absorption at 490nm.A fine linear correlation was get from 0.04mg to 0.24mg in the experiment. UltimateTM XB-NH2(5μm,4.6mm×250mm)column and Waters R401 differential refractive index detector were used in HPLC method. Chromatography was performed at 35℃, we used acetonitrile-water(85:15,v/v)for the monosaccharides ( fructose , glucose ) separation and acetonitrile - water ( 70:30 , v/v ) for the oligosaccharides (sucrose,1-kestose,nystose,1F- Fructofuranosylnystose) separation, six regression equations were acquired for quantitative determination,the six kind of suger components had a well liner correlation in the range of 0.9~16.2mg/mL(fructose),0.6~10.8mg/mL(glucose), 0.6mg/mL~ 8mg/mL (sucrose), 0.45~6mg/mL(1-kestose, nystose) and 0.27~ 3.6mg/mL (1F-Fructofuranosylnystose) separately. The experiment also examined the two main influencing factors:organic solvent ratio of the mobile phase and the column temperature. We also figured out the DM ratio in the fresh yacon tubers in this part.In the last part, Using chitosan as flocculant tranference of saccharide and transmittance at 680nm as two indexes,we examined the effect of flocculation of chitosan in yacon extracts and figured out the tranference of the six saccharides separately. The coarse yacon oligofructans we produced contained more than 20% oligofuctans (GF2~GF4).更多还原