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Presence of Phytosterol Oxides in Crude Vegetable Oils and Their Fate during Refining
Abstract
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
... 4 Refined edible oil freed of phospholipids, free fatty acids, lipophilic pigments, traces of metals, peroxides, and off-flavor components is a product acceptable for consumers. 5,6 An important refining target is the minimal degradation of beneficial compounds (e.g., polyunsaturated fatty acids, phytosterols, tocopherols, and tocotrienols) to achieve a product with good oxidative stability and high nutritional quality. 5,7 Upon chemical refining, a significant decrease of total phytosterols (3−25%) 7 and tocopherols (10−20%) 4 was observed without changes in the distribution of individual compounds. ...
... 5,6 An important refining target is the minimal degradation of beneficial compounds (e.g., polyunsaturated fatty acids, phytosterols, tocopherols, and tocotrienols) to achieve a product with good oxidative stability and high nutritional quality. 5,7 Upon chemical refining, a significant decrease of total phytosterols (3−25%) 7 and tocopherols (10−20%) 4 was observed without changes in the distribution of individual compounds. Alkali neutralization reduced a considerable part of the free phytosterols in the micellar form by liquid−liquid partitioning to the soapstock. ...
... 7,8 Stera-3,5-dienes and disteryl ethers have been identified as major degradation products and important quality markers. 5,8 Verleyen and co-workers observed a sharp increase of stigmasta-3,5-diene (3.7−102.7 mg/kg), stigmasta-3,5,22-triene (1.2−19.7 mg/kg), and campesta-3,5-diene (3.1−46.9 mg/kg) levels after bleaching of corn oil with industrial bleaching earths of a pH value below 3.6 (80−120°C, 30−60 min, 1−2 wt %). 9 Bortolomeazzi et al. investigated the impact of sunflower oil deodorization (180°C, 1 h) on the fate of phytosterols, as well as 5α-, 7α-, and 7βhydroxysitosterol derivatives. ...
Targeted analysis confirmed the presence of new phytosterol degradation products in fully hydrogenated commercial samples. EI-MS, APCI-MS and 1D-NMR experiments let to the identification of 10 novel markers of catalytic hydrogenation, among which 5α-sitostan-3-one, 5α-campestan-3-one, isomers of saturated and monounsaturated steroidal hydrocarbons were reported in edible oils for the first time. Examination of phytosterol degradation mechanism was done by catalytic transfer deuteration technique. Mitigation strategy of potentially detrimental compounds included optimization of processing parameters. The effect of catalyst dosage (≤ 0.1 % based on Ni basis) and temperature region (˂ 180 °C) were the most crucial factors in phytosterol degradation control.
... Some COP can be atherogenic, cytotoxic, mutagenic, cancerogenic . It has been shown that POPs can also have similar biological effects at higher concentrations [Bortolomeazzi et al., 2003]. This issue has become more important with the functional foods which are enriched with high levels of phytosterols due to their cholesterol lowering effects in humans [Ryan et al., 2005]. ...
... POPs are generally present at low levels in foods and vegetable oils, for examples in some samples of vegetable oils it was in the range of 1.5-67 (mg/kg oil) [Bortolomeazzi et al., 2003]. However, due to the fact that there are several prominent phytosterols in plant-based foods that have the potential to be oxidised, their levels can become quite 14 high in processed and fried foods Dutta, 2004). ...
... Phytosterols can also interfere during purification of POPs due to their polarity characteristics and high amounts in unsaponifiable material. However, unlike cholesterol, some of the POPs have similar GC retention times to phytosterols [Bortolomeazzi et al., 2003;Lampi, et al., 2002]. Therefore, if SPE is not able to separate phytosterols from the POP fraction, some of them can overlap during GC analysis [Bortolomeazzi et al., 2003;Lampi et al., 2002] and this can cause error in quantification of the POPs content of samples. ...
Suitable preparative methods in the analysis of phytosterols help to obtain accurate results. Thin-layer chromatography (TLC) is a conventional preparative method in analysis of phytosterols, but this method has some drawbacks. Solid-phase extraction (SPE) is a simple and inexpensive chromatographic method. SPE has been widely used in the preparation of lipid classes such as phytosterols prior to further analyses by GC and GC-MS. Phytosterols comprise a major proportion of the unsaponifiables in vegetable oils. They are divided into three main classes: 4-desmethylsterols, 4-monomethylsterols, and 4,4'-dimethylsterols. Methylsterols usually occur in relatively low amount and therefore it is necessary to separate and enrich them by preparative chromatographic methods before quantification by GC. Phytosterols also occur either in free form or esterified with fatty acids and other conjugates. Separation of free and esterified phytosterols by preparative methods before quantification by GC provides detailed information on their distribution and stability. Phytosterols can also oxidize in the same way as other unsaturated lipids and produce phytosterol oxidation products (POPs) which may have possible toxicity effects at higher concentrations. Separation and enrichment of POPs from bulk lipids is an essential step before quantification and identification by GC or GC-MS. In this chapter, TLC methods and their drawbacks in the preparation of phytosterols before analysis by GC and GC-MS and advantage of SPE method as an alternative method to conventional TLC methods, is discussed.
... Nourooz-Zadeh and Appelqvist (1992) and Lambelet et al. (2003) did not detect any epoxy compounds and triol in deodorized low erucic rapeseed oil. Bortolomeazzi, Cordaro, Pizzale, and Conte (2003) showed that bleaching of oil caused a reduction of hydroxyphytosterols, but triol underwent decomposition and α-epoxyderivative formed dihydroxy compounds. ...
... Among the different phytosterol oxidation products, the 7keto derivative was the most stable, whereas the hydroxyl, epoxy and triols undergo a more or less decomposition (Bortolomeazzi et al., 2003). ...
Thermo-oxidative changes were assessed during simulated storage and frying of refined rapeseed oil (RRO). When RRO was heated at 60 and 180 °C for 8h phytosterols content decreased by 10 and 40%, however tocopherols disappeared by 7 and 30%, respectively. The oxyphytosterols content increased during first 2 h of heating at both temperatures to 55 and 211 μg/g, however extended heating resulted in reduction of these compounds to 38 and 169 μg/g, respectively. Total polar compounds were observed in RRO heated at 60 and 180 °C for 8 h at 12 and 42%, respectively. Polar compounds were consisted by: free fatty acids (FFA), low molecular weight (LMW) compounds including monomers, while non-polar fraction contained LMW and monomers formed during heating at 60 °C. Non-polar fraction formed during heating of RRO at 180 °C for 8h which contained monomers and LMW compounds. Among volatile compounds, four carbonyls and an alcohol were detected in heated RRO.
... POPs were detected in vegetable oils or samples with phytosterols as ingredients. Levels in vegetable oils, being in the low ppm range, were consistent to a previously published study [15]. Up to this date, no data have been available on POPs levels in cosmetics enriched with phytosterol or the unsaponifiable matter of vegetable oils. ...
... In cosmetics, we found COPs in lanolin containing products in the low percent range (up to 2.3%) being in line with a previous study [6]. POPs levels determined in our study (1-25 ppm) were in the order of magnitudes published for food [15]. In addition, the in-house generation of LOPs and DOPs was helpful in allocating unknown peaks in lanolin containing samples. ...
Sterol oxidation products (SOPs) are linked to several toxicological effects. Therefore, investigation of potential dietary uptake sources particularly food of animal origin has been a key issue for these compounds. For the simultaneous determination of oxysterols from cholesterol, phytosterols, dihydrolanosterol and lanosterol in complex cosmetic matrices, planar solid phase extraction (pSPE) was applied as clean-up tool. SOPs were first separated from more non-polar and polar matrix constituents by normal phase thin-layer chromatography and then focussed into one target zone. Zone extraction was performed with the TLC-MS interface, followed by gas chromatography-mass spectrometry analysis. pSPE showed to be effective for cleaning up cosmetic samples as sample extracts were free of interferences, and gas chromatographic columns did not show any signs of overloading. Recoveries were between 86 and 113% with relative standard deviations of below 10% (n = 6). Results of our market survey in 2016 showed that some cosmetics with ingredients of plant origin contained phytosterol oxidation products (POPs) in the low ppm range and therefore in line with levels reported for food. In lanolin containing products, total SOPs levels (cholesterol oxidation products (COPs), lanosterol oxidation products (LOPs), dihydrolanosterol oxidation products (DOPs)) being in the low percent range exceeded reported levels for food by several orders of magnitudes.
... 7-keto, 7-α/β-hydroxy and epoxy derivatives, or 6/7-sitosterol derivatives, were also found in olive oil heated between 150°C and 200°C, up to 6 h (D'Evoli et al., 2006). Untreated olive oil showed trace amounts (2-6 mg/g) of oxysterols (Bortolomeazzi, Cordaro, Pizzale, & Conte, 2003). Oxysterols can have anti-inflammatory or pro-inflammatory roles in the human body, depending on the tissue and the cellular context. ...
Edible oils are essential energy providers in human diets. However, these oils are also a natural source of bioactive compounds and phytonutrients. This chapter will provide an overview of the bioactive lipids from olive, palm, and fish oils. The nutritional and health benefits of these oils are well documented and frequently associated with their minor lipids, which exhibit significant positive effects on human health. Each oil displays a unique set of lipid families, called lipidome, with bioactive lipids belonging to different classes and with diverse structures and functions. Bioactive lipids, such as polar lipids, prenol lipids, or polyunsaturated fatty acids, have been reported to have health benefits and to prevent the onset of several disorders. The knowledge of the bioactive compounds present in these edible oils is used to develop novel biotechnological applications that incorporate these lipids into functional foods, cosmetic or pharmaceutical innovations to improve human health and well-being.
