Aspects of milk-protein-stabilised emulsions

Food Hydrocolloids 25 (2011) 1938e1944

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Aspects of milk-protein-stabilised emulsions Harjinder Singh Riddet Institute, Massey University, Private Bag 11222, Palmerston North, New Zealand

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 January 2011 Accepted 21 February 2011

Milk proteins are widely used as ingredients in prepared foods, in which they perform a wide range of key functions, including emulsification, thickening, gelling and foaming. An important functionality of milk proteins in food colloids is their ability to facilitate the formation and stabilisation of oil droplets in emulsions. The ability of milk proteins to adsorb at the oilewater interface and to stabilise emulsions has been exploited by the food industry in the manufacture of nutritional products, specialised medical foods, dietary formulations, cream liqueurs and dairy desserts. This article provides an overview of the properties and functionalities of food emulsions formed with milk proteins, focusing on the structure and composition of adsorbed protein layers, competition between proteins and the physical and chemical stability of emulsion droplets. Of particular importance is the understanding of the behaviour of milk-protein-based emulsions under the conditions relevant to digestion in the human gastrointestinal tract. Recent relevant research in this area is reviewed and discussed. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Emulsions Milk proteins Protein adsorption Stability Lipid oxidation Lipid digestion Gastrointestinal tract

1. Introduction There are two main classes of milk proteins: caseins and whey proteins. Caseins can be fractionated into four distinct proteins, as1-, as2-, b- and k-caseins, all of which are phosphoproteins (Fox, 2009). The caseins are very flexible molecules and have been referred to as rheomorphic (Holt & Sawyer, 1993); the most unusual feature is the amphiphilicity of their primary structure. The hydrophobic residues and many of the charged residues, particularly the phosphoserine residues, in the caseins are not uniformly distributed along the polypeptide chain. Because the casein monomers cannot sufficiently remove their hydrophobic surfaces from contact with water, the caseins tend to associate with themselves and with each other. In addition, all caseins are able to bind calcium, with the extent of binding being proportional to the number of phosphoserine residues in the molecule. as1-Casein and as2-casein are most sensitive to calcium followed by b-casein, whereas k-casein is insensitive to calcium. k-Casein is capable of stabilising other caseins against calcium-induced precipitation and allows the formation of colloidal sized aggregates (Horne, 1998, 2003). In normal milk, these caseins exist mainly as colloidal particles, called casein micelles, with diameters ranging from 80 to 300 nm (average w150 nm). In addition to caseins, the micelles also contain small amounts of calcium, phosphate, magnesium and citrate, commonly referred to as micellar calcium phosphate (see Fox,

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2009). Most of the k-casein is present on the surface of casein micelles, and the hydrophilic C-terminal part of k-casein protrudes from the micelle surface into the surrounding solvent, giving it a ‘hairy’ appearance. The highly charged flexible ‘hairs’ physically prevent the approach and interactions of hydrophobic regions of the casein molecules (Horne, 1998). Whey proteins can be fractionated into b-lactoglobulin, bovine serum albumin, a-lactalbumin and immunoglobulins. b-Lactoglobulin represents about 50% of the total whey protein in bovine milk, has a molecular weight of 18,000 Da and contains two disulphide bonds and a single free thiol group. In contrast to caseins, the whey proteins possess high levels of secondary, tertiary and, in most cases, quaternary structures. For instance, b-lactoglobulin is built up of two b-sheets, formed from nine strands converging at one end to form a hydrophobic calyx or pocket, and a flanking three-turn a-helix (Edwards, Creamer, & Jameson, 2009; Kinsella & Whitehead, 1989). Because of their nutritional importance and physico-chemical properties, milk proteins are used in a wide range of prepared foods. Various types of caseins and caseinates, whey-protein concentrates and isolates, milk-protein concentrates and isolates, hydrolysed proteins and milk-protein fractions are manufactured by the dairy industry (Mulvihill & Fox, 1989). These products have applications in dairy products, meat products, beverages, baked products and infant foods. The important functional properties of protein products include water binding, emulsification, foaming and whipping, gelation and nutritional properties (Singh, 2010). In this chapter, only emulsification aspects are considered.

