METHODOLOGICAL ASPECTS OF LIPID EXTRACTION FROM BIOLOGICAL MATRICES

The paper presents the studies on the degree of raw fat extraction from meat systems by various methods. Relative differences in extraction methods are shown depending on the polarity of solvents used. Fat was extracted using the classical Soxhlet method, as well as two-step extraction. Specially prepared model systems were used as test samples including both simple and multicomponent mixtures in the form of meat-and-bone meal, bone meal, and meat-and-plant meal. It was found that when using non-polar solvents, a large amount of polar lipids was lost. The influence of the solvent on the degree of extraction for glycerides, phosphatides, free fatty acids and oxidation products was shown. A number of experiments are presented and the results are given on the degree of fat extraction using mixtures of organic solvents with mineral acid. The studies allowed to choose the best extraction method for each type of meat and meat-containing raw materials. Based on the results, methodical recommendations are proposed on the use of hydrophobic and hydrophilic solvents and their mixtures. Methods of lipid extraction from food products after alkaline and acid hydrolysis are considered. Examples of fat determination by instrumental methods (refractometry) are given. Classical methods for fat content evaluation are described (Randall method, Twisselmann method, and Rushkovsky method). УДК /UDC:637.5.072/616–0 DOI 10.21323/2414–438X-2018–3–2–4–21 Для цитирования: вострикова н.л., Кузнецова о.а., Куликовский а.в. Методические аспекты извлечения липидов и биологических матриц. Теория и практика переработки мяса. 2018; 3(2): 4–21. DOI 10.21323/2414–438X‐2018–3–2–4–21 FOr cItatIOn: Vostrikova n.L., Kuznetsova O.a., Kulikovskii a.V. Methodological aspects of lipid extraction from biological matrices. Theory and practice of meat processing. 2018; 3(2): 4–21. (In russ.). DOI 10.21323/2414–438X‐2018–3–2–4–21 Вострикова Н.Л.,* Кузнецова О.А., Куликовский А.В. Федеральный научный центр пищевых систем им. В.М. Горбатова РАН, Москва, Россия Natal’ya L. Vostrikova,* Oksana A. Kuznetsova, Andrey V. Kulikovskii V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Moscow, Russia


Introduction
Lipids (from Greek «lipos» -fat) are a complex mixture of organic compounds with similar physical and chemical properties, which is found in plants, animals and microorganisms. Lipids are widely distributed in nature. Together with proteins and carbohydrates, they make up the basic mass of organic substances in all living organisms, being an obligatory component of every cell. Lipids are widely used in obtaining many food products, and are important components of food raw materials, semi-finished and finished food products, while largely determining their nutritional and biological value and taste [1,2].
Lipids are insoluble in water (hydrophobic) and well soluble in organic solvents (gasoline, diethyl ether, chloroform, etc.).
In animals and fish, lipids are localized in subcutaneous, cerebral and nerve tissues and tissues surrounding important organs (heart, kidneys). Lipid content in sturgeon carcass may be as high as 20-25 %, in herring -10 %. In carcasses of terrestrial animals, it highly varies: 33 % (pork), 9.8 % (beef), 3.0 % (piglets). In deer milk its content is 17 to 18 %, in goat milk -5.0 %, in cow milk -3.5 to 4.0 %. Lipid content in certain types of microorganisms may be up to 60 % [3].
According to the chemical structure, lipids are derived from fatty acids, alcohols, and aldehydes, and are built using ester, ether, phosphoester, and glycosidic linkages. Lipids are divided into two main groups: simple and complex lipids. Simple neutral lipids (not containing nitrogen, phosphorus, sulfur) include derivatives of higher fatty acids and alcohols, as well as glycerides, waxes, cholesterol esters, glycopeptides and other compounds. Molecules of complex lipids contain not only the residues of high molecular weight carboxylic acids, but also phosphoric or sulfuric acids.
Lipids are important ingredients of food, as they have high energy value and are a source of building material for human body tissues. Individual components of fat, i.e. some fatty acids, phosphatides, sterols, fat-soluble vitamins, have important biological functions in the body. Lipids are substances of plant and animal origin, soluble in organic solvents and slightly soluble in water. They contain higher alkyl or acyl radicals in the molecular structure.
