Role of calcium and calcium-binding agents on the lipase digestibility of emulsified lipids using an in vitro digestion model

Food Hydrocolloids 24 (2010) 719e725

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Role of calcium and calcium-binding agents on the lipase digestibility of emulsified lipids using an in vitro digestion model Min Hu*, Yan Li, Eric Andrew Decker, David Julian McClements Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 December 2009 Accepted 18 March 2010

Controlling the digestibility of lipids within the gastrointestinal tract is important for developing food and pharmaceutical products. In vitro digestion methods are commonly used to study the influence of formulation composition and microstructure on lipid digestibility. In this paper, we focus on the impact of calcium and calcium-binding agents on the rate of lipid droplet digestion in corn oil-in-water emulsions monitored using a pH-stat method. The rate of fatty acid production increased with increasing calcium, e.g., the free fatty acids released after 20 min digestion was <12% for 0 mM CaCl2, but >95% for 20 mM CaCl2. The ability of calcium to increase the digestion rate was found for three different emulsifiers used to stabilize the initial lipid droplets: lyso-lecithin, caseinate and b-lactoglobulin. For these three systems, the initial rate of lipid digestion increased in the following order lysolecithin > b-lactoglobulin > caseinate at both 0 and 20 mM CaCl2, but the rate was considerably faster at higher calcium levels for all systems. The addition of EDTA, a calcium chelating agent, to emulsions containing 20 mM CaCl2 caused an appreciable decrease in lipid digestion rate, reducing the amount of free fatty acids produced after 20 min from around 97% to 32% when the EDTA level was increased from 0 to 5 mM. Finally, we examined the impact of two anionic polysaccharides (pectin and alginate) on the rate of lipid digestion in emulsions containing 20 mM CaCl2. High methoxy pectin, which does not bind calcium strongly, did not have a major effect on the rate of digestion, whereas alginate, which does bind calcium strongly, depressed the rate considerably. This study has important implications for designing and testing delivery systems that control lipid digestion. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Emulsions Lipid digestion Lipase Calcium Calcium-binding agents pH-Stat method

1. Introduction There is increasing interest in understanding and controlling the digestibility of emulsified lipids within the human gastrointestinal (GI) tract (McClements, Decker, & Park, 2009; Pafumi et al., 2002; Porter & Charman, 2001; Porter, Trevaskis, & Charman, 2007). This knowledge is being used by the pharmaceutical industry to develop lipid-based delivery systems to deliver active agents to specific locations within the GI tract (Porter & Wasan, 2008; Pouton, 2006). Similar systems are being developed within the food industry to encapsulate, protect and deliver bioactive food components (McClements et al., 2009; McClements, Decker, Park, & Weiss, 2007; Patten, Augustin, Sanguansri, Head, & Abeywardena, 2009). The insights gained from these studies is also being utilized in the development of foods and nutritional supplements designed to increase the digestion and absorption of lipids in individuals with health conditions that impair the normal digestive * Corresponding author. Tel.: þ1 413 545 1010; fax: þ1 413 545 1262. E-mail address: [email protected] (M. Hu). 0268-005X/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2010.03.010

process (Fave, Coste, & Armand, 2004; Pafumi et al., 2002). There is therefore considerable interest in developing analytical tools that can be used to elucidate the major physicochemical factors that impact lipid digestion and absorption under conditions that simulate the human GI tract. These tools range from physicochemical measurements using in vitro digestion tests, to cell culture models, to animal feeding studies, and ultimately to human trials. An analytical tool that is finding increasing utilization within pharmaceutical and food research for the in vitro characterization of lipid digestion is the pH-stat method (Dahan & Hoffman, 2006; Dahan & Hoffman, 2008; Nilsson & Belfrage, 1979). This method is designed to simulate lipid digestion within the small intestine (where the majority of lipid digestion normally occurs), and is based on measurements of the amount of free fatty acids released from lipids (usually triacylglcyerols) after lipase addition at pH values close to neutral. In practice, the concentration of alkali that must be titrated into the reaction chamber to neutralize the free fatty acids produced by lipid digestion, and thereby maintain the pH at the initial value, is recorded versus time. In principle, the pH-stat method is relatively simple and rapid to carry out and

