【作者】 曹文明；

【导师】 王兴国；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2013， 博士

【摘要】 三酰甘油氧化聚合物（Oxidized triacylglycerol polymers，TGP）是油脂氧化过程中产生的一类极其复杂的深度氧化产物，在油脂的加工、储藏、使用过程中TGP总量持续递增，经再次常规精炼也不衰减，是一个可以客观表征油脂氧化程度的内源性指标。本文针对目前油脂氧化指标存在的缺陷和高灵敏度TGP测定的标准技术缺失等问题，开展了TGP与油脂氧化关联性及其在油脂质量评价中应用的相关研究，主要研究内容如下：1、TGP的制备型快速柱层析-高效体积排阻色谱检测方法研究。采用制备型快速柱层析-高效体积排阻色谱（PFC-HPSEC）技术，研究建立了一种适于检测冷榨油和精炼食用植物油等低含量TGP（小于3%）油脂的检测方法，用中压Flash柱预分离油脂极性组分，经高效体积排阻色谱细分TGO、TGD、ox-TG、DG、FFA等组分，用聚苯乙烯标准相对分子量定性，根据重量法测定油脂极性组分含量及高效体积排阻色谱面积归一法定量油脂中TGO和氧化三酰甘油二聚物（TGD）百分含量，结果表明：TGO和TGD2分别在28-1800mg/L、11-2800mg/L范围内线性关系良好，相关系数（R）均高于0.99，TGO及TGD的最低检测限分别为28mg/L、11mg/L，定量下限分别为113mg/L、44mg/L；相当于油脂中TGP的最低检测限为0.01%(100μg/g)。制备型快速柱层析的3个极性组分加标水平的平均回收率为95%-98%。制备型快速柱层析-高效体积排阻色谱检测油脂TGP的重复性RSD均小于10%。制备型快速柱层析-高效体积排阻色谱方法与经典的硅胶柱-高效体积排阻色谱方法在定量油脂TGP的相对误差低于8.9%。2、TGP与油脂氧化程度关联性研究。采用高效体积排阻色谱（HPSEC）技术，对食用植物油室温储藏氧化、Schaal烘箱法加速氧化、高温煎炸氧化等三种油脂氧化过程中产生的极性组分（PCs）及其细分组分进行了比较分析，同时监测了油脂氧化过程中内源性天然抗氧化剂VE的含量变化，建立TGP与油脂氧化程度关联性，结果表明：氧化三酰甘油（ox-TG）的量与诱导期呈现良好的线性关系。氧化三酰甘油寡聚物（TGO）的出现及内源性天然抗氧化剂VE的耗尽标志诱导期结束的。TGO基本都是氧化三酰甘油的三聚体，推测主要是含环氧基、酮基、醛基、C-C键桥、C-O-C键桥的甘油酯。ln(TGO/TGO0)与大豆油氧化程度成线性关系。3、TGP的凝胶渗透色谱色谱制备与稳定性研究。采用制备型快速柱层析联用高压凝胶渗透色谱技术，从油脂极性组分制备mg量级TGO和TGD，考察了TGP的二聚体和寡聚体在多种物理状态与储藏条件下的稳定性，用高效体积排阻色谱测定了极性组分各相关组分的分子量，结果表明：高压凝胶渗透色谱制备TGP的优化条件为：采用三级串联凝胶渗透色谱柱规格Waters UltrastyragelTMLinear四氢呋喃柱（19mm×300mm）+Waters UltrastyragelTM500四氢呋喃柱（19mm×300mm）+Waters UltrastyragelTM100四氢呋喃柱（19mm×300mm），上样量500ul，流动相四氢呋喃，流速4.2mL/min，TGO、TGD的收集时间分别为29.00-32.10min、32.25-33.50min。以油脂极性组分为原料的高压凝胶渗透色谱制备TGP周期约为1h，单次分别获得0.49mgTGD与0.23mgTGO，纯度均在99%以上，TGD与TGO得率分别为29%和13%。TGD与TGO的凝胶渗透色谱制品适宜在四氢呋喃溶液稳定储藏，-20℃至20℃各温度条件下60d最大衰减均低于3%，TGP固体即使-20℃保存60d也衰减10%，在25℃室温下使用不应超过6h。极性组分中TGO、TGD、ox-TG、DG的分子量（Mw）分别为2469、1728、908、510，TGO以三聚体为主。4、TGP的细胞毒性评价。研究了不同TGP浓度对巨噬细胞Ana-1的作用，采用倒置相差镜观察细胞生长，噻唑蓝（MTT）法检测细胞增殖能力，激光共聚焦镜观测细胞凋亡，酶标定量胞外乳酸脱氢酶，流式细胞术检测胞内活性氧（ROS），结果表明：50μg/mL浓度的TGP致细胞明显损伤，该浓度下的巨噬细胞在倒置相差显微镜下存活率明显下降、增殖能力受到显著抑制、吞噬中性红的能力显著降低、乳酸脱氢酶释放量显著上升，细胞早期凋亡明显。活性氧过度产生是TGP致细胞损伤的主要原因之一。TGD与TGO细胞毒性差异不明显，支持Billek的油脂TGP低吸收率的结论，尚不能确定与恶性肿瘤相关性。5、基于TGP的油脂质量评价应用。开展了基于TGP的地沟油检测、冷榨油鉴别和煎炸油质量控制、毛油质量评价等应用研究。比较分析食用植物油与地沟油两大样本库的TGP，结论如下：12种常见食用植物油41个样本TGP含量0.69±0.41，3类典型地沟油64个样本TGP含量6.01±3.44，差异极其显著，据此可以一定程度准确鉴别地沟油；冷榨油具低TGP特征可显著区别于精炼油；煎炸油中TGP与PCs线性相关，TGP直接指示煎炸油质量；毛油的TGP反映其新鲜程度。TGP在油脂质量评价中的应用优势，依赖于分类油脂的TGP基础数据库。更多还原

【Abstract】 Oxidized triacylglycerol polymers (TGP）is a kind of extremely complex advance oxidationproducts generated in the lipid oxidation process. The growing amount of TGP in a specificoil, during the process of oil refining and storage, will never be reduced despite conventionalre-refining, which indicates that it could be regard as an objectively endogenous parameter forcharacterizing the degree of lipid oxidation. The paper aims at reviewing such issues as acouple of deficiencies in oil oxidation indicators and several technical