Quantitative determination of lipids requires the extraction of glycerides and related substances (pigments, vitamins, free fatty acids, phosphatides, etc.) from the test object [4,5].
Existing methods for determining the fat content in different types of raw materials and products may be divided into two groups, i.e. one-step and two-stage ones.
One-step methods based on the ultrasound, nuclear magnetic resonance, photometry and infrared rays allow quantitative determination of fat directly in the test object. However, this requires complex and expensive equipment. Application of some of this equipment (for example, in nuclear magnetic resonance) is recommended only in case of the impossibility of using any other method for determining the amount of the certain substance.
Most of the physical and chemical methods (extraction weight, refractometry, etc.) used to quantify fat are from second group. Their characteristic feature is a two-step process -fat extraction from the object and the quantitative determination. For fat extraction, various organic solvents are used, i.e. gasoline, petroleum ether, diethyl ether, acetone, chloroform, monobromo-and monochloronaphthalene, tricresyl orthophosphate, etc. It should be kept in mind that hydrophobic solvents (petroleum ether, gasoline, etc.) extract together with glycerides somewhat less related substances. And their extraction is selective. Glycerides are extracted more rapidly, and phosphatides, free fatty acids and oxidation products are extracted slower. In this regard, when using a hydrophobic solvent, the fat extraction process lasts for up to 2 to 3 days. To accelerate the process and extract the glycerides and related substances from the test object more completely, it is recommended to use hydrophilic solvents (methyl and ethyl ethers, etc.) or a mixture of hydrophobic and hydrophilic solvents (binary solvents) [6,7,8,9].

Methods
In practice, for lipid extraction, two basic routine extraction methods are most often used, which allow to extract lipids quantitatively from tissue and its fractions of almost all biological classes. The most common is the Folch method, according to which extraction is carried out with a chloroform: methanol mixture (2:1) with 20 parts of extracting mixture per one part of tissue. This method allows to obtain a sufficiently high yield of neutral lipids, diacyl glycerophospholipids and sphingolipids. Lysophospholipids are transferred into the solution only partially, and more polar acid lipids may be lost when the extract is washed out with salt solutions and water. However, repeated extractions and restriction of washing allow to increase the yield of lipids up to a quantitative level. Another method was proposed by Bligh and Dyer, when lipid extraction is carried out with a chloroform: methanol mixture (1:1) with two parts of the mixture per one part of tissue. However, even in this case, when washing out with water, the most polar acid phospholipids and lysophospholipids are transferred into the aqueous phase and lost.
Depending on the chemical nature of lipids, modified extraction methods are used. By replacing the chloroform: methanol mixture with a mixture of chloroform and 2 % solution of acetic acid in methanol, the yield of polar lipids can be increased. Subsequently, for the same purposes, a mixture of chloroform: methanol with 1M HCl (4:2:3) was used [10,11].
When extracting neutral and common lipids, nonpolar solvents such as chloroform, hexane, diethyl ether are often used. Obviously, in this case, a lot of polar lipids are lost.
For a more complete understanding the methodology of the lipid extraction from raw materials and finished products, the most commonly used methods are presented below.
Gerber method is used to determine fat in semi-finished meat products (minced meat, semi-finished products from minced meat), curd cheese, finished food products, bakery products, milk and dairy products, dried foods for children and healthy diet.
The method is based on the destruction of test product proteins with concentrated sulfuric acid and the dissolution of fat in isoamyl alcohol. The ester formed in the reaction of isoamyl alcohol with sulfuric acid dissolves in the latter, which enables fat extraction. The resulting mixture is centrifuged in the butyrometers. The separated fat layer is collected in the graduated part of butyrometer and is quantitatively determined.
Determination of fat is carried out in milk or cream butyrometers, which differ in size and graduation. The level of graduation mark in milk butyrometers is 0.1 %. In cream butyrometers, the level of two graduation marks corresponds to 1 % fat in the product with a weight of 5 g. The latter are used when the fat content in the product exceeds 10 %.
Weight or gravimetric method with fat extraction in a microniser. The method is used for finished food products and some canned products. Fat is extracted from the product by grinding it in a microniser. After solvent distilling, the dried fat is weighed [12,13,14].
