Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: Proposal for a standardised pH-stat method

Food Chemistry 126 (2011) 498–505

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: Proposal for a standardised pH-stat method Yan Li, Min Hu, 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

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Article history: Received 9 February 2010 Received in revised form 15 October 2010 Accepted 3 November 2010

Keywords: Emulsions Lipid digestion Lipase Calcium Calcium binding pH stat

a b s t r a c t The control of lipid digestibility within the human gastrointestinal tract is important for the development of many functional food and pharmaceutical products. The influence of product composition and microstructure on lipid digestibility is typically studied using in vitro digestion methods. This article focuses on the impact of various experimental factors on lipid digestion in oil-in-water emulsions, using a pH-stat method that simulates the small intestine. The rate and extent of lipid digestion were found to increase with: increasing lipase (from 0 to 4.8 mg/ml), decreasing bile extract (from 20 to 0 mg/ml), increasing CaCl2 (from 0 to 20 mM), decreasing lipid (from 2.5 to 0.1 wt.%); decreasing droplet diameter (from d = 800 to 200 nm), and decreasing fatty acid molecular weight (medium chain triglycerides versus corn oil). These affects are interpreted in terms of the surface area of lipid exposed to the aqueous phase, and factors affecting the accumulation of reaction products (fatty acids) at the oil–water interface. Based on our own and others’ work, we propose a standardised in vitro digestion model to test emulsified lipids, based on pH-stat titration. This study has important implications for designing and testing delivery systems that control lipid digestion using the pH-stat method. Ó 2010 Elsevier Ltd. All rights reserved.

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; Singh, Ye, & Horne, 2009). This knowledge is being used by the pharmaceutical industry to develop lipid-based delivery systems that deliver drugs to specific locations within the GI tract (Porter & Wasan, 2008; Pouton, 2006). Similar systems are now being developed by the food industry to encapsulate, protect and deliver bioactive food components (McClements, Decker, Park, & Weiss, 2007; McClements et al., 2009; Patten, Augustin, Sanguansri, Head, & Abeywardena, 2009; Singh et al., 2009). Many of these delivery systems are being designed to modulate the digestion and release of bioactive food components within the GI tract. Consequently, there is considerable interest in developing analytical tools to establish the major physicochemical and structural factors that impact lipid digestion and absorption under conditions that simulate the human GI tract. These tools range from in vitro digestion tests, to cell culture models, to animal feeding studies, and ultimately to human trials (McClements et al., 2009; Singh et al., 2009). An analytical tool that is finding increasing

⇑ Corresponding author. Tel.: +1 413 545 1019; fax: +1 413 545 1262. E-mail address: [email protected] (D.J. McClements). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.11.027

utilisation within pharmaceutical and food research for the in vitro characterisation of lipid digestion is the pH-stat method (Armand et al., 1992; Dahan & Hoffman, 2006, 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 (FFA) released from lipids (usually triacylglcyerols) after lipase addition at pH values close to neutral. The sample to be analysed is placed in a reaction chamber containing appropriate concentrations of digestive components, such as lipase, bile salts, and minerals. The concentration of alkali that must be titrated into the reaction cell to neutralise any 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 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 lipid composition (e.g., fatty acid profile, lipid type), food additives (e.g., proteins, starches, fibres, minerals), droplet characteristics (e.g., particle size distribution, physical state), and 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 standardised conditions have not yet been established that enable inter-laboratory comparisons of

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results. In a recent study, we focused on the impact of calcium and calcium-binding agents (EDTA, pectin, alginate) on the rate of lipid droplet digestion in corn oil-in-water emulsions using the pH-stat method (Hu, Li, Decker, & McClements, 2010). The objective of the present study is to establish the impact of simulated digestive fluid composition in the pH-stat reaction vessel (lipase, bile, calcium) and lipid droplet properties (size, concentration, composition) on the rate and extent of lipid digestion in oil-in-water emulsions. The information gained from this study will be useful for the development of standardised in vitro protocols for testing emulsified lipids. In addition, an understanding of the impact of various intrinsic and extrinsic factors 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.

emulsions were diluted with phosphate buffer (pH 7.0) to obtain the same final oil concentration (0.5 wt.%), but different NaCl, CaCl2 and bile concentrations, respectively. Samples were then stored overnight prior to analysis.