... It is important to note that to fully validate the above-mentioned hypotheses, a detailed fatty acid unsaturation pattern as well as the analysis of possible antioxidant properties of food ingredients have to be planned in the continuation of the study. Literature data on POPs, however, strengthen this single observation, showing how differences in POPs could be explained based on their fatty acid composition, with more saturated vegetable oils containing lower POPs, being less prone to oxidation [48]. Indeed, the oxidative instability of cholesterol could be further favored by the presence of larger amounts of polyunsaturated fatty acids (PUFA) in the cell membrane: in real food systems, it has been shown that a higher unsaturation degree of the lipid matrix tends to promote cholesterol oxidation [10,49]. ...
Cholesterol oxidation products (COPs) of non-enzymatic origin are mainly found in meat, fish, eggs and milk, mostly originating from the type of feeding, processing and storage. To verify the significance of COPs as biomarkers of cholesterol autoxidation and milk freshness, we quantified them in chocolates containing whole milk powders (WMPs) of increasing shelf-lives (i.e. 20, 120, and 180 days). Non-enzymatic total COPs (both free and esterified) ranged from 256.57 ± 11.97 to 445.82 ± 11.88 ng/g, increasing proportionally to the shelf-life of the WMPs, thus reflecting the ingredients’ freshness. Based on the expected theoretical COPs, the effect of processing was quantitatively less significant in the generation of oxysterols (41–44%) than the contribution of the autoxidation of the WMPs over time (56–59%), pointing to the shelf-life as the primary determinant of COPs. Lastly, we quantified COPs of major commercial milk chocolates on the Italian market, which followed a similar distribution (from 240.79 ± 11.74 to 475.12 ± 12.58 ng/g). Although further replications of this work are needed, this study reports preliminary results and a practical example of a first application of non-enzymatic COPs as markers to further quantify and characterize the nutritional quality and freshness, not only of ingredients but also of composite products.
... EVOO and VOO are the most expensive and healthpromoting of all the categories of olive oils. They are the supernatants of the fresh juice obtained from olives by crushing, malaxation of the formed paste, and centrifugation, which do not lead to alterations in the oil, while LOO is intended for refining or technical use, not being possible its direct use for human consumption (Bortolomeazzi et al., 2003;Commission, 1991). ...
Advances in olive oil research have blossomed in recent years, and articles on olive oil have been widely recorded. However, a comprehensive and systematic bibliometric analysis of olive oil research has not been completed. Through a systematic literature search, we consolidated 7003 papers from the Web of Science Core Collection database. This bibliometric analysis was carried out to examine the article growth, geographical distribution, and analysis of journals/research areas/authors/keywords. The results revealed the following: (1) Exponential growth was shown in the annual number of publications (R² = 0.9791). The geographical distribution is biased to a few countries, especially Spain, Italy, and Greece in the Mediterranean region, placing a larger focus on olive oil research. (2) Quality analysis‐related research acted as a link between keyword groups, the disease prevention and treatment‐related research of olive oil received decreasing interest, while authentication and quality analysis‐related research of olive oil showed the opposite trend, by‐products‐related research of olive oil got waving interests over the whole period. (3) Improvement is needed in the degree of olive oil with international collaboration. This is the first time that hotspots and trends in olive oil research have been shown.
... Although POPs are poorly absorbed in the small intestine, they are suggested, like COPs, to be atherogenic, and therefore their intake via foods requires attention [9,44]. However, it has to be noted the concentrations of total POPs in heat-processed thigh muscle being in the range between 3.5 and 4 pmol per g (equivalent to 0.875 to 1.00 µg/100 g) of thigh muscle are much lower than in other foods, such as plant oils (0.5 to 7 mg/100 g in different samples of corn oil or sunflower oil), or products baked with margarines, such as cookies or muffins (0.2-0.7 mg/100 g) [13,45]. Thus, the consumption of heat-processed broiler meat is uncritical with respect to the ingestion of POPs. ...
In this study, the hypothesis that supplementation with methionine (Met) as DL-Met (DLM) in excess of the National Research Council (NRC) recommendations improves the antioxidant system in broilers was investigated. Day-old male Cobb-500 broilers (n = 72) were divided into three groups which were fed a control diet or diets supplemented with two levels of DLM in which the concentrations of Met + Cys exceeded the recommendations of NRC by 15–20% (group DLM 1) or 30–40% (group DLM 2), respectively. The three groups of broilers did not show differences in body weight gains, feed intake, and feed conversion ratio. However, broilers of groups DLM 1 and DLM 2 had higher concentrations of glutathione (GSH) in liver and thigh muscle and lower concentrations of cholesterol oxidation products (COPs) in heat-processed thigh muscle than broilers of the control group. Concentrations of several oxidation products of phytosterols in heat-processed thigh muscle were also reduced in groups DLM 1 and DLM 2; however, the concentration of total oxidation products of phytosterols was not different between the three groups. The study shows that DLM supplementation improved the antioxidant status due to an increased formation of GSH and reduced the formation of COPs during heat-processing in thigh muscle.
... In the case of PS compounds, it is also possible that the "gap" partly originates from the formation of steradienes and -trienes, i.e., steroidal hydrocarbons with two or three double bonds in the ring structure. These structures are formed at high temperatures, e.g., through the dehydration of native sterols or 7-ketosterols, with a subsequent subtraction of the OH group from position 3, or through the elimination of water molecules from 7-hydroxysterols (Bortolomeazzi et al. 2003;Soupas et al. 2005). Moreover, it was mentioned that dimers and polymers are formed under thermal conditions. ...
Fried foods, both deep-fried and pan-fried, are enjoyed by people worldwide. Frying is one of the main factors leading to formation of phytosterols (PS) oxidation products (POP) in vegetable oils. The aim of this study was to measure the oxidation of β-sitosterol (24α-ethyl-5-cholesten-3β-ol) and campesterol (24α-methyl-5-cholesten-3β-ol) in commercial sunflower oil (SFO) during deep- and pan-frying of French fries for different periods (30, 60, 120 and 240 min). The total amount of PS in SFO was 4732 μg/g, wherein the major PS were β-sitosterol and campesterol. The results of POP were confirmed by the GC-MS analysis that monitored the formation of oxides during frying. Upon frying, total PS content decreased whereas the highest decrease was measured after 240 min of frying. The oxidative stability (OS) of different sitosterol and campesterol during both frying methods was evaluated. In general, pan frying resulted in more PS oxidation than deep frying. β-Sitosterol oxides predominated while campesterol oxides were formed to a lesser extent. 7-Ketositosterol, followed by 7β-hydroxysitosterol, 5,6-epoxy derivatives and 7α-hydroxysitosterol were the main POP induced during frying. The proportion of 7-keto derivatives decreased during frying while the proportion of 7β-hydroxy derivatives increased. The formation of POP might be a limiting factor for frying in SFO for long periods.
... The variation of phytosterol contents in commercial CBRO might be due to the different rice varieties and refining/processing parameters. 20,32,33 Although more than 200 types of phytosterols have been reported in oil from plants, 34 the major phytosterols in rice bran oil have been found to be β-sitosterol, campesterol and stigmasterol. 9,35 ...
Phytosterols, α-tocopherol and γ-oryzanol are scientifically recognized as major health promoting compounds found in cold-pressed rice bran oil (CRBO). This study aimed at encapsulating CRBO using a niosome delivery system. Water soluble CRBO niosomes approximately 200 nm in size were generated, remained stable for up to four weeks at 4 °C, and exhibited an encapsulation efficacy >80%. CRBO niosome controlled release was possible until the small intestinal phase in in vitro digestion. To our knowledge, this is the first study indicating a globular morphology of the CRBO niosomes and the location of CRBO. M0, M1, and M2 macrophage phenotypes were stimulated with 25, 50, and 100 μg ml-1 of in vitro digested CRBO niosomes. Gene expression profiles for each macrophage cell were obtained via M1 and M2 marker gene analysis. Changes of M0, M1 and M2 macrophage gene expression profiles occurred after CRBO stimulation and were visualized via principal component analysis (PCA). The results revealed a clear M1 macrophage conversion towards M0 in a digested CRBO niosome dose dependent manner, while this was not the case with M0 and M2 macrophages. Our findings indicated that CRBO niosomes have an ability to reverse M1 pro-inflammatory macrophage transformations back to resting M0 macrophages. Moreover, this study also showed future potential uses of CRBO niosomes, containing rice phytosterols and a co-surfactant, as fabricating materials to deliver and to control the release of oil soluble bioactive compounds for water soluble functional ingredient applications.
... Besides, instead of the ones we analyzed, other oxides could have been formed, such as 6- hydroxy, 20-hydroxy, 22-hydroxy, dienes, trienes . . . [29,36,63]. Moreover, SOPs derived from cholesterol and stigmasterol could have decomposed to form oligomers, polymers and other compounds characteristic of advanced stages of oxidation [29,39], yielding an overall lower balance than for campesterol and sitosterol derived oxides. ...
Dietary sterols are nutritionally interesting compounds which can suffer oxidation reactions. In the case of plant sterols, they are being widely used for food enrichment due to their hypocholesterolemic properties. Besides, cholesterol and plant sterols oxidation products are associated with the development of cardiovascular and neurodegenerative diseases, among others. Therefore, the evaluation of the particular factors affecting sterol degradation and oxysterols formation in foods is of major importance. The present work summarizes the main results obtained in experiments which aimed to study four aspects in this context: the effect of the heating treatment, the unsaturation degree of the surrounding lipids, the presence of antioxidants on sterols degradation, and at last, oxides formation. The use of model systems allowed the isolation of some of these effects resulting in more accurate data. Thus, these results could be applied in real conditions.
