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Effects of Fe3+ and Antioxidants on Glycidyl Esters Formation in Plant Oil at High Temperature and Their Influencing Mechanisms Wei Wei Cheng, Guoqin Liu, and Xinqi Liu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 7, 2017

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Journal of Agricultural and Food Chemistry

Effects of Fe3+ and Antioxidants on Glycidyl Esters Formation in Plant Oil at High Temperature and Their Influencing Mechanisms

Weiwei Cheng†, Guoqin Liu*, †, ‡, §, and Xinqi Liu⊥, #

†

School of Food Science and Engineering and ‡Guangdong Province Key Laboratory for

Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou 510640, China §

College of Food Science and Engineering, Henan University of Technology, Zhengzhou

450001, China ┴

Beijing Advanced Innovation Center for Food Nutrition and Human Health and #School of

Food and Chemical Engineering, Beijing Technology and Business University, Beijing 100048, China

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

ACS Paragon Plus Environment


1

ABSTRACT: This research investigated the effects of Fe3+ and antioxidants on glycidyl

2

esters (GEs) formation and the free radical-mediated mechanisms involving the recognition

3

of cyclic acyloxonium free radical intermediate (CAFRI) for GEs formation in both plant oil

4

model (palm oil, camellia oil, soybean oil, and linseed oil) system and chemical model

5

(dipalmitin and methyl linoleate) system heated at 200 °C. Results show that Fe3+ can

6

promote the formation of GEs, which can be inhibited by antioxidants in plant oil during

7

high-temperature exposure. Based on the monitoring of cyclic acyloxonium and ester

8

carbonyl group by Fourier transform infrared spectroscopy, the promotion of Fe3+ and the

9

inhibition of antioxidants (tert-butylhydroquinone and α-tocopherol) for GEs formation

10

occurred not only through lipid oxidation but also through directly affecting the formation of

11

cyclic acyloxonium intermediate. Additionally, a quadrupole-time of flight tandem mass

12

spectrometry measurement was conducted to identify the presence of radical adduct

13

captured by 5,5-dimethylpyrroline-N-oxide, which provided strong evidence for the

14

formation of CAFRI. Thus, one possible influencing mechanism can be that free radical

15

generated in lipid oxidation may be transferred to dipalmitin and promote CAFRI formation.

16

Fe3+ can catalyze free radical generation while antioxidants can scavenge free radical, and

17

therefore they also can directly affect CAFRI formation.

18

KEYWORDS: glycidyl esters, plant oil, iron ion, antioxidants, lipid oxidation, influencing

19

mechanisms

20 21 22 23


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24


Introduction

25

In 2010, glycidyl esters (GEs) of fatty acids, a group of new potential food-processing

26

contaminants, was first be detected in edible oils and fats due to the regular overestimation

27

of 3-monochloropropane-1,2-diol (3-MCPD) esters of fatty acids analyzed by early indirect

28

methods.1 Since then, plentiful literature has reported that GEs are mainly formed in the

29

deodorization step during oil refining and therefore widely existed in refined oils, fats, and

30

oil-based food, including infant formula.2-6 Although there is no available evidence for the

31

detrimental effect of GEs on human and animal, the hydrolysate, glycidol, has been

32

determined to be genotoxic and carcinogenic to rats7 and has already been categorized as

33

group 2A carcinogen by the Intl. Agency for Research on Cancer (IARC).8 Therefore, to

34

develop the efficient elimination and inhibition method for reducing GE levels in edible oils,

35

the further influencing factors and formation mechanisms should be investigated

36

thoroughly.

37

Up to now, it has been well known that deodorization temperature and time, type of oil, as

38

well as oil preparation method have significant effects on the formation of GEs.9

39

Furthermore, diacylglycerols (DAGs) and monoacylglycerols (MAGs) have been identified

40

as the precursors of GE formation.9 Recently, our research group has published a

41

comprehensive review of the formation, occurrence, analysis, and elimination method of

42

GEs in refined edible oils.10 To sum up, there are four proposed reaction pathways for GE

43

formation derived from DAGs and MAGs, all involving the intramolecular rearrangement via

44

charge migration and differing by means of either the nature of the intermediate or the

45

leaving group. Two of the proposed mechanisms involve a common reactive intermediate,

46

that is, cyclic acyloxonium ion, formed by either deacidification of 1,2-DAGs or dehydration



47

of MAGs (either 1-MAGs or 2-MAGs). The other 2 pathways consider a direct

48

intramolecular rearrangement followed by elimination of fatty acid for DAGs (either

49

1,2-DAGs or 1,3-DAGs) or of water for 1-MAGs. Our previous study confirmed the

50

presence of cyclic acyloxonium intermediate (CAI) during GE formation at high temperature

51

using attenuated total reflectance Fourier transform infrared spectroscopy (ATR‒FTIR),

52

which was also earlier conducted to investigate the formation of CAI during 3-MCPD ester

53

formation.11 Afterward, a novel free radical-mediated mechanism for 3-MCPD ester

54

formation from DAGs,12 triacylglycerols (TAGs),13 and MAGs14 was proposed, which was

55

involved in the formation of cyclic acyloxonium free radical intermediates but not cyclic

56

acyloxonium cations. Also, based on that, mitigating 3-MCPD esters formation by

57

antioxidant and radical scavenger was also investigated in detail.15,16 However, the issue

58

whether the free radical-mediated mechanisms are as well responsible for the formation of

59

GEs remains still unclear.

