Content Of Phytosterol Oxidation Products Research Articles

Optimization and validation of an HPLC‐HRMS method through semipreparative HPLC system for determining phytosterol oxidation products during refining processing and storage of vegetable oils

AbstractA semi‐preparative liquid chromatographic system (LC‐Prep) has been used to isolate and collect the main oxidation products generated from β‐sitosterol, campesterol, and stigmasterol, which represent the main sterols detected in vegetable oils. In less than 50 min, 15 different phytosterol oxidation products (POPs) were separated with a satisfactory resolution (R >1) and collected. From each pure phytosterol standard the 5,6α‐epoxy (α‐E), 5,6β‐epoxy (β‐E), 7‐keto (7‐K), 7α‐hydroxy (7α‐H), and 7β‐hydroxy (7β‐H) isomers were obtained. The purity of POPs was >90%. Then, the obtained pure POPs were used to validate an HPLC‐Orbitrap‐HRMS analytical method. The LOD and LOQ determined in medium chain triacylglycerols were >0.012 and >0.039 ng/mL, respectively; whereas the recoveries ranged between 82% and 98%. The suitability of the analytical method was evaluated on palm oil (PO), palm olein (POL), and high oleic sunflower oil (HOSO) during the whole refining processing (crude oil, neutralization and degumming, bleaching and deodorization) and under storage conditions (45°C, 16 days). In the refining steps, the total POPs content significantly increased (p < 0.05) by 29%, 20%, and 13% in PO, POL, and HOSO, respectively. The highest amount was found in HOSO (15.046 mg/kg), followed by POL (1.067 mg/kg) and PO (0.538 mg/kg). Under storage conditions, the content of POPs did not significantly (p > 0.05) change and was lower than 8.965 mg/kg. The developed semi‐preparative liquid chromatographic system coupled to the LC‐Orbitrap‐HRMS method demonstrated to be a useful and valid tool for a robust, precise, accurate, and sensitive determination of POPs in refined and stored vegetable oils.

Phytosterol oxidation products (POP) in foods with added phytosterols and estimation of their daily intake: A literature review.

1To evaluate the content of phytosterol oxidation products (POP) of foods with added phytosterols, in total 14 studies measuring POP contents of foods with added phytosterols were systematically reviewed. In non‐heated or stored foods, POP contents were low, ranging from (medians) 0.03–3.6 mg/100 g with corresponding oxidation rates of phytosterols (ORP) of 0.03–0.06%. In fat‐based foods with 8% of added free plant sterols (FPS), plant sterol esters (PSE) or plant stanol esters (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 a median ORP of 0.90, 0.48, and 0.06%. Hence resistance to thermal oxidation was in the order of PAE > PSE > FPS. POP formation was highest in enriched butter followed by margarine and rapeseed oil. In margarines with 7.5–10.5% added PSE oven‐heated at 140–200°C for 5–30 min, median POP content was 0.3 mg/100 g. Further heating under same temperature conditions but for 60–120 min markedly increased POP formation to 384.3 mg/100 g. Estimated daily upper POP intake was 47.7 mg/d (equivalent to 0.69 mg/kg BW/d) for foods with added PSE and 78.3 mg/d (equivalent to 1.12 mg/kg BW/d) for foods with added FPS as calculated by multiplying the advised upper daily phytosterol intake of 3 g/d with the 90% quantile values of ORP. In conclusion, heating temperature and time, chemical form of phytosterols added and the food matrix are determinants of POP formation in foods with added phytosterols, leading to an increase in POP contents. Practical applications: Phytosterol oxidation products (POP) are formed in foods containing phytosterols especially when exposed to heat treatment. This review summarising POP contents in foods with added phytosterols in their free and esterified forms reveals that heating temperature and time, the chemical form of phytosterols added and the food matrix itself are determinants of POP formation with heating temperature and time having the biggest impact. The estimated upper daily intakes of POP is 78.3 mg/d for fat‐based products with added free plant sterols and 47.7 mg/d for fat‐based products with added plant sterol esters. Phytosterols in foods are susceptible to oxidation to form phytosterol oxidation products (POP). This review summarizes literature data regarding POP contents of foods with added phytosterols that were exposed to storage and heat treatments.

Effect of Microwave Heating on Phytosterol Oxidation.

