Safety of the extension of use of plant sterol esters as a novel food pursuant to Regulation (EU) 2015/2283
2020; Wiley; Volume: 18; Issue: 6 Linguagem: Inglês
10.2903/j.efsa.2020.6135
ISSN1831-4732
AutoresDominique Turck, Jacqueline Castenmiller, Stefaan De Henauw, Karen Ildico Hirsch‐Ernst, John Kearney, Alexandre Maciuk, Inge Mangelsdorf, Harry J McArdle, Androniki Naska, Carmen Peláez, Kristina Pentieva, Alfonso Siani, Frank Thiès, Sophia Tsabouri, Marco Vinceti, Francesco Cubadda, Thomas Frenzel, Marina Heinonen, Rosangela Marchelli, Monika Neuhäuser‐Berthold, Morten Poulsen, Josef Rudolf Schlatter, Henk Van Loveren, Wolfgang Gelbmann, Helle Katrine Knutsen,
Tópico(s)Vitamin K Research Studies
ResumoEFSA JournalVolume 18, Issue 6 e06135 Scientific OpinionOpen Access Safety of the extension of use of plant sterol esters as a novel food pursuant to Regulation (EU) 2015/2283 EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), Corresponding Author EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) nda@efsa.europa.eu Correspondence: nda@efsa.europa.euSearch for more papers by this authorDominique Turck, Dominique TurckSearch for more papers by this authorJacqueline Castenmiller, Jacqueline CastenmillerSearch for more papers by this authorStefaan De Henauw, Stefaan De HenauwSearch for more papers by this authorKaren Ildico Hirsch-Ernst, Karen Ildico Hirsch-ErnstSearch for more papers by this authorJohn Kearney, John KearneySearch for more papers by this authorAlexandre Maciuk, Alexandre MaciukSearch for more papers by this authorInge Mangelsdorf, Inge MangelsdorfSearch for more papers by this authorHarry J McArdle, Harry J McArdleSearch for more papers by this authorAndroniki Naska, Androniki NaskaSearch for more papers by this authorCarmen Pelaez, Carmen PelaezSearch for more papers by this authorKristina Pentieva, Kristina PentievaSearch for more papers by this authorAlfonso Siani, Alfonso SianiSearch for more papers by this authorFrank Thies, Frank ThiesSearch for more papers by this authorSophia Tsabouri, Sophia TsabouriSearch for more papers by this authorMarco Vinceti, Marco VincetiSearch for more papers by this authorFrancesco Cubadda, Francesco CubaddaSearch for more papers by this authorThomas Frenzel, Thomas FrenzelSearch for more papers by this authorMarina Heinonen, Marina HeinonenSearch for more papers by this authorRosangela Marchelli, Rosangela MarchelliSearch for more papers by this authorMonika Neuhäuser-Berthold, Monika Neuhäuser-BertholdSearch for more papers by this authorMorten Poulsen, Morten PoulsenSearch for more papers by this authorJosef Rudolf Schlatter, Josef Rudolf SchlatterSearch for more papers by this authorHenk van Loveren, Henk van LoverenSearch for more papers by this authorWolfgang Gelbmann, Wolfgang GelbmannSearch for more papers by this authorHelle Katrine Knutsen, Helle Katrine KnutsenSearch for more papers by this author EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), Corresponding Author EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) nda@efsa.europa.eu Correspondence: nda@efsa.europa.euSearch for more papers by this authorDominique Turck, Dominique TurckSearch for more papers by this authorJacqueline Castenmiller, Jacqueline CastenmillerSearch for more papers by this authorStefaan De Henauw, Stefaan De HenauwSearch for more papers by this authorKaren Ildico Hirsch-Ernst, Karen Ildico Hirsch-ErnstSearch for more papers by this authorJohn Kearney, John KearneySearch for more papers by this authorAlexandre Maciuk, Alexandre MaciukSearch for more papers by this authorInge Mangelsdorf, Inge MangelsdorfSearch for more papers by this authorHarry J McArdle, Harry J McArdleSearch for more papers by this authorAndroniki Naska, Androniki NaskaSearch for more papers by this authorCarmen Pelaez, Carmen PelaezSearch for more papers by this authorKristina Pentieva, Kristina PentievaSearch for more papers by this authorAlfonso Siani, Alfonso SianiSearch for more papers by this authorFrank Thies, Frank ThiesSearch for more papers by this authorSophia Tsabouri, Sophia TsabouriSearch for more papers by this authorMarco Vinceti, Marco VincetiSearch for more papers by this authorFrancesco Cubadda, Francesco CubaddaSearch for more papers