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enables quantitative comparison of different lipid formulations under similar conditions. This technique can therefore be used to examine the impact of different physicochemical factors expected to affect lipid digestion, such as specific food additives (e.g., dietary fibers, minerals), food structure (e.g., particle size distribution, physical state) and lipid droplet interfacial properties (e.g., thickness, charge, composition, cross-linking). At present, our understanding of the factors that impact the rate and extent of lipid digestion of emulsified lipids using the pH-stat method is rather limited, and no standardized conditions have been established that would enable inter-laboratory comparisons of results. In the present study, we focus on the impact of calcium and calcium-binding agents on the rate of lipid droplet digestion in corn oil-in-water emulsions monitored using the pH-stat method. In particular, we focus on the affects of a number of food components that may bind calcium, including EDTA, pectin and alginate. Calcium ions may play a variety of different roles in the lipid digestion process, which impact both the rate and extent of lipid hydrolysis. Lipid digestion of emulsified lipids can be inhibited by the accumulation of long-chain free fatty acids (FFA) at the droplet surfaces, since this restricts the access of the lipase to the triacylglycerols (Fave et al., 2004). Calcium is known to precipitate these accumulated free fatty acids, thereby removing them from the lipid droplet surface and allowing the lipase to access the emulsified lipids (Patton & Carey, 1979; Patton et al., 1985. Calcium ions are able to increase the rate and extent of lipolysis by this mechanism (Armand et al., 1992; Hwang, Lee, Ahn, & Yung, 2009; Zangenberg, Mullertz, Kristensen, & Hovgaard, 2001a, 2001b). On the other hand, studies have shown that precipitates formed between calcium and long-chain saturated FFA may be absorbed less readily, thereby reducing lipid bioavailability (Karupaiah & Sundram, 2007; Lorenzen et al., 2007; Scholz-Ahrens & Schrezenmeir, 2006). Calcium has also been shown to play an important role in the activity of pancreatic lipase, acting as a cofactor required for activity (Kimura, Futami, Tarui, & Shinomiya, 1982; Mukherjee, 2003; Whayne & Felts, 1971a; 1971b). The concentration of free calcium ions in the small intestine depends on the presence of any other components present that are capable of binding calcium. These components may be naturally present within the human body such as mucins or specific proteins, or they may be present within ingested foods such as chelating agents (EDTA, phosphates) and biopolymers (proteins, peptides and polysaccharides) (Braccini & Perez, 2001; Kim & Lim, 2004; Perry, Cygan, & Mitchell, 2006; Rui, 2009). The information gained from this study may be useful in the development of standardized in vitro protocols for testing emulsified lipids. In addition, an understanding of the impact of calcium and calcium-binding agents on lipid digestion may prove useful for the design of lipid-based delivery systems that control the digestion and absorption of lipids within the GI tract. 2. Materials and methods 2.1. Materials Powdered lyso-lecithin (Solec 8160) was obtained from the Solae Company (St Louis, MO). Powdered b-lactoglobulin (b-Lg) was provided by Davisco Foods International Inc. (BioPURE, Le Sueur, MN). As stated by the manufacturer, this product contains less than 130 mg of calcium per 100 g. Powdered sodium caseinate (Lot No. 4098) was provided by American Casein Company (Burlington, New Jersey). High-methoxyl pectin (TIC Pretested Pectin 1400) with a degree of esterification of 69e77% was obtained from TIC Gums, Inc. (White Marsh, MD). Alginic acid (sodium salt, Batch 180947) was purchased from SigmaeAldrich (St. Louis, MO).

