Formation of Glycidyl Fatty Acid Esters Both in Real Edible Oils during

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Formation of Glycidyl Fatty Acid Esters both in Real Edible Oil during Laboratory-scale Refining and in Chemical Model during High Temperature Exposure Wei Wei Cheng, Guoqin Liu, and Xinqi Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01520 • Publication Date (Web): 18 Jun 2016 Downloaded from http://pubs.acs.org on June 19, 2016

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Journal of Agricultural and Food Chemistry

Target Journal: J. Agric. Food Chem. Number of main text pages: 27 TOC Graphic: 1; Number of figures: 7; Number of tables: 2; Title: Formation of Glycidyl Fatty Acid Esters both in Real Edible Oils during Laboratory-scale Refining and in Chemical Model during High Temperature Exposure

Weiwei Cheng†, Guoqin Liu*, †, ‡, and Xinqi Liu*, †

†

School of Food Science and Engineering, South China University of Technology,

Guangzhou 510640, China ‡

Guangdong Province Key Laboratory for Green Processing of Natural Products and

Product Safety, South China University of Technology, Guangzhou 510640, China

Corresponding author *G. Liu, Tel: 8620-8711-3875. Fax: 8620-8711-4262. E-mail: [email protected], and

X,

Liu,

Tel:

8620-8711-3875.

Fax:

8620-8711-4262.

[email protected].

1

ACS Paragon Plus Environment

E-mail:


1

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ABSTRACT

2

In the present study, the formation mechanism of glycidyl fatty acid esters(GEs)

3

were investigated both in real edible oils (soybean oil, and camellia oil, and palm oil)

4

during

5

(1,2-dipalmitin(DPG) and 1-monopalmitin(MPG)) during high temperature exposure

6

(160-260 °C under nitrogen). The formation process of GEs in chemical model was

7

monitored using attenuated total reflection-fourier transform infrared (ATR-FTIR)

8

spectroscopy. The results showed that roasting and pressing process could produce

9

certain amounts of GEs that were much lower than that of deodorization process. GEs

10

contents in edible oils increased continuously and significantly with increasing

11

deodorization time below 200 °C. However, when the temperature exceeded 200 °C,

12

GEs contents sharply increased in 1-2 h followed by a gradual decrease, which could

13

verify a simultaneous formation and degradation of GEs at high temperature. In

14

addition, it was also found that the presence of acylglycerol (DAGs and MAGs) could

15

significantly increase the formation yield of GEs both in real edible oils and in

16

chemical model. Compared with DAGs, moreover, MAGs displayed a higher

17

formation capacity but substantially lower contribution levels to GEs formation due to

18

their low contents in edible oils. In situ ATR-FTIR spectroscopic evidence showed that

19

cyclic acyloxonium ion intermediate was formed during GEs formation derived from

20

DPG and MPG in chemical model heated at 200 °C.

21

KEYWORDS: edible oils; chemical model; glycidyl esters; formation; refining;

22

acylglycerol; ATR-FTIR

laboratory-scale

preparation

and

refining

and

2


in

chemical

model

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23


INTYODUCTION

24

Glycidyl fatty acid esters (GEs) and 3-monochloropropane-1,2-diol fatty acid esters

25

(3-MCPDEs) are heat-induced processing contaminants most commonly occurring in

26

refined vegetable oils.1-4 Recently, renewed attention has been gained due to the

27

identification of their potential carcinogenicity and widespread occurrences in food

28

products, including infant formula.5-8 There is no available evidence that GEs and

29

3-MCPDEs have negative effects on humans and animals, but their presence in food

30

products is nevertheless worth investigating due to the toxicity of their hydrolyzates

31

which are readily released by gut lipases in vivo.9,10 Glycidol, the hydrolyzate of GEs,

32

is a genotoxic carcinogen that has been classified by International Agency for

33

Research on Cancer (IARC)11 as group 2A, ‘‘probably carcinogenic to human’’ and

34

leads to gene mutations and unscheduled DNA synthesis.6 In 2009, DAG-based oils

35

produced by Kao Corporation had to be withdrawn from the market due to “high levels”

36

of GEs.12 However, the maximum tolerable of GEs in food, especially edible oils,

37

remain still unknown. These reports have added prominence to the need of greater

38

attention on the formation of GEs in refining edible oil.

