La posición de la cámara, la maqueta y los

This current research compared the physicochemical properties between native milk (human milk and cow milk) and processed milk (liquid and powder infant formulae). For human breast milk, mature milk in the range of 13 months postpartum was investigated. Infant formulae for normal full-term newborn infants during the first six months of life were compared.

Biological milk fat is secreted from the alveolar epithelial cells of the mammary gland as milk fat globules (MFGs). These MFGs are composed of a lipid core made up to 95% or more of hydrophobic triacylglycerols (TAGs). This is surrounded by a membrane layer, comprised of amphipathic compounds (phospholipids, proteins glycolipids bipolar substances, enzymes and cholesterol) forming an emulsionstabilising membrane (Abrahamse et al., 2012, Jensen et al., 1992). The membrane of breast milk derived from apical plasma membrane of the epithelial cells, helps to maintain milk lipids in a dispersed state and prevents coalescence (Valivullah et al., 1988). In addition, some trace elements, enzymes, hormone and growth factors are found in the crescents of human milks but these are completely absent in infant formulae. On the other hand, most of the infant formula manufacturers use a mixture of vegetable oils in place of milk fat to provide the same overall fatty acid profile found in human milk. Fat droplets in infant formulae consist of a triglyceride core surrounded predominantly by surface active protein and phospholipids (Oosting et al., 2012, Michalski et al., 2005b). The above accounts for the main differences observed here between maternal and infant formulae of droplet size and ]-potential value.

The particle size of the fat droplets present in milk is important in defining properties, especially emulsion stability. Particle size could thus be one of the key factors that regulate fat digestion. Comparisons of the MFGs of human and infant formula showed that biological milks (human and cows’ milk) have a bigger droplet size than the processed infant formula milks (both powder and liquid formulae) (Table 4.2). The significant variation in size between biological and processed formula milks is due to the fact that the fat droplets in infant formulae are not native MFGs as they are produced by the emulsification of vegetable oils and passed through the homogenisation process so that the droplet size is significantly reduced and the specific surface area is larger (Michalski, 2013). However, although breast milk lipids have a bigger droplet size, they are reported as being more efficiently digested and absorbed by the infant than infant formula lipids (Michalski, 2013). Armand et al. (1999) found that a fine emulsion underwent a higher degree of lipolysis than a coarse emulsion. The rate and extent of gastric lipolysis is dependent on the available surface area of the triglyceride for the lipase to act, and this is determined not only by the initial fat content but also by the droplet size (van Aken et al., 2011); the smaller the droplet size, the larger the surface area available for the lipase to act on. However, the composition of the fat droplet membrane is also important since lipases must pass through this membrane to gain access to the TAG core. As described above the researchers have found that the much bigger MFGs in human milk which are surrounded by a native MFGM have more efficient gastric lipolysis than the smaller fat droplets present in homogenised infant formula (Armand et al., 1999, Favé et al., 2004). Possible differences in terms of fat digestion

between the infant formulae may be expected given the microstructure variation found here. These differences are the focus of following chapters.

Comparing the two native milks (human and cow’s milk), this current study showed that mature human breast milk had smaller droplet size than cow’s milk (Table 4.1 and Figure 4.1). The difference in fat droplet size of biological milks depends on many factors such as the species, the maturity of mammary gland, and the stage of lactation. Moreover, some researchers had stated that the diameter of mature human MFGs increases with advancing lactation which is opposite to bovine MFGs (Michalski et al., 2005b, Rüegg and Blanc, 1981, Mulder and Walstra, 1974).

Zeta-potential is a function of the surface charge of the particle, which depends on the adsorbed layer at the interface and the nature and composition of the surrounding medium in which the particle is suspended. The ]-potential depends strongly on pH, conductivity and concentration of components. All ]-potential results in this current study are negative. Human breast milk had the lower ]-value compared to bovine milk and infant formulae. This implies that the electrostatic repulsion forces between fat droplets of human milk were the weakest. Michalski (2013) described that a lower ]-potential value of human milk could be due to the present of glycoproteinaceous filaments at external membrane of human MFG surface which these filaments are absence in cow’s milk. In cow’s milk, the external membrane are proteins and phospholipids (Jensen et al., 1990).

