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Investigation of polymers and alcohols produced in oxidized soybean oil at frying temperatures
Food Chemistry 317 (2020) 126379
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Investigation of polymers and alcohols produced in oxidized soybean oil at frying temperatures
T
⁎
Hong-Sik Hwanga, , James C. Ballb, Kenneth M. Dolla, James E Andersonb, Karl Vermilliona a
United States Department of Agriculture1, Agricultural Research Service, National Center for Agricultural Utilization Research, Functional Foods Research, Peoria, IL, USA b Research & Advanced Engineering, Ford Motor Company, Dearborn, MI 48124, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Frying Oxidation products Polymerization Soybean oil
Although significant amounts of polymers associated with adverse health effects in oils are produced during frying, the chemical bonds forming these polymers are not well understood. This study revealed that ester bonds are responsible for the polymerization of soybean oil during frying and heating at 175 °C. The ester value of soybean oil increased during frying up to day 3 of the experiment and slightly decreased on day 4 of the experiment indicating that esterification and hydrolysis concomitantly occurred. The 13C NMR spectra showed further evidence of the formation of ester bonds. This study also examined unidentified chemical bonds in the polymer products, other than ester bonds, with NMR spectroscopy. No NMR signals indicating ether bonds were observed. The NMR study after the reaction of oxidized soybean oil with acetyl chloride clarified assignments of proton signals, confirming some previous assignments, and assigning a new proton signal as an alcohol.
1. Introduction Deep frying is a popular cooking method across the entire the food industry because it imparts desirable flavors and textures onto fried foods (Orthoefer & List, 2007b). However, oxidation of the frying oil is accelerated at high temperatures (typically, 150 °C−190 °C). Ingestion of thermally oxidized oil during frying is associated with adverse effects on skeletal and biomechanical bone properties of growing animals, glycerolipid metabolism, kidney disease, cholesterol and phospholipid levels in plasma, and cancer (Macri et al., 2019; Cam et al., 2019; Moumtaz et al., 2019; López-Varela, Sánchez-Muniz, & Cuesta, 1995). Although numerous oxidation products are formed through many different reactions during frying and heating of oil, aldehydes (Moumtaz et al., 2019) and polymers of triacylglycerols (López-Varela et al., 1995) are the compound classes of the most concern due to their potential to affect human health. Many genotoxic and cytotoxic aldehydes are volatile end up in oil fumes (Moumtaz et al., 2019), however, some aldehydes formed in frying are non-volatile and remain in the oil (Steenhorst-Slikkerveer, Louter, Janssen, & Bauer-Plank, 2000). Polymers of concern are not volatile and accumulate in oil during frying and, therefore, and are the subject of this study. An earlier study using gel permeation chromatography (GPC) (Hwang, Winkler-Moser, & Liu, 2017) showed that the peak corresponding to dimers/oligomers/
polymers (hereafter, “polymers”) accounted for about 10% of the oxidized oils, and those polymers contained 25% total polar compounds (TPC), which is the regulatory limit for TPC in frying oils in many countries (Senanayake, 2018). The polymerization reaction of oil is much faster than other oxidation reactions when oil is heated to normal frying temperatures, typically above 110 °C (Gertz, Aladedunye, & Matthäus, 2014). These polymerizations of vegetable oil can occur with or without air. Two major mechanisms were proposed for polymerization of oil, Diels-Alder reactions and radical reactions (Patrikios, Britton, Bing, & Russell, 1994). The Diels-Alder mechanism was proposed in the 1930′s and is still widely accepted although there were also several studies citing evidence against this mechanism (Gertz, Klostermann, & Kochhar, 2000). Recently, efforts are being made to confirm previously proposed mechanisms for polymerization of oil and to identify the chemical bonds forming polymers using modern analytical methods, such as NMR spectroscopy. For example, recent studies using NMR spectroscopy found no evidence of the Diels-Alder reaction mechanism as one of the major mechanisms for polymerization of oil. NMR analyses of soybean oil and fatty acid methyl esters heated at 330 °C under nitrogen showed that no Diels-Alder reaction products were formed during oxidation of these materials (Arca, Sharma, Price, Perez, & Doll, 2012; Doll & Hwang, 2013). Another study using 1H and 13C NMR
⁎ Corresponding author at: Agricultural Research Service, National Center for Agricultural Utilization Research, Functional Foods Research, 1815 N. University Street, Peoria, IL 61604, USA. E-mail address: [email protected] (H.-S. Hwang).
https://doi.org/10.1016/j.foodchem.2020.126379 Received 9 September 2019; Received in revised form 26 December 2019; Accepted 8 February 2020 Available online 10 February 2020 0308-8146/ Published by Elsevier Ltd.
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conducted. When frying started, the temperature went down to 155–160 °C and returned to the initial temperature (175 ± 1 °C) before the next frying, 30 min. later. French fries (100 g, 7.3 wt% to oil) were placed in a mesh basket and fried for 5 min. The French fries in the mesh basket were then lifted for 1 min to allow adhering oil to drain back into the fryer. Additional batches were fried in the same manner every 30 min for a total of 6 hr. After the last batch, the fryer was turned off, and the crumbs were removed with a mesh skimmer. The oil was cooled down to room temperature. Next day frying was conducted in the same way, which was repeated until 4 days. Oil samples (50 ml) were collected for analyses after the last frying of day 1, and then 100 ml of samples were collected after days 2 and 3. The day 4 sample was the remainder of the oil after the last frying on day 4. No additional fresh oil was added anytime during the 4 days.
spectroscopy, including distortionless enhancement by polarization transfer (DEPT) analysis, also showed that there was no noticeable amount of Diels-Alder reaction products in oxidized soybean oil (SBO) during frying and heating at 180 °C (Hwang, Doll, Winkler-Moser, Vermillion, & Liu, 2013). More recently, ether and ester bonds were proposed as important linkages in polymers of oil and fatty acid esters. Patrikios and Mavromoustakos (2014) studied virgin olive oil oxidized at 130 °C for 24 h with 1H NMR, 13C NMR, and 2D NMR. They demonstrated that polymerization of olive oil occurred through the formation of ether linkages between monounsaturated fatty acids. Another study with methyl esters of SBO heated at 90 °C with continuous aeration found that esters bonds contributed significantly to the polymerization (Ball, Anderson, & Wallington, 2018). Bonetti and Parker Jr. (2019) also found that significant amounts of ester bonds in oleic acid, heated at 210 °C for 3 h in an open beaker, and concluded that cross-linking in vegetable oil polymerization might occur through ester groups. Our research group focuses on frying oil and its oxidation products, particularly those that can negatively affect human health. Chemical reactions involved in lipid oxidation can vary with different oils and different oxidation conditions. Soybean oil (SBO), a representative vegetable oil, which is the most consumed oil, was used for this study (Healthiest Cooking Oil, 2019). SBO was oxidized both by frying and by heating to 175 °C. The analyses of ester value were made and NMR spectroscopy was used to determine ester bonds formation in the polymers of SBO. Oxidized SBO was also analyzed with NMR spectroscopy after the reaction with acetyl chloride to assign signals belonging to alcohols.
2.3. Oxidation of SBO by heating at 175 °C To understand the effect of frying, the fried SBO was compared with SBO oxidized by heating at 175 °C (hereafter, “heated SBO”). Heated SBO was obtained in the same fryer under the same conditions as in frying except that no frying of potatoes was conducted. 2.4. TPC, viscosity, density, oxygen content, and ester value TPC was determined following the IUPAC standard method (IUPAC, 1987). Kinematic viscosity and density at 40 °C were measured using an Anton Paar SVM 3000 Stabinger Viscometer (Anton Paar GmbH, Graz, Austria). Total oxygen content was measured by ASTM method D5622 (ASTM, 2000) at Paragon Laboratories (Livonia, MI). The ester value was quantified by measuring the saponification value following the AOCS method Cd 3–25 (AOCS, 2017) and subtracting the acid value measured by the AOCS method Cd 3d-63 (AOCS, 2010). Briefly, measuring the saponification value involved adding alcoholic KOH (25.0 ml, 0.5 mol/l) to the sample, refluxing the solution for 1 h, and titrating the remaining KOH with 0.5 mol/l HCl.
2. Materials and methods 2.1. Materials Soybean oil (SBO, Crisco®) and potatoes were purchased from a local grocery store. Silica gel 60 (70–230 mesh), used for column chromatography, was purchased from EMD Chemical Inc. (Darmstadt, Germany). Thin layer chromatography (TLC) plates (MK6F silica gel 60 Å) were purchased from Whatman (Clifton, NJ). Acetyl chloride (AcCl) and pyridine were purchased from Sigma-Aldrich (St. Louis, MO). All the solvents including ethyl acetate, hexane and dichloromethane (HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ). All materials were used without further purification. The water content of potatos used in this study was determined as 85.0 ± 1.3 wt%, according to the previously reported procedure (Ghadge, Britton, & Jayas, 1989), in quintuplicate. In brief, a potato was peeled, cut into cubes (10 mm × 10 mm × 10 mm). Approximately 15 g of potato cubes were placed on an aluminum dish and dried in a convention oven (HF4-2 Shel Lab High Performance Oven, Cornelius, OR) at 130 °C. Samples were taken out the oven, weighed after 4 h, put back into the oven for 2 more hours, and weighed again. No weight change was observed between 4 h and 6 h indicating completion of drying in 4 h. The water content was expressed by the weight loss of potato cubes after drying.
2.5. Polymerized triacylglycerols (PTAG) A previously reported method (Hwang, Winkler-Moser, Vermillion, & Liu, 2014), which was a slightly modified AOAC Official Method 993.25, was used to determine PTAG. In brief, the oil sample (50 ± 1 mg) was dissolved in dichloromethane (10 ml) in a vial and a portion (1.0 ml) of the solution was transferred to an autosampler vial. Ten µl of the solution was injected to a Shimadzu HPLC (model LC20AT, Kyoto, Japan) equipped with a column (PLGel 5 μm, 100 Å pore size, 300 × 7.5 mm, Polymer Labs, Amherst, MA), an autosampler, and a membrane degasser. The flow rate was set to 0.8 ml/min and the column temperature to 30 °C. An evaporative light-scattering detector (ELSD) was used as a detector, which was operated at 40 °C with the nebulizer gas (ultra-pure N2) pressure set to 2.5 Bar. All samples were analyzed in duplicate and peak area percentages are reported. 2.6. Separation of polar fraction of oxidized SBO
2.2. Oxidation of SBO by frying at 175 °C The polar fraction of oxidized SBO was separated using a previously reported method (Hwang et al., 2013). In brief, a silica gel (600 g) column was prepared with hexane. Fried SBO (4 d, 60.0 g) was dissolved in hexane (60 ml) and passed through the column. Elution with hexane followed by 5% ethyl acetate in hexane gave the non-polar fraction (36.9 g, 61.5%). Subsequent elution with ethyl acetate gave the polar fraction (23.1 g, 38.5% yield). The same procedure was used for 90.0 g of heated SBO (4 d) with 900 g of silica gel to give non-polar (71.6 g, 79.6%) and polar (18.4 g, 20.4%) fractions. Separation of the non-polar and polar fractions was confirmed with thin layer chromatography (TLC) using 15% ethyl acetate in hexane followed by exposure
SBO oxidized by frying (hereafter, “fried SBO”) was obtained using the following procedure. SBO was kept frozen at −20 °C until the day before the frying study to minimize oxidation before the experiments (Hwang et al., 2017). After the skin of a potato was peeled off, the potato was cut into French fries (0.6 cm × 0.6 cm cross section) using a Weston French fry cutter (Weston Products, Strongsville, OH) and dried with paper towels. Frying was carried out in Cuisinart (CDF-100) 1 L capacity fryers, which had a capacity of 1.5 L oil. Oil (1.5 L) was added in the fryer and the temperature was set to the maximum, which maintained the oil temperature at 175 ± 1 °C when no frying was 2
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level, although no similar regulations have been established in the United States (Senanayake, 2018). TPC and PTAG shown in Table 1 gradually increased with time. It is generally known that a steam layer formed over the pool of cooling oil during frying can delay deterioration of oil (Orthoefer & List, 2007a). However, in the current study, increases in TPC and PTAG during frying were greater than those for heating. Since no additional oil was added during the 4-days of frying in this study, the oil volume decrease was due to oil carryout from repeated frying batches. The faster oxidation for frying oil in this study may also have the reduced amount of oil resulting in the increased ratio of surface area to volume of oil. In four days of frying, TPC increased beyond the legally allowed amount of TPC (25–27 wt%) and a significant amounts of polymer products (8.81%, peak area in the spectrum) were formed after frying for 4 days. Kinematic viscosity of frying oil at 40 °C increased by 86% after frying for 4 days (from 28.38 mm2/s to 52.91 mm2/s) while that of heated oil increased by only 36% (to 38.58 mm2/s). Density of fried and heated SBO increased from 0.9051 g/cm3 to 0.9118 g/cm3 and 0.9104 g/cm3, respectively. Increases in kinematic viscosity and density were likely related to the increased TPC and PTAG (Ball et al., 2018). Oxygen content slightly increased during frying (from 11.32 wt% to 12.01 wt%) but did not show substantial change during heating. The results of kinematic viscosity, density, and oxygen content also reflected slower oxidation during heating compared to frying.
