Molecular Reaction Mechanism for the Formation of 3

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Molecular Reaction Mechanism for the Formation of 3-chloropropanediol Esters in Oils and Fats Yunping Yao, Ruizhi Cao, Wentao Liu, Hang Zhou, Changmo Li, and Shuo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06632 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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Journal of Agricultural and Food Chemistry

Molecular Reaction Mechanism for the Formation of 3-chloropropanediol Esters in Oils and Fats Yunping Yao, Ruizhi Cao, Wentao Liu, Hang Zhou, Changmo Li* and Shuo Wang

1. State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin 300457, China 2. Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, China 3. School of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China

*Corresponding

Author: Changmo Li

Phone: +86-22-60912419 Fax: +86-22-60912419 E-mail: [email protected]

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ACS Paragon Plus Environment


ABSTRACT 1

3-Chloro-1,2-propanediol fatty acid esters (3-MCPD esters) are a group of

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process-induced contaminants that form during the refining and heating of fats and

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oils. In this study, a combined method of simulated deodorization and computational

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simulation was used to explore the precursor substance and the generation path of

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3-MCPD esters. From the results, 3-MCPD esters reached to 2.268 mg/kg when the

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diacylglyceride (DAG) content was 4% and temperature was 220°C. A good

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correlation was observed between DAG and 3-MCPD ester contents (y = 0.0612x2 −

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1.6376x + 10.558 [R2 = 0.958]). There were three pathways for the formation of

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3-MCPD esters: A) A direct nucleophilic substitution reaction, B) an indirect

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nucleophilic substitution reaction, and C) a mechanism of intermediate (glycidyl ester)

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from the calculation of Gaussian software at the B3LYP/6-31+g** level. The data

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showed that the ester-based direct nucleophilic substitution reaction was the most

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likely reaction pathway. The energy barriers for the formation of the 3-MCPD esters

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dipalmitin, diolein, and dilinolein were 74.261, 66.017, and 59.856 kJ/mol,

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respectively, indicating that the formation process of 3-MCPD esters is a

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high-temperature endothermic process. Therefore, by controlling the introduction of

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precursor (DAG) and reducing the temperature, 3-MCPD ester formation was

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prevented.

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KEYWORDS: 3-MCPD ester, Nucleophilic substitution reaction, Diacylglyceride,

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Gaussian, Energy barrier

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INTRODUCTION

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3-Chloro-1,2-propanediol fatty acid esters (3-MCPD esters) were discovered by

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Davidek et al. in 19801,2 and are considered to be non-genotoxic carcinogens.3 The

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3-MCPD ester content in real food systems often exceeds that of free 3-MCPD

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content by 5–396 times.4-6 It has been reported that the refining process of edible oil is

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the mass production stage of 3-MCPD esters. The content of 3-MCPD esters increases

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significantly with the introduction of a large amount of water vapor, especially during

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the deodorization stage.7-9

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Studies on the toxicological properties,10,11 analytical methods of 3-MCPD and

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their fatty acid esters,12 and the approach to reduce 3-MCPD esters (screening

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materials and optimization processes) have been reviewed by several authors.13,14

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Many scholars have speculated about the formation mechanism of 3-MCPD esters,

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including the precursors and reaction pathways. Collier and Hamlet hypothesized that

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possible precursors are triacylglycerides (TAGs), diacylglyceride (DAG), and

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monoacylglyceride (MAG). Bertrand et al. found no linear relationship between TAG

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and 3-MCPD ester contents, and MAG is removed in large quantities during the

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refining process, the content of which is < 1% of refined oils.15,16, DAG content is

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also prominent in palm oil, which contains a mass of 3-MCPD esters, compared with

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other oils, so DAG is most likely its precursor material.17 According to the processing

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conditions, and the effect of chemical competition on 3-MCPD esters, there are

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currently four proposed mechanisms all involving SN2 nucleophilic attack by chloride

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ions, which are classified based on the nature of the substrate and the leaving group.

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Two of the proposed mechanisms involve the glycerol backbone carbon atoms being

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attacked by the chloride ion on the ester group (path 1) or the protonated hydroxyl

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group (path 2). The other two pathways suggest the formation of reactive

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intermediates, such as acyloxonium ion (path 3) or glycidyl ester (path 4), prior to

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chloride ion nucleophilic attack.18,19 However, the intermediate products generated

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differ according to the difference in the chloride binding sites in the path of generating

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acyloxonium ions (similar to paths 1 and 2). Therefore, they should be discussed

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separately. In addition, the acyloxonium ion is an activated complex belonging to the

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transition state, which is short-lived and unstable, and glycidyl esters are intermediate

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stable compounds. Although both have oxygen-containing polycyclic rings, their

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structural properties are quite different. Despite that many scholars have speculated on

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the formation of the 3-MCPD esters, there is no clear evidence or conclusion about

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the exact mechanism for the formation of 3-MCPD esters.

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A transition state is only 0.01 femtosecond (10-15 sec), which is difficult to

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capture with existing experimental techniques. In recent years, the electron density

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functional theory and ab initio algorithms of modern quantum chemistry have become

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important tools for studying chemical problems.20,21 Therefore, in this study, a

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Gaussian molecular simulation was used in combination with density functional

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theory. The Gaussian program used in this experiment is the most widely used

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quantum chemistry software for semi-empirical and ab initio calculations. The best

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generation mechanism is based on the energy change in the reaction site and the

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transition state structure. It is important to clarify the precursor of 3-MCPD esters and

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determine the optimal reaction path for reducing 3-MCPD ester content. The massive

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production of 3-MCPD esters can be avoided by controlling the refining conditions

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and reducing the amount of precursors.