Refractometry method is used to determine fat in finished and semi-finished bakery products, semi-finished vegetable products, and canned products.
The method is based on the fact that when the fat is dissolved, the refraction index of the solvent decreases in proportion to the amount of fat present in it. By the difference between the refraction index of the pure solvent and the fat solution, the mass fraction of the latter is determined. The greater the difference between these indices, the more precise the definition [15].
The method for fat determining with preliminary starch hydrolysis is used to determine fat in finished and semi-finished bakery products (GOST 31902-2012). It is based on fat extraction by solvent from the sample pretreated with hydrochloric acid, removing the solvent and weighing the fat.
For the qualitative oil determination, the following characteristic reactions exist.
Test for acrolein. Two to three drops of the test substance (oil, extract after solvent distillation) are heated in a test tube on a naked flame with 1.5 to 2 parts of anhydrous sodium sulfate. After foaming, the appearance of heavy white fume and pungent odor of acrolein causing lacrima-tion, indicates the presence of oil. Acrolein is unsaturated aldehyde СН 2 =СНСНО formed from glycerin upon removal of two water molecules. If the fume is transferred to a test tube with Schiff 's reagent, then the latter becomes of red color.
Test for saponification. Two to three drops of the test substance are heated in a test tube with 5 cm 3 of alcoholalkali solution, then the alcohol is distilled. The remaining product is dissolved in water (soap is soluble in water). The addition of acid for acidic pH causes the formation of aqueous solution of fatty acids floating on the surface.
Test with haloids. This reaction is qualitative for oils containing unsaturated fatty acids. One or two drops of bromine water is added in a test tube with a solution of oil and shook. The rapid disappearance of yellow color of bromine water indicates the presence of unsaturated acids [16].
Liquid extraction technique is used, for example, to determine the fat content in various objects. Soxhlet extraction is one of the most widely used analytical techniques. In recent years, it has been significantly modernized, in particular, the temperature of the solvent coming into contact with the extracted substance was increased in order to reduce the extraction time. The modifications presented by the American chemist, E. Randall, are among the most effective ones in this respect: Randall method consists of two stages: at the first stage, the sample in the sleeve is placed in a boiling solvent, and at the second stage, it is washed out with a solvent dripping from the condenser. Rapid dissolution of the sample components occurs in the first stage because of boiling solvent, which greatly reduces the time required for the entire assay. Randall method also allows the solvent to be recovered at the end of the extraction procedure.
Soxhlet extraction is a process of extraction of soluble substances from solid materials. It was developed by the German agrochemist Franz von Soxhlet in 1879. Soxhlet method is dissolution of the extracted compound using a cold solvent that drips from the condenser. Typically, a complete extraction lasts for several hours [13,14].

Discussion
Most methods for determining lipids in food can be divided into three groups.
The methods in first group are based on lipid extraction from the test product by repeated extraction with a solvent until the residual content in the product is negligible. Then, the solvent is distilled from the obtained extract, and the residue containing lipids is dried and weighed (weight method of fat determining). This operation is usually carried out in special extraction instrument, Soxhlet apparatus, that allows to produce repeated fat extraction with the same portion of ether. Non-polar solvents are used for the extraction, i.e. diethyl ether, hexane, petroleum ether.
The diverse nature of food products, which determines the different strength of lipid bonding to other parts of the product, affects the extraction efficiency. The meth-ods of this group allow to extract free and slightly sorbed lipids from food products. Strongly bound lipids are not extracted. In addition, solvents extract not only fatty acid glycerides, but also a number of other substances, i.e. free fatty acids; organic acids such as succinic, tartaric, citric, and apple acid; phosphatides; sterols; essential oils; waxy substances; resins; aldehydes; ketones; colorants. In view of this, the product extracted by the solvents is not a pure fat. That is why it is called «raw fat». Often the difference between the weight of «raw fat» and the actual fat weight is neglected. The amount of impurities in «raw fat» increases, when you use non-dehydrated diethyl ether, which dissolves up to 2 % moisture. Such ether easily extracts sugars contained in food raw materials (vegetables, cereals, etc.). Alcohol contained in diethyl ether readily dissolves many organic compounds. In view of this, diethyl ether used for fat extraction is pre-washed out with water to remove alcohol and dehydrated with annealed calcium chloride. After removing impurities, the ether is distilled.
