1、 博士学位论文（同等学力人员）驴油性质及酶法定向酯交换制备功能性油脂的研究RESEARCH ON RESEARCH ON PROPERTIES OFDONKEY FAT AND PRODUCING OFFUNCTIONAL OIL BY ENZYMATICDIRECTIONAL INTERESTERIFICATION许浮萍哈尔滨工业大学2014 年 3月国内图书分类号： TS 222.2国际图书分类号： 577.115学校代码： 10213密级：公开工学博士学位论文（同等学力人员）驴油性质及酶法定向酯交换制备功能性油脂的研究博士研究生： 许浮萍导 师： 张兰威 教授申请学位： 工学博士学 科：
2、 化学工程与技术所在单位： 食品科学与工程答辩日期： 2014 年 3 月授予学位单位： 哈尔滨工业大学Classified Index: TS 222.2U.D.C: 557.115Dissertation for the Doctoral Degree in EngineeringRESEARCH ON PROPERTIES OF DONKEY FATAND PRODUCING OF FUNCTIONAL OIL BYENZYMATIC DIRECTIONALINTERESTERIFICATIONCandidate： Fuping XuSupervisor ： Prof. Lanwei Z
3、hangAcademic Degree Applied for：Speciality ：Doctor of EngineeringChemical Engineering and TechnologySchool of Food Science and EngineeringMarch, 2014Affiliation ：Date of Defence：Degree-Conferring-Institution Harbin Institute of Technology摘 要摘要中国是世界上最大的家驴养殖国，有四千多年的养驴历史。驴油在我国华北地区是较常见的食用动物油脂。但是目前，国内外对驴
4、油的基础研究和功能性研究尚处于空白。本研究首先采用气相色谱仪和薄层色谱仪对驴油的总脂肪酸、 sn-2 位脂肪酸、反式脂肪酸的组成及含量进行了分析，利用高效液相色谱仪对驴油所含的生育酚和胆固醇进行定量分析，利用牛津核磁振动仪测定了不同温度下驴油的固体脂肪含量，对驴油基础物理化学性质中的酸价、碘价、皂化价、过氧化值、折光指数和相对密度进行了测定。经实验测得：驴油总脂肪酸中不饱和脂肪酸 59.30%，含量最高的是油酸为 36.28%。驴油反式脂肪酸含量0.206%。驴油中生育酚总含量 85.9mg/kg ，以 -生育酚含量为最高。驴油胆固醇含量甚微（ 0.03187mg/kg) ，驴油的完全融化温度
5、 39-40。为比较驴油与其他动物油成分的差异，本研究将驴油与猪油、牛油、羊油、鸡油、鸭油、鸵鸟油的理化性质和脂肪酸组成及微量元素的含量进行了比较，得出结论 :在所研究的家畜动物油中，驴油脂肪酸中的不 饱和脂肪酸含量最高，多不饱和脂肪酸的总量高过研究中所测的家畜动物油脂，接近家禽动物油。驴油中 sn-2 位键的不饱和脂肪酸含量同 样高过所测的家畜动物油脂，并且主要高在人体必需脂肪酸上。驴油的反式脂肪酸在所测试动物油中含量最低，并且驴油的胆固醇在所测动物油中含量最低。驴油的生育酚在所测试的动物油中含量最高。在驴油研究的基础上，本课题着重研究了以驴油为基料的功能性油脂的制备方法，建立了无水无溶剂环
6、境脂肪酶对底物的分解与合成在同一体系同步定向制备功能性油脂的研究模型。用驴油做底物，将驴油与共轭亚油酸（CLA）用三维运动恒速常压法提高酯交换率。实验首先寻找并筛选特异性脂肪酶，而后确定单因素反应条件，利用响应曲面分析方法对酶法定向酯交换反应条件进行优化，最终确定了最佳反应条件：用脂肪酶 novozymes435作生物催化剂，酯交换的反应温度 60，底物比为 1:3 （驴油：共轭亚油酸w/w），加酶 量为 7% （与驴油质量比），反应时间 55h，实验得到的共轭亚油酸驴油甘油三酯得率为 47%。讨论了酶法定向酯交换的反应机理，反应中的酸解反应、酸脂交换、酯-酯交换 的中间过渡态复合结构、分子内
7、反应机理，以及脂肪酶的活性中心和反应机理的协同因素。叙述了无溶剂状态下酸解、酸酯同时交换、抑制分-I-哈尔滨工业大学工学博士学位论文子间酯 -酯交换的反应机理。将复杂反应在无溶剂条件下同一体同时完成，为混合物的分离纯化创造了条件。采用二级分子蒸馏法使用转子薄膜分子蒸馏器纯化分离了酶法定向酯交换后生成的功能性甘油三酯的成分，确定出分子蒸馏除去脂肪酸的最佳条件是：一级分子蒸馏的参数为：系统真空度 2.0 Pa，蒸馏温度 230，进料流量 2.0mL/min ，刮膜转速 150rpm。二级分子蒸馏的参数为：系统真空度 0.2Pa，蒸馏温度 310，进 料流量 1.5mL/min ，刮膜转速 350r
8、pm 。为了验证产品的食用安全性，本研究对制备的共轭亚油酸驴油甘油三酯产品进行了食品安全性毒理学评价。实验方法按照国家标准食品安全性毒理学评价程序的规定，采用大鼠、小鼠对所制备的产品进行急性经口实验、遗传毒理实验（包括 Ames 实验、骨髓微核实验、精子畸形实验）和30 天喂养实验。经检测，重组的共轭亚油酸驴油甘油三酯是无毒、无遗传突变的安全性食用级油脂。采用 LNCaP 人体前列腺淋巴结癌细胞系对重组 CLA 驴油进行了增殖抑制作用验证。以具有抗癌功能的 CLA 标准样品（ 99.95%纯度）为阳性对照，无抗癌功能的驴油为阴性对照，对实验制备出的 CLA 含量为 47%的功能油脂进行对 LN
9、CaP 癌细胞增殖抑制比较实验。用 MTT 方法测定增殖情况，得出：驴油、 0.025 mol/L 乙醇溶液、 RPMI-1640+ 乙醇培养液没有显示出对于癌细胞的抑制增殖作用；而 sigma 公司的 CLA 标样、反应实验所用的 CLA 含量为 80%的原料、制备出的 CLA 含量 47%的功能性油脂，显现出对于人体前列腺淋巴结癌细胞的抑制增殖力很强。本实验的结论为：驴油不具有抑制癌细胞增殖的功能，而制备出的重组 CLA 驴油有潜在的抑制癌细胞增殖的功能。关键词：驴油；理化特性；功能性油脂；酶法定向酯交换；抗癌功能验证；安全毒理学评价。- II -AbstractAbstractChina
