La influencia del lenguaje en el desarrollo de las funciones psicológicas superiores

5.4.3.1 β-lgstabilized emulsionSGF mixture

The rates of hydrolysis of β-lg solution or β-lgstabilized emulsion mixed with SGF were monitored using SDS-PAGE (Figure 5.4-6). In β-lg emulsionSGF mixtures, the intensity of the β-lg band reduced markedly during incubation, with the simultaneous appearance of three major bands (molecular weight in the range 1015 kDa), designated as A, B and C (Figure 5.4-6 i). The intensity of band A appeared to increase slowly during the 2 h incubation period, whereas the intensities of bands B and C increased up to 30 min of incubation but decreased thereafter. Similar hydrolysis patterns were observed in the SDS-PAGE gels of the cream phases of the β-lgstabilized emulsions (Figure 5.4-6 ii).

Under the conditions of this study, about 65% of the total β-lg was adsorbed at the droplet surface. In order to examine the behaviour of unadsorbed protein during SGF treatment, the original β-lgstabilizedemulsion was centrifuged, and the subnatant was removed and mixed with SGF. SDS-PAGE showed that the intensity of the β-lg band in the continuous phase diminished gradually with incubation time (Figure 5.4-6 iii). However, the loss of the β-lg band was much less than that in the emulsion samples. Several faint bands, including those corresponding to A, B and C, were observed. Native β-lg solution, containing 0.36% β-lg (the same concentration as that in the serum phase of the emulsion), was mixed with SGF and examined using SDS-PAGE (Figure 5.4-6 iv). A slight decrease in the intensity of the β-lg band was observed during the 2 h incubation period.

Figure 5.4-6: SDS-PAGE patterns obtained from β-lgstabilized emulsions (i), cream phase of β-lg emulsions (ii), continuous phase of β-lg emulsions (iii), and native β-lg solutions [containing 0.36% β-lg (the same as the concentration of β-lg in the continuous phase of the emulsion)] (iv), after mixing with SGF, respectively as a function of incubation time.

The rate of hydrolysis of β-lg in different systems (estimated by scanning SDS- PAGE gels) is shown in Figure 5.4-7. The proportion of β-lg decreased by only about 20% in the native protein solution, compared with about 50% for the subnatant sample and 85% in the emulsion system, after 2 h of incubation. As expected, native β-lg solution was largely resistant to pepsin digestion, presumably because its aromatic amino acid side chains were buried inside the folded globular structure, confirming earlier studies (Reddy et al., 1988; Guo et al., 1995). From these results (Figure 5.4-7), it is evident that β-lg present in the serum phase (Figure 5.4-6 iii) of an emulsion is more susceptible to pepsin digestion than native β-lg in solution (Figure 5.4-6 iv).

(i) (ii)

0 20 40 60 80 100 0 20 40 60 80 100 120

Time of digestion (min)

In tact β -l g r e m a in in g ( % ) β-lg emulsion+SGF β-lg emulsion-subnatant+SGF β-lg native solution+SGF

Figure 5.4-7: Rate of hydrolysis of intact β-lg in emulsions, the continuous phase of emulsions and native protein solutions on addition of SGF.

This suggests that the conformation of unadsorbed β-lg is altered during emulsion formation, possibly as a result of high turbulence during homogenization. This change in conformation could allow greater accessibility of some aromatic amino acid residues to pepsin attack. The susceptibility of non-adsorbed β-lg to proteolytic attack has also been reported previously (Fang & Dalgleish, 1997). There is also a possibility that at least a part of continuous phase (primarily consisting of nonadsorbed protein) was also emulsified, with very fine emulsion droplets (Z-average = 0.105 μm) which were able to sequentially pass through the 0.45 μm and 0.22 μm filters (Millipore Corp., Bedford, MA, USA) and resulted in increased susceptibility to proteolysis. The presence of these sub- micronsized emulsion droplets in the nonadsorbed phases have been reported in previous studies involving bovine serum albuminstabilized emulsion systems

(Castelain & Genot, 1996; Rampon et al., 2003).

The marked increase in the hydrolysis of β-lg in the emulsion can be explained on the basis of conformational changes caused by the unfolding of the β-lg secondary structure at the droplet surface (Agboola & Dalgleish, 1996), improving the accessibility of pepsin-susceptible bonds. It appears that some relatively large fragments of β-lg (bands A, B and C) remained inaccessible to pepsin during the early stages of incubation. This could suggest that some portions of adsorbedβ-lg were not completely unfolded and retained their native structure. The hydrolysis of these relatively large fragments after prolonged

incubation suggests that these fragments were associated with the droplet surface and underwent some reorientation to allow the exposure of further cleavage sites.

5.4.3.2 Lactoferrinstabilized emulsionSGF mixture

Lactoferrin has been known to be susceptible to peptic hydrolysis in its native state (Tomita et al., 1991). However, it has been debated that considerable amounts of lactoferrin survive gastric transit and is less susceptible to peptic hydrolysis as compared to casein and transferrin at acidic pH (Britton & Koldovsky, 1989; Troost et al., 2001). An important factor influencing this hydrolysis is the degree of iron saturation of lactoferrin (Brock et al., 1976; Brines & Brock, 1983). It has been reported that 20%ironsaturated lactoferrin is more easily digested than 100%ironsaturated one.

Lactoferrin adsorbed at the droplet surface was almost fully degraded within the first 5 min (Figure 5.4-8 A and B). The proportions of intact lactoferrin in the emulsions remaining after digestion with SGF were negligible due to almost instantaneous hydrolysis of lactoferrin with > 80% within first 1 min. This indicated extensive proteolysis of the lactoferrin layer by pepsin, which generated fast moving shorter peptide fragments that probably diffused out of the 16.0 % resolving gel. The rapid rate of proteolysis of the lactoferrinstabilized emulsions and absence of larger molecular weight peptides (as seen in case of β- lgstabilized emulsions) might be the reason for higher extent of droplet aggregation and an earlier onset of coalescence as compared to β-lgstabilized emulsions, as observed earlier (Figure 5.4-1 and Figure 5.4-4 BF). Even, the SDS-PAGE patterns of unadsorbed phase and native lactoferrin during SGF treatment were similar to that of the lactoferrin emulsion (data not shown), i.e.

the lactoferrin band was depleted almost immediately (before 5 min) after the addition of SGF. This was likely due to the release of bound iron at pH 1.5 in all the lactoferrin systems (native, continuous phase or emulsions), which made the lactoferrin molecules more sensitive to pepsin attack (Baker et al., 2002; Ye & Singh, 2006a).

0 20 40 60 80 100 0 20 40 60 80 100 120

Time of digestion (min)

In tact lact of er ri n r e m a in in g ( % ) Lactoferrin emulsion+SGF

Figure 5.4-8: SDS-PAGE pattern obtained from lactoferrinstabilized emulsions (A), and rate of hydrolysis of intact lactoferrin in emulsions (B) as a function of time.

It is worth noting here, that lactoferrin and β-lgstabilized emulsions show different behaviour in presence of SGF, not because of their different initial charges, but due to their different intrinsic nature, molecular conformations and specific accessibility to pepsin.