To accelerate the extraction process and for complete fat extraction, the test product is thoroughly ground and dried, since the larger and moistier the particles, the more slowly the fat is extracted. In addition, fat is not completely extracted from moisty objects.
In this connection, and due to the significant oxidation of lipids in the extraction process, more efficient extraction methods were developed.
The methods in second group are based on the use of a mixture of polar and non-polar solvents for extraction. In this case, polar solvent (usually methanol or ethanol) breaks the bonds of lipids to proteins and other food components, and nonpolar solvent (chloroform, benzene, petroleum ether) directly dissolves the lipids. The most widely used mixtures are chloroform: methanol (2:1) and chloroform: ethanol (2:1). However, in contrast to the methods of the first group, such binary mixtures extract a significant amount of non-lipids (up to 25 % of the amount of extractable substances). Therefore, in many cases, it became necessary to remove these non-lipid substances by re-dissolving in chloroform or washing out with a 1 % solution of NaCl or KCl.
The methods in third group provide lipid extraction from food products after acid or alkaline hydrolysis. In this case, the food product is hydrolyzed by an aqueous or alcoholic solution of alkali with heating. After alkaline hydrolysis, the soaps obtained are decomposed by the acid solution, and released fatty acids are extracted with ether (petroleum, diethyl ether) and are purified by filtration. After the ether is distilled, the weight of fatty acids is determined, which is recalculated into fat weight. Theoretically, this method is unable to extract lipids in their native state. Therefore, their content in food is evaluated by the amount of fatty acids and unsaponifiable substances released from the hydrolysate. This group of methods includes the acid method of fat determining in milk, dairy products and canned foods with butyrometer. Fat is extracted by the con-centrated sulfuric acid with heating. The mixture is centrifuged. In this case, the fat is transferred into the phase of the isoamyl alcohol added. The volume of the released fat is measured in the graduated part of butyrometer.
The methods in first group are not recommended for the products rich in phospholipids firmly bound in cells (some fish species), but are suitable for foods with a predominant triglyceride content, i.e. oil seeds.
The methods in second group, almost in all cases, allow obtaining reliable quantitative results, but they are relatively labor-consuming and not always suitable for large-scale routine analyzes.
The application of the third group methods does not lead to the extraction of natural lipids, but in most cases allows obtaining results that closely correspond to the results obtained by the methods of second group. Their advantage is the use in large-scale routine analyzes [17,18].

Determination of fat by continuous extraction (Soxhlet method).
Fat is extracted from the test product with diethyl ether in a Soxhlet apparatus consisting of extractor with siphon tube, ball-shaped reflux condenser, and receiving flask. A sample of a thoroughly ground product in an amount of 5 to 10 grams (depending on the expected fat content in the product) is weighed with an accuracy of 0.0001 g and transferred into a filter paper cartridge. Before extraction, the sample of the product is dried at a temperature of 100 to 105 °C for 2 hours.
To make a filter paper cartridge, a rectangular piece of filter paper is wrapped several times around a wooden blank or glass cylinder, whose diameter is somewhat smaller than the diameter of the extractor. The part of the paper protruding beyond the edge of the blank for the length of its diameter is folded forming the cartridge bottom. A circle of filter paper and a piece of fat-free cotton wool are put on the bottom. The sample in the cartridge is closed from the top with a circle of filter paper and a piece of fatfree cotton wool. Free edges of the cartridge are folded. The height of the cartridge should be 10 to 15 mm below the upper bend of the extractor siphon tube.
Cartridge with sample is placed in extractor, which is connected to receiving flask dried up to a constant weight and condenser. The flask is previously filled up with dried distilled diethyl ether to 2/3 of its volume. The water is passed through the condenser, and the flask with diethyl ether is heated on a water bath with closed electric or steam heating on a special closed electric heater. The water temperature in the bath should not be higher than 60 °C. The solvent vapor formed in the flask during boiling go into the condenser. There it condenses into a liquid that drips down into the extractor, where the cartridge with the product sample is located. When the level of solvent in the extractor rises slightly above the upper bend of the siphon tube, the ether with dissolved fat flows into the receiving flask. After that, the entire process is repeated again. An example of equipment for fat extraction using Soxhlet method is shown in Figure 1.
