Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch

FRIN-06074; No of Pages 9 Food Research International xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Food Research International journal homepage: www.elsevier.com/locate/foodres

Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch Nuttinee Tangsrianugul a,b, Manop Suphantharika b, David Julian McClements a,c,⁎ a b c

Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Department of Biochemistry, King Abdulaziz University, Jeddah 21589, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 31 August 2015 Received in revised form 2 November 2015 Accepted 4 November 2015 Available online xxxx Keywords: Delivery system Gastrointestinal Resistant starch Lipid digestion Starch hydrogels

a b s t r a c t The influence of starch type (resistant starch (RS) versus native (NS) starch) and concentration (10 and 35 wt.%) on the potential gastrointestinal fate of digestible lipid (corn oil) droplets encapsulated within starch hydrogels was studied using a simulated gastrointestinal tract (GIT). The NS used was a normal corn starch, whereas the RS used was a high amylose corn starch. Changes in morphology, organization, size, and charge of the particles in the delivery systems were measured as they passed through each stage of the GIT model: mouth, stomach, and small intestine. The GIT fates of three types of delivery system were compared: free lipid droplets; lipid droplets in RShydrogels; and, lipid droplets in NS-hydrogels. Encapsulation of the lipid droplets in the hydrogels had a pronounced influence on their GIT behavior, with the effect depending strongly on starch type. The starch granules in the RS-hydrogels remained intact throughout the simulated GIT because their compact structure makes them resistant to enzyme digestion. The initial rate of lipid digestion in the small intestine phase also depended on delivery system type: emulsion N RS-hydrogels N NS-hydrogels. However, the lipid phase appeared to be fully digested at the end of the digestion period for all samples. These results provide useful information for designing functional foods for improved health. For example, food matrices could be developed that slowdown the rate of lipid digestion, and therefore prevent a spike in serum triacylglycerols in the blood, which may be advantageous for developing functional foods to tackle diabetes. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction An increased awareness by consumers about the relationship between a nutritious diet and human health has been one of the major reasons for the increasing popularity of foods enriched with dietary fiber (Aleixandre & Miguel, 2008; Sajilata, Singhal, & Kulkarni, 2006; Viuda-Martos et al., 2010; Warrand, 2006). Dietary fibers are defined as those polysaccharides that are indigestible within the upper part of the human gastrointestinal tract (GIT), such as cellulose, pectin, gums, and resistant starch (Biliaderis & Izydorczyk, 2007; Cui, 2005). The incorporation of dietary fibers into foods also influences their physicochemical properties and sensory attributes due to their water holding, oil holding, lightening, emulsifying, thickening and/or gelling properties (Elleuch et al., 2011; Santipanichwong & Suphantharika, 2009; Winuprasith & Suphantharika, 2015). Thus, these effects must be taken into account when formulating functional food products enriched with dietary fibers otherwise consumers will reject them. Starch has been divided into three major categories depending on its susceptibility to digestion within the human GIT: rapidly digestible ⁎ Corresponding author at: Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA. E-mail address: [email protected] (D.J. McClements).

starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) (Englyst, Kingman, & Cummings, 1992). Consumption of RDS typically leads to a rapid increase in blood glucose concentration after ingestion because it is rapidly digested by amylase within the GIT. The increase in blood glucose levels is more gradual for SDS because it is digested at a slower rate, however it is still fully digested within the small intestine. RS is not completely hydrolyzed by enzymes in the small intestine and therefore does not cause a pronounced increase in blood glucose levels, but it will subject to bacterial fermentation in the colon (Englyst et al., 1992; Singh, Dartois, & Kaur, 2010). The hydrolysis of digestible starch begins in the mouth due to the presence of salivary αamylase, but the majority of hydrolysis takes place within the small intestine due to the presence of pancreatic amylase (Lehmann & Robin, 2007). Many factors have been reported to influence the rate of starch hydrolysis, including botanical source, food processing, particle size and amylose/amylopectin ratio (Benmoussa, Moldenhauer, & Hamaker, 2007; Chung, Liu, Lee, & Wei, 2011; Lehmann & Robin, 2007; Wang & Copeland, 2013). In addition, the ability of amylase to physically interact with the starch molecules in foods is an important factor affecting the rate of starch hydrolysis, which may be impacted by the presence of other food components that can inhibit diffusion or adsorption of the enzyme, such as proteins, lipids, and dietary fibers (Colonna, Leloup, & Buléon, 1992; Singh et al., 2010).