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MATERIALS AND METHODS

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Materials. Soybean oil, rapeseed oil, corn oil, olive oil, peanut oil, sunflower oil,

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rice bran oil, and palm oil were all commercially available (available from COFCO

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Fortune). The 3-Chloro-1,2-propanediol 1-Palmitate Standard (purity 99.8%) and

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3-MCPD-d5 internal standard (purity 98.3%) were purchased from Wako Pure

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Chemical (Osaka,Japan). 1,2-dipalmitoylglycerol was procured from Sigma-Aldrich

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(St Louis, USA). Butyl ether, ethyl acetate, methanol, sodium methoxide, n-hexane,

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sodium chloride, acetic acid, phenylboronic acid, ethyl ether, 2,2,4-trimethylpentane

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were all laboratory analytically pure and were purchased from Sinopharm (Beijing,

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China).

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Detection of 3-MCPD esters in oil samples. The analysis of the 3-MCPD esters

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was carried out according to an adapted method.18,

22

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dissolved in 0.5 mL of tert-butyl methyl ether (t-BME) and the internal standard

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(3-MCPD-d5 ester) was added. Add 1 mL of 0.5 mol/L sodium methoxide, and the

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alkaline transesterification was carried out at room temperature for 10 min. The

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reaction was terminated by the addition of 3 mL of acidified sodium chloride solution,

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3 mL of n-hexane was added to separate the free fatty acids, and the supernatant was

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discarded. The derivatization reaction was carried out with 0.5 mL saturated solution

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of phenylboronic acid diethyl ether at 30°C for 20 min. After evaporation of the

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Oil sample of 100 mg was


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organic solvents, the residue was dissolved in iso-octane prior to GC-MS analysis.

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GC-MS analysis was carried out on Agilent 7890B GC Ultra equipped with

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5977C quadrupole detector (Agilent Technologies, CA, USA). Chromatographic

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separation was performed on a Quartz capillary column HP-5MS (30 m × 0.25 mm

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inner diameter, 0.25 µm film thickness, Agilent Technologies, Santa Clara, CA, USA).

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Ultra pure helium (purity: 99.9999%) was used as a carrier gas at a flow rate of 1

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mL/min, injection mode: splitless injection. The injector was held at 250°C and 1 µL

95

of the sample was injected in a splitless mode. The column temperature was

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programmed at 60°C, raised at 15°C/min to 165°C, then accelerated to 250°C at a rate

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of 60°C/min, hold for 10 min. Mass spectrometry conditions: using EI source,

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interface temperature was 250°C, ion source temperature was 200°C, solvent delay

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was 3 min. The quantitative analysis was carried out by monitoring ions at m/z 150

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and 201 for the 3-MCPD-d5, whereas the ions were m/z 147 and 196 for the

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derivatized 3-MCPD.

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Detection of DAG and MAG. 100 mg of oil sample was dissolved in 10 mL

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n-hexane/isopropanol (50:1, v/v), then micro-filtrated by the 0.25 μm organic

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microporous filter membrane, and 20 μL was taken for liquid phase analysis.

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The DAG of the oils were determined using a high performance liquid

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chromatography system, (LC-20A Shimadzu, Tokyo, Japan) equipped with Refractive

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Index Detector (RID). Chromatographic separation was performed on a waters silica

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gel column (silica 5.0 μm, 4.6 mm × 250 mm). Mobile phase was composed of

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n-hexane/isopropanol (50:1, v/v), the flow rate was 0.8 mL/min, the column

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temperature was 35°C, and the volume of injection was 20 µL of each sample.

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Detection of free fatty acids. The acid value reflected the content of free fatty

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acids using an index indirectly. The detection method was based on the national

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standard AOCS Cd 3d-63. 23

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Simulated deodorization of adding fat. In order to further determine that the

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DAG is a precursor of the 3-MCPD ester, the bleached soybean oil (the main

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component is TAG) and dipalmitin were used to prepare the content of DAG at 1%,

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2%, 4%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, respectively. 1% mixed

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oil was made of 100 mg dipalmitin and 9.9 g bleached soybean oil, and 2% ~ 80%

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mixture was made for the same reason. And the mixed oil samples were heated in an

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atmospheric open system at 180°C, 200°C, 220°C, 240°C for 2 hours to simulate the

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deodorization process of cooking oil.

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Molecular simulation of formation of 3-MCPD esters. All calculations were

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performed using the Gaussian 09 W software (Gaussian, Inc.). The calculations were

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performed using the DFT (density functional method) B3LYP for first-principles

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calculation. Using the 6-31 g all-electron basis set to describe atomic orbitals and

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valence electrons, the polarization functions were used to add a d-polarized orbital to

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a non-hydrogen atom on the 6-31 g basis set, improving the angular distribution of the

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bond-bond orbits to enhance. The softness of the key track's space promotes the bond

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formation. The spin multiplicity was set to 0 and the same method was used for the

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unit to optimize all reactants and products geometrically involved in the reaction and

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removed symmetry restrictions. The difference of convergence threshold of the

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charge density before and after SCF was set to 1.0×10-8 Ha, and the transition state

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was found using a non-restrictive open shell. Finally, the reaction path was verified

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using the IRC coordinates.

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Statistical Analyses. The data is presented as means ± standard deviations (SD)

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of duplicate technological experiments. And all analytical measurements were

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performed in triplicate. Data processing and regression analysis were performed using

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origin software (version 8.0; Microcal Software Inc., Northampton, MA).

