摘要我国豌豆(Pisum sativum)资源丰富,豌豆在食品、医药等领域具有重要的加工利用价值,近年来对其功能成份、活性物质的研究逐渐成为热点。本研究以豌豆粉为原料进行多糖超声辅助提取,采用硫酸苯酚法测定多糖含量,其多糖得率为7.26%。粗多糖经不同程度纯化处理,运用扫描电子显微镜对得到的豌豆可溶性多糖超微形貌进行观察,按照多糖不同纯化程度,将待考察的多糖种类分为:粗多糖(crude polysaccharides of pea, CPP)、脱蛋白多糖(deproteinization of polysaccharides, TPP)、DEAE Fast Flow离子交换柱层析多糖(水洗脱多糖(water elution -diethylaminoethyl fast flow- ion exchange column chromatography, W-DEPP); NaCl洗脱多糖(NaCl elution -diethylaminoethyl fast flow- ion exchange column chromatography, N-DEPP)、葡聚糖G-100凝胶柱层析多糖(water elution-G-100 sephadex gel chromatography, W-DE-GPP-a, W-DE-GPP-b; NaCl Elution-G-100 sephadex gel chromatography, N-DE-GPP1-a, N-DE-GPP1-b)四大类。通过对电镜扫描观察倍数进行调整,根据所得到的扫描电镜图对每个多糖的形貌特征从3个清晰度(×100, ×1000和×10000)进行分析和总结,解释了不同纯化程度下多糖颗粒超微结构的变化情况。本研究初步形成不同纯化程度的豌豆可溶性多糖形貌电镜照片,为豌豆多糖形貌特征及后续结构特性研究提供基础理论参考。
Abstract:China is abundant in peas (Pisum sativum), which have important processing and utilization value in food, medicine and other fields. In recent years, researches on its functional components and bioactive substances have become popular gradually. In this study, pea meal as raw material for ultrasound-assisted extraction was used to get the crude soluble polysaccharide, and the yeild of polysaccharide, measured by the method of phenol-sulfate, was 7.26%. The ultrastructural morphology of the soluble polysaccharides of pea with different purification levels was analyzed by scanning electron microscopy (SEM). By using different level of purification methods, the crude soluble polysaccharides extracted from pea were classified into 4, as follows, crude polysaccharide (CPP), deproteined polysaccharide (TPP), DEAE-sepharose Fast Flow ion-exchange chromatography polysaccharide (W-DEPP (water elution -diethylaminoethyl fast flow- ion exchange column chromatography), N-DEPP (NaCl elution -diethylaminoethyl fast flow- ion exchange column chromatography)) and sephadex G-100 chromatography polysaccharide (W-DE-GPP-a, W-DE-GPP-b, N-DE-GPP1-a, N-DE-GPP1-b). As the results, ultrastructural morphology observations were analyzed and summarized according to the characteristics of ultrastructural morphology of these different polysaccharides by changing three definitions, which were 100 fold, 1 000 fold and 10 000 fold, respectively. In this study, the soluble polysaccharides of pea with different purification levels were investigated by SEM, from which a series of photos were obtained preliminarily. The results of this study provide a reliable reference for the morphological characteristics and further studies of pea polysaccharides.
张淑杰, 权威, 姜宏伟, 康玉凡. 不同纯化程度豌豆水溶性多糖的电镜扫描分析[J]. 农业生物技术学报, 2019, 27(10): 1822-1830.
ZHANG Shu-Jie, QUAN Wei, JIANG Hong-Wei, KANG Yu-Fan. Analysis of the Polysaccharides with Different Purification Levels in Pea (Pisum sativum) by Scanning Electron Microscope. 农业生物技术学报, 2019, 27(10): 1822-1830.