10、 is the largest donkey breeding country in the world and has a donkeybreeding history of nearly 4000 years. The donkey fat is one of the commonedible animal fats in North China. But until now , the basic research andfunctional study of donkey fat is still in the blank all over the world.This researc
11、h focuses on the composition of donkey fat. With gaschromatograph and thin layer chromatograph, donkey fats total fatty acidcomposition, sn-2 fatty acid composition and trans fatty acid composition wereanalyzed. Also tocopherol and cholesterol were analyzed with the highperformance liquid chromatogr
12、aph. The efraction index and solid fat content indifferent temperatures were determinated with the nuclear magnetic vibrationinstrument and abbe refractometer instrument. Donkey fats fatty acid value,iodine value, saponification value, peroxide value, relative density weredeterminated, too. The conc
13、lusions are followings: the oleic acid content（36.28%） is the highest among all the unsaturated fatty acids（59.30%） intotal fatty acids in donkey fat，and essentidal fatty acids content in the sn-2 ishigher than that in total fatty acids in donkey fat. The trans fatty acids content（0.206%） in donkey
14、fat is far below the national standard of infantal food.Donkey fat contains all four kinds of tocopherols （85.9mg/kg ）and the alphatocopherol content is the highest in the four types of toco pherol. There is verylitter cholesterol in donkey fat（0.03187mg/kg). With the nuclear magneticvibration metho
15、d the completely melt temperature of donkey fat is determinatedas 39- 40.In order to research the differences of donkey fat and some other ani malfats, the comparision of properties, composition and trace element content withdonkey, pig, cattle, sheep, chicken, duck and ostrich fats is needed. Theco
16、nclusions are followings. Firstly, in all the studied livestock animal fats,donkey fat has the highest unsaturated fatty acid content in total fatty acids.Secondly, the total polyunsaturated acid content in donkey fat is higher than thatin all the other studied animal fats and close to the poultry f
17、ats.The sn -2unsaturated acid content in donkey fat is also higher than that in all the studiedlivestock animal fats, mainly higher in essential fatty acids. Thirdly, the transfatty acid content of donkey fat is the lowest among all the studied animal fats.The donkey fat has almost no cholesterol. I
18、n terms of tocopherol content,donkey fat is also the highest and its tocopherol types are most complete amongall the studied animal fats.-III-哈尔滨工业大学工学博士学位论文On the basis of the donkey oil research, this topic focuses on thepreparation method of functional fats with donkey fat as basic material. In t