60

Lipid oxidation is one of the vital reactions resulting in the quality deterioration of edible

61

oil and oil-based food and commonly occurs resulting from the oxidative cleavage of an

62

electron-rich double bond in unsaturated fatty acid (UFAs). It has been well known that lipid

63

oxidation involves many types of free radicals which are possible to participate in the

64

formation of CAI and even influence GE formation. Pro-oxidant and antioxidant can

65

significantly affect the lipid oxidation via the corresponding promotion and inhibition of free

66

radicals. Currently, the free radical-mediated mechanisms of 3-MCPD ester formation have

67

been identified before,12 but there is yet no report about the relationship between lipid

68

oxidation/free radical and GE formation at high temperature.


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Therefore, our present study aimed to investigate the effect of Fe3+ and antioxidants on

70

the formation of GEs and to further ascertain the free radical mediated mechanisms and the

71

influencing mechanisms of lipid oxidation for GE formation in both model oil system and

72

chemical model system. To exclude the interference of DAG and MAG discrepancy in

73

different types of oils, model oil was selected as real oil system. A series of model

74

experiments consisting of the addition of Fe2(SO4)3, tert-butylhydroquinone (TBHQ), and

75

α-tocopherol (VE) were conducted at 200 °C with dipalmitin, diolein, or methyl linoleate as

76

chemical models. FTIR was used to determine the cyclic acyloxonium and ester carbonyl

77

groups, and quadrupole−time of flight mass spectrometer (Q‒TOF MS) was used to identify

78

the presence of radical adduct captured by 5,5-dimethylpyrroline-N-oxide (DMPO).

79

Materials and methods

80

Materials

81

Crude palm oil was a gift from Yihai Kerry (Tianjin) Investment Co., Ltd. (Tianjin, China),

82

and it originates from Malaysia. Crude camellia oil was purchased from Yongxing Taiyu

83

Camellia Oil Co., Ltd. (Chenzhou, China). Crude soybean oil was provided as a gift from

84

China National Cereals, Oils and Foodstuffs Corporation (COFCO). Crude linseed oil was

85

graciously provided by Gansu Abest Plant Oil Development Co., Ltd. (Jinchang, China).

86

These crude oils were prepared by solvent extraction and free of GEs. Model palm oil

87

(MPO), model camellia oil (MCO), model soybean oil (MSO), and model linseed oil (MLO)

88

were

89

sn-1,2-Dipalmitin (DPG, 98%), sn-1,2-diolein (DOG, ≥97%), 3-MCPD (98%), DMPO (≥

90

97%), TBHQ (97%), hexadecane ( ≥ 99%), and VE ( ≥ 96%) were purchased from

91

Sigma-Aldrich Co., Ltd. (St. Louis, MO). Methyl linoleate (ML, 95%) was purchased from

prepared

by

column

chromatography

technology


in

the

present

study.


92

Aladdin Reagent Co., Ltd. (Shanghai, China). d5-3-MCPD (99%) and phenylboronic acid

93

(PBA) were bought from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Analytical

94

grade sodium thiosulfate, iron(III)sulfate (Fe2(SO4)3), n-hexane, glacial acetic acid, diethyl

95

ether, petroleum ether (bp 30−60 °C), methyl tertiary butyl ether (MTBE), and anhydrous

96

sodium sulfate were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,

97

China). 100‒200 mesh silica gel was obtained from Qingdao BANKE Separation Materials

98

Co., Ltd. (Qingdao, China). All other chemicals in this work were of analytical grade.

99

Preparation of Model Oil

100

The degumming, deacidification, and bleaching procedure of crude plant oils were

101

conducted according to the same operation in our previous report,9 which aimed to obtain

102

the refined-bleached (RB) plant oils. Subsequently, column chromatographic method was

103

carried out to prepare the corresponding model oil with the absence of polar fraction. The

104

solvent A for the column was the mixture of petroleum ether and diethyl ether (87:13, v/v),

105

and solvent B was diethyl ether. The silica gel (about 80 g) activated by 5 % deionized

106

water was loaded into a glass column fitted with a stopcock and a sintered disc with an aid

107

of a slurry in solvent A.

