**1. Introduction**

Milk phospholipids (MPLs) consist of a subclass of polar lipids, namely glycerophospholipids and sphingolipids [1]. Glycerophospholipids comprise a glycerol moiety with two fatty acids esterified at positions *sn*-1 and *sn*-2 and a hydroxyl group at *sn*-3 position, linked to a phosphate group and a polar moiety [1]. The molecular structure of the latter determines the types of glycerophospholipids, namely phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidyl-glycerol (PG), and phosphatidic acid (PA) [2]. Sphingolipids consist of a sphingosine backbone (2-amino-4-octadecene-1,3-diol) connected to a fatty acid via an amide bond and a polar head. Sphingomyelin (SM), a prominent subclass of sphingolipids, has a phosphocholine residue [1]. In raw bovine milk, the diameters of milk fat globules (MFGs) are around 0.2–15 μm; these MFGs are enveloped by an approximately 15-nm thick tri-layer MFG membrane (MFGM) [3,4]. The composition of MFGM is 30–75% polar lipids, and 25–70% protein, respectively [5]. MPLs lie within the MFGM constructing its backbone. MPLs represent 0.4–1% of the total milk lipids [6], which change with season, lactose stage, and feed [7].

MPLs have exhibited nutraceutical properties due the unique composition of this group of phospholipids. MPLs contain high proportions of SM [8] and PS [9] (24% and 12%, respectively), subclasses which are virtually absent in other sources, such as soy (0% and 0.5%, respectively) and egg yolk lecithin (1.5% and 0%, respectively) [10]. PS is associated with cognitive function and releasing stress, and is replaced by inactive cholesterol as the brain ages [11,12]. SM has been found to be effective in inhibiting colon tumors [13]. Also, MPLs have been implicated in mitigating the risks of Alzheimer's disease and repairing cognitive ability [14], restoring immunological defenses, reducing the incidence of cardiovascular diseases [15,16], and reducing cholesterol absorption and total liver lipids [17]. In addition, MPLs may narrow the gap between formula-fed and breastfed infants concerning neurodevelopment, infectious diseases, and cholesterol metabolism [18,19]. Phospholipid-coated fats, e.g., human breast MFGs, will be properly digested and absorbed, not only due to the size of the MFGs, but also due to the ratio of MFGM proteins to phospholipids [20]. Bovine MPL-enriched ingredients may be used to produce breast milk analogs. For instance, one formula recipe consists of subclasses according to a weight-relative ratio of SM > PC > PE > PS > PI, with 21.1–29.7% SM and 10.2–13.3% PS (both based on total MPLs, similar to those of human breast milk (37.5% and 9%, respectively) [21]. Another infant formula comprises 150 mg/L MPLs [22], mimicking that of breast milk (15–20 mg/dL milk [21] and 0.3–1.0% of the total lipids [23]).

Aside from nutritional value and health benefits, MPLs may provide technical functionalities in food systems, for example, MPLs have been used in the preparation of liposomes [24] and constructing vesicles of bioactive compounds [25]; they are also food emulsifiers and surfactants, foaming agents, texture improvers for bakery goods, and may improve moisture retention for yogurt [26,27].

Many research works and reviews are available on fractionation from buttermilk (BM) and beta serum (BS) [26], isolating MFGs by washing and centrifugation [5], and the membrane separation of polar lipids [8]. However, there is no standard large-scale manufacturing process adopted by the dairy industry. This is due to many reasons. First, the native MFGM is fragile. Shear and turbulent fluid flow can cause damage to the MFGM [28]. These treatments are commonly involved in handling raw milk on farms, in transportation, in silos at manufacturing plants, and during cream separation. Damage to the MFGM may cause associated materials, including MPLs, to deplete from the native MFGs to the aqueous phase of milk. Therefore, more than half of MPLs in raw milk remains in skim milk [29,30]. Second, uncertainties and variables are involved in the MPL fractionation processes. For example, cream washing for removing non-MFGM associated proteins may be performed before butter churning for increased yield or the concentration of MPLs in the resulting BM, or in the retentate of BM after tangential filtration. However, the cream washing procedure may cause a significant change to the MPL composition in BM from unwashed cream [31,32]. Although the mechanism is not clear, it may relate to the physicality of different washing processes. Zheng's group revealed that different washing procedures induce various degrees of damage to MFGM. Therefore, washing may alter the composition of MPL in the fat phase of the washed cream [4,33]. This review aimed to assess different dairy streams rich in MPLs, to evaluate their extraction processes, compare their process intensity and efficiency, and to estimate their life-cycle carbon footprints (CFs) using ISO 14067 and greenhouse gas (GHG) protocols.