19、histhesis, a research model about synchronous directional manufacture offunctional fat in the same system with lipase working on decomposition andsynthesis of the substrate in anhydrous solvent free environment isestablished.With three dimensional motion constant speed and pressure method,the ester
20、exchange rate of donkey fat and conjugated linoleic acid (CLA) isenhanced. The experimental finding and screening of specific lipase is in thefirst place and then single factor reaction conditions will be confirmed. Reactioncondition of enzymatic directional ester exchange has been optimized withres
21、ponse surface analysis. Ultimately, the best enzyme substrate reactionconditions are determined like followings: lipase novozymes435 as a biologicalcatalyst, solvent-free environment, simultaneous decomposition and synthesisreaction, ester exchange reaction temperature to be 60, the substrate ratio
22、tobe 1:3 (donkey oil: conjugated linoleic acid w/w), the enzyme added amount tobe 7% (donkey oil mass ratio), reaction time is 55 hours and the optimalforecasted exchange rate value to be 47%.The discussion focuses on the reaction mechanism of enzymatic directionalinteresterification, the middle tra
23、nsition state composite structure of acidolysisreaction, acid-ester exchange and ester-ester exchange, and reaction mechanismwithin molecule of acidolysis reaction. Moreover, synergy factors of activecentre and reaction mechanism of lipase are also involved in this disccusion. Thereaction mechanism
24、of acidolysis, acid ester exchange at the same time,inhibition of intermolecular ester-ester exchange are also narrated in thisresearched. Because several reactions complete at the same time in solvent -freeconditions, the production of the intermediate is avoided. The conditions forseparation and p
25、urification of a mixture of triacylglycerol and fatty acidcomposition are also created because of this.The functional triacylglycerol content after enzymatic directional esterexchange is purified and seperated using secondary molecular distillation withthe rotor film molecular distiller. The optimum
26、 conditions for moleculardistillation method are followings. For the first level of molecular distillation,the parameters are 2.0Pa system pressure, distillation temperature 230, thefeed rate 2.0mL/min and blown film speed 150rpm; for the second level, theparameters are 0.2Pa system pressure, distil
27、lation temperature 310, the feedrate of 1.5mL/min and blown film speed 350rpm.In order to verify the product safety, an evaluation of food safetytoxicology for conjugated linoleic acid of donkey fat triacylglycerol is needed.- IV -AbstractAccording to the national standards , in this research, mouse