108

RB oil obtained above was treated with anhydrous sodium sulfate and activated carbon

109

to remove the moisture and chlorinated substances, respectively. Subsequently, the RB oil

110

was dissolved in 2 × 30 mL of solvent A and poured into the glass column. 150 mL of

111

solvent A was used to elute the nonpolar fraction that was regarded as model oil in this

112

study. Additionally, the polar fraction remained on the silica column was also eluted by 150

113

mL of solvent B. Last, the solvent in the nonpolar or polar eluent was preliminarily removed

114

by rotary evaporator (Zhengzhou Yarong Instruments Co., Ltd., Zhengzhou, China) at


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50 °C and then evaporated completely with a gentle nitrogen flow. Both fractions were

116

stored in the nitrogen-filled bottle at ‒18 °C until analysis.

117

Heating Experiment in Oil Model System

118

A series of different levels of TBHQ and Fe2(SO4)3 were added into model oil prepared in

119

Preparation of Model Oil section and mixed completely. The final concentrations were

120

reached at 0.05, 0.12, and 0.18 mg/g for TBHQ, as well as 0.1, 0.2, and 0.4 mg/mL for

121

Fe2(SO4)3. Alternatively, the reaction bottle (10.2 cm tall, I.D. 1.8 com) was sealed by an

122

ampoule filling machine (Changsha Buyuan Pharmaceutical Machinery Co., Ltd.,

123

Changsha, China) under nitrogen aeration or exposed in air. Model oil without any addition

124

of TBHQ or Fe2(SO4)3 was set as control. The heating treatment was conducted at 200 °C

125

under air or nitrogen. After that, the samples were cooled to room temperature and stored

126

in a nitrogen-filled glass bottle at 4 °C prior to further analysis. All experiments were

127

performed in triplicate.

128

Heating Experiment in the Chemical Model System

129

DPG and DOG, dissolved in hexadecane and prepared at a concentration of 1.0 mg/mL,

130

were selected as the chemical model representing the precursors of GE formation in plant

131

oil. The antioxidants (TBHQ and VE) were prepared at 0.005 mol/L in hexadecane. 2 mg

132

aliquot of pro-oxidant (Fe2(SO4)3) or 1.0 mL of antioxidant solution, as well as 4 mL of pure

133

ML, were selectively added into chemical model system to investigate their effect on GE

134

formation. The formulas of model mixtures were exhibited in Table 1. After being blended

135

thoroughly by vortexing, the mixture was heated at 200 °C for 1 h in a drying oven

136

(Shanghai Keelrein Scientific Instruments Co., Ltd., Shanghai, China), and then stored in a



137

nitrogen-filled glass bottle at 4 °C prior to GE analysis. All experiments were performed in

138

triplicate.

139

Analytical Methods. Peroxide value (POV). POV was determined according to a modified

140

iodometric method.17 Briefly, 5 g of oil sample was dissolved completely in 50 mL of solvent

141

mixture consisting of acetic acid and isooctane (3:2, v/v), in which 0.5 mL of saturated KI

142

solution was then added. After 1 min at ambient temperature with an occasional shake, 30

143

mL of deionized water was poured into the reaction cube, shaking vigorously to release all

144

iodine from isooctane layer, and then titration was performed against 0.1 mol/L sodium

145

thiosulfate using 1% starch indicator until the blue-grey color just disappeared. All samples

146

were determined in triplicate unless otherwise mentioned. Results were presented as

147

mequiv peroxide/kg of oil.

148

p-Anisidine Value (p-AV). A measure of p-AV was done based on AOCS official Method

149

Cd 18-9018 with some modifications. Briefly, 2.0 g aliquot of oil sample was weighed into a

150

25 mL volumetric flask and dissolved by iso-octane, and then diluted to 25 mL. A Genesys

151

10S spectrophotometer (Thermo Scientific, Waltham, MA) was used to determine the

152

absorbance of this above solution (Ab) at 350 nm, against isooctane as blank.

153

Subsequently, 5 mL of oil-isooctane solution was mixed with 1 mL 2.5 g/L p-anisidine

154

reagent. After 8 min at 25 °C, the absorbance of this reaction solution (As) at 350 nm was

155

measured, and the mixture containing 5 mL isooctane and 1 mL p-anisidine reagent was

156

used as blank. p-AV was calculated based on the following formula: p-AV=25 × (1.2As‒

157

Ab)/m, where As and Ab denote the absorbance of the reaction mixture and oil-isooctane

158

solution, respectively, and m is the mass of oil. Additionally, according to the previous


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report,19 total oxidation value (TOTOX) was calculated as (2 × POV) + p-AV. All measures

160

were performed in triplicate.

