Quantitation of Sterols and Steryl Esters in Fortified Foods and Beverages by GC/FID

Direct extraction methods for the quantitation of sterols and steryl esters (SE) in foods, beverages and concentrates as free sterols (trimethylsilane ether derivatives) by gas chromatography with flame ionization detection (GC/FID) are introduced. Theoretical correction factors are used for sterol quantification relative to the internal standard, Epicoprostanol. Conversion of the acylglycerols, when present, into FFA eliminates the time-consuming acylglycerol extraction step and reduces the GC run time since no higher molecular weight acylglycerols need to elute from the column. Accuracy and precision of analytical data are important when formulating food products with sterols or SE to ensure a formulation will meet health claims, while at the same time minimizing costly over formulation. Method accuracy was determined by complete recovery of sterol and SE concentrates as free sterols with subsequent analysis of foods and beverages formulated with sterol and SE concentrates. Triplicate analysis over 5 days demonstrated repeatability for the alkaline saponification and acid extraction with alkaline saponification methods. A relative standard deviation of <5% demonstrates the repeatability of the methods. Replicate analysis of a common sample by two laboratories and replicate analysis of a common sample by multiple analysts demonstrated reproducibility of the methods.     

Pearling barley and rye to produce phytosterol-rich fractions

Because of the positive health effects of phytosterols, phytosterol-enriched foods and foods containing elevated levels of natural phytosterols are being developed. Phytosterol contents in cereals are moderate, whereas their levels in the outer layers of the kernels are higher. The phytosterols in cereals are currently underutilized; thus, there is a need to create or identify processing fractions that are enriched in phytosterols. In this study, pearling of hulless barley and rye was investigated as a potential process to make fractions with higher levels of phytosterols. The grains were pearled with a laboratory-scale pearler to produce pearling fines and pearled grains. Lipids were extracted by accelerated solvent extraction, and nonpolar lipids were analyzed by normal-phase HPLC with ELSD and UV detection. Total sterol analyses were performed by GC. After a 90-s pearling, the amounts of pearling fines from hulless barley and rye were 14.6 and 20.1%, respectively, of the original kernel weights. During pearling, higher levels of phytosterols and other lipids were fractionated into the fines. The contents of free sterols and sterols esterified with FA in the fines were at least double those in the whole grains. Pearling fines of hulless barley and rye contained >2mg/g phytosterol compounds, which makes them a good source of phytosterols and thus valuable raw materials for health-promoting foods.     

Chemical synthesis of phytosterol esters of polyunsaturated fatty acids with ideal oxidative stability

In this study, direct esterification of phytosterols with polyunsaturated fatty acid (PUFA) catalyzed by sodium bisulfate to produce sterol esters of PUFA was performed without organic solvent. Considering on both degree of esterification (DE) and oxidative stability, response surface methodology (RSM) was employed for modeling the phytosterol esters of PUFA production to obtain a food grade process. The optimal and mild reaction conditions were obtained as follows: mass ratio of PUFA:phytosterols = 4:1; amount of catalyst: 3% of the raw materials weight; reaction temperature 130°C; reaction time 8 h. Under these conditions, the degree of esterification was up to 96%, GC, TLC, NMR and GC‐MS results showed that purity of purified sterol ester was above 99%, and β‐sitosterol linolenate account for about 88%. Sterol ester of PUFA possessed low peroxide value (PV) (0.96 meq/kg) and conjugated diene (CD) value (2.15 mmol/kg), and high oxidative induction period (OIP) (10.4 h). Addition amount of sterol ester of PUFA into soybean oil, rapeseed oil, and flaxseed oil below 1, 1, and 3%, respectively, could increase OIP of the vegetable oil. The primrose phytosterol esters of PUFA possessed very low melting point, crystallization temperature, and greater solubility in oils. All the characteristics favored the wide application of sterol ester of PUFA produced by the food grade process in different formulations of functional foods.     

Analysis of free and esterified sterols in vegetable oils

In vegetable oils, phytosterols occur as free sterols or as steryl esters. Few analytical methods report the quantification of esterified and free sterols in vegetable oils. In this study, esterified and free sterols were separated by silica gel column chromatography upon elution with n-hexane/ethyl acetate (90∶10 vol/vol) followed by n-hexane/diethyl ether/ethanol (25∶25∶50 by vol). Both fractions were saponified separately and the phytosterol content was quantified by GC. The analytical method for the analysis of esterified and free sterols had a relative standard deviation of 1.16% and an accuracy of 93.6–94.1%, which was comparable to the reference method for the total sterol analysis. A large variation in the content and distribution of the sterol fraction between different vegetable oils can be observed. Corn and rapeseed oils were very rich in phytosterols, which mainly occurred as steryl esters (56–60%), whereas the majority of the other vegetable oils (soybean, sunflower, palm oil, etc.) contained a much lower esterified sterol content (25–40%). No difference in the relative proportion of the individual sterols among crude and refined vegetable oils was observed.     