... A number of studies have dealt with the prevention of fatty acid degradation through antioxidant addition, after different cooking and storage conditions [8, 14–16]. A possible interaction between cholesterol oxidation and the surrounding fatty acids has been proposed by several authors as a factor that modulates cholesterol oxidation susceptibility, although no consensus on the subject has been found [17] [18] [19] [20]. Foods are usually complex matrices where interferences among several components may hamper a clear view about the mechanisms of cholesterol oxidation. ...
Harmful health effects have been attributed to cholesterol oxidation products (COPs). Factors that modulate their formation in foods are light, oxygen, heat, and food matrix (such as antioxidants content or unsaturation degree of lipids), among others. The objective of this work was to assess the effectiveness of an extract obtained from Solanum sessiliflorum (mana‐cubiu) (MCE) as a potential inhibitor of cholesterol oxidation under heating conditions. The influence of free DHA presence in the system was also evaluated. Results showed that MCE inhibited cholesterol degradation (44 vs. 18% without and with MCE, respectively) and reduced ninefold COPs formation in the absence of DHA. However, when DHA was present, the MCE was not effective toward cholesterol oxidation. In this case, MCE showed its antioxidant effect protecting DHA from degradation (89 vs. 64%).
Practical applications: Antioxidant properties of this solvent free natural extract make MCE a potential good ingredient in food products containing highly polyunsaturated lipids to protect them from oxidation and in food products lacking polyunsaturated lipids to protect cholesterol from oxidation.
Mana‐cubiu inhibits cholesterol oxidation in absence of DHA.
... In the case of PS compounds, it is also possible that the "gap" partly originates from the formation of steradienes and -trienes, i.e., steroidal hydrocarbons with two or three double bonds in the ring structure. These structures are formed at high temperatures, e.g., through the dehydration of native sterols or 7-ketosterols, with a subsequent subtraction of the OH group from position 3, or through the elimination of water molecules from 7-hydroxysterols (Bortolomeazzi et al. 2003;Soupas et al. 2005). Moreover, it was mentioned that dimers and polymers are formed under thermal conditions. ...
... A number of studies have dealt with the prevention of fatty acid degradation through antioxidant addition, after different cooking and storage conditions[8,141516. A possible interaction between cholesterol oxidation and the surrounding fatty acids has been proposed by several authors as a factor that modulates cholesterol oxidation susceptibility, although no consensus on the subject has been found17181920. Foods are usually complex matrices where interferences among several components may hamper a clear view about the mechanisms of cholesterol oxidation. ...
... For phytosterols to be introduced into aqueous-based foods, they need to be either suspended or emulsified. Phytosterols are also susceptible to chemical degradation (oxidation) during storage (Bortolomeazzi, Cordaro, Pizzale, & Conte, 2003;Cercaci, Rodriguez-Estrada, Lercker, & Decker, 2007;Dutta, 1997;Lambelet et al., 2003;Soupas, Juntunen, Lampi, & Piironen, 2004). At present, it is unclear whether oxidized forms of phytosterols lose their bioactivity or have some toxicity as has been observed for oxidized cholesterol. ...
... During refining of fats and oils, the content of total sterols decreases due to degradation and formation of products through isomerization, polymerization, formation of hydrocarbons or sterenes and SOPs [20]. Reportedly bleaching can cause losses of sterols and POPs during vegetable oil refining [21]. The contents of SOPs in these two samples do not show any consistent pattern, and no literature data are available to compare with the present results. ...
Cholesterol and phytosterols are generally present in foods at ppm levels and they can generate many oxidation products, i.e. oxysterols. The oxysterols comprise only a small percentage of unoxidized sterols. Reliable quantitative data on these compounds requires reasonably good separation by capillary column GC. The present study attempts to overcome the difficulties involved in separating many common oxysterols generated from cholesterol, brassicasterol, campesterol, stigmasterol, and sitosterol by coupling two high-resolution GC capillary columns. The columns, DB-17MS and DB-35MS, were coupled separately to a DB-5MS column. Total separation time of the authentic samples of oxysterols was 41 min for the DB-35MS/DB-5MS and 44 min for the DB-17MS/DB-5MS coupled columns. Two oil samples EBE1 and EBE2 extracted from exhausted bleaching earth collected from Europe were analyzed for oxysterol content by using these column combination systems. Both systems showed similar quantitative results; the total levels of oxysterols in these samples ranged from 2 to 3 mg/100 g. The prominent oxysterols were as follows: 7α-hydroxysterols (0.29–0.49 mg/100 g), 7β-hydroxysterols (0.13–0.68 mg/100 g) and 7-ketosterols (0.63–0.69 mg/100 g).
... Plant sterol content has been determined in several milk beverages enriched with free or esterified PS (Laakso, 2005;Menéndez-Carreño et al., 2008;Saraiva, Castilho, Martins, Noronha da Silveira, & Ramos, 2011;Soupas et al., 2006), and in orange juices fortified with sterol esters (Mezine, Zhang, Macku, & Lijana, 2003) and with a sterol concentrate (Clement, Hansen, Costin, & Perri, 2010). However, few studies to date have identified and quantified POPs in other foods, and the existing publications mostly focus on high lipid content matrixes (Bortolomeazzi, Cordano, Pizzale, & Conte, 2003;Dutta, 2002;Soupas, Huikko, Lampi, & Piironen, 2007). In the case of dairy matrixes, 7-ketositosterol contents have been determined as an indicator of phytosterol oxidation in milk-based infant foods (Zunin, Calcagno, & Evangelisti, 1998), and several POPs have been quantified in milk-and cereal-based infant foods (García-Llatas et al., 2008). ...
Two different plant sterol (PS) sources (free PS from tall oil and esterified PS from vegetable oils) were used for manufacturing two types of functional beverages (fruit and milk-based fruit beverages), and their PS and phytosterol oxidation product (POP) contents were determined. Gas chromatography–tandem mass spectrometry (GC–MS/MS) was used for identification and gas chromatography–flame ionization detection (GC–FID) for quantitation purposes. Brassicasterol, campesterol, campestanol, stigmasterol, β-sitosterol and sitostanol were the quantified PS, conforming a profile in order with current legislation. The relative percentages of PS differed according to the enrichment source involved, though the enrichment levels (g/100 g beverage) were of the same order (1.77 from tall oil and 1.84 from vegetable oils). Only POPs from β-sitosterol (the prevalent PS in the analyzed beverages) were detected — the predominant representative being 7β-hydroxysitosterol (39–58.5% of total POP content). The following POPs were quantified: 7α-hydroxy, β-epoxy, α-epoxy, and 7-ketositosterol, yielding a total POP content ranging between 42.9 and 57.4 mg/100 g of PS. No statistically significant differences (p > 0.05) in total and individual POP content according to the source of PS were found. The mean β-sitosterol oxidation percentage was < 0.07%, which reflected a low PS oxidation extent, though manufacture was on a laboratory scale regardless of the PS source used in enrichment of the functional beverages. These functional drinks therefore can be regarded as healthy food products and as an adequate PS vehicle as well.
A 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.
Scope:
Phytosterols (PS) and sterol oxidation products are key dietary factors influencing atherosclerosis besides cholesterol, although the mechanisms remain elusive. Recently, single-cell RNA sequencing (scRNA-seq) has revealed the heterogeneity of multiple cell types associated with complex pathogenesis in atherosclerosis development.
Methods and results:
Here, we performed scRNA-seq to investigate the alterations in the aortic cells from ApoE-/- mice induced by diet-derived PS or two sterol oxidation products, phytosterols oxidation products (POPs) and cholesterol oxidation products (COPs). We identified four fibroblast subpopulations with different functions, and immunofluorescence demonstrated their spatial heterogeneity, providing evidence that suggested the transformation of smooth muscle cells (SMCs) and fibroblasts in atherosclerosis. The composition and gene expression profiles of aortic cells changed broadly in response to PS/COPs/POPs exposure. Notably, PS exhibited an atheroprotective effect where different gene expressions were mainly found in B cells. Exposure to COPs accelerated atherosclerosis and resulted in marked alternations in myofibroblast subpopulations and T cells, while POPs only altered fibroblast subpopulations and B cells.
Conclusion:
Our data elucidates the effects of dietary PS/COPs/POPs on aortic cells during atherosclerosis development, especially on the newly identified fibroblast subpopulations. This article is protected by copyright. All rights reserved.
Phytosterols have health benefits; however, they are partially removed during the bleaching of corn oil. We evaluated the chemical conversion of free phytosterols (FPs) during bleaching. FP degradation accelerated with increased time and temperature, following a first-order kinetic model. In the n-heptane system, air and activated clay promoted the chemical conversion of the FPs. Sterenes formation was analysed under different conditions using a zero-order kinetic model. The apparent activation energies revealed sterene formation decreasing in the following order: campesta-3,5-diene ≈ stigmasta-3,5,22-triene > stigmasta-3,5-diene. Isomers of the above were not detected, indicating that these sterenes were the only primary products of FPs. The desorption test indicated that the FP loss from corn oil was not only due to FPs being adsorbed the activated clay, but also FPs adsorbed at acidic activated sites being degraded. This study presents a vital scientific foundation for retaining FPs to develop healthier and more nutritious oils.