161

Total Polar Component (TPC). The amount of TPC was determined by a gravimetric

162

method. The polar fraction was isolated from model oil by column chromatography

163

separation, as described in Preparation of Model oil section. After removing the elution

164

solvent, TPC was then dried in an oven at 105 ± 2 °C and weighed accurately. All

165

measures were performed in triplicate. TPC level was presented as the percentage of

166

model oil.

167

TPC composition. High performance size exclusion chromatography (HPSEC) was

168

utilized to analyze the composition of TPC based on the method reported by Cao et al.20

169

with a minor adjustment of sample preparation. TPC sample was prepared based on the

170

procedure in Preparation of Model Oil and Total Polar Component (TPC) sections. Then,

171

this sample was dissolved in a mixture of n-hexane and isopropanol (10:1, v/v) to a

172

concentration of 10 mg/mL. After being filtered through a 0.45 µm nylon membrane, this

173

solution was transferred into the 1.5 mL test tubes and stored at ‒18 °C until further

174

analysis. TPCs, consisting of oxidized triacylglycerols (oxTAGs), triacylglycerol polymers

175

(TGPs), triacylglycerol dimers (TGDs), DAGs, MAGs, and free fatty acids (FFAs), were

176

determined by high-performance liquid chromatography using Waters 2695 system

177

equipped with a refractive index detector and two tandem Ultrastyragel columns (300 mm ×

178

4.6 mm, 5 µm; 100 and 500 Å), connected to a guard column (50 mm × 4.6 mm) (Waters

179

Co., Milford, MA). A 10 µL aliquot of TPC sample was injected and eluted with

180

tetrahydrofuran at a flow rate of 0.7 mL/min at the column temperature of 35 °C. oxTAGs,

181

TGPs, TGDs, DAGs, MAGs, and FFAs in oil samples were quantified by area normalization



182

method. All measures were performed in triplicate. The results were presented as the

183

percentage of model oil.

184

GE analysis. An indirect method for GE determination in oil sample using gas

185

chromatography−mass spectrometry (GC‒MS) was developed base on the AOCS Official

186

Method (AOCS Cd 29c-13)21 and original DGF method C-III 18 (09)22 with some

187

modifications. Briefly, two 100 mg of oil samples were weighed into test tubes containing

188

100 µL MTBE and 0.5 g d5-3-MCPD, which was labeled as assay A and assay B. Then,

189

200 µL of 25 g/L sodium methoxide-methanol solution was added into the test tubes to

190

liberate free 3-MCPD and glycidol from their bound forms, which is stop by the addition of

191

600 µL of 200 g/L acidic sodium chloride solution (assay A) and 600 µL of 600 g/L acidic

192

sodium bromide solution (assay B), respectively. After 600 µL n-hexane was added into

193

both tubes and kept at room temperature with vigorous shaking for about 5 min, both tubes

194

were centrifuged at 4000 r/min for 5 min. Subsequently, 200 µL of saturated PBA-diethyl

195

ether solution was added to both obtained organic phase above and stood at 30 °C for 20

196

min. these both assays were dried using a soft nitrogen flow and then reconstituted in 500

197

µL isooctane, which was stored at ‒18 °C until GC-MS analysis.

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3-MCPD derivatives were separated and quantified in a Shimadzu QP2010Plus GC-MS

199

system (Shimadzu, Tokio, Japan) equipped with a 30 m × 0.25 mm × 0.25 µm Agilent

200

TG-5MS column. 1 µL of sample was injected in a splitless mode with high purity helium as

201

the carrier gas at 1.18 mL/min. The oven temperature program and EI-MS conditions were

202

depicted as our previous report.9 The quantitation of GEs was conducted according to the

203

original DGF method C-III 18 (09), GE level was calculated as (Assay A ‒ Assay B) × 0.67.

204

All measures were conducted in triplicate.


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Determination of Cyclic Acyloxonium and Ester Carbonyl Groups by FTIR. The

206

variation of cyclic acyloxonium and ester carbonyl groups was monitored using ATR-FTIR.

207

The mixtures of DPG with Fe2(SO4)3 and VE, respectively, as well as single DPG, were

208

heated at 200 °C for 15 min. After that, Fe2(SO4)3 or VE was removed by solvent extraction

209

(n-hexane and methanol, respectively), centrifugation, and nitrogen blowing. The dried

210

samples were separately pressed onto the KBr pellet and scanned immediately at

211

wavenumbers ranging from 1500 to 1900 cm-1 in a VERTEX 70 spectrometer (Bruker

212

Optics, Ettlingen, Germany). DSG at 25 °C was used as negative control. Data were

213

processed using OPUS 7.2 software (Bruker Optics GmbH, Ettlingen, Germany).