by this authorThomas Frenzel, Thomas FrenzelSearch for more papers by this authorMarina Heinonen, Marina HeinonenSearch for more papers by this authorRosangela Marchelli, Rosangela MarchelliSearch for more papers by this authorMonika Neuhäuser-Berthold, Monika Neuhäuser-BertholdSearch for more papers by this authorMorten Poulsen, Morten PoulsenSearch for more papers by this authorJosef Rudolf Schlatter, Josef Rudolf SchlatterSearch for more papers by this authorHenk van Loveren, Henk van LoverenSearch for more papers by this authorWolfgang Gelbmann, Wolfgang GelbmannSearch for more papers by this authorHelle Katrine Knutsen, Helle Katrine KnutsenSearch for more papers by this author First published: 30 June 2020 https://doi.org/10.2903/j.efsa.2020.6135 Requestor: European Commission following an application by (originally) Unilever Research and Development Vlaardingen B.V (now Upfield Research and Development B.V.) Question number: EFSA-Q-2014-00865 Panel members: Dominique Turck, Jacqueline Castenmiller, Stefaan De Henauw, Karen Ildico Hirsch-Ernst, John Kearney, Helle Katrine Knutsen, Alexandre Maciuk, Inge Mangelsdorf, Harry J McArdle, Androniki Naska, Carmen Pelaez, Kristina Pentieva, Alfonso Siani, Frank Thies, Sophia Tsabouri and Marco Vinceti. Adopted: 5 May 2020 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of an extension of use of the novel food 'plant sterol esters' when added to vegetable fat spreads and to liquid vegetable fat-based emulsions for cooking and baking purposes pursuant to Regulation (EU) 2015/2283. Member States expressed concerns in relation to plant sterol oxidation products (POP) and consumption by non-target population groups. The median (0.5%) and P90 (2.28%) value of the oxidation rates of plant sterols determined by a wide range of cooking experiments were used together with exposure estimates for plant sterol when added and cooked with vegetable fat spreads and liquids. The no-observed adverse effect level (NOAEL) of a subchronic rat study and an applied default uncertainty factor of 200 served to derive levels (i.e. 0.64 mg POP/kg body weight (bw) per day) considered safe for humans. This safe level of exposure would be exceeded at the P95 by all age groups when considering the P90 oxidation rate and using EFSA's comprehensive food consumption database for assessing the potential exposure. When considering the median oxidation rate, the safe level of 0.64 mg POP/kg bw per day would be exceeded at the highest P95 intake estimates in children below 9 years of age. When considering an intake of the maximum authorised use level of 3 g plant sterols/person per day and oxidation rates of 0.5% and 2.28%, the resulting daily POP intakes per kg bw by an adult weighing 70 kg would be 0.21 and 0.98 mg/kg bw per day, respectively, the latter value exceeding 0.64 mg/kg bw per day. The Panel concludes that the safety of the intended extension of use of plant sterol esters under the proposed conditions of use has not been established. 1 Introduction 1.1 Background and Terms of Reference as provided by the requestor On 24 June 2013, the company Unilever Research and Development Vlaardingen B.V (now Upfield Research and Development B.V.) submitted a request in accordance with Article 4 of the Novel Food Regulation (EC) No1 to extend the use of phytosterols (=plant sterols, PS) esters as a novel food ingredient to be used in spreads and liquid margarines for cooking and baking purposes. On 2 April 2014, the competent authority of the United Kingdom forwarded to the Commission its initial assessment report, which came to the conclusion that the requested extension of use of plant sterol esters (PSE) meets the criteria for acceptance of a novel food defined in Article (3)1 of Regulation (EC) No 258/97. On 3 April 2014, the Commission forwarded the initial assessment report to the other Member States. Several Member States submitted comments or raised objections. The concerns of a scientific nature raised by the Member States can be summarised as follows: Heating plant sterol esters (PSE) during the production of food increases the formation of plant sterol oxidation products (POP). The formation of POP in the various food production processes is not adequately