日本枪乌贼营养分析及多糖提取分离
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摘要
枪乌贼(Teuthoidea),属软体动物门(Mollusca)头足纲(Cephalopoda)枪形目(Teuthoidea)枪乌贼科(Loliginidae),其实质为多种海生10腕头足类软体动物的统称。而现有的糖生物学研究表明,海洋生物多糖具有抗肿瘤、调节免疫、抗病毒等多种功效,且毒副作用较小,受到广大专家学者的关注。本文从日本枪乌贼提取分离多糖,并对纯化所得的多糖组分的结构进行研究,为以后此类多糖的开发利用提供参考。
本文主要通过营养学的的分析方法,测定了枪乌贼乌贼中的蛋白质、脂类等生化组分,分析了构成蛋白质的17种常见氨基酸和主要脂肪酸含量。结果表明,枪乌贼属高蛋白、低脂肪海产；其干品的蛋白总量已经超过50%,必需氨基酸和非必需氨基酸含量相当而比较平衡,因而枪乌贼的蛋白氨基酸组成是比较合乎FAO/WHO所推荐的模式的。脂肪酸分析表明其还富含多不饱和脂肪酸(PUFA)。
通过单因素实验和正交试验分析,对枪乌贼多糖的胰蛋白酶提取法的各条件进行了优化,得到最佳提取工艺如下：温度为50℃,pH 9,料液比1：3,加酶量7.5%。在分离纯化研究中,比较了三氯乙酸法、Sevag法、三氯乙酸-正丁醇法脱蛋白工艺的优劣,综合考虑,通过以蛋白质的去除率为指标,选择了三氯乙酸-正丁醇法作为脱蛋白工艺最优方法,验证了酶提法先加碱液能够有效促进多糖释放。通过咔唑法测定葡萄糖醛酸的含量为13.53%；高效凝胶色谱仪分析测得枪乌贼多糖重均分子量为162039；红外光谱表明,枪乌贼贼多糖的构型为β-多晶型,多糖为单糖是D-葡萄糖吡喃糖环构型。通过对其单糖组成分析,发现其多糖主要由甘露糖和岩藻糖组成,还有少量的氨基半乳糖、葡萄糖胺、葡萄糖、D-甘露糖、D-半乳糖、半乳糖醛酸。
Teuthoidea, is a Marine ten wrist cephalopods molluscs collectively. It belongs to Mollusca,Cephalopoda, Teuthoidea, Loliginidae, which is a special of sea anemone found in seaside in the yellow sea and bohai sea of China. With the development of glycol-biology, polysaccharides isolated from various sea animals have a great deal of attention because of the anti-tumor activity, anti-virus activity, antioxidant activity immunoregulating activity and relatively low toxicity. In this paper, extraction, isolation and structure analysis of polysaccharides from Loligo japonica were investigated that provide reference for utilization and exploration of Loligo japonica.
This paper analyzes the protein and fat in the Teuthoidea mainly through the nutrition analysis method and assays 17 kinds of common constitutes a protein content of amino acids and main fatty acids. The results show that Teuthoidea is high protein, low adipose seafood; more than 50% of dry-out sample is protein, essential amino acids and non-essential amino acid content is balance, thus Loliginidae protein amino acid composition is accord with the FAO/WHO recommend mode. Fatty acid analysis shows there is rich PUFA in Loliginidae.