to iodine vapor. 2.7. NMR analysis 1 H NMR, 13C NMR, distortionless enhancement by polarization transfer (DEPT) 135, and heteronuclear single quantum coherence spectroscopy (HSQC) experiments were carried out in deuterated chloroform (CDCl3) using a Bruker (Billerica, MA) Avance 500 spectrometer operating at 125 MHz. Chemical shifts in parts per million (ppm) were referenced using the chloroform peak. Peak analysis was conducted with SpinWorks 3.1.7 software. Coupling constants are expressed in frequency (Hz).
2.8. Reaction of fried SBO with acyl chloride (AcCl) Fried SBO (100 mg, 0.11 mmol) was weighed in a small vial and dissolved in CDCl3 (0.6 ml). The solution was transferred to an NMR tube, to which pyridine (3.9 µl, 0.054 mmol) was added, using a glass syringe, followed by AcCl (4.4 µl, 0.054 mmol). The solution in the NMR tube was briefly shaken and spectra were taken after 30 min, 3 h and 20 h using the procedures described above. 2.9. Statistical analysis Analyses for TPC, PTAG, viscosity, density, oxygen content, acid value, and saponification value were carried out at least in duplicate. Data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed with the program JMP 9 (SAS Institute, Cary, NC, USA). Means of data were compared by TukeyKramer HSD test with statistical significance at P < 0.05.
3.2. Changes in acid and ester values Acid value during frying, in general, increases with time due to the hydrolysis of triacylglycerols (Song et al., 2017). In our study, acid value during frying decreased at day 2 and day 3 and then increased to a similar or higher value than the initial value by day 4 (Table 1). Acid value during heating also decreased at day 2 and but then did not increase through day 4. According to Handel and Guerrieri (Handel & Guerrieri, 1990), acid value can initially drop due to evaporation of acidic materials and then increase by hydrolysis. In this study, the increase in acid value between day 2 and day 4 was more prominent during frying than heating, which, presumably, is due to water in the potatoes that was released in the frying process. These results generally follow the pattern of change in oxygen content. As mentioned in the method section, ester value was obtained by subtracting acid value from saponification value (Table 1). Ester value increased from 190.5 mg KOH/g to 195.6 mg KOH/g for fried SBO and to 194.5 mg KOH/g for heated SBO up to day 3, and then both slightly decreased at day 4. While the increases in ester values are small on a relative basis, it must be recognized that each SBO triacylglycerol
3. Results and discussion 3.1. Changes in TPC, PTAG, viscosity, density, and oxygen content during oxidation of SBO When oil is exposed to high temperature, complex chemical reactions occur which produce numerous products. Table 1 reports several markers generally used to assess oil oxidation including TPC, PTAG, kinematic viscosity, density, oxygen content, and acid value. While some products are volatile and do not remain in the oil, others, such as polymers and polar compounds do, therefore, their concentrations constantly increase during frying and heating experiments. Due to the potential toxicity of accumulated substances, regulations in many European countries allow 25–27 wt% TPC as the maximum acceptable Table 1 Analysis of fried and heated soybean oil (SBO) for 0, 2, 3 and 4 days at 180 °C. Properties Total polar compounds (TPC), Wt. % Polymerized triacylglycerols (PTAG), peak area % Kinematic viscosity, mm2/s at 40 °C Density, g/cm3 at 40 °C Oxygen content, Wt. % Acid value, mg KOH/g Saponification value, mg KOH/g Ester value, mg KOH/g
Day Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO
0 1
1.53 ± 0.01 d 1.53 ± 0.01 d 0e 0e 28.36 ± 0.03 d 28.36 ± 0.03 d 0.9051 ± 0.0000 d 0.9051 ± 0.0000 d 11.32 ± 0.14c 11.32 ± 0.14b 0.83 ± 0.08 bc 0.83 ± 0.08b 191.35 ± 0.90b 191.35 ± 0.90b 190.52 ± 0.74 d 190.52 ± 0.74 d
2
3
4
Polar fraction
11.47 ± 0.00c 7.54 ± 0.01c 1.84 ± 0.02 d 0.82 ± 0.01 d 34.83 ± 0.06c 31.90 ± 0.02c 0.9080 ± 0.0001c 0.9068 ± 0.0003c 11.45 ± 0.17c 11.18 ± 0.09b 0.51 ± 0.05c 0.39 ± 0.08c 193.28 ± 1.34 bc 192.48 ± 2.00b 192.77 ± 1.10c 192.09 ± 1.64 cd
17.99 ± 0.40b 11.29 ± 0.11b 4.38 ± 0.37c 2.02 ± 0.05c 41.25 ± 0.00b 34.63 ± 0.06b 0.9118 ± 0.0000b 0.9082 ± 0.0001b 11.40 ± 0.13c 11.01 ± 0.14b 0.55 ± 0.23c 0.41 ± 0.07c 196.14 ± 0.42b 194.86 ± 0.17b 195.59 ± 0.39b 194.46 ± 0.15b
28.56 ± 0.16 a 17.03 ± 0.42 a 8.81 ± 0.07b 3.88 ± 0.00b 52.91 ± 0.00 a 38.58 ± 0.00 a 0.9118 ± 0.0000 a 0.9104 ± 0.0001 a 12.01 ± 0.17b 11.37 ± 0.30b 1.09 ± 0.09b 0.43 ± 0.01c 194.59 ± 1.49 bc 193.58 ± 1.08b 193.49 ± 1.22c 193.15 ± 0.88 bc
– – 70.2 ± 0.2 a 52.3 ± 0.2 a – – – 14.88 ± 0.10 a 13.73 ± 0.06 a 5.18 ± 0.32 a 3.43 ± 0.21 a 203.56 ± 1.32 a 198.25 ± 1.82 a 198.38 ± 1.11 a 194.82 ± 1.49 a
1 Results of statistical analysis. Means not sharing the same letter(s) within a row are significantly different by Tukey-Kramer honest significant difference (HSD) test (P < 0.05).
3
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molecule contains three ester bonds so that an increase of 5 mg KOH/g represents one new ester bond for every 12–13 SBO molecules. As Ball et al. (Ball et al., 2018) reported in the study of oxidized biodiesel heated at 90 °C with aeration for 43 days, this result indicates that the formation of ester bonds contributed to the polymerization of SBO during frying. Since it is well known that alcohols and carboxylic acids are produced during oxidation of oil through a variety of reactions (Schaich, 2005), ester bonds are likely formed by reactions between these two oxidation products at frying temperatures. The decrease in ester value between day 3 and day 4 for fried SBO indicates that esterification between alcohols and carboxylic acids are in competition with hydrolysis of ester bonds. In this period, PTAG increased while ester value decreased indicating that ester bonds are partly responsible for polymerization of SBO and that there are also other chemical bonds forming. Ester values of heated SBO also decreased between day 3 and day 4. However, the difference was not statistically significant at P < 0.05.
Fig. 1. 13C NMR signals for newly formed ester bonds in polar fractions of fried and heated soybean oil (SBO).
3.3. Verification of ester bonds using NMR spectroscopy of polar fraction of oil
173.8 ppm (signal e). The previous study (Bonetti & Parker Jr., 2019) also reported a new ester carbon signal after heating oleic acid. The new signal at 32.6 ppm (signal a) may be the carbon next to the newly formed carbonyl carbon. Signals b (172.8 ppm) and c (173.2 ppm) in Fig. 1 are sn2 and sn1,3 carbonyl carbons of triacylglycerols, respectively (Mannina et al., 2000). These two peaks had small shoulders after oxidation indicating that other new ester signals may have been buried in these triacylglycerols peaks.