http://dx.doi.org/10.1016/j.foodres.2015.11.004 0963-9969/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article as: Tangsrianugul, N., et al., Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.11.004

2

N. Tangsrianugul et al. / Food Research International xxx (2015) xxx–xxx

RS has received much attention due to its potential health benefits, such as inhibiting the onset of colon cancer, diabetes, and obesity, as well as acting as a prebiotic that promotes a healthy colonic microflora (Brouns, Kettlitz, & Arrigoni, 2002; Fuentes-Zaragoza, Riquelme-Navarrete, Sánchez-Zapata, & Pérez-Álvarez, 2010; Nugent, 2005; Sharma, Yadav, & Ritika, 2008). Moreover, resistant starches containing non-gelatinized granules are often easier to incorporate into functional foods than other sources of dietary fibers because they do not cause such a large increase in viscosity due to their relatively low effective volumes (Baixauli, Salvador, Martinez-Cervera, & Fiszman, 2008; Charalampopoulos, Wang, Pandiella, & Webb, 2002). Resistant starches have been classified into five sub-types according to the major factors limiting their enzymatic degradation: RS1 — starch granules trapped in non-digestible food matrices (such as in grains, seeds, or tubers); RS2 — starch granules that have not been gelatinized (such as raw potatoes, unripe bananas, and high amylose starches); RS3 — retrograded starch formed when foods are cooked and then cooled; RS4 — chemically modified starches (Nugent, 2005; Sajilata et al., 2006; Sharma et al., 2008); RS5 — amylose-lipid complexes that inhibit amylase access to starch (Brown, Yotsuzuka, Birkett, & Henriksson, 2006; Jiang, Lio, Blanco, Campbell, & Jane, 2010; Thompson, Maningat, Woo, & Seib, 2011). There has been increasing interest in using food matrix effects to control the behavior of lipids within the human gastrointestinal tract (Golding & Wooster, 2010; McClements, Decker, & Park, 2009; McClements & Xiao, 2014; Singh & Ye, 2013). This knowledge is being utilized to design delivery systems to encapsulate, protect, and control the release of nutrients and nutraceuticals within the GIT (Kosaraju, 2005; McClements & Li, 2010), as well as to design functional foods to control satiety, satiation, and serum lipid levels (Keogh et al., 2011; Lundin, Golding, & Wooster, 2008; Steingoetter et al., 2015). Studies have reported that dietary fibers may influence the GIT fate of lipids through numerous physicochemical and physiological mechanisms: (i) they may interact with lipase and/or co-lipase, thereby reducing its enzyme activity; (ii) they may adsorb to lipid droplet surfaces and form a protective coating that prevents lipase access; (iii) they may promote or hinder lipid droplet aggregation, thereby altering the amount of lipid surface exposed to lipase; (iv) they may increase the viscosity of the aqueous solution surrounding the lipid droplets, thereby altering mass transport processes; (v) they may trap the lipids within an indigestible food matrix, thereby limiting lipase access (Beysseriat, Decker, & McClements, 2006; Espinal-Ruiz, Parada-Alfonso, Restrepo-Sanchez, Narvaez-Cuenca, & McClements, 2014; McClements et al., 2009; Pasquier et al., 1996; Torcello-Gomez & Foster, 2014; Zhang, Zhang, Zhang, Decker, & McClements, 2015). However, the impact of dietary fibers on lipid digestion depends on their molecular structure and physicochemical properties, and therefore has to be established for each type of dietary fiber. The aim of the current study was to determine the influence of starch type (native versus resistant) and concentration on the gastrointestinal fate of lipids encapsulated within starch hydrogels using a simulated GIT that included mouth, stomach and small intestine phases. In this study, normal corn starch was used as an example of a native starch, whereas high amylose corn starch was used as an example of a resistant starch (Jiang et al., 2010). We hypothesized that hydrogels formed from resistant starch would inhibit the digestion of the lipid droplets in the GIT by preventing the lipase molecules from coming into close proximity to the lipid droplet surfaces. To test this hypothesis, changes in the microstructure and physicochemical properties (particle size and charge) of the hydrogel-based delivery systems were measured as they passed through the various phases of the GIT model, and the impact of delivery system properties on the rate and extent of lipid digestion was determined. The information obtained in this study provides knowledge about the influence of starch type on the gastrointestinal fate of starch-based hydrogel delivery systems, which may be useful in the development of functional food products with enhanced health benefits.

