摘要
食用油在高温烹调时不饱和脂肪酸会发生异构化产生反式脂肪酸（trans-fatty acid，TFA）。TFA是一类含有一个或多个非共轭反式双键的脂肪酸，研究证实大量摄入TFA容易诱发心血管疾病、认知功能衰退、糖尿病和乳腺癌等疾病。长期以来学者们对TFA的研究主要围绕氢化油及其制品，而关于高温烹调食用油来源的TFA研究报道较少，特别是关于食用油在高温加热过程中TFA的形成种类、安全性及形成机理尚不明确。本文以世界上产量和消费量最多的油脂即大豆油为对象，系统研究了大豆油在高温加热形成的TFA种类及其安全性，以及热致异构化形成TFA的机理及其关键影响因素，并提出了油脂加热过程中TFA的有效调控措施。主要研究结论如下：
（1）综合考察和评价了油酸、亚油酸和亚麻酸顺反异构体的气相色谱法和大豆油样品的甲酯化前处理方法，氢氧化钾甲醇结合气相色谱法能够分离大多数顺反异构体，精密度高，检出限低，总体上满足大豆油中顺反脂肪酸的定性定量要求。在以60℃为初始柱温的条件下，油酸和亚油酸顺反异构体能够分离（分离度R≥0.8）；部分亚麻酸异构体不能完全分离，定量时采取近似分析法；各异构体的日内和日间精密度在0.68%~7.26%之间；各异构体的仪器检出限在0.071~0.129mg/L之间，最小测定限在0.237~0.432mg/L之间；各顺反异构体的仪器响应值与浓度呈良好的线性相关（R2>0.999）。氢氧化钾甲醇法操作简便，且甲酯化率大于90%，各异构体的方法检出限介于0.003~0.006g/100g之间，定量限介于0.011~0.020g/100g之间。
（2）利用衰减全反射傅立叶红外光谱和GC-MS法鉴定了大豆油不饱和脂肪酸高温异构化反式产物种类，包括1种反式油酸（C18:1-9t）、3种反式亚油酸（C18:2-9c,12t、C18:2-9t,12c和C18:2-9t,12t）和5种反式亚麻酸（C18:3-9t,12t,15c、 C18:3-9t,12c,15t、 C18:3-9c,12c,15t、C18:3-9c,12t,15c和C18:3-9t,12c,15c），均为非共轭构型。大豆油以240℃加热12h后，TFA含量由对照组的0.089g/100g增加至7.756g/100g，其中主要反式亚油酸和反式亚麻酸，分别占TFA总量的40.82%和58.52%。大豆油中的TFA种类和含量均随着加热时间的延长而不断增加。
（3）利用量子化学的密度泛函理论对油酸、亚油酸和亚麻酸顺反异构化反应进行了理论计算，提出了直接异构化和亚油酸质子转移异构化2种反应机理，通过结构和能量变化阐明了两种机理对应的反应途径。在B3LYP/6-311++G**基组水平上获得了平衡态和过渡态的键长、二面角、零点能、热焓、吉布斯自由能变、活化能和速率常数等信息，通过振动频率分析和内禀反应坐标势能曲线确证了反应过渡态和反应途径。反式双键键长（1.333）比顺式双键键长（1.337）要短，键能更大，分子更稳定；反式脂肪酸的平衡态能量要低于顺式脂肪酸，热稳定性更好。①按照直接异构化反应理论，油酸异构化包含1种过渡态和1条途径，亚油酸异构化包含4个过渡态和2条途径；亚麻酸异构化包含12种过渡态和6条反应途径。顺式亚油酸需克服236.9kJ/mol以上的能垒形成双自由基渡态，且反式双键越多，所需克服的能垒数量越多；反应温度越高，速率常数越大。②按照质子转移异构化理论，亚油酸分子中的氢原子向邻近中心碳原子迁移形成中间产物，单一反应途径需克服286.4kJ/mol的能垒，包含有2个过渡态和1个中间体，过渡态中间产物为一个包含3个碳原子和4个氢原子的π34离域体系。
（4）研究了影响加热大豆油TFA形成的3个关键因素即温度、氮气和抗氧化剂，降低加热温度和添加耐热性好的抗氧化剂是控制TFA形成的有效措施。温度是不饱和脂肪酸热致异构化的主导和最关键因素，在160℃~240℃范围内，TFA种类和含量随着温度的增加而而呈指数增加趋势，拟合方程为y=2×10~(-5)e0.0552x，拟合程度较好（R~2=0.9914）；充氮处理能够促进大豆油加热形成TFA，经240℃加热过程中，大豆油中TFA含量增加趋势比对照组更明显，加热3h后充氮组中TFA含量达4.904g/100g，显著高于对照组中TFA含量（2.816g/100g）；BHT、BHA和维生素E等抗氧化剂对大豆油加热过程中反式脂肪酸的抑制作用不明显，而TBHQ、茶多酚和迷迭香提取物能显著降低大豆油中TFA的含量，抑制率分别为12%，30%和35.66%，其中迷迭香提取物的耐热性和抗异构化能力最强。
（5）人脐静脉内皮细胞凋亡和损伤实验表明，含有大量TFA的加热大豆油和氢化大豆油能够引起内皮细胞的轻微凋亡和细胞功能的明显损伤。研究比较了大豆油加热过程中TFA与酸价、羰基价和极性组分等理化指标变化情况，结果表明TFA是最敏感最易超标的安全指标。加热大豆油（TFA：6.11%和氢化大豆油（TFA：19.26%）凋亡后期的细胞即死亡细胞数量均高于对照组，但差异不显著，对细胞凋亡的影响有限。TFA能够干扰内皮细胞的对NO的正常分泌，加热大豆油和氢化大豆油处理的内皮细胞NOS酶活力要显著低于对照组（P <0.05），前者的NO分泌量也显著低于后者（P <0.05）。加热或氢化处理过的大豆油均能显著影响人脐静脉内皮细胞的LDH活性，其中氢化大豆油对LDH活性的影响最为明显（P <0.05）。TFA含量是大豆油加热过程中最敏感的限量指标，与酸价、羰基价和极性组分等指标相比，TFA在更短的加热时间内就积累到限量值。
