Distribution Of 3-mcpd And 3-mcpde In Fats And Oils, Their Determining And Content Changing During Thermal Processing And Oil Refining Process

3-chloro-1,2-propanediol fatty acid esters(3-MCPDE), which was found widely exists in oils and fats and fatty food by researchers only in recent years, is one kind of many chloropropanols esters. Given the 3-MCPDE in vitro and cell experiments can be hydrolyzed to 3-MCPD and its manifestations of toxicity in animal studies, 3-MCPDE security issue has caused widespread concern around the world. We conducted a systematic study of the relationship between the content of 3-MCPD and 3-MCPDE, oil type, oil refining process and TFA content. In addition, HPLC method for the determination of 3-MCPDE was established and the content changing of 3-MCPDE during palm oil thermal processing was also investigated. And on this basis, the formation pathway of 3-MCPDE in palm oil thermal processing has been discussed. Further measures for reducing or removing 3-MCPDE generated during the processing of oil raw materials were also proposed. The main research content and results are as follows:1. In order to overcome the shortcomings of GC-MS which contain many steps, using large amount of solvent and with long cycle, a HPLC method was established. The oil samples were first extracted with acetonitrile to accumulate the polar and weak polar components. The isolation of 3-MCPDE was carried out on a Sun FireTM C18 column and quantitative determination was performed on Hedera Si column using HPLC method. The results showed that: components of different polarities in oil samples can be separated by Sun FireTM C18 column(4.6 mm × 150 mm, 5 μm particle) using a Waters 1525 HPLC system with Waters 2424 detector. The mobile phase was acetonitrile(solvent A) and isopropanol(solvent B). The isocratic elution was performed as follows: 50% A, 50% B. The column temperature was maintained at 30 oC. The flow rate was 1 m L/min. The quantitative determination of 3-MCPDE after pretreatment of oil sample was performed on Hedera Si column(4.6 mm × 200 mm, 3 μm particle) using a Waters 1525 HPLC system with Alltech 3300 ELSD. The mobile phase was composed of hexane(solvent A) and isopropanol(solvent B). The isocratic elution was performed as follows: 90% A, 10% B. The flow rate was 0.5 m L/min; the column temperature was maintained at 30 oC; the sample injection volume was 5 μL. Good linearity and satisfactory correlation coefficients(correlation coefficients > 0.99) were obtained for both 1,2-dioleoyl-3-chloropropanediol and 1-stearoyl-3-chloropropanediol. The intraday precision ranged from 2.1% to 4.0%, and the interday precision ranged between 3.2% and 5.6%. Therefore, the HPLC method is feasible for the measurement of 3-MCPDE in oils and fats. The relative percentage errors of intraday and interday accuracy for both 3-MCPDE standard substances were from 4.3% to 14.6% which is in an acceptable range and can be considered that the HPLC method is accurate. The recovery values of 3-MCPDE standard substances spiked canola oil sample at the concentrations of 5, 10 and 50 mg/kg were varied from 92.5% to 108.3%. The LOD and LOQ were found to be 3.43 μg/m L and 5.71 μg/m L for 1,2-dioleoyl-3-chloropropanediol, 2.55 μg/m L and 5.66 μg/m L for 1-stearoyl-3-chloropropanediol, separately.2. There are a great variety of oil crops in our country. The distribution of 3-MCPD and 3-MCPDE in sixteen different types(fourty-four samples) of commonly consumed edible oils samples purchased from the retail market in Wuxi were investigated using GC-MS. In order to clarify the proportion of mono- and diesters of 3-MCPD, a further analysis of 3-MCPD fatty acid mono- and diesters was also performed. The results showed that: free 3-MCPD was detected in all cooking oil samples and the mean levels were varied from 2μg/kg to 85μg/kg. The levels of free 3-MCPD vary enormously from one kind of oil to the other with a maximum mean value of 85μg/kg(rice bran oil with high levels of oryzanol), followed by grade 4 rice bran oil, virgin olive oil, press peanut oil and grade 1 rice bran oil(the mean value was above 40μg/kg). The levels of 3-MCPDE vary enormously from one kind of oil to the other with a maximum mean value of 325μg/kg(peanut oil), followed by sunflower oil(mean value of about 200μg/kg). 