Lipase from porcine pancreas, Type II (L3126) and bile extract (porcine, B8613) were purchased from SigmaeAldrich (St. Louis, MO). Lipase from porcine pancreas, Type II contains amylase and protease except triacylglycerol acylhydrolase and triacylglycerol lipase, and the activity of lipase from porcine pancreas, Type II is 100e400 units/mg protein using olive oil (30 min incubation) and 30e90 units/mg protein (using triacetin). The composition of bile extract (BS) has previously been analyzed as: total bile salt content ¼ 49 wt%; with 10e15% glycodeoxycholic acid, 3e9% taurodeoxycholic acid, 0.5e7% deoxycholic acid, 1e5% hyodeoxycholic acid, and 0.5e2% cholic acid; 5 wt% phosphatidyl choline (PC); Ca2þ < 0.06 wt%; CMC of bile extract 0.07  0.04 mM; the mole ratio of BS to PC is around 15 to 1. Corn oil was purchased from a local supermarket and used without further purification. Corn oil contains  99% triacylglyceride, with proportions of approximately 58.7% polyunsaturated fatty acid, 28.7% monounsaturated fatty acid, and 12.6% saturated fatty acid (Ostlund, Racette, Okeke, & Stenson, 2002). Calcium chloride (CaCl2$2H2O) was obtained from Fisher Scientific. Analytical-grade hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from SigmaeAldrich (St. Louis, MO). Analytical-grade Na2HPO4 and NaH2PO4 were purchased from SigmaeAldrich. Purified water from a laboratory water purification system (Nanopure Infinity, Barnstead International) was used for preparation of all solutions. Solution Preparation A 5 mM phosphate buffer solution (pH 7) was prepared by dispersing weighed amounts of Na2HPO4 and NaH2PO4 in water. Emulsifier solutions were prepared by dispersing 1.0 wt% of powdered emulsifier (lyso-lecithin, b-Lg or caseinate) into 5 mM phosphate buffer solution. Emulsifier solutions were stirred for at least 3 h and the pH was then adjusted to 7.0 using HCl or NaOH. Stock alginate and pectin solutions were prepared by dispersing appropriate amounts of powdered ingredients into 5 mM phosphate buffer solution (pH 7.0) and stirring overnight to ensure complete dispersion. The pH of the solutions was then adjusted back to 7.0 if required. 2.1.1. Emulsion preparation Each emulsion was prepared by homogenizing corn oil with aqueous emulsifier (lyso-lecithin, b-Lg or caseinate) solution using a high-speed blender (Tissue Tearor, Biospec Products, Inc., Bartlesville, OK) for 2 min, followed by six passes at 9000 psi through a microfluidizer (Newton, Massachusetts, Model 1101). EDTA, pectin, and alginate solutions were mixed with some of the emulsions prior to starting the in vitro lipid digestion tests. 2.1.2. Particle size and z-potential measurements Particle size and z-potential were determined using a commercial dynamic light scattering and micro-electrophoresis device (Nano-ZS, Malvern Instruments, Worcestershire, UK). The samples were diluted 100 times in buffer solution at room temperature before measurement. The particle size data were reported as the Z-average mean diameter, while the particle charge data were reported as the z-potential. The particle size distribution (PSD) of the samples before and after adjusting to pH 7 was measured using a static light scattering instrument (Mastersizer S, Malvern Instruments). A few drops of emulsions were dispersed in approximately 125 mL buffer in the same chamber with agitation until approximately 11e14% obscuration was obtained. Measurements were conducted at ambient temperature (22  C). 2.1.3. A dynamic in vitro digestion model The in vitro digestion model used in this study was a modification of those described previously (Mun, Decker, Park, Weiss, &

