Formation of Plant Sterol Oxidation Products in Foods during Baking

Article pubs.acs.org/JAFC

Formation of Plant Sterol Oxidation Products in Foods during Baking and Cooking Using Margarine without and with Added Plant Sterol Esters Yuguang Lin,*,†,§ Diny Knol,†,§ María Menéndez-Carreño,† Wendy A. M. Blom,† Joep Matthee,† Hans-Gerd Janssen,†,‡ and Elke A. Trautwein† †

Nutrition and Health, Unilever Research & Development, 3133 AT Vlaardingen, The Netherlands Analytical-Chemistry Group, van’t Hoff Institute for Molecular Sciences, University of Amsterdam, 1090 GE Amsterdam, The Netherlands

‡

S Supporting Information *

ABSTRACT: Plant sterols (PS) in foods are subject to thermal oxidation to form PS oxidation products (POP). This study measured POP contents of 19 foods prepared by typical household baking and cooking methods using margarines without (control) and with 7.5% added PS (as 12.5% PS-esters, PS-margarine). Median POP contents per portion size of cooked foods were 0.57 mg (range 0.05−1.11 mg) with control margarine versus 1.42 mg (range 0.08−20.5 mg) with PS-margarine. The oxidation rate of PS (ORP) was 0.50% (median) with the PS-margarine and 3.66% with the control margarine. Using the PSmargarine, microwave-cooked codfish had the lowest POP content, with 0.08 mg per portion, while shallow-fried potatoes had the highest POP content, 20.5 mg per portion. Median POP contents in cookies, muffins, banana bread, and sponge cake baked with the control or PS-margarine were 0.12 mg (range 0.11−0.21 mg) and 0.24 mg (range 0.19−0.60 mg) per portion, with a corresponding ORP of 1.38% and 0.06%, respectively. POP contents in all the cooked and baked foods did not exceed 20.5 mg per typical portion size. A wide variation in the distribution of individual POP among different foods existed, with 7-keto-PS and 5,6-epoxy-PS being the major oxidation products. KEYWORDS: plant sterols, phytosterols, plant sterol oxidation products, oxidation, foods, cooking, baking, frying, margarine in the body.8 However, similar to cholesterol, the steroid ring of PS is susceptible to auto-oxidation, also called non-enzymatic oxidation, to form PS oxidation products (POP). Autooxidation can take place both in the human body as well as in foods.9 Typical POP formed via auto-oxidation are 7hydroxy, 7-keto, 5,6-epoxy, and 3,5,6-triol derivatives of the major PS, which can be found in foods.9 As assessed by a number of reviews,4,10−12,16 POP may have both beneficial (e.g., anti-carcinogenic) and detrimental effects (e.g., associated with atherogenicity, inflammation, cytotoxicity, and oxidative stress) based on in vitro and animal studies, whereas data about physiological effects of POP in humans are scarce. The amount of daily POP intake from the diet may determine their circulating concentrations and subsequently the potential physiological effects, while such a relationship has not yet been established. Vegetable oil-based products such as margarines with added PS may be used by consumers to replace conventional fats and oils for home cooking and baking applications. This may potentially increase the POP contents of prepared foods.9,13 To date, only a few studies addressed POP formation in foods with added PS after heat processing, as reviewed recently by Scholz et al.13 and Lin et al.14 However, these studies have several

1. INTRODUCTION Plant sterols (PS) are naturally existing compounds found in all plant-derived foods, such as vegetable oils, grains, nuts, seeds, fruits, legumes, and vegetables. Sitosterol, campesterol, and stigmasterol are the most common PS, accounting for more than 80% of total PS in the human diet.1,2 The average intake of PS from natural sources is 200−400 mg/d, depending on the type of diet consumed.3,4 PS partially inhibit cholesterol absorption from the gut resulting in a significant lowering of serum LDL-cholesterol concentrations. At intakes between 1.5 and 3 g/d, PS lower LDL-cholesterol by 7−12%.5 Because of their well-established cholesterol-lowering effect, a variety of food products, including fat-based products like margarine, and dairy foods like milk and yoghurts, with added PS are commercially available.4,6 PS esters (PSE) are the major form of PS being added to commercial food products.6 Their substantiated cholesterol-lowering benefit forms the basis for the granted disease risk reduction health claim by the European commission based on positive opinions by the European Food Safety Authority (EFSA).7 PS are structurally similar to cholesterol having the same steroid ring structure. PS differ from cholesterol only in the sterol side chain by having a methyl (campesterol) or ethyl (sitosterol and stigmasterol) group at the C24 position and having a double bond between C22−C23 (stigmasterol). With these structural differences in the side chain, PS are poor substrates for enzymes like cytochrome P450 mono-oxygenases which are responsible for the side chain oxidation of cholesterol © XXXX American Chemical Society

Received: October 12, 2015 Revised: December 16, 2015 Accepted: December 23, 2015

A

DOI: 10.1021/acs.jafc.5b04952 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry limitations. For example, in the study by Soupas et al.,23 POP formation was only investigated under laboratory conditions using model systems containing merely fat-based products, such as vegetable oils, liquid margarine or butter, without the presence of other food ingredients. In the study by Derewiaka and Obiedzinski,26 only a small number of foods, i.e., noodles, frozen French fries, minced meats, and frozen fish products, were prepared by different cooking methods, and their POP contents were analyzed. However, the PS and POP contents in the vegetable oils used for frying the foods were not measured. Hence, data from these experiments do not reflect actual POP contents of foods with and without added PS and prepared with common household cooking and baking methods. Further, daily intake of POP from foods prepared with conventional fatbased products and those enriched with PS is largely unknown. Therefore, this study aimed to measure the amount of POP in a wide range of foods, including vegetables, potato, meats, fish, and egg, which were prepared using margarines without and with added PSE, and using various common household cooking methods such as shallow- and stir-frying, stewing, roasting, and microwave cooking as well as baking.