NMR spectroscopy is one of the most powerful tools used to elucidate the molecular structure of organic substances. As mentioned in the introduction section, there are numerous oxidation products that have yet to be identified in these materials. Recently, NMR spectroscopy has been intensively used to identify previously unrecognized compounds in oxidized oil (Alexandri et al., 2017; Goicoechea & Guillen, 2010; Guillén & Ruiz, 2001; Martínez-Yusta & Guillén, 2014a, 2014b, 2016; Patrikios & Mavromoustakos, 2014). In this study, we used the NMR spectroscopy to understand chemical bonds forming polymers. Signal assignments were conducted based on previous reports, which were further confirmed with the simulations. Newly formed ester bonds can be detected in NMR spectra. First, we attempted to find signals of ester bonds in 1H NMR spectra and 13C NMR spectra of SBO samples fried and heated for 4 days. There were a few tiny signals in 13C NMR spectra of fried and heated SBO samples that possibly indicated ester bond formation, but they were too small to make solid conclusions. This indicated that the amount of newly formed ester bonds in fried and heated SBO’s having 8.81 and 3.88% PTAG, respectively, was lower than the detection limit of NMR spectroscopy. Our previous report showed that the polar fraction of the oxidized oil had a high content of polymeric materials (Hwang et al., 2013). Following the previous method, column chromatography was conducted with SBO samples fried and heated for 4 days to give polar fractions consisting of 70.2% and 52.3% PTAG, respectively (Table 1). Acid values of polar fractions were much higher than the oil before separation indicating acidic compounds including free fatty acids and polymers containing an acid group(s) were concentrated in the polar fraction. In addition, saponification and ester values of the polar faction were also significantly higher than oil before separation. Oxygen content in the polar fractions of fried and heated SBO were also significantly higher than oxidized SBO before separation. The increased ester value and oxygen content in the polar fraction where TPAG was concentrated is another indication that at least some of the polymerization occurred through the formation of new ester bonds. The 8 mg KOH/g ester value increase implies approximately one new ester bond per 7–8 SBO molecules. The increase in oxygen content in the fried SBO polar fraction, 3.5%, is about 1/3 of the initial oxygen content, indicating that most of the oxygen is assimilated into other functional groups besides esters. The NMR spectroscopy study was conducted on these polar fractions of oxidized SBO. While the 1H NMR spectra did not give much information on newly formed ester bonds due to signal overlapping, 13C NMR spectrum of the polar fraction of fried SBO clearly showed new signals for ester bonds at 173.6 and 173.8 ppm (signal d and e in Fig. 1). The polar fraction of heated SBO showed only the signal at
3.4. New 1H NMR signals at 3.61 and 3.71 ppm – ether bonds or alcohols? As discussed above, while the ester value slightly deceased from day 3 to day 4, PTAG significantly increased in this period of frying (Table 1) indicating that there are possibly chemical bonds in polymers other than ester bonds. New NMR signals are often used as indicators for the formation of new chemical bonds and new products (Han, Kang, Bai, Xue, & Shi, 2018). Therefore, we attempted to find other possible chemical bonds in polymers by scrutinizing the new NMR signals of oxidized oils. A triplet peak at 3.61 ppm and a doublet peak at 3.71 ppm are the most prominent new signals in 1H NMR spectra of polar fractions of fried and heated SBO. Fig. 2a shows these new proton peaks in the spectra of fried SBO (4 d) and its polar fraction. The heated SBO spectrum also had the same signals. Slight changes in their chemical shifts to upfield in fried SBO and its polar fraction might have been caused by different environments, such as an increased number and amount of oxidation products in the SBO samples. Patrikios and Mavromoustakos (2014) found proton signals in the region of 3.65–3.7 ppm in olive oil oxidized at 130 °C for 24 h and concluded that these peaks are protons of newly formed ether linkages, CH–O–CH, which formed oligomers of fatty acids. In contrast, Martínez-Yusta and Guillén (2014a), assigned the triplet signal at 3.61 ppm and the doublet signal at 3.71 ppm as a primary alcohol CH2, (–CH2OH), and –CH2OH of 1,2-diacylglycerol, respectively, in the 1H NMR spectrum of olive oil subjected to deep-frying at 190 °C. To examine if these signals correspond to ethers that may be responsible for polymerization of SBO during frying, the new NMR signals of oxidized SBO were closely investigated in this study. The triplet and doublet peaks in Fig. 2a had the different coupling constants (6.6 and 5.2 Hz, respectively) indicating carbons of these protons were not connected to each other. Therefore, these signals represent two different substances or separate functional groups. Fig. 2b shows 13C NMR spectra of fried SBO (4 d) and its polar fraction compared to the spectrum of fresh SBO. The 13C NMR spectrum of heated SBO also has a similar pattern to that of fried SBO. Although no clear new signals were shown in the fried SBO, four new signals at 4
Food Chemistry 317 (2020) 126379
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Fig. 2. The most prominent new signals in a) 1H NMR and b) 13C NMR spectra in fried soybean oil (SBO) (4 d) and its polar fraction and c) heteronuclear single quantum coherence spectroscopy (HSQC) spectrum of the polar faction.
valuable tool in discerning the differences in structures. Fig. 3a-c show reaction schemes and changes in 1H NMR and in 13C NMR of the polar fraction of fried SBO after the reaction with AcCl. As shown in Fig. 3b, the triplet signal at 3.61 ppm (signal b) and the doublet signal at 3.71 ppm (signal c) completely disappeared after 3 h indicating these signals were protons of alcohols rather than ethers. The proton signal e also disappeared while new signals a and d appeared. Signals were assigned according to the previous reports (Martínez-Yusta & Guillén, 2016) along with simulation with ChemDraw to confirm the assignments. The signal d was assigned to be the methylene group (–CH2) (2 in Fig. 3a) of the acetylated product that was shifted from 3.61 ppm, signal b, after the reaction. The proton signal e corresponded to protons at C2 of 1,2-diacylglycerol backbone (4 in Fig. 3a), which moved under the big signal at 3.9–4.4 ppm of the triacylglycerols after the reaction. The new signal a was assigned to the methyl group (–CH3) of the newly formed acetyl group (9 in Fig. 3a). Therefore, the 1H NMR experiment after the reaction with AcCl strongly supported the assignments by Martínez-Yusta and Guillén (2014a), that the triplet signal at 3.61 ppm and the doublet signal at 3.71 ppm are a primary alcohol and 1,2-diacylglycerol, respectively. 13 C NMR was also used to study the polar fraction of fried SBO oil after the reaction with AcCl. Fig. 3c shows the region of 13C NMR spectrum where the most obvious changes were observed. Assignment of 13C NMR signals was conducted using the previous report (Vlahov, 1996) along with the simulated 13C NMR spectra, by ChemDraw, and the database provided by AIST (2018). Distortionless enhancement by polarization transfer (DEPT) was also used to confirm the assignment. Signal A, at 61.4 ppm, in Fig. 3c corresponding to C1 of 1,2-diacylglycerol backbone (3 in Fig. 3a) which moved to 62.3 ppm (signal C
61.4, 65.0, 68.2, and 72.1 ppm appeared in the polar fraction of fried SBO. According to Vlahov (1996), signals at 61.4, 65.0, 68.2, and 72.1 ppm can be assigned to C1 of 1,2-diacylglycerol backbone, C1 and C3 of 1,3-diacylglycerol backbone, C2 of 1,3-diacylglycerol backbone, and C2 of 1,2-diacylglycerol backbone, respectively. The proton signal at 3.71 ppm and carbon signals at 61.4 and 72.1 ppm are strong evidence of the existence of 1,2-diacylglycerol. Furthermore, a 2D NMR study using heteronuclear single quantum coherence spectroscopy (HSQC) showed the correlation between the proton signal at 3.71 ppm and the carbon signal at 61.4 (Fig. 2c), which supports the assignments by Martínez-Yusta and Guillén (2014a). The signal at 3.61 ppm correlated with the tiny carbon peak at 62.8 ppm in HSQC, which confirmed the previous assignment as a methylene group carbon of a primary alcohol, –CH2OH. In contrast, the study by Patrikios and Mavromoustakos (2014) found the correlation of proton signals at 3.65–3.7 ppm with carbon peaks at the region of 50 ppm in the HSQC spectrum, which were thought to be ether bonds. 3.5. Reaction of hydroxyl group with acetyl chloride (AcCl) To further confirm if the most prominent new signals at 3.61 and 3.71 ppm in Fig. 2a are of a primary alcohol CH2, (–CH2OH), and –CH2OH of 1,2-diacylglycerol, respectively, the reaction of the polar fraction of fried SBO with AcCl was conducted. It was hypothesized that the assignments of signals in Fig. 2a and 2b as primary alcohols and 1,2diacylglycerol could be confirmed if these signals moved after the reaction with AcCl which reacts with an alcohol at room temperature to form an ester (Ishihara, Kurihara, & Yamamoto, 1993; Smith & Bryant, 1935). However, AcCl does not decompose ether bonds making it a 5
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Fig. 3. a) Reactions of a primary alcohol and 1,2-diacylglycerols in the polar fraction of fried soybean oil (SBO) with acetyl chloride (AcCl) for 3 h. Changes in b) 1H NMR spectrum and c) 13C NMR after the reaction. d) Expanded 1H NMR spectrum for the peak at 4.07 ppm. Assignments of proton signals are: a = 9; b = 1; c = 5; d = 2; e = 4. Assignments of carbon signals are: A = 3; B = 8, C1 and C3 of triacylglycerol backbone; C = 6; D = C1 and C3 of 1,3-diacylglycerol backbone; E, F = C2 of 1,3-diacylglycerol backbone G = 7, H = C2 of triacylglycerol backbone; I = 4 for 13C NMR.
in Fig. 3c, 6 in Fig. 3a) after the reaction. Signal B at 62.1 ppm is the signal of C1 and C3 of triacylglycerol backbone including those of newly formed triacylglycerols by the reaction with AcCl. Signal I at 72.1 ppm corresponds to C2 of 1,2-diacylglycerol backbone (4 in Fig. 3a) that shifted to 68.8 ppm (signal G) after the reaction with AcCl. Signal H (68.9 ppm) is C2 of triacylglycerol backbone including the reaction products. All the changes in 13C NMR spectrum supported the presence of 1,2-diacylglycerols in the fried SBO, which were converted to triacylglycerols after the reaction with AcCl. Signals E and F, at around 68.2 ppm, were shown at slightly different chemical shifts. However, after repeated experiments, it was found that the signal E was the same signal as signal F and was shown at slightly different field, 68.15 ± 0.05 ppm, presumably, due to the different environments caused by the excess AcCl. Signals E and F can be assigned as C2 of 1,3-diacylglycerol backbone and signal D as C1 and C3 of 1,3-diacylglycerol backbone based on the previous report (Vlahov, 1996). All of these points were in agreement with the simulated 13C NMR spectra in this study. The carbon signal at 68.2 ppm also correlated with the proton signal at 4.07 ppm in HSQC. The proton signal at 4.07 ppm was previously assigned as the CH proton at C2 of 1,3-diacylglycerols (Martínez-Yusta & Guillén, 2014a). These results indicate the presence of 1,3-diacylglycerols in the oxidized SBO, which did not completely react with AcCl due to the steric hindrance by two long fatty acid groups. In general, sterically hindered secondary alcohols react significantly slower than primary alcohols with AcCl (Ishihara et al., 1993). Carbon signals D and F did not completely disappear even after a 20-h reaction. The proton signal at 4.07 ppm in the 1H NMR seemed to completely disappear in Fig. 3b. However, a closer observation (Fig. 3d) revealed that the large peak at 4.1 ppm was broadened and the peak at 4.07 ppm was merely overlapped with the large peak.
