【作者】 曾凡逵；

【导师】 宁正祥；

【作者基本信息】 华南理工大学 ， 食品科学， 2010， 博士

【摘要】 本研究采用酶法生产单油酸甘油酯,以甘油和茶籽油为底物,对催化过程进行了工艺优化。通过对三种商品化的脂肪酶Lipozyme RM IM、Lipozyme TL IM和Novozym 435进行筛选,发现:在叔丁醇溶剂体系中Novozym 435是催化茶籽油甘油解反应合成单油酸甘油酯(Glycerol monooleate, GMO)的最适合的酶。在批次反应中,研究了不同的反应条件如底物摩尔比、底物浓度和温度对单油酸甘油酯生成量和甘油三酯的转化率的影响。最佳的反应条件为底物摩尔比6:1(甘油/茶籽油),底物浓度为40%,反应温度为50℃。在这个最佳反应条件下,经过10h反应,茶籽油的转化率为98.7%,甘油酯中MAG的含量为82.0%。用一根装有4.5g Novozym 435酶的填充床反应器(Packed bed reactor,PBR)连续催化茶籽油甘油解合成单油酸甘油酯,当进料速度为0.25 mL/min时,产物中单脂肪酸甘油酯(Monoacylglycerol, MAG)的含量为80.74%,经过38天长期运行填充床反应器,平均MAG的产能为0.698 Kg MAG/(Kg酶·h)。针对茶籽油为底物的甘油解反应,建立了酶促反应动力学方程。Novozym 435催化茶籽油甘油解合成单油酸甘油酯符合非连续性多底物酶促反应机制即乒乓机制,根据反应机理推导出动力学方程,将实验数据代入推导出来的动力学方程求得动力学常数并对动力学方程进行验证。改变反应体系中酶、水、甘油和茶籽油的添加量,发现实验值和所建立的动力学方程比较吻合。本课题还通过模拟探讨了甘油和茶籽油的添加量对GMO最大生成初速率及反应达到平衡时GMO的产率的影响的协同作用,从模拟出来的结果可以看出高浓度的茶籽油可以获得较高的GMO最大生成初速度,但是会导致GMO的产率下降。当甘油和茶籽油的添加量分别为91.62 mM和15.27mM,Novozym435和水的添加量分别为1.09g和12.59mM时,MAG的最大生成初速率为9.773 mM·h-1,对应的MAG的产率为1.8379 mM MAG·mM-1 TAG。采用分子蒸馏对酶法合成的产物GMO进行分离。正交实验结果表明在蒸发温度(ET)、进料流速(Q)、进料温度(FT)和冷凝温度(CT)四个因素当中ET和Q是影响分子蒸馏分离纯化GMO最重要的两个因素。通过响应面实验设计建立了轻相中MAG、DAG的含量对蒸发温度和进料流速的回归方程:MAG=-577.41+7.44ET-34.13Q+0.24ET×Q-0.02ET2-5.28Q2;DAG=801.97-8.51ET-1.64Q+0.01ET×Q+0.02ET2-0.16Q2,实验值和模型比较吻合。GMO的回收率与其预期的纯度相关,当采用分子蒸馏条件为:Q=0.71mL/min,FT=70℃,CT=60℃,ET为170-190℃之间,可以得到纯度为80%以上的GMO,对应的GMO的回收率为60-80%。通过二级分子蒸馏,Q=1mL/min,ET=180℃,FT=90℃,CT=60℃,得到纯度为98.9%的GMO,对应的回收率为33.42%。对GMO的理化性质分析,并进行了冰淇淋的应用试验研究。结果表明,GMO的含量为82.42% (w/w),酸价为2.1,游离甘油含量为3.21% (w/w),羟基价为314,碘值为78,铅含量为0.08 mg/Kg,灰分为0.03% (w/w),皂化值为175,完全符合美国食品化学品法典第五版(FCC-V)对GMO的质量指标要求。酶法合成的GMO的脂肪酸组成为C12:0,0.06%;C14:0,0.08%;C16:0,10.59%;C16:1,0.13%;C18:0,3.36%;C18:1,73.91%;C18:2,1.87%;其他,3.13%。GMO在室温下是一种蜡状固体,有轻微的油脂味,能溶于热酒精和氯仿,在常温的酒精、乙醚和石油醚中的溶解度很低,不溶于水。GMO的融点约为35-36℃,HLB值为3.7。抗氧化剂BHT、Vc棕榈酸酯和TBHQ能有效地抑制GMO的氧化,添加0.015%BHT、0.015%TBHQ和0.015%Vc棕榈酸酯的GMO在常温下酸价(AV)上升到FCC-V规定的6.0所需要的时间为33-36个月,是空白对照样品的1.38-1.71倍。单油酸甘油酯作为乳化剂在一定程度上能弥补单硬脂酸甘油酯低脂冰淇淋由于脂肪含量降低给冰淇淋口感带来的不利影响。单油酸甘油酯将冰淇淋内部的酪蛋白从脂肪球表面分散开,并将更多的脂肪球簇集到空气泡周围的能力更强,使得最终产品的抗融性效果更好。更多还原