considering some minor PS and stanols also present in the PS formulation. Sitosterol and campesterol accounted for the major proportion of PS in both margarines. Measured total amounts of POP in the two margarines were low and consisted mainly of 7-keto-PS (Table 1). The method used for the POP measurements is described in section 2.2. Fresh food ingredients including different vegetables (green beans, cabbage, and onions), fish (cod, salmon, and frozen fish fingers), meats (pork, beef, and chicken), and eggs next to some other food ingredients (e.g., flour, sugar, and salt) needed for the baking recipes, were purchased from whole sale (Zegro, The Netherlands). Precooked small potatoes and minced meat (50/50 beef/pork mixture) were purchased from a local supermarket in The Netherlands. 2.1.2. Cooking Methods. Onions, green beans, and cabbage were cut into typical small pieces of ca. 3 cm or into pieces of 0.5 × 0.5 cm before cooking. Chicken used for stir-frying and beef used for stewing were cut into small pieces of ca. 2 × 2 cm. Minced meat was mixed with a small amount of egg, onion, and bread crumbs to make a meatball of approximately 150 g. The portion sizes of fresh food ingredients prepared varied between 100 and 250 g (Table 2), representing a typical portion customarily consumed per eating occasion by 1 or 2 individuals. Roasted beef was prepared using 1 kg of meat, and the portion size was defined as 200 g roasted (cooked) meat. The food ingredients were prepared according to the following cooking methods: Shallow-f rying of onion, potatoes, egg, salmon, fish fingers, codfish, steak, pork fillet, and minced meat: 20 g of control or PS-margarine was put into a pan, and the pan was placed on an induction stove and pre-heated for 5 min to reach 180 °C. The pre-weighed food was then added to the pan, and the temperature was maintained at about 180 °C for another 4−18 min, depending on the food ingredient. A stainless steel spoon was used to turn the foods over during the shallow-frying process to prevent burning. For frying eggs, two eggs were put in the pan and shallow-fried with “sunny side up” for a total of 4 min. Stir-f rying of green beans, cabbage, and chicken: the method was similar to that of shallow-frying, but using a wok and applying higher temperatures of about 200−250 °C and shorter frying times of 3 min. The amount of fat used was 20 g of control or PS-margarine, preheated for 3 min before the food was added. During the stir-frying process, the food ingredients were constantly stirred to prevent burning. Stewing of beef: A 200 g piece of beef was cut into pieces of ca. 2 × 2 cm. It was first shallow-fried with 20 g of pre-heated control or PSmargarine for about 4 min at ca. 150 °C. Then, 100 mL of water was added to cover the beef, and the meat was further cooked on an induction stove for a total of 90 min with the pan covered with a lid. After 30 and 60 min, another 100 mL of water was added, for a total of 300 mL added water. Roasting of beef: 70 g of control or PS-margarine was added to a pan and pre-heated on an induction stove. After 5 min, 1 kg of meat was added and shallow-fried for 9 min at ca. 180 °C, browning the meat on all sides. The pan with the meat was then placed on the middle rack of a preheated electric oven at 140 °C, allowing hot air to circulate around the beef. The meat was roasted at 140 °C for 30 min while frequently basting with the residual fat from the pan. Microwave cooking of codfish: A codfish fillet was placed with 20 g control or PS-margarine into a microwave-safe dish, and the dish was placed into a microwave oven. The fish was cooked for 5 min at a power level of 600 W. 2.1.3. Baking Procedures. Four different recipes were used to prepare sponge cake, banana bread, muffins, and cookies. The different batters were prepared by mixing the control or PS-margarine with the other ingredients according to the following recipes: For cookies (507 g batter in total): 150 g sugar, 220 g self-rising flour, 10 g mixed eggs, and 2 g salt were mixed with 125 g control or PS-margarine. Each cookie (ca. 6 cm in diameter) was made with about 20 g batter. Using half of the batter, ca. 10 cookies were baked on an oven tray for 12 min in a 170 °C pre-heated oven.

2. MATERIALS AND METHODS 2.1. Cooking and Baking Methods. 2.1.1. Margarines Used for Cooking and Baking. Two types of margarine, one without (“control margarine”) and one with added PSE (“PS-margarine”), were manufactured at Unilever R&D, Vlaardingen, The Netherlands. The margarines had an identical fatty acid composition and differed only in the addition of 12.5% PSE (equivalent to 7.5% free PS) that replaced part of the water content in the PS-margarine (Table 1). The control and PS-margarine contained 0.14 and 6.4 g PS (free PS equivalent) per 100 g, respectively, based on the sum of the four major PS and not

Table 1. Composition of the Control and PS-Margarine per 100 g of Product energy, kJ energy, kcal total fatty acid (FA), g saturated FA, g monounsaturated FA, g polyunsaturated FA, g added PS, ga measured PSb, mg sitosterol, mg campesterol, mg stigmasterol, mg brassicasterol, mg measured POP, mg 7-OHe, mg 7-ketoe, mg 5,6-epoxye, mg triolse, mg water, g othersf, g total, g

control margarine

PS-margarine

2243 544 60.4 12.4 17.9 29.6 − 144.8 ± 5.84c 92.0 ±2.5 33.5±2.3 10.3 ±0.9 9.0 ± 0.2 0.38 ± 0.02 ND 0.38 ± 0.02 ND ND 38.5 1.1 100.0

2239 543 60.3 12.3 17.9 29.5 7.5 6429.2 ± 396.6d 5176.4 ± 314.8 1030.2 ± 100.4 37.2 ± 3.3 185.4 ± 16.2 1.12 ± 0.02 0.17 ± 0.003 0.95 ± 0.08 ND ND 31.1 1.1 100.0

a

PS were added as PSE (12.5 g, representing 7.5 g PS and 5 g fatty acids). bOnly the four major plant sterols were measured, while stanols and minor plant sterol were not accounted for. cRepresents the mean ± SD of three measurements. dRepresents the mean ± SD of 14 measurements. eRepresents the sum of individual oxidation products of sitosterol, campesterol, and stigmasterol. fIncludes small amounts of protein, carbohydrates, vitamins, and minerals. ND = not detectable. B


Article

Journal of Agricultural and Food Chemistry

Table 2. Weights of Foods before and after Cooking by Different Methods and Amounts of Residual Fat Left after Cooking weight (g) before cookinga cooking method

after cookinga

ingredient

raw ingredient

amount of margarine used

stir-frying

green beans cabbage chicken

150.5 ± 0.4 150.1 ± 0.1 150.6 ± 1.0

20.0 ± 0.0 20.0 ± 0.0 20.0 ± 0.0

shallow-frying

egg onions codfish fish fingers pork fillet steak (beef) salmon potatoes minced meat

125.9 100.0 150.4 150.2 152.1 150.5 151.4 251.5 150.7

stewing roasting microwave cooking

beef beef codfish

± ± ± ± ± ± ± ± ±

2.8 0.0 2.1 2.4 2.5 2.2 1.9 1.2 0.8

20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0 20.0

199.6 ± 0.9 1005.9 ± 15.4 151.3 ± 2.1

± ± ± ± ± ± ± ± ±

cooked ingredient 134.0 ± 8.7 119.2 ± 5.1 120.3 ± 2.6

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

88.1 59.6 129.5 141.8 123.9 110.2 121.2 206.9 123.7

20.0 ± 0.0 70.0 ± 0.0 20.0 ± 0.0

± ± ± ± ± ± ± ± ±

3.5 2.7 3.9 3.5 3.6 6.2 4.1 5.1 2.3

127.5 ± 4.1 925.2 ± 14.8b 115.8 ± 4.8

total amount of residual fat 3.1 ± 0.7 − 5.5 ± 0.5 7.3 0.9 7.1 4.5 9.6 18.9 17.6 5.4 12.7

± ± ± ± ± ± ± ± ±

1.5 0.1 1.1 0.7 0.8 2.4 2.0 0.7 0.9

18.8 ± 4.9 59.1 ± 3.2 21.5 ± 3.8

a Means ± SD of five experiments with the control margarine and five with the PS-margarine combined (n = 10). bPortion size of roasted beef was defined as 200 g cooked beef.