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Emulsion-type products, e.g. coffee whiteners, whipped toppings, cream liqueurs, dietary formulatons, liquid nutritional products and medical foods, are an important application of caseinates and whey proteins in the food industry. The basis for the emulsifying ability of milk proteins is the amphiphilicity of their primary structure, which determines their ability to adsorb to the oilewater interface. Major advances have been made in understanding the adsorption process, the composition and structure of adsorbed layers of proteins and how they influence the physical and chemical properties of emulsions (Dickinson, 1998, 1999, 2008; McClements, 1999; Singh & Ye, 2009). In recent years, there has been considerable research activity on understanding how the adsorbed layers and the physical structures of food emulsions influence the rates of lipid digestion (Golding & Wooster, 2010; Le Révérend, Norton, Cox, & Spyropoulos, 2010; McClements, Decker, & Park, 2009; Singh, Ye, & Horne, 2009). This research activity is aimed at developing novel foods that regulate calorie intake, control satiety responses, provide controlled lipid digestion and/or deliver bioactive molecules. This chapter provides an overview of the properties of emulsions formed with milk proteins, and discusses latest advances in understanding and controlling the behaviour of milk-protein-based emulsions under physiological conditions. 2. Properties of milk-protein-stabilised emulsions Extensive studies have been carried out on purified milk proteins, in particular b-casein and b-lactoglobulin. Two major caseins, as1-casein and b-casein, are distinctly amphiphilic and have strong tendencies to adsorb at oilewater interfaces and stabilise oil-in-water emulsions. b-Casein has been shown to adsorb with an extensive hydrophobic region anchored directly at the surface and a hydrophilic region (40e50 residues at the N-terminus) protruding extensively into the aqueous phase. For as1-casein, a loop-like conformation has been predicted as it does not have such a pronounced inequality in the distribution of hydrophobic and hydrophilic residues in its primary structure (Dickinson, 1992). It has been suggested that as1-casein adsorbs to the oilewater interface via peptides towards the middle of its sequence, rather than at the end, as in b-casein, and it may be this that causes the protein to form a thinner adsorbed layer than does b-casein (Dalgleish, 1996; Dickinson, 1992). Because of its relatively higher surface activity, b-casein can displace as1-casein from the oilewater interface (Dickinson, Rolfe, & Dalgleish, 1988; Dickinson & Stainsby, 1988). Because of its amphiphilic nature, b-lactoglobulin readily adsorbs at the oilewater interface, where it partially unfolds. In contrast to caseins, a closely packed, dense and rather thin (2e3 nm at neutral pH) adsorbed layer of b-lactoglobulin is formed (Dalgleish, 1996). Additionally, the partial unfolding of the globular whey-protein structure following adsorption causes exposure of the reactive sulphydryl group, leading to slow polymerisation of the adsorbed protein in the aged adsorbed layer via sulphydryldisulphide interchange (Dickinson & Matsumura, 1991; McClements, Monahan, & Kinsella, 1993). Milk proteins manufactured by the dairy industry are complex mixtures of individual proteins. Moreover, the processes of the isolation and conversion into protein powder modify the native protein structures and consequently influence their functional properties, including emulsification. The most commonly used commercial milk-protein product, i.e. sodium caseinate, is made using the following procedure. Casein is extracted from skim milk via isoelectric precipitation by the addition of mineral acid. The casein curd is then heated to 50e55  C, washed with water, followed by a dewatering process to reduce the moisture content to