According to the single factor experiment and orthogonal experiment, the optimal extraction parameters are established as follow: ratio of material to solvent is 1:3 (w/v); extraction temperature is 50℃; enzyme load is 7.5%; and initial pH is 9.In the purification study, compares the different methods of removing proteins,through comprehensive consideration of protein as the index, Chose trimethoxysilane n-buoh acetic acid-laws as Deproteinization method, Verify lye before enzymatic hydrolysis can effectively promote to release of polysaccharides.Chemical methods are used to analysis the constituents of Teuthoidea, it show that the glycuronic acid is13.53%. HP GPC analysis shows that the molecular weight of Actiniaria Purificata is 162039. IR spectrum indicate tha Teuthoidea polysaccharides configuration for Beta much crystalline, polysaccharide monosaccharide glucose is D-pyranoid sugars ring configurations. Through its composition analysis, found polysaccharide of Teuthoidea is mainly composed of D-Man and D-Fuc, there is also some D-(+)-Gal Hcl, GlcN, D-Glu, D-Gal, GluA
本文主要通过营养学的的分析方法,测定了枪乌贼乌贼中的蛋白质、脂类等生化组分,分析了构成蛋白质的17种常见氨基酸和主要脂肪酸含量。结果表明,枪乌贼属高蛋白、低脂肪海产；其干品的蛋白总量已经超过50%,必需氨基酸和非必需氨基酸含量相当而比较平衡,因而枪乌贼的蛋白氨基酸组成是比较合乎FAO/WHO所推荐的模式的。脂肪酸分析表明其还富含多不饱和脂肪酸(PUFA)。
通过单因素实验和正交试验分析,对枪乌贼多糖的胰蛋白酶提取法的各条件进行了优化,得到最佳提取工艺如下：温度为50℃,pH 9,料液比1：3,加酶量7.5%。在分离纯化研究中,比较了三氯乙酸法、Sevag法、三氯乙酸-正丁醇法脱蛋白工艺的优劣,综合考虑,通过以蛋白质的去除率为指标,选择了三氯乙酸-正丁醇法作为脱蛋白工艺最优方法,验证了酶提法先加碱液能够有效促进多糖释放。通过咔唑法测定葡萄糖醛酸的含量为13.53%；高效凝胶色谱仪分析测得枪乌贼多糖重均分子量为162039；红外光谱表明,枪乌贼贼多糖的构型为β-多晶型,多糖为单糖是D-葡萄糖吡喃糖环构型。通过对其单糖组成分析,发现其多糖主要由甘露糖和岩藻糖组成,还有少量的氨基半乳糖、葡萄糖胺、葡萄糖、D-甘露糖、D-半乳糖、半乳糖醛酸。
Teuthoidea, is a Marine ten wrist cephalopods molluscs collectively. It belongs to Mollusca,Cephalopoda, Teuthoidea, Loliginidae, which is a special of sea anemone found in seaside in the yellow sea and bohai sea of China. With the development of glycol-biology, polysaccharides isolated from various sea animals have a great deal of attention because of the anti-tumor activity, anti-virus activity, antioxidant activity immunoregulating activity and relatively low toxicity. In this paper, extraction, isolation and structure analysis of polysaccharides from Loligo japonica were investigated that provide reference for utilization and exploration of Loligo japonica.
This paper analyzes the protein and fat in the Teuthoidea mainly through the nutrition analysis method and assays 17 kinds of common constitutes a protein content of amino acids and main fatty acids. The results show that Teuthoidea is high protein, low adipose seafood; more than 50% of dry-out sample is protein, essential amino acids and non-essential amino acid content is balance, thus Loliginidae protein amino acid composition is accord with the FAO/WHO recommend mode. Fatty acid analysis shows there is rich PUFA in Loliginidae.
According to the single factor experiment and orthogonal experiment, the optimal extraction parameters are established as follow: ratio of material to solvent is 1:3 (w/v); extraction temperature is 50℃; enzyme load is 7.5%; and initial pH is 9.In the purification study, compares the different methods of removing proteins,through comprehensive consideration of protein as the index, Chose trimethoxysilane n-buoh acetic acid-laws as Deproteinization method, Verify lye before enzymatic hydrolysis can effectively promote to release of polysaccharides.Chemical methods are used to analysis the constituents of Teuthoidea, it show that the glycuronic acid is13.53%. HP GPC analysis shows that the molecular weight of Actiniaria Purificata is 162039. IR spectrum indicate tha Teuthoidea polysaccharides configuration for Beta much crystalline, polysaccharide monosaccharide glucose is D-pyranoid sugars ring configurations. Through its composition analysis, found polysaccharide of Teuthoidea is mainly composed of D-Man and D-Fuc, there is also some D-(+)-Gal Hcl, GlcN, D-Glu, D-Gal, GluA
引文
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[3]李桂芬.鱿鱼的营养与开发利用[J]科学养鱼,2003,(07)：9-10
[4]方尔笠,顾洛,田苏平,等.海螵蛸防治胃溃疡作用的机理探讨[J].中国中西医结合杂志,1994,12(4)：101-103
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[6]魏江洲,张建鹏,刘军华,徐红丽,冯伟华,焦炳华,.海螵蛸多糖CPS-1对小鼠实验性溃疡性结肠炎作用的初步观察[J].第二军医大学学报,2006,(1).