Table 3. Weights of Batters before and after Baking, Defined Portion Sizes, and Calculated Amounts of Margarine per Portion weight (g) batter according to recipe

baked food cookies muffins banana bread sponge cake

total batter (a) 503.9 647.2 703.0 402.3

± ± ± ±

0.6 12.3 2.0 0.1

a

margarine content of total batter (b) 125.0 50.1 75.1 100.1

± ± ± ±

0.0 0.1 0.0 0.1

batter/product during actual bakinga batter used for bakingb (c) 198.2 302.0 692.4 398.0

± ± ± ±

5.5 2.4 6.4 0.8

product after baking (d) 177.8 278.4 667.5 370.0

± ± ± ±

6.9 8.0 5.0 4.5

defined portion size (e)

amount of margarine per portionc (f)

18 44 80 50

5.0 3.7 8.8 13.4

a Means ± SD of five experiments with the control margarine and five with the PS-margarine combined (n = 10). bOnly part of the prepared batter, according to the recipe, was used for baking 10 cookies, 6 muffins, 1 banana bread, and 1 sponge cake. cCalculated as f = (e*c/d)*b/a.

For muffins (654 g batter in total): 125 g sugar, 125 g milk, 250 g self-rising flour, 100 g mixed eggs, and 4 g baking powder were mixed with 50 g control or PS-margarine. Each muffin was made with about 48 g of batter, put into a muffin pan to make 6 muffins. The muffins were baked for 25 min in a 180 °C pre-heated oven. For banana bread (702 g batter in total): 125 g sugar, 250 g selfrising flour, 100 g mixed eggs, 2 g salt, and 150 g chopped banana were mixed with 75 g control or PS-margarine. Two equal-sized banana breads were formed from the batter, placed in a baking tin, and baked for 60 min in a 155 °C pre-heated oven. For sponge cake (402 g batter in total): 100 g sugar, 100 g selfrising flour, 100 g mixed eggs, and 2 g salt were mixed with 100 g control or PS-margarine. Two equal-sized sponge cakes were formed from the batter, placed in a baking tin, and baked for 60 min in a 155 °C pre-heated oven. The portion sizes of the baked products were defined as one typical slice of banana bread (80 g) or sponge cake (50 g), one muffin (44 g), and one cookie (18 g), based on customary serving sizes typically consumed by one person. The corresponding amounts of control and PS-margarines used for each portion of baked products were calculated according to the weights of the batter before and after baking and the percentage margarine add to the batter, which are summarized in Table 3. 2.1.4. Standardization of Cooking and Baking Methods. All cooking and baking was performed by one chef in a professional kitchen at Unilever R&D Vlaardingen, The Netherlands. The different

cooking methods were standardized regarding the applied pre-heating and main heating temperatures and times, which were set up in pilot experiments. All used baking and cooking utilities and equipment, e.g., a stir-frying wok (cast iron, bottom diameter 16 cm, and upper side diameter 36 cm), a frying pan (Teflon-coated cast iron, bottom diameter 22 cm), an electromagnetic (induction) stove, a microwave oven, and an electric oven (all Pallux, Germany), were domestic appliances. Each cooking and baking method was repeated five times. In total, 19 different foods based on the described cooking and baking methods were prepared. To control and monitor the temperatures during the different cooking methods, a digital infrared thermometer measuring the temperature on the surface of the pan or wok was used. The temperatures were measured three times per cooking occasion during the main cooking procedure: (1) at the end of pre-heating, just before the ingredient was put into the pan, (2) halfway through, and (3) at the end of the cooking process. The mean value and SD of the three measured temperatures is reported as an indication of the applied cooking temperatures. The baking temperatures were according to the temperature setting of the oven. For microwave cooking, the exact cooking temperature was not monitored. For each food, the weights before (raw materials) and after baking or cooking were measured. For cooked foods (except roasted beef), the total amounts of prepared food and the total amounts of residual fat remaining in the pan or wok were separately collected. For roasted beef, one-quarter of the total roasted meat was sampled by cutting the C


Article

Journal of Agricultural and Food Chemistry Table 4. Cooking and Baking Times and Measured Temperatures during Cooking and Baking mean temperaturea ± SD (°C)

cooking time (min) ingredient

pre-heating

with control margarine

with PS-margarine

stir-frying

cooking method


3 3 3

main cooking 3 3 3

200.4 ± 15.0 248.6 ± 35.5 228.4 ± 13.6

199.5 ± 16.9 260.0 ± 16.2 241.1 ± 16.4

shallow-frying


5 5 5 5 5 5 5 5 5

4 6 6 8 8 10 10 15 18

170.8 ± 6.4 161.4 ± 7.2 173.2 ± 5.8 184.9 ± 4.7 177.0 ± 4.8 168.9 ± 3.2 192.9 ± 5.5 195.2 ± 6.2 168.8 ± 12.0

167.8 161.5 175.3 188.9 177.1 171.2 194.3 198.5 170.9

stewingb

beef

5

shallow-frying: 4 stewing: 90

155.2 ± 21.6 85.4 ± 6.7

149.5 ± 20.0 84.3 ± 2.7

roastingb

beef

5

shallow-frying: 9 roasting: 30

174.3 ± 10.7 140

185.4 ± 8.8 140

microwave cooking baking

codfish baked food

−

5 baking time (min)

cookies muffins banana bread sponge cake

12 25 60 60

± ± ± ± ± ± ± ± ±

5.9 3.6 4.0 7.6 3.3 6.2 7.7 3.9 8.6

600 W 600 W fixed oven temperature (°C) 170 140 155 155

170 140 155 155

a

Mean temperature refers to the average temperature of three recorded temperatures taken during the main cooking (i.e., just before adding the ingredient into the pan, halfway through, and at the end of the cooking) in five repeated cooking procedures (so 15 measurements in total). b Stewing and roasting had an initial shallow-frying step; see Cooking Methods. 19-Hydroxycholesterol (purity ≥99%) from Steraloids (Wilton, NH, USA) was used as an internal standard, which was added to each food/residual fat sample. The conditioned samples were saponified overnight using a 95% KOH ethanol solution in the dark at room temperature. Saponified POP were extracted with dichloromethane. Dichloromethane was evaporated, and samples were dissolved in nhexane:isopropanol (4:1, v/v) solution. The POP were separated from the non-oxidized sterols on a 5 μm Nucleosil 50 analytical column (100 × 4 mm i.d., Macherey Nagel, Düren, Germany). Derivatization of POP was performed by adding TriSil reagent, and the samples were silylated for 45 min at 80 °C. The excess of silylation reagent was evaporated under a stream of nitrogen at 30 °C, and the residues were re-dissolved in 50 μL of n-hexane before GC-MS analysis. Analysis of POP was done once in each of the five independent samples per food ingredient. Because POP standards are not commercially available, commercial cholesterol oxidation products were used as model substances: cholestanetriol (purity ≥98%), 5,6β-epoxycholesterol (purity ≥80%), 7β-hydroxycholesterol (purity ≥95%), and 7-ketocholesterol (purity ≥90%), all obtained from Sigma-Aldrich (Saint Louis, MO, USA), and 7α-hydroxycholesterol (purity ≥99%) and 5,6α-epoxycholesterol (purity ≥99%), which were from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). POP separation and analysis was performed on a model 6890N gas chromatograph coupled to a model 5975 mass-selective detector (Agilent Technologies, Santa Clara, CA, USA) using a CP-Sil-13CB capillary column (25 m × 0.25 mm i.d. × 0.25 μm film thickness) (Agilent Technologies). Mass spectra were acquired over the mass-tocharge (m/z) range of 50−650. Integration was performed with an Agilent G1701DA GC/MSD ChemStation instrument. 2.3. Data Processing and Statistical Analysis. GC-MS data were first integrated using ChemStation software, version E.02.00. The

