# A Comprehensive Review of Milk Components: Recent Developments on Extraction and Analysis Methods
## 3 Extraction Methods for Milk Macro-Constituents  
Milk macro-constituents can be extracted through different methods characterized by advantages and drawbacks, which make them more or less suitable for the purpose. All the proposed methods are summarized in Table 2, along with advantages and drawbacks. Lipids are typically extracted from milk using Blich [38] and Folch [39] methods, which involve solvent extraction using mixtures of chloroform, methanol and water. Soxhlet extraction is also used, but it is more complex due to the aqueous nature of milk [40; 41]. For milk, Soxhlet extraction could provide a high yield of lipids. However, continuous heating at the boiling temperature could lead to lipid oxidation and degradation of heat-liable compounds [42]. Other studies report the use of the single-phase liquid extraction, which is a type of liquid extraction in which the extracted substance forms a single, homogeneous phase with the feed solution (or solid material), as it mixes uniformly with the extraction solvent [43]. Typically, mixtures of chloroform/methanol (2:1) are used for these purposes [43], allowing the efficient extraction of both polar and nonpolar lipids. In order to avoid hazardous problems, other alcohols (ethanol or isopropanol) can be used to replace methanol [44]. Recently, supercritical CO\({}_{2}\) extraction, supercritical liquid-liquid extraction and accelerated solvent extraction (ASE) have received more attention [45; 46; 47; 48], as they are often based on automated instrumentation, which makes it possible to minimize extraction times and chemical consumption. Since triglycerides with short-chain are, in general, more volatile than long-chain molecules, they are more soluble in pressurized carbon dioxide at low pressure. With regard to ASE, different solvents have been proposed mainly for the extraction of fatty acids and polar lipids from milk-based products (cheese and mozzarella), such as azeotropic mixtures of cyclohexane and ethyl acetate [47], petroleum ether [48], or mixture of tert-butyl methyl ether, cyclohexane and isopropanol [46]. Another example of a newly introduced technique for lipid extraction from milk is the dispersive micro-solid phase extraction (D-\(\upmu\)-SPE). D-\(\upmu\)-SPE involves the dispersion of a small amount of solid sorbent directly into the liquid sample to adsorb specific analytes. Then, after enough contact time, the mixture is centrifuged to separate the sorbent (with the adsorbed analyte) from the liquid. To retrieve the analyte, a small amount of solvent is added to the sorbent pellet, releasing the analyte into the elution solvent, which then can be analysed by chromatographic techniques like HPLC and GC coupled to different detectors, such as mass spectrometer. For the extraction of milk lipids, mainly C18 and zirconia-coated silica gel have been used as a sorbent [49; 50]. The extracted lipids are almost triglycerides, while free fatty acids are typically the minor components [51; 52]. Milk polysaccharides, such as lactose, can be easily extracted through a few steps aimed at removing interfering fats and proteins. Lipid interferers are removed by centrifuging milk and collecting the aqueous (non-fat) portion. Then, proteins such as casein and whey proteins, are precipitated through the addition of acetonitrile and removed to minimize contamination in the final polysaccharide extract. Once proteins and fats are removed, the remaining aqueous solution is treated with alcohols (ethanol or isopropanol) to isolate polysaccharides [51]. Other research reports the use of molecular imprinted polymers (MIPs) for the extraction of milk polysaccharides. MIPs are synthetic polymers engineered to recognize and bind specific molecules, such as lactose, in complex mixtures. For lactose extraction from milk, MIPs offer a powerful and selective approach by mimicking the \"lock-and-key\" mechanism, allowing to selectively target and isolate lactose even in mixtures containing various other components like proteins, fats and other sugars [52].