# A Comprehensive Review of Milk Components: Recent Developments on Extraction and Analysis Methods
## 3 Extraction Methods for Milk Macro-Constituents
In addition, also water extraction is suggested as a green extraction method [53] for milk sugars. Some other green solvents used in the extraction of polysaccharides are ionic liquids based on organic cations, such as imidazolium, pyridinium, pyrrolidinium and organic or inorganic anions such as chloride, dicyanamide and trifluoroacetate [54]. On the other hand, solid phase extraction (SPE) is often applied to extract polysaccharides from milk, while concentrating and purifying them. Typically, cartridges packed using octadecyl silica gel, i.e., reversed phase (C18), are used. Common solvents for conditioning, washing and elution steps include methanol, acetonitrile, water and buffer solutions [55]. As regards proteins, they are extracted from milk mainly with enzymatic methods [56]. These methods rely on the use of specific enzymes to modify, separate, or concentrate proteins and offer a gentle and highly selective approach, often preserving the structure and functional properties of proteins better than harsh chemical or physical methods. A pre-treatment step to remove fats and adjust protein concentration is always carried out before enzyme addition [57]. Another innovative method proposed for milk proteins extraction involves the use of nano micelles that have an aqueous core capable of solubilizing polar substances. These nanostructures can capture and isolate proteins due to their high surface area, selective binding capabilities and ability to work under mild conditions that preserve protein structure [58]. Other research proposes microwave assisted extraction (MAE) for milk proteins, as MAE accelerates the extraction process by uniformly and quickly heating the sample, at the same time enhancing protein yield [59]. It is remarkable to consider that after their extraction, proteins can be separated before further analysis by different techniques, such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). 2D-PAGE allows proteins separation according to their molecular mass and isoelectric point [60]. \begin{table}
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**Extraction Method** & **Advantages** & **Drawbacks** & **Macro-Constituents** & **Used Solvents** & **Refs.** \\ \hline \multirow{4}{*}{Liquid extraction} & \(\bullet\) & Available standard methods & \(\bullet\) & Laborious multistep process & Lipids & Chloroform Methanol Water & [38; 39] \\  & \(\bullet\) & High yield & \(\bullet\) & Use of toxic solvents & & Ethanol & [51] \\  & & & \(\bullet\) & Solvent residues in the extract & & Ionic liquids & [54] \\ \hline \multirow{4}{*}{Supercritical CO\({}_{2}\) and liquid–liquid extraction} & \(\bullet\) & Environment-friendly, non-toxic Food quality grade extract & \(\bullet\) & High instrumentation cost & & \\  & \(\bullet\) & Minimum/zero post-extraction & \(\bullet\) & High energy requirements & Lipids & CO\({}_{2}\) & [45] \\  & & & processing & & requirements & & \\ \hline \multirow{4}{*}{Soxhlet extraction} & \(\bullet\) & Available standard methods high yield & \(\bullet\) & Time-consuming & & & \\  & & & \(\bullet\) & Use of toxic solvents & & & \\  & & & \(\bullet\) & High extraction temperature & & & \\ \hline \multirow{4}{*}{Accelerated solvent extraction (ASE)/Microwave assisted extraction (MAE)} & \(\bullet\) & Automated and rapid & \(\bullet\) & High extraction temperature & & Cyclohexane/ethyl acetate & \\  & & & & temperature & & Petroleum ether, & \\  & & & of solvents & \(\bullet\) & ASE/MAE Instruments required & & Ternt-butyl methyl ether/cyclohexane/ & \\ \cline{1-1}  & & & & required & & isopropanol & \\ \cline{1-1}  & & & & & & \\ \cline{1-1}  & & & & & & \\ \hline \hline \end{tabular}
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Table 2: Commonly used methods for milk macro-constituents’ extraction, i.e., lipids, polysaccharides and proteins, with their advantages and drawbacks.