2. Materials and methods 2.1. Materials The starches used in this study were a resistant corn starch (RS) and a native corn starch (NS). The native corn starch was provided by Tate & Lyle (Decatur, IL, USA), while the resistant corn starch (Hi-Maize® 260) was purchased from Ingredion (Bridgewater, NJ, USA). The manufacturer reported that the resistant starch ingredient was derived from high amylose corn starch. Mucin (from porcine stomach), α-amylase (1254 units/mg protein from porcine pancreas), pepsin (from porcine gastric mucosa), pancreatin (from porcine pancreas 8× USP specifications), porcine bile extract, Tween 80 and Nile Red dye were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Corn oil was purchased from a local supermarket. All other chemicals were of analytical grade. Double distilled water was used for the preparation of all solutions and emulsions which was obtained using a Milli-Q water purification system (Millipore, Billerica, MA, USA). 2.2. Methods 2.2.1. Emulsion preparation A stock oil-in-water emulsion was prepared by homogenizing 10 wt.% lipid phase (corn oil) with 90 wt.% aqueous phase (1 wt.% Tween 80 in 10 mM phosphate buffer, pH 7.0) using a high shear blender (M133/1281-0, Biospec Products, Inc., ESGC, Basle, Switzerland) for 2 min. The coarse emulsion was then passed through a microfluidizer (M110Y, Microfluidics, Newton, MA, USA) four times at a homogenization pressure of 12,000 psi. 2.2.2. Starch-hydrogel preparation An emulsion containing 2 wt.% lipid was prepared by diluting the stock emulsion (10 wt.% lipid) with 10 mM phosphate buffer solution (pH 7.0), and then weighed amounts of starch (10 or 35 wt.% RS or NS) were dispersed into the diluted emulsion and stirred at 400 rpm for at least 10 min to ensure they were homogenous. The emulsionstarch dispersions were then heated at 100 °C for 10 min with continuous stirring at 400 rpm. After heating, the samples were cooled in an ice water bath, and then the samples were stored at 4 °C overnight. 2.2.3. Simulated gastrointestinal tract model Each sample was passed through a three-step simulated gastrointestinal tract (GIT) model, which included mouth, gastric, and small intestinal phases, which was slightly modified from that used in a previous study (Mun, Kim, & McClements, 2015): the starch-hydrogel samples were crushed into small pieces before samples were exposed to the simulated mouth system to simulate fragmentation during mastication. 2.2.3.1. Mouth phase. Simulated saliva fluid (SSF), containing mucin and various salts, was prepared according to a previous study (Sarkar, Goh, Singh, & Singh, 2009). The digestive enzyme α-amylase was added to the SSF before mixing with the samples. Amylase was added at an activity level of 100 units/mL, which is the average activity reported during mastication (Yamaguchi et al., 2004). The samples (emulsion or starch-hydrogels) were mixed with SSF at a 50:50 mass ratio and the mixture was adjusted to pH 6.8. The mixture was incubated at 37 °C for 10 min with continuous agitation at 100 rpm in a temperature controlled incubator (Innova Incubator Shaker, Model 4080, New Brunswick Scientific, New Jersey, USA). 2.2.3.2. Gastric phase. Simulated gastric fluid (SGF) was prepared using a method reported previously (Sarkar et al., 2009) by dissolving 2 g of NaCl, and 7 mL of HCl (37%) in 1 L of water, adjusting the pH to 1.2 using 1.0 M HCl, and then adding 3.2 g of pepsin. The sample from the mouth phase was mixed with SGF at a 50:50 mass ratio and the pH of the sample was adjusted to 2.5. The sample was then incubated at 37 °C for 2 h with continuous agitation at 100 rpm.

