【作者】 钮琰星；

【导师】 江木兰；

【作者基本信息】 中国农业科学院 ， 农业微生物学， 2013， 博士

【摘要】 随着育种技术的进步，菜粕中的毒性物质硫苷的含量显著降低，酚类物质成为影响菜粕利用的重要的抗营养因子。芥子碱是菜粕中最重要的简单酚类物质，占菜粕简单酚类的70-85%，而目前国内外关于芥子碱的降解研究尚不深入，存在产物具有潜在食用安全风险和降解效率不高的问题。针对这一现状，本论文通过研究加工工艺对芥子碱含量的影响、芥子碱酶解动力学和降解机理、芥子碱在生物发酵过程中的变化等，探明芥子碱在热和酶作用下的变化规律，为高效安全的芥子碱降解提供理论指导，为推动菜粕生物改良技术的进步提供理论支撑。具体研究结果如下:1、采用两样本配对t检验法对回流萃取法和超声辅助提取法对芥子碱提取效率的影响进行了研究,发现超声辅助提取法效率更高。研究确定了芥子碱提取的最佳溶剂比为100:1的无水甲醇。建立了用于芥子碱含量快速测定的紫外分析方法和精确测定的高效液相色谱法。2、进行了微波处理对菜粕芥子碱含量和菜籽细胞结构影响的研究。通过透射电镜观察发现菜籽经微波处理后大部分油小体消失，油小体和其他细胞器的膜结构被破坏，为油脂的提取提供了良好的条件，经过4mins的微波处理菜籽饼中的油分含量与未处理相比减少了50.8％。微波处理对菜籽芥子碱的含量也产生了显著影响，微波处理7分钟后，芥子碱的含量下降了16.7%。通过了双样本配对t检验发现压榨和溶解提取两种油脂提取方法对饼粕中芥子碱含量具有显著影响（p≤0.05），芥子碱和芥子酸更易于被溶剂浸出。3、采用紫外、薄层层析等研究了漆酶和酪氨酸酶对芥子碱的酶解作用，发现漆酶可快速降解芥子碱，而酪氨酸酶对芥子碱基本无作用。选取了两种结构与芥子碱类似的底物化合物Ⅰ（1-(4-hydroxy-3,5-dimethoxyphenyl)ethanone）和底物化合物Ⅱ（sodium (E)-3-(4-hydroxy-3,5-dimethoxyphenyl)acrylate）进行漆酶酶解对比实验，发现漆酶作用芥子碱的位点为苯环上的酚羟基。通过TLC、UPLC和NMR的分析发现漆酶酶解芥子碱后生成了以低聚物为主的产物。4、根据酶解产物的特点结合文献报道，推测漆酶酶解芥子碱的过程为：首先漆酶与氧气结合形成活性中间体，然后中间体与芥子碱中较为活泼的羟基氢反应，得到一个芳基氧自由基；少部分自由基发生迁移和重排，得到结构较为简单的产物；大部分的芳基氧自由基发生了聚合反应，形成结构较为复杂的低聚物。5、芥子碱酶解速率受酶用量、底物浓度、温度和pH等的显著影响，通过不同条件下的酶解全过程监测，得出芥子碱在底物和酶的质量比20：1、温度55℃、pH值5.5、底物浓度0.01895mg/mL的条件下可以实现芥子碱的快速酶解。氧气作为芥子碱酶解的底物之一对反应速率也产生显著影响，但是通常水中溶解的氧气已可满足反应的需要。漆酶酶解菜粕芥子碱的Km值为0.00013mol/L，表明漆酶与芥子碱亲和力强，反应迅速。6、通过接种酿酒酵母的含芥子碱培养基的液态和固态发酵发现，引起固态发酵过程中芥子碱降解的一个重要原因是热的作用。芥子碱在湿热条件下易于降解，其含量降低速率和水分的流失呈显著正相关。云芝菌所产漆酶对芥子碱具有显著的酶解作用，在云芝菌发酵过程中，随着漆酶的产生，芥子碱含量显著下降。更多还原