explained and discussed in the application documents. Information regarding the analytical methods applied in the stability studies and their suitability for analyses of specific POP and/or specific chemical substance classes should be provided. For spreads with added PSE for cooking and baking (CB), the applicant investigated POP oxidation using various different conditions to mimic the variety of cooking and frying practices at home. The information on the variation of the results obtained for each test condition is not presented in the dossier (this also holds for the vegetable oils used as controls), which makes it difficult to interpret these data. The oxidation stability studies of the applicant were reported in 2002. The applicant should review also recent relevant literature on this topic, with particular attention to the results published by Soupas et al. (2007). This report deals with a systematic investigation on the effect of parameters like temperature, duration of heating/frying, type of lipid matrix and water content on the formation of POP. The data presented by Soupas et al. (2007) seem to indicate a more extensive oxidation while using less severe heat-stress conditions compared to the applicant's tests. The requested extension of use can be expected to cause an exposure of the non-targeted population (children amongst others) to the product. While the applicant recommends that the product is not to be used for deep frying, fats offered for cooking and baking purposes cannot be excluded from also being used for roasting and frying. The report on post launch marketing (PLM) data by Europanel in 2013 shows that households consisting of two members have a much higher daily average of PS use compared with the average of all households. A more detailed analysis of the daily consumption by loyal consumers in the two member-households e.g. the 95th percentile in The Netherlands or in the United Kingdom is desirable. The PLM study mentioned did not cover the other product categories that have also been authorized, although more recently, i.e. sauces, cheese type products, soya drinks and rye bread. Apparently, such products seem not (yet) widely available, but it is recommendable to closely monitor consumption of phytosterol-enriched foods on a regular basis, because convincing scientific evidence that chronic exposure gives no cause for concern is still absent. Data are lacking on the intestinal absorption of POP from the consumption of heated foods with added PSE. An assessment of the toxicological tests involving the phytosterol oxide concentrate (POC) is only possible after the full study reports have been submitted. When a chronic toxicity study is not available, an additional uncertainty factor is normally included in the risk assessment, and therefore the safety margin for the expected absorption would decrease accordingly. There are studies which showed undesirable effects of POP on endothelial functions in experimental systems. However, there is a lack of studies that determine the content of POP in human plasma after the consumption of heated foods added with PSE and their effects on vascular parameters. The data available to assess the atherogenic potential of POP is insufficient and inconsistent and such effects of PS and POP cannot be ruled out (Kelly et al., 2011; Weingärtner et al., 2008, 2009, 2011). Experimental studies with animals:Yang et al. (2013) showed an undesirable influence of POP on the endothelial function of rats. Tomoyori et al. (2004), on the other hand, did not discover any undesirable effects of POP on the endothelial function of rats.A study by Liang et al. (2011) showed that the plaque-reducing effects of PSE were cancelled out by their oxidation products in apoE-deficient hamster.Plat et al. (2014) found POP significantly increased the development of atherosclerotic lesions in LDL receptor deficient (LDLR+/−) mice, although oxysterols (oxidised cholesterol) were shown to cause a greater increase.Studies in humansPOP can be identified in human serum; however, the question of whether the consumption of foods with added PSE increases the content of POP in plasma has not yet been answered. Baumgartner et al. (2013), for example, found no