Optimization of Phytosterols Recovery from Soybean Oil Deodorizer Distillate

A large amount of phytosterols in the bound form remains in the waste residue during the traditional process of recovering tocopherols and sterols from soybean oil deodorizer distillate (SODD). In order to avoid the loss of natural resources, we developed a process to recover the maximum amount of phytosterols from SODD. The process includes saponification, methyl esterification, and crystallization. The purpose of saponification and methyl esterification were to decompose the bound phytosterols and to esterify the fatty acids, respectively. The yield of sterols was dependent on saponification and solvent crystallization. The conditions of saponification and solvent crystallization were optimized by single-factor tests and response surface methodology, respectively. The sterol yield obtained under the optimized conditions was 6.64%. This value is much greater than 4.43% obtained by the traditional industrial process. The purity of the recovered sterols was 94.7%.     

Simultaneous quantification of serum phytosterols and cholesterol precursors using a simple gas chromatographic method

Determination of the main phytosterols (Ps, β‐sitosterol and campesterol) and cholesterol precursors (desmosterol and lathosterol) in human serum using a simple GC‐FID method has been validated. Direct saponification, without lipid extraction, sterols extraction, and further derivatization was applied to samples prior to GC analysis. To evaluate the method, a pool of serum samples from eight healthy women was used. Good linearity (r>0.99) was found in the assay range: β‐sitosterol (0.99–17.82 µg/mL), campesterol (0.14–10.8 µg/mL), desmosterol (0.17–2.6 µg/mL), and lathosterol (0.6–5.97 µg/mL). Limits of detection (ng/mL) were: 86 (β‐sitosterol), 42 (campesterol), 4 (desmosterol), and 44 (lathosterol). Accuracy, estimated by recovery assays (%), were: 113 (β‐sitosterol), 114 (campesterol), 111 (desmosterol), and 102 (lathosterol). Within and between precision values (%), expressed as the relative SD (RSD), were: 2.6 and 8.1 (β‐sitosterol), 1.6 and 7.2 (campesterol), 2.1 and 7.9 (desmosterol), and 4.1 and 5.8 (lathosterol), respectively. The developed methodology allowed fast (1‐day analysis) and reliable quantification of sterols in serum, required a small volume of sample and reduced use of solvents. It therefore could be used in clinical assays for the determination of serum sterols, as in evaluating the pharmacological response to lipid‐lowering agents, and in assessing biological responses to Ps‐enriched diets. Practical applications : This methodology allows fast and reliable quantification of sterols in serum, requiring a small volume of sample and reduced use of solvents. It can be used as a routine method for the quantification of phytosterols and cholesterol precursors in clinical assays, and it is also suitable for monitoring biological responses to health‐promoting phytosterol‐enriched diets.     

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

To 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.     

Phytosterols in human nutrition: Type,formulation, delivery,and physiological function

Phytosterols are a family of compounds similar to cholesterol which have been shown to lower cholesterol levels when supplemented in the diet. A daily dose of 2–3 g of phytosterols has been shown to reduce LDL‐cholesterol levels by 5–15%. Phytosterol supplementation can be undertaken using phytosterol enriched functional foods or nutraceutical preparations. The type of phytosterol supplemented, such as plant sterol or saturated plant stanol appear to be equally effective in lowering cholesterol levels. Phytosterols, whether in esterified or free form have both been shown to lower cholesterol levels, with esterified phytosterol formulations having a greater number of clinical trials demonstrating efficacy. The functional food or nutraceutical matrix which is used to deliver supplemental phytosterols can significantly affect cholesterol lowering efficacy. Effective cholesterol lowering by phytosterols depends on delivery of phytosterols to the intestine in a form which can compete with cholesterol for absorption. New phytosterol functional food and nutraceuticals products should always be tested to demonstrate adequate delivery of phytosterol dose and effective total and LDL‐cholesterol lowering. Phytosterol products which do not effectively lower cholesterol will negatively impact the perception and use of phytosterols, and must not be allowed on the marketplace.     