【Abstract】 With the development of breeding technology, the content of glucosinolates in rapeseed meal weresignificantly decreased, but phenols have been become the important factors on the value of rapeseedmeal. Sinapine is one of the most important simple pehenols in rapeseed meal, accounting for70-85%of simple phenols in rapeseed meal. So the degradation of sinapine should be studied further to avoidthe potential food security risks. The objective of this dissertation is to reveal the dynamics andmechanisms involved in the degradation of sinapine through the heat and enzymatic treatment; andtherefore to provide theoretical guidance for the sinapine safety degradation. This work will focus onthe study of microwave treatment on the content of sinapine, enzymatic kinetics and mechanism ofsinapine catalyzed by polyphenol oxidase，the effect of fermentation on the content of sinapine. Theresults are as follows:1. Two-sample paired t-test was used to evaluate the efficiency of reflux and ultrasonic assistedextraction on sinapine extraction from rapeseed meal. It was found that ultrasound-assisted extractionmethod is most efficient. The best solvent ratio to extract sinapine was100:1anhydrous methanol.Ultraviolet (UV) analysis method was established for rapid determination of the content of sinapine.Ultra Performance Liquid Chromatography was established to determine the content of sinapineprecisely.2. The content of sinapine in rapeseed meal and microsturcture of rapeseed effected by microwavetreatment was assessed. Microscopic work showed that most of the cellular oil bodies were notobserved, and the membranes of organelles damaged and the cell wall folded after microwave treatment.Almost all the oilbodies were absent in rapeseed cakes treated by microwave. It was shown thatmicrowave treatment provided good conditions for pressing and oil extraction. Four minutesmicrowave treatment resulted in the decreased oil content by50.8%. Microwave treatment also hassignificant effect on the content of sinapine in rapeseed and rapeseed meal. By seven minutes ofmicrowave treating, the content of sinapine in rapeseed exhibited the largest decrease of16.7%. Weemployed two sample paired t-tests for the means to analyze the difference between the meal anddefatted cold-pressed cake. The results indicated that the content of sinapine and sinapic acid indefatted cake was significantly higher than that in meal (p≤0.05).3. UV and Thin layer chromatography (TLC) was used to evaluate the effect of laccase andtyrosinase on the content of sinapine. It was found that laccase can catalyse sinapine quickly whiletyrosinase has no effect on the content of sinapine.1-(4-hydroxy-3,5-dimethoxyphenyl) ethanone) andsodium (E)-3-(4-hydroxy-3,5–dimethoxyphenyl was selected to judge the group site of laccasecatalyzed on sinapine. It was found that phenolic hydroxyl group was the right site and the product ofsinapine catalyzed by laccase was oligomers which was confirmed by analyses of TLC, UPLC andNMR.4. Combined the literature data and the characteristics of the catalysates of sinapine, the pathwayof sinapine catalysed by laccase could be as follows: First, after the combination of oxygen with the enzyme, the reactive intermediates was formed and reacted with hydroxyl hydrogen in sinapine, thenthe oxygen free radicals were obtained. Some of them were rearranged to form a relatively simplestructure compound. Most of the aryloxy radicals were polymerized forming subsequent oligomers.5. The catalytic velocity of sinapine catalysed by laccase was affected by enzyme concentration,substrate concentration, temperature and pH significantly. The reaction conditions were: substrate andenzyme ratio20:1,55°C, pH5.5. The oxygen is one of the substrates affecting the reaction speedsignificantly, and the dissolved oxygen in water could meet the needs of the reaction. Michaelisconstant was determined. The Km value is0.00013mol/L. The results indicated the strong affinitybetween laccase and sinapine.6. Liquid and solid fermentation was used to evaluate the effect of heat and laccase on the contentof sinapine. It was found that heat and moisture have significant effects on the content of sinapine; thedecreased content of sinapine was well related to the loss of moisture (with positive correlation).Laccase produced by Trametes sp. catalyses the degradation of sinapine in a fast manner. With theproducing of laccase, the content of sinapine was decreased rapidly.更多还原