214

Determination of DMPO Radical Adducts by Q‒TOF MS/MS. The determination of

215

DMPO radical adducts was conducted according to the previous report by Zhang et al.12

216

with a minor modification. 2 mL of 1.0 mg/mL DPG in toluene was mixed with 2 mL of 5.0

217

mg/mL DMPO, which was then heated at 200 °C for 15 min. Likewise, 4 mL of 0.5 mg/mL

218

DPG-toluene solution was also heated at the same conditions, which was set as control.

219

After both cooling immediately to room temperature in the freezing chamber, the DPG

220

solution was diluted 50-fold with toluene and then injected into the Bruker Maxis Impact Q‒

221

TOF mass spectrometer (Bruker Co., Bremen, Germany) combined with tandem mass

222

spectrometry (MS/MS) for analysis. Electrospray ionization positive mode (ESI+) was

223

selected as the LockSpray source. High purity nitrogen was used for drying (4.0 L/min), and

224

nebulization (0.6 Bar). The drying heater was set as 180 °C, and the voltage of capillary

225

was 3.5 kV. MS spectra were recorded at the range of 50‒1000 mass/charge (m/z) and

226

processed using a Bruker Compass DataAnalysis 4.1 software (Bruker Co., Bremen,

227

Germany).



228

Statistical analysis. All tests were carried out in triplicate independently. Data were

229

reported as mean ± standard deviation (SD). Statistical significance was measured by

230

Duncan's multiple range tests using SPSS 22.0 software (IBM Corp. Armonk, NY, USA),

231

and the difference between data was considered to be significant at P < 0.05.

232

RESULTS AND DISCUSSION

233

The relationship between GE formation and lipid oxidation and radical scavenging in

234

plant oil was investigated and demonstrated in the present work. Previously, we have get a

235

result that 200 °C is a turning point for the formation rate of GEs in laboratory-scale

236

deodorization, and thus it was used as the heating temperature. To eliminate the

237

interference of polar fractions containing the precursors of GE formation, that is, DAGs and

238

MAGs, model oil without polar fractions was prepared and used as the research subject.

239

Based on the fatty acid composition of plant oil that is essentially and closely linked to lipid

240

oxidation, palm oil, camellia oil, soybean oil, and linseed oil were selected in this study. The

241

fatty acid composition of their corresponding model oil was shown in Figure 1. The effects

242

of Fe3+, TBHQ, and VE on GE formation were investigated in both model oil and chemical

243

model systems via GC‒MS and FTIR. At last, in order to ascertain their influencing

244

mechanisms, the free radical in model systems was captured by DMPO and identified by

245

Q‒TOF MS/MS. The results presented in this work will provide evidence for the free

246

radical-mediated GE formation mechanism and show that lipid oxidation/free radical

247

promotes for the formation of GEs in plant oil. Further, this work will lay a foundation for the

248

development of efficient elimination methods of GEs.

249

Evolution of GE Level, POV, TOTOX, and TPC Level in Plant Oil Model at High

250

Temperature. It is well known that high temperature is not only largely responsible for the


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formation of GEs,23,24 but also can easily lead to lipid oxidation. According to our previous

252

work,9 the experimental temperature was set at 200 °C in this study. When MPO, MCO,

253

MSO, and MLO were heated directly at this temperature, GE levels were rather low and

254

varied insignificantly during heating (data not shown). This observation is explained based

255

on the previous finding that TAG is not the precursor of GE formation.24 Therefore, 4% DPG

256

was added into the model oil to ensure the feasible formation of GEs in model oil. GE level,

257

POV, TOTOX, and TPC level were monitored every 30 or 60 min during heating and the

258

results were shown in Figure 2. From Figure 2A, GE levels in all tested model oils

259

increased significantly with time. Interestingly, GE level in MLO was rather higher than that

260

in other model oils. Based on the fact that the highest GE content was detected in palm oil

261

and even palm oil-based fat.1,25-27, it was interesting to find that the formation of GEs in

262

MPO was lowest in the tested model oils, which suggested the great contribution of TPC

263

involving precursors to the formation of GEs. To our knowledge, this is the first report that

264

when plant oil possesses the same amount of precursors, such as DAG, the type of plant

265

oil seems responsible for the difference of GE levels in different tested oils. Lipid oxidation

266

is one of the most common thermal reaction at high temperature, which is largely related to

267

the degree of unsaturation of plant oil. It was well expected that MLO had the highest POV

268

and TOTOX, followed by MSO, then MCO and MPO (Figure 2B and Figure 2C), which

269

accounted for the difference of unsaturated fatty acid in tested oils (Figure 1). These results

270

seemingly coincided with GE formation in tested oils (Figure 2A). However, it has also been

271

reported that conventional heating enabled the increasing DAG level in edible oil.28 In the

272

present study, as shown in Figure 2D, it was indeed found that TPC contents increased

273

linearly with time up to 3 h, regardless of the type of oil. this finding was in agreement with



274

the results reported by Correia et al.29 Additionally, TPC content in tested oil was ranked as

275

MLO > MSO > MCO > MPO, which seemed also to be positively related to GE formation.

