Sterols 301 unsaponifiable matter

There are cases where HPLC separation is performed not in order to quantify the alcohols but as a technique for the purification of the analytes to be subjected to further instrumental analysis. This is the case, for example, with the identification and determination of the structure of an abscisic acid in starfruit extract (Averrhoa carambola L.). The separation and purification of the analytes was carried out also with HPLC using a mobile phase of diethyl ether, whereas the structure was elucidated by H and UC-NMR (6). In a similar way, to separate the sterols and alkanols from the unsaponifiable matter from olive oils on a silica column, a gradient composed of hexane/diethyl ether was chosen in an offline system (7), whereas an online HPLC-HRGC system uses as its mobile phase hexane/isopropanol (8). 

Sterols, which are very abundant in some fruits, can be removed by precipitating them in different solvents. The unsaponifiable matter is dissolved in a minimum volume of methanol, petroleum ether, or acetone. The precipitation is completed overnight at — 20°C following centrifugation, the sterol-free supernatant is used for analysis (5). 

Frega, N., Bocci, E, Giovannoni, G. and Lercker, G. (1993) High resolution GC of unsaponifiable matter and sterol fraction in vegetable oils. Chromatographia, 36, 215-217. 

Unsaponifiable Matter in Fats and Oils, Including Marine Oils (Ca bb-5, 3) determines substances dissolved in fats and oils that cannot be saponified (turned into sodium salts) by the usual caustic treatment, including higher aliphatic alcohols, sterols, pigments, and hydrocarbons. This method is not suitable for marine oils or feed grade fats. 

Unsaponifiable matter. Oils and fats contain variable amounts of sterols, hydrocarbons, tocopherols, carotenoids, and other compounds, collectively referred to as unsaponifiable matter because they do not produce soaps upon hydrolysis (Table 6). The sterol and tocopherol composition of commodity oils is discussed in another chapter. Some of these minor components are removed during refining, and the resulting concentrates may be useful byproducts, for example, tocopherol antioxidants. Characteristic fingerprints of minor components, particularly phytosterols and tocopherols, are also used to authenticate oils and detect adulteration (18). 

Lipases liberated from the testa and the cross cells promote rapid hydrolysis of the oil, and therefore, it should be extracted within hours of milling. Attempts have been made to upgrade oil with 30% free acid by reaction with glycerol and the enzyme Lipozyme Mucor miehei lipase) followed by neutralization. The major acids in rice bran oil are palmitic (12-18%, typically 16%) oleic (40-50%, typically 42%), and linoleic acid (29 2%, typically 37%). The oil contains phospholipids ( 5%), a wax that may be removed and finds industrial use, and unsaponifiable matter including sterols, 4-methylsterols, triterpene alcohols, tocopherols, and squalene among others. 

Avocado Persea americana). The avocado grows in tropical and subtropical countries between 40°N and 40°S and is available particularly from California, Florida, Israel, New Zealand, and South Africa. Like the palm and the olive, lipid is concentrated in the fruit pulp (4—25%) from which it can be pressed. There is very little oil in the seed (2%). The oil is used widely in cosmetic products as it is easily absorbed by the skin, and its unsaponifiable material is reported to provide some protection from the sun. It is also available as a high-oleic speciality oil for food use. It is rich in chlorophyll, making it green before processing. It contains 16 0 (10-20%), 18 1 (60-70%), and 18 2 (10-15%) as its major fatty acids. Its unsaponifiable matter, total sterol, and tocopherol levels have been reported (74-78). 

Approximately 0.5% of crude coconut oil is not saponified by caustic treatment. The unsaponifiable matter consists mainly of tocopherols, sterols, squalene, color pigments, and carbohydrates. The odor and taste of coconut oil is largely due to 5- and y-lactones, which are present in trace quantities (24). Among the unsaponifi-ables, tocopherol contributes to the oxidative stability of crude coconut oil. A typical sample of crude coconut oil contained 55 ppm total tocopherols of which 40.7 ppm is cx-tocopherol (25). Most of the unsaponifiables are removed in the process of refining, bleaching, and deodorizing of crude coconut oil. 

Refined oil usually retains little phospholipid, but damaged beans can have a significant content of phosphatidic acid, and the amount of iron in the oil is related to the amount of phosphorus (24). During deodorization, considerable amounts of sterol and tocopherol may be removed from the oil. The proportion removed depends on deodorization conditions, but a 30% to 40% decrease is not unusual (25). Much of the hydrocarbons and squalene are lost to the deodorizer distillate as well. Free fatty acids in fully refined oil are required to be <0.05% and unsaponifiable matter < 1.5% (26). 

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). 

