Estado de la cuestión

proteinstabilized emulsion

A schematic diagram is presented to illustrate the physical state of both β-lg and lactoferrinstabilized emulsions when they are exposed to an in vitro gastric condition (Figure 5.4-9). The net charge (-ve or +ve) of the protein used to stabilize the emulsion had no significant impact when exposed to SGF. Due to the acidic pH of the SGF (pH 1.2), the net charges of both the β-lg and

(A)

lactoferrinstabilized emulsions were positive. So initially at pH 7.0 and even after the addition of SGF (pH 1.5), both types of emulsions were stabilized by strong positive charges and steric effects were provided by chains of β-lg or lactoferrin protruding into the aqueous phase in corresponding emulsion systems. Hence, the pH did not influence the stability of the emulsions but only provided suitable conditions for the pepsin activity.

Figure 5.4-9: Schematic diagram of interaction of a proteinstabilized emulsion with SGF. The big shaded circles represent either lactoferrin or

β-lgstabilized emulsion droplets, the blue long coil structures represent the proteins at the interfacial layer and the smaller coil structures represent the peptides formed later.

The emulsions were very stable at the low pH of SGF, but once pepsin is introduced into the system, several physico-chemical changes occur in both the emulsions. The hydrolysis of the interfacial protein layer by pepsin causes a gradual loss of positive charge at the droplet surface and presumably also leads to a reduction in the thickness of the adsorbed layer. The peptides that remain at the interfacial layer are unable to create strong interfacial layers with sufficient electrostatic repulsion and steric barriers. Consequently, the emulsions become susceptible to flocculation, and the presence of high concentrations of salt in the SGF further enhances the process of flocculation. The peptide adsorbed layers on some of the droplets are obviously not effective at preventing coalescence of the droplets.

β-Lg, which is largely resistant to pepsin attack in its native state, is hydrolysed by pepsin when present as the adsorbed layer in emulsions. The change in conformation as a result of unfolding at the emulsion interface exposes the peptic cleavage sites and thus significantly decreases the resistance of β-lg to pepsin. Lactoferrin, which is susceptible to proteolysis by pepsin in its native state, is also hydrolysed when present as an interfacial layer coating the oil droplets. This study showed that both β-lg and lactoferrinstabilized emulsions undergo peptic hydrolysis, leading to flocculation and coalescence of droplets. Such a phenomenon is likely to occur in the stomach in vivo. This type of flocculation followed by coalescence might reduce the overall surface area of the emulsions, potentially influencing the accessibility of gastric lipase and finally affecting the rate of lipid digestion (Armand et al., 1999). In addition, the coalesced droplets might interact with the receptors to send signals to the brain, giving a feeling of satiety (Norton et al., 2007).

5.5 Conclusions

This study provided a better understanding of the mechanisms of flocculation in both positively and negatively charged milk proteinstabilized emulsion systems in the presence of SGF containing pepsin at acidic pH. It was observed that both lactoferrin and β-lgstabilized emulsions underwent flocculation followed by some degree of coalescence on being exposed to simulated gastric environments depending upon protein conformation at the adsorbed layer and incubation time with pepsin. It is recognized that the substrate:enzyme ratio (3:1) used in this model is considerably higher than that in some of the previous substrateenzyme studies (Dalgalarrondo et al., 1995; Guo et al., 1995). However, it is difficult to select an optimal substrate:pepsin ratio that exactly mimics the secretion found physiologically in humans because a wide variation (up to about 10,000 fold) in gastric and pancreatic secretions has been suggested depending on the individual’s health and the type of food intake (da Silva Gomes et al., 2003; Moreno, 2007).

Another interesting finding of this study was that β-lg, which is usually resistant to pepsin attack in its native state, became susceptible to proteolysis when present

as the interfacial layer in emulsions. A change in the conformation of the β-lg molecules upon adsorption at the oilwater interface exposes the peptic cleavage sites for proteolysis, leading to emulsion instability and coalescence.

