【作者】 张岩；

【导师】 刘洪章；

【作者基本信息】 吉林农业大学 ， 作物资源学， 2007， 硕士

【摘要】 杏仁、榛子、山核桃、松籽在分类上属于坚果中的树坚果，在我国种植面积广、产量高、也是在国际上享有盛誉的果仁品种。杏仁、榛子、山核桃、松籽的营养价值高，生理、药理活性明确，经济价值高，随着我国经济树种的开发，树坚果的种植面积还将大幅度提高。因此深入研究它们的活性成分具有重要意义。目前对果仁的研究比较集中于果仁油脂这方面。本实验采用目前应用较广的冷浸法、超声清洗法、超临界流体萃取法提取果仁油脂并进行了比较研究。结果显示，三种方法对果仁油脂的提取率由高到低依次为：超临界流体萃取法＞超声清洗法＞冷浸法。四种果仁含油率的高低依次为：松籽＞山核桃＞杏仁＞榛子。另外，用正交分析法优化了超声清洗法提取果仁油脂的工艺。在选定的因素中，最活跃的影响因素是溶剂，其次是料液比，最小的影响因素是时间。最后确定松籽的最佳提取工艺为：以石油醚为溶剂，料液比1：10，超声提取时间为40min；杏仁：以石油醚为溶剂，料液比1：8，超声提取时间为30min；山核桃：以正己烷为溶剂，料液比1：10，超声提取时间为40min；榛子：以正己烷为溶剂，料液比1：10，超声提取时间为30min。应用GC／MS对四种果仁油脂的脂肪酸组成进行了分析，结果表明：四种果仁的脂肪酸组成有很大差异，但是均含有9-十八碳烯酸甲酯和9，12-十八碳二烯酸甲酯，且含量都很高。杏仁肽是杏仁蛋白质经酶水解、精制后所得到的短肽混合物。本研究以脱脂杏仁为原料，经蛋白质提取、酶解、分离等过程制备有生物活性的杏仁蛋白质多肽。杏仁经搅拌、调pH值、离心等一系列过程，分离出杏仁蛋白质。分别用胰蛋白酶、木瓜蛋白酶、菠萝蛋白酶对其进行水解，以水解度为指标，确定木瓜蛋白酶为最佳酶制剂。通过单因素试验和正交试验确定了木瓜蛋白酶的最佳水解条件：pH值为11.3，底物浓度为30mg／mL，酶与底物比为6.5％，温度60℃，水解时间2h，可以得到水解度为65％的杏仁肽。本实验采用凝胶色谱法分离多肽，凝胶型号为SephadexG-25，洗脱液为pH值8.5的Tris-HCl，洗脱速度为2ml／4min，收集各管洗脱液，加入靠马斯亮蓝G-250，在595nm下检测各管吸光度，作洗脱曲线。根据测得内水体积、外水体积，排除大分子量的洗脱峰和分子量约为单个氨基酸的洗脱峰，则中间部分为分子量为5000左右的多肽混合物。此多肽混合物再经过浓缩、干燥即成为成品。应用凯氏定氮法测定果仁中的蛋白质含量，结果为：山核桃＞杏仁＞松籽＞榛子；蒽酮比色法测定果仁中可溶性总糖含量，结果为：杏仁＞山核桃＞松籽＞榛子；DNFB柱前衍生化RP-HPLC法测定果仁中氨基酸，测得果仁中含有丰富的氨基酸，其种类至少有16种，且必需和半必需氨基酸的含量很高；用电感耦合等离子体发射光谱仪及火焰原子吸收法对果仁中的Sr、Ti、Mn、Mo、V、Zn、Cr、Fe、Co、Cu、Ni、Al、B、Ca、Mg、Ba、Be、Cd、La、P、Pb、Y、K、Na等24种微量元素进行了检测；用索氏提取法提取苦杏仁苷并采用二次重结晶的方法提纯苦杏仁苷，苦杏仁苷含量为3.13％；用水蒸气蒸馏法提取杏仁精油，精油得率1.85％，经GC／MS分析，共分离出21种化合物，鉴定出了其中的18种，其中14种为首次从杏仁中获得，鉴定出的成分占精油总成分的99.99％，明显高于前人。更多还原