Ochratoxin A (OTA) is an important mycotoxin detected in edible oil, and it can be effectively removed by classical edible oil refining processes. However, the fate of OTA in the refining process has not been reported. In this study, we systematically tracked the OTA changes during the oil refining process by fortifying 100 μg/kg OTA in crude rapeseed oil. Results showed that about 10.57%, 88.85%, and 0.58% of OTA were removed during the degumming, deacidification, and decolorization processes. Among them, 16.25% OTA was transferred to the byproducts, including 9.85% in degumming wastewater, 5.68% in soap stock, 0.14% in deacidification wastewater, and 0.58% in the decolorizer; 83.75% OTA was found to transform into the lactone ring opened OTA (OP-OTA) during the deacidification stage, which is attributed to the hydrolysis of the lactone ring of OTA in the alkali refining. The OP-OTA was verified to distribute in the soap stock, and small amounts of OP-OTA could be transferred to deacidified wastewater when the OTA pollution level reached 500 μg/kg in crude rapeseed oil. The OP-OTA exhibited strong toxicity, especially nephrotoxicity, as reflected by the cell viability assay and in silico toxicity. Therefore, the safety of the soap stock processing products from OTA-contaminated rapeseed deserves attention.
Rice bran oil contains a significant quantity of phytosterols that have various active functions and are natural active substances beneficial to humans. It is well known that deodorization during refining affects the quality of rice bran oil. However, changes in phytosterols fraction caused by stripping with nitrogen compared to water vapor remain unexplored. We measured phytosterols in rice bran oil after deodorization with nitrogen and water vapor. The variations in sitosterol fraction, which accounts for the highest percentage of phytosterols in rice bran oil, were analyzed by Gas chromatography (GC), Fourier transform infrared spectrometer (FTIR), and Nuclear magnetic resonance (NMR). Results showed that using nitrogen as the stripping gas was more suitable for deodorization. It promoted the formation of phytosterol esters, reduced the production of phytosterol oxidation products and improved the oil quality. This study provides a theoretical basis for improving the industrial production quality of rice bran oil.
Plant sterols, also called phytosterols, are plant-derived compounds that structurally and functionally resemble cholesterol in mammals. These compounds are known to lower the total and low-density lipoprotein (LDL-C) fractions of cholesterol in humans. For a number of decades, various food products have been enriched with phytosterols and phytostanols as free compounds, or as their esters with fatty acids. The quality of the raw material, as well as the conditions of food processing and storage, affects the degradation of phytosterols and the formation of the various derivatives. The speed at which they degrade in model systems, as well as in raw materials and food products, is associated with autoxidation and photooxidation reactions. Phytosterol oxidation products (POPs) are derivatives formed during thermo-oxidation of sterols; they then undergo decomposition to volatile compounds and oligomers. The toxic properties of POPs in humans have been demonstrated, and their biological properties are based on literature data. The chemical structure of phytosterols, their biological properties, and their content in food products are presented in this work.
The study reports the changes in fatty acid and sterol composition; as well as volatile and 3-MCPD ester content of two commercial vegetable oils at different steps of refining process. Canola and corn oils were collected from neutralization, bleaching, winterization and deodorization stages of a chemical refining plant and oil samples were evaluated for fatty acid, sterol, volatile compounds and 3-MCPD ester contents. Results have shown that total sterol contents decreased throughout the process by 15.22% and 42.57% for canola and corn oils, respectively. Total volatiles of canola oil decreased gradually during refining, however corn oil had significant increases at bleaching step. 3-MCPD ester contents were between 0.19-0.26 mg kg-1 for corn and 0.20-0.48 mg kg-1 for canola oils. Deodorization was found to be the most influential refining stage for formation of 3-MCPD esters.
Oxidative deterioration of vegetable oils is of great importance in the food industry. In China, vegetable oils produced via thermal pretreatment are popular owing to their strong oil flavor and enhanced yield. Here, we review: (i) the currently employed thermal treatment methods of oilseeds before oil extraction; (ii) effects of thermal treatments on the physicochemical properties, contents of minor lipid components, and oxidative stability of vegetable oils; and (iii) Maillard model systems that are related to oil and oilseed chemistry. Among the thermal pretreatment technologies, microwave and infrared radiations are promising, but these are not performed on the same large production scales as roasting. For most oilseeds, thermal treatments increase the yield of extracted oil and content of minor lipid compounds in the oil, such as polyphenols, tocopherols, and phytosterols. In addition, some Maillard reaction products (MRPs) generated by heating oilseeds have been extracted. The presence of both minor lipids and MRPs in the oil confers improved oxidative stability. However, the mechanism or relationship between thermal treatment and oxidative stability is yet to be clearly elucidated because vegetable oil oxidation is dependent on variables such as unsaturation, concentration and types of minor lipid components, MRPs, and the potential synergistic effects of these components. Recently, several Maillard reaction models related to thermally treated oilseeds have been established, suggesting that MRPs play a critical role during oxidation. However, comprehensive identification of antioxidants and the mechanism by which they inhibit oxidation are lacking. Future research can be performed to establish models that would help elucidate the antioxidative mechanisms of MRPs for more oilseeds. Using these models, it will be possible to predict the oil quality after processing, based on the presence of MRPs and oil chemistry.
Phytosterols are commonly found in vegetable oils and possess health benefits for humans. While investigating the chemical conversion of stigmasterol at deodorisation temperatures, gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) experiments led to the identification of 5-ethyl-6-methyl-3-heptene-2-one, 3-hydoxy-steroid, 3-ketostigmasterol, and 3,7-diketostigmasterol as by-products. The identification of these compounds assisted in the interpretation of the stigmasterol oligomers characterised by high-pressure size exclusion chromatography (HPSEC). A similar analysis was conducted in stripped corn oil at the deodorisation temperatures. As such, 5-ethyl-6-methyl-3-heptene-2-one, 3-hydoxy-steroid, 3-ketostigmasterol and 3,7-diketostigmasterol were also detected in stripped corn oil, while the contents of 3-hydoxy-steroid and 5-ethyl-6-methyl-3-heptene-2-one were higher than those of 3-ketostigmasterol, as revealed by quantum chemical simulations. In addition, stripped corn oil exhibited the characteristic of preventing stigmasterol degradation below 200 °C, whereas it enhanced the chemical conversion (such as esterification and degradation) of stigmasterol at higher temperatures.
Phytosterols, which are naturally occurring in plants, have excellent nutritional and health values on lowering both the blood cholesterol level and the risk of cardiovascular diseases. Edible oils are the main source of daily intake of phytosterols, whereas the properties of phytosterols may vary a lot depending on their sources. During the processing of edible oil including refining and frying, phytosterol's content fluctuates, which influences the properties of the final product. Phytosterols and their derivatives undergo physical migration between different phases and chemical conversion during the processing, which reduces the quality and the commercial value of edible oils. Therein, the loss of phytosterols is the major concern in the process of neutralization and deodorization. In addition, oxidation and thermal degradation of phytosterols occur simultaneously during frying, which also reduces the content of phytosterols. Nevertheless, the oil matrix has a promoting or an inhibitory effect on the thermal oxidation of phytosterols. Therefore, various efforts have been devoted to analyzing and improving the remaining contents of phytosterols in edible oil. Regardless of the processing method, temperature plays an important role in the loss of phytosterols. At present, the main analysis methods of phytosterols include gas chromatography and liquid chromatography, in which the pretreatment of different types of phytosterols is also a crucial step. This review focused on the following topics comprehensively: (i) the distribution of phytosterols in the oil-containing plants and edible oils during the refining processing; (ii) the pretreatment and analysis methods of various phytosterols (free phytosterols, phytosteryl fatty acid esters, phytosteryl glycosides and acylated phytosteryl glycosides); (iii) the variation of phytosterols in process of esterification and oxidation, storage and so on. The study also proposed that the investigation in the loss and safety of phytosterols during processing of the vegetable oils should be proceeded further in combination with efficient and accurate chromatography methods.
Arachis hypogaea, known as the groundnut or peanut, is an annual herbaceous legume grown in tropical and temperate areas of the world. Peanuts are a composite food consisting of a wide variety of nutrients, such as carbohydrates, proteins, lipids, vitamins, minerals and a good dose of fiber. Bioactive compounds have also been isolated from peanuts that include flavonoids, phytosterols, amino acids, and stilbenes. Large-scale clinical studies have shown that regular peanut consumption has a positive effect on cardiovascular diseases, and Alzheimer's. These bioactive compounds also have anti-inflammatory, antioxidants, anticancer, and antitumor activities. Potential health concerns regarding peanuts include allergies and contamination with aflatoxins. Peanuts are widely used in the food industry for the production of flour, protein concentrates and isolates, confectionaries, oils and beverages.
Considering the importance of the edible oils in the diet and the increasing demand for row and unprocessed oils, in this research the chemical properties of oils obtained from the cold press and solvent extraction of three different varieties of sesame seed including fatty acids composition, chlorophyll, carotenoids, sterol, tocopherol and aflatoxin values were determined. The peroxide value of oils in two conditions of thermal accelerated and optical oxidation was also compared. The results indicated that the extraction efficiency was the highest by using solvent for extraction (58.13%) and oleic acid and linoleic acid are the predominant fatty acids in sesame. There was a significant difference in total sterol, chlorophyll and carotenoid contents of oils extracted by cold pressing, solvent extraction and refined oils (P <0.05). The highest and lowest levels of total tocopherol were related to solvent extracted and refined oils, which were equivalent to 759.18 and 437.4 mg/kg, respectively. The peroxide value increased in accelerated light and temperature conditions over time, and the effect of light on the increasing of the peroxide value was higher than the temperature. The amount of aflatoxin in the extracted oil samples was below the detection limit (ppb 0.01). The results showed that the oils produced by cold pressing methods contain more micronutrients and antioxidants than the refined oils but have shorter shelf lives in unfavorable conditions.