2. Materials and methods

2.6. In vitro digestion model (pH-stat)

2.1. Materials

The in vitro digestion model used in this study was a modification of those described previously (Mun, Decker, Park, Weiss, & McClements, 2006; Zangenberg, Mullertz, Kristensen, & Hovgaard, 2001a). The basic procedure used was: (i) 30.0 ml of emulsion containing 0.5% oil 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 using NaOH or HCl solutions; (ii) 5.0 ml of bile extract solution (187.5 mg of bile extract dissolved in phosphate buffer, pH 7.0, 37.0 °C) and 1.0 ml of CaCl2 solution (188 mM CaCl2 in double-distilled water, 37.0 °C) were added to the emulsion under stirring and the system was adjusted back to pH 7.0 if required; (iii) 1.5 ml of freshly prepared lipase suspension (60 mg lipase powder dispersed in phosphate buffer, pH 7, 37.0 °C) were added to the above mixture. The final composition of the sample in the reaction cell was therefore 100 mg of lipid, 5 mg/ml of bile extract, 1.6 mg/ml of lipase, 5 mM CaCl2 unless otherwise stated. A 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 concentrations of NaOH solution. The volume of NaOH added to the emulsion was recorded, and used to calculate the concentration of free fatty acids generated by lipolysis. To analyse the effects of the composition of the simulated digestion medium on the rate of lipid digestion, we independently varied the concentrations of CaCl2, bile extract, and lipase in the reaction cell, while keeping the other variables constant. To analyse the effects of oil droplet properties (size, concentration and composition), we used the following final composition of the simulated digestion medium in the reaction cell: 5 mM CaCl2; 150 mM NaCl; 20 mg/ml of bile; and, 2.4 mg/ml of lipase. Unless otherwise stated, the reaction cell contained 100 mg of lipid, and the titration solution was 0.1 M NaOH solution.

Powdered lactoglobulin (b-Lg) was obtained from Davisco Foods International (Lot # JE 002-8-415, Le Sueur, MN). Corn oil was purchased from a local supermarket and was used without further purification. Medium chain triglyceride (MIGLYOL 812N) (MCT) was purchased from Sasol Germany Gmbh, Witten, Germany. Lipase from porcine pancreas, Type II (L3126), and bile extract (porcine, B8613) were purchased from Sigma–Aldrich (St. Louis, MO). The supplier reported that the activity of pancreatic lipase was 100–400 units/mg protein, using olive oil. Here, an activity unit is defined as 1.0 microequivalent of fatty acids released from olive oil after 1 h of incubation at pH 7.7 at 37 °C. Calcium chloride (CaCl2
2H2 O) and sodium chloride (NaCl) were obtained from Fisher Scientific. Analytical grade hydrochloric acid (HCl) and sodium hydroxide (NaOH) were purchased from Sigma. Purified water from a Nanopure water system (Nanopure Infinity, Barnstead International, Dubuque, IA) was used for the preparation of all solutions. 2.2. Solution preparation Emulsifier solution was prepared by dispersing 1.0 wt.% of powdered b-Lg into 5 mM phosphate buffer solution and stirring for at least 2 h. The protein solution was kept overnight at 4 °C to ensure complete hydration. NaCl and CaCl2 solutions were prepared by dissolving a certain amount of powder in phosphate buffer and double-distilled water, respectively. 2.3. Emulsion preparation A stock emulsion was prepared by homogenising 10 wt.% oil with 90 wt.% aqueous emulsifier solution (1.0 wt.% b-Lg, pH 7.0), using a high-speed blender for 2 min (M133/1281-0, Biospec Products, Inc., ESGC, Switzerland) followed by passage through a microfluidizer (Microfluidics M-110Y, F20Y 75 lm interaction chamber, Newton, MA). Three emulsions, with different mean droplet sizes, were prepared: (i) only the high-speed blender was used (coarse emulsion, CE); (ii) a blender and a microfluidizer at 4000 psi for 3 passes was used (medium emulsion, ME); (iii) a blender and a microfluidizer at 9000 for 5 passes was used (fine emulsion, FE). Emulsions containing different lipids were prepared by using different oil phases (corn oil or medium chain triglycerides). 2.4. Effect of NaCl, CaCl2, and bile on emulsion stability In some experiments, we examined the influence of the in vitro digestion reaction vessel contents on emulsion stability. Stock