M. Hu et al. / Food Hydrocolloids 24 (2010) 719e725

 30.0 mL of emulsion containing 0.5 wt% corn oil and 0.1 wt% emulsifier was placed into a glass beaker that was placed in a water bath at 37.0  C for 10 min, and then adjusted to pH 7.0 using NaOH or HCl solutions.  4.0 mL of bile extract solution (187.5 mg bile extract dissolved in phosphate buffer, pH 7.0) and 1.0 mL of CaCl2 solution (110 mg CaCl2 dissolved in phosphate buffer, pH 7) were then added to the emulsion under stirring (speed 4) and then the system was adjusted back to pH 7.0 if required.  2.5 mL of freshly prepared lipase suspension (60 mg lipase powder dispersed in phosphate buffer, pH 7) was added to the above mixture. The pH-stat automatic titration unit (Metrohm, USA Inc.) was then used to automatically monitor the pH and maintain it at pH 7.0 by titrating appropriate amounts of NaOH solution (0.05 M). The volume of NaOH added to the emulsion was recorded and used to calculate the concentration of free fatty acids generated by lipolysis. The final standard composition of the reaction vessel (37.5 mL) was therefore: 0.4 wt% corn oil, 0.08 wt% emulsifier, 20 mM CaCl2, 5 mg/mL bile salts, and 1.6 mg/mL lipase. In some experiments different levels of CaCl2, lipase or bile salt were used. In these cases, the amount of these substances added to the appropriate buffer solution was varied, but the total volume of the buffer solution was kept constant, which meant that the final volume of the test samples was always 37.5 mL. For the EDTA tests, a certain amount of EDTA and CaCl2 was dissolved in phosphate buffer before adding to the digestion mixture. The final lipid concentration in the reaction vessel (0.4 wt%) was selected because it represents a typical value that might be found in the small intestine after an ingested emulsion was diluted with digestive juices in the mouth, stomach and small intestine (Hur, Decker, & McClements, 2009). The percentage of free fatty acids released was calculated from the number of moles of NaOH required to neutralize the FFA divided by the number of moles of FFA that could be produced from the triacylglycerols if they were all digested (assuming 2 FFA produced per 1 triacylglycerol molecule):

VNaOH  mNaOH  MLipid wLipid  2

a 100

80

2.1.4. Data analysis Differences among the treatments were evaluated by one-way ANOVA with post hoc mean ranking test using Tukey by SPSS v 16.0 (SPSS Inc., Chicago, IL). Mean values with statistical difference of P < 0.05 were considered as significant. 3. Results and discussion 3.1. Effect of lipase concentration on lipid digestion Initially, we examined the influence of lipase concentration on the rate of lipid digestion in corn oil-in-water emulsions stabilized by b-Lg. The purpose of these experiments was to identify an

Lipase (mg/mL) 0.8

40

1.2 1.6 2 2.4

!

Here VNaOH is the volume of sodium hydroxide required to neutralize the FFA produced (in mL), mNaOH is the molarity of the sodium hydroxide solution used (in M), wLipid is the total weight of oil initially present in the reaction vessel (0.15 g), and MLipid is the molecular weight of the oil (assumed to be 900 g mol1). Blanks were carried out in the absence of oil and subtracted from the reported values.

60

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%FFA ¼ 100 

appropriate lipase concentration to use in the in vitro digestion studies of the effect of calcium and calcium-binding agents. The initial mean particle diameter of the droplets in these emulsions was 178  2 nm and their initial z-potential was 50  1 mV (5 mM phosphate buffer, pH 7.0). The final bile salt amount (5 mg/mL) and calcium concentration (20 mM CaCl2) in the reaction vessel were held constant (37.5 mL, pH 7.0, 37  C). As expected, the initial rate of lipid digestion increased as the lipase concentration in the reaction vessel increased (Fig. 1a). The impact of lipase concentration on digestion rates is highlighted in Fig. 1b, which shows the time required to reach 50% of complete triacylglycerol digestion. At lower lipase concentrations (0.8 and 1.2 mg/mL), a lag time was observed during which the lipid digestion rate remained relatively low, followed by a steep increase in fatty acid production at longer times. This phenomenon may be attributed to the finite time required for lipase to adsorb to the lipid droplet surfaces and gain access to the triacylglyercols within the droplet core (Macierzanka, Sancho, Mills, Rigby, & Mackie, 2009). Presumably at higher lipase concentrations the adsorption and displacement processes occurred much more rapidly so that digestion could begin almost immediately. Previous studies have shown that bile salts may be required in order to facilitate the displacement of protein emulsifiers by lipase (Macierzanka et al.,

% FAA released

McClements, 2006; Zangenberg et al., 2001a). The standard procedure for the in vitro digestion model was as fololows:

721

8

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c

c

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2 0

0.8

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Lipase Concentration in Reaction Vessel (mg/mL) Fig. 1. Influence of lipase concentration on digestion rate for in vitro digestion of corn oil-in-water emulsions stabilized with b-Lg (20 mM CaCl2, pH 7, 37  C). (b) Influence of lipase concentration on half-time for enzymatic hydrolysis in in vitro digestion of corn oil-in-water emulsions stabilized with b-Lg (20 mM CaCl2, pH 7, 37  C).