276

Accordingly, these facts raised a question: what led to the discrepancy of GE levels among

277

tested oils, lipid oxidation or TPC formation?

278

Evaluation of Contribution of formed TPC, Pro-Oxidant, and Antioxidant to the

279

Formation of GEs in Plant Oil Model at High Temperature. As depicted above, both

280

TPC content and degree of lipid oxidation seemingly increased with the same trend with GE

281

formation in tested oils. To further explain this phenomenon, first, TPC, consisting of oxTAG,

282

TGP, TGD, FFA, MAG, and DAG, was analyzed every hour for a period of 3 h, and the

283

results were presented in Table 2. In spite of the linear increase of TPC contents in all

284

tested oils (Figure 2D), DAG and MAG levels were relatively low compared to

285

oxidized/polymerized products, that is, oxTAG, TGP, and TGD, suggesting that oxidative

286

polymerization was the main chemical reaction in heated oil exposed in air, as reported by

287

Choe and Min.30 Previously, Craft et al.31 have reported that GE content increases

288

exponentially when DAG level exceeds 3‒4% of oil but varied slightly with below 3% of

289

DAG level. Thus, DAG and MAG (altogether 0.042‒0.139% of oil) from pyrolyzation of TAG

290

contributed mildly to the formation of GEs during heating. In addition, as shown in Figure 3,

291

the total levels of DAG and MAG did almost not present significant difference (P > 0.05) in

292

tested oils heated for the same time. These findings seemingly indicated that the

293

remarkable increase of TPC content did not necessary contribute to GE formation and the

294

discrepancy of GE levels in different tested oils (Figure 2A) compared with our previous

295

report.9


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Subsequently, the second experiment was to confirm the effects of pro-oxidants and

297

antioxidants on the formation of GEs in four tested oils. For this sake, Fe2(SO4)3 and TBHQ

298

were added into the model oil to promote or inhibit the lipid oxidation evaluated by POV and

299

TOTOX levels (data not shown). Meanwhile, GE level was detected in these model oils

300

heated at 200 °C for 1 h. The results were shown in Figure 4. It is well known that Fe3+ can

301

initiate oil peroxidation by catalyzing the generation of alkyl radical.32,33 From Figure 4A, GE

302

levels in tested model oils heated at 200 °C for 1 h increased significantly with the addition

303

of Fe2(SO4)3 (P < 0.05). However, when EDTA-2Na, a chelating agent, together with

304

Fe2(SO4)3 was added to model oil, GE level was significantly lower than that without

305

EDTA-2Na (P VE. These results were consistent with that in the presence of ML and

355

therefore indicated that Fe3+, TBHQ, and VE might also directly acting on the formation of

356

GEs, which could involve the free radical mechanisms based on the promotion or inhibition

357

mechanisms of these substances for lipid oxidation as depicted above.

358

Monitoring of Cyclic Acyloxonium and Ester Carbonyl Groups in Chemical Model

359

System Using ATR-FTIR. As depicted above, pro-oxidants and antioxidants could

360

influence the formation of GEs not only through lipid oxidation but also through the direct

361

effect on its formation pathway (for example, accelerated or inhibited the formation of CAI)

362

in plant oil. Accordingly, to further investigate whether and how the pro-oxidant and

363

antioxidant affect the formation reaction of GEs, cyclic acyloxonium and ester carbonyl

364

groups were monitored in DPG system using ATR-FTIR. Due to the absence of ester



365

carbonyl group in VE and without formation of cyclic acyloxonium group at high

366

temperature,16 VE was just selected as the antioxidant in FTIR test. From Figure 6, two

367

different carbonyl absorbance bands centered at 1709 and 1733 cm-1, representing two

368

different chemical environments for the ester carbonyl groups (at sn-2 and sn-1 position,

369

respectively),11 were observed in the spectra of DPG at 25 °C. Upon heating to 200 °C, just

370

a carbonyl band centered at 1736 cm-1 was observed and the absorbance band of cyclic

371

acyloxonium group centered at 1640 cm-1 appeared. When Fe2(SO4)3 and VE were

372

respectively added into two same DPG model systems and heated at 200 °C for 1 h.

373

Compared with the bands of carbonyl band and cyclic acyloxonium group of heated DPG

374

system without any addition, for the former, the carbonyl band at 1736 cm-1 was weaker but

375

the band of cyclic acyloxonium group was stronger; for the latter, on the contrary, DPG

376

system heated together with VE still exhibited two carbonyl peaks and stronger one at 1733

377

cm-1 as well as weaker band of cyclic acyloxonium group. These findings suggested that

378

Fe3+ could accelerate the formation of cyclic acyloxonium intermediate, which was

379

nevertheless slowed by VE. Previously, it has been reported that free radical, determined

380

using electron spin resonance (ESR) spectrometry, was formed in sn-1,2-stearoylglycerol

381

(DSG) system during heat exposure and a higher temperature seemingly resulted in an

382

increasing ESR signal.12, 16 As a consequence, these observations led to an interesting

383

hypothesis that the formation of GEs involved free radical-mediated mechanism, and more

384

likely, occurred via a free radical intermediate.