The oxidative stability of phytosterols during microwave heating was evaluated. Two different model systems (a solid film made with a phytosterol mixture (PSF) and a liquid mixture of phytosterols and triolein (1:100, PS + TAG (triacylglycerol))) were heated for 1.5, 3, 6, 12, 20, and 30 min at 1000 W. PS degraded faster when they were microwaved alone than in the presence of TAG, following a first-order kinetic model. Up to 6 min, no phytosterol oxidation products (POPs) were generated in both systems. At 12 min of heating, the POP content reached a higher level in PSF (90.96 μg/mg of phytosterols) than in PS + TAG (22.66 μg/mg of phytosterols), but after 30 min of treatment, the opposite trend was observed. 7-Keto derivates were the most abundant POPs in both systems. The extent of phytosterol degradation depends on both the heating time and the surrounding medium, which can impact the quality and safety of the food product destined to microwave heating/cooking.

Stabilisation of phytosterols by natural and synthetic antioxidants in high temperature conditions

The aim of the study was to assess the potential applicability of natural antioxidants in the stabilisation of phytosterols. A mixture of β-sitosterol and campesterol was incorporated into triacylglycerols (TAGs). The following antioxidants were added to the prepared matrix: green tea extract, rosemary extract, a mix of tocopherols from rapeseed oil, a mix of synthetic tocopherols, phenolic compounds extracted from rapeseed meal, sinapic acid and butylated hydroxytoluene (BHT). Samples were heated at a temperature of 180°C for 4h. After the completion of heating, the losses of phytosterols were analysed, as well as the contents of β-sitosterol and campesterol oxidation products. The total content of phytosterol oxidation products in samples ranged from 96.69 to 268.35μg/g of oil. The effectiveness of antioxidants decreased in the following order: phenolic compounds from rapeseed meal>rosemary extract>mix of tocopherols from rapeseed oil>mix of synthetic tocopherols>green tea extract>sinapic acid>BHT.

Plant sterol oxides in functional beverages: Influence of matrix and storage

Three plant sterol (PS)-enriched beverages, milk based fruit juice (MFJPS), fruit juice (FJPS) and milk beverage (MPS), were stored at 4, 24, or 37°C and analysed at regular time intervals of 2months until 6months. PS stability was analysed from the production of phytosterol oxidation products (POPs). The β-sitosterol oxides (7α/7β-hydroxy, β/α-epoxy, triol, and 7-keto) and campesterol oxides (β/α-epoxy, and 7-keto) were detected in all beverages and at all storage times and temperatures. Total POP contents followed the order MPS≫FJPS>MFJPS. In general, the beverages showed low PS oxidation levels (<0.17%). Predictive models of POP content versus storage time were established. These models explain total POP content by over 75% and individual POP content by over 50%. We propose 7-ketositosterol and 7-ketocampesterol as PS oxidation markers during storage of beverages of this kind.

Phytosterol oxides content in selected thermally processed products

Heat treatments are very popular methods of food preparation in European countries. Phytosterol present in foodstuffs undergoes oxidative changes during heat treatment, and phytosterol oxides (e.g., 7α-, 7β-hydroxysterol, 5α,6α-, 5β,6β-epoxysterol, 7-ketosterol and triol) are formed. Phytosterol oxidation products (POPs) have been associated with cytotoxic and pro-apoptotic effects in humans. On the other hand, several studies conducted on animals revealed that some phytosterol oxides lower serum triacyglycerol and blood glucose levels. The aim of this study was to evaluate the effect of heat treatment on formation of phytosterol oxidation products in selected foodstuffs. The following products were taken into considerations: minced meat (pork and beef), frozen French fries, frozen fish fillets, frozen fish products (e.g., fish sticks), wheat and egg noodles. Sterols and POPs content was evaluated by GC–MS working in total and selected ion monitoring modes. The phytosterol oxidation rate was higher in French fries and fish fillets (0.20–1.69% of total phytosterol content) than in noodles, minced meats and readymade fish products (in 0.04–0.36% range). Method of POPs determination using GC–MS is reported in this study. Results of this study show also that products of the animal origin might be considered as sources of the phytosterol oxides in the human diet.

β-Sitosterol and campesterol stabilisation by natural and synthetic antioxidants during heating

The aim of this study was to estimate the effect of natural and synthetic antioxidants in protecting phytosterols during heating at 180°C. Green tea extract, rosemary extract, a mix of tocopherols from rapeseed oil, a mix of synthetic tocopherols, phenolic compounds extracted from rapeseed meal, sinapic acid and BHT were used. After 4h of heating in oxygen atmosphere β-sitosterol and campesterol oxidation products (7α- and 7β-hydroxysterol, 5α,6α- and 5β,6β-epoxysterol, 7-ketosterol and triols) were estimated by GC. Total content of phytosterol oxidation products in samples ranged from 137 to 374mg/kg of sample. The effectiveness of antioxidants decreased in the following order: synthetic tocopherols>green tea extract>natural tocopherols from rapeseed oil>rosemary extract>phenolic compounds extracted from rapeseed meal>sinapic acid>BHT.

