Free,esterified and glucosidic sterols in cocoa butter

Sterol lipids of cocoa butter (cocoa beansLome Tongo) were fractionated into free sterols, steryl esters (SE), steryl glucosides and acylated steryl glucosides (ASG). 4-Desmethyl, 4-methyl and 4,4′-dimethyl sterols or triterpene alcohols, which were isolated as free sterols or which resulted from hydrolysis, were determined by thin layer chromatography-flame ionization detection and identified by gas chromatography and combined gas chromatography-mass spectroscopy. Free sterols comprise the main sterol fraction in cocoa butter. Esterified sterols amount to 11.5% of total sterols and glucosidic sterols to 16.3%. Fatty acids and D-glucose from hydrolysis of esters and glucosides were analyzed. The fatty acids of SE and ASG are richer in unsaturated fatty acids than cocoa butter total fatty acids.     

Novel Method for the Synthesis of Cholesteryl Glucosides starting from Disaccharides

Steryl glucosides (SG) and acylated steryl glucosides (ASG) are natural components of plant cell membranes and present in different concentrations in various plant foods. Currently, their positive effects on human health are under investigation. The present work presents a new and efficient synthesis method for cholesteryl glucosides starting from disaccharides. A five‐step synthesis protocol is done to obtain the desired product in 35% overall yield. In the first step, the hydroxy groups of the starting material sucrose are protected using benzyl ethers. After the subsequent acidic hydrolysis the obtained pyranosyl moiety of the disaccharide is transformed to its trichloroacetimidate derivative. Next, the formation of the glycosidic bond to cholesterol is performed and catalytic transfer hydrogenation in order to remove the protecting groups leads to the desired product. In this context, APCI‐MS‐TOF has turned out to be an excellent analytical tool for the high sensitive analysis of SG as well as intermediates. Practical Applications: Due to the comparatively high amounts of SG and ASG in seeds and oils, not only the food industry but also in biodiesel production, these natural compounds are of increasing interest. However, analysis of the compounds is difficult, commercially available pure standard materials are costly and their synthesis often requires time‐consuming work‐up procedures. The described preparation method allows the synthesis of cholesteryl glucosides which can be used as reference or standard material for the quantitative analysis of phytosteryl glucosides in plant derived samples. The general synthesis method could be also applied to other SG and ASG derivatives. Cholesteryl glucosides are synthesized using a new and efficient five‐step synthesis protocol starting from disaccharides. The preparation method provides products with good overall yield and high purity and, therefore, the synthesized glucosides can be used as reference or standard material for the quantitative analysis of phytosteryl glucosides in plant derived samples. APCI‐MS‐TOF is extensively used as analytical tool for the sensitive analysis of cholesteryl glucosides as well as intermediates.     

Steryl esters in a cell suspension culture of celery (Apium graveolens)

Arange of analytical techniques was used to investigate the composition of the steryl fatty acyl esters in a cell suspension culture of celery (Apium graveolens). Gas chromatography (GC) and GC-mass spectrometry (GC-MS), using electron ionization (EI) and negative ion chemical ionization (NICI), were employed to characterize the intact steryl esters. Assignments were supported by analysis of the sterol and fatty acid moieties released from the intact molecular species by alkaline hydrolysis. A selectivity for sterol esterification was noted, with the major free sterol, stigmasterol, occurring only in a very small amount in the esterified form. Instead, the precursors to Δ⁵-phytosterols, particularly cycloartenol, predominated in the ester fraction. The pentacyclic triterpene, β-amyrin, was also found as the palmitate and linoleate esters. Changes in composition and abundance of the steryl esters during the different growth phases of a celery cell suspension culture were investigated. The total amount of esterified sterols exceeded that of free sterols throughout the growth cycle. The changes observed during growth highlighted differences between the esters of precursor sterols and those of the 4-desmethyl-sterols, and it is postulated that the various steryl esters perform different functions in cell metabolism.     

