1. Introduction

Milk is nature's most complete food because it is an essential source of nutrients and bioactive compounds such as immunoglobulins, antimicrobial proteins, oligosaccharides, phospholipids, hormones, minerals and vitamins ^[1][2]. Milk is an oil-in-water emulsion, and milk lipids are organized as lipid droplets composed of a hydrophobic core surrounded by a hydrophilic membrane. These two components are called milk fat globule (MFG) core and milk fat globule membrane (MFGM), respectively ^[3][4][5]. Milk lipids exert many bioactive functions in newborns and acts as a “natural carrier” for the milk compounds that retain these bioactive properties ^[6]. These compounds are mainly phospholipids, sphingolipids and proteins of the MFGM, lipophilic vitamins, minerals and cholesterol. 

In the last decades, research has focused on the characterization of MFGM and on the exploitation of its properties to design and produce value-added products. Alongside the progress in this area, the research has been less concerned both with the industrial exploitation of the MFGM proteome and with the comprehension of the content of minor components of the milk lipid fraction. In this review we discussed the similarities and differences regarding the composition of the MFGM proteome of various species, highlighting the properties of the human and bovine MFGM proteome. Then, we revised the existing literature about the content and the distribution of micronutrients in the milk lipid fraction of human and bovine milk. The aim of the review is to provide an overview of the minor constituents of the milk lipid fraction, discussing their bioactive properties and suggesting innovative attempts of implementing the findings present in the literature about these topics.

2. MFGM Proteome and Comparative Proteomics

The use of proteomics techniques enabled a better comprehension of the role of MFGM proteome in milk and allowed to identify a higher number of MFGM proteins across all species. In addition, comparative proteomics analyses were performed to elucidate the variations and the similarities of MFGM proteome among species and phases of lactation ^[7].

Human and cow MFGM proteomes show several similarities. In terms of quantity, human and cow proteomes share the highest number of MFGM proteins and the highest linear correlation coefficient (166 proteins, R = 0.71) if compared to the MFGM proteome of goat (95 proteins, R = 0.60) and yak (76 proteins, R = 0.62) ^[7]. These data are also reported in Figure 1. 

Figure 1. Number of individual and common milk fat globule membrane (MFGM) proteins in human, cow, goat and yak, and Pearson correlation coefficient (R) between the MFGM proteomes of human–cow, human–goat and human–yak. Adapted from ^[8].

In 2011, Hettinga and collaborators ^[9] observed slight variations in the number of proteins responsible for various molecular functions in human and cow MFGM proteomes. The authors observed that the number of MFGM proteins related to the immune defense was comparable between cows (44) and humans (51). The cow’s MFGM proteome was enriched in antibacterial proteins, named cathelicidins and mucins, related to innate immunity. Instead, the human MFGM proteome was enriched in four proteins involved in the mucosal immune system: IgA, CD14, lactoferrin and lysozyme ^[9].

Moreover, colostral MFGM proteome contains a higher number of proteins related to the immune defense than milk of later phases of lactation ^[10][11][12]. A recent study ^[12] identified a total of 411 MFGM proteins in human and bovine colostrum. Of the 26 differentially expressed proteins, 9 were upregulated and 17 downregulated in humans if compared to bovine colostrum. These 9 proteins, such as neutrophil defensin 1, protein S100, immunoglobulin K and lactadherin, were mostly involved in the innate and adaptive immune defense. The authors assessed that the MFGM proteome of human colostrum was more enriched in immune-related proteins than bovine colostrum ^[12].

All the results reported lead to two main conclusions: (i) the immune-related proteins are high in human colostral MFGM proteome and (ii) the molecular function and protein expression levels vary among species and different phases of lactation.

3. Milk Minerals and Vitamins

Milk and dairy products are crucial to the intake of minerals and vitamins. In fact, they provide 20–40% of total dietary intake of vitamins and 10–20% of total dietary intake of minerals in Western countries ^[13][14].

3.1 Minerals

The major minerals in milk are calcium, magnesium, phosphorus, sodium, zinc and potassium ^[13]. The various minerals are distributed in milk according to their chemical characteristics. The milk lipid fraction contains mainly iron, copper, zinc and calcium, even though some of these minerals are present in trace amounts (Table 1).