385

Monitoring of DMPO Radical Adducts in Chemical Model Using Q‒TOF MS/MS. To

386

confirm the occurrence of a free radical intermediate during GE formation at high

387

temperature, Q‒TOF MS and MS/MS were carried out to determine the DMPO radical


Page 18 of 41

Page 19 of 41


388

adducts. The mixtures of DPG and DMPO heated at 200 °C for 15 min were injected into

389

Q‒TOF MS/MS system. The MS and MS/MS spectra were recorded at the range of m/z

390

100‒800 and shown in Figure 7 and Figure 8, respectively. As exhibited in Figure 7A, m/z

391

314.5035 and 553.9145, attributed to [CAI’]+ (calcd 314.5017) and [CAI’’]+ (calcd 553.9172),

392

respectively, shown in Figure 9A, was observed in the heated DPG system without DMPO,

393

suggesting the formation of two different CAI. Glycidyl palmitate (PGE) (m/z 312.4838,

394

calcd 312.4870) was also detected, which was consistent with FTIR measurement.9

395

Analysis of the mixture of DPG and DMPO under the same conditions as above found two

396

pairs of sodiated adduct ions, that is, at m/z 466.6428 most likely attributed to [CAI’ +

397

DMPO + O + Na]+ (calcd 466.6489) and m/z 706.0681 for [CAI’’ + DMPO + O + Na]+ (calcd

398

706.0644), as well as their corresponding hydrogenated ions (Figure 7B). However, neither

399

[CAI’]+ nor [CAI’’]+ were observed in Figure 7B due to their complete reaction with DMPO,

400

suggesting that the CAI existed in a free radical form, readily captured by DMPO. In

401

addition, [PGE]+ was detected in the heated mixture of DPG and DMPO, which indicated

402

the absence of GE radical intermediate formation at high temperature. This observation

403

was not consistent with the speculative description of Zhang et al.13 that GEs might be

404

formed through epoxide ring free radical intermediate. These findings provided support for

405

that the DMPO radical adducts were involved in the formation of GEs derived from DPG in

406

this model system at high temperature.

407

To further confirm the formation of DMPO radical adducts, Q‒TOF MS/MS of the

408

selected parent ion at m/z 706.0681 was also conducted. From Figure 8, the fragment ions

409

at m/z 553.9152 corresponding to [CAI’’]+, as well as m/z 685.0949 and 686.0984

410

consistent with [CAI’’ + DMPO + O + H]+ (calcd 685.0904) and [CAI’’ + DMPO + OH + H]+



411

(calcd 685.0983), respectively, were detected upon collisionally activated dissociation of

412

parent oil. Furthermore, [DMPO + Na]+ and [DMPO + O + Na]+ were also found in the

413

fragment ions, which suggested that the parent ion at m/z 706.0681 was indeed the

414

sodiated adduct of CAI and DMPO with one oxygen atom bound. Hence, these above data

415

adequately demonstrated that GEs were formed through the free radical intermediate at

416

high temperature.

417

Proposed Influence Mechanism of Lipid oxidation on the Formation of GEs at High

418

Temperature. According to the above data, a free radical-mediated mechanism for the

419

formation of GEs from DAGs at high temperature was demonstrated in the present study

420

(Figure 9A). The reaction process involved in the formation of cyclic acyloxonium free

421

radical intermediates (CAI’ and CAI’’), which might be initiated by the removal of either fatty

422

acid radical (R•) at the sn-2 position or hydroxyl radical at the sn-3 position combining with

423

the thermally resulting radical (such as ROO•) that most likely originated from oil

424

peroxidation, or the alternatively direct fracture of covalent bond under high-temperature

425

condition. The ester carbonyl group at the sn-1 position might be immediately attacked by

426

the sn-2 carbon-centered radical to form CAI’ free radical. Also, the ester carbonyl group at

427

the sn-2 position might also be attacked by the sn-3 carbon-centered radical to form CAI’’

428

free radical. As shown in Figure 9A, subsequently, CAI free radical could perform a further

429

intramolecular rearrangement to form GEs in which the free radical might participate in the

430

termination stage of oil autoxidation.36 As a consequence, the generated free radical during

431

lipid oxidation could be in favor of the formation of cyclic acyloxonium free radical

432

intermediates, which explained why lipid oxidation promoted GE formation (Figure 9B).