3.6. Other proton signals of alcohols Due to the many types and relatively small amounts of oxidation products, it was difficult to assign all the small peaks in 1H NMR of oxidized oil. Tremendous efforts have been made by several research groups to assign many of these small peaks during the last decade. However, some signals remain unidentified, unclear, or contradictory. In this study, in addition to the above assignments, including proton signals at 3.61 and 3.71 ppm, we clarified previously uncertain assignments, confirmed previous assignments, and found one new alcohol signal from the NMR study of acetylated fried SBO. Signals a-p in Fig. 4a are newly formed or grown proton signals found in fried SBO and its polar fraction. 1H NMR spectrum of heated SBO showed a very similar pattern to that of fried SBO. Fig. 4b shows these signals of oxidation products before and after the reaction with AcCl. It cannot be definitively determined if signals a and b moved after the reaction with AcCl due to overlapping other signals. Signals c and d, which were previously thought to be epoxy or alcohol (Goicoechea & Guillen, 2010; Martínez-Yusta & Guillén, 2014a), were assigned as alcohols in this study. Signal e, a very small but obvious signal, disappeared after the reaction with AcCl. This result confirmed the previous assignment of this signal as a secondary alcohol (Martínez-Yusta & Guillén, 2014a). Since this secondary alcohol completely reacted with AcCl in 3 h, this alcohol does not appear to be sterically hindered. As discussed above, signals f and g were verified as signals of alcohols instead of ethers. Signals h-j were also shifted after the reaction confirming the previous assignment as alcohols (MartínezYusta & Guillén, 2014a,b). To the best of our knowledge, no information was previously reported for signal k shown at 5.07 ppm. This signal was newly assigned as an alcohol signal in this study although further study is needed to verify the detailed molecular structure. Signals l (many peaks at 5.52–7.08 ppm) did not move or change after the reaction, which is consistent with the previous assignments of these 6
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Fig. 4. a) Newly appearing or intensified proton signals after oxidation of soybean oil (SBO). Intensity of signals m-p were magnified four fold compared to other peaks. b) Changes in proton signals of fried SBO polar fraction after reaction with acetyl chloride (AcCl) for 3 h. Table 2 Signals assigned as alcohols in this study from the reaction with acetyl chloride (AcCl) compared to previous assignments (Alexandri et al., 2017; Goicoechea & Guillen, 2010; Guillén & Ruiz, 2001; Martínez-Yusta & Guillén, 2014a, 2014b, 2016; Patrikios & Mavromoustakos, 2014). Signal
Chemical shift (ppm)
Splitting pattern
Previous assignments
Assignments in this study
c d e f g h i j k o
2.89 3.09 3.50 3.61 3.71 3.92 4.07 4.41 5.07 9.57
Multiplet Multiplet Multiplet Triplet Doublet Triplet Multiplet Multiplet Triplet d
Epoxy or alcohol Epoxy or alcohol Secondary alcohol Ether CH or primary alcohol Ether CH2 or –CH2OH (1,2-diacylglycerol) ROCH2-CHOH-CH2OH in 1-monoacylglycerol ROCH2-CHOH-CH2OR′ in 1,3-diacylglycerol CHO-CH]CH-CHOH– Not assigned –CHO (4-hydroxy-(E)-2-alkenals)
Alcohol, correlated to a carbon at 56.9 ppm Alcohol, –CH-CH-OH Secondary alcohol Primary alcohol –CH2OH (1,2-diacylglycerol) Agreed Confirmed with HSQC* Confirmed with HSQC Alcohol, correlated to a carbon at 72.2 ppm Agreed with reaction with AcCl
*HSQC: heteronuclear single quantum coherence spectroscopy.
shifting due to functional groups give the conclusion that the assignment is in agreement. A carbon peak at 68.2 ppm correlated with signal i confirmed it as ROCH2-CHOH-CH2OR′ in 1,3-diacylglycerol. The correlation of signal j with the carbon signal at 62.1 ppm as well as the simulated NMR experiment strongly supported the assignment as CHOCH]CH-CHOH–. HSQC experiments also provided further information for a few signals that will help future studies in elucidating detailed molecular structures. For example, signals c, d, and k correlated with carbon signals at 56.9, 32.8, and 72.2 ppm, respectively. The carbon signal at 32.8 ppm correlated with the proton signal d indicates that these protons are on the beta position carbon to the hydroxy group.
signals as conjugated diene double bonds (Alexandri et al., 2017). Signals m-p were assigned as a variety of aldehydes and the detailed molecular structures of these aldehydes were reported by MartinezYusta and Guillen (2016). Among them, signal o at 9.57 ppm was assigned as 4-hydroxy-(E)-2-alkenals. In this study, the signal o seemed to imply two aldehyde signals that overlapped. One of them disappeared after the reaction confirming the previous assignment, but also indicates that there was another kind of aldehyde having a very similar chemical shift. Signals that were assigned as protons of alcohols in this study are summarized in Table 2 along with previous assignments. HSQC experiments confirmed or strongly supported these assignments. For example, signal e correlated with a carbon signal at 71.6 ppm, which is a good indication of a secondary alcohol. As discussed above, assignments of signal f and g were well confirmed with HSQC. Signal h correlated with a carbon at 72.2 ppm, which is slightly different chemical shift than the reported one as ROCH2-CHOH-CH2OH in 1-monoacylglycerol (70.3 ppm) (Vlahov, 1996). However, possible
4. Conclusions The increased ester value and 13C NMR signals provides evidence that ester bonds are responsible for the formation of polymers in oxidized SBO during frying and heating at 175 °C, consistent with that in a 7
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recent study with oxidized biodiesel. The fact that PTAG constantly increased while the ester value slightly decreased from day 3 to day 4 of frying indicated that there are also other chemical bonds formed during polymerization. In this study, no NMR signals corresponding to ether bonds were found. The NMR study after the reaction of oxidized SBO with AcCl showed that proton signals at 3.61 and at 3.71 ppm were indicative of alcohols produced in oxidized SBO. From the NMR study, several signals were assigned to be those of alcohols confirming literature signal assignments, verifying uncertain assignments, and finding one previously unreported alcohol signal. Further studies are needed to definitively describe the chemical bonds forming polymers other than ester bonds.
102:8/9<543::AID-EJLT543>3.0.CO;2-V. Ghadge, A. D., Britton, M. G., & Jayas, D. S. (1989). Moisture content determination for potatoes. Transactions of the American Society of Agricultural Engineers, 32, 1744–1746. Goicoechea, E., & Guillen, M. D. (2010). Analysis of hydroperoxides, aldehydes and epoxides by 1H nuclear magnetic resonance in sunflower oil oxidized at 70 and 100 °C. Journal of Agricultural and Food Chemisty, 58, 6234–6245. https://doi.org/10.1021/ jf1005337. Guillén, M. D., & Ruiz, A. (2001). High resolution 1H nuclear magnetic resonance in the study of edible oils and fats. Trends in Food Science and Technology, 12, 328–338. https://doi.org/10.1016/S0924-2244(01)00101-7. Han, X., Kang, J., Bai, Y., Xue, M., & Shi, Y.-C. (2018). Structure of pyrodextrin in relation to its retrogradation properties. Food Chemistry, 242, 169–173. https://doi.org/10. 1016/j.foodchem.2017.09.015. Handel, A. P., & Guerrieri, S. A. (1990). Evaluation of heated frying oils containing added fatty acids. Journal of Food Science, 55, 1417–1420. https://doi.org/10.1111/j.13652621.1990.tb03948.x. Healthiest Cooking Oil. Soybean Oil: Is it Healthy. (2019). https://healthiestcookingoils. com/soybean-oil/ Accessed 11 December 2019. Hwang, H.-S., Doll, K. M., Winkler-Moser, J. K., Vermillion, K., & Liu, S. X. (2013). No evidence found for Diels-Alder reaction products in soybean oil oxidized at the frying temperature by NMR study. Journal of the American Oil Chemists' Society, 90, 825–834. https://doi.org/10.1007/s11746-013-2229-9. Hwang, H.-S., Winkler-Moser, J. K., & Liu, S. X. (2017). Reliability of 1H NMR analysis for assessment of lipid oxidation at frying temperatures. Journal of the American Oil Chemists' Society, 94, 257–270. https://doi.org/10.1007/s11746-016-2945-z. Hwang, H. S., Winkler-Moser, J. K., Vermillion, K., & Liu, S. X. (2014). 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Food and Chemical Toxicology, 33, 181–189. https://doi.org/10.1016/0278-6915(94)00133-9. Macri, E. V., Ramos, C., Bozzini, C., Zago, V., Giacomino, S., Pellegrino, N., ... Friedman, S. M. (2019). Fried sunflower oil intake affects bone quality, in growing rats. Current Research in Nutrition and Food Science, 7, 52–65. https://doi.org/10.12944/CRNFSJ.7. 1.06. Mannina, L., Luchinat, C., Patumi, M., Emanuele, M. C., Rossi, E., & Segre, A. (2000). Concentration dependence of 13C NMR spectra of triglycerides: Implications for the NMR analysis of olive oils. Magnetic Resonance in Chemistry, 38, 886–890. https://doi. org/10.1002/1097-458X(200010)38:10<886::AID-MRC738>3.0.CO;2-J. Martínez-Yusta, A., & Guillén, M. D. (2014a). Deep-frying food in extra virgin olive oil: A study by 1H nuclear magnetic resonance of the influence of food nature on the evolving composition of the frying medium. Food Chemisty, 150, 429–437. https:// doi.org/10.1016/j.foodchem.2013.11.015. Martínez-Yusta, A., & Guillén, M. D. (2014b). Deep-frying. A study of the influence of the frying medium and the food nature, on the lipidic composition of the fried food, using 1 H nuclear magnetic resonance. Food Research International, 62, 998–1007. https:// doi.org/10.1016/j.foodres.2014.05.015. Martínez-Yusta, A., & Guillén, M. D. (2016). Monitoring compositional changes in sunflower oil-derived deep-frying media by 1H Nuclear Magnetic Resonance. European Journal of Lipid Science and Technology, 118, 984–996. https://doi.org/10.1002/ejlt. 201500270. Moumtaz, S., Percival, B. C., Parmar, D., Grootveld, K. L., Jansson, P., & Grootveld, M. (2019). Toxic aldehyde generation in and food uptake from culinary oils during frying practices: Peroxidative resistance of a monounsaturate-rich algae oil. Scientific Reports, 9(4125), 1–21. https://doi.org/10.1038/s41598-019-39767-1. Orthoefer, F. T., & List, G. R. (2007a). Dynamics of frying. In M. D. Erickson (Ed.). Deep frying (pp. 253–275). Champaign, IL: AOCS Press. Orthoefer, F. T., & List, G. R. (2007b). Evaluation of used frying Oil. In M. D. Erickson (Ed.). Deep frying (pp. 329–342). Champaign, IL: AOCS Press. Patrikios, I. S., Britton, O., Bing, D. K., & Russell, C. S. (1994). Heating unsaturated fatty acids in air produces hemagglutinins. Biochimica et Biophysica Acta, 1212, 225–234. https://doi.org/10.1016/0005-2760(94)90257-7. Patrikios, I. S., & Mavromoustakos, T. M. (2014). Monounsaturated fatty acid ether oligomers formed during heating of virgin olive oil show agglutination activity against human red blood cells. Journal of Agricultural and Food Chemistry, 62, 867–874. https://doi.org/10.1021/jf403745x. Schaich, K. M. (2005). Lipid oxidation: Theoretical aspects. Bailey's industrial oil and fat products (pp. 269–355). Hoboken, NJ: John Wiley & Sons Inc. Senanayake, N. (2018). Enhancing oxidative stability and shelf life of frying oils with antioxidants. Inform Magazine, 29, 6–13. Smith, D. M., & Bryant, W. M. D. (1935). Rapid determination of hydroxyl by means of acetyl chloride and pyridine. Journal of the American Chemical Society, 57, 61–65. https://doi.org/10.1021/ja01304a018. Song, J., Kim, S., Kim, J., Kim, M.-J., Lee, S.-M., Jang, M.-I., & Lee, J. (2017). Oxidative properties and moisture content in repeatedly used oils for French fries and breaded chickens during frying. European Journal of Lipid Science and Technology, 119. https:// doi.org/10.1002/ejlt.201600279.