1 程浩然. 2015. 淫羊藿多糖的分离纯化和结构鉴定及生物活性研究 [D]. 硕士学位论文, 四川农业大学, 导师: 丁春邦, pp: 25 - 27. (Cheng H R. 2015. Extraction, structural characterization and biological activities of polysaccharides from Epimedium acuminatum[D]. Thesis for M.S., Sichuan Agricultural University, Supervisor: Ding C B, pp: 25-27.) 2 崔再兴, 李玲. 2010. 豌豆的特征特性及开发利用价值[J]. 杂粮作物, 30(2): 154-155. (Cui Z X, Li L.2010. The utilization value of features and developments in pea[J]. Rain Fed Crops, 30(2): 154-155.) 3 代晶. 2016. 笃斯越橘(Vaccinium uliginosum L.)果实多糖的提取纯化、结构鉴定及生物活性研究[D]. 硕士学位论文, 东北农业大学, 导师: 于泽源, 徐雅琴, pp: 35-43. (Dai J.2016. Studies on extraction, purification,structure analysis and biological activity of polysaccharides in Vaccinium uliginosum[D]. Thesis for M.S., Northeast Agricultural University, Supervisor: Yu Z Y, Xu Y Q, pp: 35-43.) 4 郭琦. 2012. 枸杞多糖的提取、分离纯化_溶液性质及其结构的初步研究[D]. 硕士学位论文, 陕西师范大学, 导师: 孙润广, pp: 31-39. (Guo Q.2012. The extraction, isolation and purification, solution properties and structural analysis of Lycium barbarum polysaccharide[D]. Thesis for M.S., Shaanxi Normal University, Supervisor: Sun R G, pp: 31-39.) 5 黎英, 陈雪梅, 严月萍, 等. 2015. 超声波辅助酶法提取红腰豆多糖工艺优化[J]. 农业工程学报, 31(15): 293-301. (Li Y, Chen X M, Yan Y P, et al.2015. Optimal extraction technology of polysaccharides from red kindey bean using ultrasonic assistant with enzyme[J]. Transactions of the Chinese Society of Agricultural Engineering, 31(15): 293-301.) 6 马茜. 2011. 枸杞多糖的结构及细胞凋亡诱导活性研究[D]. 硕士学位论文, 天津科技大学, 导师: 张民, pp: 18-45. (Ma Q.2011. Study on structure and apoptosis inducing activity of Lycium barbarum polysaccharide[D].Thesis for M.S., Tianjin University of Science and Technology, Supervisor: Zhang M, pp: 18-45.) 7 王绍清, 曹红, 曹宝森. 2013. 扫描电镜法观察鸡蛋壳超微结构形貌[J]. 食品科学, 34(13): 110-114. (Wang S Q, Cao H, Cao B S.2013. Ultrastructure of chicken eggshell observed by scanning electron microscopy[J]. Food Science, 34(13): 110-114.) 8 王发春, 陈志, 王文颖, 等. 2011. 豌豆多糖的抗氧化与抑菌活性研究[J]. 安徽农业科学, 39(26): 16431-16432. (Wang F C, Chen Z, Wang W Y, et al.2011. Research on antioxidant and antibacterial activity of pea polysaccharides[J]. Anhui Agricultural Sciences, 39(26): 16431-16432.) 9 王发春. 2011. 青海豌豆多糖提取工艺的研究[J]. 青海师范大学学报, 01: 67-70. (Wang F C.2011. Study on extraction process of Qinghai pea polysaccharide[J]. Journal of Qinghai Normal University (Natural Science), 01: 67-70. 10 王绍清, 王琳琳, 范文浩等. 2011. 扫描电镜法分析常见可食用淀粉颗粒的超微形貌[J]. 食品科学, 32(15): 74-79. (Wang S Q, Wang L L, Fan W H, et al.2011. Morphological analysis of common edible starch granules by scanning electron microscopy[J]. Food Science, 32(15): 74-79.) 11 王夏梅. 2016. 姬松茸子实体多糖分离纯化_结构表征和免疫活性分析[D]. 硕士学位论文, 江南大学, 导师: 戴军, pp: 20-25. (Wang X M.2016. The isolation, purification, structure characterization and immunological activity analysis of polysaccharides from the fruiting bodies of Agaricus blazei Murill[D]. Thesis for M.S., Jiangnan University, Supervisor: Dai J, pp: 20-25.) 12 徐铮铮. 2015.草菇多糖的分离纯化、生物活性研究及结构分析[D]. 扬州大学,导师: 汪志君, pp. 51-54. (Xu Z Z.2015. Isolation, purification, biological activity and structural analysis of Volvariella volvacea polysaccharides[D]. Thesis for M.S., Yangzhou University, Supervisor: Wang Z J, pp. 51-54.) 13 杨永利, 郭守军, 叶文斌, 等. 2012. 银合欢种子多糖微结构的原子力显微镜观察[J]. 食品科学, 33(15): 99-102. (Yang Y L, Guo S J, Ye W B, et al.2012. Microstructural observation of leucaena glaucal seed polysaccharides under atomic force microscope[J]. Food Science, 33(15): 99-102.) 14 余稳稳. 2014. 去皮绿豆水溶性多糖的分离纯化_结构与抗氧化活性研究[D]. 硕士学位论文, 华南理工大学, 导师: 吴晖, pp: 29-39. (Yu W W.2014. Extraction, purification, structural and antioxidant properties of water-soluble polysaccharide from dehulled Mung bean[D]. Thesis for M.S., South China University of Technology, Supervisor: Wu H, pp: 29-39.) 15 张敏, 施大文. 1998. 八角莲及其近缘属植物叶表面的电镜扫描观察[J]. 中国中药杂志, 23(8): 451-453. (Zhang M, Shi D W.1998. The scanning electron microscopy on leaf surface octagon and its proximal genus[J]. Journal of Chinese Materia Medica, 23(8): 451-453.) 