Principales estructuras sociales de aprendizaje

The mechanism involved in the interaction of β-lgstabilized emulsions in the presence of SGF, pepsin and mucin can be postulated based on the results from particle size analysis, confocal microscopy, SDS-PAGE quantification, and ζ- potential measurement. A schematic representation of the possible interactions is given in Figure 6.4-13. When SGF without pepsin and mucin is added to a β-lg emulsion, immediate charge reversal takes place because of the acidic pH (pH of 1.2 for SGF) but the emulsion remains stable at this low pH. However, the presence of salts promotes aggregation of the oil droplets, which can be attributed to the screening of the positive charges of the interfacial β-lg molecules by the negatively charged electrolytes, such as chlorides, in the SGF. Low levels of mucin (0.1 wt%) appear to bind strongly to the β-lg emulsion droplets, causing droplet flocculation.

Figure 6.4-13: Schematic diagram illustrating the behaviour of β- lgstabilized emulsions in a simulated gastric environment containing both pepsin and mucin. The big shaded circles represent emulsion droplets, the small blue dots represent salts (NaCl), the long thin blue curved lines represent the interfacial β-lg, small blue curved lines represent peptides formed later and the pink thick flexible coil structures represent mucin. Finally, introducing a significant amount of pepsin (0.32 wt%) into the system causes proteolysis of the interfacial layer, leading to the loss of net positive charge and steric repulsion. As the molecular weights of the adsorbed peptides

are much lower than that of the intact protein, the interfacial layers are rather weak and are prone to rupture, resulting in coalescence of the droplets. Furthermore, this emulsion destabilization is accelerated by the presence of high concentrations of salt and low levels of mucin in the SGF.

6.5 Conclusions

This study unravelled some of the key interactions of β-lgstabilized emulsion systems in the presence of simulated gastric fluid at physiological pH and ionic strengths in presence of pepsin and mucin. It was clearly understood that not only pepsin, but the variable pH and ionic strengths prevailing in the gastric tract may potentially lead to emulsion destabilization. The presence of mucin also promotes the flocculation of the β-lgstabilized emulsion through bridging mechanism. It is well known that, at physiological concentration (> 2.0 wt%) and in an acidic environment, mucin forms a gel (Lee et al., 2005; Bansil & Turner, 2006), which protects the stomach lining; thus, mucin might not be available for binding to the

β-lg emulsion droplets. However, mucin (approximately 0.020.1 wt% concentration) coming from the saliva into the gastric tract could have some potential aggregation effects at low pH, which could subsequently influence the rate of proteolysis and thus the characteristics of the emulsion droplets. Hence, the interactions of β-lg emulsion droplets in the complicated in vitro gastric model clearly identify the physiological variables, which might influence the physicochemical behaviour of a proteinstabilized emulsion droplets during their gastric transit.

After passing through the gastric tract, food emulsions are exposed to alkaline pH, various electrolytes, enzymes (lipases, proteases, amylases) as well as highly surface active bile salts, etc. as they enter the intestine. Bile salts are generally known to displace proteinstabilized interfaces by virtue of their higher surface activities (Maldonado-Valderrama et al., 2008). The interfacial composition of emulsion droplets in the intestine might be highly complicated due to the surface active and enzymatic pancreatic secretions. Hence, a control study involving only the interaction of individual pancreatic variable, such as bile salts, with proteinstabilized emulsions is required to shed light on fundamental principles

underlying the interfacial interactions in the upper intestine, which might dictate the lipid digestion of emulsions by pancreatic lipase. Therefore, the behaviour of two oppositely charged proteinstabilized emulsions (lactoferrin and β- lgstabilized emulsion) in the presence of simulated intestinal fluid containing intestinal electrolytes and bile salts (without added enzymes) was investigated in the next chapter.

Chapter Seven: Colloidal Interactions of Milk

ProteinStabilized Emulsions with Intestinal Bile

Salts

7.1 Abstract

The behaviour of milk proteinstabilized emulsions (1.0 wt% protein) as influenced by the addition of bile salts was studied in simulated intestinal conditions (37 °C; pH 7.5; 39 mM K2HPO4, 150 mM NaCl, 30 mM CaCl2; with

continuous agitation at ~ 95 rpm for 2 h). Oil-in-water emulsions (20.0 wt% soy oil) stabilized by lactoferrin or β-lactoglobulin (β-lg) were prepared at pH 6.8 to produce cationic or anionic interfaces, respectively. Varying physiological concentrations of bile salts (0.025.0 mg/mL) were added to each emulsion. The changes in droplet size, ζ-potential and confocal microstructures were monitored as a function of incubation time. Pre-heat treatment of simulated intestinal buffer containing bile salts was performed to eliminate any residual enzymatic activities.

