植物甾醇酯的酶法合成

发布时间：2018-05-03 03:00

本文选题：植物甾醇酯 + 酶法合成　；参考：《哈尔滨商业大学》2017年硕士论文

【摘要】：植物甾醇是一种具有生物活性的物质,由于水溶性和脂溶性都不是很好,限制了其应用。通过酶催化的方式,与油酸反应生成植物甾醇油酸酯,它不仅具有植物甾醇与脂肪酸两部分的营养价值,而且改善了植物甾醇的脂溶性和降胆固醇效果,应用范围更广。大豆油脱臭馏出物(SODD)是植物油精炼过程中的副产物,富含游离脂肪酸、植物甾醇、天然维生素E等具有应用价值的物质。因此从大豆油脱臭馏出物中提取出植物甾醇和脂肪酸,并通过酶法合成植物甾醇脂肪酸酯具有重大意义。本实验构建了在非水相中酶法合成植物甾醇酯的反应体系,优化了合成工艺,并对催化酶进行了固定化研究。从大豆油脱臭馏出物中提取出植物甾醇和脂肪酸,根据优化的工艺酶法合成植物甾醇脂肪酸酯,并进行了定性分析。主要研究内容如下:(1)在非水相体系中进行酶促反应,合成植物甾醇酯。分别筛选脂肪酶和有机溶剂介质,实验结果表明,皱褶假丝酵母(Candida rugosa)脂肪酶催化合成植物甾醇酯的酯化率最高;在正己烷中,酶法合成植物甾醇酯的酯化率效果最好。确定了酯化反应的反应体系后,对反应产物进行了薄层色谱分析,在薄层色谱中有新物质生成。对分离纯化后的产物进行红外光谱分析,在1734cm-1处有明显吸收峰,是酯键的特征吸收峰。因此证明反应合成的产物为植物甾醇油酸酯。(2)研究了影响酯化反应的因素,对油酸与植物甾醇的物质的量的比,酶用量,酯化的温度和时间4个因素进行了探讨。经单因素实验考察后,通过甾醇酯化率的响应曲面分析,优化了反应条件:底物摩尔比(油酸/植物甾醇)为3:1,酶用量为底物总质量的7.2%,反应温度为45.3℃,反应时间为25.3h,最优条件下酯化率可达71.95%。(3)采用物理吸附法对CRL脂肪酶(Candida rugosa lipase)进行固定化,以植物甾醇转化率和酶活力为考察指标,实验表明:CRL脂肪酶分别在磷酸缓冲溶液中和有机溶剂正己烷中固定化,前者效果更好;CRL脂肪酶在磷酸缓冲溶液中分别以几种不同载体固定化,进行批式反应,对几种不同固定化酶载体进行筛选,大孔吸附树脂HP-20操作稳定性最好,性能最优。连续反应3次后,酶活力为509.92U/g,酯化反应的酯化率为62.99%,仍有较高的催化能力。(4)研究了溶剂冷却结晶法从大豆油脱臭馏出物中提取出植物甾醇,实验确定最佳提取工艺:丙酮-甲醇混合溶剂的比例为1:2,溶剂原料比为3:1(V/W),养晶时间为18h,洗涤粗甾醇的溶剂为正己烷,洗涤粗甾醇的温度为25℃,洗涤粗甾醇的时间为15min。然后从提取植物甾醇后的滤液中提取出脂肪酸,根据前三章优化好的合成工艺酶法合成植物甾醇酯。通过薄层色谱法鉴定分析,在色谱中有新的物质出现,可初步判定是植物甾醇脂肪酸酯。再通过红外光谱法表征,经红外图谱分析,图中在1737cm-1处有明显的伸缩振动吸收峰,是酯键的特征吸收峰,因此证明了从大豆油脱臭馏出物中提取的植物甾醇和脂肪酸,在非水相中经脂肪酶催化合成的产物为植物甾醇脂肪酸酯。
[Abstract]:Phytosterol is a bioactive substance. Because of its water solubility and fat solubility are not very good, it restricts its application. The plant sterol oleate is produced by enzyme catalyzed reaction with oleic acid. It not only has the nutritional value of two parts of plant sterols and fatty acids, but also improves the fat solubility and cholesterol lowering of plant sterols. Soybean oil deodorization distillate (SODD) is a by-product of plant oil refining, which is rich in free fatty acids, phytosterols, natural vitamin E and other substances. Plant sterols and fatty acids are extracted from soybean oil deodorizer distillate, and phytosterol fatty acid esters are synthesized by enzyme method. In this experiment, the reaction system of phytosterol ester synthesis in non aqueous phase was constructed, the synthesis process was optimized, and the catalytic enzyme was immobilized. Plant sterol and fatty acid were extracted from the deodorizer distillate of soybean oil, and the phytosterol fatty acid ester was synthesized according to the optimized process enzyme method, and the qualitative analysis was carried out. The main contents are as follows: (1) enzymatic reaction in non aqueous phase and synthesis of phytosterol esters. Lipase and organic solvent medium are screened respectively. The results show that the esterification rate of phytosterol esters catalyzed by Candida rugosa lipase catalyzed by crease Candida folds is the highest; esterification efficiency of phytosterol ester in n-hexane After determining the reaction system of esterification, the reaction product was analyzed by TLC, and the new substance was formed in the