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2009). In the remainder of the experiments we therefore selected a final lipase concentration of 1.6 mg/mL in the reaction vessel. In reality, ingested lipid droplets have to pass through the mouth and stomach before they reach the small intestine, where they may interact with other digestive enzymes (e.g., gastric lipase and proteases) and other types of biological molecules (e.g., mucin). These interactions have been ignored in this study, since we wanted to focus on the physicochemical processes that occur within the small intestine, where the majority of lipid digestion normally occurs. Nevertheless, in a more complete in vitro digestion model it is important to simulate the various stages of the gastrointestinal tract to obtain a more accurate picture of the complex events occurring (Hur et al., 2009; Versantvoort, Oomen, Van de Kamp, Rompelberg, & Sips, 2005). 3.2. Effect of calcium concentration on lipid digestion The influence of calcium concentration on the rate and extent of lipid digestion was examined in corn oil-in-water emulsions stabilized by b-Lg (d ¼ 178  2 nm, z ¼ 50  1 mV). The lipase amount (1.6 mg/mL) and bile salt concentration (5 mg/mL) in the reaction vessel (37.5 mL, pH 7, 37  C) were kept constant, so that we could focus on the influence of added calcium. Visual observation of the emulsions indicated that they underwent extensive aggregation at higher calcium levels (>10 mM) as evidenced by the formation of a clear creamed layer after 24 h storage. The rate and extent of free fatty acid production increased as the initial calcium concentration in the reaction vessel was increased (Fig. 2). For example, the free fatty acids released after 20 min digestion was <12% for 0 mM CaCl2, but was >95% for 20 mM CaCl2. These measurements clearly demonstrate that calcium ions have a major impact on the lipid digestion process, and are in agreement with previous studies of the influence of Ca2þ on in vitro digestion models (Zangenberg et al., 2001a). Calcium ions may increase the rate and extent of lipid digestion through a number of physicochemical mechanisms. A certain level of calcium is required as a cofactor to activate pancreatic lipase (Kimura et al., 1982; Whayne & Felts, 1971b). Consequently, the slow rates of FFA production at low calcium levels may have been at least partly due to the fact that the enzyme was not in its most active form. Alternatively, calcium ions can increase the rate of lipid digestion by binding and precipitating the long-chain fatty acids produced during the lipolysis of emulsified triacylglyercols (Armand et al., 1992; Hwang et al.,

0 mM

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Time (min) Fig. 2. Effect of CaCl2 levels on the in vitro digestion of corn oil-in-water emulsions stabilized with b-Lg.

