Detection, Evaluation And Its Application Of Oxidized Triacylglycerol Polymers

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.