The Identification and Quantification of Steryl Glucosides in Precipitates from Commercial Biodiesel

There have been several discoveries of unexpected precipitates in manufacturing facilities, transport vessels, and storage tanks containing commercial biodiesel. In some cases these have been formed during storage at temperatures above the cloud point of the fuel. High performance liquid chromatography (HPLC) and mass spectrometry (MS) methods were applied to 24 field receipt samples of solids from such biodiesels. The analyses revealed the presence of steryl glucosides (SG), common phytosterols (plant sterol) found in crude soybean oil and many other plant materials, in these biodiesel precipitates. Quantitative analysis of the solids revealed SG concentrations as high as 68 wt% of the provided material (which had not been previously washed with solvent to remove entrained biodiesel). In some samples no SG were present. In others they constituted all of the non-biodiesel material present. Monacylglycerols and diacylglycerols, the products of incomplete transesterification of triacylglycerols, were also present in some samples. The normal phase and reverse phase methods described in this report could be used to analyze SG quantitatively from biodiesel precipitates with an HPLC instrument equipped with either an evaporative light-scattering detector (ELSD) or a more common UV detector operating at 205 nm. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Comparing Process Efficiency in Reducing Steryl Glucosides in Biodiesel

A significant obstacle to the commercial acceptance of biodiesel is the potential for filter plugging due to precipitates in the fuel. The majority of these precipitates can be attributed to either steryl glucosides (SGs) or monoacylglycerols in biodiesel. A GC–FID method to quantify minor components content in biodiesel is presented. The effectiveness of room temperature and cold soak filtration, adsorbent treatment, centrifuge, and vacuum distillation processes for SG removal was studied. The vacuum distillation process is the most effective method of removing the SG from biodiesel.     

Sterol lipids in finger millet(Eleusine coracana)

Neutral lipids, glycolipids, and phospholipids (1.3%, 0.25%, and 0.10% of seed weight) were isolated from the total lipids (chloroform-methanol) of finger millet seeds(Eleusine coracana), and four sterol-containing lipids further isolated from neutral and glycolipids by preparative column and thin layer chromatography. On seed weight, these comprised: free sterols (S) 0.091%, sterol esters (SE) 0.013%, sterol glycosides (SG) 0.025%, acyl sterol glycosides (ASG) 0.020%, and total 0.149%. The major fatty acids, totaling 85-90%, were the same in both esterified sterols, but the proportions varied: 16:0, 18:1, and 18:2 comprising 24, 49, and 17% in SE (calculated iodine value 75) and 43, 36, and 7% in ASG (calculated iodine value 46). All four sterol lipids contained 80-84% of β-sitosterol, the remainder being stigmasterol. The only sugar in SG and ASG was D-glucose. It is deduced that the major representative species are: SE, β-sitosterol oleate/palmitate; SG, β-D-glucopyranosyl-(l → 3)-β-sitosterol; and ASG, 6-0-palmitoyl-β-D-glucopyranosyl-(l → 3)-β-sitosterol.     

Glycosidic Bond Cleavage is Not Required for Phytosteryl Glycoside-Induced Reduction of Cholesterol Absorption in Mice

Phytosteryl glycosides occur in natural foods but little is known about their metabolism and bioactivity. Purified acylated steryl glycosides (ASG) were compared with phytosteryl esters (PSE) in mice. Animals on a phytosterol-free diet received ASG or PSE by gavage in purified soybean oil along with tracers cholesterol-d₇ and sitostanol-d₄. In a three-day fecal recovery study, ASG reduced cholesterol absorption efficiency by 45 ± 6% compared with 40 ± 6% observed with PSE. Four hours after gavage, plasma and liver cholesterol-d₇ levels were reduced 86% or more when ASG was present. Liver total phytosterols were unchanged after ASG administration but were significantly increased after PSE. After ASG treatment both ASG and deacylated steryl glycosides (SG) were found in the gut mucosa and lumen. ASG was quantitatively recovered from stool samples as SG. These results demonstrate that ASG reduces cholesterol absorption in mice as efficiently as PSE while having little systemic absorption itself. Cleavage of the glycosidic linkage is not required for biological activity of ASG. Phytosteryl glycosides should be included in measurements of bioactive phytosterols.     