Esterification affects phytosterol oxidation

The effect of esterification with rapeseed oil fatty acids on the oxidation reactions of sitosterol, campesterol and sitostanol was investigated, as well as the interactions between phytosterol/stanol compounds and the saturated lipid matrix at 100 °C and 180 °C. Free and esterified phytosterols differed in their reactivity in terms of the formation and profile of secondary oxidation products. Phytosteryl esters were more reactive than free phytosterols during prolonged heating at 100 °C. In contrast, free phytosterols were slightly more reactive than phytosteryl esters at 180 °C. The oxidation reactions of phytostanol compounds were low under all conditions studied. Changes in the phytosterol compounds during heating were also studied via the losses in the original phytosterol contents. This study revealed that the formation of secondary oxides did not account for all the phytosterol losses; this indicates the presence of other oxidation products, especially at 180 °C, and during the heating of free sitosterol. Thus, in order to understand the overall deterioration of phytosterol and phytostanol compounds, both the secondary oxide formation and the sterol loss need to be studied. The deterioration of the saturated lipid matrix used in this study was rather low and was mainly associated with the heating temperature and time.     

Phytosterols and pectin added to a high‐saturated fat diet do not show hypocholesterolemic activity in female guinea pigs

This paper presents the results of a study whose aim was to test the effects of several doses of pectin and phytosterols on the sterol content in plasma, liver and feces of guinea pigs, when added to a high‐saturated fat diet. The treatments followed a 3×3 factorial design, with three levels of pectin (0, 3.67 and 6.93%) and three levels of phytosterols (0, 1.37 and 2.45%). Seventy‐two female Dunkin Hartley guinea pigs were randomly assigned to the treatment groups (eight animals per group). The duration of the treatment was 4 weeks. No differences were found in plasma cholesterol concentrations, while in liver we saw a reduction in cholesterol concentration after phytosterol feeding. Moreover, we found no pectin effects. Plant sterol concentration increased in plasma and liver after phytosterol ingestion, with the highest concentrations being obtained with the intermediate pectin dose. Our results suggest that a high‐saturated diet may impair the cholesterol‐lowering properties of plant sterols and pectin.     

Sterols and Stanols in Foods and Dietary Supplements Containing Added Phytosterols: A Collaborative Study

An international, multilaboratory collaborative study was carried out to evaluate the performance of Official Method Ce 12‐16 of the American Oil Chemists’ Society (AOCS) for the determination of plant sterols and stanols, collectively referred to as phytosterols, in foods and dietary supplements containing added phytosterols and in the phytosterol food additive concentrates used to prepare such products. AOCS Official Method Ce 12‐16 involves the extraction of free sterols/stanols and saponified steryl/stanol esters followed by the gas chromatographic separation and flame ionization detection of phytosterol trimethylsilyl ether derivatives. A total of 14 laboratories from six countries successfully completed the analysis of collaborative samples of foods (e.g., baked goods, beverages, margarines; n = 9), dietary supplements (n = 5), and phytosterol concentrates (n = 4). Study results for the contents of total phytosterols (weight/weight) were 0.19–8.4% for foods, 8.7–49% for dietary supplements, and 57–97% for concentrates. AOCS Official Method Ce 12‐16 showed acceptable performance for total and individual phytosterols, indicating that this method was suitable for the determination of added phytosterols in a wide variety of market products and concentrates. AOCS Official Method Ce 12‐16 is appropriate for the determination of the five major phytosterols (i.e., campesterol, stigmasterol, β‐sitosterol, campestanol, and sitostanol) that are the subject of the United States Food and Drug Administration's health claim for phytosterols and the reduced risk of coronary heart disease.     

Phytostanol Supplementation Through Frying Dough in Enriched Canola Oil

The objectives of this study were to determine a suitable level of phytostanols for addition to canola oil and to investigate the performance of the supplemented oil during frying. The frying oil was supplemented with 5, 10, 15, 20 % w/w phytostanols and two suitable levels (5 and 10 %) were selected. Dough frying was performed for 5 consecutive days at 180 °C for 5 h/day. The ranges of analytical measurements in the treatment groups were; free acidity (0.12–10.07 %), conjugated dienes (0.47–1.37 %), total polar material with probe (9.00–51.25 %), viscosity (46.27–195.51 cP), turbidity (0.82–1.80 NTU), and smoke point (202.75–274.25 °C). The results indicated that 5 % phytostanol enriched oil was superior in terms of oil stability and sensory quality of the fried dough among all the enriched oils. Samples with 10 % added phytostanols were high in free acidity, conjugated dienes and smoke points. Sterol composition analysis showed that the fried dough absorbed total sterols of 49.9 and 95 g/kg in 5 and 10 % supplemented oils, respectively. Hence, some health benefits could be achieved through consuming products which have been fried in phytostanol supplemented canola oil.     