2.5. Particle size and f-potential measurements Particle size distribution and f-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 appropriate buffer solution at room temperature before measurement. The particle size data are reported as the Z-average mean diameter, while the particle charge data are reported as the f-potential.

2.7. Data analysis All experiments were performed at least twice on freshly prepared samples. The results were then reported as averages and standard deviations of these measurements. 3. Results and discussion 3.1. General The overall objective of these experiments was to identify the relative importance of some of the major factors influencing the rate of lipid digestion in oil-in-water emulsions, using the pH-stat method. This information is important for designing realistic protocols for testing and comparing emulsion formulations with different compositions and structures.

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3.2. Effect of lipase concentration on lipid digestion The concentration of pancreatic lipase in the human small intestine depends on many factors, including the individual, the time of day, and the amount and type of food consumed (Bauer, Jakob, & Mosenthin, 2005; Fave, Coste, & Armand, 2004). Initially, we therefore examined the influence of lipase concentration on the digestion of emulsified lipids. Corn oil-in-water emulsions, stabilized by b-Lg, were used in this study, which had an initial mean particle diameter of 178 ± 5 nm and an initial f-potential of 69 ± 4 mV (5 mM phosphate buffer, pH 7.0). The final corn oil (100 mg), bile extract (5 mg/ml), and calcium (20 mM CaCl2) concentrations in the reaction vessel were held constant (37.5 ml, pH 7.0, 37 °C). The initial rate of lipid digestion and the final amount of free fatty acids (FFA) released increased as the lipase concentration in the reaction vessel increased from 0 to 4.8 mg/ml (Fig. 1). At relatively low lipase concentrations (60.2 mg/ml), the amount of FFA released increased only slowly with time and most of the corn oil within the droplets remained undigested after 30 min of digestion (<35% FFA released). At intermediate lipase concentrations (e.g., 0.4 and 0.8 mg/ml), there was an initial period during which the rate of FFA release was relatively slow (from 0 to 10 min), followed by another period when the rate increased appreciably. At relatively high lipase concentrations (P2.4 mg/ml), the amount of FFA released increased rapidly with time almost immediately after digestion started, and then levelled off at longer times because all the corn oil within the droplets had been fully digested. One would expect the rate of lipid digestion to increase as the lipase concentration increased because there would be more total enzyme present in the system to catalyse the conversion of triacylglycerols to free fatty acids (Reis, Holmberg, Watzke, Leser, & Miller, 2009). In addition, the amount of lipase present at the oil–water interface, where the lipolysis reaction occurs, will increase as the total lipase concentration increases. Lipase is a surface-active protein that can compete for the oil–water interface with other surface-active components (Reis et al., 2008), such as the b-lactoglobulin initially coating the lipid droplets or the bile salts added to the reaction vessel. At low lipase concentrations, there may be insufficient lipase present to displace b-lactoglobulin and/or bile from the oil–water interface, and so the enzyme cannot come into close contact with the triacylglycerol substrate within the lipid droplets. The observation of an initial slow rate of FFA release at intermediate lipase concentrations may be explained by