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RESULTS AND DISCUSSION

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Effect of trace components on the formation of 3-MCPD esters. The

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3-MCPD ester contents in commercially available soybean oil, rapeseed oil, corn oil,

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olive oil, peanut oil, sunflower oil, rice bran oil, and palm oil were analyzed by gas

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chromatography-mass spectrometry. The results show that the 3-MCPD ester contents

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in palm oil and rice bran oil were 3.736 ± 0.083 and 1.002 ± 0.028 mg/kg (Table 1),

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which is far below the maximum allowable intake as specified by the Joint

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FAO/WHO Expert Committee on Food Additives (JECFA).

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The content of DAG, MAG, and free fatty acids in different oils was detected by

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high performance liquid chromatography equipped with a RID detector, as shown in

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Table 1. The DAG and 3-MCPD ester contents showed a similar trend, in accordance

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with the equation y = 0.1507x2 − 0.5676x + 0.803. A significant correlation (R2 =

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0.998) was detected, as shown in Figure 1. The correlation coefficient between

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3-MCPD esters and MAG was only 0.012, and the correlation coefficient between

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3-MCPD esters and free fatty acids was 0.030 (data not shown). Lower MAG and free

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fatty acid contents were found in oils and fats, which also limits their effects on

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3-MCPD ester production. Similar results have been reported by other researches.

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Sveikovska simulated deodorization using palmitic triglyceride and diglycidyl

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palmitate, proposing that DAG was more efficient for 3-MCPD ester formation than

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TAG.24 Bertrand et al. detected no significant correlation between MAG and TAG

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contents and that of 3-MCPD esters, while there was linear correlation (R2 = 0.962)

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between DAG and 3-MCPD ester contents.17 A large amount of 3-MCPD esters (2.5

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mg/kg) was produced when DAG exceeded 4% of the oil. Judging from the results,

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DAG is widely recognized as a precursor substance in the production of 3-MCPD

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esters, which were largely found in bleaching oils.

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Changes in 3-MCPD ester content during the bleaching and deodorization

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process. Edible vegetable oils are refined through four processes: degumming,

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neutralization, bleaching, and deodorization. To further document the 3-MCPD ester

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precursor, an intrinsic link was set by examining the changes in the contents of

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possible precursor substances and the contents of 3-MCPD esters at different stages of

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the process. As most of the 3-MCPD esters was produced during deodorization, the

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bleaching and deodorization processes were monitored. The relationship between the

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content of components (DAG, MAG, and free fatty acids) and 3-MCPD ester content

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during bleaching and deodorization of oils (palm oil and soybean oil) is discussed in

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this study.

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DAG in palm oil decreased from 9.220% to 6.675% during the deodorization

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process. At the same time, the corresponding 3-MCPD ester content in palm oil

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increased from 1.368 to 3.736 mg/kg, as shown in Table 1. MAG content remained

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almost unchanged and 3-MCPD ester contents increased 2.74 times. DAG content in

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soybean oil changed from 1.816 ± 0.021 to 1.801 ± 0.017 mg/kg by deodorization,

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and the 3-MCPD esters increased from 0.040 ± 0.000 to 0.262 ± 0.008 mg/kg. The

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change in this process was not clear, but the extent also reflected a change in DAG

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and an increase in 3-MCPD esters. The MAG and free fatty acid contents also

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changed, mainly due to removal of deodorization, and Bertrand et al. demonstrated

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that MAG and free fatty acids were not the main influencing factors.17 In summary,

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DAG was partially consumed during the refining process, particularly deodorization,

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which was changed in total with an increase in 3-MCPD esters. Therefore, DAG is the

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main precursor of 3-MCPD esters.

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Effect of DAG on 3-MCPD esters. The effect of DAG and heating temperature

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on the amount of 3-MCPD esters formed was investigated. Figure 2 shows the amount

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of 3-MCPD esters formed at different DAG levels and heating temperatures. The

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results show that both DAG content and heating temperature had a significant effect

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on 3-MCPD ester content. This finding indicates that as the temperature reached

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220°C and above, the generation of 3-MCPD was more obvious as DAG content

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increased. Notably, when MAG content was < 4%, the amount of 3-MCPD esters

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formed was not obvious, and there was no significant increase in 3-MCPD esters at

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the deodorization temperature. As DAG content exceeded 4% (as shown in Table 3),

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3-MCPD ester content increased significantly, so 4% DAG was the critical value for

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forming 3-MCPD esters. These results show that contaminants were generated in

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large quantities when the critical value was reached or exceeded, which was

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consistent with the results of Masao Shimizu’s study.25 3-MCPD ester content showed

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an increasing trend with the increase in DAG content in oils and fats, and the

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promoting effect was more obvious with the increase in temperature, especially at

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220°C and above. The amounts of 3-MCPD ester formed at 180°C, 200°C, 220°C,

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and 240°C were in accordance with the quadratic equation shown in Figure 2. In

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addition, a good correlation was detected between 3-MCPD ester content, DAG

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content, and heating temperature (Figure 2), which was sufficient to demonstrate that

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DAG is a precursor of 3-MCPD esters. These data are in agreement with the report of

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Masao et al.25

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Molecular simulation of the reaction mechanism. The mechanisms of

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formation of 3-MCPD esters involve SN2 nucleophilic attack by chloride ions,

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distinguished from each other based on either the nature of the substrate or the leaving

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group. Three mechanisms have been proposed: A) Direct nucleophilic attack by a

