*3.1. Solvent Extraction*

Many kinds of polar solvents have been used to extract MPLs, such as ethanol and alkanes [21,66]. To separate casein from MPLs, proteins can also be thermally denatured or in an acid solution (pH 4.6) [48,81], the aggregated particles are subsequently filtrated. Regarding fractionation of MPL from WPPC, ethanol (70% *v*/*v*) at 60–80 ◦C denatures proteins; after filtration the MPL concentration is ≈45.8% in the filtrate in Figure 2a [48]. This notable method uses no toxic solvent. However, the incompleteness of the phospholipid recovery may be a concern [48].

**Figure 2.** Process flow diagram of solvent extraction unit: (**a**) adapted from Price et al. [48], (**b**) Ota et al. [93], and (**c**) Shulman et al. [21]. BMC, buttermilk concentrate; MPL, milk phospholipid; AI, acetone insoluble; WPPC, whey protein phospholipid concentrate (liquid, reconstituted from powder).

Compared to 58.1% recovery by ethanol, the tertiary amine CyNMe2 (*N*,*N*-dimethylcycloexylamine) yielded a 99.96% recovery rate of MPLs. At various solvent–sample weight ratios, the lipid extraction was conducted at ambient temperature. The dissolved MPLs in the amine were released by bubbling

CO2 at atmospheric pressure, which converts CyNMe2 into the carbonate salt in Figure 2b. By injecting nitrogen and removing CO2, the carbonate salt regenerated into the amine form for reuse (Figure 2b). Though the recovery rate for BM was as high as 99.96 ± 1.2%, the extraction rates for BS and concentrated BM were only 7.57 ± 0.59% and 77.27 ± 4.51%, respectively. Aside from the input sensitivity, the amine may interact with dairy components and cause toxic consequences [93], and the chemical facilities required may be incompatible in a dairy factory setting.

MPLs can be dissolved in ethanol and alkanes [21,67,68], and may not dissolve in acetone, ethyl acetate, and 2-pentanone [21,67,68]. Lipid BMP (100 g) dissolved in ethanolic hexane (1:4 *v*/*v*, 800 mL) under constant agitation at 45 ◦C for 2 h will produce an extract. The permeate of vacuum filtration (repeated twice) can then be vacuum-dried at 1 kPa (Figure 2c). The residue (≈20 g) is then defatted twice with 120 mL acetone, and the resulting acetone is insoluble (AI, ≈7 g), composed of mainly polar lipids, and in the final step vacuum, is dried again at 1 kPa [21]. However, acetone poses a degree of toxicity, as acetone residue in defatted MPLs may reach 5–10 ppm. Further, acetone can form a mesityl oxide via a condensation reaction, causing an off flavor [94]. Hence, toxic residues in acetone-insoluble fractions need analysis and monitoring.