Trans-fatty acids (TFA) would be produced when edible oil is cooked under high temperature. TFAis a kind of fatty acid that contains at least one non-conjugate trans double bond. Large intake of TFAmay easily cause cardiovascular disease, cognitive decline, diabetes and breast cancer. For a long time,study of TFA focused on hydrogenated oil and its products, but there had been less reports aboutresearches on TFA that from cooking oils heated under high temperature, especially about the types ofTFA formed, its damange to health and the formation mechanism. Since soybean oil is the mostlyconsumed and produced edble oil in the world, we systematically studied the formation of TFA insoybean oil under high temperature including TFA types and its harmness, and isomerizationmechanism of the TFA and key influence factors, and we also put forward the effective measures tocontrol TFA in heated soybean oil.The main results of this research are as follows:
(1) GC analysis of oleic acid, linoleic acid and linolenic acid cis-trans isomers and methyl ester ofsoybean oil sample pretreatment method were comprehensively inspected and evaluated. Potassiumhydroxide methanol combined with gas chromatography can separate most cis trans isomer with highprecision and low detection limit, and this method generally can satisfy quantitative requirements of cistrans fatty acid in soybean oil. The chromatographic column temperature program was determined as:60℃(5min)→25℃/min to160℃(5min)→2℃/min→225℃(15min); Oleic acid andlinoleic acid cis-trans isomers were separated well (R≥0.8); Some types of linolenic acid isomercannot be completely separated, thus approximately method was adopt for quantitative analysis; Thecis-trans isomers of interday precisions among0.68%~0.68%and intraday precisions were among1.82%~1.82%; The detection limits of cis-trans isomers were among0.071~0.129mg/L, determination ofminimum limits were among0.237~0.432mg/L; The cis-trans isomers of instrument response valueand the concentration has good linear correlation (R2>0.999). Potassium hydroxide methanol method issimple with high methyl rate over90%, the detection limit is0.003~0.006g/100g, and quantitativelimit is0.011~0.020g/100g.