the finite time required for lipase to adsorb to the lipid droplet surfaces and displace the b-Lg and/or bile coating them, so as to get access to the triacylglycerols within the droplet core (Macierzanka, Sancho, Mills, Rigby, & Mackie, 2009). Presumably, at higher lipase concentrations, the adsorption and displacement processes occur rapidly so that digestion could begin almost immediately after the enzyme is added to the reaction vessel. In reality, ingested lipid droplets have to pass through the mouth and stomach before they reach the small intestine (McClements et al., 2009; Singh et al., 2009). Consequently, the physicochemical properties of the originally ingested lipid droplets may have been altered appreciably due to their interactions with other molecules in the mouth and stomach (e.g., salts, acids, mucin, gastric lipase and proteases). For example, a limited amount of lipid digestion may occur in the stomach due to the action of gastric lipase, and the interfacial properties may be altered due to competitive displacement or digestion processes. In particular, proteases in the stomach or small intestine may digest any protein molecules adsorbed to the lipid droplet surfaces, which may alter the surface area of lipid exposed to lipase (through droplet coalescence) and/or the ability of lipase to adsorb to the droplet surfaces (through competitive adsorption), thereby affecting the rate and extent of lipid digestion. These factors were ignored in the present study, since we wished 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, Decker, & McClements, 2009; Versantvoort, Oomen, Van de Kamp, Rompelberg, & Sips, 2005). 3.3. Effect of bile concentration on lipid digestion The concentration of bile salts within the small intestine depends on a number of factors, including the genetics and health status of an individual, the time of day, and the nature and amount of food consumed (Tso, 2000). In this section, we therefore examined the influence of bile concentration on the rate and extent of lipid digestion, using the pH-stat in vitro digestion model for corn oil-in-water emulsions stabilized by b-Lg (d = 178 ± 5 nm, f = 69 ± 4 mV). The final corn oil (100 mg) and calcium concentration (20 mM CaCl2) in the reaction vessel were held constant, and two levels of lipase were used to simulate either fasting (0.4 mg/ml)

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Digestion Time (min) Fig. 1. Impact of lipase concentration in the reaction vessel on the rate and extent of lipid digestion measured in a pH-stat in vitro digestion model.

Fig. 2a. Impact of bile concentration in the reaction vessel on the rate and extent of FFAs released in a pH-stat in vitro digestion model using conditions that mimic the fasting state.

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or fed (2.4 mg/ml) conditions (37.5 ml, pH 7.0, 37 °C). These levels of lipase are consistent with the ranges reported in the literature for lipase in the small intestine for the fasting and fed states (Kalantzi et al., 2006). The impacts of bile extract level on FFA release during digestion were fairly similar for the fasting and fed conditions (Figs. 2a and 2b). The rate and extent of FFA release decreased as the concentration of bile extract in the reaction vessel was increased from 0 to 20 mg/ml. The suppression of lipid digestion by bile salts is well established in the literature, where it has been attributed to the ability of surface-active bile salts to displace lipase from the oil– water interface, thereby preventing the enzyme from coming into close contact with the lipid substrate (Bauer et al., 2005; Lowe, 2002; Patton & Carey, 1981; Reis, Holmberg, Watzke et al., 2009). Addition of co-lipase has been shown to reverse this effect and restore lipase activity (Bauer et al., 2005). In this study, we used a porcine lipase, rather than a crude pancreatin extract that contained both lipase and co-lipase, which accounts for the observed decrease in lipase activity with increasing bile salt concentration. The ability of bile to displace proteins from droplet surfaces is demonstrated in Fig. 3, which shows the change in particle

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f-potential and size when increasing amounts of bile are added to an oil-in-water emulsion stabilized by b-lactoglobulin (pH 7). We did not include lipase in these experiments, so there would have been no lipid digestion. In the absence of bile, the proteincoated droplets have a high negative charge ( 70 mV) because the proteins are above their isoelectric point. When increasing amounts of bile were added to the emulsions, the droplets became increasingly more negative, which can be attributed to displacement of proteins by bile. There was also a slight increase in the mean particle diameter when bile was added to the emulsions, which may have been because bile formed a thicker layer around the lipid droplets than proteins, or because it induced a limited amount of droplet aggregation. These results are in agreement with recent structural, surface tension and surface rheology studies that have also demonstrated the ability of bile salts to displace globular proteins from interfaces (Maldonado-Valderrama et al., 2008). 3.4. Effect of calcium concentration on lipid digestion A certain level of calcium is naturally present in human digestive juices and additional amounts may also arise from ingested foods, particularly those containing high levels of this mineral (Zangenberg et al., 2001a). It is therefore useful to examine the impact of calcium levels on the digestion of emulsified lipids. 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 = 178 ± 5 nm, f = 69 ± 4 mV). Experiments were carried out using in vitro conditions to simulate both fasting and fed conditions. The final corn oil (100 mg) and calcium concentration (20 mM CaCl2) in the reaction vessel were held constant for both systems, but the levels of bile and lipase were varied: fasting = 5 mg/ml of bile and 0.4 mg/ml of lipase; fed = 20 mg/ml of bile and 2.4 mg/ml of lipase (37.5 ml, pH 7.0, 37 °C). These levels were selected from the values reported by previous researchers (Dahan & Hoffman, 2008; Sek, Porter, Kaukonen, & Charman, 2002; Wright, Pietrangelo, & MacNaughton, 2008). The influence of calcium addition on lipid digestion depended on whether fasting or fed conditions were used. For fasting conditions, the rate and extent of FFA production increased as the calcium concentration in the reaction vessel was increased from 0 to 20 mM (Fig. 4a). At lower calcium levels, lipid digestion was not complete (FFA < 100%) within the experimental time frame, which can be attributed to the fact that the lipolysis reaction can

