Phytosterol fatty acid esters

Com germ oil contains two phytosterol lipid classes, free phytosterols and phytosterol fatty acid esters. Phytosterols have been recognized as one of the most important classes of phytonutrients (Ostlund et al., 2002). Most chemical identification processes of phytosterols in vegetable oils have been carried out by saponifying the oil and by measuring the resulting free phytosterols, usually trough GLC (Table 6). 

As the vast majority of human studies on the effects of phytosterols on serum cholesterol levels have been done using free sterols and sterol fatty acid esters, there has been a debate about the efficacy of the other natural sterol conjugates in lowering cholesterol. However, recent studies have demonstrated that other conjugates (ferulic acid... 

Figure 10.1 Examples of structural formulas of phytosterol and sterol conjugates. FS = free sterol alcohol, SE = sterol fatty acid ester, SF = steryl ferulate, SG = steryl glycoside, ASG = acylated steryl glycoside.

Abbreviations nr—not reported TAG—triacylglycerols FFA—free fatty acids St —phytosterol fatty acyl esters St—free phytosterols FPE—phytosterol ferulate esters tocols-tocopherols and tocotrienols GL—glycolipids PL—phospholipids RBD—refined, bleached, and deodorized oil. 

Soybean tocopherols are the major source of natural fat-soluble antioxidants and Vitamin E. The Vitamin E activity of natural d-cx-tocopherol is much greater that that of synthetic Vitamin E, which is a mixture of eight stereoisomers (203). Phytosterols are used as raw materials for over 75% of the world s steroid production. The more recent application of phytosterol, phytostanol, and their fatty acid esters in margarine and table spreads is based on the blood cholesterol-lowering effect of these compounds (204,205). The recent development of functional foods containing phytosterols has been reviewed by Hollingsworth (206) and Hicks and Moreau (207). 

The preparation of high-purity tocopherols and phytosterols involves steps such as molecular distillation, adduct formation, liquid-liquid extraction, supercritical fluid extraction, saponification, and chromatography (175). The extraction of tocopherols from soybean oil deodorizer distillate by urea inclusion and saponification of free fatty acids resulted in good recovery of tocopherols (208). To improve the separation of sterols and tocopherols, Shimada et al. (209) used a lipase to esterify sterols with free fatty acids. Then the steryl esters and tocopherols were separated better by molecular distillation. Chang et al. (210) used supercritical fluid CO2 extraction to recover tocopherols and sterols from soybean oil deodorizer distillate. A patent by Sumner et al. (211) advocated treatment of the distillate with methanol to converted free fatty acids and other fatty acid esters to methyl esters that can then be removed by a stripping operation. Then separation of sterols and tocopherols could be carried out by molecular distillation. 

In the DOD, phytosterols are present in both the free and esterified forms with fatty acids. Therefore, the first step in the extraction of phytosterols is conversion of phytosterol fatty esters into free phytosterols. This is achieved either by hydrolysis or trani-esterification. Hydrolysis could be carried out under strong basic conditions (saponification with further acidulation), under strong acidic conditions, or under chemical or enzyme (specific or nonspecific) catalyzation. Re-esterification of phytosterols occurs during methyl ester distillation as a result of the high temperatures involved therefore, a further trani-esterification step for free sterols is required. Esterification of phytosterols or trani-esterification of sterol fatty acid esters is the second step in this process. Methanol is the most commonly used alcohol, and it leads to methyl esters, which are characterized by a higher volatility, however, other Ci to C4 alcohols may also be used. Esterification and trans-esterification of fatty acids or phytosterols can be catalyzed by metal alcoholates, or hydroxide, by organic catalysts, or by enzymes (Table 7). 

Abbreviations nr, not reported TAG, triacylglycerols FFA, free fatty acids St E, phytosterol fatty acyl esters St, free phytosterols FPE, phytosterol ferulate esters Tocols, tocopherols and tocotrienols GL, glycolipids PL, phospholipids and RBD, refined, bleached and deodorized oil. 

Non-specific esterification of wood sterols can be performed chemically (www. freshpatents.com/Phytosterol-esterification-product-and-method-of-make-same-dt-20070628ptan20070148311.php) however, enzymatic esterification with lipases has the potential advantages of higher specificity and mild reaction conditions which are desirable, both from process and environmental perspectives. More than 20 lipases were previously screened for their ability to catalyze the transesterification of wood sterols and fatty acid esters (Martinez et al. 2004). The goal was now to screen among them those specific for stanol esterification, so as to obtain a product consisting in mostly esterified stanols and mostly free sterols (see Fig. 6.3.4) amenable for separation through short-path distillation. 