(2) Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and GC-MSwere used to determine the types of TFA, which were1trans oleic acid (C18:1-9t),3trans linoleic acid(C18:2-9c,12t、C18:2-9t,12c and C18:2-9t,12t) and5linolenic acid (C18:3-9t,12t,15c, C18:3-9t,12c,15t,C18:3-9c,12c,15t, C18:3-9c,12t,15c and C18:3-9t,12c,15c). TFA content in soybean oil increased from0.089g/100g to7.756g/100g after heat treatment at240℃for12h. Trans linolenic acid and linoleicacid were the main TFA formed in soybean oils, where were40.82%and58.52%, respectively.
(3) The quantum chemistry density functional theory (DFT) was used for the theoretical calculationof cis-trans isomerization reaction of oleic acid, linoleic acid and linolenic acid. Two mechanism (directisomerization and proton transfer isomerization) was proposed by illustrating the change of structureand energy approach. Bond length, dihedral Angle, zero correction energy, enthalpy, gibbs free energychange, the reaction activation energy and rate constant of the equilibrium and transition states were caculated at the B3LYP/6-311++G**level. And the reaction transition state and the reaction pathwaywas confirmed through the analysis of the vibration frequency of intrinsic reaction coordinate (IRC)potential energy curve. The bond length of trans double bond was1.333, which was shorter than thecis one (1.337). This meaned that trans double bond had higher bond energy and the molecular oftrans fatty acid was more stable to heat.①According to the direct isomerization reaction theory, theoleic acid isomerization contains one transition state and one isomerizaion path, linoleic acidisomerization contains four transition state and two isomerizaion paths; Linolenic acid isomerizationcontains12types of transition states and6types of isomerization paths. The transition state containsbiradical structure. Single cis double bond had to overcome more than200kJ/mol enery to become thetransition state. More trans double bond to form, more energy barrier needed. The higher the reactiontemperature, the larger the reaction rate constant.②According to proton transfer isomerization theory,the hydrogen atom of linoleic acid was migrated to the center adjacent carbon atoms in the molecule.Each reaction pathway contained a two transition state and one intermediate. Transition state has freeradical structure that is not stable. The intermediates contains3carbon atom and four hydrogen atoms,which seems like a π34delocalized molecular system.
(4) Three key factors (temperature, nitrogen and antioxidants) which influence the TFA contents inheated soybean oil and effective measures was proposed to control the TFA. Temperature is the mostimportant key factors to the isomerization of unsaturated fatty acids in heated soybean oil. Within thescope of the160℃~240℃, TFA types and content increased with the increase of temperature byexponential increase trend, with fitting equation y=2×10-5e0.0552x(R=0.9914); Sufficient nitrogenprocessing can promote soybean oil heating form TFA. The TFA content increased more significantlythan the control group in soybean oil after240℃heating process. TFA content in heating nitrogenfilled group after3h up to4.904g/100g, the TFA content is significantly higher than control group(2.816g/100g); The inhibitory effect of BHT, vitamin E and BHA and trans fatty acids in soybean oilheating process is not obvious, while TBHQ, tea polyphenol and rosemary extract can significantlyreduce the TFA content in soybean oil, with inhibition rate of12%,30%and35.66%. The rosemaryextract shows the best heat resistance and maximum isomerization resistance.
(5) The security features of heated soybean oil and hydrogenated soybean oil were studied throughthe endothelial cell damage experiment analysis and studies. TFA and acid value, carbonyl value andpolar components during the heating process of soybean oil were compared. Endothelial cell damageexperiments showed that6.11%TFA content heated soybean oil and19.26%TFA content ofhydrogenated soybean oil at a later stage of apoptosis cells called dead cells were higher than controlgroup, but there were no significant difference, which means the effect of cell apoptosis was limited.Heated and hydrogenated soybean oil both decreased the endothelial NOS enzyme activity. NOproduction of the former is significantly lower than the latter, that TFA can interfere with the normalsecretion of endothelial cells on the NO. Heat treated or hydrogenated soybean oil could significantlyinfluence the LDH activity of human umbilical vein endothelial cells, and hydrogenated soybean oilwas most evident influence on the activity of LDH (P <0.05). TFA content is the most sensitive indicators of soybean oil heating process. And compared with acid value, carbonyl value and polarcomponents, TFA is easy to accumulate to the limit value in much shorter time.
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