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chloride ion at the glycerol carbon atoms carrying either a protonated hydroxyl group

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(pathway a.1) or an ester group (pathway a.2), B) Acylglycerol is induced by

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nucleophiles to form the acyloxonium ion transition state, which is attacked by

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chloride ions to produce a 3-MCPD diester. Similarly, this path can be divided into

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hydroxyl indirect nucleophilic substitution (b.1) or ester indirect nucleophilic

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substitution (b.2), C) The reactive intermediates before nucleophilic attack by chloride

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ions, such as a glycidyl ester, undergo ring opening to generate 3-MCPD esters. The

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newer three 3-MCPD ester reaction pathways are shown in Figure 3. Notably, the

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acyloxonium ion is in a free-radical state, which is also an activated complex, whereas

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the glycidyl ester is a stable intermediate compound. Consequently, this study did not

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categorize it into the same reaction pathway but defined two different reaction

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pathways. The attacking ester and hydroxyl groups are classified into different

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reaction modes within the same path. These changes differ from the previous

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classification. The intermediates, transition states, and generation pathways were

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simulated by Gaussian software using energy barriers.

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Pathway A: 3-MCPD ester formation through direct nucleophilic attack (pathways a1

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and a2)

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a.1) Hydrophilic direct nucleophilic substitution

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1,2-Diacylglyceride played an important role in the formation of 3-MCPD esters.

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As shown in Figure 3A in the reaction system in which proton hydrogen was present,

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the chlorine ion directly attacked the hydroxyl group at the sn-3 position of the

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glycidyl ester, the carbon and oxygen bonds were broken, and the protonated

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hydroxyl was released as water, which was efficient in the hydrolyzable lipid system.

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Direct nucleophilic substitution without formation of the intermediate acyloxonium

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ion was still considered a plausible mechanism by many groups and is expected to

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proceed more rapidly in an acid medium containing partially hydrolyzed lipids as

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water serves as a superior leaving group compared to the preferred fatty acid.16 Water

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dissolves more easily in the deodorizing vapor, which promoted the forward reaction.

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The 3-chloropropane dicarboxylate was further hydrolyzed to form 3-MCPD esters.

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3-Chloropropane dipalmitate, 3-chloropropane dioleate, and 3-chloropropanol

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dilinoleate were chosen as reactants during formation of the 3-chloropropanediol ester,

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and the corresponding energy barriers were 81.012, 83.896, and 79.109 kJ/mol,

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respectively, (p > 0.05) in the first-step of the nucleophilic reaction. In the second step,

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the energy barriers for hydrolysis were 24.978, 22.803, and 18.710 kJ/mol,

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respectively, as shown in Figure 4A. 3-Chloropropanol dilinoleate formed easiest

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because it crossed the energy barrier easier at high temperatures, resulting in a stable

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compound.

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a.2) Direct nucleophilic substitution of ester groups

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The ester group of dipalmitin, diolein, and dilinolein at the sn-1 position was

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directly attacked by a chloride ion (Figure 3A), resulting in formation of 3-MCPD

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esters, and the energy barriers were 74.261, 66.017, and 59.856 kJ/mol, respectively

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in the first step. The difference in polarity increased in non-polar inert media, such as

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lipids, which increased nucleophilicity. The ester bond was hydrolyzed at the sn-2 site

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to form 3-MCPD and free fatty acids, with energy barrier values of 83.509, 74.407,

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and 66.345 kJ/mol. Finally, in the third step, free 3-MCPD was esterified with a single

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free fatty acid at the sn-3 site to form the 3-MCPD ester with energy barriers of

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76.704, 73.886, and 62.661 kJ/mol, respectively. We speculate that the energy barrier

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tends to decrease as the carbon chain grows and the degree of unsaturation increases.

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Although the steps of the two pathways are different, the first step was a rate-limiting

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reaction. Therefore, the energy barrier of the first step was the point of examination.

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Pathway B: Indirect nucleophilic substitution

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b.1) Hydroxyl indirect nucleophilic substitution

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The hydroxyl group interacts with other nucleophiles, such as HCl, to form an

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acyloxonium ion with its neighboring groups at the sn-3 position of diacylglycerol,15

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as shown in Figure 3B. The oxygen bond was attacked by a chloride ion at the

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original hydroxyl group position and the epoxy ring opened to form a

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3-chloropropylene ester. The barriers were 101.202, 80.441, and 70.926 kJ/mol,

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respectively (Figure 4B). The ester bond at the sn-2 position of 3-chloropropane

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diester was hydrolyzed to form the 3-MCPD ester. The alkyl and hydroxymethyl

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groups, which were hydrolyzed from a previous step, acted as electron-donating

272

groups to promote stability of the transition state. Accordingly, preferential loss of

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water may not only indicate the presence of a better leaving group but also a

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preference for the formation of a more stable acyloxonium ion. Furthermore, the

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release of leaving groups is influenced by the polarity of the medium in which they

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are released. Water may be a better leaving group than a long carbon chain of a

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carboxylic acid, as a small molecular weight molecule is easier to remove.