[7]魏江洲,张建鹏,刘军华,王顺春,冯伟华,焦炳华,.海螵蛸多糖的提取分离及活性组分CPS-1的纯化[J].第二军医大学学报,2006,(1).
[8]王夙博,刘纯慧,.硫酸化乌贼墨多糖对肿瘤细胞克隆抑制作用观察[J].山东医药,2008,(23).
[9]雷晓凌,赵树进,吴红棉,彭晓娜,杨志娟,.弯斑蛸多糖纯化及对小鼠脾细胞增殖的影响[J].华南理工大学学报(自然科学版),2007,(11).
[10]雷晓凌,赵树进,蒋琳兰,.弯斑蛸多糖对免疫抑制小鼠的调节作用[J].中国海洋药物,2007,(5).
[11]雷晓凌,范秀萍,赵树进,陈敏,何启兰,.章鱼干糖蛋白的提取与分级分离的初步研究(英文)[J].陕西科技大学学报,2006,(4).
[12]文松松,赵峡,高英霞,于广利.短蛸章鱼多糖的提取分离和基本性质分析[J].中国海洋药物,2010,(1).
[13]Hokput sa S, Harding S E, Inngjerdingen K, et al. Bioactive polysaccharides from the stems of the Thai medicinal plant Acanthusebracteatus:their chemical and physical features[J]. Carbohy drate Research,2004,339 (4):753.
[14]吴东儒.糖类的生物化学[M).北京：高等教育出版社,1987
[15]张维杰.糖复合物生化研究技术[M].第2版.杭州：浙江大学出版社,1999：36
[16]陈士国.鱿鱼墨多糖和黑色素的研究[D].中国海洋大学：,2007.
[17]殷红玲,杨静峰,李冬梅,董秀萍,姜丹,李韬,朱蓓薇,.酶法提取鲍鱼多糖的研究[J].食品与发酵工业,2006,(12).
[18]方升平,王维香,雒小龙,.川芎多糖除蛋白方法研究[J].时珍国医国药,2009,(9).
[19]冯颖,孟宪军,王建国,李振华,.大孔吸附树脂法去除无梗五加果粗多糖中蛋白的研究[J].食品研究与开发,2008,(5).
[20]YOUNG MYOUNG KIM, YONG SEOK CHOI, JONG HYUK PARK. Parl'fication and Chemical Characterization of Laminaran from Eisenia bicyclis in Korea[J]. Korean Society of Food Science and Nutrition,2006,35(1):78-86.
[21]M.R.S.MELO, J.PA.FEITOSA, A.L.P.FREITAS, et al. Isolation and characterization of soluble sulfated polysaccharide from the red seaweed Gracilaria cornea[J]. Carbohydrate Polymers,2002,49:491-498.
[22]WANG J, YAO Y, ZHANG JP, et al.Preparation and biological activity of polysaccharides from Ostrea rivularis in the east China sea[J]. Medical Postgraduates,2006,19(3):217-220.
[23]TATIANA N, ZVYAGINTSEVA A, NATALIYA M, et al. A new procedure for the separation of water-soluble polysaccharides from brown seaweeds[J]. Carbohydrate Research,1999,322:32-39.
[24]VOLPI NICOLA, MACCARI FRANCESEA. Purification and characterization of hyaluronic acid from the mollusk bivalve mytilus gal-loprovincialis[J]. Biochimie,2003,85: 619-625.
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