Please cite this article as: Tangsrianugul, N., et al., Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.11.004

N. Tangsrianugul et al. / Food Research International xxx (2015) xxx–xxx

2.2.3.3. Small intestinal phase. A pH-stat automatic titration unit (Metrohm USA Inc., Riverview, FL, USA) was used to simulate the conditions in the small intestinal phase of the GIT (Salvia-Trujillo, Qian, Martín-Belloso, & McClements, 2013). An aliquot of 30 mL of sample from the gastric phase was placed in a temperaturecontrolled (37 °C) chamber and the pH was set at 7.0 using NaOH solution. Then 3.5 mL of bile extract solution (187.5 mg/3.5 mL) and 1.5 mL of salt solution (10 mM of calcium chloride and 150 mM of sodium chloride) were added to the sample and the mixture was adjusted to pH 7.0. Afterwards, 2.5 mL of freshly prepared pancreatin suspension (187.5 mg/2.5 mL) dissolved in phosphate buffer was added into the mixture. The pH of the mixture was monitored and the volume (mL) of 0.1 M NaOH necessary to neutralize the free fatty acids (FFA) released from the lipid digestion (i.e., to keep pH at 7.0) was recorded during two hours. The percentage of FFA released from the system was then calculated from the volume of alkaline titrated into the reaction vessel using the following equation: %FFA ¼ 100 

VNaOH  mNaOH  MLipid WLipid  2

ð1Þ

where VNaOH is the volume of titrant in liters, mNaOH is the molarity of the NaOH solution (0.1 M), MLipid is the molecular weight of corn oil (872 g/mol), and WLipid is the weight of oil in the reaction vessel (grams). Blanks (samples without oil) were run under the same conditions as the samples, and the amount of alkali for the control was subtracted from the samples that contained oil. For the emulsions, a simple buffer solution was used as a blank, whereas for the filled starch-hydrogels the same starch-hydrogels containing no oil droplets were used as blanks so as to account for any digestible matter associated with the starch. Samples were also collected for physicochemical and structural characterization after the 2 h digestion period in the small intestinal stage. The micelle phase was obtained after the samples had been passed through the full simulated GIT model. An aliquot of each sample (10 mL) was centrifuged at 4000 rpm (2674 g) for 40 min at 25 °C (CL10 centrifuge, Thermo Scientific, Pittsburgh, PA, USA). The supernatant was assumed to be the “micelle” and was collected to measure the electrical charge (in Section 2.2.4). 2.2.4. Particle characterization The particle size, particle size distribution, and electrical charge (ζ-potential) of the samples were measured before, during, and after the simulated GIT process. The particle size distribution was measured using static light scattering (Mastersizer 2000, Malvern Instruments Ltd., Worcestershire, UK). The samples were diluted in 10 mM phosphate buffer to avoid multiple scattering effects, and were stirred in the dispersion unit (1200 rpm) to ensure sample homogeneity throughout the measurements. The particle size was reported as the surface-weighted mean diameter (d32). The ζ-potential of the particles in the samples was determined by particle micro-electrophoresis (Zetasizer NanoZS, Malvern Instruments Ltd., Worcestershire, UK). Samples were diluted with appropriate buffer solutions prior to analysis: initial, small intestine and micelle — phosphate buffer solution (pH 7.0); mouth — simulated saliva fluid without mucin (pH 6.8); stomach — simulated gastric fluid (pH 2.5) and placed in a capillary cell equipped with two electrodes to assess the electrophoretic mobility of the particles. Samples were equilibrated for 60 s in the instrument (25 °C) before they were measured using 10–100 runs per analysis. 2.2.5. Microstructure measurement The microstructure of the samples was examined by conventional optical microscopy and/or confocal scanning laser microscopy using a