piece of meat lengthwise and crosswise. Of the baked products, a quarter of a baked sponge cake and banana bread was sampled by cutting the cake or bread lengthwise and crosswise, ensuring equal proportions of crust and inner baked product. Of the small baked goods, two muffins and 10 cookies were sampled. All food samples were put into 500 mL pre-labeled dark glass containers, and the residual fat was put into 20 mL glass containers, which were flushed with N2 to prevent oxidation and then stored at −20 °C before analysis. 2.1.5. Heating Temperatures and Times of the Cooking and Baking Procedures. The means and SD of the actually measured temperatures during the different cooking and baking procedures are presented in Table 4. Pre-heating for 3−5 min allowed to reach the desired pan or wok temperatures. In general, measured temperatures during cooking were similar using either the control or the PSmargarine. Only for stir-frying cabbage and chicken as well as roasting beef, the recorded temperatures were 11−13 °C higher when using the PS-margarine than the control margarine. 2.2. Lipid Extraction and Chemical Analysis of POP. POP analyses were carried out using GC-MS according to a validated method as recently described in detail.15 Briefly, before lipid extraction, the entire amount of each food sample was homogenized using an Ultra-Turrax 153 T25 homogenizer (Jankel & Kunkel GmbH, Staufen, Germany). Lipids in the homogenized samples were then extracted using different extraction methods modified and validated for the specific food matrices as follows: Folch extraction method for all meat samples, Bligh and Dyer method for fish samples, and the Hara and Radin extraction method for vegetable and potato samples. Egg samples were first freeze-dried to avoid emulsification during extraction, and then lipids were extracted by using acid hydrolysis with petroleum ether/diethyl ether. For the baked products, acid hydrolysis and Soxtec (automated Soxhlet) extraction were applied. D


E

beef (128) beef (200) codfish (116)

stewing roastingb microwave cooking

29 22 29

29 29 29 29 29 29 29 29 29

29 29 29

PS (mg) (A)

± ± ± ± ± ± ± ± ±

0.25 0.04 0.46 0.29 0.93 1.88 1.01 1.35 1.06 0.71 ± 0.36 0.52 ± 0.06 0.10 ± 0.04

0.29 ± 0.09 0.30 ± 0.04 0.21 ± 0.05 1.11 ± 0.46 0.05 ± 0.0.03 0.97 ± 0.39 0.57 ± 0.15 0.99 ± 0.14 0.74 ± 0.46 0.75 ± 0.25 0.60 ± 0.18 0.05 ± 0.01 0.57 0.05−1.11

0.23 ± 0.04 − 0.53 ± 0.15

0.55 ± 0.08 0.67 ± 0.11 0.53 ± 0.15

0.52 0.04−1.88

0.14 0.004 0.08 0.05 0.28 0.47 0.17 0.06 0.33

POP in RF (mg) (C)

POP in food (mg) (B)

control margarine

1.06 0.15−2.85

1.46 1.12 0.15

0.54 0.34 0.67 1.40 0.98 2.85 1.58 2.34 1.80

0.78 0.67 1.06

total POP (mg) (B+C)

3.66 0.51−9.82

5.05 5.09 0.51

1.86 1.17 2.30 4.82 3.37 9.82 5.45 8.05 6.19

2.69 2.32 3.66

ORP (%) ((B+C)/A × 100)

1286 964 1286

1286 1286 1286 1286 1286 1286 1286 1286 1286

1286 1286 1286

PS (mg) (D)

± ± ± ± ± ± ± ± ± 0.08 0.17 0.23 1.11 0.16 0.55 0.51 1.3 0.51

1.42 0.08−20.50

1.32 ± 0.22 1.42 ± 0.48 0.08 ± 0.01

0.55 0.92 1.11 4.51 0.66 7.34 1.15 20.50 1.88

2.27 ± 1.21 6.48 ± 1.50 11.78 ± 3.3

POP in food (mg) (E)

± ± ± ± ± ± ± ± ± 1.06 0.08 3.01 0.42 4.29 2.36 2.70 2.83 3.27

6.98 0.22−23.9

2.16 ± 0.28 2.51 ± 0.59 0.22 ± 0.10

3.08 0.32 10.87 1.04 20.44 22.74 19.41 23.90 13.86

0.41 ± 0.0.47 − 16.48 ± 2.79

POP in RF (mg) (F)

PS-margarine

6.48 0.30−44.40

3.48 3.93 0.30

3.63 1.24 11.98 5.52 21.10 30.08 20.56 44.40 15.74

2.68 6.48 28.26

total POP (mg) (E+F)

0.50 0.02−3.45

0.27 0.41 0.02

0.28 0.10 0.93 0.43 1.64 2.34 1.60 3.45 1.22

0.21 0.50 2.20

ORP (%) ((E+F)/D × 100)

Weights (in grams) of the cooked ingredients (see Table 2) are indicated in parentheses, except for roasted beef (see note b). b200 g of roasted beef was defined as portion size, corresponding to about 21.6% of the total weight (925 g, Table 2) of the roasted beef after cooking. Accordingly, the corresponding amount of RF was calculated by multiplying the total amount of RF by 21.6% (59.1 g × 21.6% = 12.8 g), from which the amount of POP was calculated. cAmount of PS measured from the used margarine (20 g), except for roasted beef, in which the amount (15 g) of margarine used was calculated according to 70 g × 21.6% (see note b).

a

median range

egg (88) onions (60) codfish (130) fish fingers (142) pork fillet (124) steak (beef) (110) salmon (121) potatoes (207) minced meat (124)

shallow-frying

ingredient

green beans (134) cabbage (119) chicken (120)

cooking method

stir-frying

a

c

Table 5. POP Contents per Portion Size (Mean ± SD) of Cooked Foods and per Actual Amount of Residual Fat (RF), and Oxidation Rate of PS (ORP)