connection between POP concentration in plasma and the consumption of products with added PS, whereas Husche et al. (2011) found that plasma POP concentration doubled after consumption of these types of products.Schött et al. (2014) analysed the PS and POP content of the plasma and aortic valve cusp tissue of patients with severe aortic stenosis. They discovered increased amounts of POP in the aortic tissue that did not correlate to the content found in the plasma. The authors discuss the possibility of POP developing locally from PS in the valve tissue, which could lead to increased inflammatory reactions with increased plaque formation. On 27 November 2014 and in accordance with Article 29(1)(a) of Regulation (EC) No 178/20022, the Commission asked the European Food Safety Authority to provide a scientific opinion by carrying out the additional assessment for the NF in the context of Regulation (EC) No 258/97 and to consider the elements of a scientific nature in the comments raised by the other Member States. According to Article 35 (1) of Regulation (EU) 2015/22833, any request for placing a novel food on the market within the Union submitted to a Member State in accordance with Article 4 of Regulation (EC) No 258/97, and for which the final decision has not been taken before 1 January 2018, shall be treated as an application under this Regulation. (Note: This is the case for this application). In accordance with Article 10 (3) of Regulation (EU) 2015/2283, EFSA shall give its opinion as to whether the update of the Union List referred to in Article 10 (1) is liable to have an effect on human health. 2 Data and methodologies 2.1 Data The safety assessment of this NF is based on data supplied in the original application, the initial assessment by the competent authority of the United Kingdom, the concerns and objections of a scientific nature raised by the other Member States and information submitted by the applicant in response to the Member States' comments and following an EFSA request for supplementary information as well as additional data identified by the Panel. Administrative and scientific requirements for NF applications referred to in Article 10 of Regulation (EU) 2015/2283 are listed in Commission Implementing Regulation (EU) 2017/24694. A common and structured format on the presentation of NF applications is described in the EFSA guidance on the preparation and presentation of a NF application.5 As indicated in this guidance, it is the duty of the applicant to provide all of the available (proprietary, confidential and published) scientific data, including both data in favour and not in favour to support the safety of the proposed NF. This NF application includes a request for protection of proprietary data in accordance with Article 26 of Regulation (EU) 2015/2283. The applicant claims proprietary rights for a human pharmacokinetic study (i.e. Title: The effect of oxidised PS Intake on serum concentrations of PS oxidation products. Global Study Number: FDS-SCC-2838). 2.2 Methodologies The assessment follows the methodology set out in the EFSA guidance on NF applications (EFSA NDA Panel, 2016) and the principles described in the relevant existing guidance documents from the EFSA Scientific Committee. The legal provisions for the assessment are laid down in Article 11 of Regulation (EU) 2015/2283 and in Article 7 of Commission Implementing Regulation (EU) 2017/2469. 3 Assessment 3.1 Introduction In 2000, the Scientific Committee on Food (SCF) assessed the safety of PSE in yellow fat spreads on the basis of an application from Unilever under Regulation (EU) No 258/97 (SCF, 2000). This application specified that this NF was not intended for use in cooking. The toxicological information provided in the application included a 13-week feeding study with rats, a two-generation feeding study with rats, studies on oestrogenic potential and tests on genotoxicity. In its assessment, the SCF noted the target population 'adults above 50 years of age, who try to control their elevated blood cholesterol', but considered that also children would not be expected to experience any adverse effect on metabolism when their blood cholesterol is lowered. The SCF noted also that PS absorption