3

60× objective lens (Nikon D-Eclipse C1 80i, Nikon, Melville, NY, USA). A small aliquot of sample was placed on a microscope slide and covered by a cover slip prior to analysis. For confocal microscopy measurements, samples were stained with a fat-soluble fluorescent dye (Nile red) that had previously been dissolved at 0.1% (w/v) in ethanol. An air-cooled argon ion laser (Model IMA1010BOS, Melles Griot, Carlsbad, CA, USA) was used to excite the Nile red at 488 nm. All images were taken and processed using the instrument software program (EZ-CS1 version 3.8, Nikon, Melville, NY, USA). 2.2.6. Statistical analysis All measurements were performed in at least duplicate using freshly prepared samples. The mean and standard deviations were calculated from these data. A one-way analysis of variance (ANOVA) was performed using commercial statistical software (SPSS). The Duncan's test was used to establish the significance of difference among the mean values at the 0.05 significance level. 3. Results and discussion 3.1. Influence of delivery system type on gastrointestinal fate GIT Initially, the influence of the initial composition and structure of the delivery systems on their behavior within the simulated GIT was determined. Emulsions (free lipid droplets) and filled starch-hydrogels (lipid droplets dispersed in starch hydrogels) were passed through the mouth, stomach, and small intestine stages, and then changes in the particle size, microstructure, and particle charge were measured. Starchhydrogels were formed using different types (RS and NS) and levels (10 and 35 wt.%) of starch. 3.1.1. Particle size and microstructure 3.1.1.1. Free droplets. The droplets in the initial oil-in-water emulsions had a mean particle diameter (d32) of 0.18 μm (Fig. 1), a monomodal particle size distribution (Fig. 2a), and were evenly distributed throughout the samples (Fig. 3a). These measurements indicated that small lipid droplets could be successfully produced using microfluidization. After passing through the simulated mouth and gastric phases, there was a slight increase in the mean particle diameter (Fig. 1), and evidence of a small population of large particles in the particle size distribution (Fig. 2a). The confocal microscopy images indicated there were some large lipid droplet aggregates in the emulsions under simulated mouth conditions, but these were largely disrupted under simulated

Fig. 1. Influence of simulated gastrointestinal conditions on the mean droplet diameter (d32) of corn oil-in-water emulsions and filled starch-hydrogels stabilized by Tween 80 (* represents not detected).

Please cite this article as: Tangsrianugul, N., et al., Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.11.004

4

N. Tangsrianugul et al. / Food Research International xxx (2015) xxx–xxx

Fig. 2. Influence of simulated gastrointestinal conditions on the particle size distributions of: (a) corn oil-in-water emulsions; (b) 10 wt.% RS-hydrogel; (c) 35 wt.% RS-hydrogel; and (d) NS-hydrogels stabilized by Tween 80.

stomach conditions (Fig. 3a). According to previous studies, aggregation in the mouth can be attributed to depletion and/or bridging flocculation induced by interactions of mucin from the saliva with the lipid droplets (Sajilata et al., 2006; Salvia-Trujillo et al., 2013; Vingerhoeds, Blijdenstein, Zoet, & van Aken, 2005). The decrease in aggregate size when the emulsions moved from the mouth phase to the stomach phase is similar to that reported in previous studies using small lipid droplets stabilized by nonionic surfactants (Zhang et al., 2015). Dissociation of the flocculated droplets in the stomach may have occurred due to a number of physicochemical changes that altered the strength of the attractive forces between the droplets: (i) dilution; (ii) changes in pH and ionic strength; (iii) mechanical agitation (Dickinson, 2003; McClements, 2005; Salvia-Trujillo et al., 2013). Dilution will reduce the concentration of non-adsorbed biopolymers (mucin) in the aqueous phase, which will reduce the strength of the depletion attraction. Alterations in pH and ionic strength will alter the magnitude and range of any electrostatic interactions, which may also change the tendency for droplet aggregation to occur. Mechanical agitation (stirring) may disrupt any flocculated droplets held together by relatively weak attractive forces. After incubation in the small intestine phase, particle size analysis indicated that there was a broad range of different sized particles within the emulsion samples (Fig. 2a). In addition, confocal fluorescence microscopy indicated that lipid-rich particles of various sizes and morphologies were present (Fig. 3a). These samples were likely to have contained a complex mixture of different kinds of colloidal particles, such as non-digested lipid droplets, micelles, vesicles, insoluble matter, and calcium soaps (Salvia-Trujillo et al., 2013; Yang & McClements, 2013). There were some important differences between the results obtained by static light scattering and those obtained by microscopy. In particular, large particles were observed in the mouth using microscopy (Fig. 3a), but not using light scattering (Figs. 1 and 2a). A possible reason