Journal of Agricultural and Food Chemistry Article


Article

Journal of Agricultural and Food Chemistry Table 6. POP Contents (Mean ± SD) per Portion Size of Baked Products, and Oxidation Rate of PS (ORP) control margarine baked fooda

PSb (mg) (A)

cookies (18) muffins (44) banana bread (80) sponge cake (50)

7.2 5.4 12.7 19.4

median range

POP in food (mg) (B) 0.12 0.11 0.12 0.21

± ± ± ±

PS-margarine ORP (%) (B/A × 100)

0.02 0.01 0.03 0.02

0.12 0.11−0.21

1.67 2.04 0.94 1.08

PSb (mg) (C) 321.5 237.9 565.8 861.5

1.38 0.94−2.04

POP in food (mg) (D) 0.20 0.19 0.27 0.60

± ± ± ±

0.06 0.07 0.13 0.06

0.24 0.19−0.60

ORP (%) (D/C × 100) 0.06 0.08 0.05 0.07 0.06 0.05−0.08

a

Weights (in grams) per portion size (for details, see Table 3) of the baked food are indicated in parentheses. bAmount of PS calculated from the amount of margarine used for a portion of the baked food (Table 3).

output file was then exported into a spreadsheet (Microsoft Office Excel 2013). Total POP was defined as the sum of individual POP including 7-OH (7α/β-hydroxy-), 7-keto-, 5,6α/β-epoxy-, and triol (3β,5α,6β-triol-) derivatives of sitosterol, campesterol, and stigmasterol. Total 7-OH (7α/β-hydroxy), total 7-keto, total 5,6-epoxy, and total triols were defined as the sum of the corresponding derivatives of sitosterol, campesterol, and stigmasterol. To avoid underestimation of the amount of total POP, the representative values for the limit of detection (LOD) and limit of quantification (LOQ) for each POP were taken into account. When the signal of an individual POP was below the LOD, the amount of the individual POP was defined to be “not detectable”. When the signal was above the LOD but below the LOQ, the amount of the individual POP was estimated to be equal to the LOD. For 7-OH-POPs, LOD = 0.0007 mg/100 g and LOQ = 0.0024 mg/100 g. For 5,6-epoxy POPs, LOD = 0.0014 mg/100 g and LOQ = 0.0048 mg/100 g. For 7-keto and triols POPs, LOD = 0.002 mg/100 g and LOQ = 0.0068 mg/100 g.15 Results are reported as mean ± standard deviation (SD) of five independent experiments (n = 5), or as otherwise indicated. Medians and min−max ranges are used to summarize overall data of individual cooking and baking results due to the skewed distribution of the data.

residual fat was 0.52 mg (range 0.04−1.88 mg) per actual amount of residual fat (Table 5). Using the PS-margarine for cooking, the median POP content of the residual fat was 6.98 mg (range 0.22−23.9 mg) per actual amount of residual fat (Table 5). Sum of POP Contents of Foods plus Residual Fat. The total amounts of POP formed during the different cooking methods are reflected by the sum of POP measured in the food plus in the residual fat (Table 5). Using the control or PS margarine, median total POP contents were 1.06 mg (range 0.15−2.85 mg) and 6.48 mg (range 0.30−44.4 mg), respectively. Shallow-fried potatoes had the highest total POP formation compared to other processed foods using both of the margarines. Distribution of POP between Foods and Residual Fat. The distribution of POP found in the food itself and in the residual fat ranged widely among the foods. POP in foods per se accounted for 5−100% (median 50%) and 3−100% (median 37%) of the total POP when prepared with the control or with the PS-margarine, respectively. Most of the cooked animal foods and fish, e.g., pork fillet, steak, minced meat, salmon, and codfish, contained only about 3−41% of the total amount of POP, while in other foods, e.g., green beans, cabbage, and onions, but also fish fingers that absorb more fat 71−100% of the POP, the POP was mostly in the food itself. Oxidation Rate of PS (ORP). On average, the control and PS-margarines contained 29 mg and 1286 mg PS per 20 g of margarine, respectively (Table 5). ORP represents the percentage of PS in the margarine that was oxidized based on the amount of total POP measured in the food plus the residual fat. In foods prepared with the control margarine, ORP values ranged from 0.51% (in microwave-cooked codfish) to 9.82% (in shallow-fried beefsteak) with a median of 3.66% (Table 5). For foods prepared with the PS-margarine, the ORP values ranged from 0.02% (in microwave-cooked codfish) to 3.45% (in shallow-fried potatoes), with a median of 0.50%. 3.3. POP Contents per Portion Size of Baked Foods. POP Contents of Baked Foods. In baked products prepared with the control margarine and PS-margarine, median total POP contents were 0.12 mg/portion (range 0.11−0.21 mg/ portion) and 0.24 mg/portion (range 0.19−0.60 mg/portion), respectively (Table 6). Oxidation Rate of PS (ORP). On average, the control and PS-margarine delivered 11 mg and 497 mg PS, respectively, per portion size of baked foods (Table 6). In the baked foods made with the control margarine, 1.38% (median) of the PS were oxidized, while the ORP was 0.06% in the baked foods made with the PS-margarine (Table 6).

3. RESULTS 3.1. Weights of Prepared Foods before and after Cooking and Baking. The weights of different foods before and after cooking and the total amount of residual fat left in the pan or wok are summarized in Table 2. After shallow-frying of cabbage, all the fat was absorbed by the food, leaving no residual fat in the pan for that sample only. Weights of the baked products before and after baking are presented in Table 3. 3.2. POP Contents per Portion Size of Cooked Foods. POP Contents of Cooked Foods. POP contents per portion size of foods prepared by the different cooking methods using the control or PS-margarine are summarized in Table 5. POP contents of foods prepared with the control margarine were low, ranging from 0.05 mg (in shallow-fried pork fillet and microwave-cooked codfish) to 1.11 mg (in shallow-fried fish fingers), with a median of 0.57 mg/portion size. Foods prepared with the PS-margarine contained POP in a range of 0.08 mg (in microwave-cooked codfish) to 20.5 mg (in shallowfried potatoes), with a median of 1.42 mg/portion size. The amounts of POP in stir-fried cabbage and chicken and shallowfried steak and potatoes were all higher than in the other foods. Shallow-fried onions, egg, and pork fillet as well as microwavecooked codfish contained less than 1 mg/portion of POP. POP Contents of Residual Fat. Various amounts of residual fat remained in the pan or wok after cooking (Table 2). When the control margarine was used, median POP content of F