The particles in formula emulsions possessed greater negative charge than the biological milks, which is considered a consequence of protein adsorption during homogenisation as mentioned above. The negative surface charge generates

electrostatic repulsion keeping the emulsion droplets apart and preventing them from aggregating. Milk emulsions, especially liquid infant formulae had all good electrostatic stability. The emulsifier type used in the infant formulae may be crucial for stabilisation of the fat droplets by influencing the ]-potential value. Moreover, the emulsifier might protect the triglyceride surface against enzymatic lipolysis as the type of polar lipid at the surface of MFG modifies the TAGs hydrolysis by gastric lipase depending on the biding strength of the enzyme onto the surface (Michalski, 2013). As stated earlier, the area of the triglyceride surface available to the lipase may also affect the rate and extent of gastric lipolysis (van Aken et al., 2011).

Commonly used emulsifiers in infant formulae include lecithin, monoglycerides and/or diglycerides. McSweeney, Healy and Mulvihill (2008) have studied the concentration effect of monoglycerides or lecithin, used as emulsifiers on the ]- potential of oil droplets in model infant formula emulsions. They found that when the concentration of monoglycerides increased the ]-potential became less negative due to the displacement of protein molecules from the interface of the fat droplet by monoglycerides. On the other hand, lecithin had no effect on the ]-potential value because the negative charge of lecithin may displace or interact with protein at the interface. As it can be seen from the ingredient lists of commercial infant formulae in this study (Table 2.6) the most used emulsifier is lecithin. The ]-potential value (Table 4.1) measured in infant formulae showed no drastic differences among the samples.

In processed milk, the fat droplet size distributions among the infant formulae both liquid and powder varied. The different heat processing of liquid infant formulae, UHT and retort showed no obvious differences in terms of droplet size (d43), being in the range of 0.320.37 mm. However, some powder formulae had bigger droplet size, presenting a bimodal size distribution, probably due to processing. The microstructure of different infant formula milks clearly showed very fine emulsions for the liquid formulae versus coarser emulsions for powder formulae. The differences in the extensive protein adsorption at the interface of fat droplets were observed (Table 4.2). The fat membranes in infant formulae are new membranes formed from whey proteins and caseins during processing or from phospholipids and proteins, depending on the formulation (Michalski et al., 2005b). In the aqueous phase of the emulsion system non adsorbed protein is present.

Infant formula uses vegetable oils instead of milk fat and is subjected to severe mechanical processes. The variation of fat globule size can be due to different processing conditions during blending, pasteurising, homogenising, and spray drying. The homogenisation is the extreme process where fat globules are subjected to such high shear stress, thus component redistribution and interactions such as proteinprotein, proteinlipid and proteinlipidmineral may occur (Guo et al., 1998, Guo et al., 1996, Hendricks et al., 1994). Interactive forces such as electrostatic, steric, and bridging between droplets can induce the aggregation and coalescence of fat droplets (Britten and Giroux, 1993). Some proteins such as denatured whey proteins can become clustered during pasteurisation or sterilisation and precipitated with micelles during processing. Also casein micelles and denatured whey protein clusters cover small fat globules during homogenisation. Powdered

milks which undergo a drying process after homogenisation showed secondary 10

μm particles probably due to a high degree of whey protein aggregation and more damaged globules compared to liquid milks as shown in the micrographs of powder formulae in Table 4.2. The differences in fat globule size distribution among brands (Figure 4.1 and Table 4.1) is likely due to variations in the manufacturing of the processed milk powder, especially homogenisation pressures conditions, heat treatment and the use of different ingredients in their formulations.

In powder infant formulae, the observed bimodal peak maybe the result of the spray- drying process. Thus, the fat globules are disrupted during spray drying and then (i) caseins/heat denatured whey proteins complexes adsorb onto the new expanded lipid droplet interface (Michalski and Januel, 2006) or (ii) the fat globules are attached to each other by casein micelledenatured whey aggregates and a short-chain-like structure is formed (Guo et al., 1998). All this results in the formation of larger particles affecting the surface protein coverage, given the incidence of coalescence of “uncovered fat” occurring at the surface of powder particles (Ye et al., 2007). Moreover, newly uncovered surface on the fat globules may come into contact at the air-water surface of the drying droplet, with the result of some fat spreading easily on the powder particles surface (Ye et al., 2007, Walstra, 1995). The amount of damage fat in the powder samples may have an impact on the digestion in the infant.