Steryl glycoside biosynthesis

The existence of steroid glycosides has been known for many years and, more recently, their derivatives have been described. Steroid glycosides have been isolated from a number of organisms, however, the largest number of these compounds are found in plants. As to glycoside biosynthesis, the sterols are the most extensively studied steroid group. Of the sterols, only the 4-demethyl sterols have been isolated as glycosides. The glycosidic bond formation is mediated through nucleotide sugars, and UDP-glucose appears to be the most active glycosyl donor. In cell-free studies, the pH of the incubation medium is quite critical and depends on the tissue under investigation, but generally the optimum is near pH 7.0. Formation of steryl glycosides is particulate in nature and is stimulated by ATP, Ca²⁺, and Mg²⁺. Similar results are obtained, regardless whether the sterol or the sugar moiety is labeled. Formation of acylsteryl glycosides could occur via two pathways: through the acylation of steryl glycosides or through the transfer of an acylglycosyl unit to a sterol moiety. Results from in vitro experiments suggest that acylsteryl glycoside formation occurs via steryl glycosides. Two acyl transfer reactions have been demonstrated; one is microsomal in nature and involves phosphatidylethanolamine, while the other reaction involves a soluble enzyme and requires galactolipids. In vivo experiments, however, indicate that a second pathway may also exist. If cholesterol-4-¹⁴C is used as substrate, a highly radioactive component can be isolated which is readily converted to acylsteryl glycoside, but not to free sterol or steryl glycoside. It is suggested that this component is an intermediate in acylsteryl glycoside biocynthesis. At present, the nature of the component is unknown. It is quite stable, and acid hydrolysis produces free sterol. Saponification produces two products which in thin layer chromatograms closely resemble acylsteryl glycoside.     

Role of glycolipids in breadmaking

In wheat flour used for breadmaking, glycolipids are essential “endogenous surfactants” (1). Bread volume and crumb structure of wheat flour‐based bakery products are influenced by glycolipids, without the decisive mode of action being conclusively known. Little information on how glycolipids from other plant sources perform as wheat flour improvers is available. In this paper we review our work done on revealing the baking performance of the four major glycolipid classes present in commercial lecithins (sterol glucosides (SG), acylated sterol glucosides (ASG), cerebrosides and digalactosyl diacylglycerides (DGDG). Our purpose is to better understand the role of these substances in the baking process. Our results suggest that there are two groups of glycolipids with excellent baking performance. Both groups were found to have an impact on the dough liquor rather than on the gluten‐starch matrix. The cerebrosides and DGDG most likely influenced the overall baking result by directly stabilizing the dough liquor/gas cell interface, while ASG and SG did this indirectly through a synergistic effect with a dough liquor constituent, most probably the endogenous flour lipids.     

Quantification of free and esterified steryl glucosides in vegetable oils and biodiesel

Steryl glucosides (SG) are minor components that dramatically modify the low temperature performance of fatty acid methyl esters (FAME) used as biodiesel. SG are naturally present in vegetable oils but they may also be the result of the transesterification of esterified steryl glucosides (ESG). These are present in vegetable oils at a level of a few hundred milligrams per kilogram, depending on the nature of the feedstock. We developed an analytical method to quantify SG and ESG in vegetable oils and in FAME. The purification of SG and ESG was performed by liquid chromatography on silica gel, and the analysis of the trimethylsilyl derivatives was achieved by gas chromatography and flame ionization detection. The filterability of biodiesel is affected when the SG content is higher than 20 mg/kg. Therefore, the sensitivity of this new method is adapted for this purpose since the quantification limit is 10 mg/kg of SG and ESG. The recoveries are acceptable, between 75% and 90% depending on the species and content, and the reproducibility relative standard deviation, evaluated at 10%, is comparable to other studies.     

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

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

Formation of Insolubles in Palm Oil-, Yellow Grease-, and Soybean Oil-Based Biodiesel Blends After Cold Soaking at 4 °C

The insolubles formed in biodiesel blends can cause operation problems because they can plug the fuel lines and filters. The formation of insolubles in soybean oil (SBO-), yellow grease (YG-), and palm oil (PO-) based biodiesel blends after cold soaking at 4 °C was investigated. PO-based biodiesel blends displayed a much higher time to filter (TTF) and greater insoluble mass, compared to SBO-, and YG-based biodiesel blends. Fourier transform infrared (FTIR) spectra and gas chromatography-flame ionization detector (GC-FID) chromatograms indicated that PO-based biodiesel insolubles can be attributed to monoglycerides, while SBO-based biodiesel insolubles are due to steryl glucosides (SG). A simple analytical method for identification of SG in biodiesel samples was established by GC-FID.     