39

Unlike 3-MCPDEs, the formation mechanisms of GEs in oil refining remain

40

unverified experimentally. Destaillats13 proposed that GEs were formed by

41

intramolecular elimination of a fatty acid from diacylglycerols (DAGs) and a water from

42

monoacylglycerols (MAGs), but not from triacylglycerols (TAGs), and oxopropyl esters,

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isomer of GEs, were formed from DAGs heated at over 140 °C. Additionally, Several

44

authors speculated that GEs and 3-MCPDEs were formed through common 3



45

intermediates which had been thought as acyloxonium ion.1,14,15 However, the

46

predominant formation mechanisms of GEs during oil refining have not yet been

47

demonstrated individually. Although it has been demonstrated that high temperature,

48

DAGs and MAGs contents in crude oils play key roles in the formation of GEs, further

49

influence of high temperature for long residence time (formation and degradation) on

50

GEs formation and formation capacity of GEs from DAGs and MAGs remain still

51

unclear. Consequently, the development of highly effective measures for inhibiting the

52

formation of GEs are restricted.

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The present study aims to investigate the factors of influencing GEs formation

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during oil extracting and laboratory-scale refining in soybean oil (SO), camellia oil

55

(CO), and palm oil (PO). Additionally, a series of confirmatory experiments with a

56

single factor design were performed to further ascertain the precursors of GEs and

57

compare their formation capacity. Finally, attenuated total reflection Fourier transform

58

infrared (ATR-FTIR) spectroscopy, a well-established detection technique which

59

provides highly specific molecular information of a wide range of compounds, was

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used to identify the possible reaction intermediates in chemical models mimicking oil

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deodorization conditions. The results may serve as the scientific basis for the

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elimination of GEs in refined vegetable oils, functional lipids, and oil-based food

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products.

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MATERIALS AND METHODS

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Chemicals. 3-monochloropropane-1,2-diol (3-MCPD, purity 99%), isotopically

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labelled 3-monochloropropane-1,2-diol (d5-3-MCPD, purity 99%), Tripalmitin (TPG, 4


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purity 98%), 1,2-dipalmitin (DPG, purity 98%), 1-monopalmitin (MPG, purity 98%),

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hexadecane, and chromatographic grade tetrahydrofuran were purchased from

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Sigma-Aldrich

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molybdophosphoric acid, sodium bromide, sodium methoxide, n-hexane, diethyl ether,

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methyl tertiary butyl ether(MTBE), ethyl acetate, petroleum ether (bp 30-60 °C),

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iso-octane, ethanol, chloroform, anhydrous sodium sulfate, and glacial acetic acid of

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analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd.

74

(Shanghai, China). Silica gel (100-200 mesh) was purchased from QingDao BANKE

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Separation Materials Co., Ltd. (Qingdao, China). All other reagents used in the study

76

were of analytical grade.

Co.,

Ltd.

(St.

Louis,

MD).

Phenylboronic

acid

(PBA),

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Preparation of Crude Oil. Soybean, Camellia oleifera fruit and oil palm fruit were

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supplied from a local market in Guangzhou, China. They were from Heilongjiang,

79

Guangxi and Malaysia, respectively. Three methods, hot pressing, cold pressing, and

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solvent extraction, were used for the preparation of crude oil. For hot pressing process,

81

the washed and dried raw materials were roasted at 200 °C for 30 min in an automatic

82

roasting machine (Sanyou co., Zhengzhou, China). The roasted samples were ground

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and steamed over boiling water for 5 min and finally pressed to produce the

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hot-pressed crude oil. And the cold-pressed crude oils were prepared by the same

85

procedure as described above but without roasting step. Prior to the pressing process,

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n-hexane was used as the extracting solvent to produce the solvent-extracted oil. All

87

the oil samples were kept in the dark bottles under nitrogen at 4 °C until analysis.

5



88

Preparation of Model Oil. The model oils were prepared by column

89

chromatography based on the previous report with some adaption.16 The solvents for

90

the column were: A: mixture of petroleum ether/ diethyl ether (87:13 by volume); B:

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diethyl ether. About 80 g of silica gel, adjusted to contain 5% water, were loaded into a

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2.5×40 cm glass column with a sintered disc and a stopcock by means of a slurry in

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solvent A.

