Droplet size distribution and microstructure

Figure 5.4-1 illustrates the mean hydrodynamic diameter (μm) of the emulsionSGF mixtures, determined using dynamic light scattering. Both β-lg and lactoferrin formed a stable emulsion with a Z-average diameter of approximately 0.35 µm at pH 7.0. However, on mixing with SGF, both the emulsions showed an almost linear increase in hydrodynamic diameter with

incubation time up to 2 h (p < 0.05). The Z-average diameters of the lactoferrinstabilized emulsion droplets were significantly higher than that of the

β-lgstabilized emulsions after treatment with SGF (p < 0.05). The lactoferrin emulsionSGF mixture showed roughly a linear increase in hydrodynamic diameter to ~ 5.0 μm as a function of time. In contrast, β-lg emulsion droplets showed a comparatively smaller increase in diameter with a maximum of ~ 2.5

μm under the same conditions.

0.0 1.0 2.0 3.0 4.0 5.0 0 0.5 1 1.5 2 2.5 Time (hours) Z -a ve ra ge di a m et er ( µ m ) β-lg emulsion Lactoferrin emulsion

Figure 5.4-1: Change in Z-average diameter (µm) of emulsion droplets [20.0 wt% soy oil, 1.0 wt% β-lg or lactoferrin respectively] after mixing with SGF as a function of time. The error bars represent standard deviations.

The droplet size distributions of the emulsions studied using static light scattering (Malvern Mastersizer) showed that uniformly dispersed emulsions (with monomodal size distributions) were formed (Figure 5.4-2) immediately after homogenization in case of both lactoferrin and β-lg interfacial layers. However, after treatment with SGF for 1 h, β-lgstabilized emulsions showed bimodal distributions, with a second peak in the region 2–10 μm and a corresponding decrease in the area of the first peak (Figure 5.4-2 A). After 2 h of incubation, the size distribution of the β-lg emulsionSGF mixture droplets became multimodal with a third peak appearing in the range 950 μm. Lactoferrinstabilized emulsions showed a higher proportion of larger sized droplets than those stabilized by β-lg after SGF treatment (Figure 5.4-2 B), which is in agreement with the dynamic light scattering results. When the emulsionSGF mixtures (showing multiple peaks) were dispersed in 2.0% SDS buffer, the proportions of

larger droplets in both the emulsions were reduced, although the size distribution remained bimodal (Figure 5.4-2 A and B). This indicates that, in addition to droplet flocculation, some coalescence of the emulsion droplets in these systems had occurred. Similar effects have been seen in oil-in-water emulsions formed with extensively hydrolysed whey proteins (Ye & Singh, 2006b).

0 2 4 6 8 0.01 0.1 1 10 100 Particle diamter (μm) Vo lu me (% ) β-lg emulsion β-lg emulsion+SGF (1h) β-lg emulsion+SGF (1h)+2% SDS β-lg emulsion+SGF (2h) β-lg emulsion+SGF (2h)+2% SDS 0 2 4 6 8 0.01 0.1 1 10 100 Particle diamter (μm) Vol u m e ( % ) Lactoferrin emulsion Lactoferrin emulsion+SGF (1h) Lactoferrin emulsion+SGF (1h)+2% SDS Lactoferrin+SGF (2h) Lactoferrin emulsion+SGF (2h)+2% SDS

Figure 5.4-2: Droplet size distribution of emulsions after mixing with SGF at various incubation periods, without or with the addition of 2.0% SDS solution. Each data point is the average of measurements on duplicate samples.

Confocal laser scanning microscopy (CLSM) showed that the freshly prepared β- lg (Figure 5.4-3 A) and lactoferrinstabilized emulsions (Figure 5.4-4 A) before mixing with SGF had fine and uniform droplet distributions, confirming the light scattering results. However, the β-lgstabilized droplets appeared to flocculate when the emulsions were treated with SGF (Figure 5.4-3 BF). Some distinct large droplets (> 10 μm) were observed after 1 h of incubation (Figure 5.4-3

(A)

DF), in agreement with the bimodal droplet size distribution. This confirms that some coalescence had occurred in these flocculated β-lgstabilized emulsions.

Figure 5.4-3: Changes in the microstructure of β-lgstabilized emulsions (A) after mixing with SGF (mixture pH 1.5) as a function of incubation time: 30 min (B), 45 min (C), 1 h (D), 1h 30 min (E), and 2 h (F). Scale bar corresponds to 10 μm.

On the other hand, the emulsions stabilized by lactoferrin appeared to be more densely flocculated on addition of SGF (Figure 5.4-4 BF). This can be viewed from larger emulsion droplets (~ 10 µm) and irregular flocs in the confocal micrographs immediately after 30 min of digestion with SGF. There was almost

(A) (B)

(C) (D)

complete absence of discrete lactoferrin emulsion droplets after addition of SGF as compared to some degree of uniformly dispersed droplets in β-lg emulsionsSGF mixtures.

Figure 5.4-4: Changes in the microstructure of lactoferrinstabilized emulsions (A) after mixing with SGF (mixture pH 1.5) as a function of incubation time: 30 min (B), 45 min (C), 1 h (D), 1h 30 min (E), and 2 h (F). Scale bar corresponds to 10 μm.

Clusters of many small droplets were also observed surrounding larger lactoferrinstabilized emulsionSGF droplets, which appeared to have flocculated (Figure 5.4-4 DF). Onset of coalescence appeared to occur between

(A) (B)

(C) (D)

these large droplets (> 10 µm) and the adjacent droplets due to the probable rupture of the adsorbed lactoferrin film at the oil–water interface. The stronger and higher rate of flocculation observed in lactoferrinstabilized emulsions could be attributed to the higher susceptibility of lactoferrin moleculesto hydrolysis by pepsin.

Although, there was an obvious increase in droplet diameter, the light scattering studies did not show a dramatic rise in average size (> 10 µm) as expected due to the coalescence of droplets. It could be possible that the coalesced droplets were so large (considerable ‘oiling-off’ clearly visible by eye) that they immediately creamed to the top of the cuvette and thus were not detected by the laser (Mun et al., 2007).