【Abstract】 Almond, hazelnut, walnut, pinenut are tree-nut spicies.They are widely planted, high-yield in China,and great reputation in the world.These four nut spicies are full of nutritious compositions and higheconomy value, whose pharmacologic action and physiological activity are definite.With China’seconomic trees development and returning farmland to forest policy implementation, the cultivate area ofthese four tree-nut will increase quickly in the near future.Therefore it’s quite sigmificant to deeply studyand research their active component.Present researches of nutlet are mostly about fatty oil.In this experiment, applied cold solventextraction, ultrasonic cleaning method and SCF-CO~2 of the current extraction methods to extract fatty oiland compare.The result showes that the production of nutlet fatty oil obtained from the 3 ways issequenced:cold solvent extraction＞ultrasonic cleaning method＞SCF-CO~2.The fatty oil proportion of the 4nutlets in order: pinenut＞walnut＞almond＞hazel ut.In addition, I have improved the ultrasonic cleaningmethod of fatty oil extraction from nutlets with orthogotal test.In selected factors, the most athleticinfluencing factor is dissolvent, then dosage liquor ratio, the weakest is time.The most effective extractiontechnology is as below.Pinenut: petroleum ether as dissolvent, dosage liquor ratio 1:10, ultrasonic cleaningmethod time 40 min.Almond, petroleum ether as dissolvent, dosage liquor ratio 1:8, ultrasonic cleaningmethod time 30 min.Walnut, N-hexane as dissolvent, dosage liquor ratio 1:10, ultrasonic cleaning methodtime 40 rain.Hazelnut, N-hexane as dissolvent, dosage liquor ratio 1:10, ultrasonic cleaning method time30 min.Analysis for composition of fatty acid in the 4 nutlets by GC/MS showed that: Fatty acidcomposition of the four nutlets are very different, but they contained 9-Octadecenoic acid (Z)-, methyl esterand 9,12-Octadecadienoic acid, methyl ester, which have a high content.Almond peptide is the mixture of short peptides that the almond protein is hydrolyzed by enzymes andagain handled specially.The process of this experiment is this: proteins derived from almonds werehydrolyzed, and peptides were separated from the hydrolysate.Almond protein was obtained after the stepsof whisking, adjusting pH, and centrifugalizing.The substrate of experiment was defatted almond meal thatwas individually hydrolyzed by trypsin, papain and bromelain.It was determined by the degree ofhydrolysis that the papain was the best one.Through the single factor experiments and orthogotal test, theoptimum hydrolyzing condition has been determined:temperature 60℃, pH11.3, substrate concentration30mg/mL, the ratio of enzyme and substrate at 6.5%, the time 2 hours, and it can get the degree ofhydrolysis 65%.The gel filtration was adopted in this process of seperation.The gel is SephadexG-25.Theeluate is Tris-HC1 ofpH value 8.5, and the elution speed is 2ml/4min.The eluates was collected, detect theabsorbance of the eluates under 595nm, and draw the elution curve.Remove the eluates of very largemolecular weight and the eluates of molecular weight equal to the pure amino acid according to the Vo andVi, and the left is the peptide mixture which the average molecular weight is about 5000.These peptidesproduct was obtained after concentration and dry.Protein Content of nutlets was determined by the Kjeldahl Nitrogen Determination method, results:walnut＞almond＞pinenut＞hazelnut.Determined nutlets total soluble sugar content by Anthronecolorimetric method, results: almond＞walnut＞pinenut＞hazelnut.To develop a pre-column derivatizationprocedure for the determination of the content of amino acid in nutlets by reversed-phase high performanceliquidchromatography, the results show that 16 kinds of amino acids were assayed in every nutlet at leastand a majority of all was necessary or half-necessary amino acids.Content of 24 kinds of trace elements(Sr、Ti、Mn、Mo、V、Zn、Cr、Fe、Co、Cu、Ni、Al、B、Ca、Mg、Ba、Be、Cd、La、P、Pb、Y、K、Na)in nutlets was determined by ICP atomic emission spectroscopy and Flame AtomAbsorbability Spectrum(FAAS).Essential oil of almond was obtained by steam distillation, yield 1.85%.Chemical composition was analyzed by mean s of GC/MS technique, 18 components of 21separatedcomponents,14 components of them is the first gained from almond, which constitute about 99.99% thetotal oil.更多还原