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b.2) Indirect nucleophilic substitution of ester groups

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The ester group located at the sn-1 site of diacylglycerol reacts with an ortho

280

ester group to form an acyloxonium ion as an intermediate transition state under

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acidic conditions. The sn-1 position (original ester group position) was attacked by

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chloride ions; consequently, the ring of the epoxy group opened to generate

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3-chloropropanediester with activation energies of 132.415, 96.453, and 82.223

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kJ/mol, respectively (Figure 4B). The carbon atoms and groups were attacked by the

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nucleophiles, and the ester bond at the sn-2 position was hydrolyzed to form 3-MCPD

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esters. The 3-MCPD esters were produced by hydrolysis and then reacted with one

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molecule of free fatty acid at sn-3. The 3-MCPD esters were produced by an

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esterification reaction. Finally, increasing fatty acid chain length also increased its

289

electron donating effect; however, the steric effect may overtake the electronic effect

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as the carbon chain is extended. Accordingly, long chain fatty acids stabilize the

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resulting acyloxonium ion through its electron donating ability.

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Pathway C: Glycidyl ester intermediates

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As mentioned above, MAG can be generated from 1,2 DAG when the ester bond

294

is broken at the sn-2 position, in which the energy barriers were 245.407, 156.190,

295

and 210.878 kJ/mol, respectively (Figure 4C). Internal dehydration of MAG occurred

296

with the release of glycidyl esters in which the energy barriers were 290.868, 292.304,

297

and 292.284 kJ/mol, respectively. The halide ion preferentially attacked the least

298

hindered carbon of the glycidyl esters to exclusively form the 3-MCPD monoester.

299

The glycidyl esters were alkylated due to the electrophilic epoxy structure and were

300

prone to react directly with nucleophiles.16 The glycidyl ester was converted to the

301

3-MCPD ester through an opening of the epoxide ring by chloride ions, with energy

302

barriers of 151.444, 155.478, and 150.791 kJ/mol, respectively (Figure C). Due to the

303

high temperature environment, paths B and C present a similar situation.

304

The possibility of breaking old bonds and forming new substances was explored

305

by examining bond length, bond energy, and the reaction energy barrier (Table 2).

306

Dipalmitate was taken as an example. The length of a Cα-Oα bond was 1.433 Å, and

307

the distance between a hydrogen atom in HCl and the hydroxyl oxygen atom Oα-Hα

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308

was 1.809 Å, and the Cα-Cl distance was 4.024 Å in length. The Cα-Oα bond stretched

309

to 2.277 Å in the path of direct nucleophilic substitution of the hydroxyl group

310

(pathway a.1), indicating that these bonds were about to break. As Hα approached Oα,

311

the distance between them decreased from 1.809 Å to 1.001 Å. The Oα-Hα bond

312

became possible, and the distance between Cα-Cl shortened to 2.825 Å. These two

313

atoms were attracted to each other and showed a tendency to bond. In the final stage,

314

the Cα-Oα bond was completely broken, and the distance between the two atoms was

315

3.240 Å. A new bond formed between Cα-Cl and Oα-Hα. These two new bonds

316

formed with lengths of 1.822 Å and 0.968 Å, respectively, as shown in Table 2.

317

Because of the strong negative chlorine, the C-Cl bond was longer and significantly

318

larger than the C-O bond. The reaction eventually released a molecule of water, which

319

was removed by the deodorizer vapor. Initial C-O bond length was longer and the

320

bond energy was smaller and easier to break during direct nucleophilic substitution of

321

b.1 ester groups. Therefore, path b.1 was superior to path a.1, which was consistent

322

with the energy calculation results.

323

The acyloxonium ion was a transition state during the indirect nucleophilic

324

substitution reaction, which involves dissociation of the C-O bond and the generation

325

of H-O bonds. The ester group in the sn-1 position or hydroxyl group in the sn-3

326

position was more likely to be attacked by a nucleophile, such as HCl. However, there

327

was a hindrance of bond energy in the sn-2 position due to the steric hindrance effect

328

and the electron effect, and the greater the bond energy the more difficult it was to be

329

attacked. Thus, the sn-2 position was less likely to participate in the reaction. The

16


Page 16 of 35

Page 17 of 35


330

Cγ-Oγ bond length was 1.458 Å as HCl approached the ester group in pathway b.2,

331

which was slightly longer than that of Cγ-Oγ (bond length: 1.435 Å) in the a.2

332

nucleophilic substitution because the increase in the ester-based carbon chain makes

333

that ester group have a greater tendency to leave. The longer bond length and greater

334

activity resulted in easier breaks. The Cγ-Oγ bond length elongated to 3.730 Å in the

335

transition state, and the increase in the distance indicated that a fracture was imminent.

336

Finally, the distance between the products reached 3.425 Å, and the C-O bond was

337

broken. At the same time, Oγ-H gradually moved from 1.925 Å to 1.026 Å, and then

338

to the final bond length of 0.974 Å. Similarly, the length of the Cγ-Cl was shortened

339

from 4.033 to 3.336 Å, the resulting in a bond length of 1.836 Å. As the acyloxonium

340

ion transition state was required during the indirect substitution process, the dihedral

341

angle also changed significantly. The dihedral angle Cδ-Oβ-Cβ-Cγ was deflected from

342

the original 58.089° to −10.740° while generating the epoxy ring, as shown in Table 2.

343

The chlorine atom, which was electroneutral, attacked the binding site, and the

344

previously generated ring was forced to break. This process repelled the ester moiety

345

in the entire mechanism, and the dihedral angle eventually reached 58.209°.

346

In summary, the energy barrier in the direct reaction pathway (path A) became

347

predominant from the indirect reaction pathway (path B), indicating that DAG was

348

more likely to be attacked by a nucleophile at the ester or hydroxyl position instead of

349

forming an acyloxonium ion or other intermediate while forming the 3-MCPD ester

350

from DAG.16 The energy barrier for direct nucleophilic substitution of ester groups

351

was 74.261 kJ/mol, while that for the indirect nucleophilic substitution of ester groups

17



352

was 132.415 kJ/mol, leaving a 58.154 kJ/mol energy gap between the two pathways.