Their capacity to decrease serum low-density lipoprotein (LDL) cholesterol levels and, thus, in protecting against CVD, has led to the development of functional foods enriched with plant sterols. At present, several functional products, such as spreadable fats, yoghurts, and milk, with free phytosterols or phytosteryl fatty acid esters or phytostanyl fatty acid esters added at high levels, are available in the market, especially in several European countries. ... 

Figure 1. HPLC-ELSD chromatogram of corn fiber oil. Insert shows the structure of sitostanyl-ferulate, the major ferulate-phytosterol ester in corn fiber oil (Norton, 1995). Abbreviations StE, phytosterol fatty acyl ester TAG, triacylglycerol, a-Toc, a-tocopherol FFA, free fatty acids CE, p-coumarate-phytosterol esters y-Toc, y tocopherol ST, free phytosterols 1,3-D, 1,2-diacylglycerols FE, ferulate-phytosterol esters 1,2-D, 1,2-diacylglycerols.

Since free plant sterols can be used in margarines at a concentration of up to 3% (w/w) without causing quality problems (Sierksma et al., 1999), an average margarine consumption of 25 g/day would provide only 0.75 g of plant sterol (and even less stanol). This limitation has been the main stimulus for the development of the more lipid-soluble fatty acid esters of phytosterols and phytostanols. 

Cmde com oil contains principally triglycerides and minor nonpolar and polar lipid components such as free fatty acids, phytosterol fatty acyl esters, free phytosterols, phytosterol femlate esters, tocols, tocopherols, tocotrienols glycolipids, phospholipids, pigments, volatile compoimds, waxes etc. (Gimstone, 2011). 

Sterols are minor constituents of most fats. Those of animal origin contain cholesterol and traces of other sterols, whereas plants contain phytosterols, of which p-sitosterol is the most common. Sterols occur in the free form or, after esterification to fatty acids through the 3-OH group, as steryl esters. The presence of sterols in archaeological residues can be a useful indicator of a plant or animal origin or an indicator of both if cholesterol and phytosterols are detected in the same sample. That noted, cholesterol is a potential contaminant of all archaeological samples subjected to handling. 

Naturally occurring fats contain small amounts of soluble minor consituents pigments (carotenoids, chlorophyll, etc.), sterols (phytosterols in plant fats, cholesterol in animal fats), vitamin A (from carotenes), vitamin D (calciferol), waxes (esters of long-chain alcohols and fatty acids), ethers, and degradation products of fatty acids, proteins, and carbohydrates. Most of these minor compounds are removed in processing, and some are valuable by-products. 

Lipophilic extracts of sabal fruit (Serenoa repens syn. Sabal seruulata) are used in the treatment of benign prostatic hyperplasia (BPH) (see Chapter 55). The hexane extract of the pulp and seed contains a complex mixture of free fatty acids and their esters, small quantities of phytosterols (such as [3-sitosterol), aliphatic alcohols, and polyprenic compounds. The efficacy of this extract was recently compared with that of the 5a-reductase inhibitor finasteride for 6 months, in a randomized, double-blind trial involving almost 1100 men with moderate BPH. Dwarf palm and finasteride reduced the International Prostate Symptom Score by 37 and 39% and increased urinary peak flow by 2.7 and 3.2 ml/sec, respectively. In contrast to finasteride, dwarf palm had little effect on prostate volume. 

Deodorizer distillate is the material collected from the steam distillation of oils. It is a mixture of free fatty acids (especially during physical refining) tocopherols, phytosterols and their esters, hydrocarbons, and hpid oxidation products. The quality and composition of deodorizer distillate depends on the feedstock oil composition and processing conditions. Tocopherols and sterols are the most valuable components that can be recovered from the distillate, and they are used in the nutrition supplement and pharmaceutical industries (201). Typical soybean deodorizer distillate contains about 33% unsaponifiable matters, of which 11% is tocopherol and 18% sterol (202). 

The recovery of phytosterols or their concentration is the third step in their isolation. Methyl esters of fatty acids are distilled to raise the phytosterol content up to 50% by weight. Molecular distillation can be used for phytosterols. Phytosterol concentrates are used as raw materials for steroid drug preparation via microbial fermentation, in which the pure form of phytosterols are not necessary. The final recovery of phytosterols can be achieved by crystallization (physical), solvent extraction (chemical), or crystallization with additives via adduct formation and separation (physicochemical). Organic solvents or solvent mixtures composed of low- and high-polarity solvents and water are used for crystallization of phytosterols... 

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