28、 and rats were put in acute experiment throughthe mouth, genetic toxicology experiment (including the Ames test, bone marrowmicro nuclear test and sperm malformation test) and 30 days feeding test. Aftertesting, it is seen that all indicators of the triacylglycerol products in thisresearch meet the
29、national standards, so it could be concluded that CLA functionoil is a kind of non-toxic, safe oil without genetic mutations and could be usedin food and pharmaceutical industries.In order to verify the functionality of the product, a preliminary study ofantitumous effect about recombined CLA donkey
30、 triacylglycerol is alsonecessary. Human prostate lymph node cancer cells LNCaP is needed to testanti-proliferative effect of recombined CLA donkey triacylglycerol. Using anti -cancer functional CLA standard sample (99.95% purity) as a positivecomparison and normal donkey fat as a negative compariso
31、n, a comparative testabout antitumous effect to human prostate lymph node cancer cells between bothof them and functional fat from experiment with 47% CLA content is done. TheMTT method is used every 24 hours to see proliferation of tested cells in 96h.We fourd in the first 4 determinations in 72h,
32、donkey fat, ethanol solution of0.025mol/L density and RPMI -1640 cell culture fluid could not inhibit cancercell growth while CLA sample from Sigma Ltd., but raw material with 80%content CLA in molecular rearrangement experiment and the anticancerfunctional oil with 47% content CLA showed ability of
33、 inhibiting cancer cellgrowth. In summary, donkey fat does not have a capacity of cancer cellproliferation. 99.95% CLA sample could inhibit human prostate lymph nodecancer cells growth while recombined CLA donkey triacylglycerol also showedthe potential, similar capacity like CLA sample.Keywords: do
34、nkey fat, physicochemical property, function oil and fat, enzymaticdirectional interesterification, anti-cancer function, safetytoxicology evaluation.- V -哈尔滨工业大学工学博士学位论文目录摘要. IAbstract III第 1章绪论 11.1 课题背景及研究的目的和意义 . 11.2 驴油 . 21.2.1 驴油的产量 . 21.2.2 驴油的研究现状 21.3 功能性油脂 31.3.1 脂肪对人体的作用 41.3.2 功能性油脂的意义
35、41.3.3 功能性油脂分类 51.3.4 功能性油脂的研究状况 51.4 功能性油脂的制备 61.4.1 化学合成法 . 61.4.2 生物酶定向酯交换法 71.4.3 酶法定向酯交换反应 81.4.4 酶法定向酯交换技术的产品 91.5 共轭亚油酸 111.5.1 共轭亚油酸的简介 111.5.2 共轭亚油酸的生理功能 111.5.3 共轭亚油酸的来源 131.5.4 共轭亚油酸制备现状 131.5.5 共轭亚油酸直接食用的害处 141.6 共轭亚油酸功能性油脂 . 141.6.1 国内研究状况 . 141.6.2 国外研究状况 151.7 本论文主要研究内容 15第 2章实验材料与方法
36、. 162.1 实验材料与仪器设备 162.1.1 主要仪器和设备 16-VI-目 录2.1.2 主要化学试剂 . 172.1.3 动物油脂样品的采集 182.1.4 菌株和细胞 . 202.1.5 主要溶液配制 . 202.2 测定方法 212.2.1 脂肪含量的测定及制备中性油 . 212.2.2 油脂物理化学性质的测定 222.2.3 油脂中脂肪酸组成的测定 222.2.4 反式脂肪酸组成的测定 232.2.5 Sn-2 甘油一酯脂肪酸组成的测定 . 232.2.6 生育酚的测定 . 242.2.7 油脂中胆固醇含量测定 252.3 实验方法 262.3.1 甘油一酯脂肪酸分析方法的改进