It is recognized that the ionic strength and pH varies significantly in real physiological circumstances (Kalantzi et al., 2006). Furthermore, the role of mucin, which is a high molecular weight glycosylated protein (molecular weight about 2.04.0 × 106 _{Da) and forms a self-associated networked structure under}

gastric conditions (low pH and at high mucin concentrations) (Nordman et al., 2002; Lee et al., 2005; Bansil & Turner, 2006), has not been explored. Therefore, the interactions of emulsions in SGF media containing pepsin and mucin under different gastric pH and ionic strength conditions need to be determined to better understand their effects on gastric digestion of proteinstabilized emulsions. Since, the initial charges of the emulsions have been shown to play an insignificant role in determining the interactions with SGF (as both the emulsions acquired net positive charge at gastric pH), the behaviour of only β-lgstabilized emulsions as affected by different pH, ionic strength in presence of mucin and pepsin was investigated in the next chapter.

Chapter Six: Factors Influencing the Interactions of

β-lactoglobulinStabilized Emulsions with Simulated

Gastric Fluid

6.1 Abstract

The effects of pH (6.51.5), ionic strength (0150 mM NaCl) and the presence of mucin (0.1 wt%) on the properties of oil-in-water emulsions [20.0 wt% soy oil, stabilized by 1.0 wt% β-lactoglobulin (β-lg)] under simulated gastric conditions (with/without 0.32 wt% pepsin at 37 °C, with continuous shaking at approximately 95 rev/min for 2 h) were investigated. Changes in Z-average diameter, ζ-potential and microstructure were determined as a function of incubation time. The emulsions mixed with simulated gastric fluid (SGF) (without added pepsin) were stable over a wide pH range (except at pH 5 close to the isoelectric point of β-lg) and at low ionic strengths (≤ 50 mM NaCl). Extensive droplet flocculation with some degree of coalescence was observed in emulsions with 0.32 wt% added pepsin, the flocculation being potentially accelerated in the presence of NaCl. The addition of 0.1 wt% mucin resulted in a greater extent of flocculation, possibly because of binding of mucin to the positively charged β-lgstabilized emulsion droplets. Ionic strength, pH and the presence of mucin had a significant influence on the rate of hydrolysis of β-lg by pepsin. The behaviour of the emulsion in SGF was predominantly driven by electrostatic interactions, which varied as a function of digestion time, pH, ionic strength and the presence of pepsin and mucin.

6.2 Introduction

In Chapter 5, it was found that an almost instantaneous change in the electrostatic charge of β-lactoglobulin (β-lg)stabilized emulsions droplets (from negative charge to positive charge) occurs in a simulated gastric fluid (SGF) environment.

3 _{Part of the contents presented in this chapter has been published previously as a peer-reviewed}

paper: Sarkar, A., Goh, K. K. T., and Singh, H. (2010), Properties of oil-in-water emulsions stabilized

by β-lactoglobulin in simulated gastric fluid as influenced by ionic strength and presence of mucin.

β-Lg, which is largely known to be resistant to pepsin attack in its native state because of its folded structure, became accessible to proteolysis by pepsin when present as the adsorbed layer in emulsions. A change in the conformation of the

β-lg molecules upon adsorption at the oilwater interface exposes the peptic cleavage sites for proteolysis, leading to emulsion instability and some degree of coalescence. However, the emulsionSGF interaction studies were limited to pH 1.5 and ionic strength of 34 mM NaCl. Generally, the pH and ionic strengths vary significantly during real gastric processing conditions (Kalantzi et al., 2006). Moreover, the presence of high molecular weight glycosylated mucin in the gastric system might influence the behaviour of emulsions and proteolysis patterns by pepsin. Mucin has been shown to play an important role in the flocculation of emulsions in the oral environment because of its negative charge at neutral pH (Chapter 4). However, the influence of mucin at acidic pH and at higher ionic strengths on the stability of emulsions, particularly in the presence of a proteolytic enzyme, has not been explored to date.

Hence, the aim of this chapter was to elucidate the interactions of β-lgstabilized emulsions in SGF media taking into consideration the role of different gastric variables, such as pH and ionic strength in presence of pepsin and mucin. The focus was on the changes in the physico-chemical properties, microstructures and proteolysis patterns (indicated by SDS-PAGE) of the emulsions.

6.3 Materials and Methods

6.3.1 Materials

Pepsin and mucin from porcine gastric mucosa were used (details mentioned in sections 4.3.1 and 5.3.1).