353

In addition, there were three steps in the pathway C reaction, which involved

354

hydrolysis of DAG, formation of an epoxy group, ring-opening of the glycidyl ester,

355

and release of the 3-MCPD ester. The energy hindrance was 245.407 kJ/mol in the

356

first step of the pathway C, consequently this path was not considered the optimal

357

mechanism. Chloropropanol esters mainly formed from pathways A and B.

358

The energy barrier for path A was 81.012 kJ/mol for hydroxyl direct nucleophilic

359

substitution (Path a.1) in the first step. The energy barrier of the direct nucleophilic

360

substitution of ester groups (Path a.2) was reduced to 74.261 kJ/mol. The position of

361

the ester group changed from the sn-2 site to the sn-3 site on the glycerol backbone.

362

Pathway a.1 needed to be completed in two steps, and the pathway a.2 involved three

363

reaction steps, which were close to the energy barriers for the second and third steps.

364

Path a.2 was better than path a.1 from an energy perspective. Although the protonated

365

hydroxyl group was a better leaving group in terms of molecular weight, the oil

366

system was a hydrophobic environment, in which carboxylic acid groups have a

367

stronger tendency to leave. The first-step in the path B reaction was opposite to that of

368

path A. The energy barrier of path b.1 was somewhat lower than that of path b.2. The

369

energies were similar to the first step in the second part of the hydrolysis process.

370

After comparing the diacylglycerols, such as dipalmitin, diolein olein, and dilinoleic

371

olein, the overall energy barrier tended to decrease as the degree of unsaturation

372

increased and the carbon chain increased, but the difference was not significant. There

373

was a high temperature for deodorization, which made it easy to push the reaction

18


Page 18 of 35

Page 19 of 35


374

over the energy barrier. The greater the energy difference between the product and the

375

reactant, the more stable the product.

376

Chloropropanol esters can be divided into 3-MCPD esters and 2-MCPD esters

377

depending on the chlorine binding sites. Collier and Hamlet et al. proposed that

378

3-MCPD content was 3–7 times that of 2-MCPD.16 They concluded that the sn-1,3

379

positions are more likely to be attacked by chlorine atoms; therefore, 3-MCPD esters

380

were the main research subjects here.

381

The nucleophilic attack of chloride ions plays a crucial role converting lipids into

382

chloropropanoates in food. The different attack sities of 1,2-diacylglycerol (1,2-DAG)

383

produced various chloropropanolate esters. Sites with less steric hindrance are more

384

vulnerable to be attacked. The sn-1 and sn-3 sites are considered to be less sterically

385

hindered by secondary carbon atoms, and sn-2 ester bonds, especially long-chain fatty

386

acids, may hinder the approaching chlorine atom as it undergoes molecular

387

rearrangement. Thus, the precursor should be 1,2-DAG, and the sn-1 and sn-3

388

positions of the hydroxyl or ester group were the probable reaction site, as shown in

389

Figure 5. In addition, the reaction was also affected by the choice of diacylglyceride

390

precursors. In this study, a clear judgement was made by comparing the energies of

391

1,2-DAG and 1,3-DAG. In the direct nucleophilic substitution, the energy barrier was

392

118.271 kJ/mol using 1,3 dipalmitoyl diglyceride as the precursor. The energy barrier

393

of indirect nucleophilic substitution was 127.465 kJ/mol, which was significantly

394

higher than that of path a.2, as shown in Figure 6. 1,3-DAG is not common in oils and

395

fats, so it is unlikely to be a precursor. 1,2-DAG was identified as the suitable

19



396

precursor.

20


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Page 21 of 35


398

REFERENCES

399

(1) Davidek, J.; Velisek, J.; Kubelka, V.; Janicek, G.; Simicova, Z. Glyceral C

400

hlorohydrins and Their Esters as Products of the Hydrolysis of Tripalmitin, Tri

401

stearin and Triolein with Hydrochloric Acid. Z. Lebensm. Unters. Forsch. 1980,

402

171, 14–17.

403

(2) Velisek, J.; Davidek, J.; Kubelka, V.; Janicek, G.; Svobodova, Z.; Simicova,Z

404

. New chlorine-containing organic-compounds in protein hydrolysates. J. Agric.

405

Food Chem. 1980, 28, 1142–1144.

406

(3) Hamlet, C. G.; Sadd, P. A.; Crews, C.; Velíšek, J.; Baxter, D. E. Occurren

407

ce of 3-chloro-propane-1,2-diol (3-MCPD) and related compounds in foods: a r

408

eview. Food Addit. Contam. 2002, 19, 619–631.

409

(4) Svejkovska, B.; Novotny, O.; Divinova, V.; Reblova, Z.; Dolezal, M.; Velis

410

ek, J. Esters of 3-chloropropane-1,2-diol in foodstuffs. Czech J. Food Sci. 2004,

411

22, 190–196.

412

(5) Karšulínová, L.; Folprechtová, B.; Doležal M.; Dostálová, J.; Velíšek, J. An

413

alysis of the lipid fractions of coffee creamers, cream aerosols, and bouillon cu

414

bes for their health risk associated constituents. Czech. J. Food Sci. 2007, 25,

415

257–264.

416

(6) Doležal, M.; Chaloupská, M.; Divinová, V.; Svejkovská, B.; Velišek, J. Oc

417

currence of 3-chloropropane-1,2-diol and its esters in coffee. Eur. Food Res. Te

418

ch. 2005, 221, 221–225.