### **2. Milk Phospholipid Extraction from Dairy Products**

#### *2.1. Dairy By-Products Rich in Phospholipids*

Commercial MPL products are usually derived from dairy products, such as BM [34], BS [8], acid cheese whey BM [35,36], whey protein phospholipids concentrate (WPPC) [37], or whey BM [38]. The dairy streams in Table 1 comprise 2.29%–26.02% MPLs on a dry matter (DM) basis, varying with sources and processes.

BM is the product that remains after the removal of butter by churning cream, which may have been concentrated and/or dried as butter milk powder [39], as illustrated in Figure 1. Acid

BM, a by-product of lactic butter, is made by churning cultured cream. Furthermore, whey BM is produced via the churning of whey cream during cheese making [40]. WPPC is a by-product produced during the microfiltration (MF) of whey for manufacturing whey protein isolate (WPI). The permeate phase (milk-fat-discriminated phase) from this process goes forward for WPI manufacturing and the fat-remaining phase (retentate phase) containing residual whey proteins is further concentrated for producing WPPC. A typical WPPC is comprises more than 12% fat and 50% protein (DM), and less than 8% ash and 6% moisture [37].

BM, the serum phase resulting from the churning of cream, comprises milk proteins and residual fat [34]. In terms of protein, lactose, ash, and DM contents, BS and BM are very similar to those of cream products (Table 1) [41]. For instance, BM (FDC ID 454974) protein content is 3.33%, which is the same as that of cream (FDC ID 495516). Though the fat content of BM is only one-tenth of cream, MPLs of BM are 4–27-fold that of raw milk, as shown in Table 1. The empirical equation MPL = 0.0137 × FC provides an estimation of the MPL content (g/L) of a dairy product, where FC is the fat content of cream [42]. For instance, the estimated BM MPL content of anhydrous milk fat (AMF) from 80% cream, and of butter from 40% cream, is 1.1 and 0.55 g/kg, respectively. Whey BM, a by-product of whey butter, comprises sixfold the MPL content of raw milk, as seen in Table 1 [38].

BM and BS, the most abundant source of MPLs [43], have been underexplored or even treated as a waste stream [44]. For instance, a New Zealand-based dairy manufacturer used two-thirds of their BM for standardization, only one-tenth for BM powder (BMP), and their annual output of MPL concentrate is 320 metric tons [44]. The annual BM output in Canada was 14.1 metric kilotons (18% of butter and 0.5% of bulk liquid [45]), compared to 20 kilotons in Belgium, 16 kilotons in Denmark, and 124.5 kilotons in Germany [46]. In 2013, approximately 5.2 million tons of BM was produced worldwide, similar to that of butter [34]. Worldwide, the annual BMP production was estimated at 410 kilotons (≈9.5% of butter), which has downstream applications for producing ice cream, ingredients-baked foods, low-fat Cheddar cheese, reduced-fat cheese, pizza cheese [40], or in the replacement of skim milk powder for low-fat yogurt [47].



MPLs, milk phospholipids; BS, beta serum; BM, buttermilk; BMP, BM powder; WPPC, whey protein phospholipid concentration; CM, cream; DM, dry matter; United States Department ofAgriculture(USDA)FoodDataCentralID[41];b wetbasis;c lactose;Ref.,reference.

### *2.2. Commercialized Milk Phospholipid Products and Concentrate*

Phosphoric 500/600/700 and Gangolac 600 (products manufactured by Fonterra) comprise 34%, 75%, 62%, and 15% MPLs, respectively, representing one source of highly-purified MPLs [52,53]. Arla Foods Amba have developed phospholipid-rich, concentrated dairy milk commodities for infant milk formulas and skin care. It has been claimed that Lacprodan® MFGM 10 supports physiological development of the infant gut and provides infants with similar health benefits to breast milk because of their similarities in fatty acid profile [54]. Arla dairy products PL 20/75 consist of 20% and 75% MPLs, respectively [55].