Fat is extracted for 10-12 hours, while the heating and boiling of the ether should be adjusted for 6 to 8 drains per hour with an extractor volume of 100 ml. For a more complete fat extraction, the sample of the product is placed into the solvent for 6-8 hours before the extraction. The infusion is performed in the extractor filled with ether below the siphon tube.
When the extraction is completed, the cartridge with the sample is removed from the extractor and the solvent is distilled from the receiving flask into an empty extractor. The residual fat in the flask is dried in a desiccator to constant weight at a temperature of 100 to 105 °C. For the first time, the flask with fat is weighed after 1 hour of drying and then every 0.5 hours. Before weighing, the flask is cooled down in a desiccator for 30 to 35 minutes and then weighed with an accuracy of 0.0001 g.
The amount of fat (X,%) is calculated by the following equation: where G is the weight of the flask with fat, g; G 1 is the weight of the empty flask, g; g is the weight of the test product sample, g.
The final result is expressed as the arithmetic mean of two determinations. The difference between two parallel determinations should not exceed 0.3 %.
Completeness of fat extraction from the sample of the test object should be verified as follows. Apply a drop of miscella (solvent) on a clean, degreased glass. Upon the complete fat extraction, a greasy stain should not appear on the glass after evaporation of the solvent [19,20,21].
Determination of fat by infusion with solvent. A sample of the test product in an amount of 2 g is weighted with an accuracy of 0.01 g in a conical flask with volume of 50 to 100 ml. Then, 10 ml of a solvent (gasoline or dichloroethane) are added, the flask is closed with a cork stopper and weighed again to determine the solvent weight. Fat is extracted for 1 hour, while periodically shaking the sample with the solvent. Then, the contents of the flask are filtered through a paper filter into a dry flask previously weighed with an accuracy of 0.001 g. The flask with filtrate is weighed with an accuracy of 0.01 g and the filtrate weight is determined by the difference.
The solvent is distilled on a sand bath with appropriate precautions. The flask with the residue is placed in a desiccator and dried at a temperature of 100 to 105 °C. Then, the flask with fat is cooled down in a desiccator and weighed with an accuracy of 0.001 g.
The fat content (X,%) is determined by the following equation: where G is the amount of solvent, g; G 1 is the amount of fat, g; G 2 is the amount of filtrate, g; g is the weight of the test product sample, g.
The final result is expressed as the arithmetic mean of two determinations. The difference between two parallel determinations should not exceed 0.3 % [22].
Determination of fat by refractometry method. The method is based on the determination of refraction index of a fat solution in а-monobromonaphthalene, motor oil or a mixture of monobromonaphthalene with motor oil, by which fat is previously extracted from the test product. Dissolving fat in any of these solvents causes a decrease in refraction index of the latter directly proportional to fat concentration in the extract.
а-Bromonaphthalene has high refraction index of 1.65; it is slightly volatile and does not dissolve water. All these properties make it the most suitable solvent for determining the amount of fat using a refractometer. When performing the analysis, a universal refractometer with a scale graded up to nD = 1.75 is used. A sugar refractometer with a scale of only up to nD = 1.54 is not suitable for working with monobromonaphthalene.
Method for determination of fat with monobromonaphthalene as a solvent. A sample of a thoroughly ground product in amount of about 2 g is weighted with an accuracy of 0.0001 g in a small porcelain mortar (not more than 5 cm in diameter), 1.3 ml of fine annealed sand are added, and about 6 g of monobromonaphthalene is weighted with an accuracy of 0.01 g. The sample with sand and solvent is thoroughly pestle for 5 minutes. Then, the contents of the mortar are transferred to a small folder filter with diameter of 7 cm. The fat extract obtained after filtration is collected in a small laboratory glass.
After stirring the extract with a glass rod, 2-3 drops of it is applied to the refractometer prism and the refraction index is determined. Determination of refraction is repeated at least 3 times with new portions of the extract and arithmetic mean is calculated.
The refraction indices of pure monobromonaphthalene and the fat extract are determined at the same temperature. The temperature during the determination is kept constant, which is achieved by passing water through the prism casing.