433

Thus, the effects of Fe3+, TBHQ, and VE on the formation of GEs, as shown above, was well


Page 20 of 41

Page 21 of 41


434

understood. More importantly, Fe3+ facilitated the formation of free radical, and therefore

435

also positively (+) affected CAI formation. On contrary, antioxidant, such as TBHQ, VE, et

436

al., could negatively (‒) affect CAI formation by scavenging free radical. In brief, the

437

relationship between GE formation and lipid oxidation was exhibited in Figure 9B.

438

In summary, the significant effect of Fe3+, TBHQ, and VE on GE formation at high

439

temperature was demonstrated in both model oil system and chemical model system. The

440

formation of GEs involved the free radical-mediated mechanisms and the formation of

441

cyclic acyloxonium free radical intermediates was confirmed, which explained why the

442

radical-generated lipid oxidation was in favor of GE formation. Therefore, it was well

443

understood that Fe3+ promoted the formation of GEs, which was also inhibited by TBHQ

444

and VE, by not only indirectly regulating lipid oxidation/free radical, but also by directly

445

affecting CAI formation. These results will enlighten the development of effective

446

elimination and inhibition methods for GE formation in the production of refined edible oil.

447

AUTHOR INFORMATION

448

Corresponding Author

449

*Phone: +86 20 87113875. Fax: +86 20 87114262. E-mail: [email protected].

450

ORCIDID

451

Guoqin Liu: 0000-0001-9513-1115

452

Funding

453

The work was supported by the National Hi-tech Research and Development Project of

454

China (Project No. 2013AA102103), the National Key Research and Development Program

455

of China (Project No. 2016YFD0400401‒5), the National Natural Science Fund of China



Page 22 of 41

456

(Project No. 31471677), the Public Welfare (Agriculture) Research Project (Project No.

457

201303072), and the Fundamental Research Funds for the Central Universities of China.

458

Notes

459 460 461

The authors declare no competing financial interest.

ABBREVIATIONS USED GEs, glycidyl esters; 3-MCPD, 3-chloropropane-1,2-diol; MPO, model palm oil; MCO,

462

model camellia oil; MSO, model soybean oil; MLO, model linseed oil; DAGs, diacylglycerols;

463

MAGs, monoacylglycerols; CAI, cyclic acyloxonium intermediate; TPC, total polar

464

component; FFAs, free fatty acids; PGE, glycidyl palmitate; PBA, phenylboronic acid;

465

TBHQ, tert-butylhydroquinone; VE, α-tocopherol; DMPO, 5,5-dimethylpyrroline-N-oxide;

466

oxTAGs, oxidized triacylglycerols; TGPs, triacylglycerol polymers, TGDs, triacylglycerol

467

dimers; POV, peroxide value; p-AV, p-anisidine value; TOTOX, total oxidation value; SFA,

468

saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid;

469

ATR‒FTIR, attenuated total reflectance Fourier transform Infrared; GC‒MS, gas

470

chromatography-mass

471

chromatography; Q‒TOF, quadrupole−time of flight; ESI, electrospray ionization. DGF,

472

German Society for Fat Science; AOCS, American Oil Chemists’ Society.

spectrometry;

HPSEC,

high

473


performance

size

exclusion

Page 23 of 41

474 475 476


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575



576

Figure captions

577

Figure 1. Fatty acid composition of model palm oil (MPO), model camellia oil (MCO) ,

578

model soybean oil (MSO), and model linseed oil (LO). SFA, saturated fatty acid; MUFA,

579

monounsaturated fatty acid; PUFA: polyunsaturated fatty acid.

580

Figure 2. Evolution of GE levels (A), POV (B), and TOTOX (C), as a function of time (0.5‒

581

3.0 h) in tested model oils with the addition of 4% DPG heated at 200 °C, and evolution of

582

TPC contents (D) in tested model oils heated at 200 °C. Data are mean ± standard

583

deviation (SD) (n=3).

584

Figure 3. Changes of total contents of DAGs and MAGs in MPO, MCO, MSO, and MLO

585

during heating at 200 °C up to 3 h. All data are mean ± SD (n=3). Different letters at the

586

same time represents significant difference between total levels of DAG and MAG in

587

different types of model oil (P < 0.05)

588

Figure 4. Effects of Fe2(SO4)3 (A), TBHQ (B), isolating oxygen (C) on GE formation in

589

tested model oils with addition of 4% DPG heated at 200 °C for 1 h. aequimolar quantities of

590

Fe3+ and EDTA-2Na were added into model oils. All data are mean ± SD (n=3). Values

591

labeled by a different letter are significantly different for the same model oil (P < 0.05).

592

Figure 5. GE levels in different chemical model systems heated at 200 °C for 1 h. All data

593

are mean ± SD (n=3). Values labeled by different letters are significantly different (P

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