for this discrepancy is the influence of sample preparation on microstructure. The samples analyzed by light scattering were highly diluted and stirred prior to measurement, and so any flocs held together by weak attractive forces in the mouth may have been disrupted (Salvia-Trujillo et al., 2013; Zou et al., 2015). Hence, flocs held together by weak depletion forces in the mouth would have been disrupted during light scattering measurements, but not during microscopy analysis. 3.1.1.2. Encapsulated droplets. The filled starch-hydrogels behaved differently than the emulsions within the simulated GIT. In addition, the type of starch used had a major impact on the fate of the filled starchhydrogels. The initial particle size distributions were roughly monomodal for filled starch-hydrogels containing different types and amounts of starch granules (Fig. 2b-d). However, the NS-hydrogels contained particles with larger mean diameters (≈35 to 39 μm) than the RS-hydrogels (≈ 10 μm) (Fig. 1). The optical microscopy images confirmed that the granules in the NS-hydrogels were considerable larger than those in the RS-hydrogels (Fig. 3b and c). The RS-hydrogel was derived from high amylose corn starch, which has been reported to have a gelatinization temperature appreciably higher than most other types of starch. Indeed, previous studies report that most corn starches gelatinize around 62 to 72 °C, whereas high amylose corn starches gelatinize at temperatures exceeding 130 °C (Cai, Zhao, Huang, Chen, & Wei, 2014; Schirmer, Höchstötter, Jekle, Arendt, & Becker, 2013; Wang, Wang, Yu, & Wang, 2014). Thus, the small size of the granules in the RS-hydrogels can be attributed to the fact that they did not swell appreciably during the heat-treatment (100 °C for 10 min) used in this study, whereas those in the NS-hydrogels did. Interestingly, the small lipid droplets could not be observed in the particle size distributions (Fig. 2), which can be attributed to the fact that the large starch granules scattered light much more strongly and therefore

Please cite this article as: Tangsrianugul, N., et al., Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.11.004