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Journal of Agricultural and Food Chemistry Table 7. POP Contents per 100 g of Prepared Foods and Residual Fat (RF) POP content (mg) control margarine cooking methods

in food

in RF

in food

in RF

stir-frying


0.41 ± 0.06 0.55 ± 0.09 0.45 ± 0.13

8.52 ± 1.58 − 10.80 ± 3.60

1.68 ± 0.89 5.58 ± 1.29 9.66 ± 2.71

11.74 ± 1.34 − 272.35 ± 46.17

shallow-frying


0.33 0.50 0.16 0.77 0.04 0.87 0.47 0.48 0.59

stewing roasting microwave cooking

beef beef codfish

0.59 ± 0.20 0.30 ± 0.09 0.04 ± 0.01

4.08 ± 2.04 4.10 ± 0.50 0.52 ± 0.20

1.03 ± 0.17 0.71 ± 0.24 0.07 ± 0.01

10.77 ± 1.38 19.58 ± 4.63 0.96 ± 0.45

median range

0.47 0.04−0.87

7.12 0.52−26.70

1.52 0.07−9.83

76.86 0.96−415.58

cookies muffins/cupcakes banana bread sponge cake

0.66 0.24 0.15 0.42

median range

0.33 0.15−0.66

baking

food

PS-margarine

± ± ± ± ± ± ± ± ±

± ± ± ±

0.10 0.06 0.04 0.32 0.02 0.35 0.12 0.07 0.37

4.14 4.71 7.03 7.20 10.40 10.40 6.18 26.70 8.83

0.11 0.02 0.03 0.03

± ± ± ± ± ± ± ± ±

2.39 0.45 1.15 1.18 3.20 2.60 1.05 1.10 2.79

0.61 1.54 0.87 3.21 0.50 6.75 0.95 9.83 1.52

1.13 0.43 0.34 1.20

± ± ± ± ± ± ± ± ±

± ± ± ±

0.09 0.28 0.18 0.79 0.10 0.51 0.42 0.62 0.41

46.23 35.85 140.36 20.76 197.34 116.40 117.78 415.58 107.50

± ± ± ± ± ± ± ± ±

12.93 9.41 38.87 8.67 41.42 12.10 16.39 49.18 25.41

0.31 0.16 0.16 0.12

0.78 0.34−1.20

3.4. POP Contents per 100 g of Baked and Cooked Foods and Residual Fat. POP contents of foods are often presented per 100 g food. To allow comparison with reported data, total POP contents are also presented per 100 g (Table 7). Using the control margarine, the median POP content of cooked foods was 0.47 mg/100 g food), while using the PSmargarine, the median POP content was 1.52 mg/100 g food. The median POP amounts in the residual fat were 7.12 mg/100 g fat and 76.86 mg/100 g fat with use of the control margarine and PS-margarine, respectively, for cooking. The highest amount of POP was found in residual fat (415.58 mg/100 g fat) when the PS-margarine was used for shallow-frying potatoes. The median amounts of POP per 100 g residual fat were much high than the amounts of POP in the control margarine (0.62 mg/100 g fat) and in the PS-margarine (1.62 mg/100 g fat) before cooking (see Table 1). The amounts of POP per 100 g of baked products were ≤1.20 mg/100 g, with a median of 0.33 mg/100 g (range 0.15− 0.66 mg/100 g) with the control margarine and a median of 0.78 mg/100 g (range 0.34−1.20 mg/100 g) with the PSmargarine (Table 7). 3.5. Compositions of Individual POP in Cooked and Baked Foods and in Residual Fat. The individual POP contents of the different foods and the residual fats are summarized in (Supplemental Table) The compositions of individual POP, expressed as percent of total POP, varied widely among the foods and their corresponding residual fat. However, when control margarine was used, the distribution of individual POP in the foods and in the residual fats was on

average consistently in the order of 7-keto-PS (47.2−45.1%, medians in foods and in residual fat), 5,6-epoxy-PS (25.0− 29.9%), 7-OH-PS (22.1−21.3%), and PS-triols (5.6−3.9%), with 7-keto-PS being the dominant individual POP. When PSmargarine was used for cooking, 5,6-epoxy-PS and 7-keto-PS each accounted for 35.8−37.8% (median) of the total POP, with 7-OH-PS and PS-triols accounting for 23.9% and 1.9%, respectively, in foods. In the residual fat, 7-OH-PS, 5,6-epoxyPS, 7-keto-PS, and PS-triols accounted for about 38.4%, 30.7%, 28.1%, and 1.2% of the total POP, respectively, with 7-OH-PS being the dominant individual POP. For baked foods prepared with the control and PSmargarines, the distribution of individual POP was in the order of 7-keto-PS > 5,6-epoxy-PS > 7-OH-PS > PS-triols.

4. DISCUSSION To the best of our knowledge, this study is the first that investigated POP formation in a wide range of foods, which were prepared by typical household cooking methods next to baking under well-monitored conditions. In addition, this study investigated POP formation in foods by comparing the use of a margarine without and with added PS side-by-side. The results of this study therefore describe realistic POP contents of foods as they could be prepared and consumed in consumer households. However, taking into account that cooking and baking habits of consumers can vary considerably, it is unavoidable that the variation of POP formation in foods made in households might be larger than the POP contents described in our study. G


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or without added PSE and showed ORP in the fat with added PSE being about 6 times lower than in the fat without added PSE (0.07% versus 0.41%).21 The underlying reason for the lower ORP in foods with added PS is unclear. One possible explanation related to thermal oxidation of PS requiring the presence of reactive oxygen species.22 An increase in the PS content of foods lowers the ratio of oxygen species to PS and thus may reduce the likelihood of PS to be in contact with reactive oxygen. Alternatively, PS in the PS-margarine were in the form of PSE. It has been suggested that PSE are less prone to oxidation than free PS when heated under the same conditions.23 Further studies are required to understand the underlying mechanisms. Since POP are hydrophobic compounds they preferably reside in the fat phase. For all cooked foods, except for the stirfried cabbage where all fat was absorbed, a variable amount of residual fat ranging from 0.9 to 59.1 g remained in the pan or wok after cooking. This residual fat represents a mix of margarine used for cooking, some fat released from the food itself, especially from meats and fish, and some small amounts of water. When the total amount of residual fat was greater than 5 g, more than 50% of the total amounts of POP were found in the residual fat. Consequently, and despite having a higher ORP, the POP contents of the fried meats and fish were similar to those of fried vegetables due to the larger amounts of POP residing in the residual fat. The estimation of daily POP intake as described above from the cooked foods is based on the assumption that the residual fat is not consumed at all. However, a part or even the total amount of residual fat could still be used for gravy making and so consumed. If two portions of cooked foods together with the total amount of residual fats would indeed be consumed, the estimated daily POP intake would be about 13 mg, which is still about 680 times lower than the NOEL obtained in rats. Soupas et al.23 reported a study in which rapeseed oil, liquid margarine, and butter with 8% added PS (in the form of PSE) were pan-fried at 160−200 °C for 5−10 min to measure POP formation. The study conditions in view of temperature and time applied in their study and in our study are comparable except that no foods were added to the pan in the Soupas et al. experiments. They report median POP contents of the fatbased products after pan-frying of 38 mg/100 g fat. In our study, the median POP contents of the residual fats after cooking with the PS-margarine was 76.86 mg/100 g fat (Table 7). These data may thus indicate that pan-frying foods with the PS-margarine may lead to POP contents higher than those of pan-frying fats alone. An explanation for this finding is that the foods were constantly stirred during stir- or shallow-frying, which may increase exposure of PS to air oxygen and thus may enhance PS oxidation. Among the applied cooking methods, microwave cooking was associated with the lowest ORP (0.02% with use of the PSmargarine) and the lowest POP content of the prepared food. This finding is supported by a recent study,24 in which free PS alone or in a mixture with triolein were heated in a microwave oven at 1000 W for up to 6 min. Formation of POP was below the detectable level. The low ORP with microwave cooking can be explained by the fact that microwave radiation penetrates into the food and the heat is transferred from the inside to the surface of the food. Thus, PS located at the surface of the food may have less heat exposure during microwave cooking compared to other cooking procedures. As reported by others, during microwaving, the temperature in the food itself was