【Abstract】 Three commercial immobilized lipases, Lipozyme RM IM, Lipozyme TL IM and Novozym 435, were screened for the production of GMO (Glycerol monooleate)by glycerolysis of camellia oil in a solvent medium of tert-butyl alcohol. Novozym 435 showed the best performance and was selected to catalyze the subsequent glycerolysis reaction. For the batch reaction, different reaction conditions, substrate mole ratio, substrate concentration and temperature, for the GMO concentration and conversion rate of TAG, were investigated. The optimal reaction conditions were determined as 6:1 mole ratio of glycerol to camellia oil at 40% (w/v) of substrate concentration in tert-butyl alcohol at a reaction temperature of 50℃. Under these optimal conditions, the conversion rate of camellia oil was 98.7% (10 h), and the mixture of acylglycerols contained 82.0% of MAG. A packed-bed reactor (PBR) system with 4.5 g Novozym 435 was employed in continuous production, the resulting product mixture of acylglycerols contained 80.74% of MAG and was obtained at a flow rate of 0.25 mL/min of substrates. The long-term operation of the PBR system gave an average productivity of 0.698 Kg MAG/(Kg enzyme·h) after 38 days of operation.Kinetic study of GMO production by glycerolysis of camellia oil using Novozym 435 has also been investigated, and a mathematical model that took into account for mechanism of glycerolysis reaction using camellia oil as substrate was developed. The kinetic parameters were estimated by fitting experimental data of glycerolysis reaction of camellia oil by Novozym 435 into proposed model equations. There was a good agreement between experimental results and those proposed model predicted equations under conditions of various enzyme, water, glycerol and camellia oil concentrations. From proposed model equations, the synergic effect of glycerol and palm olein concentrations was simulated. The simulation results show that the behavior of high substrate concentrations obtained high initial production rates but low yields of MAG. Thus, with a concentration of 15.27mM of camellia oil and 91.62 mM of glycerol, the concentrations of Novozym 435 and water were 1.09g and 12.15 mM, respectively, the high initial production rate of MAG of 9.733mM·h-1 and corresponding yield of 1.8379 mM MAG·mM-1 TAG were obtained.In this study, molecular distillation (MD) was used to purify GMO synthesized lipase-catalyzed reaction. Orthogonal design was carried out in order to study the effect of operating conditions on the mass ratio (D/F) using MD process to purifying GMO. Among the four studied variables, evaporator temperature (ET), feed flow rate (Q), feed temperature (FT) and condenser temperature (CT), only ET and Q are important at the studied experimental conditions. The model equations of MAG and DAG concentration in the outlet stream to the operating conditions ET and Q was proposed: MAG=-577.41+7.44ET-34.13Q+0.24ET×Q-0.02ET2-5.28Q2; DAG=801.97-8.51ET-1.64Q+0.01ET×Q+0.02ET2-0.16Q2, there was a good agreement between experimental results and those predicted equations. The MAG recovey rate depends strongly on the purity of MAG was desired, GMO with a purity of 80% can be obtained by the operating conditions of Q=0.71ml/min,FT=70℃,CT=60℃,ET 170-190℃, the corresponding recovery rate of GMO is 60-80%. A strategy that consisting of two distillation steps was developed to obtain distilled MAG, Q=1mL min, ET=180℃, FT=90℃, CT=60℃, MAG with a purity of 98.9% can be obtained, the corresponding recovery rate of MAG was 33.42%.Physical and chemical analysis of products indicated that, the GMO synthesized by lipase-catalyzed reaction contain 82.42% of MAG, acid value 4.10, free glycerin 3.21%, hydroxyl value 314, iodine value 78, lead 0.08 mg/kg, residue on ignition 0.03%, saponification value 175 , which met the requirements of FCC-V very well. The fatty acid composition is C12:0, 0.06%; C14:0, 0.08%; C16:0, 10.59%; C16:1, 0.13%; C18:0, 3.36%; C18:1, 73.91%; C18:2, 1.87% and others 3.13%. GMO occurs as a waxy solid at room temperature. It has a mild, fatty taste. It is soluble in hot alcohol and in chloroform; very slightly soluble in cold alcohol, in ether, and petroleum ether; and insoluble in water. It melts at 35-36℃, The hydrophilic-lipophilic balance (HLB) of GMO is 3.7. The storage stability of GMO with added antioxidants BHT, L-ascorbyl palmitate and TBHQ could be significantly improved, the sample with added antioxidants mixed by 0.015% BHT, 0.015% L-ascorbyl palmitate and 0.015% TBHQ had the highest stable degree of 33-36 month guarantee in room temperature, which was 1.38-1.71 times higher than that of control group. In certain sense, GMO used as emulsifier in low fat ice cream was able to compensate for the decrease of sensory quality of low fat GMS ice cream. Unsaturated GMO was more powerful than saturated GMS at displacing caseins from the fat interface and brought more partially coalesced fat to air interface, giving the final product better melting resistance.更多还原