Effects of cholestyramine and squalene feeding on hepatic and serum plant sterols in the rat

Hepatic and serum phytosterol concentrations were compared in the rat under basal conditions and during activated cholesterol and bile acid production due to squalene and cholestyramine feeding. Both treatments consistently decreased hepatic and serum levels of sitosterol and campesterol and, unlike esterified cholesterol, esterified plant sterols were not increased in liver during squalene feeding. Serum levels of phytosterols were decreased quite proportionately to those in the liver. The hepatic levels of sitosterol and campesterol closely correlated with each other, but not with cholesterol levels. The percentage esterification of both phytosterols was lower than that of cholesterol. The results indicate that activation of hepatic sterol production leads to depletion of hepatic plant sterols. It is suggested that poor esterification of plant sterols may contribute to this decrease.     

Phytosterols in 17 Turkish hazelnut (Corylus avellana L.) cultivars

The phytosterol contents of the oils from 17 Turkish hazelnut cultivars were determined by gas chromatography with a flame ionization detector. The total phytosterol content varied from 1180.4 (Uzunmusa‐Ordu) to 2239.4 mg/kg (Cavcava), and the average was 1581.6 ± 265.1 mg/kg. One of the most significant commercial cultivars, Tombul, contained quite low total phytosterols (1297.7 mg/kg). Total and individual phytosterol contents of hazelnut cultivars were significantly different at p <0.01, except for phytostanol and campestanol. The main component was β‐sitosterol which ranged from 82.8 to 86.7% in all cultivars. This was followed by campesterol, Δ⁵‐avenasterol, sitostanol and stigmasterol. Interestingly, the same cultivars from different regions showed similar total phytosterol contents, and fall almost within the same range according to Duncan's test, which may indicate that the phytosterol content is highly related to the cultivar.     

Influence of the vegetable oil refining process on free and esterified sterols

The influence of the refining process on the distribution of free and esterified phytosterols in corn, palm, and soybean oil was studied. Water degumming did not affect the phytosterol content or its composition. A slight increase in the content of free sterols was observed during acid degumming and bleaching due to acid-catalyzed hydrolysis of steryl esters. A significant reduction in the content of total sterols during neutralization was observed, which was attributed to a reduction in the free sterol fraction. Free sterols probably form micelles with soaps and are transferred into the soapstock. The steryl ester content remained constant during all neutralization experiments, indicating that hydrolysis of steryl esters did not take place during neutralization. During deodorization, free sterols are distilled from the oil, resulting in a gradual reduction in the total sterol content as a function of the deodorization temperature (220–260°C). A considerable increase in the steryl ester fraction was found during physical refining, probably owing to a heat-promoted esterification reaction between free sterols and FA.     

Phytosterol Ester Constituents Affect Micellar Cholesterol Solubility in Model Bile

Plant sterols and stanols (phytosterols) and their esters are nutraceuticals that lower LDL cholesterol, but the mechanisms of action are not fully understood. We hypothesized that intact esters and simulated hydrolysis products of esters (phytosterols and fatty acids in equal ratios) would differentially affect the solubility of cholesterol in model bile mixed micelles in vitro. Sodium salts of glycine- and taurine-conjugated bile acids were sonicated with phosphatidylcholine and either sterol esters or combinations of sterols and fatty acids to determine the amount of cholesterol solubilized into micelles. Intact sterol esters did not solubilize into micelles, nor did they alter cholesterol solubility. However, free sterols and fatty acids altered cholesterol solubility independently (no interaction effect). Equal contents of cholesterol and either campesterol, stigmasterol, sitosterol, or stigmastanol (sitostanol) decreased cholesterol solubility in micelles by approximately 50% compared to no phytosterol present, with stigmasterol performing slightly better than sitosterol. Phytosterols competed with cholesterol in a dose-dependent manner, demonstrating a 1:1 M substitution of phytosterol for cholesterol in micelle preparations. Unsaturated fatty acids increased the micelle solubility of sterols as compared with saturated or no fatty acids. No differences were detected in the size of the model micelles. Together, these data indicate that stigmasterol combined with saturated fatty acids may be more effective at lowering cholesterol micelle solubility in vivo.     