Considering the importance of the edible oils in the diet and the increasing demand for row and unprocessed oils, in this research the chemical properties of oils obtained from the cold press and solvent extraction of three different varieties of sesame seed including fatty acids composition, chlorophyll, carotenoids, sterol, tocopherol and aflatoxin values were determined. The peroxide value of oils in two conditions of thermal accelerated and optical oxidation was also compared. The results indicated that the extraction efficiency was the highest by using solvent for extraction (58.13%) and oleic acid and linoleic acid are the predominant fatty acids in sesame. There was a significant difference in total sterol, chlorophyll and carotenoid contents of oils extracted by cold pressing, solvent extraction and refined oils (P <0.05). The highest and lowest levels of total tocopherol were related to solvent extracted and refined oils, which were equivalent to 759.18 and 437.4 mg/kg, respectively. The peroxide value increased in accelerated light and temperature conditions over time, and the effect of light on the increasing of the peroxide value was higher than the temperature. The amount of aflatoxin in the extracted oil samples was below the detection limit (ppb 0.01). The results showed that the oils produced by cold pressing methods contain more micronutrients and antioxidants than the refined oils but have shorter shelf lives in unfavorable conditions.
Phytosterols (plant sterols and stanols) and waxes are a part of the minor components of vegetable fats and oils. Stanols are the saturated form of plant sterols. Phytosterols (PS) are isoprenoid derivatives, which are essential compounds of biological membranes. They are structurally similar to cholesterol and control fluidity of eukaryote membranes and have role in the synthesis of hormonal sterols. PS are a major part of the unsaponifiable fraction of vegetable oils. They can be found either in free or esterified forms. PS are classified into three groups according to the presence or absence of methyl group at their C4 position, namely desmethylsterols (without methyl group), monomethylsterols (with one methyl group), and dimethylsterols (with two methyl groups). Generally, desmethylsterols comprise a high proportion of PS. PS have antioxidative properties and also can be used in vegetable oil authenticity. Consumption of PS through diet has beneficial effects on human health, such as lowering of LDL cholesterol and prevention of various types of cancers. However, PS like other lipids can be oxidized, and their oxidation products are important from the nutritional point of view. Waxes naturally occur in vegetable oils. They are present in saponifiable fraction and can be removed with dewaxing/winterization process, which is necessary in salad oils. Waxes have different edible and nonedible applications. This article reviews plant sterols, stanols and waxes structure, content in vegetable oils, changes in the effects of various processes, applications, analytical aspects, and future prospects.
Oxyphytosterols are similar to oxycholesterols in structure, and they exhibit pro-atherogenic properties. Recently, more interests were focused on the metabolism of oxyphytosterols for their increasing intake from phytosterol-enriched food. In this review, we discussed the origin, absorption, distribution, and transport of oxyphytosterols in vivo and their biological effects in humans. The two dominant oxyphytosterols in human plasma are 7-keto-sitosterol and 7-keto-campesterol, but their origins are unclear. It is suggested that oxysitosterols are formed to eliminate sitosterol from tissue to the blood stream. Aside from the pro-atherogenic, oxyphytosterols also exhibit pro-inflammatory properties and antiviral activity against equine herpesvirus 1. Further research is needed to investigate the physiological and pathological role of oxyphytosterols in humans.
We examined the effect of the butter and margarine contents in dough on the lipid composition of deep-fried doughnuts. An increasing content of butter or margarine in the dough increased the absorption of frying oil and the efflux of butter or margarine from the dough during the deep-frying procedure, and also increased the efflux of cholesterol from the dough. Differing butter or margarine content in the dough did not result in a differing fatty acid composition in the resulting doughnuts due to the higher efflux of fatty acids from the dough.
Sterols, minor compounds present in dietary fat, comprise a major portion of the unsaponifiable matter of most vegetable oils. They are mainly present as free sterols and esters of fatty acids, in addition to sterol glucosides and acetylated. Vegetable oil sterols are collectively known as plant sterols or phytosterols. Sterols vary with the origin of the fat and are affected by food processing. Cholesterol is the main animal sterol, while β-sitosterol, campesterol, stigmasterol, brassicasterol, avenasterol, and stigmastenol are major plant sterols present in vegetable oils at much higher levels than cholesterol is in animal fats. Sterols share a similar chemical structure that undergoes oxidation in the presence of oxygen. This chapter provides an overview of the formation, analysis, and health effects of oxidized sterols in frying fat. It reviews the techniques used for the analysis of sterol oxidation products in food and biological matrices and discusses the health implications of phytosterol-oxidized products in addition to those reported for cholesterol-oxidized products. Oxidation products of cholesterol are of considerable interest because of their possible effects on human health. As more vegetable oil is used for cooking, it is necessary to consider the occurrence and effect of plant sterol oxides. This became particularly apparent when it was observed that vegetarians absorbed more phytosterols than did those on non-vegetarian diets. This implies that the effects could be greater than previously perceived. Work with oxidation products of plant sterols has expanded due to the availability of analytical methods.
During thermal processing of sterols, complex mixtures of sterol oxidation products may be formed. Here, a new method for the separation and detection of such products is described. The method is based on normal-phase liquid chromatography (NPLC) for separation and atmospheric-pressure photoionization–mass spectrometry (APPI–MS) for detection. The method was optimized using commercial cholesterol oxidation products and tested on an experimentally derived mixture of phytosterol oxidation products. The investigated parameters include solvent and dopant selection, dopant concentration, polar modifiers, the type of stationary phase, and flow rate.
Best chromatographic separation and highest sensitivity were achieved using a diol-bonded silica column, employing a solvent system consisting of hexane and isopropanol. The dopant of choice was chlorobenzene, added post-column to the solvent stream at 10% of the flow rate.
The developed NPLC–APPI–MS method proved to be a valuable tool for the separation and detection of sterol oxidation products.
Conversion of agricultural wastes into biofuel and other value-added bio-products has not only benefited the environment but also helped reduce the dependency on natural resources leading to improved lifestyle of humankind. Palm oil wastes (OPW) comprising both solid and liquid residues from the palm oil industry are among the most abundant agricultural wastes in the world. The inefficient method of disposal and management of OPW has necessitated the need to recover value-added products from them. This study reviews the vital characteristics of OPW which present them suitable for the synthesis of value-added bio-products. Simultaneous production of palm oil fresh fruit bunches (FFB) and the utilisation of the plantation wastes for food and animal feed with integration of animal husbandry in the palm oil plantation are discussed. Moreover, the simultaneous conversion of FFB into palm oil and the utilisation of the mill’s residues for value-added bio-products are also discussed.
Hypercholesterolemia is an important risk factor for the development of cardiovascular diseases. Dietary intake of phytosterols/phytostanols and their fatty acid esters results in a reduction of the LDL and total plasma cholesterol levels. Therefore, these constituents are added to a broad spectrum of foods. As in the case of cholesterol, thermo-oxidative treatment of phytosterols may result in the formation of phytosterol oxidation products (POPs), i.e. keto-, hydroxy- and epoxy- derivatives. This review summarizes and evaluates the current knowledge regarding POPs in the light of the potentially increasing dietary exposure to these constituents via the consumption of foods enriched with phytosterols/phytostanols and their esters. Data on the occurrence of POPs and approaches to assess the potential intake of POPs resulting from the consumption of enriched foods are described. The knowledge on the uptake of POPs and the presently available data on the impact of the consumption of enriched foods on the levels of POPs in humans are discussed. Biological effects of POPs, such as potential pro-atherogenic properties or the loss of the cholesterol-lowering effects compared to non-oxidized phytosterols are discussed. Finally, knowledge gaps are outlined and recommendations for further research needed for a safety assessment of POPs are presented. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Phytosterols/-stanols and their fatty acid esters are able to reduce the serum cholesterol level and are therefore added to an increasing number of products claiming cholesterol lowering properties. An undesirable reaction that may be expected in these products is the formation of so-called phytosterol oxidation products, i.e. keto-, hydroxy- and epoxy derivatives of phytosterols/-stanols. The Senate Commission on Food Safety (SKLM) of the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) summarized and evaluated the current knowledge on the formation, intake and biological effects of phytosterol oxidation products and identified gaps in knowledge as well as research needs for a safety assessment, particularly in light of the potentially increasing dietary exposure to such compounds via the consumption of foods enriched with phytosterols/-stanols and their esters. The following opinion was adopted on the 5th of December 2014.