defects in highsensitivity TGP determination standard, studies on the correlation between TGP and oiloxidation as well as on the applications of TGP in oil quality evaluation. The main contentsare as follows:1.Studies on the correlation between TGP and the extent of lipid oxidation. HPSEC methodwas employed to analyze the polar compounds (PCs) and its subdivision components whichwere formed in three oxidizing processes, at room temperature storage, Schaal ovenaccelerated oxidation, and high temperature frying. The content changes of endogenousnatural antioxidant tocopherols were monitored at the same time. Correlation between TGPand the extent of lipid oxidation had been built. The results proved that ox-TG contentshowed good linear relationship with oil induction period. The emergence of TGO and thedepletion of endogenous natural antioxidant tocopherols marked the end of induction period.TGO were almost ox-TG trimers, which were supposed to be glycerol esters mainlycontaining epoxy, ketone, aldehyde, C-C bridge, C-O-C bridge groups. Ln (TGO/TGO0)varied linearly to the degree of soybean oil oxidation.2. Studies on PFC-HPSEC method for TGP determination. A kind of PFC-HPSECdetermination method was studied and established, which was suitable for detecting eithercold pressing edible vegetable oil or refined oil with low content of TGP (less than3%). PCsin oil were separated firstly by medium pressure FLASH column. The PCs was fractionizedfurther into TGO，TGD，ox-TG，DG，FFA，etc. by HPSEC. By means of the relativemolecular weight of polystyrene standards for qualitative analysis, gravimetric method fordetermining PCs content in oil and HPSEC area normalization method for quantifying thepercentage of TGO and TGD within oil, the results showed that: the calibration curves forTGO and TGD were linear in the concentration ranges of28-1800mg/L and11-2800mg/L,respectively, with correlation coefficients (r2) more than0.99. Their LODs were28mg/L and11mg/L, respectively, and LOQs were113mg/L and44mg/L, respectively, which wasequivalent to0.01%for LOQ of TGP in oil. The recoveries at3different spiked levels rangedfrom95%-98%. The RSDs of TGP in oil detected by PFC-HPSEC were less than10%. Therelative error of quantifying TGP in oil between the silica gel column-HPSEC method andthe classical PFC-HPSEC method was less than8.9%.3. Studies on TGP preparation by GPC and the TGP stability. Preparation of mg level TGOand TGD from oil polar compounds was achieved by PFC-high pressure GPC technology.The stability of the TGO and TGD in a variety of physical state and storage conditions wasstudied. The molecular weight of each relevant polar component was determined by HPSEC.The results showed that: the optimum conditions for TGP preparation by high pressure GPC were as follows: three GPC columns in