2009; Zangenberg et al., 2001a; 2001b). If these fatty acids are not removed from the lipid droplet surfaces, then they accumulate there, so that the ability of the lipase to contact and hydrolyze the triacylglycerols is inhibited (Fave et al., 2004). Previous experiments suggest that the precipitation of fatty acids from the droplet surfaces is the most important mechanism (MacGregor et al., 1997). These workers studied the effects of calcium concentration on the rate of lipid digestion in emulsified short and long-chain triglycerides. They found that the rate of FFA production was independent of calcium concentration for short chain triglycerides because the FFA generated were water-dispersible and easily moved away from the droplet surfaces. On the other hand, the rate of FFA production increased with increasing calcium concentration for long-chain triglycerides because the FFA produced were not readily waterdispersible and so had to be removed from the droplet surfaces. It should also be stressed that calcium levels could influence other system characteristics that might affect lipid digestion, such as promoting droplet flocculation, biopolymer aggregation or bile salt precipitation. Finally, we should mention that there may have been some level of endogenous calcium present in the emulsions, due to the presence of calcium impurities in the various ingredients and reagents added to the reaction cell, such as emulsifiers, lipase, and bile extract. Nevertheless, these levels would be expected to be relatively low. 3.3. Effect of emulsifier type on lipid digestion The influence of calcium concentration (0 or 20 mM CaCl2) on the rate of lipid digestion in emulsions containing oil droplets stabilized by three different kinds of emulsifier was also examined: lyso-lecithin, b-Lg, and caseinate (Fig. 3a, b). The mean particle diameters of the droplets in the emulsions stabilized by lyso-lecithin, b-lactoglobulin, and caseinate were, respectively, 210  5, 178  2, and 227  2 nm, while their z-potentials were 82  2, 50  1 and 66  1 mV. All three emulsions therefore initially contained highly anionic droplets and had particle sizes (and therefore specific surface areas) within the same order of magnitude (30%). However, after CaCl2 was respectively added into each emulsion (final concentration of CaCl2 was 20 mM), the mean particle size diameters of the droplets in the emulsions dramatically increased, with 7198  1428 nm for lyso-lecithin stabilized emulsion, 22,400  12,800 nm for b-lactoglobulin stabilized emulsion and 23,840  17,630 nm for caseinate stabilized emulsion, while their z-potentials decreased to 29  1, 13  1 and 1.2  1 mV. Meanwhile, the aggregation and separation occurred for each of the emulsions. It was not possible to determine the size and charge of the droplets during and after digestion because of the structural and compositional complexity of the in vitro digestion fluids. The initial rate of lipid digestion decreased in the following order: lyso-lecithin > caseinate > b-lactoglobulin for 0 mM CaCl2 (Fig. 3a); lyso-lecithin > b-lactoglobulin > caseinate for 20 mM CaCl2 (Fig. 3b). For all three emulsifiers, the initial rate of lipid digestion was considerably faster at the higher calcium level. The dependence of the initial digestion rates on emulsifier type may be attributed to the ability of the original emulsifier coating to restrict the access of lipase to the oil within the droplets. Our results suggest that the two proteins were more effective at inhibiting the initial stages of lipid digestion than lecithin, which is in agreement with earlier studies (Mun, Decker, & McClements, 2007). It may be possible for lipase to directly adsorb to lecithin-coated droplet surfaces and/or for bile salts to more easily displace lecithin from oil droplet surfaces. On the other hand, protein coatings must first be disrupted or displaced from the oil droplet surfaces before lipase can adsorb and digest the lipids. The reason that caseinate-coated

M. Hu et al. / Food Hydrocolloids 24 (2010) 719e725

20 Lyso-lecithin Caseinate B-Lg

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complex digestion media, which could lead to differences in digestion rates. The fact that calcium increased the digestion rate in all systems can be attributed to the fact that the original emulsifier coating became displaced from the oil droplet surfaces during the in vitro digestion process. The reaction vessel contained bile salts and phospholipids, which are highly surface-active substances known to displace proteins and lecithin from oilewater interfaces (Macierzanka et al., 2009; Maldonado-Valderrama et al., 2008; Mun et al., 2007). Thus, once the lipid digestion process begins, the oil droplets are likely to be surrounded by a compositionally and structurally complex mixture of surface active molecules regardless of the initial emulsifier used (McClements et al., 2009). This mixture will contain lipid digestion products (free fatty acids and monoacylglycerols), phospholipids and bile salts, with perhaps some of the initial emulsifier remaining. It is also possible that desorbed emulsifier molecules impacted the lipid digestion process, e.g., by binding enzymes, bile salts, phospholipids, or mineral ions. It is clear that the lipid digestion process is extremely complex, and that further studies are needed to fully elucidate the various physicochemical mechanisms involved. 3.4. Effect of EDTA on the lipid digestion