The amount of fat (X,%) is calculated by the following equation: Х = 10 4 · а ·(Н о -Н) · g / g 1 , where H 0 is the refraction index of pure solvent; H is the refraction index of the fat extract; g is the weight of the solvent, g; g 1 is the weight of the test product, g; а is the ratio of the fat percentage in the solvent to the difference between the refraction indices of the solvent and the fat extract. For food concentrates, a is 0.0368 [17]. When calculating fat percentage, the refraction index and the fat density indicated in Table 1 are used. Method for determination of fat using motor oil or a mixture of monobromonaphthalene and motor oil as solvents. About 5 grams of well-ground product is weighed with an accuracy of 0.01 g into a small porcelain mortar and 4 grams of motor oil or a mixture of 25 volume parts of monobromonaphthalene and 75 volume parts of motor oil are added. Then, 3 g of fine annealed sand are introduced into the mortar and the resulting mixture is thoroughly ground for 5 to 10 minutes. Longer grinding is recommended for food concentrates with meat [24].
When mixture of monobromonaphthalene and motor oil is used as a solvent, the ground mass is transferred from the mortar to a folded filter, 2 to 3 drops of the filtered fat extract are applied to refractometer prism and the refraction index is determined. In the case of using motor oil as a solvent, the mixture in a mortar is pestle with heating, while immersing the mortar in a vessel with hot water. The ground mass is transferred from the mortar to a filter of two gauze fabric layers with a thin interlayer of cotton wool. Then, several drops of fat extract are squeezed out, cooled, applied to the refractometer prism and the refraction index is determined.
The fat content is calculated by the equation given in the procedure for determination of fat with monobromonaphthalene as a solvent. Value of а is equal to: in the case of using the motor oil, 0.3; in the case of using the mixture of monobromonaphthalene and motor oil, 0.19. The difference between two parallel determinations should not exceed 0.3 % [22].
The refractometry method of determining the amount of fat is one of the most rapid methods. It is widely used in analysis of food concentrates and other products containing fat.
Determination of fat by centrifuging. The method is used to determine fat in dried dairy products, i.e. milk, cream, butter, as well as in egg powder and milk formulas for infants. In this method, the test product is treated with concentrated sulfuric acid in the presence of isoamyl alcohol with heating and centrifuging. Under the action of sulfuric acid, the protein of dairy products is converted into a soluble compound with chemical formula: H 2 SO 4 -NH 2 R(COOH) 6 resulting in fat separation. The isoamylsulfuric ether formed by the addition of isoamyl alcohol reduces surface tension of fat globules and promotes their aggregation. Heating and centrifuging accelerate this process. Glass instruments, butyrometers, are used for determination, in the graduated part of which the separated fat is collected. The volume of fat is measured directly on the butyrometer scale. Butyrometer, or a lactoscope, is designed to determine fat mass fraction in milk and dairy products. The most common butyrometer for milk is a glass cylindrical vessel with a scale, on which the amount of fat in milk is determined: one graduation mark is 0.1 % by mass.
The determination procedure is given for dried dairy products. Butyrometers for cream or milk may be used ( Figure 2).
Determination of fat in a cream butyrometer (for analyzing dried whole milk, dried cream, dried butter). A sample of 2.5 g (dried milk, dried cream) or 2 g (dried butter) is weighed with an accuracy of 0.01 g in 25-50 ml laboratory glass with a spout. Then, 4 to 5 ml of sulfuric acid are added (relative density 1.5 to 1.55) and thoroughly ground with a glass rod. The resulting homogeneous mass is quantitatively transferred through a small funnel into the butyrometer and the glass is washed several times with an acid of the same density in 3 to 4 ml portions ensuring that the total volume of acid in the butyrometer is 18 to 19 ml, and the content of the butyrometer is 7 to 8 mm below the neck. Then, 1 ml of isoamyl alcohol is added.