Other Components of the Unsaponifiable Matter The unsaponifiable matter in a cmde regular sunflower oil is usually in the range of 0.5-1.5% (9, 17), or lower than 15 g/kg according to the Codex-Stan 210-1999. In addition to sterols (around 2.4. 6 g/kg) and tocopherols and tocotrienols (0.4-1.5 g/kg), there are minor components of sunflower oil. Aliphatic compounds and terpenoids occur naturally in oils. Of the terpenoid family, squalene is the most widely occurring compound. The occurrence of squalene in regular sunflower oil is fairly low 0.008-0.019% (5) or 15-20 mg/100 g (9). The aliphatic alcohol content is 100-mg/lOO-g oil (9). 

Part of the unsaponifiable matter (such as tocopherols, sterols, and sterolesters) is distilled together with the free fatty acids during deodorization/deacidification, as... 

The cashew (Anacardium occidentale L.) is an evergreen species native to tropical America and contains 47% oil (w/w) (1, 68). Other components of cashew nuts include carbohydrate (27.1%), protein (18.2%), water (5.2%), and ash (2.5%) The predominant fatty acid in cashew nut oil is oleic acid (57.3-65.1%), followed by linoleic (15.6-18.6%), and palmitic (9.0-14.2%) acids (Table 10) (68). Cashew nut oil contains 1.4% unsaponifiable matter (w/w), of which 76.2-82.7% is p-sitosterol. Other sterols present in cashew nut oil include A -avenasterol, campesterol, fucosterol, cholesterol, and stigmasterol (68). Cashew nut oil contains 45.3-83.5 mg/lOOg y-tocopherol other tocopherols present are a-tocopherol (2.8-8.2 mg/lOOg) and 5-tocopherol (2.0-5.9 mg/100 g) (68). 

The unsaponifiable matter of soy, corn, canola/rapeseed, sunflower, cottonseed, peanut, and palm ranges from 10-30%, and it is composed of 40% phytosterols and 15% tocopherols (199-202). The temperature, duration, quantity of skimming vapor, and the extent of vacuum used for deodorization are the parameters that greatly influence the quality and quantity of DOD. The low content of tocopherols and sterols in the DOD often requires a concentration step however, if the starting material is soybean, this is not an issue because of the high concentration of these compounds. 

Preparative HPLC was used to separate sterols and triterpene alcohols from the unsaponifiable matter in plant oils from Camellia weiningensis L., Brassica juncea L., and Microula sikkimensis. The isolated compounds were acetylated and further purified by AgN03-impregnated silica gel preparative thin layer chromatography (TLC). The identification was done by IR and MS. 

Table III gives the physical and chemical properties of the M. oleifera oil. Some of the properties of the oil depend on the extraction medium. The M oleifera oil is liquid at room temperature and pale-yellow in colour. Electronic nose analysis shows that it has a flavor similar to that of peanut oil. The melting point estimated by differential scanning calorimetry is 19°C (15). The chemical properties of the oil depicted in Table III below are amongst the most important properties that determines the present condition of the oil. Free fatty acid content is a valuable measure of oil quality. The iodine value is the measure of the degree of unsaturation of the oil. The unsaponifiable matter represents other lipid- associated substances like, sterols, fat soluble vitamins, hydrocarbons and pigments. The density, iodine value, viscosity, smoke point and the colour of Moringa oil depends on the method of extraction, while the refractive index does not. Varietal differences are significant in all physical characteristics apart from refractive index and density (2). The heating profile of the M. oleifera seed oil using the differential scanning calorimetry (DSC) conventional scan rate shows that there is one major peak B and, two small shoulder peaks A and C...

In addition to triacylglycerols and free fatty acids, crude CNO contains about 0.5% of unsaponifiable matter, though the Malaysian standard (MS239 1987) allows a maximum level of 0.8% and Codex (2001) up to 1.5%. This material consists mainly of sterols, tocols, squalene, colour compounds, carbohydrates and odour compounds (such as lactones). The pleasant odour and taste of CNO when the oil is extracted from fresh material is mainly due to y- and 8-lactones, which are present in trace amounts (Young 1983). 

The unsaponifiable matter in peanut oil is mainly sterols (largely fi-sitostcrol and campesterol). 

The Codex Alimentarius standard includes limits for acidity, volatile matter, insoluble impurities, peroxide value, colour, odour, taste, iron, copper, K27o, AK, permitted additives, contaminants (lead, arsenic, halogenated solvents), refractive index, saponification value, iodine value and unsaponifiable matter. Physical and chemical constants, such as iodine value and saponification value, are not found in the EU regulation. This is explained by the fact that more definite information is obtained by determining fatty acid composition, sterol and wax composition, trans fatty acid content, stigmastadiene, and so on. 

The unsaponifiable matter includes lipids of natural origin such as sterols, higher aliphatic alcohols, pigments, hydrocarbons as well as any foreign organic matter non-volatile at 103 °C (e.g. mineral oils). 

Saponification of the oil or fat, then separation of the unsaponifiable matter. Isolation of the sterols from the unsaponifiable matter by TLC. Analysis by GLC of the sterol fraction so isolated (or of the trimethyl-silyl ethers prepared from the sterol fraction) and interpretation of the chromatograms. 

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