For β-lgstabilized droplets, ζ-potential significantly changed from 63.1 ± 0.5 mV to 37.2 ± 0.3 mV in presence of bile salts due to competitive interfacial displacement of β-lg by bile salts as characterized by SDS-PAGE analysis of the continuous phase. On the other hand, lactoferrinstabilized emulsion droplets showed considerable aggregation in presence of intestinal electrolytes alone at pH 7.5. The ζ-potential values of lactoferrin emulsion decreased gradually from +53.5 ± 0.6 mV to 12.2 ± 0.2 mV in presence of bile salts due to certain electrostatic effects (e.g. pH shift towards the isoelectric point, binding of anionic bile salts to cationic interfacial lactoferrin layer).

4 _{Part of the contents presented in this chapter has been accepted for publication as a peer-reviewed}

paper: Sarkar, A., Horne, D. S., and Singh, H. (2010), Interactions of milk proteinstabilized oil-in-

water emulsions with bile salts in a simulated upper intestinal model. Food Hydrocolloids, 24(12),

7.2 Introduction

The majority of the lipid digestion (~ 7090% ) in healthy human adults occurs in the small intestine by pancreatic lipases (Fave et al., 2004; Bauer et al., 2005; Mun et al., 2006; Singh et al., 2009). When the partially digested emulsion enters the small intestine from the stomach, it is generally subjected to a wide range of physicochemical conditions, including mixing with various enzymes, such as trypsin, chymotrypsin and lipases, co-enzymes, such as co-lipases, surface active agents, such as bile salts and phospholipids. Moreover, it undergoes a drastic change in pH (from ~ 1.53.0 in the stomach to ~ 6.07.5 in the intestine) and a variation in ionic strength due to the presence of different electrolytes such as Na+_{, Ca}2+_{, HCO}

3 etc. in the pancreatic secretions

(McClements et al., 2008; Singh et al., 2009). Thus the interfacial composition of the emulsion droplets in the small intestine could be extremely complicated and largely dependent on the concentrations and surface activities of the intestinal components at any given period of time. In order to understand such complex conditions, model systems are beneficial because they allow separate investigation of interactions of emulsions in the context of individual physiological components.

In this chapter, lactoferrin or β-lgstabilized oil-in-water emulsions were exposed to biochemical conditions using an in vitro intestinal model at pH 7.5, 39 mM K2HPO4,150 mM NaCl, 30 mM CaCl2 with or without the addition of bile

salts, for up to 2 h. Bile salts are highly surface active mixtures consisting mainly of cholic acid derivatives conjugated to glycine and/or taurine; these can possibly displace the adsorbed materials from the surface of emulsion droplets promoting the accessibility of active site of lipase to the hydrophobic lipid core (Wickham et al., 1998; Fave et al., 2004; Mun et al., 2006). Broadly, the surface activities of interfacial material adsorbed on the emulsion droplets and the thickness of the adsorbed layer influence the displacement mechanism of bile salts but very little attention has been paid to the initial electrostatic charge of interfacial material coated on the emulsion droplets, which might potentially affect the bile saltinduced displacement mechanism.

In this chapter, an extremely controlled case study is envisaged in which proteinstabilized emulsion remains intact before entering the intestine ignoring the important physicochemical changes that might have occurred to the oil phase during oral and gastric processing (as described previously in Chapters 46). Also, pancreatic enzymes (lipases, trypsin etc.) have been deliberately excluded in the simulated intestinal fluid (SIF) in this study to focus on understanding the kinetics of interfacial exchange between the adsorbed protein layer and intestinal bile salts, and its influence on emulsion stability. Particularly, the aim of this chapter is to unravel the mechanisms of interactions between proteinstabilized oilwater interfaces and small molecular surface active bile salts and some of the electrolytes present in the intestine.

7.3 Materials and Methods

7.3.1 Materials

Dried un-fractionated bovine bile salts (B3883) was purchased from Sigma- Aldrich Chemical Company (St. Louis, MO, USA). The bile acid standards i.e.

sodium salts of glycocholic acid (GCA), glycodeoxycholic acid (GDCA), taurocholic acid (TCA) and taurodeoxycholic acid (TDCA) were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Methanol was HPLC- grade from BDH Merck Ltd., Poole, England. The reagents used for making the SIF and all other chemicals were purchased from BDH Chemicals (BDH Ltd, Poole, England) unless otherwise specified. All the chemicals used were of analytical grade.