was found to be higher in children than in adults. The SCF concluded that PSE in yellow fat spreads at a maximum level corresponding to 8% free PS (FPS) are safe for human use. The SCF noted that ingestion of 20 g/day for 1 year of products containing 8% PS reduced plasma β-carotene concentrations in adults by 20% and that the reduced plasma β-carotene levels might become relevant when the vitamin A status is not optimal, which may be the case for pregnant and lactating women as well as younger children. Consequently, in 2000, the European Commission adopted the Decision 2000/500/EC authorising the placing on the market of 'yellow fat spreads with added PSE' as a novel food or novel food ingredient under Regulation (EC) No 258/97 (European Commission, 2000). According to the specifications of that marketing authorisation, the margarine/vegetable oil spreads may contain up to 8% w/w of added PS (equivalent to 14% w/w PSE) with a relative content among PS of 10–40% campesterol, 6–30% stigmasterol, 30–65% β-sitosterol and 0–5% other PS. The marketing authorisation included labelling requirements specifying (1) the target population, i.e. 'people who want to lower their cholesterol levels', (2) that patients on cholesterol lowering medication should only consume the product under medical supervision, (3) that the product may not be nutritionally appropriate for pregnant and breastfeeding women and children under the age of 5 years and (4) that the product should be used as part of a healthy diet, including regular consumption of fruit and vegetables to help maintain carotenoid levels. In 2002, the SCF assessed the long-term effects of the intake of elevated levels of PS from multiple dietary sources, with particular attention to the effects on β-carotene (SCF, 2002a). Noting that no additional effect on cholesterol levels is derived from an intake of PS above the 3 g per person per day and that the consequences of a persistent decrease of blood concentrations of β-carotene on human health are largely unknown, the SCF considered it prudent to avoid PS intakes exceeding 3 g/day. In 2004, an extension of this marketing authorisation was authorised for the uses of PSE in milk- and yoghurt-type products by Commission Decision 2004/335/EC, (European Commission, 2004a). Also in 2004, Commission Regulation (EC) No 608/2004 concerning the labelling of foods and food ingredients with added phytosterols, phytosterol esters, phytostanols and/or phytostanol esters, laid down labelling requirements and limiting the consumption of added PS to a maximum of 3 g per person per day (European Commission, 2004b). At their 69th meeting, JECFA established a group acceptable daily intake (ADI) of 0–40 mg PS/kg body weight. This corresponds to a daily intake of 2.8 g PS/day for a 70-kg individual (JECFA, 2009). This conclusion was based on an overall NOAEL derived from several subchronic (90-day) studies, supported by studies on reproductive toxicity. Noting the absence of a chronic toxicity study, JECFA found the application of an uncertainty factor of 100 sufficient taking into account the availability of a range of human studies including two 1-year studies. In this application, the applicant seeks the authorisation to extend the uses of PSE, i.e. the addition to vegetable fat spreads and to liquid vegetable fat-based emulsions for cooking and baking purposes excluding deep-frying. The applicant indicated that the production process and specifications do not change. This assessment concerns the risks that might be associated with the consumption of the NF used for cooking purposes. In accordance with Article 5(6) Commission Implementing Regulation (EU) 2017/2469, the safety of the NF is assessed for the general population. It is not an assessment of the efficacy of the NF with regard to any claimed benefit. 3.2 Identity of the NF The NF concerns PSE added to vegetable fat spreads ('PS Spreads') and liquid vegetable fat-based emulsions ('PS Liquids') which meet the specifications laid down in Commission Decisions 2000/500/EC and 2004/335/EC and subsequently included in Commission Implementing Regulation (EU) 2017/2470 establishing the Union list of novel foods in accordance with Regulation (EU) 2015/2283. 