In this study, process optimization of an ultrasonic-assisted organosolv/liquid oxidative pretreatment (SOP) of oil palm fronds (OPFs) for the simultaneous recovery of cellulose, bioethanol and biochemicals (i.e. phenolic compounds) in a biorefinery concept was carried out. The effects of time (30–60 min.), temperature (40–80 °C), NaOH concentration (1–5%) and sample:solvent ratio (1:10–1:50 g/ml) on cellulose content, bioethanol yield and total phenolics contents (TPC) after SOP were investigated. At optimum conditions of pretreatment (i.e. 60 °C, 40 min, 3% w/v aq. NaOH and 1:20 g/ml sample to solvent ratio), the recovered cellulose (55.30%) which served as substrate for enzymatic hydrolysis and subsequent fermentation yielded about 20.1 g/l glucose, 11.3 g/l xylose and 9.3 g/l bioethanol (yield of 0.769 g/g). The pretreatment liquor (mostly regarded as wastes) obtained at the optimum pretreatment conditions contained about 4.691 mg gallic acid equivalent (GAE)/g OPFs of TPC, 0.297 mg vanillic acid (VA)/g OPFs, 1.591 mg gallic acid (GA)/g OPFs and 0.331 mg quercetin (QU)/g OPFs. The pretreatment liquor was again analyzed to possess high antiradical scavenging activity (about 97.2%) compared to the synthetic antioxidant, 3,5-di-tert-butyl-4-hydroxytoluene (BHT) (80.7%) at 100 ppm. Thus one sustainable way of managing wastes in biorefinery is the recovery of multi-bioproducts (e.g. bioethanol and biochemicals) during the pretreatment process.
Commercially available refined rice bran oil (RBO) contained high contents of phytosterol and γ-oryzanol, that is, 858–1034 and 248–887 mg per 100 g oil, respectively. Although the crude rice bran oil (CRBO) had high contents of both phytochemicals (1362–1376 mg phytosterol per 100 g and 1599–1666 mg γ-oryzanol per 100 g), physical refining process resulted in their reduction to 820–895 mg phytosterol per 100 g and 933–960 mg γ-oryzanol per 100 g in RBO (P < 0.05). Both phytochemicals evaporated and accumulated in the deodoriser distillate (DD) to some extent during the deodorising step. Further distillation of DD using molecular distillation (MD) evaporated the free fatty acids (FFAs), resulting in the unevaporated fraction (UMD) having the acid values of 5.6–9.3 mg KOH g−1. The UMD also contained 1585–3391 mg phytosterol per 100 g and 781–848 mg γ-oryzanol per 100 g. Results suggest potential use of DD as the source of phytochemicals for further concentration by MD.
For a hectare of oil palm plantation, about 21.63 tonnes of biomass comprising 20.43 % empty fruit bunches, 5.09 % palm kernel shells, 11.65 % oil palm trunks, 50.30 % oil palm fronds and 12.53 % palm pressed fibre is produced per year as wastes which keep raising many environmental concerns as most of them are incinerated and dumped at open sites. Oil palm wastes are found to contain phytochemicals which have anti-cancer, antioxidants and other vital biological activities. About 17–65 kg of carotenoids, 0.1–60 kg phenolic compounds, 0.6–39 kg sterols and 4.0–62 kg tocols could be extracted from these wastes which would not only boost the economy but also help improve human health and promote clean environments. This study assesses the phytochemistry of oil palm wastes and their pharmacological activities beneficial to the nutraceutical industry with the view of utilizing oil palm wastes for sustainable development.
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.
Potato chips and french fries were analyzed by high performance liquid chromatography and thin layer chromatography for cholesterol and (β-sitosterol oxidation products. Chips stored for 150 d at 23°C in unopened foil bags contained no detectable sitosterol oxidation products, but those held at 40°C contained 7α-hydroxysitosterol, 7β-hydroxysitosterol, and sitosterol (β-epoxide only after an extended storage of 95 d. French fries as purchased contained sterol α- and β-epoxides, and 7α- and 7β-hydroxysterols. These sterol oxidation products were present in repeat samples from five different fast food restaurants. Ingestion of sterol oxides from potato chips is unlikely, whereas ingestion of sterol oxides from french fries is possible.
The toxicological significance of oxidized cholesterol has been well documented in numerous studies. This review focuses on the analysis of dietary sterol oxides in the foodstuffs examined to date with particular emphasis on isolation and characterization techniques. Eight common oxidation products of cholesterol have been identified in certain cholesterol-rich foods subjected to oxidative stress during food processing and/or storage. These products include 25-hydroxycholesterol, α or β 5,6-epoxycholesterol, α or β 7-hydroxycholesterol, 7-ketocholesterol, cholesta-3,5-dien-7-one and cholestane-3β, 5α, 6β-triol. A limited number of studies on the biological effects of dietary phytosterol oxides indicate these products may also be of nutritional concern. Four common autoxidation products of β-sitosterol have been identified in edible oils; these include α or β 7-hydroxysitosterol, 7-ketositosterol and setosta-3,5-dien-7-one. Few quantitative data are available on the sterol oxide content of foods. Moreover,...
Cholesterol oxidation products (COPs) may produce adverse biological effects, and there is increasing concern regarding the potential health implications of these products in the diet. We have developed a GC/MS method allowing the quantification of COPs in commercially available milk powders and infant formulas. Since oxidation artifacts from cholesterol are essentially impossible to avoid, we have used a deuteriated cholesterol probe to precisely monitor the formation of cholesterol oxidative artifacts during all stages of sample analysis. This strategy allowed the determination of the artifact-free cholesterol oxide content of the samples. We found that freshly opened full cream powders which had been packed under inert gas contained only traces of COPs (<250 ng/g total). Infant formulas were also found to contain very low levels of COPs. Our values were considerably lower than those previously reported, especially for 7-ketocholesterol. This difference is at least in part explained by the correction of the oxidation artifacts applied to our samples. We conclude that the exposure to COPs from commercial milk powders and infant formulas, and consequently their potential health hazard, may have been previously overestimated.
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.
The objective of this study was to determine the composition of sterols in vegetable oils used in industrial frying operations, and sterols and sterol oxides in the fried potato products. The sterols and sterol oxides were enriched by saponification of oils and by solid phase extraction. Preparative thin layer chromatography, capillary gas chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectroscopy, were used to give qualitative and quantitative data. The results revealed that the content of desmethylsterols in palm oil, sunflower oil, high oleic sunflower oil, and rapeseed oil/palm oil blend were, 790, 4501, 3550, and 4497 ppm, respectively. Sitosterol was the major desmethylsterol in all samples. Palm oil also contained the lowest levels of total unsaponifiables. The sterols and unsaponifiable contents in sunflower oil were, to some extent, higher than in higholeic sunflower oil. The compositions of sterols after two days of frying were neither markedly different in the oils nor in the potato products fried in these oils compared with the original oils. Isomerised sterols were tentatively quantified to account for 10 ppm, 50 ppm and 20 ppm, in rapeseed oil/palm oil blend, sunflower oil, and high-oleic sunflower oils, respectively. Lipids extracted from French fries prepared in rapeseed oil/palm oil blend contained the highest levels of total sterol oxides, 191 ppm, and epoxides of both sitosterol and campesterol were the major contributors, together at a level of 172 ppm. On the other hand, lipids extracted from French fries prepared in sunflower oil and high-oleic sunflower oil contained 7α-hydroxy-, 7β-hydroxy-, 7-keto- and both epimers of epoxysitosterol, generally in equal amounts. All samples also contained small amounts of different oxidation products of campesterol and stigmasterol.
The formation of stigmasta-3,5-diene (STIG) in vegetable oils from beta-sitosterol was investigated. Previously, analytical methods for STIG determination were developed and verified. For virgin olive oil and crude vegetable oils, the usual oil production processes (pressure, centrifuging and solvent extraction) and long term storage did not produce measurable amounts of STIG, except in the case of crude olive pomace oils where small quantities arose as a result of the high temperature applied to the solid residues during the drying operation. The influence on STIG generation of variables affecting the refining processes was studied. Although minor amounts of STIG appeared after only heating the oil, this compound was produced mainly during the bleaching earth treatment. The decoloration temperature and the bleaching earth activity were the most important variables involved in STIG formation. After deodorising, carried out under normal conditions, the refined olive oils retained measurable amounts of STIG. The refining of other vegetable oils with high beta-sitosterol content (such as sunflower, rapeseed and soya oils) also rendered considerable amounts of STIG. These results support the method based on STIG determination for detecting low percentages of refined vegetable oils in virgin olive oils and crude seed oils.
Cholesterol oxidation products (COPs) may accumulate in foods of animal origin during processing or storage. As they represent a potential health risk, a need exists for rapid and efficient methods to monitor the occurrence of COPs in the human diet. Methods involving saponification to remove triacylglycerols suffer from artifact formation; therefore, alternatives involving solid-phase extraction (SPE) cartridges have been proposed. The efficiencies of several SPE methods for the cleanup of COPs were compared, and a combination of a silica cartridge followed by an NH2 cartridge was found to be optimal for the removal of matrix components. COPs added at the 150 ppb level to milk fat were recovered by the method proposed ≥90% except cholestanetriol, for which the recovery rate was only 52%. Separation and quantification of COPs were done by GC/MS in full-scan mode. Splitless injection of COPs at a concentration of 0.3 ng/μL produced signal-to-noise ratios between 1:10 and 1:200 for epoxycholesterol and 7β-hydroxycholesterol, respectively. Keywords: Cholesterol oxidation products; oxysterols; solid-phase extraction; milk fat; GC/MS
A simple method to prepare silver nitrate loaded silica gel for low pressure silver ion column chromatography is presented. To prepare the packing material, activated silica gel is homogenised with a small volume of aqueous solution of silver nitrate. The effects of water and silver content on the column efficiency, and of elution solvent and light on the recovery, were studied. The procedure was applied to the isolation of steroidal hydrocarbons in olive oils. Separation between 3,5-, 2,4- and 2,5-sterene isomers was achieved.