In this study, control and PS-enriched margarines were used for baking and cooking. The PS content of the control margarine was low (0.14 g per 100 g) and within the typical range found in commercial vegetable oils and margarines (0.07−0.73 g/100 g).2 POP contents of all the cooked foods prepared with the control margarine were smaller than 1.1 mg per portion size, with a median POP content of 0.57 mg per portion (median 0.47 mg/100 g). Using the control margarine for baking, POP contents of the baked goods were even lower, with a median POP content of 0.12 mg per portion (median 0.33 mg/100 g). Although the PS-margarine contained a higher amount of PS (6.4 g per 100 g), the POP contents of foods prepared with the PS-margarine were still in a low range. The median POP content was 1.42 mg/portion size in cooked foods (median 1.52 mg/100 g) and 0.24 mg per portion size in baked products (median 0.78 mg/100 g). These data indicate that using the PS-margarine for cooking and baking resulted in a modest increase in the absolute amount of POP in foods as compared to using the control margarine. In relative terms, using the PS-margarine as compared to the control margarine resulted in an about 2-fold increase in POP contents in the cooked and baked foods. Based on the median POP content of cooked foods, consuming per day two portions made with the control margarine would result in a daily POP intake of 1.14 mg. If one would consume each day two portions of baked products, e.g., two pieces of cake prepared with the control margarine, the daily POP intake would be by 0.24 mg even lower. Consuming daily two portions of cooked foods prepared with the PSmargarine, would result in a daily intake of POP of 2.84 mg. The daily intake of POP from two portions of baked products, e.g., a piece of cake and a cookie made with the PS-margarine, would be just 0.48 mg. An estimated daily POP intake of 2.84 mg from cooked foods prepared with PS-margarine is of the same magnitude as the reported average 3 mg/d intake of cholesterol oxidation products from a typical Dutch diet.18 These data indicate that humans have a typical exposure to about 3 mg/d of cholesterol oxidation products with their habitual diet, and thus an exposure to the same amount of POP seems unlikely to raise concerns. In addition, the estimated daily POP intake from cooked and baked foods prepared with the PS-margarine is equivalent to ∼0.04 mg/kg body weight/ day for an adult of 70 kg body weight. A 90-day feeding study in rats established the “no observed effect level” (NOEL) as being 128 mg/kg BW/d for males and 144 mg/kg BW/d for females.19 Therefore, the estimated daily POP intake of 2.8 mg/d is about 3200 times lower than the NOEL obtained in rats. The low amounts of POP formed in the prepared foods using the PS-margarine are a result of the low oxidation of PS as indicated by the ORP and of larger amounts of formed POP remaining in the residual fat after cooking. With use of the PSmargarine, the ORP values after cooking and baking were about 2−28 times lower than when using the control margarine, with a median ORP of 0.5% versus 3.66% in cooked foods and of 0.06% versus 1.38% in baked products. Other investigators have also observed that an increase in the PS contents of margarine was associated with a decrease in ORP. For example, the ORP calculated for dark chocolate bars with 7% added PS (in the form of PSE) was 0.1% compared to an ORP of 3.25% in dark chocolate bars containing only 0.1% of natural PS, all after 5 months storage at temperatures of 20−30 °C.20 Another study investigated the POP contents of vegetable oil-based fats with H


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Journal of Agricultural and Food Chemistry 200 °C for up to 10 min). The ORP values of fish products (including frozen fish fillets, fish fingers, fish burgers, salmon in pastry with spinach) after pan-frying were in the range of 0.57−0.74%. The ORP values of the pan-fried minced pork and minced beef were 0.04% and 0.36%, respectively. These data are comparable to our data, for which the ORP values of the different fish products were in the range of 0.43− 1.60%, and those of the pan-fried meats (minced meat, pork, and beef) were in the range of 0.27−1.64%, using the PSmargarine for cooking (Table 5). Baking is another cooking method that resulted in low ORP and low POP contents in the baked products. The low ORP in baked products is probably due to the lower exposure of PS to oxygen since the batter has a higher volume-to-surface ratio and PS are within the core of the batter. In contrast, during stir- or shallow-frying, fat was present in a thin layer in the pan or wok. Furthermore, constant stirring during frying also increases the exposure of PS to air and thus oxygen. Our study further revealed that a long heating time of up to 60 min during baking did not have a strong effect on POP formation. Previous reported data as reviewed by Lin et al.14 show that margarine with added PSE heated in the absence of a batter in an oven at 140−200 °C for 60−120 min, resulted in a 50 times higher ORP (on average 3.7%) as compared to the ORP of 0.1% calculated in our study in the 60 min baked products with PSmargarine. Whether the PS are surrounded by the batter during the baking process or not probably explains these conflicting data. Clearly, reported data using merely fats without the presence of other ingredients to make a batter seem to overestimate POP formation under oven heating conditions. A noticeable finding was that POP formation, as indicated by ORP, during shallow-frying potatoes using either the control or the PS-margarine was high, as compared to the other fried foods, especially the vegetable foods. Derewiaka and Obiedzijski26 reported POP formation in commercial samples of frozen French fries, of which POP were not detectable before heating. When these frozen French fries were then oven-heated without the addition of vegetable oil at 225 °C for 15 min, POP contents increased up to 3.5 mg/100 g French fries, and the ORP value (1.69%) was higher than those of any other cooked foods (e.g., meats and fish products, ORP < 0.74%). Reasons leading to this high ORP in shallow-fried potatoes or ovenbaked French fries are unclear; it can neither be explained by the difference in heating temperatures and times nor by an increase of exposure to air oxygen. It warrants further study to explore whether especially potatoes contain any pro-oxidative compounds that stimulate thermal oxidation. Despite the high ORP, and since 66% of the formed POP resided in the residual fat, the mean POP content in shallow-fried potatoes using the control margarine was 0.5 mg/100 g. This is in the range of results from reported studies in which French fries were either oven-heated at 225 °C for 15 min without vegetable oil addition (POP content 0.03−0.25 mg/100 g French fries)26 or deep-fried in vegetable oils with unknown PS contents at 200 °C for 15 min (POP content 2.4−4.0 mg/100 g).27 Measurements by others of various commercial potato crisp products revealed that POP contents ranged from 0.05 mg to 0.26 mg per 100 g.28 Taken these data together, the high ORP during shallow-frying potatoes observed in this study is also

