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菜粕芥子碱的提取、分离纯化、鉴定及酶解研究

Extraction, Purification, Determination and Enzyme Hydrolysis of Rapeseed Meal Sinapine

作者: 专业:食品科学 导师:黄凤洪 年度:2010  院校: 华中农业大学

Keywords

        菜粕是数量上仅次于豆粕的植物蛋白源,我国每年产生大约有600-700万吨菜粕。随着菜籽双低(低硫甙、低芥酸)化进程的推进,酚类物质成为菜粕中重要抗营养因子之一。芥子碱是菜粕中最重要的酚类物质之一。菜粕中芥子碱含量1.2%-2.3%,溶于水易发生水解反应,生成芥子酸和胆碱。芥子碱是使菜粕产生苦味的主要原因,亦能使褐壳鸡蛋产生鱼腥味。对菜粕芥子碱的提取、分离纯化、酶解工艺进行研究,不仅能解决菜粕饲料适口性差的问题,而且为菜粕的精深加工与利用提供基础依据。1.通过精密度和回收率试验,比较紫外分光光度法(326nm)和可见光光度法(388nm)测定菜粕芥子碱的含量,确定在本研究中采用可见光光度法(388nm)。2.对菜粕芥子碱的提取工艺进行了初步探讨,通过单因素试验和Box-Behnken优化试验,确定了热醇回流提取的最佳工艺组合为:提取时间3.9h,提取温度85.5℃,溶剂比3:5,料液比1:26,此时菜粕芥子碱得率达1.59%。通过单因素和正交试验,确定了加速溶剂萃取仪(Accelerated Solvent Extractor,简称ASE)最佳提取工艺为:萃取温度为140℃,静态萃取时间为15min,萃取次数为3次,上样量为3g,此时菜粕芥子碱得率达1.88%。并对经两种不同提取工艺提取后菜粕的理化指标进行了检测,结果表明:主要抗营养因子含量均大大降低,蛋白和氨基酸含量显著提高。经ASE萃取后的菜粕品质最优,其中粗蛋白含量较原料粕提高了18.46%,粗纤维提高了26.65%,硫甙、总酚未检出,植酸降低了17.05%,芥子碱含量仅为原料的10.14%。经索氏提取后菜粕总氨基酸含量比原料粕提高了22.60%,经ASE提取后总氨基酸含量提高了26.72%;经索氏提取后菜粕总必需氨基酸含量提高了17.70%,经ASE提取后的菜粕总必需氨基酸含量提高了21.25%。3.通过对D-101、DK-16、NKA-9、AB-8四种大孔树脂吸附率和解吸率的测定,筛选出DK-16是比较适合纯化菜粕芥子碱的树脂。确定了DK-16纯化菜粕芥子碱的最佳工艺:pH值7.0,上样流速2BV/h,洗脱剂浓度90%(v/v),洗脱流速2BV/h,最佳吸附时间120min,最佳洗脱时间90min。产品真空干燥后浓度可达49.89%。通过硫氰酸盐沉淀法制备菜粕芥子碱,可得到纯度较高的芥子碱硫氰酸盐样品,重结晶次数越多,纯度越高,试验表明最佳结晶次数为3次,纯度可达94.54%。4.采用UV、IR、HPLC、HPLC-ESI/MS等现代分析检测手段对菜粕芥子碱纯化样品进行研究。5.从多酚氧化酶、漆酶、β-葡萄糖苷酶、脂肪酶中筛选出对菜粕芥子碱降解率较高的漆酶,通过单因素和Box-Behnken优化试验,得最佳工艺参数为:酶解时间25min, pH5.10,酶添加量为2mL,酶解温度为28℃,此时漆酶降解菜粕芥子碱降解率达81.93%。研究了漆酶酶解菜粕芥子碱的米氏常数:漆酶在30℃时,在最适pH值条件下对菜粕芥子碱底物的Km值分别为1.16mol/L,最大酶反应速率Vmax=2.38A/(U*min)。
    Rapeseed meal are the second most widely traded protein ingredients after soybean meal, there are about 600-700 million tons of rapeseed meal in china each year. With the advance of double-low rapeseed, rapeseed meal phenolics become one of the most important anti-nutritional factors. Sinapine is one of the most important polyphenols in rapeseed meal, rapeseed meal content 1.2%-2.3% sinapine,it’s prone to dissolved in water, sinapine acid and choline are easy formed by hydrolysis.Sinapine is the main reasons of bitterness, but also can produce brown-shell eggs, fish smell. I studied the extraction process, not only can solve the rapeseed meal feed, the problem of poor palatability, but also for deep processing and utilization of rapeseed meal, the biological activity of rapeseed meal sinapine provide the basis for research and application.(1) Ultraviolet spectrophotometry(326nm) method and visible light spectrophotometry (388nm) method were used to determine the content of rapeseed-meal-sinapine. Through the comparison of precision and recovery, the visible light spectrophotometry (388nm) was selected.(2)I conducted a preliminary study through rapeseed meal sinapine extraction process, by single-factor test and Box-Behnken optimization tests to determine the hot alcohol extract optimum combination of reflux as follows:extraction time is 3.9h, extraction temperature is 85.5℃, solvent ratio is 3:5, solid-liquid ratio is 1:26, under those conditions, rapeseed meal sinapine extraction rate is 1.59%.Through the single factor and orthogonal tests, I determined the optimal extraction process of the accelerated solvent extraction instrument (Accelerated Solvent Extractor, for short ASE) as follows: extraction temperature is 140℃, static extraction time is 15min, extraction times are 3 times,the sample amount is 3g,under those conditions, rapeseed meal sinapine extraction rate is 1.88%.The quality of rapeseed meal that extracted by two different post-extraction was tested, results showed that:The main anti-nutritional factors greatly reduced, while the protein and amino acid content was significantly increased.The ASE extraction of rapeseed meal quality is best, in which the crude protein content of raw materials increased by 18.46% compared with the crude fiber increased by 26.65%, glucosinolate, total phenols were not detected, phytic acid reduced 17.05%, sinapine content only10.14% of raw materials.Soxhlet extracted by the total amino acid content of rapeseed meal than the raw materials increased 22.60%, after the total amino acid content after ASE extraction increased 26.72%; by Soxhlet extracted by the total essential amino acid content of rapeseed meal 17.70%, extracted by the ASEThe total amino acid content of rapeseed meal increased 21.25%.(3) By D-101, DK-16, NKA-9, AB-8 macroporous resin adsorption rate of four and desorption rate measurement, I filtered out the DK-16 is more suitable for purification of rapeseed meal sinapine resin.Selected the DK-16 purified rapeseed meal sinapine the optimum:70%(v/v) ethanol solution to do eluant, pH value is 7.0, the sample flow rate is 2BV/h, elution concentration of 90%(v/v), flow rate is 2BV/h, the best adsorption time is 120min, the best elution time is 90min.The concentration of product after vacuum drying up to 49.89%.Thiocyanate precipitate through the dregs mustard base, I obtained purity sinapine thiocyanate samples, re-crystallization of the more the number the higher the purity, the best crystallization of the frequency of three times, the purity is 94.54%.(4) Using UV, IR, HPLC, HPLC-ESI/MS and other means of modern analytical testing purified rapeseed meal sinapine conducted in-depth study of the identification.(5) From the polyphenol oxidase, laccase,β-glucosidase, lipase enzymes, I found that laccase have a higher degradation rate of rapeseed meal sinapine.by a single factor and Box-Behnken optimization tests, I got the optimum parameters were:enzymolysis time 25min, pH5.10, enzymes adding amount 2mL, hydrolysis temperature is 28℃, at this time laccase degradation of rapeseed meal sinapine degradation rate of 81.93%.We have studied the laccase enzyme Michaelis-Menten constant of rapeseed meal sinapine:laccase at 30℃, the optimum pH, under the condition of rapeseed meal sinapine substrate KM values were 1.16mol/L, maximum enzyme reaction rateVmax= 2.38A/(U* min).
        

