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Once the emulsion passes from the stomach into the small intestine, the partially digested emulsified lipids are subjected to various salts, pancreatic enzymes, coenzymes and surface active agents (bile salts, phospholipids) secreted by the liver, gall bladder and pancreas in an alkaline condition (Tso, 2000; McClements et al., 2009; Singh et al., 2009).

2.7.3.1 Alkaline pH and ionic strength

Generally, sodium bicarbonate is secreted in the small intestine to cause the pH to increase from highly acidic conditions (pH 13) in the stomach to nearly alkaline conditions (pH 67.5) in the duodenum, where the pancreatic enzymes can act effectively (Bauer et al., 2005). During this drastic increase in pH, most of the proteinstabilized emulsions would be expected to be reverted back to their anionic form, with some possible aggregation while passing through their pI

zone.

The osmolality and ionic strength of fasted duodenum is 180 mOsm/kg and 140 mM, respectively (Lindahl et al., 1997). The osmolality of duodenum increases from ~ 180 mOsm/kg in fasted state to ~ 290 mOsm/kg in fed state (after ingestion of a nutrient drink), with the osmolality gradually decreasing over time

(Kalantzi et al., 2006). Both pH and ionic strengths are particularly important parameters as they might affect the electrostatic interactions of charged emulsified lipid droplets.

2.7.3.2 Biochemical factors

The possible interactions between emulsion droplets and intestinal components can be highly complicated as the intestine is the house to many enzymes, such as proteases and peptidases (trypsin, chymotrypsin, carboxypeptidases, etc.), lipases and esterases (pancreatic lipase, cholesterol esterase, phopholipase A2, etc.) and

pancreatic amylases (Singh et al., 2009). Furthermore, the adsorbed layer surrounding the lipid droplets may change significantly, on entering the small intestine due to the secretion of not only surface active bile salts, but also the generation of various hydrolytic products of acidic nature, such as surface active monoacylglycerols and fatty acids (generated by lipolysis of triglycerides), peptides and oligosaccharides (Bauer et al., 2005; Kalantzi et al., 2006).

2.7.3.2.1 Intestinal proteases

Proteinstabilized emulsions may undergo substantial alteration due to proteolysis of the interfacial layer by trypsin and/or chymotrypsin. Serine proteases, particularly trypsin predominantly catalyses the peptide chains at

chymotrypsin favours large aromatic residues, such as phenylalanine, tyrosine, and tryptophan (Vajda & Szabó, 1976; Honey et al., 1984; Olsen et al., 2004; Ma et al., 2005). Over the last few decades, several studies have reported the effects of hydrolysis of proteinstabilized oil-in-water emulsions by trypsin (Shimizu et al., 1986; Kaminogawa et al., 1987; Leaver & Dalgleish, 1990), detailing on peptides generated and their respective functionalities (Turgeon et al., 1992). The inability of the peptide films to provide effective surface coverage, resulting in coalescence of droplets, has also been explored (Agboola & Dalgleish, 1996). However, the complex interactions of these proteases with the adsorbed proteins in presence of pancreatic lipases and bile salts have not been unravelled as yet.

2.7.3.2.2 Bile salts

Bile salts originate from the liver via gall bladder, which basically facilitate emulsification by adsorbing to the droplet surface (Tso, 2000). They are synthesized from cholesterol and contain cholic acid as the “backbone” conjugated with amino acids taurine or glycine to form taurocholates and glycocholates respectively (Bauer et al., 2005). In humans, bile salts are the surface active mixtures of mainly sodium salts of taurocholic, taurodeoxycholic, taurochenodeoxycholic, glycocholic and glycodeoxycholic acids respectively, and these can possibly displace the adsorbed materials from the surface of emulsion droplets, thus 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 dictate the displacement mechanism of bile salts (Singh et al., 2009).