unlikely due to experimental error. The high ORP in shallowfried potatoes prepared with the PS-margarine led to the highest amount of POP in the potatoes themselves (9.83 mg/ 100 g and 20.50 mg/portion) and in the residual fat (415.58 mg/100 g or 23.90 mg per total amount of residual fat) among all the different foods prepared. In a worst-case scenario with a daily consumption of two portions of shallow-fried potatoes made with the PS-margarine together with the entire amount of residual fat, the daily POP intake could reach up to 88.8 mg. This is still about 100 times lower than the NOEL obtained in rats. Although a wide variation was found in the composition of individual POP measured in the different cooked and baked foods, on average (median) 7-keto-PS accounted for ≥37.8% of total POP. These data are comparable to findings by others showing that 7-ketocholesterol is the major thermal oxidation product of cholesterol, accounting for 30−70% of total oxidized cholesterol in most model system studies as reviewed by Rodriguez-Estrada et al.29 However, in a study23 in which PScontaining fat-based products were pan-fried at 180 °C for 10 min, 7-OH-PS were the major POP formed accounting for about 50% of the total POP. In our study, the presence of 7OH-PS (38.4%) was higher than that of the other individual POP in the residual fat samples when the PS-margarine was used. In another study using margarine enriched with PSE heated at 180 °C for 90 min in an oven, 5,6-epoxy-PS contributed 51% to the total POP formed.17 Clearly, there is inconsistency in the reported composition of individual POP formed during thermal treatments including cooking and baking, which might be due to the different experimental conditions applied in the studies. In conclusion, using the PS-margarine for typical household cooking and baking applications increases POP contents of foods as compared to using the control margarine. However, when PS-margarine is used, the amounts of POP in prepared foods remained in a low range, with a median POP content of 1.42 mg and a range of 0.08−20.50 mg per typical portion size of consumed foods.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04952. Supplemental table presenting the contents and percentages of individual POPs of the 19 cooked and baked foods and of the residual fat (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +31-10-460-6405. Fax: +31-10-460-5993. E-mail: [email protected]. Author Contributions §

Y.L. and D.K. contributed equally.

Notes

The authors declare the following competing financial interest(s): All authors are employed by Unilever R&D Vlaardingen. Unilever markets food products with added plant sterols.

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ACKNOWLEDGMENTS We gratefully acknowledge Edward de Jongh for performing all cooking and baking experiments, Sander Verduyn for making I


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and phytosterol oxidation products in cooked and baked food products. J. Chromatogr. A 2015, DOI: 10.1016/j.chroma.2015.09.073. (16) Ryan, E.; McCarthy, F. O.; Maguire, A. R.; O’Brien, N. M. Phytosterol Oxidation Products: Their Formation, Occurrence, and Biological Effects. Food Rev. Int. 2009, 25 (2), 157−174. (17) Scholz, B.; Wocheslander, S.; Lander, V.; Engel, K. H. On-line liquid chromatography-gas chromatography: A novel approach for the analysis of phytosterol oxidation products in enriched foods. J. Chromatogr. A 2015, 1396, 98−108. (18) Van de Bovenkamp, P.; Kosmeijer-Schuil, T. G.; Katan, M. B. Quantification of oxysterols in Dutch foods: egg products and mixed diets. Lipids 1988, 23 (11), 1079−1085. (19) Lea, L. J.; Hepburn, P. A.; Wolfreys, A. M.; Baldrick, P. Safety evaluation of phytosterol esters. Part 8. Lack of genotoxicity and subchronic toxicity with phytosterol oxides. Food Chem. Toxicol. 2004, 42 (5), 771−783. (20) Botelho, P. B.; Galasso, M.; Dias, V.; Mandrioli, M.; Lobato, L.; Rodriguez-Estrada, M.; Castro, I. Oxidative stability of functional phytosterol-enriched dark chocolate. LWT–Food Sci. Technol. 2014, 55 (2), 444−451. (21) Conchillo, A.; Cercaci, L.; Ansorena, D.; Rodriguez-Estrada, M. T.; Lercker, G.; Astiasaran, I. Levels of phytosterol oxides in enriched and nonenriched spreads: application of a thin-layer chromatographygas chromatography methodology. J. Agric. Food Chem. 2005, 53 (20), 7844−7850. (22) Ansorena, D.; Barriuso, B.; Cardenia, V.; Astiasaran, I.; Lercker, G.; Rodriguez-Estrada, M. T. Thermo-oxidation of cholesterol: effect of the unsaturation degree of the lipid matrix. Food Chem. 2013, 141 (3), 2757−2764. (23) Soupas, L.; Huikko, L.; Lampi, A. M.; Piironen, V. Pan-frying may induce phytosterol oxidation. Food Chem. 2007, 101, 286−297. (24) Leal-Castaneda, E. J.; Inchingolo, R.; Cardenia, V.; HernandezBecerra, J. A.; Romani, S.; Rodriguez-Estrada, M. T.; Galindo, H. S. Effect of Microwave Heating on Phytosterol Oxidation. J. Agric. Food Chem. 2015, 63 (22), 5539−5547. (25) Medina-Meza, I. G.; Barnaba, C. Kinetics of Cholesterol Oxidation in Model Systems and Foods: Current Status. Food Eng. Rev. 2013, 5, 171−184. (26) Derewiaka, D.; Obiedzinski, M. Phytosterol oxides content in selected thermally processed products. Eur. Food Res. Technol. 2012, 234 (4), 703−712. (27) Dutta, P. C. P. Studies on phytosterol oxides. II: Content in some vegetable oils and in French fries prepared in these oils. J. Am. Oil Chem. Soc. 1997, 74 (6), 659−666. (28) Tabee, E.; Jaegerstad, M.; Dutta, P. C. Lipids and phytosterol oxidation products in commercial potato crisps commonly consumed in Sweden. Eur. Food Res. Technol. 2008, 227 (3), 745−755. (29) Rodriguez-Estrada, M. T.; Garcia-Llatas, G.; Lagarda, M. J. 7Ketocholesterol as marker of cholesterol oxidation in model and food systems: when and how. Biochem. Biophys. Res. Commun. 2014, 446 (3), 792−797.

the margarines, Mireille Blommaert for her input in defining the cooking and baking experiments, and Dr. Rouyanne Ras for her critical comments and helpful suggestions during manuscript preparation.

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ABBREVIATIONS USED EFSA, European Food Safety Authority; NOEL, no observed effect level; ORP, oxidation rate of plant sterols; POP, plant sterol oxidation products; PS, plant sterols; PSE, plant sterol esters

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REFERENCES

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Formation of Plant Sterol Oxidation Products in Foods during Baking

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