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Fig. 4a. Impact of calcium concentration in the reaction vessel on the rate and extent of FFAs released in a pH-stat in vitro digestion model using conditions that mimic the fasting state.

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be self-limiting due to accumulation of free fatty acids at the droplet surface that inhibit enzyme activity (Reis, Holmberg, Miller, Leser, Raab et al., 2009; Reis, Holmberg, Watzke et al., 2009). For fed conditions, the rate and extent of FFA production increased when the calcium concentration was increased from 0 to 10 mM, but decreased when 20 mM calcium was added (Fig. 4b). Moreover, two different digestion regimes could be distinguished at 20 mM calcium: (i) an initial period, from 0 to 30 min, when digestion was relatively slow; (ii) another period when the digestion rate increased appreciably. Overall, the initial rate of FFA production was higher for fed state conditions (Fig. 4b) than for fasting state conditions (Fig. 4a), which can be attributed to the higher levels of lipase in the fed state. These measurements clearly demonstrate that calcium has a major impact on the lipid digestion process, and are in agreement with previous studies of the influence of Ca2+ on in vitro digestion models (Armand et al., 1992; Hu et al., 2010; Zangenberg et al., 2001a). Calcium ions are believed to increase the rate of lipid digestion by binding and precipitating long chain FFA that accumulate at the oil–water interface due to lipolysis of emulsified triacylglycerols. If these fatty acids are not removed from the lipid droplet surfaces, they then accumulate, thereby limiting the ability of lipase to access and hydrolyse the emulsified triacylglycerols (Fave et al., 2004; Reis et al., 2008; Reis, Holmberg, Miller et al., 2009). The dramatic suppression of FFA production at high calcium levels (20 mM) in the fed state (Fig. 4b) suggests that calcium ions were able to restrict the access of lipase to the lipid droplet surfaces. Calcium may have promoted extensive droplet flocculation, which meant that lipase had to diffuse between droplets at the exterior of the flocs before it could reach droplets in the interior. Once these outer droplets were digested, then it would have been easier to digest the inner ones. The ability of calcium to induce flocculation in protein-stabilized oil-in-water emulsions is highlighted in Fig. 5, which shows the change in mean particle diameter and f-potential with increasing calcium levels. The f-potential became much less negative as the calcium concentration was increased, which can be attributed to binding of cationic Ca2+ ions to the surfaces of the anionic protein-coated droplets, as well as some electrostatic screening effects (McClements, 2005). The mean particle diameter of the emulsions increased appreciably upon addition of calcium, particularly at 15 and 20 mM Ca2+, which is indicative of extensive droplet flocculation. This finding confirms that extensive droplet flocculation may occur in the presence of high calcium levels, which could potentially slow down fat digestion. Nevertheless, it does not account for the observed difference in behaviour between the fasting and