series (Waters UltrastyragelTMLinear THF column(19mm×300mm)+Waters UltrastyragelTM500THF column (19mm×300mm)+WatersUltrastyragelTM100THF column (19mm×300mm)), sample loading volume of500ul,mobile phase THF at a volume flow rate of4.2mL/min,29.00-32.10min and32.25-33.50min forTGO eluent collecting and for TGD eluent collecting respectively. It takes1h for TGP preparation fromthe raw material of oil PCs by high pressure GPC, yielding0.49mg TGD and0.23mg TGO withevery sample’s purity above99%, the productivity of29%and13%respectively. TGO andTGD prepared by GPC were suitable for stable storage in THF solution,with the maximum attenuationlower than3%under the condition of-20℃to20℃for60d. The TGP solid samplesattenuated by10%even in the storage state of-20℃for60d, suggesting shorter than6h at25℃use. Molecular weights (Mw) for TGO、TGD、ox-TG、DG in PCs were2469、1728、908、510respectively. TGO was mainly comprised of trimer.4. Evaluation on the cytotoxicity of triacylglycerol polymers. The effect of differentconcentrations of TGP on macrophages Ana-1had been studied. After observing live cells byinverted phase contrast microscope, evaluating phagocytic activity by MTT, observingphagocytic apoptosis by LSCM, determining the LDH extracellular released by ELISA andintracellular ROS level by flow cytometry, the results showed that:50mg/mL of TGPresulted in macrophages notable apoptosis and necrosis, obvious decline in Ana-1survivalrate, viability and phagocytic ability, marked LDH release and early stage apoptosis.Excessive ROS production was one of the main mechanisms via which TGP induced cellinjury. There was no significant difference between TGD and TGO, which may confirmBillek’s conclusion about TGP low intestinal absorption rate, whereas the correlation betweenTGP and malignancy remained cloudy.5. Application on oil quality evaluation. By means of TGP, researches carried out a seriesof studies on distinguishing waste cooking oil from cool pressing oil, controlling frying oilquality, evaluating crude oil quality, comparatively analyzing TGP contents in ediblevegetable oil and waste cooking oil. The conclusions were as follows: average TGP of12kinds of common edible vegetable oil samples (n=41) and three kinds of typical waste cookingoil samples (n=64) is0.69±0.41and6.01±3.44, respectively. The difference was significant enoughthat this method could be utilized to distinguish waste cooking oil to some extent. Low TGPcharacteristics of cold pressing oil were very different from the refined oil. Linear correlation existsbetween TGP and PCs in frying oil. TGP content directly indicates quality of frying oil andreflects the freshness of crude vegetable oil. The TGP advantage in application of oil qualityevaluation is on the simple calibration of TGP database for various kinds of oil.更多还原