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About 50 g of crude oils were mixed with ultrapure water (1:1 by volume) and

95

treated by ultrasound for 30 min at 50 °C. The mixtures were centrifuged, and then the

96

separated oil were dried by anhydrous sodium sulfate. Subsequently, the

97

organochlorines in oils were adsorbed with activated carbon. This process is repeated

98

until chlorinated substances was removed completely in crude oils. The chlorine-free

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oils, dissolved in 2×30 mL of solvent A, were placed at the top of the column. The

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nonpolar fraction was eluted with 150 mL solvent A, and the polar fraction of the oil

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was eluted from the silica gel with 150 mL of solvent B afterwards. Subsequently, the

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solvents were rotoevaporated at 50 ℃ using a rotary evaporator (Zhengzhou Yarong

103

Instruments Co., Ltd., Zhengzhou, China) and then evaporated by a gentle flow of

104

nitrogen until the solvents were removed completely. The nonpolar fraction was used

105

as model oil in our study. Both fractions were kept frozen at -18 °C prior to analysis.

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Laboratory-scale Refining Process. Three different kinds of crude oils were

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refined with standard conditions that are widely applicable to the industry. To avoid

108

the oil oxidation, the refining processes were proceeded under nitrogen. About 800 g

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of crude oils were degummed by adding 0.10% w/w phosphoric acid and 3% w/w hot 6


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water, stirred for 30 min at 85 °C. Gums were separated by centrifugation at 3500

111

r/min for 15 min in L530 centrifuge (Hunan Xiangyi Laboratory Instrument

112

Development Co., Ltd, Hunan, China). Degummed oils were neutralized by adding the

113

required amounts of 3 mol/L NaOH solution at 85 °C with stirring for 20 min to form

114

soaps with free fatty acid. The resulting soaps were washed twice with deionized

115

water (20% m/m of oil) and removed by gravity with the help of a separating funnel.

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After a drying step at reduced pressure (30-40 mbar), subsequent bleaching was

117

performed at 95 °C with a 1% w/w dosage of activated bleaching earth (Tonsil

118

Optimum 214 FF) which was filtered out after 30 min treatment to obtain bleached oils.

119

The gums and bleaching earth were removed by vacuum filtration on a 0.45 µm

120

membrane. In the last step of refining, in order to ascertain how the deodorization

121

influences the formation of GEs, about 500 g of bleached oils were deodorized at six

122

different temperatures and times in the range of 160-260 °C and 0.5-3.0 h,

123

respectively. Simultaneously, a constant strip steam with the speed of 0.15 mL/min

124

was continuously passed through the bottom. The deodorizer was equipped with a

125

three-necked flask and thermal-controlled heating mantle. Three necks were

126

connected with an electric thermometer from the heating mantle, an inlet tube from

127

steam generator and a vacuum pump (Shanghai Jingke Industrial Co., Ltd., Shanghai,

128

China), respectively. Sampling was performed after each step and the testing

129

samples were stored at -18 °C until analysis.

130

Analytical Methods. Total Polar Components (TPC). The gravimetric method was

131

used to determine the total amounts of polar fraction after removing the nonpolar 7



132

components by column chromatography separation, as depicted in Preparation of

133

Model Oil section. The TPC was analyzed qualitatively for composition by thin-layer

134

chromatography(TLC), according to the previous report with some modification.17 The

135

developing solvents consisted of a mixture of n-hexane/diethyl ether/acetic acid

136

(90:10:2 by volume) system. To visualize the lipids, the plates were sprayed with

137

10%(w/w) solution of molybdophosphoric acid in ethyl alcohol. After evaporation of

138

alcohol, the plate was heated in drying oven (Shanghai Keelrein instruments Co., Ltd.,

139

Shanghai, China) at 120 °C.

140

TPC Composition. The composition of TPC was analyzed using high performance

141

size exclusion chromatography (HPSEC) as described previously with slight

142

modifications in sample preparation.18 The TPC was obtained as described in Total

143

Polar Components (TPC) section above. 0.1 mL aliquot fraction was 10-fold serially

144

diluted with n-hexane and isopropanol (10:1 by volume). 1.5 mL aliquot of this solution

145

was transferred into the testing tube and stored at -18°C before analysis. Oxidised

146

triacylglycerols (oxTAG), triacylglycerol polymers (TGP), triacylglycerol dimers (TGD),

147

DAGs, and MAGs were analyzed using the high-performance liquid chromatography

148

(Waters 2695) equipped with a refractive index detector. These components were

149

separated over a Ultrastyragel columns (300×4.6 mm, 5 µm; 100 Å and 500 Å) that

150

were connected in tandem, and equipped with a guard column (50 × 4.6 mm) (Waters

151

Co., Milford, MA) preceding the main column. The column temperature was kept at

152

35 °C. Tetrahydrofuran was used as the mobile phase at a flow-rate of 0.7 mL/min.