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50e60%. Drying of casein curd is most commonly carried out on horizontal vibrating fluid bed driers. Sodium caseinate is usually prepared by solubilising acid casein with NaOH by increasing the pH to 6.7e7.0 to produce a solution of about 23% solids. This solution is then spray dried. In calcium caseinate manufacture, a similar process is used except that Ca(OH)2 is used to neutralise the acid casein curd. The type of commercial heating equipment and the drying conditions affect the extent of proteineprotein interactions, resulting in different levels of aggregation in caseinate products. For instance, it has been shown that casein molecules in aqueous dispersions of commercial sodium caseinate exist as a polydisperse mixture of monomers, complexes and aggregates (Lucey, Srinivasan, Singh, & Munro, 2000). The most important commercial whey-protein products are whey-protein concentrates (WPCs) (up to about 85% protein) and whey-protein isolates (WPIs) (approximately 95% protein). The manufacture of whey-protein products involves combinations of several processes such as ultrafiltration, diafiltration, ion exchange, evaporation and drying (see Singh, 2010). Depending on the process used, whey-protein products tend to contain various levels of lipids, lactose and minerals, as well as different extents of wheyprotein denaturation and aggregation. These variations in composition and denaturation level have a major influence on the emulsifying and other functional properties of whey proteins. Because of their importance in food emulsion applications, several studies have been carried out to understand the adsorption behaviour of caseinates and whey proteins in model oil-in-water emulsions and its relationship to emulsion stability. Both sodium caseinate and whey proteins (particularly WPI) show excellent emulsifying ability; generally a protein surface coverage of 1e2 mg/ m2 is sufficient is produce a fine stable emulsion (Singh, 2005). The composition of the interfacial layer is determined by the quantities and structures of the proteins present at the moment the emulsion is formed (Dalgleish, 1997), although rapid exchanges between adsorbed protein and unadsorbed protein could occur after emulsion formation. Emulsions formed with sodium caseinate show that the casein composition of the adsorbed layer is different from that of the original material. When the ratio of protein to oil is very low (about 1:60), b-casein is preferentially adsorbed at the emulsion droplet surface but, when the total amount of protein is greatly in excess of the amount needed for full surface coverage, as1-casein is adsorbed in preference to the other caseins. At all concentrations, k-casein from sodium caseinate appears to be less readily adsorbed (Srinivasan, Singh, & Munro, 1999a, 1999b). Addition of CaCl2 at above a certain critical concentration to a sodium-caseinate solution before homogenisation increases the surface protein coverage and alters the casein composition of the adsorbed layer; the adsorption of as1-casein at the droplet surface is markedly enhanced whereas the adsorption of b-casein is hardly affected (Ye & Singh, 2001). The effects of calcium and protein concentrations on the composition of the adsorbed layer reflect the state of aggregation of the casein molecules in a sodium-caseinate solution prior to homogenisation. The binding of calcium to the phosphoserine residues of caseins reduces electrostatic repulsions between the protein molecules and increases the potential for intermolecular associations. Similarly, the possibility of interaction between casein aggregates/complexes becomes more pronounced at higher caseinate concentrations. Therefore, under a given set of homogenisation conditions, the composition of the adsorbed layer in emulsions formed with sodium caseinate is determined by the surface activities and flexibilities of the casein aggregates and complexes that exist at the time of emulsification. The underlying physics of the self-association of caseins in complex systems, such as sodium or calcium caseinate, under different environmental conditions (particularly at high concentrations) is not well understood.

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Lactoferrin protein isolated from bovine milk is now available commercially and is used in nutritional beverages and infant formulae. As the isoelectric point of this protein is about pH 8.0, it is possible to prepare oil-in-water emulsions containing cationic emulsion droplets, through adsorption of lactoferrin, over a wider pH range (Ye & Singh, 2006). This provides an opportunity for electrostatic interactions with other milk proteins that are mostly negatively charged around neutral pH. Adsorption layers with different protein compositions can be formed using mixtures of b-lactoglobulin and lactoferrin as emulsifiers or through interactions at the emulsion surface (Ye & Singh, 2007). As long as sufficient protein is present during homogenisation to cover the oil droplets, emulsions stabilised by milk proteins are generally very stable to coalescence over prolonged storage. However, these emulsions are susceptible to different types of flocculation, which in turn leads to enhanced creaming or serum separation. The creaming stability of sodium-caseinate emulsions shows a complex dependence on protein content. At low protein content, the emulsion is destabilised by bridging flocculation because of low surface protein coverage. At a caseinate content of about 2.0 wt%, the emulsions are stable against flocculation, coalescence and creaming for several weeks. However, when the protein content is increased to above 3.0 wt%, unadsorbed caseinate gives rise to depletion flocculation (Dickinson & Golding, 1997; Euston & Hirst, 1999; Srinivasan, Singh, & Munro, 2001). Further increasing the protein content above 6.0 wt% results in very high depletion flocculation, leading to a strong emulsion droplet network that is stable to creaming. Depletion flocculation has not been observed in whey-protein-based emulsions. It appears that depletion flocculation in sodium-caseinate emulsions is caused by the presence of casein aggregates formed from the self-assembly of sodium caseinate in the aqueous phase of the emulsion at concentrations above 2 wt%. The addition of moderate amounts of CaCl2 to emulsions containing excess sodium caseinate has been shown to eliminate depletion flocculation and to improve the creaming stability (Ye & Singh, 2001). This effect appears to be due to an increase in the average size of the casein aggregates in the aqueous phase, resulting in a large increase in the molecular mass of the caseins (Dickinson et al., 2001). In addition, there is a reduction in the concentration of unadsorbed caseinate. The interfacial characteristics and the composition of emulsions can also influence the oxidative stability of oil-in-water emulsions. This is particularly relevant in emulsions containing polyunsaturated oils. The presence of casein or whey proteins in oil-inwater emulsions has been shown to suppress lipid oxidation. This effect is largely dependent on protein concentration, oil droplet size and droplet charge (McClements & Decker, 2000). Positively charged proteins (milk proteins below their isoelectric point or lactoferrin at neutral pH) have been shown to retard the rate of lipid oxidation. Lipid oxidation is, in general, lowered by an increase in the protein concentration, but contrasting results on the effect of droplet size on lipid oxidation have been reported; some workers have reported greater lipid oxidation for small droplets (Gohtani, Sirendi, Yamamoto, Kajikawa, & Yamano, 1999; Lethuaut, Metro, & Genot, 2002) whereas others have found greater lipid oxidation for large droplets (Let, Jacobsen, Sorensen, & Meyer, 2007; Nakaya, Ushio, Matsukawa, Shimizu, & Ohshima, 2005). Our recent work has shown that the oxidative stability of both WPI- and sodium-caseinate-stabilised linoleic acid emulsions with smaller droplet size was greater than that of emulsions with larger droplet size (Ries, Haisman, & Singh, 2010). Caseinate appears to be a better antioxidant than WPI in emulsions with large droplet size. At high protein concentrations, the antioxidative effect of the protein in the emulsions appears to offset the effects of emulsion droplet size and protein type.