Phytosterols in cereal by-products

Phytosterols are hypocholesterolemic. Like corn fiber oil, the lipid extracts of certain cereal by-products may be rich sources of these health-promoting compounds. The objective of this research was to examine the phytosterol content and composition of various cereal by-products. Total lipids in rice bran, wheat bran, wheat germ, durum wheat (bran and germ mixture), oat bran, oat hull, and corn fine fiber were extracted, and the sterol profiles of the extracted lipids were analyzed by GC. Rice bran contained the most lipids (22.2%), followed by wheat germ, durum wheat, oat bran, wheat bran, and oat hull; corn fine fiber contained the least amount of lipids (1.7%). Sitosterol, campesterol, and stigmasterol were the major phytosterols in these lipid extracts, whereas brassicasterol was detected only in wheat samples. Rice bran oil contained considerable amounts of cycloartenol and 24-methylenecycloartanol, which were unique to these samples. Total sterol concentrations in extracted lipids were similar for rice bran, wheat bran, wheat germ, and durum wheat (21.3–15.1 mg/g), but they were very low in oat bran lipids and oat hull lipids (3.4 and 8.2 mg/g, respectively). Corn fine fiber lipids contained the highest amount of sterols (48.3 mg/g). Rice bran appears to be the best source of phytosterols, with the highest oil content and high concentration of sterols.     

Oxidation of phytosterols in a test food system

The oxidative stability of phytosterols in canola, coconut, peanut, and soybean oils was examined under simulated frying conditions of 100, 150, and 180°C for 20 h. The degree of oxidative decomposition was assessed by the loss of phytosterols, accumulation of phytosterol oxides, and the change in fatty acid profiles. The phytosterol oxides produced in the oils were identified using mass spectroscopy. Oils with higher levels of polyunsaturated fatty acids showed greater amounts of sterol loss; however, the sterol loss was less complete than in the more saturated oils. A greater variety of sterol oxides was observed at the lower temperatures of 100 and 150°C compared to 180°C. This study demonstrates that under conditions similar to frying, there is a loss of phytosterols and polyunsaturated fatty acids. The accumulation of phytosterol oxides may be temperature-limited because of further break-down into products not measurable by typical gas chromatography-mass spectrometry techniques.     

Fucosterol Causes Small Changes in Lipid Storage and Brassicasterol Affects some Markers of Lipid Metabolism in Atlantic Salmon Hepatocytes

Several feeding trials with Atlantic salmon fed naturally high phytosterol concentrations due to dietary rapeseed oil inclusion have shown changes in lipid metabolism and increased hepatic lipid storage in the fish. An in vitro trial with Atlantic salmon hepatocytes was, therefore, performed to study the possible direct effects of phytosterols on lipid storage and metabolism. The isolated hepatocytes were exposed to seven different sterol treatments and gene expression, as well as lipid accumulation by Oil Red O dyeing, was assessed. Fucosterol, a sterol found in many algae species, had an effect on the size of individual lipid droplets, leading to smaller lipid droplets than in the control without added sterols. A sterol extract from soybean/rapeseed led to an increase in the percentage of hepatocytes with visible lipid droplets at 20× magnification, while hepatocytes of both the sterol extract‐treated groups and fucosterol‐treated groups had a larger proportion of their area covered with lipids compared to control cells. Brassicasterol, a sterol characteristic of rapeseed oil, was the only sterol treatment leading to a change in gene expression, affecting the expression of the nuclear receptors, peroxisome proliferator‐activated receptor gamma (pparg) and retinoid X receptor (rxr). The current study thus shows that phytosterols can have direct, although subtle, effects on both hepatic lipid storage and gene expression of Atlantic salmon in vitro.     

Degradation of Phytosterols During Near-Ambient Drying of Rapeseeds in a Thick Immobile Layer

The effect of the drying method applied and subsequent rapeseed storage on changes in phytosterols was determined. After harvest, rapeseeds were dried by the near-ambient method in a thick immobile layer of 2 m and using air heated to a temperature of 60, 80 and 100 °C. Analyses of phytosterol contents were performed immediately after drying and after 6 and 12 months of storage at a temperature of 10 ± 2 °C. Results showed a significant effect of drying conditions, cultivar-specific differences and storage time on the contents of phytosterols. Near-ambient drying of seeds resulted in a reduction in total sterol contents by 6–20 %, while for drying with hot air it was by 14–40 %. The level of sterols decreased by 13–18 % after a 1 year storage of seeds dried by the near-ambient methods. A reduction in 12–22 % in sterols for seeds dried by high temperature occurred after 1 year of storage.