16 章文琴, 刘成梅, 刘伟. 2010. 动态高压微射流技术对可溶性大豆多糖结构的影响[J]. 食品科学, 31(09): 30-34. (Zhang W Q, Liu C M Liu W, et al.2010. Effect of dynamic high-pressure microfluidization on morphology of soluble soybean polysaccharides[J]. Food Science, 31(09): 30-34.) 17 Anneke H M, Douglas G, Alexandra S.2006. Immobilization of casein micelles for probing their structure and interactions with polysaccharides using scanning electron microscopy (SEM)[J]. Food Hydrocolloids, 20(6): 817-824. 18 Benjamin T, Christel G.2016. Volume scanning electron microscopy for imaging biological ultrastructure[J]. Biology of the Cell, 108(11): 307-323. 19 Dubois M, Gilles K A, Hamilton J K, et al.1956. Colorimetric method for determination of sugars and related substances[J]. Analytical Chemistry, 28(3): 350-356. 20 Hakimeh J A, Masoud R, Bahareh S, et al.2018. Effects of sulfated polysaccharides from green alga Ulva intestinalis on physicochemical properties and microstructure of silver carp surimi[J]. Food Hydrocolloids, 74: 87-96. 21 Hassana A N, Franka J F, Elsodab M.2003. Observation of bacterial exopolysaccharide in dairy products using cryo-scanning electron microscopy[J]. International Dairy Journal, (13): 755-762. 22 Holbrook R D, Wagner M S, Mahoney C M, et al.2006. Investigating activated sludge flocs using microanalytical techniques: Demonstration of environmental scanning electron microscopy and time-of-flight secondary ion mass spectrometry for wastewater applications[J]. Water Environment Research, 78(4): 381-391. 23 Imjongjairak S, Ratanakhanokchai K, Laohakunjit N, et al.2016. Biochemical characteristics and antioxidant activity of crude and purified sulfated polysaccharides from Gracilaria fisheri[J]. Bioscience Biotechnology and Biochemistry, 80(3): 514-532. 24 Ji L, Xue Y, Zhang T, et al.2017. The effects of microwave processing on the structure and various quality parameters of Alaska pollock surimi protein-polysaccharide Gels[J]. Food Hydrocolloids, 63: 77-84. 25 Liu F, Zhu Z Y, Sun, X L, et al.2017. The preparation of three selenium-containing Cordyceps militaris polysaccharides: Characterization and anti-tumor activities[J]. International Journal of Biological Macromolecules, (99): 196-204. 26 Lin X, Yang W, Xu D, et al.2015. Improving gel properties of hairtail surimi by electron irradiation[J]. Radiation Physics and Chemistry, 110: 1-5. 27 Li S Q, Shah N P.2014. Antioxidant and antibacterial activities of sulphated polysaccharides from Pleurotus eryngii and Streptococcus thermophilus ASCC 1275[J]. Food Chemistry, 165: 262-270. 28 Li S Q, Shah N P.2016. Characterization, antioxidative and bifidogenic effects of polysaccharides from Pleurotus eryngii after heat treatments[J]. Food Chemistry, 197: 240-249. 29 Ludĕk F, Miloš H, Šárka M, et al.2012. Scanning electron microscopy with samples in an electric field[J]. Materials, 5: 2731-2756. 30 Maeda N, Miao J, Simmons T J, et al.2014. Composite polysaccharide fibers prepared by electrospinning and coating[J]. Carbohydrate Polymers, (102): 950-955. 31 Martin A H, Goff H D, Smith A, et al.2006. Immobilization of casein micelles for probing their structure and interactions with polysaccharides using scanning electron microscopy (SEM)[J]. Food Hydrocolloids, 20(6): 817-824. 32 Peng Y F, Han B Q, Liu W S, et al.2016. Deproteinization and structural characterization of bioactive exopolysaccharides from Ganoderma sinense mycelium[J]. Separation Science and Technology, 51(2): 359-369. 33 Sun Q, Sun Y L, Juzenas K.2017. Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls[J]. Journal of Experimental Botany, 68(9): 1-14. 34 Zhang T, Xue Y, Li Z, et al.2015. Effects of deacetylation of konjac glucomannan on Alaska pollock surimi gels subjected to hightemperature (120 ℃) treatment[J]. Food Hydrocolloids, 43: 125-131.