Table 1. Distribution of minerals in whole milk and in milk lipid fraction of human and cow’s milk. Data from Zamberlin et al. (2011) ^[13]. Mulder and Walstra (1974) ^[15] , Fransson and Lonnerdal (1983) ^[16], Soliman (2005) ^[17], Walton and Flynn (2013) ^[18], Chassaing et al. (2016) ^[19] and Pietrzak-Fie´cko and Kamelska-Sadowska (2020) ^[20].

It is important to underline that milk usually undergoes several industrial processes before consumption. For what concerns minerals, their content is unaffected by heating because minerals are both very heat-stable, and in milk they are usually bound with other components, such as enzymes ^[21].

Since this fraction is rich in bioactive polar lipids and proteins, and it also contains certain amounts of minerals, the inclusion into value-added products could be beneficial.

3.2 Vitamins

Milk contains both hydrophilic and lipophilic vitamins. Hydrophilic vitamins are vitamins C and those belonging to the B family, whereas lipophilic vitamins are A, D, E and K. According to their chemical features, they are distributed either in the aqueous phase or in the milk lipid fraction ^[2][22]. The milk lipid fraction is a major source of lipophilic vitamins (Figure 2).

Figure 2. Schematic representation of the major minerals and lipophilic vitamins present in the milk lipid fraction.

The main lipophilic vitamins in milk are present either in the MFGM or in the MFG core. Vitamin A, vitamin D3, α-tocopherol and riboflavin are mainly located in the MFGM, whereas β-carotene and γ-tocopherol are mainly located in the MFG core (Figure 2). Table 2 reports the content of lipophilic vitamins in human and cow milk lipid fractions.

Table 5. Content of lipophilic vitamins in human and cow milk. Data from Baldi and Pinotti (2008) ^[6], Öste et al. (1997) ^[23], Park (2007) ^[24], Fantuz et al. (2016) ^[25] and Biadala and Konieczny (2018) ^[26].

Regarding the effect of heating on the milk vitamin content, there is a distinction between hydrophilic and lipophilic vitamins. The levels of the hydrophilic vitamins B1, B12 and C decrease by 10–20% after pasteurization. Instead, vitamin B2 is unaffected, and this is valid also for the lipophilic vitamins. Indeed, vitamins A, D, E and  -carotene appear to suffer no loss after pasteurization or microwave heating ^[21][27].

4. Prospective Opportunities for the Future

The well-established higher content of immune-related proteins in MFGM of colostrum across all species could be used to enhance the immune functions of specific subjects by potentially decreasing the number of infective episodes in newborns ^[28]. Moreover, the human MFGM proteome is enriched in proteins capable of protecting the integrity of the mucosal immune system, thus decreasing the risk of pathogen adhesion and infection ^[9]. The industrial exploitation of this knowledge could lead to the design of glycoprotein-rich MFGM mixtures that exert an antiadhesive effect at the intestinal mucosal level ^[29] or an antiviral effect, for example against rotavirus infection ^[30].

Recently, the isolation and separation procedures of MFGM moved forward ^[31][32], increasing the possibility to isolate and separate specific bioactive lipid or protein components. The human milk lipid fraction has a higher content of minerals if compared to cow’s milk. This suggests that the human milk lipid fraction is more effective as being a “natural solvent” for micronutrients, especially for minerals. This feature could be crucial to face new necessities, such as deficient conditions regarding the intake of certain minerals or vitamins. The innovative element of this application of the milk lipid fraction lies in the fact that the bioactive role of micronutrients would be associated with the bioactive role of MFGM phospholipids and the MFGM proteome. The affinity between MFMG and vitamins can be exploited to design and produce stable delivery systems for molecules.

5. Conclusions

The complex architecture of MFGs ensures a stable dispersion for the bioactive compounds of milk. The MFGM proteome varies among species but retains its antimicrobial and antiadhesive properties. Further, the MFGM proteome can act as a scaffold for many of the micronutrients such as minerals and lipophilic vitamins, which can indirectly join the
milk lipid fraction. The presence of minerals and lipophilic vitamins enhances the bioactive role of the milk lipid fraction.