419

(7) Myher, J. J.; Kuksis, A.; Marai, L.; Cerbulis, J. Stereospecific analysis of f

21



420

atty-acid esters of chloropropanediol isolated from fresh goat milk. Lipids. 1986,

421

21, 309–314.

422

(8) Seefelder, W.; Varga, N.; Studer, A.; Williamson, G.; Scanlan, F. P.; Stadl

423

er, R. H. Esters of 3-chloro-1,2-propanediol (3-MCPD) in vegetable oils: Signif

424

icance in the formation of 3-MCPD. Food Addit. Contam. 2008, 25, 391–400.

425

(9) Hrncirik, K.; Van Duijn, G. An initial study on the formation of 3-MCPD

426

esters during oil refining. Eur. J. Lipid Sci. Tech. 2015, 113, 374–379.

427

(10) Abraham, K.; Appel, K. E.; Berger-Preiss, E.; Apel, E.; Gerling, S.; Miel

428

ke, H.; Creutzenberg,O.; Lampen, A. Relative Oral Bioavailability of 3-MCPD

429

from 3-MCPD Fatty Acid Esters in Rats. Arch. Toxicol. 2013, 87, 649–659.

430

(11) Bakhiya, N.; Abraham, K.; Gürtler, R.; Appel, K. E.; Lampen, A. Toxicol

431

ogica Assessment of 3-Chloropropane-1,2-diol and Glycidol Fatty Acid Esters i

432

n Food. Mol. Nutr. Food Res. 2011, 55, 509–521.

433

(12) Masukawa, Y.; Shiro, H.; Nakamura, S.; Kondo, N.; Jin, N.; Suzuki, N.;

434

Ooi, N.; Kudo, N. A New Analytical Method for the Quantification of Glycido

435

l Fatty Acid Esters in Edible Oils. J. Oleo Sci. 2010, 59, 81–88.

436

(13) Franke, K.; Strijowski, U.; Fleck, G.; Pudel, F. Influence of chemical refi

437

ning process and oil type on bound 3-chloro-1,2-propanediol contents in palm

438

oil and rapeseed oil. LWT Food Sci. Technol. 2009, 42, 1751–1754.

439

(14) Bornscheuer, U.; Hesseler, M. Enzymatic removal of 3-monochloro-1,2-pro

440

panediol (3-MCPD) and its esters from oils. Eur. J. Lipid Sci. Technol. 2010,

441

112, 552–556.

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(15) Collier, P. D.; Cromie, D. D. O.; Davies, A. P. Mechanism of formation

443

of chloropropanols present in protein hydrolysates. J. Am. Oil Chem. Soc. 1991,

444

68, 785–790.

445

(16) Hamlet, C. G.; Sadd, P. A.; Gray, D. A. Generation of monochloropropan

446

ediols (MCPDs) in model dough systems. 2. Unleavened doughs. J. Agric. Foo

447

d Chem. 2004, 52, 2067–2072.

448

(17) Matthaus B.; Pudel F.; Fehling P.; Vosmann, K.; Freudenstein, A. Strategi

449

es for the reduction of 3-MCPD esters and related compounds in vegetable oils.

450

Eur. J. Lipid Sci. Technol. 2011, 113, 380–386.

451

(18) Hrncirik K.; Zelinkova Z.; Ermacora A. Critical factors of indirect determi

452

nation of 3-chloropropane-1,2-diol esters. Eur. J. Lipid Sci. Technol. 2011, 113,

453

361–367.

454

(19) Velíšek J.; Calta P.; Crews C.; Hasnip, S.; Doležal, M. 3-Chloropropane-1,

455

2-diol in models simulating processed foods: Precursors and agents causing its

456

decomposition. Czech. J. Food Sci. 2003, 21, 153–161.

457

(20) Li, C.; Zhang, Y.; Li, S.; Wang, G.; Xu, C.; Deng, Y.; Wang, S. Mecha

458

nism of Formation of Trans Fatty Acids under Heating Conditions in Triolein.

459

J. Agric. Food Chem. 2013, 61, 10392–10397.

460

(21) Li, A.; Yuan, B.; Li, W.; Wang, F.; Ha, Y. Thermally induced isomerizat

461

ion of linoleic acid in soybean oil. Chem. Phys. Lipids. 2013, 166, 55–60.

462

(22) Kuhlmann J. Determination of bound 2,3-epoxy-1-propanol (glycidol) and

463

bound monochloropropanediol (mcpd) in refined oils. Eur. J. Lipid Sci. Tech, 2

23



464

011, 113, 335–344.

465

(23) AOCS. Official methods and recommended practices of the American Oil

466

Chemists’ Society. In Acid Value (Cd 3d-63), AOCS Press, Champaign, 1997.

467

(24) Svejkovska, B.; Dolezal, M.; Velisek, J. Formation and decomposition of

468

3-chloropropane-1, 2-diol esters in models simulating processed foods. Czech. J.

469

Food Sci. 2006, 24, 172–179.

470

(25) Shimizu, M.; Vosmann, K.; Matthaus, B. Generation of 3-monochloro-1,2-

471

propanediol and related materials from tri-, di-, and monoolein at deodorization

472

temperature. Eur. J. Lipid Sci. Technol. 2012, 114, 1268–1273.

24


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474

FIGURE CAPTIONS

475

Figure 1. Relationships between DAG content and 3-MCPD ester content in different

476

oils.