The inhibition of lipid oxidation by milk proteins in oil-in-water emulsions has been shown to be mostly due to metal ion chelation and free-radical-scavenging (Benjelloun, Talou, Delmas, & Gaset, 1991). The presence of iron and copper ions in emulsions is closely related to the development of lipid-based radicals. These ions may be present in the lipid phase, in the continuous phase or in the interfacial region. The specific antioxidative feature of caseins has been attributed to their metal-binding ability as a result of their high content of phosphoseryl groups (Cervato, Cazzola, & Cestaro, 1999; Gaucheron, Famelart, & LeGraet, 1996). Binding of prooxidative iron and copper ions reduces their chemical reactivity and removes them from their location in proximity to lipids. Whey proteins have no phosphoseryl residues but other metal-binding mechanisms exist, such as involvement of carboxyl groups (Vegarud, Langsrud, & Svenning, 2000) in aspartic and glutamic acid residues. Free-radical-mediated reactions are a substantial element of lipid autooxidation. The ability of proteins to scavenge free radicals derives from their constituent amino acids, such as cysteine, tyrosine, tryptophan, phenylalanine and histidine. WPI exhibits its freeradical-scavenging activity as a result of free sulphydryl groups (Faraji, McClements, & Decker, 2004; Hu, McClements, & Decker, 2003; Kiokias, Dimakou, & Oreopoulou, 2007; Ostdal, Daneshvar, & Skibsted, 1996; Tong, Sasaki, McClements, & Decker, 2000) that contribute to its antioxidative effects. However, these characteristics, phosphoseryl groups and free sulphydryl groups, do not contribute solely to the total antioxidative capacity of the respective protein. It was shown that the dephosphorylation of as1- and b-caseins only partially suppressed their antioxidative activity in a liposome system (Cervato et al., 1999) and caseinophosphopeptides, high in phosphoseryl groups, did not show the greatest antioxidative capability in a corn oil-in-water emulsion compared with other casein-derived protein fractions (Diaz, Dunn, McClements, & Decker, 2003). When sulphydryl groups of the high molecular weight fraction of whey were blocked with N-ethylmaleimide in aqueous solution, its amidinopropane-hydrochloride-radical-scavenging activity was reduced by only 20% (Tong et al., 2000). The concept of the stabilisation of fish oil emulsions against primary oxidation with the use sodium caseinate and whey proteins as antioxidants and emulsifiers was exploited in a patent by Singh, Zhu, and Ye (2006) to produce a commercial fish emulsion product. Fish oil emulsions (about 30% oil), with an average droplet size of 400 nm, were prepared by a two-stage homogenisation process, using mixtures of caseinate and whey proteins. The mixtures of proteins were processed in such a way that antioxidant and free-radical-scavenging activities were optimised. 3. Interactions of protein-stabilised emulsions under physiological conditions There is currently limited knowledge of the physico-chemical and structural changes that an emulsion may undergo during consumption, i.e. physiological processing. It is considered that a better understanding of the gastrointestinal processing of emulsions would allow the manipulation of physico-chemical and interfacial properties to modulate lipid ingestion, improve the bioavailability of lipid-soluble nutrients and reduce the absorption of saturated fats, cholesterol and trans fats. Over the last 5 years, a number of studies on the in vitro digestion of emulsions have been published. In general, on ingestion, an emulsion is exposed to a relatively narrow range of physical (e.g. shear and temperature) and biochemical (e.g. dilution, pH, pepsin, pancreatin, mucins and bile salts) environments as it passes through the mouth into the stomach and then the intestines. In many cases, proteins have been used to stabilise the emulsions, and