37、 . 262.3.2 脂肪酶的筛选 . 292.3.3 重组油脂中 CLA 交换率的确定 . 292.3.4 酶法定向酯交换实验设计 302.3.5 混合物分离纯化实验 322.3.6 分子蒸馏分离甘油三酯与脂肪酸 . 332.3.7 重组 CLA 驴油甘油三酯的毒理学评价 . 342.3.8 重组 CLA 驴油甘油三酯对 LNCaP 细胞的增殖抑制作用 . 362.4 数据处理与统计分析 39第 3章驴油特性及与其它动物油的比较 403.1 引言 . 403.2 驴油的脂肪酸组成 403.2.1 驴油总脂肪酸组成 413.2.2 驴油的反式脂肪酸组成 423.2.3 甘油一酯脂肪酸分析方法的
38、改进 . 433.3 驴油中 、 、 生育酚含量 473.4 驴油的胆固醇含量 483.5 驴油与其他动物油脂肪酸组成比较 . 493.5.1 总脂肪酸含量的比较 493.5.2 反式脂肪酸含量的比较 51-VII-哈尔滨工业大学工学博士学位论文3.5.3 Sn-2 位甘油一酯脂肪酸组成和含量的比较 523.6 驴油与动物油生育酚含量的比较 . 533.7 驴油与动物油胆固醇含量的比较 . 543.8 驴油与动物油基本物理化学性质比较 553.9 驴油与其他动物油 SFC 值的比较 573.10 驴油特性及与其它动物油比较的讨论 593.11 本章总结 60第 4章酶法定向酯交换制备 CLA甘
39、油三酯的研究 . 624.1 引言 . 624.2 脂肪酶的筛选 634.3 CLA 甘油三脂的酯交换率 . 644.3.1 样品中 CLA 交换率的确定 644.3.2 交换率的计算 . 654.4 CLA 酶法定向酯交换反应 . 654.4.1 CLA 酶法定向酯交换反应单因素实验 . 654.4.2 CLA 甘油三脂酶法定向酯交换反应优化实验 694.4.3 响应曲面图和等值线图 704.5 分子蒸馏分离纯化甘油三脂 . 764.5.1 一级蒸馏影响因素 764.5.2 二级蒸馏影响因素 794.6 重组 CLA 驴油与天然驴油性质比较 824.6.1 外观比较 . 824.6.2 总脂
40、肪酸和 sn-2 位脂肪酸组成比较 834.7 酶法定向酯交换制备 CLA 甘油三酯反应的讨论 864.8 本章小结 90第 5章重组驴油的毒理学实验及其功能初探 925.1 引言 . 925.2 重组 CLA 驴油甘油三酯食用毒理学评价 935.2.1 小鼠急性经口毒性实验 935.2.2 遗传毒性实验 . 935.2.3 30 天喂养实验 . 965.3 重组驴油甘油三酯对 LNCaP 癌细胞增殖抑制作用 . 1015.3.1 细胞培养 . 102-VIII-目 录5.3.2 细胞最适接种量的确定 1025.3.3 重组驴油水解物对 LNCaP 癌细胞最适作用浓度的确定 1035.3.4
41、MTT 实验检测样品对前列腺癌细胞（ LNCaP 细胞）增殖的影响1055.4 重组驴油毒理学评价及对 LNCaP 癌细胞增殖抑制作用讨论 1085.5 本章小结 109结论 110展望 112参考文献 113附录 124攻读博士学位期间发表的学术论文及其它成果 . 127哈尔滨工业大学学位论文原创性声明及使用授权说明 . 128致谢 129个人简历 130-IX-哈尔滨工业大学工学博士学位论文ContentsAbstract (In Chinese) IAbstract (In English) IIIChapter 1 Introduction . 11.1. Background, ob
42、jective and significance of the subject . 11.2. Introduction of donkey fat 21.2.1. Estimation of donkey fat 21.2.2. Research significance of donkey fat 21.3. Functional oils and fats 31.3.1. Role of fat in human body . 41.3.2. Significance of functional oils and fats 41.3.3. Classification and physi
43、ology of Functional oils and fats 51.3.4. Current research status of Functional oils and fats. 51.4. Technology research status of functional oils and fats . 61.4.1. Method of Chemical synthesis . 61.4.2. Method of Enzymatic directional interesterification 71.4.3. Reaction of mechanism Enzymatic dir
44、ectional interesterification 81.4.4. Product of Enzymatic directional interesterification 91.5. Conjugated linoleic acid oil . 111.5.1. Brief introduction of conjugated linoleic acid 111.5.2. Physiological functions of conjugated linoleic acid . 111.5.3. Natural source of conjugated linoleic acid .
45、131.5.4. Preparation of Conjugated linoleic acid . 131.5.5. Directly eating harm of conjugated linoleic acid . 141.6. Research present of conjugated linoleic acid triacylglycerol 141.6.1. Domestic situation . 141.6.2. Abroad situation 151.7. Mainly research content of thesis . 15Chapter 2 experimental materials and methods . 162.1. Experimental materials and equipment . 162.1.1. Instruments and equipment 1