Sterol composition ofPhaeodactylum tricornutum as influenced by growth temperature and light spectral quality

In a detailed sterol analysis of the marine diatomPhaeodactylum tricornutum, free sterols as well as esterified and glycosylated conjugates were found. When the alga was grown under standard conditions (i.e., at 13°C under white light), 64% of total sterols were steryl glycosides. In all sterol classes, except steryl esters, (24S)-24-methylcholesta-5,22E--dien-3β-ol (epibrassicasterol) was the major (80 to 99%) sterol component. Eight other sterols were identified. Growth under different light spectral quality (red, blue, yellow, and green) at 13 and 23°C was examined. At 23°C, a dramatic decrease in total sterol content was observed, especially under blue light. The distribution of sterols between free and conjugated forms as well as sterol profile inside each class was found to be strongly dependent on the light spectral quality at both temperatures.     

Identification of Δ<Superscript>7</Superscript> phytosterols and phytosteryl glucosides in the wood and bark of several <Emphasis Type="Italic">Acacia</Emphasis> speciesphytosterols and phytosteryl glucosides in the wood and bark of several <Emphasis Type="Italic">Acacia</Emphasis> species

The wood and bark of four Acacia species growing in Portugal, namely, A. longifolia, A. dealbata, A. melanoxylon, and A. retinodes, were investigated for their sterol content. The lipids fractions of the different wood and bark samples were isolated, and the sterols were identified and quantified by GC-MS. Two Δ⁷ sterols, specifically, spinasterol and dihydrospinasterol, were the main sterols found in considerable amounts, particularly in wood tissues (more than 0.5 g/kg of dry wood in the case of A. melanoxylon and A. retinodes). The corresponding unusual steryl glucosides were also identified in significant amounts in the wood and bark extracts.     

Steryl ferulates,bioactive compounds in cereal grains

Steryl ferulates are minor bioactive phytochemicals present in various cereal grains and other seeds. They possess well‐established health promoting properties, such as antioxidant and cholesterol lowering activities. However, the health promoting potential of steryl ferulates extracted from different sources can vary depending on their characteristic sterol composition.     

Fractionation of Canola Biodiesel Sediment for Quantification of Steryl Glucosides with HPLC/ELSD

Steryl glucosides (SG) and other trace contaminants in biodiesel may cause filter plugging and engine performance issues, most notably in temperate regions that experience low temperatures. While sediments have been characterized from palm, soybean, and European rapeseed biodiesel, identification of the causative agents and sediment components derived from North American canola (Brassica napus) feedstocks is lacking. Analytic methods used to quantify sediment constituents are time consuming and sample heterogeneity may lead to decreased lab precision. The objectives of this research were to develop a method to fractionate biodiesel sediment in order to confirm and quantify the presence of SG in canola biodiesel. A reverse phase HPLC method with evaporative light scattering detection was modified to confirm the presence of SG from sediments collected at three North American canola biodiesel processing facilities. SG was confirmed in two of three sediment samples where 25.1 and 9.5 wt% of total sediment was SG. FTIR spectroscopy confirmed the presence of SG and provided a rapid method for qualitative confirmation of sediment composition. A third sediment had no detectable SG, but contained a clay filter aide as confirmed by ATR-FTIR spectroscopy.     

Specific procedure for analysing steryl glucosides in olive oil

Avoiding any kind of oil sample pretreatment, a fraction containing the steryl glucosides (SG) was directly isolated by SPE. This fraction was derivatised and analysed by GC in order to quantify the SG content. The limit of detection of the method was 0.37 mg/kg and the recovery 90%. Additionally, the identity of the SG was confirmed by MS. We applied the procedure to oil samples of different categories and origins indicating that the only SG that could be quantified in olive oil was β‐sitosteryl glucoside, which was present at concentrations not higher than 3.00 mg/kg. Practical applications: Olive oil is of upmost importance from both the nutritional point of view and from the economic repercussion that fraud may have. Among the many parameters required by the EU in order to determine olive oil characteristics, SG are completely overlooked and no specific methodology has been elaborated so far. We have developed a straightforward protocol for the determination of SG in olive oil. This method can be used to provide an additional identity parameter and a new blend indicator. This method is fast, cheap and easy to perform. Besides, it has a great potential for automation which would allow its routine application. The possibility of applying the procedure to study vegetable oils used in the biodiesel industry is also pointed out.     