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it would be expected that, for such emulsions, the proteolysis of interfacial protein by pepsin (under gastric conditions) and by trypsin/chymotrypsin would play a critical role in emulsion stability, which could in turn affect lipid digestibility. Only milkprotein-based emulsions are considered in the following discussion. When a liquid food emulsion is consumed, it resides for a few seconds in the mouth, where it is diluted with saliva. There are also changes in pH and temperature (around 37  C) and some shear, as a result of friction between the tongue and the oral mucosa (Bardow, Moe, Nyvad, & Nauntofte, 2000; Glantz, 1997; Malone, Appelqvist, & Norton, 2003). Human saliva is a highly complex biological fluid, consisting mainly of water (w99.5%), various proteins (w0.3%) and small organic compounds and inorganic compounds that contribute to its buffering capacity, and it has a pH of around 6.8 (Humphrey & Williamson, 2001; Schipper, Silletti, & Vingerhoeds, 2007; Zalewska, Zwierz, Zólkowski, & Gindzienski, 2000). The proteins in saliva mainly include salivary enzymes, immunoglobulins, antibacterial proteins, proline-rich proteins, lysozyme, lactoferrin, peptides (such as histatins and cystatins) and highly glycosylated mucin (Amado, Vitorino, Domingues, Lobo, & Duarte, 2005; Humphrey & Williamson, 2001). The behaviour of protein-based emulsions in the mouth is largely driven by the interactions of salivary components with the adsorbed layer on emulsion droplets. Mucin proteins have been shown to play an important role in the flocculation of emulsions because of their negative charge at neutral pH. Negatively charged protein-stabilised emulsions, i.e. b-lactoglobulin emulsions, do not interact with artificial saliva because of strong repulsive forces between anionic mucin and the anionic b-lactoglobulin interfacial layer at neutral pH, but undergo depletion flocculation on the addition of higher concentrations of mucin (1.0 wt%) (Sarkar, Goh, & Singh, 2009). In contrast, positively charged milk-protein emulsions (lactoferrin-based or whey-protein emulsions at low pH) interact with mucin via electrostatic interactions, which may lead to bridging type flocculation under certain conditions (Sarkar, Goh, & Singh, 2009). However, some irreversible flocculation has been observed in protein-based emulsions when mixed with parotid saliva (Vingerhoeds, Blijdenstein, Zoet, & van Aken, 2005), in which the mucin concentration is almost negligible, suggesting that salivary components other than mucin (salivary salts, proline-rich proteins etc.) might also contribute to flocculation. Clearly, there is little knowledge about the influence of each salivary component and physico-chemical factors such as pH, ionic strength and shear on the behaviour of emulsion droplets during consumption. Moreover, there are large variations in the quantity, composition and biochemical properties of saliva for a given individual at different times, as well as between different individuals. After residing for a few seconds in the mouth, the emulsions are swallowed, which involves exposure to intense shear effects in the pharynx, the oesophagus and finally the stomach. In the gastric tract, the emulsion is mixed with the digestive juices at a highly acidic pH (typically between 1 and 3) and containing various minerals and enzymes (both proteolytic and lipolytic). There is also mechanical agitation caused by peristalsis in the stomach (Kalantzi et al., 2006; Weisbrodt, 2001). Protein-stabilised emulsions would be expected to undergo major changes in the stomach because of the possible action of pepsin on the adsorbed layers, the effects of low pH and ionic strength on the droplet charge and the interactions of mucin with interfacial protein. Because of their flexible random structures, caseins are highly susceptible to hydrolysis by pepsin in aqueous solutions (Guo, Fox, Flynn, & Kindstedt, 1995), but whey proteins, particularly b-lactoglobulin because of its highly folded globular conformation, are largely resistant to peptic hydrolysis in their native state (Schmidt & Poll, 1991). Relatively