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fed states. The fed state has a higher level of lipase and bile than has the fasting state. It is possible that the cationic calcium ions formed an insoluble complex with the anionic bile salts (Zangenberg, Mullertz, Kristensen, & Hovgaard, 2001b), resulting in the formation of a shell around the lipid droplets that inhibited access of lipase to the triacylglycerols. However, further experiments are needed to establish the precise physicochemical mechanism involved. In the remainder of the experiments, we used fed conditions with a calcium concentration of 5 mM in the reaction vessel to avoid this effect. This level of calcium falls within the range of that reported in the literature for the small intestine in fed conditions (Zangenberg et al., 2001a). 3.5. Effect of lipid concentration on lipid digestion The concentration of free emulsified fat within the small intestine depends on the total amount of food ingested, the fat content of that food, and the physicochemical processes occurring within the mouth, stomach and small intestine. In this section, we therefore examined the influence of fat content on the digestion of emulsified lipids. Different amounts of corn oil-in-water emulsions stabilized by b-Lg (d = 178 ± 5 nm, f-potential = 69 ± 4 mV) were added to the reaction vessel to give final corn oil contents of 150, 300, and 850 mg. The final composition of the digestion media within the reaction vessel was designed to simulate the fed state: lipase = 2.4 mg/ml; bile extract = 20 mg/ml; 5 mM CaCl2; 150 mM NaCl; pH 7.0; total volume = 37.5 ml; 37 °C. The rate and extent of lipid digestion decreased noticeably for the system that contained the highest amount of fat (850 mg), but they were fairly similar for lower fat contents (Fig. 6). It should be noted that, even though the percentage of total fatty acids originally present within the droplets released was less in the 850 mg sample, the absolute amount of FFA released would have been higher. A number of physicochemical mechanisms may explain the observed impact of fat content on the lipid digestion process. The ratio of lipase-to-fat in the reaction vessel decreases as the fat content increases, which may slow the initial rate of lipolysis of the triacylglycerol molecules. In addition, the ratio of calciumto-FFA will also decrease at higher calcium levels, which may mean that the calcium is less effective at precipitating the free fatty acids that accumulate at the oil–water interface during lipolysis, thereby inhibiting the reaction. Finally, the ratio of calcium-to-bile salts

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also decreases at higher calcium levels, which may mean that less of the FFA released during digestion can be solubilised within micelles, again inhibiting the lipolysis reaction by not removing FFA from the droplet surfaces. This result shows that it is important to use a fixed fat content in the pH-stat model when comparing the interfacial or structural properties of one sample with those of another.

3.6. Effect of droplet size on lipid digestion The size of the oil droplets reaching the small intestine may vary widely, depending on the nature of the ingested food and the physicochemical processes occurring within the mouth, stomach and small intestine, e.g., droplet digestion, flocculation, coalescence and disruption (Armand et al., 1992; Armand et al., 1999). It is therefore useful to examine the effect of particle size on the rate of lipid digestion. Corn oil-in-water emulsions, stabilized by b-Lg, were prepared, with three different sizes: coarse emulsion (CE) with d = 760 ± 100 nm; medium emulsion (ME) with d = 252 ± 1 nm; and, fine emulsion (FE) with d = 178 ± 5 nm. The final composition of the digestion media within the reaction vessel was designed to simulate the fed state: corn oil = 100 mg/ml; lipase = 2.4 mg/ml; bile extract = 20 mg/ml; 5 mM CaCl2; 150 mM NaCl; pH 7.0; total volume = 37.5 ml; 37 °C.

The initial rate of lipid digestion increased as the size of the droplets decreased (Fig. 7). This effect can be attributed to the fact that the surface area of the lipid exposed to the surrounding aqueous phase increased as the droplet size decreased (McClements, 2005). Consequently, it is possible for a higher fraction of lipase molecules to accumulate at the oil–water interface and come into direct contact with the emulsified lipid (Reis, Holmberg, Watzke et al., 2009). In addition, a greater amount of FFA needs to be produced before the surface area becomes saturated with lipid digestion products that may inhibit lipase activity (Armand et al., 1992; Armand et al., 1999). This result shows that it is important to use a fixed droplet size in the pH-stat model when comparing one sample with another when a particular experimental parameter (other than particle size) is to be compared. In reality, the droplets in the small intestine may be considerably larger than the ones used in this study (Armand et al., 1996), either because the fat droplets in the ingested foods were larger or because some droplet coalescence normally occurs in the mouth and stomach prior to passage of a food into the small intestine. We would expect larger droplets to be digested more slowly than the ones used in our study because the surface area of lipid exposed to the surrounding aqueous phase would be less.