153

The injection volume was 10 µL. The results were expressed as percentage of TPC. 8


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154

Glycidyl Esters Analysis. According to the AOCS Official Method (AOCS Cd

155

29c-13),19 GEs in the oils were identified. An indirect method, based on the

156

determination of 3-MCPDEs levels, was used for estimation of GEs contents, which

157

involved the release of 3-MCPD and glycidol from their esters, PBA derivatization and

158

quantitation using gas chromatography-mass spectrometry (GC-MS). The internal

159

standard d5-3-MCPD and a stoichometric conversion factor (0.67) were used for

160

quantitation of GEs based on the original DGF method C-III 18 (09).20 In sample

161

preparation, two 100 mg aliquots of lipid samples were dissolved in MTBE and spiked

162

with d5-3-MCPD (Assay A and B). Subsequently, free 3-MCPD and glycidol were

163

released from the esters by adding 200 µL of 25 g/L sodium methoxide in methanol.

164

The reaction was stopped by the addition of 600 µL acidic sodium chloride solution

165

(200 g/L) (Assay A) and 600 µL acidic sodium bromide solution (600 g/L) (Assay B),

166

respectively. A quantitative analysis of the 3-MCPD derivatives was performed in a

167

Shimadzu QP2010Plus system (Shimadzu, Tokio, Japan) equipped with a capillary

168

column Agilent TG-5MS (30 m×0.25 mm×0.25 µm). High purity helium was used as

169

carrier gas with a constant flow rate of 1.18 mL/min.1 µL of sample was injected in a

170

splitless mode. GC oven temperature program was as follows: 80 °C with an increase

171

of 5 °C/min to obtain 155 °C, followed by increase by 60 °C/min to obtain 300 °C that

172

was held for 5 min. The mass spectrometer was operated in selected ion monitoring

173

mode with positive electron ionization (EI+) at an ionization voltage of 70 eV.

174

Temperature of ion source and interface in mass spectrometer was 200 and 280 °C,

175

respectively. The ion traces m/z 147 and 196 were selected as the quantitative ions 9



176

for the quantitation of GEs. The dwell time was set as 200 ms each. Quantification of

177

the 3-MCPDEs was conducted by multiplying the ratio of peak areas of the analyte

178

and the internal standard d5-3-MCPD based on corresponding ion traces with the

179

spiking levels of d5-3-MCPD. The levels determined by Assay A were sum of

180

3-MCPDEs and GEs and only 3-MCPDEs contents for Assay B, so GEs levels could

181

be calculated as the difference between the two determinations multiplying a

182

stoichiometric conversion factor, that is, (Assay A-B) × 0.67, and were expressed as

183

glycidol equivalent units. The determination was run in triplicate.

184

In-situ Monitoring of GEs Formation by ATR-FTIR. About 5 mg of pure TPG,

185

DPG, and MPG for this study were heated at 200 °C for 30 min under nitrogen. The

186

samples were taken every 5 min and immediately snap-frozen in liquid nitrogen to

187

stop the further reaction. The samples were analyzed for the variations of functional

188

groups using ATR-FTIR. The spectra were collected in a VERTEX 70 spectrometer

189

(Bruker Optics, Ettlingen, Germany) equipped with an attenuated total reflectance

190

(ATR) attachment and submitted to 4 cm-1 of resolution and 64 scans per sample. All

191

spectra were recorded from 1800 to 800 cm−1 and processed with the computer

192

software program OPUS 7.2 for Windows (Bruker Optics GmbH, Ettlingen, Germany).

193

Statistical Analysis. Statistical analysis was performed with MATLAB R2015a

194

program (The Mathworks, Natick, MA). Each analytical measurement was carried out

195

in triplicate and the data are expressed as mean ± standard deviation (SD). Tukey–

196

Kramer multiple comparisons tests were used to determine the lowest statistically

197

significant differences (LSD) between the means at a confidence level of 95%(P < 10


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198

0.05). Figure preparation was carried out using Origin 2015 (MicroCal Software Inc.,

199

MA).

200

RESULTS AND DISCUSSION

201

Effect of refining degrees on the formation of GEs in the tested oils. Three

202

kinds of vegetable oil samples, that is, SO, CO, and PO, with different refining

203

degrees were prepared. The data of GEs levels were depicted in Table 1. No

204

statistical differences were observed on GEs contents in the tested oils with different

205

refining degrees before deodorization. Nevertheless, deodorized oils showed

206

significantly higher contents of GEs compared with corresponding bleached oils

207

(P

Formation of Glycidyl Fatty Acid Esters Both in Real Edible Oils during

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