3-MCPDE were not detected in edible vegetable blending oil, natural cereal blending oil, phytosterol corn oil, virgin olive oil and rapeseed oil. The content of 3-MCPDE in refined oil samples with 3-MCPDE >100 μg/kg was significantly higher than 3-MCPD. The ratio of 3-MCPDE:3-MCPD varied from 4.1:1 to 99.8:1. In 3-MCPDE detected oil samples, most 3-MCPDE exists in the form of 3-MCPD diesters. In addition to sunflower oil and corn oil, the ratio of 3-MCPD diesters:3-MCPD monoester varied from 3.6:1 to 19.0:1 with a maximum value of 19.0:1 in peanut oil, followed by camellia blending oil(16.9:1) and grade 1 rice bran oil(8.0:1 to 8.7:1). The PMTDI established by JECFA was 2 μg/kg BW. In addition, according to ’Dietary Guidelines for Chinese Residents’, the recommended daily intake of fat is no more than 30 g. Taking this recommended amount, 3-MCPD level of 100 μg/kg, 3-MCPDE level of 700μg/kg and BW 60 kg as a reference, the calculated daily intake of 3-MCPD and 3-MCPDE was 0.05 and 0.35 μg/kg BW separately, both were lower than PMTDI recommended by JECFA. It can be considered that the toxicity of 3-MCPD and 3-MCPDE in oil samples investigated in our study do not pose a risk to human health.3. The influence of oil refining process on 3-MCPD and 3-MCPDE formation is rarely reported. Thus the distribution of TFA, 3-MCPD and 3-MCPDE in four batches of different processing section(i.e. degumming, neutralization, bleaching and deodorization in oil refining process) oil samples and the relationship between TFA and 3-MCPD and 3-MCPDE in forty-four commonly consumed edible oil samples were investigated in this study. The results showed that: three batches were detected in the presence of chlorine content of 9.41~20.87 mg/kg. The 3-MCPD levels showed a decreasing trend overall after oil refining process. The content of 3-MCPDE showing an increasing trend overall in refined oils containing chlorine. However, the content of 3-MCPDE showing a decreasing trend overall in refined oils without chlorine. There was a significant positive linear correlation between trans linolenic acid and 3-MCPDE in C peanut oil containing chlorine(p<0.01). The mean value of trans oleic acid and total TFA was generally high in samples with 3-MCPD higher than 10 μg/kg, 3-MCPDE higher than 100 μg/kg, total 3-MCPD higher than 100 μg/kg and 3-MCPD diesters higher than 10 μg/kg, varied from 2.6% to 2.7% and 3.4% to 3.8%, respectively. However, the content of 3-MCPD, 3-MCPDE, total 3-MCPD and 3-MCPD diesters were not change regularly in samples with higher content of trans oleic acid(>3.1%) and total TFA(>3.1%). To reduce or decrease the generation amount of 3-MCPDE in fats and oils, we recommend major two-pronged approach: First, remove or reduce chlorine from oil raw materials; Second, remove or reduce 3-MCPDE formed during oil refining by optimizing the refining process.4. The research work on 3-MCPDE variation during thermal processing is still relatively limited. Therefore, the influence of Na Cl addition, water content, heating temperature and heating time on the changing of 3-MCPDE in palm oil during thermal processing were examined using HPLC method. In addition, the formation pathway of 3-MCPDE in palm oil during thermal processing was also discussed. The results in our study showed that all those four factors had significant effects on the generation of 3-MCPDE in palm oil during deep fat frying. The generation amount of 3-MCPDE reached the maximum value in palm oil when heating time was 2 h. The heating process imparted an accretion of up to nearly three times when increasing the water dosage from 1% to 10%. When the heating temperature was increased from 100 to 200 oC, the maximum value of 3-MCPDE decreased approximately one times higher than that formed at 100 oC. Moreover, 3-MCPDE was decomposed when heated at high temperatures for a long time. Increasing the heating time had resulted in the reduction of 3-MCPDE drastically. The findings supported the acyloxonium ion formation mechanism of 3-MCPDE: TAG would react with water to produce DAG through hydrolysis reaction when heating with water and Na Cl during thermal processing. Consequently, the DAG will react with Na Cl presented in the reaction system and finally result in the generation of 3-MCPDE in palm oil through the nucleophilic reaction of chloride ions to the acyloxonium ion.