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little information is available on the pepsin susceptibility of these proteins when they are adsorbed at oilewater interfaces in emulsion systems. Recent work has shown that b-lactoglobulin becomes susceptible to proteolysis by pepsin when present as the interfacial layer in an emulsion (Macierzanka, Sancho, Mills, Rigby, & Mackie, 2009; Sarkar, Goh, Singh, & Singh, 2009). This could be attributed to a possible change in the conformation of the b-lactoglobulin molecules upon adsorption at the oilewater interface, which exposes the peptic cleavage sites for proteolysis. Hydrolysis of the adsorbed protein layer by pepsin is the most important factor in determining the stability of protein-based emulsions under gastric conditions. Hydrolysis of the adsorbed layers by pepsin generally results in a loss of positive charge on the droplet surface as well as a reduction in the thickness of the adsorbed layer. The peptides that remain at the interface are unable to provide sufficient electrostatic repulsions and/or steric effects. As a result, these emulsions are highly susceptible to flocculation and coalescence. This has been demonstrated for lactoferrin- and b-lactoglobulin-stabilised emulsions, which undergo flocculation followed by some degree of coalescence on exposure to simulated gastric conditions (Sarkar, Goh, Singh, & Singh, 2009). In addition to the action of pepsin on protein-stabilised emulsions, the potential of the impact of gastric lipase on emulsion stability must also be considered. Human gastric fluid contains acid-stable gastric lipase, which accounts for 10e30% of the overall lipid digestion of ingested triacylglycerols (Armand, 2007; Armand et al., 1994; Carriere, Barrowman, Verger, & Laugier, 1993). These acid-stable lipases could act on emulsions, resulting in the generation of surface-active fatty acids and monoacylglycerols from the hydrophobic lipid core. These gastric lipase digestion products could displace intact or hydrolysed protein from the interface of protein-stabilised emulsions, affecting the droplet size and consequently the overall stability of the emulsion. However, there are no reported studies on how protein-stabilised emulsions are affected by the action of gastric lipase. The presence of highly glycosylated mucin (molecular weight > 106 kDa), which forms a self-associated gel-like structure at gastric pH, has an important physiological role of protecting the stomach from digesting itself (Bansil & Turner, 2006). There is no information on how emulsion droplets with different interfacial compositions interact with gastric mucin gelled layers and whether or not these interactions influence the pepsin accessibility of the adsorbed protein layers. Our recent work indicates that the addition of a low level of soluble mucin appears to promote the flocculation of b-lactoglobulin-stabilised emulsions, possibly through a bridging mechanism, but does not significantly affect the action of pepsin on the adsorbed protein layer (Sarkar, Goh, & Singh, 2010). After passing through the stomach, food emulsions enter the small intestine and this stimulates contraction of the gall bladder, resulting in the delivery of digestive enzymes and bile into the duodenum. The presence of various lipolytic as well as proteolytic enzymes at different concentrations, inorganic salts and surfaceactive bile acids and the neutralealkaline pH (6.0e7.5) of the upper intestinal fluid together with the remnants of oral and gastric digestion make the overall intestinal system highly complex (McClements et al., 2009; Singh et al., 2009). There are at least three important aspects of small intestinal processing of protein-stabilised emulsions. Firstly, the action of serine proteases on the adsorbed protein layer would be expected to cause substantial alterations. Trypsin predominantly catalyses the peptide chains at the C-terminal of aliphatic amino acids, mainly lysine and arginine, whereas chymotrypsin favours large aromatic residues, such as phenylalanine, tyrosine and tryptophan (Ma, Tang, & Lai, 2005; Olsen, Ong, & Mann, 2004). Over the last few decades, several studies have reported the effects of the hydrolysis