The 5α-hydroperoxides of β-sitosterol, campesterol, stigmasterol, and brassicasterol were obtained by photooxidation of the respective sterols in pyridine in the presence of hematoporphyrine as sensitizer. The reduction of the hydroperoxides gives the corresponding 5α-hydroxy derivatives. The 7α- and 7β-hydroperoxides of the sterols were obtained by allowing an aliquot of the 5α-hydroperoxides to isomerize to 7α-hydroperoxides, which in turn epimerize to 7β-hydroperoxides. The reduction gave the corresponding 7α- and 7β-hydroxy derivatives. The 5α-, 7α-, and 7β-hydroxy derivatives of β-sitosterol, campesterol, stigmasterol, and brassicasterol were identified by comparing thin-layer chromatography mobilities, specific color reactions, and mass spectral data with those of the corresponding hydroxy derivatives of cholesterol, which were synthesized in the same manner. The phytosterols had the same behavior to photooxidation as cholesterol and, moreover, the different phytosterols photooxidized at about the same rate. The mass spectra of the trimethylsilyl ethers of the hydroxy derivatives of the phytosterols investigated and of the corresponding hydroxy derivatives of cholesterol have the same fragmentation patterns and similar relative ion abundances. Keywords: Sterols; phytosterols; hydroxysterols; photooxidation; gas chromatography/mass spectrometry; GC/MS; mass spectra; trimethylsilyl ether
Monitoring cholesterol oxidation products is important in the evaluation of the potential health risks associated with lipid oxidation. In the present study, a method allowing quick and reliable analysis of polar cholesterol oxidation products was evaluated. After Soxhlet-lipid extraction, the fat was transesterified under mild conditions, thereby minimizing degradation and allowing determination of the free and esterified cholesterol oxides. Sample fractionation was achieved with aminopropyl solid phase extraction cartridges and a stepwise elution with hexane, hexane/methyl tert-butyl ether, and acetone to separate polar cholesterol oxides from cholesterol and other lipid products. Further analysis of the trimethylsilyl derivatives was performed by gas chromatography with detection by flame ionization or mass spectrometry. A phytosterol oxide such as sitosterol α-epoxide (24α-ethyl-5α,6α-epoxycholestan-3β-ol) was employed for the first time as an internal standard for the quantification of cholesterol oxides in foodstuffs of animal origin. Keywords: Cholesterol oxides; oxysterols; solid phase extraction; amino phase; gas chromatography
A new method for the determination of the autoxidation products from free cholesterol, viz., the isomeric 5,6-epoxycholestanols, the C-7 oxidized cholesterol, 20α-hydroxycholesterol, 25-hydroxycholesterol, and cholestanetriol, has been developed. The method is also useful in combination with an enzymatic method to release esterified cholesterol to facilitate total cholesterol oxide determination. For analysis of total cholesterol oxides, the lipids were dispersed in phosphate buffer by using bovine serum albumin as a detergent and incubated with triacylglycerol lipase under nitrogen atmosphere. The phospholipids and the released fatty acids were removed by column chromatography. The remaining lipids were dispersed again and incubated with cholesterol ester hydrolase under nitrogen atmosphere. Cholesterol oxides were isolated in three enrichment steps. The isolated cholesterol oxides were separated as trimethylsilyl ether derivatives on a capillary methyl silicone column.
In der vorliegenden Arbeit wird nachgewiesen, daß beim normalen, industriellen Raffinationsprozeß der Pflanzenöle wesentliche Änderungen in der Zusammensetzung der Steroide eintreten. Bei der Raffination entstehen Autoxydationsprodukte der Steroide und daraus Sterin-Kohlenwasserstoffe. Es bilden sich Umwandlungsprodukte der Sterine, die in natürlichen unbehandelten Pflanzenölen nicht vorkommen.The Formation and Removal of Different Steroid-Derivatives during the Refining of Vegetable Edible Oils In the present work it has been shown that appreciable changes occur in the composition of steroides during normal industrial refining of vegetable oils. The autoxidation products of steroides and the sterol-hydrocarbones which are formed from the former appear during the refining. There are other products too, formed from the sterols, which do not occur in natural untreated vegetable oils.
Durch Einwirkung von aktivierter Bleicherde auf Cholesterin, β-Sitosterin, Stigmasterin und Brassicasterin in Hexan bilden sich unpolare Steroide. Die gebildeten Kohlenwasserstoffe wurden durch Säulen-Chromatographie und präparative Dünnschicht-Chromatographie isoliert und untersucht. Die durch die Behandlung von Cholesterin mit aktivierter Bleicherde gebildeten Kohlenwasserstoffe wurden nach mehrmaliger Kristallisation aus Äthylalkohol als Δ3,5-Cholestadien identifiziert.Alteration of Sterols in Fats and Oils During the Industrial ProcessingNonpolar steroids are formed by the action of bleaching earth on cholesterol, β-sitosterol, stigmasterol and brassicasterol in the presence of hexane. The hydrocarbons formed were isolated and investigated by column chromatography and preparative thin-layer chromatography. The hydrocarbons formed by the action of activated bleaching earth on cholesterol were repeatedly crystallized from ethyl alcohol and identified as Δ3,5-cholestadiene.
Die Untersuchungen der Vff. ergaben, daß beim Erhitzen von sterinhaltigem Sojaöl bei Anwesenheit von Sauerstoff u. a. 7-Hydroxysterine gebildet werden. Kohlenwasserstoffe und Disteryläther entstanden bei der Bleicherde-Behandlung von Cholesterin, β-Sitosterin, Stigmasterin und Brassicasterin. 7-Hydroxysterin ergibt bei der Behandlung mit Bleicherde Kohlenwasserstoffe und Ketosterine. Während der Desodorierung wurden bei unzureichendem Vakuum die Sterine teilweise oxydiert. Frisch extrahiertes Rüböl enthielt keine Disteryläther, die aber in Handelsölen und -fetten nachgewiesen wurden. Dicholesteryläther zeigte im Rattenversuch keine cancerogenen Eigenschaften.Changes in Sterols During the Industrial Processing of Fats and Oils IThe investigations of the authors have shown that 7-hydroxy sterols are formed when soybean oil containing sterols is heated in the presence of oxygen. Hydrocarbons and disteryl ethers are formed by bleaching earth treatment of cholesterol, β-sitosterol, stigmasterol and brassicasterol. Treatment of 7-hydroxy sterol with bleaching earth leads to the formation of hydrocarbons and keto-steroids. The sterols are partly oxidized during deodorization under inadequate vacuum. Freshly extracted rapeseed oil does not contain any disteryl ether. However, these substances could be detected in commercial fat samples. In experiments on rats, no carcinogenic properties of dicholesteryl ether could be found.
Samples of maize, rapeseed, soyabean, sunflower, and palm oils, collected at different stages in their respective refining processes, have been analyzed for the presence of dehydrated sterols (sterenes). A total of fifteen compounds were detected. Four have been characterized using model systems designed to investigate their formation. Their elemental composition, and that of the other eleven sterenes, was determined by high resolution mass spectrometry and structures assigned to a further six compounds. These included the -3,5-, -2,5-, -4,6-,-3,5,22-, and -4,6,22- sterenes. Sterenes were not present in crude or alkali neutralized oils. Bleaching introduced a maximum concentration of 5 μg g−1 of total sterenes into the edible oils. Deodorization is the dominant stage in sterene production resulting in a maximum total concentration of 163 μg g−1. This increased concentration arises due to dehydration of the sterols and considerable isomerism of the sterenes.
Several extraction, separation and detection methods for cholesterol oxidation products (COPS) were developed and evaluated using liquid chromatography. The results showed that extraction of COPS from lard by cold saponification at 25°C or a Sep-Pak C18 cartridge resulted in higher recoveries than those given by a silica gel Sep-Pak cartridge. With HPLC, by using a cyano-bonded column and hexane-2-propanol (95:5, v/v) as the mobile phase at a flow-rate of 1.0 ml/min, and a C18 column with a gradient flow system with acetonitrile-methanol (55:45, v/v), it was possible to separate eight and nine COPS within 17 and 60 min, respectively. All the COPS with one, two and three double bonds could be detected by UV spectrophotometry at 212, 234 and 280 nm, respectively. UV detection had a 1000 times higher sensitivity than refractive index detection. Ten COPS were separated from heated lard using a cyano-bonded column, and five of them were identified.
Disterylether entstehen in geringer Konzentration (wenige mg/kg) durch Dehydratisierung von Sterinen whrend der blichen Bleichung von Fetten und len an sure-aktivierten Bleicherden. Ein gaschromatographisches Verfahren zum Nachweis und zur quantitativen Erfassung der Disterylether wird beschrieben. Die Probenvorbereitung besteht aus der Gewinnung des unverseifbaren Anteils, aus dem die Sterine durch Extraktion mit Dimethylformamid weitgehend entfernt werden, und einer chromatographischen Reinigung durch SC, DC oder HPLC. Zur Trennung der Disterylether werden kurze, dnn belegte, kufliche Quarz-Capillarsulen verwendet. Dicholesterylether lt sich von der Gruppe der Diphytosterylether gut abtrennen, whrend die verschiedenen Diphytosterylether eine Serie von dicht nebeneinanderliegenden Peaks ergeben. Disterylether lassen sich analytisch als Indikatoren fr die Bleichung von Fetten und len nutzen.Disteryl ethers are formed in low concentration (a few mg/kg) by dehydration of sterols during the conventional bleaching of fats and oils with acid-activated bleaching earths. A gas Chromatographie method for the detection and quantitative determination of disteryl ethers is described. Sample preparation consists of isolation of the unsaponifiable matter, from which most of the sterols are eliminated by extraction with dimethylformamide, and Chromatographie purification by CC, TLC, or HPLC. Short, thin-film fused silica capillaries, commercially available, are used for the separations. Dicholesteryl ether is well separated from the group of diphytosteryl ethers, whereas the various diphytosteryl ethers form a series of some closely separated peaks. Disterylethers are useful as analytical indicators of the bleaching process.