CRediT authorship contribution statement Hong-Sik Hwang: Conceptualization, Methodology, Investigation, Writing - original draft. James C. Ball: Conceptualization, Investigation, Writing - review & editing. Kenneth M. Doll: Conceptualization, Writing - review & editing. James E Anderson: Investigation, Writing - review & editing. Karl Vermillion: Methodology, Formal analysis. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors would like to express appreciation to Lynne Copes for excellent technical work. Conflict of interest The authors declare that they have no conflict of interest. References AIST. Spectral database for organic compounds (2018). https://sdbs.db.aist.go.jp/sdbs/ cgi-bin/direct_frame_top.cgi. Accessed 3 August 2019. Alexandri, E., Ahmed, R., Siddiqui, H., Choudhary, M. I., Tsiafoulis, C. G., & Gerothanassis, I. P. (2017). High resolution NMR spectroscopy as a structural and analytical tool for unsaturated lipids in solution. Molecules, 22, 1663. https://doi.org/ 10.3390/molecules22101663. AOCS (2010). Acid value of fats and oils. AOCS official method Cd 3d–63. Urbana, IL: American Oil Chemists' Society. AOCS (2017). Saponification value of fats and oils. AOCS official method Cd 3–25. Urbana, IL: American Oil Chemists' Society. Arca, M., Sharma, B., Price, N. J., Perez, J., & Doll, K. (2012). Evidence contrary to the accepted Diels-Alder mechanism in the thermal modification of vegetable oil. Journal of the American Oil Chemists' Society, 89, 987–994. https://doi.org/10.1007/s11746011-2002-x. ASTM (2000). Standard test methods for determination of total oxygen in gasoline and methanol fuels by reductive pyrolysis. ASTM D5622. Annual book of ASTM standards. West Conshohocken, PA: ASTM International. Ball, J. C., Anderson, J. E., & Wallington, T. J. (2018). Depolymerization of polyester polymers from the oxidation of soybean biodiesel. Energy & Fuels, 32, 12587–12596. https://doi.org/10.1021/acs.energyfuels.8b03169. Bonetti, R., & Parker, W. O., Jr. (2019). Insights into polymerization of vegetable oil: Oligomerization of oleic acid. Journal of the American Oil Chemists' Society, 96, 1181–1184. https://doi.org/10.1002/aocs.12274. Cam, A., Oyirifi, A. B., Liu, Y., Haschek, W. M., Iwaniec, U. T., Turner, R. T., ... Helferich, W. G. (2019). Thermally abused frying oil potentiates metastasis to lung in a murine model of late-stage breast cancer. Cancer Prevention Research, 12, 201–210. https:// doi.org/10.1158/1940-6207.Capr-18-0220. Doll, K. M., & Hwang, H.-S. (2013). Thermal modification of vegetable oils. Lipid Technology, 25, 83–85. https://doi.org/10.1002/lite.201300269. Gertz, C., Aladedunye, F., & Matthäus, B. (2014). Oxidation and structural decomposition of fats and oils at elevated temperatures. European Journal of Lipid Science and Technology, 116, 1457–1466. https://doi.org/10.1002/ejlt.201400099. Gertz, C., Klostermann, S., & Kochhar, S. P. (2000). Testing and comparing oxidative stability of vegetable oils and fats at frying temperature. European Journal of Lipid Science and Technology, 102, 543–551. https://doi.org/10.1002/1438-312(200009)
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Vlahov, G. (1996). Improved quantitative 13C nuclear magnetic resonance criteria for determination of grades of virgin olive oils. The normal ranges for diglycerides in olive oil. Journal of the American Oil Chemists' Society, 73, 1201–1203. https://doi. org/10.1007/bf02523385.
Steenhorst-Slikkerveer, L., Louter, A., Janssen, H.-G., & Bauer-Plank, C. (2000). Analysis of nonvolatile lipid oxidation products in vegetable oils by normal-phase high-performance liquid chromatography with mass spectrometric detection. Journal of the American Oil Chemists' Society, 77, 837–845. https://doi.org/10.1007/s11746-0000134-1.
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Investigation of polymers and alcohols produced in oxidized soybean oil at frying temperatures
T
⁎
Hong-Sik Hwanga, , James C. Ballb, Kenneth M. Dolla, James E Andersonb, Karl Vermilliona a
United States Department of Agriculture1, Agricultural Research Service, National Center for Agricultural Utilization Research, Functional Foods Research, Peoria, IL, USA b Research & Advanced Engineering, Ford Motor Company, Dearborn, MI 48124, United States
A R T I C LE I N FO
A B S T R A C T
Keywords: Frying Oxidation products Polymerization Soybean oil
Although significant amounts of polymers associated with adverse health effects in oils are produced during frying, the chemical bonds forming these polymers are not well understood. This study revealed that ester bonds are responsible for the polymerization of soybean oil during frying and heating at 175 °C. The ester value of soybean oil increased during frying up to day 3 of the experiment and slightly decreased on day 4 of the experiment indicating that esterification and hydrolysis concomitantly occurred. The 13C NMR spectra showed further evidence of the formation of ester bonds. This study also examined unidentified chemical bonds in the polymer products, other than ester bonds, with NMR spectroscopy. No NMR signals indicating ether bonds were observed. The NMR study after the reaction of oxidized soybean oil with acetyl chloride clarified assignments of proton signals, confirming some previous assignments, and assigning a new proton signal as an alcohol.
1. Introduction Deep frying is a popular cooking method across the entire the food industry because it imparts desirable flavors and textures onto fried foods (Orthoefer & List, 2007b). However, oxidation of the frying oil is accelerated at high temperatures (typically, 150 °C−190 °C). Ingestion of thermally oxidized oil during frying is associated with adverse effects on skeletal and biomechanical bone properties of growing animals, glycerolipid metabolism, kidney disease, cholesterol and phospholipid levels in plasma, and cancer (Macri et al., 2019; Cam et al., 2019; Moumtaz et al., 2019; López-Varela, Sánchez-Muniz, & Cuesta, 1995). Although numerous oxidation products are formed through many different reactions during frying and heating of oil, aldehydes (Moumtaz et al., 2019) and polymers of triacylglycerols (López-Varela et al., 1995) are the compound classes of the most concern due to their potential to affect human health. Many genotoxic and cytotoxic aldehydes are volatile end up in oil fumes (Moumtaz et al., 2019), however, some aldehydes formed in frying are non-volatile and remain in the oil (Steenhorst-Slikkerveer, Louter, Janssen, & Bauer-Plank, 2000). Polymers of concern are not volatile and accumulate in oil during frying and, therefore, and are the subject of this study. An earlier study using gel permeation chromatography (GPC) (Hwang, Winkler-Moser, & Liu, 2017) showed that the peak corresponding to dimers/oligomers/
polymers (hereafter, “polymers”) accounted for about 10% of the oxidized oils, and those polymers contained 25% total polar compounds (TPC), which is the regulatory limit for TPC in frying oils in many countries (Senanayake, 2018). The polymerization reaction of oil is much faster than other oxidation reactions when oil is heated to normal frying temperatures, typically above 110 °C (Gertz, Aladedunye, & Matthäus, 2014). These polymerizations of vegetable oil can occur with or without air. Two major mechanisms were proposed for polymerization of oil, Diels-Alder reactions and radical reactions (Patrikios, Britton, Bing, & Russell, 1994). The Diels-Alder mechanism was proposed in the 1930′s and is still widely accepted although there were also several studies citing evidence against this mechanism (Gertz, Klostermann, & Kochhar, 2000). Recently, efforts are being made to confirm previously proposed mechanisms for polymerization of oil and to identify the chemical bonds forming polymers using modern analytical methods, such as NMR spectroscopy. For example, recent studies using NMR spectroscopy found no evidence of the Diels-Alder reaction mechanism as one of the major mechanisms for polymerization of oil. NMR analyses of soybean oil and fatty acid methyl esters heated at 330 °C under nitrogen showed that no Diels-Alder reaction products were formed during oxidation of these materials (Arca, Sharma, Price, Perez, & Doll, 2012; Doll & Hwang, 2013). Another study using 1H and 13C NMR
⁎ Corresponding author at: Agricultural Research Service, National Center for Agricultural Utilization Research, Functional Foods Research, 1815 N. University Street, Peoria, IL 61604, USA. E-mail address: [email protected] (H.-S. Hwang).
https://doi.org/10.1016/j.foodchem.2020.126379 Received 9 September 2019; Received in revised form 26 December 2019; Accepted 8 February 2020 Available online 10 February 2020 0308-8146/ Published by Elsevier Ltd.
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conducted. When frying started, the temperature went down to 155–160 °C and returned to the initial temperature (175 ± 1 °C) before the next frying, 30 min. later. French fries (100 g, 7.3 wt% to oil) were placed in a mesh basket and fried for 5 min. The French fries in the mesh basket were then lifted for 1 min to allow adhering oil to drain back into the fryer. Additional batches were fried in the same manner every 30 min for a total of 6 hr. After the last batch, the fryer was turned off, and the crumbs were removed with a mesh skimmer. The oil was cooled down to room temperature. Next day frying was conducted in the same way, which was repeated until 4 days. Oil samples (50 ml) were collected for analyses after the last frying of day 1, and then 100 ml of samples were collected after days 2 and 3. The day 4 sample was the remainder of the oil after the last frying on day 4. No additional fresh oil was added anytime during the 4 days.
spectroscopy, including distortionless enhancement by polarization transfer (DEPT) analysis, also showed that there was no noticeable amount of Diels-Alder reaction products in oxidized soybean oil (SBO) during frying and heating at 180 °C (Hwang, Doll, Winkler-Moser, Vermillion, & Liu, 2013). More recently, ether and ester bonds were proposed as important linkages in polymers of oil and fatty acid esters. Patrikios and Mavromoustakos (2014) studied virgin olive oil oxidized at 130 °C for 24 h with 1H NMR, 13C NMR, and 2D NMR. They demonstrated that polymerization of olive oil occurred through the formation of ether linkages between monounsaturated fatty acids. Another study with methyl esters of SBO heated at 90 °C with continuous aeration found that esters bonds contributed significantly to the polymerization (Ball, Anderson, & Wallington, 2018). Bonetti and Parker Jr. (2019) also found that significant amounts of ester bonds in oleic acid, heated at 210 °C for 3 h in an open beaker, and concluded that cross-linking in vegetable oil polymerization might occur through ester groups. Our research group focuses on frying oil and its oxidation products, particularly those that can negatively affect human health. Chemical reactions involved in lipid oxidation can vary with different oils and different oxidation conditions. Soybean oil (SBO), a representative vegetable oil, which is the most consumed oil, was used for this study (Healthiest Cooking Oil, 2019). SBO was oxidized both by frying and by heating to 175 °C. The analyses of ester value were made and NMR spectroscopy was used to determine ester bonds formation in the polymers of SBO. Oxidized SBO was also analyzed with NMR spectroscopy after the reaction with acetyl chloride to assign signals belonging to alcohols.
2.3. Oxidation of SBO by heating at 175 °C To understand the effect of frying, the fried SBO was compared with SBO oxidized by heating at 175 °C (hereafter, “heated SBO”). Heated SBO was obtained in the same fryer under the same conditions as in frying except that no frying of potatoes was conducted. 2.4. TPC, viscosity, density, oxygen content, and ester value TPC was determined following the IUPAC standard method (IUPAC, 1987). Kinematic viscosity and density at 40 °C were measured using an Anton Paar SVM 3000 Stabinger Viscometer (Anton Paar GmbH, Graz, Austria). Total oxygen content was measured by ASTM method D5622 (ASTM, 2000) at Paragon Laboratories (Livonia, MI). The ester value was quantified by measuring the saponification value following the AOCS method Cd 3–25 (AOCS, 2017) and subtracting the acid value measured by the AOCS method Cd 3d-63 (AOCS, 2010). Briefly, measuring the saponification value involved adding alcoholic KOH (25.0 ml, 0.5 mol/l) to the sample, refluxing the solution for 1 h, and titrating the remaining KOH with 0.5 mol/l HCl.
