| 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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