As illustrated in the patents in Table 2, both filtration and solvent extraction are validated processes for manufacturing MPLs. Acetone and supercritical CO2 are effective solvents for de-fatting. Tatua [56] and Synlait [57] have concentrated MPLs to 5–12.8% (*w*/*w*, DM basis). Lecico has used membrane separation to produce Lipamine M20 (20% purity) [58].



MPLs, milk phospholipids; BM, buttermilk; BMP, BM powder; BS, beta serum; BSP, BS powder; MF/UF, micro/ultra-filtration; DME, dimethyl ether; PI, phosphatidylinositol; PS, phosphatidylserine; SM; sphingomyelin; SFE, supercritical fluid extraction.

#### *2.3. Laboratory Extraction of Milk Phospholipids*

Intact MFGM makes up 2–6% of the total mass of MFG [26]. However, MFGM represents 60%–70% of the total milk phospholipids [69]. Raw bovine milk comprises 0.2–0.4 g MPLs/kg, and raw milk is generally a laboratory source of MPLs [5,70]. Intact MFGs can be isolated with low-speed centrifugation. The cream layer from raw milk skimming can be washed with phosphate buffered saline (PBS; pH 6.8, 0.1 M, 1:10, *v*/*v*) and centrifuged at 390 g for 10 min at 10 ◦C. The final cream layer after three washes is the large MFG fraction [71]. Different from isolating intact MFGM, Sanches-Juanes et al. [72] ruptured MFGM and recrystallized milk lipids, and starting from raw milk, they washed cream with a 0.15 M NaCl solution and precipitated casein using centrifugation at 5000× *g*.

Cream washing is a step used to remove casein and other non-MFGM materials from cream [44]. After centrifugation, casein will precipitate, with lipid stratification at the top layer [73]. Also, calcium, naturally present in casein micelles, can form a complex between MFGM and the casein micelles through its binding to the phospho-casein and phospholipids of MFGM, leaving impurities with MPLs [74]. In addition, washing causes a severe loss of phospholipids, almost 60% per wash [32]. Hence, washing facilities for separating MPLs are costly and energy-intensive [44], thereby they are mainly only used for laboratory purposes [5,73,75].

In addition to washing and centrifugation, the microfiltration of raw milk has been applied to produce MFGM material. It has been found that a 1.4-μm ceramic membrane was superior to 0.8 μm, yielding a high-purity MFGM material, which was composed of 7% phospholipids and 30% protein [76].

For analysis purposes, MPL samples are usually prepared using solvent extraction. The Folch [77] and Bligh [78] methods use chloroform and methanol to dissolve lipids. Other lipophilic extraction formulas include the Mojonnier solvents [79], dichloromethane [80], and the ammoniacal ethanolic solution of lipids with dimethyl ether and light petroleum in the Röse–Gottlieb extraction [81,82]. The total lipid content in samples can be determined with a gravimetric assay, Gerber-van Gulik butyrometer, infrared spectroscopy according to an International Dairy Federation (IDF) method [81], or gas chromatography equipped with a flame ionization detector [83].

To determine the MPLs and their subclasses, solid-phase extraction can fractionate polar lipids from non-polar lipids. Silica-gel-bonded cartridges or silica gel plates can be used for such a purpose [84]. The obtained MPLs can be solvent dried using a vacuum and stored at −20 ◦C before using [85]. In addition, chloroform and methanol are also valid elution solvents [86]. Total MPLs can be measured using the IDF molybdate assay [87], Fourier transform infrared spectroscopy [88], or a fluorescence assay on cleaved choline group [89]. Both nuclear magnetic resonance of 31P and chromatography can quantify MPLs and their subclasses [90,91]. High-performance liquid chromatography coupling with detectors as a charged aerosol detector, evaporative light-scattering detector, and mass spectroscopy is more acceptable than thin layer chromatography [92].