Adulteration of expensive edible oils, such as olive oil, often involves desterolized oils in order to render the adulteration undetectable. Sunflower oil contains characteristic Delta 7-sterols, which are readily removed upon strong bleaching. It is shown that these Delta 7-sterols do not primarily dehydrate (as do Delta 5-sterols), but isomerize to Delta 8 (14)- and Delta 14-sterols. These compounds can be analysed by LC on silica gel or GC on capillary columns with stationary phases of intermediate to high polarity.
Soybean oil and wheat flour were analyzed for the content of sitosterol oxides. The method involved chromatography on a Lipidex-5000
column and enrichment on a disposable NH2-column, yielding a sterol fraction and a sterol oxide fraction. Each fraction was separated as trimethylsilyl-ethers on a
methyl silicone capillary column. Analysis of crude and freshly refined soybean oil showed no detectable levels of the isomeric
5,6-epoxysitosterols, the epimeric 7-hydroxysitosterols and 5,6-dihydroxysitosterol at the detection limit of 0.2 ppm. Storage
of a refined soybean oil for one year at 4°C caused no significant increase in the level of free sitosterol oxides when compared
to the freshly refined soybean oil. Analysis of three wheat flours (at 2, 8 and 36 months) revealed that the samples contained
variable levels of 5α,6α-epoxysitosterol (5.4–55 ppm in the lipids), 5β,6β-epoxysitosterol (0.2–29 ppm), 7α-hydroxysitosterol
(9.3–118 ppm) and 7β-hydroxysitosterol (9.7–126 ppm).
Hydrogenated rapeseed oil/palm oil blend, sunflower oil and high-oleic sunflower oil, and French fries fried in these oils
were assessed for contents of sterol oxidation products. Different oxidation products of phytosterols (7α- and 7β-hydroxy-sito-and
campesterol, 7-ketosito- and 7-ketocampesterol, 5α,6α-epoxy-sito- and campesterol, 5β,6β-epoxy-sito-and campesterol, dihydroxysitosterol
and dihydroxycampesterol) were identified and quantiated by gas chromatography (GC) and GC-mass spectroscopy. Rapeseed oil/palm
oil blend contained 41 ppm total sterol oxides before frying operations. After two days of frying, this level was increased
to 60 ppm. Sunflower oil and high-oleic sunflower oil had 40 and 46 ppm sterol oxides, respectively, before frying operations.
After two days of frying operations, these levels increased to 57 and 56 ppm, respectively. In addition to campesterol and
sitosterol oxidation products, small amounts of 7α- and 7β-hydroxystigmasterol were detected in the oil samples. Total sterol
oxides in the lipids of French fries fried at 200°C in rapeseed oil/palm oil blend, sunflower oil, and high-oleic sunflower
oil were 32, 37, and 54 ppm, respectively. The levels of total oxidized sterols, calculated per g sample, ranged from 2.4
to 4.0 ppm. In addition to the content of phytosterol oxides, full scan mass spectra of several oxidation products of stigmasterol
are reported for the first time.
Two methods for routine analysis of free 7-ketocholesterol (7-k) in foods by high-performance liquid chromatography (HPLC)
were compared. The analyzed samples were egg noodles, biscuits prepared with eggs, sweet snacks, grated Parmesan cheeses,
and some ingredients utilized in the food industry (whole-milk and whole-egg powder). The enrichment of cholesterol oxides
was carried out by solid-phase separation of the total lipids with florisil and silica cartridges for methods A and B, respectively.
The 7-k analyses were run in normal-phase HPLC for method A, and a reverse-phase process was used in method B. Identification
of the 7-k peak was confirmed by gas chromatography/mass spectrometry analysis and peak purity checkvia spectral analysis with a diode array detector. The quantitation of 7-k was carried out with an internal standard for method
A and with a calibration curve for method B. The limit of quantitation (LQ) was 3 × 10−9 g/injection; the limit of detection was ten times lower than the LQ for both methods. The two methods showed good recovery
(99%) and good repeatability (coefficient of variation of 3.9 and 3.7% for methods A and B, respectively). These methods allow
a fast, sensitive, and reliable determination of one cholesterol oxidation product, which is present at a high concentration
level in the first stages of the oxidation process.
Potato chips fried in palm oil, sunflower oil, and high-oleic sunflower oil were studied for the content of different phytosterol
oxides during 0 to 25 weeks of storage in the dark. Oxidation products of sitosterol (24α-ethyl-5-cholesten-3β-ol) and campesterol
(24α-methyl-t-cholesten-3β-ol) were synthesized to help identify the phytosterol oxides. The oxides of phytosterols were analyzed
by preparative thin-layer chromatography, solid-phase extraction, capillary column gas chromatography (GC), and GC-mass spectrometry.
Epimers of 7-hydroxysitosterol and 7-hydroxycampesterol; 7-ketositosterol and 7-ketocampesterol; epimers of 5,6-epoxy-sitosterol
and 5,6-epoxy-campesterol; 24α-ethylcholestane-3β,5,6β-triol (dihydroxysitosterol) and 24α-methylcholestane-3β,5,6β-triol
(dihydroxycampesterol) were detected and quantitated in the samples of chips fried in different vegetable oils. Potato chips
fried in palm oil had the lowest level of total sterol oxides, ranging from 5 to ca. 9 ppm in the lipids from time 0 to 25 wk of storage. The level of total sterol oxides in chip samples fried in sunflower
oil ranged from 46 to 47 ppm, and the lipids in samples fried in high-oleic sunflower oil ranged from 35 to 58 ppm from 0
time to 25 wk of storage. During 25 wk of storage no considerable increase in sterol oxides was observed in the samples of
chips fried in palm oil and sunflower oil. The chip samples fried in high-oleic sunflower oil had slightly higher levels of
sterol oxides after 10 and 25 weeks of storage. In addition to the levels of individual sterol oxides, a new method for enrichment
of phytosterol oxides from the unsaponifiables and full-scan mass spectra of various oxidation products of sitosterol and
campesterol are reported in this paper.
To clarify conflicting information regarding 7-ketocholesterol (7-KC) recovery from saponification, we evaluated the stability
of 7-KC in methanolic alkaline medium. We added 1 N alcoholic KOH to 7-KC or lard spiked with 7-KC and held the mixtures at
45, 55, 65, and 75°C for different times to simulate various saponification conditions. Gas-chromatographic determination
of residual 7-KC with 5α-cholestane as the internal standard (IS) showed that the higher the saponification temperature, the
greater the 7-KC degradation. Hot saponification at 75°C for 30 min caused extensive destruction and left only 31% 7-KC. 7-KC
loss during saponification could be described by pseudo first-order kinetics, and the dependence of degradation rate on temperature
(T, K) byk (h−1)=(2.6×1017) exp (−1.36×104/T). As predicted by the kinetic equation, 7-KC loss during room-temperature saponification (21°C) was practically negligible;
following the exposure of 7-KC or lard spiked with 7-KC to 1 N alcoholic KOH for 18 h, about 97% 7-KC was recovered. 6-Ketocholestanol,
when used as an IS, should be looked at carefully because of potential tautomerization, leading to the formation of two enol
isomers when in extended contact with trimethylsilyl derivatization reagents.
Deuterium-labeled cholesterol is used to monitor for artifactually produced cholesterol oxidation products during analysis. [2H9]Cholesterol, labeled on the side chain, is added to the sample immediately upon isolation, and the ratios of labeled to unlabeled oxides and of labeled to unlabeled cholesterol are monitored by capillary gas chromatography-mass spectrometry. The analytical methodology involves an initial solvent extraction followed by silica gel LC and reversed-phase HPLC to isolate and concentrate the oxide fraction. The feasibility of the technique for analysis of cholesterol oxides in foods and biological samples at the part per million level with an accuracy of better than +/- 5% is demonstrated.
Literature published between 1980 and 1986 dealing broadly with the topic of cholesterol autoxidation is reviewed. The review builds on the detailed 1981 monographic treatment of the topic by the author and covers new items of chemistry, analysis, and metabolism.
The dehydration of sterols during the refining process of vegetable oils results in the formation of steroidal hydrocarbons (sterenes or steradienes) with two double bonds in the ring system. Other steroidal hydrocarbons whose structures were in agreement with the presence of three double bonds in the ring system were detected in the sterene fractions of refined vegetable oils. The 5alpha-, 7alpha-, and 7beta-hydroxy derivatives of cholesterol and phytosterols have been dehydrated in n-butanol/H(3)PO(4) to form steroidal hydrocarbons with three double bonds at the 2, 4, and 6 positions in the ring system. These hydrocarbons had the same relative retention time and mass spectra as those present in the sterene fractions of refined oils. The dehydration of the hydroxy sterols dissolved in extra virgin olive oil and in the presence of 1% bleaching earths at 80 degrees C for 1 h results in the formation of the same steroidal hydrocarbons found in the refined oils.
Steroidal hydrocarbons formed by dehydration of oxidized sterols in refined oils
- R Bortolomeazzi
- L Pizzale
- L Novelli
- L S Conte
Bortolomeazzi, R.; Pizzale, L.; Novelli, L.; Conte, L. S. Steroidal hydrocarbons formed by dehydration of oxidized sterols in refined oils. RiV. Ital. Sostanze Grasse 1996, 73, 457-460.