2. Materials and methods 2.1. Materials Soybean oil (SBO, Crisco®) and potatoes were purchased from a local grocery store. Silica gel 60 (70–230 mesh), used for column chromatography, was purchased from EMD Chemical Inc. (Darmstadt, Germany). Thin layer chromatography (TLC) plates (MK6F silica gel 60 Å) were purchased from Whatman (Clifton, NJ). Acetyl chloride (AcCl) and pyridine were purchased from Sigma-Aldrich (St. Louis, MO). All the solvents including ethyl acetate, hexane and dichloromethane (HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ). All materials were used without further purification. The water content of potatos used in this study was determined as 85.0 ± 1.3 wt%, according to the previously reported procedure (Ghadge, Britton, & Jayas, 1989), in quintuplicate. In brief, a potato was peeled, cut into cubes (10 mm × 10 mm × 10 mm). Approximately 15 g of potato cubes were placed on an aluminum dish and dried in a convention oven (HF4-2 Shel Lab High Performance Oven, Cornelius, OR) at 130 °C. Samples were taken out the oven, weighed after 4 h, put back into the oven for 2 more hours, and weighed again. No weight change was observed between 4 h and 6 h indicating completion of drying in 4 h. The water content was expressed by the weight loss of potato cubes after drying.
2.5. Polymerized triacylglycerols (PTAG) A previously reported method (Hwang, Winkler-Moser, Vermillion, & Liu, 2014), which was a slightly modified AOAC Official Method 993.25, was used to determine PTAG. In brief, the oil sample (50 ± 1 mg) was dissolved in dichloromethane (10 ml) in a vial and a portion (1.0 ml) of the solution was transferred to an autosampler vial. Ten µl of the solution was injected to a Shimadzu HPLC (model LC20AT, Kyoto, Japan) equipped with a column (PLGel 5 μm, 100 Å pore size, 300 × 7.5 mm, Polymer Labs, Amherst, MA), an autosampler, and a membrane degasser. The flow rate was set to 0.8 ml/min and the column temperature to 30 °C. An evaporative light-scattering detector (ELSD) was used as a detector, which was operated at 40 °C with the nebulizer gas (ultra-pure N2) pressure set to 2.5 Bar. All samples were analyzed in duplicate and peak area percentages are reported. 2.6. Separation of polar fraction of oxidized SBO
2.2. Oxidation of SBO by frying at 175 °C The polar fraction of oxidized SBO was separated using a previously reported method (Hwang et al., 2013). In brief, a silica gel (600 g) column was prepared with hexane. Fried SBO (4 d, 60.0 g) was dissolved in hexane (60 ml) and passed through the column. Elution with hexane followed by 5% ethyl acetate in hexane gave the non-polar fraction (36.9 g, 61.5%). Subsequent elution with ethyl acetate gave the polar fraction (23.1 g, 38.5% yield). The same procedure was used for 90.0 g of heated SBO (4 d) with 900 g of silica gel to give non-polar (71.6 g, 79.6%) and polar (18.4 g, 20.4%) fractions. Separation of the non-polar and polar fractions was confirmed with thin layer chromatography (TLC) using 15% ethyl acetate in hexane followed by exposure
SBO oxidized by frying (hereafter, “fried SBO”) was obtained using the following procedure. SBO was kept frozen at −20 °C until the day before the frying study to minimize oxidation before the experiments (Hwang et al., 2017). After the skin of a potato was peeled off, the potato was cut into French fries (0.6 cm × 0.6 cm cross section) using a Weston French fry cutter (Weston Products, Strongsville, OH) and dried with paper towels. Frying was carried out in Cuisinart (CDF-100) 1 L capacity fryers, which had a capacity of 1.5 L oil. Oil (1.5 L) was added in the fryer and the temperature was set to the maximum, which maintained the oil temperature at 175 ± 1 °C when no frying was 2
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level, although no similar regulations have been established in the United States (Senanayake, 2018). TPC and PTAG shown in Table 1 gradually increased with time. It is generally known that a steam layer formed over the pool of cooling oil during frying can delay deterioration of oil (Orthoefer & List, 2007a). However, in the current study, increases in TPC and PTAG during frying were greater than those for heating. Since no additional oil was added during the 4-days of frying in this study, the oil volume decrease was due to oil carryout from repeated frying batches. The faster oxidation for frying oil in this study may also have the reduced amount of oil resulting in the increased ratio of surface area to volume of oil. In four days of frying, TPC increased beyond the legally allowed amount of TPC (25–27 wt%) and a significant amounts of polymer products (8.81%, peak area in the spectrum) were formed after frying for 4 days. Kinematic viscosity of frying oil at 40 °C increased by 86% after frying for 4 days (from 28.38 mm2/s to 52.91 mm2/s) while that of heated oil increased by only 36% (to 38.58 mm2/s). Density of fried and heated SBO increased from 0.9051 g/cm3 to 0.9118 g/cm3 and 0.9104 g/cm3, respectively. Increases in kinematic viscosity and density were likely related to the increased TPC and PTAG (Ball et al., 2018). Oxygen content slightly increased during frying (from 11.32 wt% to 12.01 wt%) but did not show substantial change during heating. The results of kinematic viscosity, density, and oxygen content also reflected slower oxidation during heating compared to frying.
to iodine vapor. 2.7. NMR analysis 1 H NMR, 13C NMR, distortionless enhancement by polarization transfer (DEPT) 135, and heteronuclear single quantum coherence spectroscopy (HSQC) experiments were carried out in deuterated chloroform (CDCl3) using a Bruker (Billerica, MA) Avance 500 spectrometer operating at 125 MHz. Chemical shifts in parts per million (ppm) were referenced using the chloroform peak. Peak analysis was conducted with SpinWorks 3.1.7 software. Coupling constants are expressed in frequency (Hz).
2.8. Reaction of fried SBO with acyl chloride (AcCl) Fried SBO (100 mg, 0.11 mmol) was weighed in a small vial and dissolved in CDCl3 (0.6 ml). The solution was transferred to an NMR tube, to which pyridine (3.9 µl, 0.054 mmol) was added, using a glass syringe, followed by AcCl (4.4 µl, 0.054 mmol). The solution in the NMR tube was briefly shaken and spectra were taken after 30 min, 3 h and 20 h using the procedures described above. 2.9. Statistical analysis Analyses for TPC, PTAG, viscosity, density, oxygen content, acid value, and saponification value were carried out at least in duplicate. Data are expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was performed with the program JMP 9 (SAS Institute, Cary, NC, USA). Means of data were compared by TukeyKramer HSD test with statistical significance at P < 0.05.
3.2. Changes in acid and ester values Acid value during frying, in general, increases with time due to the hydrolysis of triacylglycerols (Song et al., 2017). In our study, acid value during frying decreased at day 2 and day 3 and then increased to a similar or higher value than the initial value by day 4 (Table 1). Acid value during heating also decreased at day 2 and but then did not increase through day 4. According to Handel and Guerrieri (Handel & Guerrieri, 1990), acid value can initially drop due to evaporation of acidic materials and then increase by hydrolysis. In this study, the increase in acid value between day 2 and day 4 was more prominent during frying than heating, which, presumably, is due to water in the potatoes that was released in the frying process. These results generally follow the pattern of change in oxygen content. As mentioned in the method section, ester value was obtained by subtracting acid value from saponification value (Table 1). Ester value increased from 190.5 mg KOH/g to 195.6 mg KOH/g for fried SBO and to 194.5 mg KOH/g for heated SBO up to day 3, and then both slightly decreased at day 4. While the increases in ester values are small on a relative basis, it must be recognized that each SBO triacylglycerol
3. Results and discussion 3.1. Changes in TPC, PTAG, viscosity, density, and oxygen content during oxidation of SBO When oil is exposed to high temperature, complex chemical reactions occur which produce numerous products. Table 1 reports several markers generally used to assess oil oxidation including TPC, PTAG, kinematic viscosity, density, oxygen content, and acid value. While some products are volatile and do not remain in the oil, others, such as polymers and polar compounds do, therefore, their concentrations constantly increase during frying and heating experiments. Due to the potential toxicity of accumulated substances, regulations in many European countries allow 25–27 wt% TPC as the maximum acceptable Table 1 Analysis of fried and heated soybean oil (SBO) for 0, 2, 3 and 4 days at 180 °C. Properties Total polar compounds (TPC), Wt. % Polymerized triacylglycerols (PTAG), peak area % Kinematic viscosity, mm2/s at 40 °C Density, g/cm3 at 40 °C Oxygen content, Wt. % Acid value, mg KOH/g Saponification value, mg KOH/g Ester value, mg KOH/g
Day Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO Fried SBO Heated SBO
0 1
1.53 ± 0.01 d 1.53 ± 0.01 d 0e 0e 28.36 ± 0.03 d 28.36 ± 0.03 d 0.9051 ± 0.0000 d 0.9051 ± 0.0000 d 11.32 ± 0.14c 11.32 ± 0.14b 0.83 ± 0.08 bc 0.83 ± 0.08b 191.35 ± 0.90b 191.35 ± 0.90b 190.52 ± 0.74 d 190.52 ± 0.74 d
2
3
4
Polar fraction
11.47 ± 0.00c 7.54 ± 0.01c 1.84 ± 0.02 d 0.82 ± 0.01 d 34.83 ± 0.06c 31.90 ± 0.02c 0.9080 ± 0.0001c 0.9068 ± 0.0003c 11.45 ± 0.17c 11.18 ± 0.09b 0.51 ± 0.05c 0.39 ± 0.08c 193.28 ± 1.34 bc 192.48 ± 2.00b 192.77 ± 1.10c 192.09 ± 1.64 cd
17.99 ± 0.40b 11.29 ± 0.11b 4.38 ± 0.37c 2.02 ± 0.05c 41.25 ± 0.00b 34.63 ± 0.06b 0.9118 ± 0.0000b 0.9082 ± 0.0001b 11.40 ± 0.13c 11.01 ± 0.14b 0.55 ± 0.23c 0.41 ± 0.07c 196.14 ± 0.42b 194.86 ± 0.17b 195.59 ± 0.39b 194.46 ± 0.15b
28.56 ± 0.16 a 17.03 ± 0.42 a 8.81 ± 0.07b 3.88 ± 0.00b 52.91 ± 0.00 a 38.58 ± 0.00 a 0.9118 ± 0.0000 a 0.9104 ± 0.0001 a 12.01 ± 0.17b 11.37 ± 0.30b 1.09 ± 0.09b 0.43 ± 0.01c 194.59 ± 1.49 bc 193.58 ± 1.08b 193.49 ± 1.22c 193.15 ± 0.88 bc
– – 70.2 ± 0.2 a 52.3 ± 0.2 a – – – 14.88 ± 0.10 a 13.73 ± 0.06 a 5.18 ± 0.32 a 3.43 ± 0.21 a 203.56 ± 1.32 a 198.25 ± 1.82 a 198.38 ± 1.11 a 194.82 ± 1.49 a
1 Results of statistical analysis. Means not sharing the same letter(s) within a row are significantly different by Tukey-Kramer honest significant difference (HSD) test (P < 0.05).
