本文選題：植物甾醇酯 + 酶法合成　； 參考：《哈爾濱商業(yè)大學》2017年碩士論文

【摘要】：植物甾醇是一種具有生物活性的物質,由于水溶性和脂溶性都不是很好,限制了其應用。通過酶催化的方式,與油酸反應生成植物甾醇油酸酯,它不僅具有植物甾醇與脂肪酸兩部分的營養(yǎng)價值,而且改善了植物甾醇的脂溶性和降膽固醇效果,應用范圍更廣。大豆油脫臭餾出物(SODD)是植物油精煉過程中的副產物,富含游離脂肪酸、植物甾醇、天然維生素E等具有應用價值的物質。因此從大豆油脫臭餾出物中提取出植物甾醇和脂肪酸,并通過酶法合成植物甾醇脂肪酸酯具有重大意義。本實驗構建了在非水相中酶法合成植物甾醇酯的反應體系,優(yōu)化了合成工藝,并對催化酶進行了固定化研究。從大豆油脫臭餾出物中提取出植物甾醇和脂肪酸,根據優(yōu)化的工藝酶法合成植物甾醇脂肪酸酯,并進行了定性分析。主要研究內容如下:(1)在非水相體系中進行酶促反應,合成植物甾醇酯。分別篩選脂肪酶和有機溶劑介質,實驗結果表明,皺褶假絲酵母(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。確定了酯化反應的反應體系后,對反應產物進行了薄層色譜分析,在薄層色譜中有新物質生成。對分離純化后的產物進行紅外光譜分析,在1734cm-1處有明顯吸收峰,是酯鍵的特征吸收峰。因此證明反應合成的產物為植物甾醇油酸酯。(2)研究了影響酯化反應的因素,對油酸與植物甾醇的物質的量的比,酶用量,酯化的溫度和時間4個因素進行了探討。經單因素實驗考察后,通過甾醇酯化率的響應曲面分析,優(yōu)化了反應條件:底物摩爾比(油酸/植物甾醇)為3:1,酶用量為底物總質量的7.2%,反應溫度為45.3℃,反應時間為25.3h,最優(yōu)條件下酯化率可達71.95%。(3)采用物理吸附法對CRL脂肪酶(Candida rugosa lipase)進行固定化,以植物甾醇轉化率和酶活力為考察指標,實驗表明:CRL脂肪酶分別在磷酸緩沖溶液中和有機溶劑正己烷中固定化,前者效果更好;CRL脂肪酶在磷酸緩沖溶液中分別以幾種不同載體固定化,進行批式反應,對幾種不同固定化酶載體進行篩選,大孔吸附樹脂HP-20操作穩(wěn)定性最好,性能最優(yōu)。連續(xù)反應3次后,酶活力為509.92U/g,酯化反應的酯化率為62.99%,仍有較高的催化能力。(4)研究了溶劑冷卻結晶法從大豆油脫臭餾出物中提取出植物甾醇,實驗確定最佳提取工藝:丙酮-甲醇混合溶劑的比例為1:2,溶劑原料比為3:1(V/W),養(yǎng)晶時間為18h,洗滌粗甾醇的溶劑為正己烷,洗滌粗甾醇的溫度為25℃,洗滌粗甾醇的時間為15min。然后從提取植物甾醇后的濾液中提取出脂肪酸,根據前三章優(yōu)化好的合成工藝酶法合成植物甾醇酯。通過薄層色譜法鑒定分析,在色譜中有新的物質出現(xiàn),可初步判定是植物甾醇脂肪酸酯。再通過紅外光譜法表征,經紅外圖譜分析,圖中在1737cm-1處有明顯的伸縮振動吸收峰,是酯鍵的特征吸收峰,因此證明了從大豆油脫臭餾出物中提取的植物甾醇和脂肪酸,在非水相中經脂肪酶催化合成的產物為植物甾醇脂肪酸酯。
[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.

【學位授予單位】：哈爾濱商業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：TS202.1

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