Effects of α‐Tocopherol on Oxidative Stability and Phytosterol Oxidation During Heating in Some Regular and High‐Oleic Vegetable Oils

AbstractThe first part of this study evaluated oxidative stability in high‐oleic rapeseed oil, palm olein, refined olive oil, low erucic acid rapeseed oil and sunflower oil. The results showed oxidative stability in the order: palm olein &gt; high‐oleic rapeseed oil &gt; refined olive oil &gt; low erucic acid rapeseed oil &gt; sunflower oil, as determined by the Rancimat method. Addition of α‐tocopherol at high levels of up to 0.2% increased the oxidative stability of refined olive oil, whereas the opposite effect was generally observed in the other oil samples. In the second part of the study, high‐oleic rapeseed oil, palm olein, refined olive oil and refined olive oil containing 0.2% α‐tocopherol were heated for 3, 6, 9 and 12 h at 180 °C. The peroxide and p‐anisidine values generally increased over time in the samples, including olive oil containing 0.2% α‐tocopherol. High‐oleic rapeseed oil contained the highest amount of total sterols and total phytosterol oxidation products (POPs), but during heating the total POPs content increased moderately (~10%), in contrast to the threefold increase after 12 h of heating in palm olein and refined olive oil. Very high levels of 6‐hydroxy derivatives of brassicastanol, campestanol and sitostanol and of 7‐ketobrassicasterol were observed in high‐oleic rapeseed oil samples. Addition of 0.2% α‐tocopherol during heating significantly decreased POPs formation in refined olive oil (P &lt; 0.05).

Stability of Minor Lipid Components with Emphasis on Phytosterols During Chemical Interesterification of a Blend of Refined Olive Oil and Palm Stearin

AbstractMinor compounds such as tocopherols and phytosterols in vegetable oils play an important role in their stability and nutritional value. This study monitored the effects of chemical interesterification on the levels of tocopherols, tocotrienols, phytosterols and phytosterol oxidation products (POPs) in an olive oil and palm stearin blend (50/50 w/w). Tocopherols and tocotrienols were dominated by α‐tocopherol (192 ppm) and γ‐tocotrienol (70 ppm) and decreased during interesterification. Among the tocopherols, δ‐tocotrienol had the highest decrease (35%) at 120 °C. During interesterification at 90 and 120 °C, total sterol content in the oil blend (509 ppm) declined slightly, by 3 and 5%, respectively. Phytosterols were esterified at a higher level at 120 °C (7%) than at 90 °C (4%) during this process. Distribution of fatty acids in the esterified sterols followed the fatty acid composition of the oil blend. Total POP content was 4.3 ppm, and remained generally unchanged during interesterification. Among the nine POPs tentatively identified by their mass spectra, 6‐hydroxysitostanol and 6‐hydroxycampestanol dominated in the oil blend and in the interesterified product. The formation pathways of these saturated di‐hydroxyphytosterols have yet to be identified. Although the interesterification process comprised several treatments, there were only minor losses of tocopherols and phytosterols and virtually no increases in the POPs.

Lipids and phytosterol oxidation products in commercial potato crisps commonly consumed in Sweden

The stability of frying oils and fried foods is mainly affected by the fatty acids present and by the types and levels of minor components such as phytosterols and tocopherols. This study assessed the current status of lipid composition and the occurrence of oxidised phytosterols as a parameter of lipid oxidation in potato crisps available in the Swedish market. Fatty acid composition and concentrations of tocopherols, sterols and phytosterol oxidation products (POPs) were determined in 16 commercial potato crisp samples of two types, distinguished by a high or low fat content. The fatty acid composition in most samples was dominated by saturated and monounsaturated fatty acids. The sum of α-tocopherol and γ-tocopherol content varied from undetectable levels to 10.2 mg/100 g potato crisps, with α-tocopherol dominating. Among the tocotrienols, α-tocotrienol and γ-tocotrienol were present in almost equal proportions, while δ-tocotrienol was present in all samples but in smaller amounts. Fatty acid composition, tocopherol and tocotrienol content, showed that all potato crisp samples were prepared in palm oil or a blend of palm oil and unspecified fats and oils. Total sterol content ranged from 10.2 to 93.1 mg/100 g sample, with β-sitosterol being the major sterol in all samples. The content of POPs ranged from 0.05 to 0.68 mg/100 g potato crisps. In general, there were no significant differences in content of POPs between high and low fat samples, and generally no correlations could be established between content of POPs and fatty acid, tocopherol, tocotrienol, and sterol content among the potato crisp samples.