Identification and Occurrence of Steryl Glucosides in Palm and Soy Biodiesel

A problem of excessive sedimentation was detected in soy and palm biodiesel, preventing the product from complying with requirements on contamination/filterability. The objective of the study was to determine the nature of the sediment by different analytical techniques and to obtain data on the typical range of its components in industrially produced biodiesel samples. The sediment was investigated and the appearance of haze is linked to the presence of free steryl glucosides (FSG) above a certain concentration. This paper focuses on the original analytical approach, taking into account particular physical properties of FSG. Nuclear magnetic resonance and mass spectrometry were used as fast and reliable identification methods, without the need for a prior hydrolysis of the glucosidic bond. A GC method, including optimised sample preparation, was developed for the quantification of the FSG in biodiesel as well as in filter residues. The FSG concentrations in biodiesel produced by different processes ranged between 55 and 275 mg/kg for palm and from not detectable to 158 mg/kg for soy biodiesel.     

Purification of tocopherols and phytosterols by a two-step <Emphasis Type="Italic">in situ</Emphasis> enzymatic reaction

The purification of tocopherols and phytosterols (referred to as sterols) from soybean oil deodorizer distillate (SODD) was attempted. Tocopherols and sterols in the SODD were first recovered by short-path distillation, which was named sODD tocopherol/sterol concentrate (SODDTSC). The SODD-TSC contained MAG, DAG, FFA, and unidentified hydrocarbons in addition to the two substances of interest. It was then treated with Candida rugosa lipase to convert sterols to FA steryl esters, acylglycerols to FFA, and FFA to FAME. Methanol (MeOH), however, inhibited esterification of the sterols. Hence, a two-step in situ reaction was conducted: SODDTSC was stirred with 20 wt% water and 200 U/g mixture of C. rugosa lipase at 30°C, and 2 moles of MeOH per mole of FFA was added to the reaction mixture after 16h. The lipase treatment for 40 h in total achieved 80% conversion of the initial sterols to FA steryl esters, complete hydrolysis of the acylglycerols, and a 78% decrease in the initial FFA content by methyl esterification. Tocopherols did not change throughout the process. To enhance the degree of steryl and methyl esterification, the reaction products, FA steryl esters and FAME, were removed by short-path distillation, and the resulting fraction containing tocopherols, sterols, and FFA was treated with the lipase again. Distillation of the reaction mixture purified tocopherols to 76.4% (recovery, 89.6%) and sterols to 97.2% as FA steryl esters (recovery, 86.3%).     

Quantification and Determination of Composition of Steryl Ferulates in Refined Rice Bran Oils Using an UPLC-MS Method

Gamma-oryzanol contains a mixture of steryl ferulates found in rice bran oil. Several studies have attributed nutraceutical properties to this mixture, such as hypocholesterolemic and anti-inflammatory activities. A method based on ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry was developed and evaluated for the simultaneous quantification of gamma-oryzanol and identification of five major steryl ferulates directly in refined rice bran oils (RBO) samples. The proposed method was evaluated according to linearity by obtaining standard curves with R ² values above 0.990, and limit of detection values ranged from 1.9 to 5.9 µg/mL, whereas limits of quantification ranged from 5.9 to 17.9 µg/mL; inter- and intraday accuracy and precision were within the range required by the US Food and Drug Administration guidelines; recovery levels ranged from 78 to 85% for gamma-oryzanol, and from 84 to 119% for steryl ferulates. The method can be considered robust in relation to the NH₄OH (ammonium hydroxide) content and cone voltage variations, with coefficient of variation and average relative percentage deviation values lower than 7.0 and 4.4%, respectively. The stability during the storage test was maintained in concentrated samples (18.5 µg/mL), with recovered values between 93 and 113%. This method was successfully applied to the analysis of RBO samples, demonstrating that it could be easily used for quality control purposes.