3.7. Effect of lipid composition on lipid digestion Finally, we examined the impact of lipid type on digestion, since different kinds of lipids are commonly used to prepare food emulsions and delivery systems. Oil-in-water emulsions stabilized by bLg were prepared that had similar droplet sizes and concentrations, using either corn oil or medium chain triglycerides (MCT) as the lipid phase. These emulsions were then tested with the pH-stat digestion model, using conditions to simulate the fed state: lipid = 100 mg; lipase = 2.4 mg/ml; bile extract = 20 mg/ml; 5 mM CaCl2; 150 mM NaCl; pH 7.0; total volume = 37.5 ml; 37 °C. The rate and extent of lipid digestion were clearly higher when MCT was used as the lipid phase than when corn oil was used (Fig. 8). This effect can be attributed to the fact that the medium chain FFA digestion products arising from MCT have a higher dispersibility in aqueous media than the long chain FFA digestion products arising from corn oil (Porter et al., 2007; Pouton & Porter, 2006). The medium chain fatty acids produced during digestion of MCT are able to migrate rapidly into the surrounding aqueous phase and therefore do not inhibit the interfacial lipase reaction. On the other hand, the long chain fatty acids produced by corn oil tend to accumulate at the oil–water interface and inhibit lipase

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Digestion Time (min) Fig. 8. Impact of lipid type (corn oil or medium chain triglycerides) in the reaction vessel on the rate and extent of FFAs released in a pH-stat in vitro digestion model using conditions that mimic the fed state.

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activity until they are removed by being solubilised in micelles or precipitated by calcium ions. 3.8. Proposal for a standardised pH-stat model It would be useful to have standardised test conditions for in vitro digestion models so that the results from one study can easily be compared to those of another. Ideally, this model should give a measure of lipid digestibility that correlates well with that found for similar systems passing through the human digestive tract. In addition, it would be useful for the test procedure to be relatively straightforward so that it can easily be implemented by many different laboratories. The pH-stat method is relatively straightforward to set up and carry out, only requiring an automatic titration unit cable of controlling the pH (Dahan & Hoffman, 2006). Based on the results of our own and others’ work we propose the experimental conditions outlined in Table 1 as a basis for a standardised pH-stat digestion model. One of the main sources of potential variation with in vitro digestion models is the nature of the bile and lipase used in the experiments. For bile, it is possible to use either bile extract or pure bile salts. Bile extract is a complex mixture of various kinds of molecules typically found in the GI tract (such as bile salts, phospholipids, salts); and, therefore, more accurately reflects actual GI conditions; however, it tends to be more variable and inconsistent in composition. On the other hand, individual bile salts can be obtained in purer form, but they are less representative of the complex composition of real small intestine fluids. Similarly for lipase, it is possible to use either pancreatin or pure pancreatic lipase. Pancreatin is a complex mixture of digestive enzymes (e.g., lipase, protease) and other components, whereas pancreatic lipase can be obtained with a more reliable purity. If pure lipase is used, it is important also to use an appropriate amount of co-lipase. For the above reasons, we propose that bile extract and pancreatin be used in standardised in vitro digestion models, rather than pure bile salts and lipase, since their components more accurately represent the complex composition of the GI tract. Nevertheless, it is still important that the composition and activity of bile extract and pancreatin be reported when presenting the results of any in vitro digestion studies. The model proposed in Table 1 is certainly too simplistic to accurately reflect the complex physiological and physicochemical processes that occur in the human gastrointestinal tract, but it does contain the major factors that would be expected to impact lipid digestion and it is relatively simple to implement. Ideally, the rate of FFA release should fall within a reasonably rapid timeframe (e.g., 30 min) so that multiple samples can be conveniently screened. The above conditions should lead to a release rate that falls within this timeframe. Nevertheless, there are often variations in the activity of the lipase within pancreatin from Table 1 Proposed standardised pH-stat method for testing emulsified lipids using the in vitro digestion model under fed state conditions. The amounts of the different substances added to the reaction vessel are highlighted in Section 2. Experimental parameter