3
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molecule contains three ester bonds so that an increase of 5 mg KOH/g represents one new ester bond for every 12–13 SBO molecules. As Ball et al. (Ball et al., 2018) reported in the study of oxidized biodiesel heated at 90 °C with aeration for 43 days, this result indicates that the formation of ester bonds contributed to the polymerization of SBO during frying. Since it is well known that alcohols and carboxylic acids are produced during oxidation of oil through a variety of reactions (Schaich, 2005), ester bonds are likely formed by reactions between these two oxidation products at frying temperatures. The decrease in ester value between day 3 and day 4 for fried SBO indicates that esterification between alcohols and carboxylic acids are in competition with hydrolysis of ester bonds. In this period, PTAG increased while ester value decreased indicating that ester bonds are partly responsible for polymerization of SBO and that there are also other chemical bonds forming. Ester values of heated SBO also decreased between day 3 and day 4. However, the difference was not statistically significant at P < 0.05.
Fig. 1. 13C NMR signals for newly formed ester bonds in polar fractions of fried and heated soybean oil (SBO).
3.3. Verification of ester bonds using NMR spectroscopy of polar fraction of oil
173.8 ppm (signal e). The previous study (Bonetti & Parker Jr., 2019) also reported a new ester carbon signal after heating oleic acid. The new signal at 32.6 ppm (signal a) may be the carbon next to the newly formed carbonyl carbon. Signals b (172.8 ppm) and c (173.2 ppm) in Fig. 1 are sn2 and sn1,3 carbonyl carbons of triacylglycerols, respectively (Mannina et al., 2000). These two peaks had small shoulders after oxidation indicating that other new ester signals may have been buried in these triacylglycerols peaks.
NMR spectroscopy is one of the most powerful tools used to elucidate the molecular structure of organic substances. As mentioned in the introduction section, there are numerous oxidation products that have yet to be identified in these materials. Recently, NMR spectroscopy has been intensively used to identify previously unrecognized compounds in oxidized oil (Alexandri et al., 2017; Goicoechea & Guillen, 2010; Guillén & Ruiz, 2001; Martínez-Yusta & Guillén, 2014a, 2014b, 2016; Patrikios & Mavromoustakos, 2014). In this study, we used the NMR spectroscopy to understand chemical bonds forming polymers. Signal assignments were conducted based on previous reports, which were further confirmed with the simulations. Newly formed ester bonds can be detected in NMR spectra. First, we attempted to find signals of ester bonds in 1H NMR spectra and 13C NMR spectra of SBO samples fried and heated for 4 days. There were a few tiny signals in 13C NMR spectra of fried and heated SBO samples that possibly indicated ester bond formation, but they were too small to make solid conclusions. This indicated that the amount of newly formed ester bonds in fried and heated SBO’s having 8.81 and 3.88% PTAG, respectively, was lower than the detection limit of NMR spectroscopy. Our previous report showed that the polar fraction of the oxidized oil had a high content of polymeric materials (Hwang et al., 2013). Following the previous method, column chromatography was conducted with SBO samples fried and heated for 4 days to give polar fractions consisting of 70.2% and 52.3% PTAG, respectively (Table 1). Acid values of polar fractions were much higher than the oil before separation indicating acidic compounds including free fatty acids and polymers containing an acid group(s) were concentrated in the polar fraction. In addition, saponification and ester values of the polar faction were also significantly higher than oil before separation. Oxygen content in the polar fractions of fried and heated SBO were also significantly higher than oxidized SBO before separation. The increased ester value and oxygen content in the polar fraction where TPAG was concentrated is another indication that at least some of the polymerization occurred through the formation of new ester bonds. The 8 mg KOH/g ester value increase implies approximately one new ester bond per 7–8 SBO molecules. The increase in oxygen content in the fried SBO polar fraction, 3.5%, is about 1/3 of the initial oxygen content, indicating that most of the oxygen is assimilated into other functional groups besides esters. The NMR spectroscopy study was conducted on these polar fractions of oxidized SBO. While the 1H NMR spectra did not give much information on newly formed ester bonds due to signal overlapping, 13C NMR spectrum of the polar fraction of fried SBO clearly showed new signals for ester bonds at 173.6 and 173.8 ppm (signal d and e in Fig. 1). The polar fraction of heated SBO showed only the signal at
3.4. New 1H NMR signals at 3.61 and 3.71 ppm – ether bonds or alcohols? As discussed above, while the ester value slightly deceased from day 3 to day 4, PTAG significantly increased in this period of frying (Table 1) indicating that there are possibly chemical bonds in polymers other than ester bonds. New NMR signals are often used as indicators for the formation of new chemical bonds and new products (Han, Kang, Bai, Xue, & Shi, 2018). Therefore, we attempted to find other possible chemical bonds in polymers by scrutinizing the new NMR signals of oxidized oils. A triplet peak at 3.61 ppm and a doublet peak at 3.71 ppm are the most prominent new signals in 1H NMR spectra of polar fractions of fried and heated SBO. Fig. 2a shows these new proton peaks in the spectra of fried SBO (4 d) and its polar fraction. The heated SBO spectrum also had the same signals. Slight changes in their chemical shifts to upfield in fried SBO and its polar fraction might have been caused by different environments, such as an increased number and amount of oxidation products in the SBO samples. Patrikios and Mavromoustakos (2014) found proton signals in the region of 3.65–3.7 ppm in olive oil oxidized at 130 °C for 24 h and concluded that these peaks are protons of newly formed ether linkages, CH–O–CH, which formed oligomers of fatty acids. In contrast, Martínez-Yusta and Guillén (2014a), assigned the triplet signal at 3.61 ppm and the doublet signal at 3.71 ppm as a primary alcohol CH2, (–CH2OH), and –CH2OH of 1,2-diacylglycerol, respectively, in the 1H NMR spectrum of olive oil subjected to deep-frying at 190 °C. To examine if these signals correspond to ethers that may be responsible for polymerization of SBO during frying, the new NMR signals of oxidized SBO were closely investigated in this study. The triplet and doublet peaks in Fig. 2a had the different coupling constants (6.6 and 5.2 Hz, respectively) indicating carbons of these protons were not connected to each other. Therefore, these signals represent two different substances or separate functional groups. Fig. 2b shows 13C NMR spectra of fried SBO (4 d) and its polar fraction compared to the spectrum of fresh SBO. The 13C NMR spectrum of heated SBO also has a similar pattern to that of fried SBO. Although no clear new signals were shown in the fried SBO, four new signals at 4
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Fig. 2. The most prominent new signals in a) 1H NMR and b) 13C NMR spectra in fried soybean oil (SBO) (4 d) and its polar fraction and c) heteronuclear single quantum coherence spectroscopy (HSQC) spectrum of the polar faction.
valuable tool in discerning the differences in structures. Fig. 3a-c show reaction schemes and changes in 1H NMR and in 13C NMR of the polar fraction of fried SBO after the reaction with AcCl. As shown in Fig. 3b, the triplet signal at 3.61 ppm (signal b) and the doublet signal at 3.71 ppm (signal c) completely disappeared after 3 h indicating these signals were protons of alcohols rather than ethers. The proton signal e also disappeared while new signals a and d appeared. Signals were assigned according to the previous reports (Martínez-Yusta & Guillén, 2016) along with simulation with ChemDraw to confirm the assignments. The signal d was assigned to be the methylene group (–CH2) (2 in Fig. 3a) of the acetylated product that was shifted from 3.61 ppm, signal b, after the reaction. The proton signal e corresponded to protons at C2 of 1,2-diacylglycerol backbone (4 in Fig. 3a), which moved under the big signal at 3.9–4.4 ppm of the triacylglycerols after the reaction. The new signal a was assigned to the methyl group (–CH3) of the newly formed acetyl group (9 in Fig. 3a). Therefore, the 1H NMR experiment after the reaction with AcCl strongly supported the assignments by Martínez-Yusta and Guillén (2014a), that the triplet signal at 3.61 ppm and the doublet signal at 3.71 ppm are a primary alcohol and 1,2-diacylglycerol, respectively. 13 C NMR was also used to study the polar fraction of fried SBO oil after the reaction with AcCl. Fig. 3c shows the region of 13C NMR spectrum where the most obvious changes were observed. Assignment of 13C NMR signals was conducted using the previous report (Vlahov, 1996) along with the simulated 13C NMR spectra, by ChemDraw, and the database provided by AIST (2018). Distortionless enhancement by polarization transfer (DEPT) was also used to confirm the assignment. Signal A, at 61.4 ppm, in Fig. 3c corresponding to C1 of 1,2-diacylglycerol backbone (3 in Fig. 3a) which moved to 62.3 ppm (signal C
61.4, 65.0, 68.2, and 72.1 ppm appeared in the polar fraction of fried SBO. According to Vlahov (1996), signals at 61.4, 65.0, 68.2, and 72.1 ppm can be assigned to C1 of 1,2-diacylglycerol backbone, C1 and C3 of 1,3-diacylglycerol backbone, C2 of 1,3-diacylglycerol backbone, and C2 of 1,2-diacylglycerol backbone, respectively. The proton signal at 3.71 ppm and carbon signals at 61.4 and 72.1 ppm are strong evidence of the existence of 1,2-diacylglycerol. Furthermore, a 2D NMR study using heteronuclear single quantum coherence spectroscopy (HSQC) showed the correlation between the proton signal at 3.71 ppm and the carbon signal at 61.4 (Fig. 2c), which supports the assignments by Martínez-Yusta and Guillén (2014a). The signal at 3.61 ppm correlated with the tiny carbon peak at 62.8 ppm in HSQC, which confirmed the previous assignment as a methylene group carbon of a primary alcohol, –CH2OH. In contrast, the study by Patrikios and Mavromoustakos (2014) found the correlation of proton signals at 3.65–3.7 ppm with carbon peaks at the region of 50 ppm in the HSQC spectrum, which were thought to be ether bonds. 3.5. Reaction of hydroxyl group with acetyl chloride (AcCl) To further confirm if the most prominent new signals at 3.61 and 3.71 ppm in Fig. 2a are of a primary alcohol CH2, (–CH2OH), and –CH2OH of 1,2-diacylglycerol, respectively, the reaction of the polar fraction of fried SBO with AcCl was conducted. It was hypothesized that the assignments of signals in Fig. 2a and 2b as primary alcohols and 1,2diacylglycerol could be confirmed if these signals moved after the reaction with AcCl which reacts with an alcohol at room temperature to form an ester (Ishihara, Kurihara, & Yamamoto, 1993; Smith & Bryant, 1935). However, AcCl does not decompose ether bonds making it a 5
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Fig. 3. a) Reactions of a primary alcohol and 1,2-diacylglycerols in the polar fraction of fried soybean oil (SBO) with acetyl chloride (AcCl) for 3 h. Changes in b) 1H NMR spectrum and c) 13C NMR after the reaction. d) Expanded 1H NMR spectrum for the peak at 4.07 ppm. Assignments of proton signals are: a = 9; b = 1; c = 5; d = 2; e = 4. Assignments of carbon signals are: A = 3; B = 8, C1 and C3 of triacylglycerol backbone; C = 6; D = C1 and C3 of 1,3-diacylglycerol backbone; E, F = C2 of 1,3-diacylglycerol backbone G = 7, H = C2 of triacylglycerol backbone; I = 4 for 13C NMR.