3.3 Compositional data The content of PSE is 12.5 g/100 g, corresponding to a content of PS of approximately 7.5 g/100 g. The applicant provided information on the compositions of PS-Spreads and PS-Liquids for CB. Both product categories (spreads and liquids) contain proteins, varying levels of emulsifiers, stabilisers, salt and vitamins. 3.3.1 Stability Upon request of EFSA, the applicant provided data from a publication on the amounts of POP in 19 foods prepared by typical household cooking and baking methods using margarine without (control) and with 7.5% added PS (as 12.5% PSE; further named PS-margarine) (Lin et al., 2016a). Various foods, including vegetables (green beans, cabbage, onions), potatoes, meat (pork, beef and chicken), fish (cod, salmon and frozen fish fingers) and eggs, were subjected to shallow-frying, stir-frying, stewing, roasting and microwave cooking (details given in Annex 1). In addition, different baked foods (cookies, muffins, banana bread and sponge cake) were investigated for POP content (Annex 2). Methodologies for the quantification of PS and POP in these cooked and baked foods, in particular extraction procedures, have been developed and validated (Menéndez-Carreno et al., 2016). The time and temperature conditions of the applied cooking and baking procedures are summarised in Annex 3. The portion sizes prepared varied between 100 and 250 g. Roast beef was prepared from 1 kg of meat, and the portion size was defined as 200 g roast (cooked) meat. The portion sizes of the baked products were defined as one typical slice of banana bread (80 g) or sponge cake (50 g), one piece of muffin (44 g) and one cookie (18 g). The experimental data demonstrate that the increase of POP contents in cooked and baked foods, resulting from the use of the PS-margarine as compared to the control margarine, was heavily influenced by the applied cooking/baking procedures (Annex 2). The median contents of POP per portion size of cooked foods were 0.57 mg (range 0.05–1.11 mg) with control margarine and 1.42 mg (range 0.08–20.5 mg) with PS-margarine. The increase of POP in the cooked foods prepared with the PS-margarine compared to those prepared with the control margarine ranged from 1.8-fold (shallow-fried egg, stewed beef and microwave-cooked codfish) to 21-fold (stir-fried chicken and shallow-fried potatoes). In foods prepared with the control margarine, the oxidation rate of phytosterols (ORP) ranged from 0.51% (microwave-cooked codfish) to 9.82% (shallow-fried beefsteak) with a median of 3.66%. For foods prepared with the PS-margarine, the oxidation rates ranged from 0.02% (microwave-cooked codfish) to 3.45% (shallow-fried potatoes), with a median of 0.50%, the P90 oxidation rate across the whole range of cooking and baking experiments for all foods was 2.28%. For all cooked foods, except for the stir-fried cabbage where all fat was absorbed, a variable amount of residual fat remained in the pan or the wok after cooking. When the control margarine was used, the median POP content of residual fat was 0.52 mg (range 0.04–1.88 mg) per amount of residual fat observed for the different foods and cooking methods. Using the PS-margarine, the median POP content of the residual fat was 6.98 mg (range 0.22–23.9 mg) per observed amount of residual fat. Regarding the sum of POP contents of foods plus residual fat, the median total POP content was 1.06 mg (range 0.15–2.85 mg) with the control margarine and 6.48 mg (range 0.30–44.4 mg) with the PS-margarine. The distribution of POP between the food itself and the corresponding residual fat widely ranged among the foods. Most of the cooked animal foods and fish, like pork fillet, steak, minced meat, salmon and codfish contained only about 3–41% of the total amount of POP, whereas in other foods, e.g. green beans, cabbage, onions but also fish fingers (which absorb more fat), 71–100% of the POP were found within the food matrix. Whenever the total amount of residual fat was higher than 5 g, more than 50% of the total amounts of POP were found in the residual fat. The POP contents per portion size of baked products and the respective oxidation rates are presented in Annex 3. Median POP contents across cookies, muffins, banana bread and