Proposed value

pH Reaction cell volume Temperature Stirring speed [NaOH] in titration unit Lipid content in reaction cell NaCl in reaction cell CaCl2 in reaction cell Bile extract in reaction cell Pancreatin (100–400 units/mg protein) in reaction cell Mean droplet diameter

7 37.5 ml 37 °C 4s 1 0.1 mM 300 mg 150 mM 10 mM 20 mg/ml 2.4 mg/ml 500 nm

batch-to-batch or during storage, which means that it may be necessary to adjust its concentration so as to obtain a FFA release profile in the appropriate timeframe. The mean particle diameter and the emulsifier used in an experiment should always be reported, since the rate of FFA release in the pH-stat method depends on both of these parameters. Again, for standardization purposes, it would be useful to use a fixed mean particle diameter. We recommend a mean particle diameter (d32) of 500 nm, since this can be produced using standard high pressure valve or sonication methods, and represents a typical size of emulsion droplet used in the food industry. If different droplet diameters are used, then it is possible to normalise the digestion rate, using a recently developed mathematical model (Li & McClements, 2010). Finally, it should be stressed that the results obtained with the proposed method should be correlated with those obtained using similar systems and in vivo tests in animals and/or humans so as to establish in vitro – in vivo correlations. 4. Conclusions In vitro digestion models are being increasingly used in the food and pharmaceutical industries to screen the digestibility of products with different compositions and structures. This study has highlighted some of the major factors influencing the rate and extent of lipid digestion using the pH-stat method, which is increasing being employed to monitor lipid digestion. The rate and extent of lipid digestion were found to increase with increasing lipase concentration, decreasing bile extract concentration, increasing CaCl2 concentration, decreasing droplet size, and decreasing lipid droplet concentration. These effects are interpreted in terms of the surface area of lipid exposed to the aqueous phase per unit amount of digestive substances (lipase and bile), and factors that impact the accumulation of lipid digestion reaction products (free fatty acids) at the oil–water interface. Factors that increase the contact between lipase and emulsified lipid tend to increase lipid digestion, including increasing the oil–water surface area, decreasing competition with other surface active molecules (bile), and removing free fatty acids from the droplet surfaces (calcium). This study has important implications for designing and testing delivery systems that control lipid digestion. We have proposed some standardised conditions for testing the in vitro digestibility of emulsified lipids using the pH-stat method that may be useful for comparing results from different studies. Acknowledgements This material is partly based upon work supported by United States Department of Agriculture, CREES, NRI Grants and AFRI Grants, and Massachusetts Department of Agricultural Resources CTAGR7AGI UMA 00 Grant. We also acknowledge funding from the University of Massachusetts (CVIP and Hatch). Finally, we thank the Chinese government for providing funding for Yan Li. References Armand, M., Borel, P., Ythier, P., Dutot, G., Melin, C., Senft, M., et al. (1992). Effects of droplet size, triacylglycerol composition, and calcium on the hydrolysis of complex emulsions by pancreatic lipase – An invitro study. Journal of Nutritional Biochemistry, 3(7), 333–341. Armand, M., Hamosh, M., Mehta, N. R., Angelus, P. A., Philpott, J. R., Henderson, T. R., et al. (1996). Effect of human milk or formula on gastric function and fat digestion in the premature infant. Pediatric Research, 40(3), 429–437. Armand, M., Pasquier, B., Andre, M., Borel, P., Senft, M., Peyrot, J., et al. (1999). Digestion and absorption of 2 fat emulsions with different droplet sizes in the human digestive tract. American Journal of Clinical Nutrition, 70(6), 1096–1106. Bauer, E., Jakob, S., & Mosenthin, R. (2005). Principles of physiology of lipid digestion. Asian-Australasian Journal of Animal Sciences, 18(2), 282–295. Dahan, A., & Hoffman, A. (2006). Use of a dynamic in vitro lipolysis model to rationalize oral formulation development for poor water soluble drugs:

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