in Fig. 3c, 6 in Fig. 3a) after the reaction. Signal B at 62.1 ppm is the signal of C1 and C3 of triacylglycerol backbone including those of newly formed triacylglycerols by the reaction with AcCl. Signal I at 72.1 ppm corresponds to C2 of 1,2-diacylglycerol backbone (4 in Fig. 3a) that shifted to 68.8 ppm (signal G) after the reaction with AcCl. Signal H (68.9 ppm) is C2 of triacylglycerol backbone including the reaction products. All the changes in 13C NMR spectrum supported the presence of 1,2-diacylglycerols in the fried SBO, which were converted to triacylglycerols after the reaction with AcCl. Signals E and F, at around 68.2 ppm, were shown at slightly different chemical shifts. However, after repeated experiments, it was found that the signal E was the same signal as signal F and was shown at slightly different field, 68.15 ± 0.05 ppm, presumably, due to the different environments caused by the excess AcCl. Signals E and F can be assigned as C2 of 1,3-diacylglycerol backbone and signal D as C1 and C3 of 1,3-diacylglycerol backbone based on the previous report (Vlahov, 1996). All of these points were in agreement with the simulated 13C NMR spectra in this study. The carbon signal at 68.2 ppm also correlated with the proton signal at 4.07 ppm in HSQC. The proton signal at 4.07 ppm was previously assigned as the CH proton at C2 of 1,3-diacylglycerols (Martínez-Yusta & Guillén, 2014a). These results indicate the presence of 1,3-diacylglycerols in the oxidized SBO, which did not completely react with AcCl due to the steric hindrance by two long fatty acid groups. In general, sterically hindered secondary alcohols react significantly slower than primary alcohols with AcCl (Ishihara et al., 1993). Carbon signals D and F did not completely disappear even after a 20-h reaction. The proton signal at 4.07 ppm in the 1H NMR seemed to completely disappear in Fig. 3b. However, a closer observation (Fig. 3d) revealed that the large peak at 4.1 ppm was broadened and the peak at 4.07 ppm was merely overlapped with the large peak.
3.6. Other proton signals of alcohols Due to the many types and relatively small amounts of oxidation products, it was difficult to assign all the small peaks in 1H NMR of oxidized oil. Tremendous efforts have been made by several research groups to assign many of these small peaks during the last decade. However, some signals remain unidentified, unclear, or contradictory. In this study, in addition to the above assignments, including proton signals at 3.61 and 3.71 ppm, we clarified previously uncertain assignments, confirmed previous assignments, and found one new alcohol signal from the NMR study of acetylated fried SBO. Signals a-p in Fig. 4a are newly formed or grown proton signals found in fried SBO and its polar fraction. 1H NMR spectrum of heated SBO showed a very similar pattern to that of fried SBO. Fig. 4b shows these signals of oxidation products before and after the reaction with AcCl. It cannot be definitively determined if signals a and b moved after the reaction with AcCl due to overlapping other signals. Signals c and d, which were previously thought to be epoxy or alcohol (Goicoechea & Guillen, 2010; Martínez-Yusta & Guillén, 2014a), were assigned as alcohols in this study. Signal e, a very small but obvious signal, disappeared after the reaction with AcCl. This result confirmed the previous assignment of this signal as a secondary alcohol (Martínez-Yusta & Guillén, 2014a). Since this secondary alcohol completely reacted with AcCl in 3 h, this alcohol does not appear to be sterically hindered. As discussed above, signals f and g were verified as signals of alcohols instead of ethers. Signals h-j were also shifted after the reaction confirming the previous assignment as alcohols (MartínezYusta & Guillén, 2014a,b). To the best of our knowledge, no information was previously reported for signal k shown at 5.07 ppm. This signal was newly assigned as an alcohol signal in this study although further study is needed to verify the detailed molecular structure. Signals l (many peaks at 5.52–7.08 ppm) did not move or change after the reaction, which is consistent with the previous assignments of these 6
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Fig. 4. a) Newly appearing or intensified proton signals after oxidation of soybean oil (SBO). Intensity of signals m-p were magnified four fold compared to other peaks. b) Changes in proton signals of fried SBO polar fraction after reaction with acetyl chloride (AcCl) for 3 h. Table 2 Signals assigned as alcohols in this study from the reaction with acetyl chloride (AcCl) compared to previous assignments (Alexandri et al., 2017; Goicoechea & Guillen, 2010; Guillén & Ruiz, 2001; Martínez-Yusta & Guillén, 2014a, 2014b, 2016; Patrikios & Mavromoustakos, 2014). Signal
Chemical shift (ppm)
Splitting pattern
Previous assignments
Assignments in this study
c d e f g h i j k o
2.89 3.09 3.50 3.61 3.71 3.92 4.07 4.41 5.07 9.57
Multiplet Multiplet Multiplet Triplet Doublet Triplet Multiplet Multiplet Triplet d
Epoxy or alcohol Epoxy or alcohol Secondary alcohol Ether CH or primary alcohol Ether CH2 or –CH2OH (1,2-diacylglycerol) ROCH2-CHOH-CH2OH in 1-monoacylglycerol ROCH2-CHOH-CH2OR′ in 1,3-diacylglycerol CHO-CH]CH-CHOH– Not assigned –CHO (4-hydroxy-(E)-2-alkenals)
Alcohol, correlated to a carbon at 56.9 ppm Alcohol, –CH-CH-OH Secondary alcohol Primary alcohol –CH2OH (1,2-diacylglycerol) Agreed Confirmed with HSQC* Confirmed with HSQC Alcohol, correlated to a carbon at 72.2 ppm Agreed with reaction with AcCl
*HSQC: heteronuclear single quantum coherence spectroscopy.
shifting due to functional groups give the conclusion that the assignment is in agreement. A carbon peak at 68.2 ppm correlated with signal i confirmed it as ROCH2-CHOH-CH2OR′ in 1,3-diacylglycerol. The correlation of signal j with the carbon signal at 62.1 ppm as well as the simulated NMR experiment strongly supported the assignment as CHOCH]CH-CHOH–. HSQC experiments also provided further information for a few signals that will help future studies in elucidating detailed molecular structures. For example, signals c, d, and k correlated with carbon signals at 56.9, 32.8, and 72.2 ppm, respectively. The carbon signal at 32.8 ppm correlated with the proton signal d indicates that these protons are on the beta position carbon to the hydroxy group.
signals as conjugated diene double bonds (Alexandri et al., 2017). Signals m-p were assigned as a variety of aldehydes and the detailed molecular structures of these aldehydes were reported by MartinezYusta and Guillen (2016). Among them, signal o at 9.57 ppm was assigned as 4-hydroxy-(E)-2-alkenals. In this study, the signal o seemed to imply two aldehyde signals that overlapped. One of them disappeared after the reaction confirming the previous assignment, but also indicates that there was another kind of aldehyde having a very similar chemical shift. Signals that were assigned as protons of alcohols in this study are summarized in Table 2 along with previous assignments. HSQC experiments confirmed or strongly supported these assignments. For example, signal e correlated with a carbon signal at 71.6 ppm, which is a good indication of a secondary alcohol. As discussed above, assignments of signal f and g were well confirmed with HSQC. Signal h correlated with a carbon at 72.2 ppm, which is slightly different chemical shift than the reported one as ROCH2-CHOH-CH2OH in 1-monoacylglycerol (70.3 ppm) (Vlahov, 1996). However, possible
4. Conclusions The increased ester value and 13C NMR signals provides evidence that ester bonds are responsible for the formation of polymers in oxidized SBO during frying and heating at 175 °C, consistent with that in a 7
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recent study with oxidized biodiesel. The fact that PTAG constantly increased while the ester value slightly decreased from day 3 to day 4 of frying indicated that there are also other chemical bonds formed during polymerization. In this study, no NMR signals corresponding to ether bonds were found. The NMR study after the reaction of oxidized SBO with AcCl showed that proton signals at 3.61 and at 3.71 ppm were indicative of alcohols produced in oxidized SBO. From the NMR study, several signals were assigned to be those of alcohols confirming literature signal assignments, verifying uncertain assignments, and finding one previously unreported alcohol signal. Further studies are needed to definitively describe the chemical bonds forming polymers other than ester bonds.
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CRediT authorship contribution statement Hong-Sik Hwang: Conceptualization, Methodology, Investigation, Writing - original draft. James C. Ball: Conceptualization, Investigation, Writing - review & editing. Kenneth M. Doll: Conceptualization, Writing - review & editing. James E Anderson: Investigation, Writing - review & editing. Karl Vermillion: Methodology, Formal analysis. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors would like to express appreciation to Lynne Copes for excellent technical work. Conflict of interest The authors declare that they have no conflict of interest. References AIST. Spectral database for organic compounds (2018). https://sdbs.db.aist.go.jp/sdbs/ cgi-bin/direct_frame_top.cgi. Accessed 3 August 2019. Alexandri, E., Ahmed, R., Siddiqui, H., Choudhary, M. I., Tsiafoulis, C. G., & Gerothanassis, I. P. (2017). High resolution NMR spectroscopy as a structural and analytical tool for unsaturated lipids in solution. Molecules, 22, 1663. https://doi.org/ 10.3390/molecules22101663. AOCS (2010). Acid value of fats and oils. AOCS official method Cd 3d–63. Urbana, IL: American Oil Chemists' Society. AOCS (2017). Saponification value of fats and oils. AOCS official method Cd 3–25. Urbana, IL: American Oil Chemists' Society. Arca, M., Sharma, B., Price, N. J., Perez, J., & Doll, K. (2012). Evidence contrary to the accepted Diels-Alder mechanism in the thermal modification of vegetable oil. Journal of the American Oil Chemists' Society, 89, 987–994. https://doi.org/10.1007/s11746011-2002-x. ASTM (2000). Standard test methods for determination of total oxygen in gasoline and methanol fuels by reductive pyrolysis. ASTM D5622. Annual book of ASTM standards. West Conshohocken, PA: ASTM International. Ball, J. C., Anderson, J. E., & Wallington, T. J. (2018). Depolymerization of polyester polymers from the oxidation of soybean biodiesel. Energy & Fuels, 32, 12587–12596. https://doi.org/10.1021/acs.energyfuels.8b03169. Bonetti, R., & Parker, W. O., Jr. (2019). Insights into polymerization of vegetable oil: Oligomerization of oleic acid. Journal of the American Oil Chemists' Society, 96, 1181–1184. https://doi.org/10.1002/aocs.12274. Cam, A., Oyirifi, A. B., Liu, Y., Haschek, W. M., Iwaniec, U. T., Turner, R. T., ... Helferich, W. G. (2019). Thermally abused frying oil potentiates metastasis to lung in a murine model of late-stage breast cancer. Cancer Prevention Research, 12, 201–210. https:// doi.org/10.1158/1940-6207.Capr-18-0220. Doll, K. M., & Hwang, H.-S. (2013). Thermal modification of vegetable oils. Lipid Technology, 25, 83–85. https://doi.org/10.1002/lite.201300269. Gertz, C., Aladedunye, F., & Matthäus, B. (2014). Oxidation and structural decomposition of fats and oils at elevated temperatures. European Journal of Lipid Science and Technology, 116, 1457–1466. https://doi.org/10.1002/ejlt.201400099. Gertz, C., Klostermann, S., & Kochhar, S. P. (2000). Testing and comparing oxidative stability of vegetable oils and fats at frying temperature. European Journal of Lipid Science and Technology, 102, 543–551. https://doi.org/10.1002/1438-312(200009)
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