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Time (min) Fig. 3. (a) Impact of emulsifier type on fatty acids released from corn oil-in-water emulsions in an in vitro digestion model (pH 7, 37  C, 0 mM CaCl2). (b) Impact of emulsifier type on fatty acids released from corn oil-in-water emulsions in an in vitro digestion model (pH 7, 37  C, 20 mM CaCl2).

droplets digested more slowly at higher calcium levels than b-Lgcoated droplets, and vice versa, may have been because calcium restricted the access of lipase to the lipid droplets in the caseincoated system. There are a number of physicochemical mechanisms that might account for this effect: (i) calcium promoted more droplet flocculation in the casein system; (ii) calcium made the proteins at the interface more difficult to displace and disrupt; and (iii) calcium formed an interfacial complex with casein that was difficult for the lipase to penetrate. As mentioned earlier, in reality lipid droplets must pass through the mouth and stomach before reaching the small intestine, and some digestion and displacement of the adsorbed protein coatings are likely to occur because of the presence of digestive enzymes and surface active components in these regions of the GI tract (Macierzanka et al., 2009; McClements et al., 2009). Consequently, the composition and properties of the interfacial layer surrounding the oil droplets may be very different from the original values. A number of previous studies have shown that calcium ions are highly effective and promoting droplet flocculation in oil-in-water emulsions stabilized by anionic emulsifiers, such as lecithin, b-lactoglobulin and caseinate (Gu, Regnier, & McClements, 2005; Ogawa, Decker, & McClements, 2003; Surh & McClements, 2008; Wooster & Augustin, 2006). The nature of the flocs formed (e.g., their size, porosity and bond strength) may vary for emulsions stabilized by different emulsifiers, particularly in a compositionally

We hypothesized that if calcium plays a crucial role in the lipid digestion process, then the rate of lipid digestion may be influenced by any food components that can bind calcium. EDTA is a foodgrade chelating agent that is capable of strongly sequestering divalent mineral ions, such as Ca2þ, in a 1:1 molar ratio (Haahr & Jacobsen, 2008). We therefore examined the influence of EDTA concentration on the rate of lipid digestion in corn oil-in-water emulsions stabilized by b-Lg (d ¼ 178  2 nm, z ¼ 50  1 mV). The in vitro digestion reaction vessel contained a fixed Ca2þ ion concentration of 20 mM. The addition of EDTA to the reaction vessel clearly had a pronounced impact on the rate of lipid digestion in the emulsions (Fig. 4). When the EDTA concentration was increased there was a pronounced decrease in the lipid digestion rate, e.g., the %FFA produced after 20 min digestion was 98, 80 and 32% for 0, 1 and 5 mM EDTA, respectively. This decrease may be attributed to the ability of the EDTA to bind to some of the calcium ions, and thereby preventing them from either activating the lipase and/or precipitating the long-chain fatty acids at the droplet surface. One would expect that the addition of 5 mM EDTA would only bind 5 mM Ca2þ ions, thereby leaving 15 mM Ca2þ ions free. However, we found that

100 0 mM 1 mM 5 mM

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Time (min) Fig. 4. Effect of EDTA level on fatty acids released from corn oil-in-water emulsions stabilized by b-Lg in an in vitro digestion model (pH 7, 37  C, 20 mM CaCl2).

M. Hu et al. / Food Hydrocolloids 24 (2010) 719e725

the initial lipid digestion rate was considerably less in the system containing 20 mM Ca2þ and 5 mM EDTA (Fig. 4) than in the one containing 10 mM Ca2þ and no EDTA (Fig. 2). For example, only 32% of the free fatty acids were released after 20 min digestion in the system containing 20 mM Ca2þ and 5 mM EDTA (Fig. 4), whereas over 71% free fatty acids were released in the system containing 10 mM Ca2þ (Fig. 2). The physicochemical origin of this apparent discrepancy is currently unknown. It suggests that the EDTA is able to have some other effects apart from just binding calcium ions, e.g., it may be able to bind multivalent cations that are essential for lipase activity.

a

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3.5. Effect of anionic polysaccharides on lipid digestion