thin layer chromatography. The infrared spectrum analysis of the purified product was carried out by the infrared spectrum analysis. The absorption peak of the ester bond was found at 1734cm-1, and the product of the reaction was phytosterol oleic acid. (2) the factors affecting the esterification were studied. The ratio of the amount of oleic acid to phytosterol, the amount of the enzyme, the temperature and the time of the esterification were discussed. After a single factor experiment, the reaction strip was optimized by the response surface analysis of sterol esterification rate: the molar ratio of the substrate (oleic acid / phytosterol) was 3:1, the amount of the enzyme was The total mass of the substrate is 7.2%, the reaction temperature is 45.3, the reaction time is 25.3h, the esterification rate can reach 71.95%. (3) under the optimal condition. The physical adsorption method is used to immobilize the CRL lipase (Candida rugosa lipase), and the conversion rate of phytosterol and the activity of the enzyme are considered as the indexes. The results show that the CRL lipase is neutralized in the phosphate buffer solution and the organic matter respectively. The former is immobilized in n-hexane, the former is better, CRL lipase is immobilized on several different carriers in the phosphate buffer solution, batch reaction is carried out, and several different immobilized enzyme carriers are screened. The macroporous adsorption resin HP-20 has the best stability and the best performance. After 3 times the continuous reaction, the enzyme activity is 509.92U/g and esterification reaction. The esterification rate is 62.99% and still has high catalytic ability. (4) the extraction of phytosterol from the deodorizing distillate of soybean oil is studied by the solvent cooling crystallization method. The optimum extraction process is determined by the experiment: the proportion of acetone methanol mixed solvent is 1:2, the ratio of solvent to raw material is 3:1 (V/W), the crystallization time is 18h, the solvent washing crude sterol is n-hexane, and the washing is coarse. The temperature of sterol is 25 degrees C, the time of washing crude sterol is 15min. and then the fatty acid is extracted from the filtrate of phytosterol extraction. The plant sterol ester is synthesized by the optimized synthetic process enzyme method based on the first three chapters. A new substance in the chromatogram is identified and analyzed by TLC, and the phytosterol fatty acid ester can be preliminarily determined. It was characterized by infrared spectroscopy. After the analysis of infrared spectrum, there was an obvious absorption peak of expansion and vibration at 1737cm-1, which was the characteristic absorption peak of ester bond. Therefore, phytosterol and fatty acids extracted from soybean oil deodorizer distillate were proved to be phytosterol fatty acid esters in non aqueous phase.

【学位授予单位】：哈尔滨商业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS202.1

【参考文献】