Lipids and phytosterol oxidation in commercial French fries commonly consumed in Sweden

Industrial frying oils continuously evolve as efforts are made to improve their overall quality because of health concerns. The objectives of this study were to determine fatty acids, tocopherols, sterols, and phytosterol oxidation products (POPs) in French fries, which are commonly consumed in Sweden. Five different commercial prefried French fries and four samples of French fries prepared in different fast food restaurants were studied. POPs were determined with transesterification, purification of oxides by solid-phase extraction, quantification by GC and identification by GC–MS. The method of POPs analysis was validated by spiking with standard samples of cholesterol oxidation products which showed good recoveries of those compounds. The results showed that the samples were generally prepared in frying oils dominated by saturated and monounsaturated fatty acids, except for two prefried samples which were dominated by a di-unsaturated fatty acid. In prefried samples, only α-tocopherol could be quantified whereas restaurant samples generally contained both α- and γ-tocopherols. Sitosterol was the dominating sterol in the all samples. The POPs content in prefried commercial French fries ranged from 0.01 to 0.21 mg/100 g, and the values in restaurant samples were considerably higher, ranging from 0.15 to 0.81 mg/100 g. The increased levels of POPs in the restaurant samples may be partially due to higher contents of fat, saturated and monounsaturated fatty acids and sterols. Further studies are required to identify other factors for the formation of POPs in French fries and their long-term implications to human health.

Levels of Phytosterol Oxides in Enriched and Nonenriched Spreads: Application of a Thin-Layer Chromatography−Gas Chromatography Methodology

The content of phytosterol oxidation products (POPs) in enriched and nonenriched commercial spreads was evaluated by thin-layer chromatography-gas chromatography (TLC-GC). Oxides of beta-sitosterol, campesterol, and stigmasterol were produced by thermo-oxidation (7-hydroxy, 7-keto, and epoxy derivatives) and chemical synthesis (triol derivatives), which were then separated and identified by TLC-GC. Their identification was further confirmed by GC-mass spectrometry (GC-MS). The total amounts of phytosterols found were 6.07 and 0.33 g/100 g of sample in phytosterol-enriched and nonenriched spread, respectively, whereas the total POPs contents were 45.60 and 13.31 mg/kg of sample in the enriched and nonenriched products. The main POPs found were the 7-keto derivatives of all phytosterols analyzed; 7-ketositosterol was the most abundant one (14.96 and 5.93 mg/kg of sample in phytosterol-enriched and nonenriched spread). No beta-epoxy and triol derivatives were detected in both types of samples. The enriched spread presented a lower phytosterol oxidation rate (0.07%) than the nonenriched one (0.41%).

Presence of Phytosterol Oxides in Crude Vegetable Oils and Their Fate during Refining

The content of phytosterol oxidation products was determined in samples of crude vegetable oils: peanut, sunflower, maize, palm nut, and lampante olive oils that were intended for refining and not for direct consumption. The 7 alpha- and 7 beta-hydroxy derivatives of beta-sitosterol, stigmasterol, and campesterol and the 7-keto-beta-sitosterol were the principal phytosterol oxides found in almost all of the oils analyzed. In some oils, the epoxy and dihydroxy derivatives of beta-sitosterol were also found at very low levels. The highest total concentrations of phytosterol oxides, ranging from 4.5 to 67.5 and from 4.1 to 60.1 ppm, were found in sunflower and maize oils, respectively. Lower concentrations were present in the peanut oils, 2.7-9.6 ppm, and in the palm nut oil, 5.5 ppm, whereas in the lampante olive oils, only three samples of the six analyzed contained a low concentration (1.5-2.5 ppm) of oxyphytosterols. No detectable levels of phytosterol oxides were found in the samples of palm and coconut oils. Bleaching experiments were carried out on a sample of sunflower oil at 80 degrees C for 1 h with 1 and 2% of both acidic and neutral earths. The bleaching caused a reduction of the hydroxyphytosterol with partial formation of steroidal hydrocarbons with three double bonds in the ring system at the 2-, 4-, and 6-positions (steratrienes). The same sunflower oil was deodorized at 180 degrees C under vacuum for 1 h, and no dehydration products were formed with a complete recovery of the hydroxyphytosterols. A bleaching test with acidic earths was carried out also with an extra virgin olive oil fortified with 7-keto-cholesterol, dihydroxycholesterol, and alpha-epoxy-cholesterol. There was no formation of steratrienes from these compounds, but dihydroxycholesterol underwent considerable decomposition and alpha-epoxycholesterol underwent ring opening with formation of the dihydroxy derivative, whereas 7-ketocholesterol was rather stable