Formation and Hazard Control of TBBQ during Oil Frying and Molecular Mechanism of Its Cytotoxicity

Author:Ye Qin

Supervisor:meng xiang he


Degree Year:2019





In the process of frying at high temperature,a series of reactions such as hydrolysis,oxidation,polymerization and thermal cracking will occur when the oil is exposed to oxygen and water,and the interaction of fats and oils with other food components will also occurs simultaneously.It will affect the quality and nutrition of frying oil and fried food,even bring safety hazards.Tert-butylhydroquinone(TBHQ)has lower price and higher efficacy in comparison to other antioxidants so that it is widely used to protect the oil oxidation.However,TBHQ is easily to oxidate degratied,and has lots of oxidation and degration products.Stuides have shown that2-tert-butyl-1,4-benzoquinone(TBBQ)was the one of the main oxidation products,while the generation pathway,residual level,biological toxicity characteristics and molecular mechanism are still unknown.Therefore,in order to improve the quality,nuturion and safety of frying oil and fried oil,and provide the theoretical support for the establishment of quality control standard of frying process,it is necessary to study the formation and hazard control of TBBQ during oil frying and molecular mechanism of its cytotoxicity.Studies on safety evaluation and control on TBBQ,the main oxidation products of TBHQ during frying with edible oil,would be carried out by means of multidisciplinary technology.The oxidated products were identified by GC/MS,and the main formation pathway of TBBQ was elucilated.The rules on TBBQ accumulation in frying oil and fried food,its environmental parameters effects and selectively removal methods would be explored.This will underlie the theoretical foundation for the development of safe and scientific frying process.Meanwhile,the residual level of TBBQ in frying oil and fried food and its exposure level to human were evaluated to confirm the cytotoxicity and dose-effect relationship of TBBQ.To disclose the signal pathway and key targets genes functioned by TBBQ,the transcriptome sequencing(RNA-Seq)technology was used to screen the differentially expressed genes.The expression of key genes and its products were verified by using quantitative real-time reverse-transcriptase-polymerase chain reaction(qRT-PCR)and western blot.The clarification of the cytotoxic mechanism of TBBQ at the molecular level would provide fundermental data for the safety assessment of TBBQ in fried food.(1)Firstly,this study identified the main products of TBHQ by GC/MS at high temperature in the tripalmitate simulated frying system,the results showed that TBBQ was the main degradion product.Meanwhile,this study compared the changes of TBHQ and TBBQ concentration in the oil of the open and closed system under 120,170 and 190oC.Results showed that TBHQ loss was increasd gradually as the heating temperature was increased and the time was prolonged.The maximum residual level of TBBQ(164.67±6.02 mg/kg)was observed with closed heating after 8 h at 170 oC,rather than at 120oC(120.16±6.03 mg/kg for heating after 5 h with reflux).Although the loss of TBHQ was reduced at low temperature(120oC),the residual level of TBBQ in the oil was higher.The final TBBQ concentrations were 52.6162.93 mg/kg,10.2346.95 mg/kg,and 07.74 mg/kg after heating at 120,170,and 200°C,respectively,with or without reflux for 24 h.The above results showed that TBBQ concentration is not only a function of heating temperature and time,but also affected by volatilization intensity and the presence or absence of oxygen.By comparing the change rules under open system and closed system,it can be found that the loss of TBHQ under high temperature heating condition is mainly dominated by oxidative degradation.In addition,the dynamic equation of TBHQ loss and TBBQ generation at high temperature was preliminarily fitted in this study.It was assumed that the volatilization of TBHQ and TBBQ could be ignored under reflux conditions,therefore,the degradation of TBHQ and the generation of TBBQ were consistent with the first-order reaction kinetic equation under high temperature heating.The results showed that the first-order kinetic reaction rate constants of TBHQ loss at 120,170and 200oC in open and closed systems were 0.018,0.23,0.31 h-1;0.019,0.22,0.32 h-1respectively,and the first-order kinetic reaction rate constants generated by TBBQ were 0.0091,0.090,0.16 h-1;0.031,0.13,0.30 h-1,respectively,and the root mean square error(RMS)are all less than 5%,indicating that the model value has a good consistency with the experimental value.