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of protein-stabilised oil-in-water emulsions by trypsin on their physical stability (Kaminogawa, Shimizu, Ametai, Lee, & Yamauchi, 1987; Leaver & Dalgleish, 1990). As expected, extensive hydrolysis of adsorbed protein layers leads to emulsion instability, mainly because of the coalescence of droplets (Agboola & Dalgleish, 1996). However, the behaviour of protein-based emulsions in a more complex environment, simulating intestinal conditions, has not yet been reported. Secondly, we must consider the action of bile salts on the composition of the adsorbed layer. Bile salts, which originate from the liver via the gall bladder, consist mainly of sodium salts of taurocholic, taurodeoxycholic, taurochenodeoxycholic, glycocholic and glycodeoxycholic acids. These surface-active compounds could possibly displace the adsorbed proteins/peptides from the surface of emulsion droplets, thus promoting the accessibility of the active site of lipase to the hydrophobic lipid core (Fave, Coste, & Armand, 2004; Mun, Decker, & McClements, 2005, 2007; Wickham, Garrood, Leney, Wilson, & Fillery-Travis, 1998). The nature of the adsorbed layer appears to dictate the displacement mechanism of bile salts (Singh et al., 2009). For instance, bile salts have been shown to displace whey proteins more readily than caseinates from the interface of emulsion droplets during storage (Mun et al., 2005, 2007). In emulsions stabilised by b-lactoglobulin, displacement of protein was observed even at the lowest concentration of bile salts, but bile salts appeared to bind to the positively charged lactoferrin emulsion droplet with no protein displacement (Sarkar, Horne, & Singh, 2010a). Thirdly, the action of pancreatic lipase on emulsions is critical in determining the final state of emulsion droplets in the small intestine and ultimately the course of lipid digestion. Once the lipid droplets enter the intestine, pancreatic lipase adsorbs to the droplet interface, usually via complexation with colipase and/or bile salts (Bauer, Jakob, & Mosenthin, 2005). Colipase is a short polypeptide of molecular weight w10 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 oilewater interface (Erlanson-Albertsson, 1992). Bile salts may either facilitate or inhibit the activity of pancreatic lipase depending on their concentration (Bauer et al., 2005; Lowe, 2002). At low concentrations, bile salts promote pancreatic lipase activity mainly by allowing the adsorption of lipase to the oilewater interface (Gargouri, Julien, Bois, Verger, & Sarda, 1983; Mun et al., 2007) as well as solubilising and removing the inhibitory reaction products from the oilewater interface. At high concentrations, the bile salts generally compete with lipases for the oilewater interface, inhibiting the point of contact between the non-polar lipid core and the lipase (Gargouri et al., 1983), thus retarding lipase activity. Pancreatic lipase cleaves triacylglycerols to form 2-monoacylglycerols and fatty acids; some of these digestion products are surface active and could potentially displace the initial adsorbed material from the droplet surface (McClements et al., 2009; Singh et al., 2009). Most studies of lipid digestion in protein-based emulsions have used in vitro intestinal models containing pancreatic lipase and bile salts. The hydrolysis of lipids is greatly enhanced in most systems in the presence of bile salts. The extent of lipid hydrolysis was found to be similar in caseinate- and whey-protein-stabilised emulsions, although the oil droplets in the whey-protein emulsions were less stable (Mun et al., 2007). Lactoferrin- and b-lactoglobulin-stabilised emulsions showed a significant degree of coalescence on the addition of physiological concentrations of pancreatin and bile salts (Sarkar, Horne, & Singh, 2010b). For both emulsions, this emulsion destabilisation in simulated intestinal fluid was largely attributed to the lipolysis of the lipid hydrophobic core by the lipase fractions of the pancreatin as well as the proteolysis of the adsorbed protein

layer by the trypsin or other proteolytic fractions present in pancreatin. There is a clear need for further research in this area to have a better understanding of the competitive displacement mechanisms occurring in the intestine, characterisation of the final state of droplets and the products of lipid hydrolysis. Future studies also need to consider how the emulsion droplets (and/or products of lipid hydrolysis) move through and interact with the mucus layer in the small intestine and are transferred from the lumen through the unstirred water layer to the enterocyte for absorption. There are some other issues in this field of protein-stabilised emulsions that need to be addressed. Most of the published studies have attempted to understand the interactions of protein-stabilised emulsions at a particular site (mouth, stomach or duodenum) to gain fundamental insights into the influence of individual physiological variables. However, significant changes that might have occurred during pre-processing in the mouth, followed by sequential gastric digestion, have not been explored when studying lipid digestion in the intestine. This is particularly important in dealing with proteinbased emulsions as they would be destabilised to a large extent in the presence of pepsin and low pH in the stomach. More complete in vitro digestion models to simulate various physiological processes occurring in the mouth, stomach and small intestine need to be developed. Further research in this area is likely to lead to new knowledge that can be used to develop novel food products with health and sensory attributes, aimed at satiety and reducing fat intake.

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