Recently, Mun et al. (2006) reported that bile salts displace whey proteins more readily than caseinates from the interface of emulsion droplets during storage. Dependence of bile saltinduced displacement mechanisms on the nature of adsorbed layer was further supported by another study, where the digestibility of adsorbed milk proteins in simulated gastrointestinal conditions was investigated

caseinadsorbed surfaces was compared with or without the addition of surfactants (bile salts and phosphatidyl choline). Macierzanka and others (2009)

reported that the final structure of the β-lgstabilized interface was largely driven by competitive displacement by bile salts and/or phosphatidyl choline in contrast to adsorbed β-casein, where the gastric hydrolysis by pepsin was the major cause of destabilization for β-caseinstabilized interface. In another study, the competitive displacement of β-lg by bile salts from airwater and oilwater interfaces was investigated in an in vitro duodenal model (Maldonado- Valderrama et al., 2008). These authors suggested that bile salts might completely displace the β-lg from the interface, when passing through the duodenum in vivo, which in turn would affect the rate of lipid digestion in case of

β-lgstabilized emulsified droplets.

A typical range of bile salt concentrations found in the healthy human small intestine varies from 4 to 6 mM and from 10 to 20 mM in the fasted and fed states respectively (Porter & Charman, 2001; Rich et al., 2003; Wright et al., 2008). At low concentrations, bile salts promote lipase activity mainly by allowing the adsorption of lipase to the oilwater interface (Gargouri et al., 1983; Mun et al., 2007). Moreover, bile salts also solubilize the lipid hydrolytic products (fatty acids, monoacylglycerols, etc.) and remove these inhibitory reaction products from the oilwater interface, thus allowing the lipase to have access to the triacylglycerols within the emulsion droplets (McClements et al., 2009). However, at higher concentrations, the bile salts generally compete with lipases for the oilwater interfaces, inhibiting the point of contact between the nonpolar lipid core and lipase (Gargouri et al., 1983), thus retarding lipase activity. Hence, the presence of bile salts may either facilitate or inhibit the activity of pancreatic lipase depending on their concentration (Lowe, 2002; Bauer et al., 2005). Monoacylglycerols, diacylglycerols and fatty acids generated from the hydrophobic core of the emulsion droplets by the action of pancreatic lipase also play an important role in the interfacial displacement mechanisms (Mun et al., 2007; McClements et al., 2009; Singh et al., 2009).

2.7.3.2.3 Pancreatic lipase

The majority of lipid digestion (~ 7090%) occurs in the upper part of the intestine (Fave et al., 2004; Bauer et al., 2005; Mun et al., 2006). Partially hydrolysed lipids enter the small intestine as fine droplets less than 0.5 µm, largely due to the peristaltic shearinduced disruption in the stomach (Carey et al., 1983). However, it has been suggested that the lipid droplet size in the duodenum typically varies from 150 μm (Armand et al., 1996a). Once the lipid droplets enter the intestine, pancreatic lipase adsorbs to the droplet interface usually by a complexation with colipase and/or bile salts (Bauer et al., 2005). Colipase is a short polypeptide of molecular weight ~ 10 kDa, which forms a stoichiometric complex with lipase in the ratio of 1:1 w/w, allowing the pancreatic lipase to anchor firmly to the substrate (hydrophobic lipid core) at the oilwater interface (Patton et al., 1978; Erlanson-Albertsson, 1992). Colipase is a coenzyme, which is an amphiphilic molecule; its hydrophobic residues are assumed to be bound to the oil phase of the emulsified droplets and hydrophilic side is bound to the lipase (Bläckberg et al., 1979). This orientation facilitates anchoring of the lipase’s active site to its substrate. Presence of this coenzyme has been also assumed to remove the inhibitory effects of bile salts and phospholipids (Crandall & Lowe, 2001).

As compared to gastric lipase in the stomach, pancreatic lipase has a pH optimum of ~ 89 (Patton & Carey, 1981), but acts even at pH 6.5 and has little specificity for fatty acid chain length (McClements et al., 2009). Pancreatic lipase cleaves triacylglycerols to form 2monoacylglycerols and fatty acids. The diacylglycerols, monoacylglycerols, free fatty acids, thus released as digestion products, might be adsorbed at the droplet surface displacing the initial adsorbed moieties from the interface (McClements et al., 2008; McClements et al., 2009; Singh et al., 2009). This competitive exchange of interfacial layer by the lipid digestion products, which in turn depends on the relative concentrations and surface activities of the adsorbed layer, makes the composition of the adsorbed layers even more complicated to understand. In general, the compounds with higher surface activities would be expected to dominate the interface.

Currently, there is very limited understanding about the fundamental mechanisms of interactions of different intestinal surface active agents, which might influence the kinetics of interfacial exchange of emulsified lipids, and thereby affect lipid digestion. Hence, further research is clearly required.