The butyrometer is tightly closed with a dry rubber stopper, wrapped with a towel to protect the hands from heat and vigorously shaken while holding the stopper and at the same time turned over several times for better mixing the contents. Then, the butyrometer is placed with a stopper down into a water bath at a temperature of 65 to  °C, while the water level in the bath should be slightly higher than the liquid level in the butyrometer. After complete dissolution of the protein substances of the product, about 7 to 8 minutes, butyrometers are removed and fat fraction is placed in the graduated part by turning the stopper upward or downward. Then, the butyrometers are placed symmetrically into the centrifuge with narrow ends toward the center and centrifuged for 5 minutes at 800 to 1000 rpm (500 to 1000 g).
After that, the butyrometers are put again in a water bath at a temperature of 65 to 70 °C for 5 minutes, re-centrifuged for 5 minutes and after staying in bath for 5 minutes at the same temperature, the number of graduation marks occupied by the fat is quickly counted. When counting, the butyrometer is held upright against the light. For convenient reading, the lower boundary of fat is placed on any graduation mark by stopper. The fat percentage is calculated by multiplying the butyrometer reading by 2 when sample weight is 2.5 g and by 2.5 when sample weight is 2 g. Fat is determined in two parallel samples. The difference between two parallel determinations should not exceed 0.5 %.
Determination of fat in a milk butyrometer. When analyzing whole and skim milk or dried cream, the sample of the product is weighed in an amount of 1.5 g with an accuracy of 0.01 g in 25-50 ml laboratory glass with a spout, 4 ml of hot water with a temperature of 70 to 75 °C are added, the mixture is thoroughly ground and the resulting homogeneous product is transferred without losses through a small funnel into a butyrometer, in which 10 ml of sulfuric acid with relative density of 1.81 to 1.82 g/cm 3 are preliminarily added. The glass is washed 2 times with distilled water in 3 ml portions, while adding it to the contents of the butyrometer. Then, 1 ml of isoamyl alcohol is added. In all other respects, the analysis is performed as described above. When analyzing dried skim milk, threefold centrifuging is used.
The fat content (X,%) is calculated by the following equation: where а is the butyrometer reading; К 1 is the coefficient for translating the butyrometer readings into percentages; g is the weight of the product.
To determine fat in dough and finished products, the All-Russian Research Institute of the Bakery Industry, Moscow, Russia, developed rapid butyrometric method. It is based on dissolving the sample in 60 % sulfuric acid and determining the fat in milk butyrometer by centrifuging in the presence of isoamyl alcohol, which forms isoamyl sulfuric ether with sulfuric acid. The former reduces the surface tension of fat globules and promotes their aggregation into continuous fat layer. When analyzing finished products, all inclusions and surface finish are removed, while analyzing only the crumb. Two average samples of 2 g each are taken from dough or finished products. They are carefully ground to better dissolve starch and protein in sulfuric acid. At the same time, samples are taken to determine the moisture content of the dough (by the VNIIHP-VC instrument, Chizhova's device) and finished products. Dough or finished product samples are placed in porcelain cups with volume of 20 to 30 ml and 9 ml of 60 % sulfuric acid are added. The cups are immersed in a water bath with a water temperature of 80 °C and the samples are dissolved in sulfuric acid for 20 minutes with periodic stirring with a glass rod. After dissolving the sample, the dark liquid is transferred to the milk butyrometer by means of a glass rod, and the residue is washed out from the cup with 10 ml of 60 % H 2 SO 4 .
Carefully, not to soak the neck, 1 ml of isoamyl alcohol is added into the butyrometer, which is then tightly closed with rubber stopper. The mixture is gently stirred for 3 minutes and the butyrometers are placed in a water bath with a water temperature of 80 °C for 5 minutes (with stopper down). After 5 minutes, the butyrometers are removed from the water bath, placed in a Gerber milk centrifuge and centrifuged for 5 minutes at a speed of 1200 rpm. After centrifuging, the butyrometers are again placed in 80 °C water bath (with stopper down) for 5 minutes. Then, they are remove and the height of yellow fat layer above the dark liquid is measured according to the number of small graduation marks on the butyrometer graduated part [29].
