Food Lipids: Chemistry, Nutrition, and Biotechnology

solvents without being allowed to thaw [4]. Lipolytic enzymes of animal and plant
tissues are usually deactivated irreversibly by homogenization with polar solvents.
Use of high temperatures should be avoided; it is also advisable, when possible, to
maintain an inert atmosphere during sample preparation and extraction which may
minimize oxidation reactions of unsaturated lipids.
B. Pretreatments
1. Drying
Sometimes nonpolar solvents, such as diethyl ether and hexane, do not easily penetrate
the moist tissues (>8% moisture); therefore, effective lipid extraction does not
occur. Diethyl ether is hygroscopic and becomes saturated with water and thus inefficient
for lipid extraction. Therefore, reducing moisture content of the samples
may facilitate lipid extraction. Vacuum oven drying at low temperatures or lyophilization
is usually recommended. Predrying facilitates the grinding of the sample,
enhances extraction, and may break fat-water emulsions to make fat dissolve easily
in the organic solvent and helps to free tissue lipids. Drying the samples at elevated
temperatures is undesirable because lipids become bound to proteins and carbohydrates,
and such bound lipids are not easily extracted with organic solvents [5].
2. Particle Size Reduction
The extraction efficiency of lipids from a dried sample also depends on the size of
the particles. Therefore, particle size reduction increases surface area, allowing more
intimate contact of the solvent, and enhances lipid extraction (e.g., grinding of oilseeds
before lipid extraction). In some cases, homogenizing the sample together with
the extracting solvent (or solvent system) is carried out instead of performing these
operations separately.
3. Acid/Alkali Hydrolysis
To make lipids more available for the extracting solvent, food matrices are often
treated with acid or alkali prior to extraction. Acid or alkali hydrolysis is required
to release covalently and ionically bound lipids to proteins and carbohydrates as well
as to break emulsified fats. Digestion of the sample with acid (usually 3–6 M HCl)
under reflux conditions converts such bound lipids to an easily extractable form.
Many dairy products, including butter, cheese, milk, and milk-based products, require
alkali pretreatment with ammonia to break emulsified fat, neutralize any acid, and
solubilize proteins prior to solvent extraction [6]. Enzymes are also employed to
hydrolyze food carbohydrates and proteins (e.g., use of Clarase, a mixture of �amylase
and protease) [2].
C. Lipid Extraction with Solvents
The insolubility of lipids in water makes possible their separation from proteins,
carbohydrates, and water in the tissues. Lipids have a wide range of relative hydrophobicity
depending on their molecular constituents. In routine food analysis,
‘‘fat’’ content (sometimes called the ether extract, neutral fat, or crude fat) refers
to ‘‘free’’ lipid constituents that can be extracted into less polar solvents, such as
light petroleum ether or diethyl ether. The ‘‘bound’’ lipid constituents require more
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