sponge cake were 0.12 mg per portion (range 0.11–0.21 mg) with the control margarine and 0.24 mg per portion (range 0.19–0.60 mg) with the PS-margarine. For baked foods, the POP content increase ranged from a factor 1.7 (for cookies) up to a factor 2.9 (for sponge cake) as compared to the control margarine. The applicant also expressed the POP contents as mg per 100 g prepared food and residual fat (Annex 4). Using the control margarine, the median POP content of cooked foods was 0.47 mg/100 g food, while using the PS-margarine, 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 76.68 mg/100 g fat, with the use of the control margarine and PS-margarine, respectively. The highest amount of POP was found in the residual fat (415.58 mg/100 g fat) from shallow-frying of potatoes with the PS-margarine. The median amount of POP/100 g of baked products was 0.33 mg/100 g (range 0.15–0.66 mg/100 g) with the control margarine and 0.78 mg/100 g (range 0.34–1.20 mg/100 g) with the PS-margarine. When control margarine was used, the median values from the distributions of individual POP compounds (expressed as percent of total POP) in the foods and in the residual fat, respectively were 47.2% and 45.1% for 7-keto-PS, 25.0% and 29.9% for 5,6-epoxy-PS, 22.1% and 21.3% for 7-hydroxy-PS and 5.6% and 3.9% for PS-triols, with 7-keto-PS being the dominant individual POP. When PS-margarine 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-hydroxy-PS and PS-triols accounting for 23.9% and 1.9%, respectively, in foods. In the residual fat, 7-hydroxy-PS, 5,6-epoxy-PS, 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-hydroxy-PS being the dominant individual POP. For baked foods prepared with the control and PS-margarines, the distribution of individual POP was in the order of 7-keto-PS > 5,6-epoxy-PS > 7- hydroxy-PS > PS-triols. The applicant also performed a series of frying experiments without the use of foods. Heating of the PS-Spread for CB in tub format at 180°C for 15 min resulted in amounts of POP ranging from 34.3 to 48.5 mg/100 g product. In addition, two samples of the PS-Liquid for CB (containing 70% of total fat), with 12.5% PSE (equivalent to 7.5% plant sterols) were shallow-fried at 205°C ± 5°C for 30 min. On average 74.2 mg/100 g (range 70.4–80.0 mg/100 g) POP was formed. The occurrence of POP in foods with added PSE has been recently been reviewed (Scholz et al., 2015). Heating temperature and time, the chemical form in which the phytosterols are added (free phytosterols, PSE or phytostanol esters), and the food matrix were shown to be critical parameters determining the formation of POP. For example, in heat-treated milk, the POP contents ranged from 0.2 mg/kg (milk with added plant stanol esters (PAE), corresponding to 0.5% free phytostanols; pasteurised at 127°C for 2 s) (Soupas et al., 2006) to 6.4 mg/kg (milk with added PSE, corresponding to 0.3% PS; microwave heated at 900 W for 1.5 min) (Menéndez-Carreno et al., 2008). Pan-frying of a liquid spread (with added PSE corresponding to 5% PS) at 180°C for 5 and 10 min, resulted in an increase of the content of POP from 255 mg/kg to 291 mg/kg and 668 mg/kg POP, respectively (Soupas et al., 2007). Storage of a dark chocolate with added PSE at 30° C for 5 months resulted only in a minor increase of POP from 68.6 to 71 mg/kg (Bothelo et al., 2014). On the other hand, in spread with added PAE, the contents of POP were reported to increase upon storage for 6 weeks at 4°C and 20°C from 255 mg/kg to 354 and 734 mg/kg, respectively (Rudzinska et al., 2014). In a systematic review, the applicant evaluated 14 studies measuring POP contents of foods with added FPS, PSE and PAE (Lin et al., 2016b). In non-heated or stored foods, POP contents (medians) ranged from 0.03 to 3.6 mg/100 g with corresponding ORP of 0.03–0.06%. In fat-based products with 8% of added FPS, PSE or PAE pan-fried at 160–200°C for 5–10 min, median POP contents were 72.0, 38.1 and 4.9 mg/100 g, respectively, with median oxidation rates of 0.90%, 0.48% and 0.06%, respectively, indicating that the resistance to thermal oxidation was in the order of PAE > PSE > FPS. POP formation was highest in butter followed by margar
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