菜粕芥子碱的提取、分离纯化、鉴定及酶解研究

摘要7-9
Abstract9-10
第一章 绪论11-20
    1 芥子碱研究现状11-18
        1.1 物理化学性质11-12
        1.2 芥子碱检测方法12-14
        1.3 提取方法14
        1.4 分离、纯化方法14-15
        1.5 毒性研究15-16
        1.6 芥子碱的脱除方法研究16-18
    2 论文研究目的、意义及内容18-20
        2.1 研究目的和意义18
        2.2 研究的基本思路与内容18-20
第二章 菜粕芥子碱提取工艺优化20-39
    1 材料与方法20-23
        1.1 材料20-21
        1.2 试验方法21-23
    2.结果与分析23-38
        2.1 芥子碱检测方法的建立23-24
        2.2 索氏提取工艺24-31
        2.3 ASE萃取工艺31-35
        2.4 不同芥子碱提取方法提取后菜粕主要指标比较35-37
        2.5 芥子碱常用提取方法比较37-38
    3.本章小结38-39
第三章 菜粕芥子碱的分离纯化39-48
    1.实验材料与仪器39-40
        1.1 材料与试剂39
        1.2 仪器39-40
    2.实验方法40-42
        2.1 树脂的预处理40
        2.2 装柱40
        2.3 大孔树脂型号的选择40-42
        2.4 芥子碱硫氰酸盐的制备42
    3 实验结果与讨论42-47
        3.1 静态吸附与解吸42-43
        3.2 上样pH值的确定43-44
        3.3 上样流速的确定44
        3.4 洗脱剂浓度的确定44-45
        3.5 洗脱剂流速的确定45
        3.6 最佳吸附时间的确定45-46
        3.7 洗脱终点的确定46-47
        3.8 菜粕芥子碱纯度检测47
        3.9 芥子碱硫氰酸盐样品纯度检测47
    4 本章小结47-48
第四章 菜粕芥子碱的鉴定48-54
    1 材料与方法48-50
        1.1 材料、试剂与仪器48
        1.2 试验方法48-50
    2 结果与分析50-53
        2.1 显色反应50
        2.2 菜粕芥子碱样品的紫外、可见光谱检测50-51
        2.3 菜粕芥子碱样品的红外光谱测定51
        2.4 菜粕芥子碱的液相图谱分析51-52
        2.5 菜粕芥子碱的ESI/MS分析52-53
    3.结论53-54
第五章 菜粕芥子碱酶解研究54-70
    1 材料与方法54-57
        1.1 材料54
        1.2 主要化学试剂54
        1.3 实验仪器及设备54-55
        1.4 工艺流程55
        1.5 实验方法55-56
        1.6 工艺优化设计56-57
    2 结果与分析57-68
        2.1 酶活57-58
        2.2 单因素试验58-62
        2.3 Box-Behnken实验62-66
        2.4 漆酶降解过程中现象与分析66-67
        2.5 漆酶酶解米氏方程67-68
    3.结论68-70
结论70-72
参考文献72-79
致谢79-80
附录80
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