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The studies with EDTA suggest that if a calcium-binding substance can maintain its activity until it reaches the small intestine, where the majority of lipid digestion occurs, then it may be able to decrease the lipid digestion rate. Some dietary fibers (e.g., alginate) can bind calcium strongly and are resistant to digestion in the stomach and small intestine, and therefore they may be able to play this role. We therefore examined the impact of two ionic polysaccharides on lipid digestion in corn oil-in-water emulsions stabilized by b-Lg: alginate and highmethoxyl (HM) pectin. These two polysaccharides were chosen because of differences in their electrical characteristics and calcium-binding properties (Cui, 2005). Alginate is an anionic linear polysaccharide with a high linear charge density that binds calcium ions strongly (Fang et al., 2007). HM pectin has an anionic linear backbone with a relatively low linear charge density, with some neutral side groups (“hairy regions”) along the chain, and does not bind calcium strongly (Fang et al., 2008). The presence of relatively small amounts of alginate (0.05 or 0.1 wt%) in the reaction vessel caused a large decrease in the rate and extent of lipid digestion in the emulsions (Fig. 5a). On the other hand, the presence of similar quantities of HM pectin had little effect on the rate or extent of lipid digestion (Fig. 5b). Previous studies have shown that alginates are capable of binding calcium ions much more strongly than HM pectins (Donati, Benegas, & Paoletti, 2006; Fang et al., 2008, 2007). This effect has been attributed to the differences in the arrangement of anionic groups along the polysaccharide backbone: alginates normally have a blocky pattern of guluronate groups, whereas HM-pectins have a random pattern of galacturonate groups. As a consequence, alginates bind calcium ions to form mono-complexes, then egg-box dimers, and then egg-box multimers, whereas HM-pectins only bind calcium ions to form mono-complexes (Fang et al., 2008, 2007). The observed decrease in lipid digestion rate in the system containing alginate may therefore be attributed to the ability of the alginate molecules to strongly bind calcium ions in the reaction vessel, thus preventing them from precipitating the free fatty acids produced at the oil droplet surfaces. Nevertheless, the molar concentration of monosaccharide groups within the reaction vessel coming from the alginate molecules is relatively small, being about 3 or 6 mM for 0.05 and 0.1 wt% alginate, respectively. Only a fraction of the monosaccharide groups along the alginate chains carry a negative charge (the guluronate groups), and so the anionic group concentration of the alginate is relatively low compared to the total calcium ion concentration present (20 mM). This suggests that the alginate molecules may also have been able to influence the lipid digestion process by other physicochemical mechanisms, e.g., by forming a coating around the lipid droplets that inhibited the enzyme from reaching the surface, by binding lipase/co-lipase and preventing it reaching the droplet surface, or by binding multivalent cations required for lipase activity. Further work is required to identify the reason why alginate is so much more effective at

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Time (min) Fig. 5. Impact of alginate concentration on fatty acid release from corn oil-in-water emulsions stabilized with b-Lg in an in vitro digestion model (pH 7, 37  C, 20 mM CaCl2). (b) Impact of HM-pectin level on fatty acid release from corn oil-in-water emulsions stabilized with b-Lg in an in vitro digestion model (pH 7, 37  C, 20 mM CaCl2).

retarding lipid digestion than HM-pectin. Interestingly, the concentration of charged groups in the alginate capable of inhibiting lipid digestion was fairly similar to the concentration of EDTA required. 4. Conclusions This study has shown that calcium ions play an important role in the rate and extent of lipid digestion in oil-in-water emulsions. This effect was observed in emulsions stabilized by the three different emulsifiers used in this study: lecithin, b-lactoglobulin and caseinate. The addition of components known to strongly bind calcium ions, such as EDTA or alginate, was found to substantially decrease the lipid digestion rate. These results have important implications for understanding and controlling the digestion of lipids in the human diet. It may be possible to increase the rate of lipid digestion by ensuring that there is a sufficiently high quantity of free calcium ions present in the food, which may be important for humans with compromised digestive systems. On the other hand, it may be possible to decrease the rate of lipid digestion by incorporating components that bind calcium strongly in fatty foods,

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