The method for determination of fat content by the fat-free residue (according to Rushkovsky). The amount of fat in the product is determined by decreasing the weight of the dried sample after extraction with the solvent. Test sample of 2 to 5 g weighed with an accuracy of 0.001 g is dried in a desiccator at a temperature of 100 to 105 °C and transferred to a bag of filter paper with a size of 8 x 9 cm. Weighing cup walls are wiped with a small amount of cotton wool wetted in ether. Cotton wool with sample is put into a bag of filter paper. The bag with sample is put into the second bag with a size of 9 x 10 cm so that the folding lines do not match, and tied with a thread. The outer bag is numbered with a simple graphite pencil, placed in the same weighing cup, in which the sample was previously dried, and placed in desiccator. The sample is dried to a constant weight at a temperature of 100 to 105 °C. The sample may be dried directly in the bag. The dried bag with the sample is placed in the extractor of the Soxhlet apparatus. Several bags may be placed in one device provided that they are completely immersed in ether and well washed by it. Extraction time is 10 to 12 hours. The end of the process is established as follows. The drop of the solution (miscella) flowing from the extractor is applied to a watch glass. Upon full fat extraction, there should be no greasy stain on the glass after evaporation of the solvent. Bags with fatfree sample are transferred into the same weighing cup and held in exhaust fume hood for 20 to 30 min to remove ether, and then dried in a desiccator at 100 to 105 °C to constant weight. The duration of the process is 1 to 3 hours.
The fat content (X,%) is calculated by the following equation: Х = (m 1 -m 2 ) · 100 / g, where m 2 is the weight of the dried weighing cup, the bag and the sample of the product before extraction, g; m 1 is the weight of the dried weighing cup, the bag and the sample of the product after fat extraction; g is the weight of the sample.
The difference between parallel determinations should not exceed 0.5 % [30].
Randall method. As the process in the Soxhlet extractor, the method is carried out in 3 stages: extraction, washing out and drying (Figure 3).
A cup with sample is placed in a solvent. The solvent is heated. The solvent vapor is transferred to condenser. Then, condensed solvent drops enter the cup with sample. The sample is extracted and the solvent with the analyte is collected in the sample cup. Washing out consists of 2 stages: • the solvent is evaporated and collected in a special container until the sample level is above the solvent level; • the solvent remaining in the cup is evaporated; after condensation, the solvent enters the cup with the sample carrying out the washing. The drying stage is similar to the Soxhlet method. The solvent remaining in the cup is evaporated and enters the container with a solvent. The analyte remains in the cup, and the residual sample remains in the cup.
Twisselmann method (cost-efficient continuous extraction). Extraction by Twisselmann method is a costefficient alternative to Soxhlet extraction, which allows to reduce analysis time and solvent costs. The extraction process is about 60 minutes.
The principle of Twisselmann extractor (cost-efficient continuous extraction): in contrast to the methods considered earlier, it is carried out in two stages: extraction and drying (Figure 4).
Extraction. The solvent is placed in a cup and is heated. The solvent vapor passes through the extraction chamber with the sample and enters the condenser. The condensed solvent drips into the extraction chamber with the sample where the extraction process proceeds. Simultaneously, the solvent with the analyte is transferred from the extraction chamber to the cup with solvent. The extraction time is set preliminarily.
Drying. At the drying stage, the cup with the solvent is heated, the solvent is evaporated and collected in a suitable container [32,33].

Conclusion
Determination of fat content in food raw materials, as well as the determination of fat content in food products, is the urgent analytical problem for control laboratories of industry and regulatory bodies. Increasing requirements for the quality and safety of food, as well as the reproducibility and effectiveness of traditional fat extraction techniques force modern laboratories to search for different methods for extraction and analysis of fats.
Extraction of individual lipids from the raw material usually involves several steps. The first step is the destruction of the tissue by grinding of dried raw material, followed by the extraction of neutral lipids, and then the extraction of total phospho-and glycolipids followed by fractioning and extraction of pure substances. The degree of lipid extraction depends on the grinding of raw material. Polar solvents, such as methanol and ethanol, which destroy hydrogen bonds and weaken the electrostatic interaction of lipids with proteins, are most effective in extracting the lipids. The use of alcohols for the extraction of phospholipids is also convenient because the former deactivate the majority of lipolytic enzymes that cause lipid degradation. The extraction time and completeness, as well as the method and conditions, are determined in each particular case [34].
Thus, in this review, the authors attempted to highlight all available and well-known methods for lipid (fat) extraction from various biological matrices of both plant and animal origin.