(2)The effects of different frying temperatures(140,170,190oC),frying oil types(crude soybean oil,refined soybean oil,rapeseed oil,palm oil,sesame oil)and frying mode(continuous and discontinuous frying)on TBBQ formation in frying oil and fries were studied.The results showed that the residue level of TBBQ in both frying oil and fries exceeded 10 mg/kg after batch 1st to 5th frying when different types of edible oils containing 204.89±1.63 mg/kg TBHQ were fried in potato chips under different frying conditions.For the frying temperature,from the perspective of TBBQ content,170oC was better than 190oC and 140oC,and there was no effect on TBBQ concentration using different types of edible oils(including saturated palm oil and antioxidant sesame oil).Compared with the continuous frying mode,discontinuous frying is more beneficial to reduce the content of TBBQ.Furthermore,FTIR is an effective means to monitor the absorption of functional groups in frying oil,especially the O-O absorption at 845 cm-1,which can effectively evaluate the oxidation performance of frying oil containing TBHQ.(3)The cytotoxicity of TBBQ to RAW 264.7 cells was evaluated.MTT cell viability assay showed that different concentrations of TBBQ(0,0.5,1,5,10,20,30μg/mL)inhibited the growth of RAW 264.7 cells with different degrees,and the inhibitory effect on cells showed a TBBQ dose and action time dependent relationship.The IC50 concentration was 10.71μg/mL when TBBQ treated RAW 264.7 cells for 24h.It was observed that the nuclei showed dense hyperchromatism,shrinkage or fragmentation,and the fluorescence staining was enhanced with the increase of TBBQ concentration,presenting typical morphological characteristics of apoptosis,after Hoechst 33258 fluorescence staining.In addition,flow cytometry was used to detect the cell cycle and apoptosis of RAW 264.7 cells stimulated by TBBQ,and it was found that the apoptosis rate increased with the increase of TBBQ concentration,and TBBQ inhibited the proliferation of RAW 264.7 cells by inhibiting the synthesis of DNA in RAW 264.7 cells in S phase.Besides,TBBQ effects on RAW 264.7 cells can cause the increase of intracellular ROS level and the decrease of GSH content,thus causing oxidative stress and inducing apoptosis.(4)RAW 264.7 cells exposed to 1μg/mL TBBQ produced a total of 758differentially expressed genes was revealed based on transcriptome sequencing analysis,including 442 up-regulated genes and 316 down-regulated genes.The possible mechanism of RAW 264.7 cells toxic response to TBBQ and the related signal pathway were preliminarily revealed based on the differential gene analysis of transcriptome sequencing.Verification results of RT-qPCR showed that the mRNA expressions of caspase-3,8,9 and 12 in the treated group were all increased,and the mRNA expressions of Fas,Jnk,Ask1,Jun,Bax,CtsL and Capn2 were also increased compared with the control group,while the mRNA expressions of Bcl-2 were decreased;Meanwhile,verification results of western blot showed that Bcl-2 showed a down-regulation trend(p≤0.01),and Fas,Cytochrome c,Cleaved caspase-12,-8,and-3 showed an increased trend(p≤0.01)compared with the blank control group after 12h of RAW 264.7 cells treated with different concentrations of TBBQ(0.5,1,2μg/mL).Thus,we suggest that death receptor/Fas pathway,lysosomal pathway,endoplasmic reticulum stress and mitochondrial pathway are all involved in the TBBQ-induced apoptosis of RAW 264.7 cells,and the mitochondria-mediated apoptosis pathway is the main pathway of TBBQ-induced apoptosis of RAW 264.7 cells.(5)The removal of low concentration TBBQ(10 mg/kg)and high concentration TBBQ(50 mg/kg)from oils using ionic liquid as a new extraction agent was studied.The removal effects of 10 different ionic liquids were compared in this study,and N-butyl pyridine tetrafluoroborate and 1-ethyl-3-methyl imidazole sulfate were selected to optimize the extraction conditions,including extraction temperature,oil and ionic liquid mass ratio and other extraction conditions.Results showed that the mass ratio of lipid to ionic liquid was 1:1 and the extraction temperature was 70oC,the removal efficiency of TBBQ was the best.The removal rate of low concentration TBBQ(10mg/kg)was 52.98±1.34%and 53.71±2.49%,respectively,and the removal rates of high concentration TBBQ(50 mg/kg)were 55.22±2.09%and 53.66±2.08%,respectively.Besides,the removal rates of low concentration TBBQ(10 mg/kg)and high concentration TBBQ(50 mg/kg)of n-butylpyridine tetrafluoroborate in oil can reach 100%after secondary extraction,and the residue level of ionic liquid in oil was below 0.1 ppm.