477

Figure 2. The relationship between the amount of DAG added and the formation

478

amount of 3-MCPD esters at different temperatures (180°C (a), 200°C (b), 220°C (c),

479

240°C (d)).

480

Figure 3. The newer three reaction pathways for 3-MCPD esters (TS: transition

481

state).

482

Figure 4. Energy barriers for palmitic acid 3-MCPD ester in all pathways. (A): Direct

483

nucleophilic attack (black indicates pathway a.1 and red indicates pathway a.2), (B):

484

indirect nucleophilic substitution (green indicates pathway b.1 and blue indicates

485

pathway b.2), (C): glycidyl ester intermediates. TS1, TS2, and TS3 represent the

486

transition states in the first, second, and third steps, respectively.

487

Figure 5. The reaction path for formation of 2-CPD diesters and 3-MCPD esters from

488

1,3-DAG.

489

Figure 6. Energy barriers for the first step from 1,2-DAG and 1,3-DAG (black

490

indicates 1,2-DAG, red indicates hydrophilic direct nucleophilic substitution of

491

1,3-DAG, blue indicates direct nucleophilic substitution of ester groups from

492

1,3-DAG).

25



Page 26 of 35

Table 1. 3-MCPD esters, DAG, MAG, and FFA contents in different oils MCPD esters

DAG content

MAG content

Acid value

Content (mg/kg)

(%)

(%)

(mg/g)

soybean oil 1a

0.040±0.000

1.816±0.021

0.242±0.008

0.302±0.002

soybean oil 2b

0.262±0.008

1.801±0.017

0.221±0.007

0.112±0.005

palm oil 1a

1.368±0.039

9.220±0.083

0.149±0.002

0.469±0.010

palm oil 2b

3.736±0.083

6.675±0.097

0.141±0.004

0.220±0.003

rapeseed oil

0.340±0.010

2.195±0.054

0.188±0.003

0.123±0.007

corn oil

0.910±0.023

3.889±0.049

0.149±0.001

0.304±0.006

olive oil

0.262±0.007

1.729±0.023

0.070±0.001

0.434±0.002

peanut oil

0.326±0.004

1.732±0.029

0.135±0.005

0.976±0.012

sunflower oil

0.201±0.004

1.652±0.051

0.195±0.004

0.279±0.003

rice bran oil

1.002±0.028

4.174±0.074

0.081±0.001

3.211±0.035

Samples

a

Bleaching oil. b Deodorization oil.

Data are means ± standard deviations (SD) of duplicate technical experiments. The free fatty acid content is expressed

by

the

26


acid

value

Page 27 of 35


Table 2. Direct nucleophilic substitution bond length and dihedral angle. A

a.1

b.1

a.2

b.2

Bond length (Å)

Dihedral angle (°)

Cα-Oα

Cα-Cl

Oα-Hα

Cδ-Oβ-Cβ-Cα

DAG+HCl

1.433

4.024

1.809

154.773

TS

2.277

2.825

1.001

129.959

CP+H2O

3.240

1.822

0.968

156.302

DAG+HCl

1.435

3.876

1.803

145.277

TS

3.249

3.407

0.973

22.589

CP+H2O

3.211

1.822

0.967

141.726

Cγ-Oγ

Cγ-Cl

Oγ-H

Cδ-Oβ-Cβ-Cγ

DAG+HCl

1.448

4.037

1.914

-93.487

TS

2.396

2.921

1.001

-127.487

CP+FA

4.327

1.825

0.972

-131.742

DAG+HCl

1.458

4.033

1.925

58.089

TS

3.730

3.364

1.026

-10.740

CP+FA

3.425

1.836

0.974

58.209

Diacylglyceride (DAG), Transition state (TS), Chloropropane diester (CP), Fat acid (FA).

27



Page 28 of 35

Table 3. 3-MCPD ester (mg/kg) content produced by different oil samples at different heating temperatures DAG (%)

180°C

200°C

220°C

240°C

1

0.086

0.116

0.253

0.259

2

0.156

0.197

0.387

0.454

4

1.011

1.464

2.268

3.156

10

1.597

1.804

2.963

7.573

15

2.005

3.111

5.444

9.679

20

2.318

4.541

14.423

27.541

28


Page 29 of 35


Figure 1

29



Figure 2

30


Page 30 of 35

Page 31 of 35


Figure 3

31



Figure 4 Path A

120

Path a.1 Path a.2

100

Energy (kJ/mol)

80 60 40 20 0 -20 -40 -60 R1

TS1

P1

R2

TS2

P2

R3

TS3

P3

Reaction Coordinate

Path B

160 140

Path b.1 Path b.2

120 Energy (kJ/mol)

100 80 60 40 20 0 -20 -40 -60

R1 TS1 P1

R2 TS2 P2

R3 TS3 P3

Reaction Coordinate

Path C

300

Path C

250

Energy (kJ/mol)

200 150 100 50 0 -50 -100

R1 TS1 P1

R2 TS2 P2

R3 TS3 P3

Reaction Coordinate

32


Page 32 of 35

Page 33 of 35


Figure 5

33



Figure 6

140 120

Energy (kJ/mol)

100 80 60 40 20 0 -20

R

TS

P

Reaction Coordinate

34


Page 34 of 35

Page 35 of 35


TOC graphic

(A)

Energy (kJ/mol)

(B)

C R1 C R2 C OH2

(C)

C R1

H2C

C OH

Reactant

C HC

C R2

Transition state

Product

R1 O

C

R1

C

OH

C

Cl

Reaction Coordinate

35


Molecular Reaction Mechanism for the Formation of 3

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