N. Tangsrianugul et al. / Food Research International xxx (2015) xxx–xxx

dominated the overall light scattering signal. Nevertheless, confocal microscopy was able to show that the lipid droplets were fairly evenly dispersed in the regions surrounding the starch granules in both types of filled hydrogel system (Fig. 3b and c). There was little change in the mean particle diameter (Fig. 1) or particle size distribution (Fig. 2b and c) of the RS-hydrogels after they were exposed to the different phases of the simulated GIT, which can be attributed to the fact that this type of starch granule is resistant to enzyme digestion. It was not possible to measure the particle size of the NShydrogels using static light scattering after exposure to mouth and stomach phases because they formed a semi-solid gel that would not disperse in the buffer solution used for dilution. However, measurements could be made after they were exposed to small intestine conditions because the samples became more fluid, probably because of the digestion, dilution, and shearing that occurred during this phase. The measured particle size was higher for the 35 wt.% NS-hydrogel than for the 10 wt.% one (Fig. 2d), which may have been because there were more non-digested starch granules present or because of differences in the nature of the lipid digestion products formed between the two samples. In addition, there appeared to be a population of smaller particles (d b 1 μm) in the 10 wt.% NS-hydrogel after digestion, which was possibly due to the formation of mixed micelles (micelles and vesicles). Optical and confocal microscopy was used to investigate changes in the microstructure of the hydrogels as they passed through the GIT. The confocal images showed that extensive lipid droplet aggregation occurred in both systems after passing through the mouth and gastric phases (Fig. 3b and c). As mentioned earlier, this effect may have been due to bridging or depletion flocculation by mucin present in the simulated saliva (Sarkar et al., 2009; Singh & Ye, 2013). In addition, the presence of non-adsorbed polysaccharides derived from ingested foods within the gastrointestinal fluids may also promote bridging or depletion flocculation (Espinal-Ruiz et al., 2014). In our case, there may have been some starch molecules (amylose and amylopectin) released from the granules due to the action of amylase within the mouth. Confocal microscopy indicated that there were lipid droplets distributed between any remaining starch granules after exposure to the small intestine phase (Fig. 3b). These lipid droplets may have been undigested fat droplets or mixed micelles (vesicles and micelles) arising from the lipid digestion process (Singh & Ye, 2013). The optical microscopy images indicated that the starch granules in the NS-hydrogels were progressively disrupted as they passed through the simulated GIT, which can be attributed to digestion of the starch by amylase from the simulated saliva and intestinal fluids. On the other hand, the starch granules in the RS-hydrogels remained intact after exposure to mouth, stomach, and small intestine phases (Fig. 3b) because they are resistant to digestive enzymes in the upper GIT. 3.1.2. Electrical charge characteristics The electrical characteristics of the particles in the different delivery systems were measured to provide additional information about their properties within the GIT (Fig. 4). 3.1.2.1. Free droplets. For the emulsions, the particles in the initial system were only slightly anionic, i.e. ζ ≈ −5 mV. The lipid droplets in the initial emulsions were coated by a nonionic surfactant (Tween 80), and so would not be expected to have a charge. Nevertheless, earlier studies have shown that this type of droplet may have some negative charge due to the presence of anionic impurities in the oil, water, or surfactant (Espinal-Ruiz et al., 2014; Mayer, Weiss, & McClements, 2013; Salvia-Trujillo et al., 2013). The particles in the emulsions became more negative (≈− 10 mV) after passing through the mouth phase, suggesting that either adsorbed or free anionic mucin molecules contributed to the electrical signal (Mayer et al., 2013; Salvia-Trujillo et al., 2013). There was a decrease in the magnitude of the negative charge on the particles in the emulsion after exposure to the stomach

5

phase, which can be attributed to changes in surface charge and/or electrostatic screening effects. For example, protonation of carboxyl groups (–COOH) and amino groups (–NH+ 3 ) would reduce the net negative charge on the droplets and mucin molecules, whereas the high ionic strength would cause electrostatic screening of surface charges (Israelachvili, 2011). The ζ-potential measurements indicated that the particles in the emulsion had high negative charges (≈−37 mV) after exposure to the small intestine phase. In addition, the mixed micelle phase collected by centrifugation of the digesta (the material resulting after the small intestine phase) also had a strong negative charge (≈−43 mV). This high negative charge can be attributed to the presence of various types of anionic substances in the digesta, including bile salts, phospholipids, and free fatty acids (Salvia-Trujillo et al., 2013; Yang & McClements, 2013). Other studies have also reported similar trends in the electrical characteristics of the particles formed by the digestion of oil-in-water emulsions (Mayer et al., 2013; Zou et al., 2015) 3.1.2.2. Encapsulated droplets. For the starch-hydrogels, we examined the influence of starch type and concentration on the electrical characteristics. It was not possible to measure the ζ-potential of the particles in the NS-starch hydrogels exposed to mouth and stomach conditions because they had semi-solid structures that could not be dispersed in buffer solutions. For the other samples, the ζ-potential of the particles followed fairly similar trends after exposure to the various regions of the simulated GIT (Fig. 4). The initial charge of the particles in the filled starch-hydrogels was slightly more negative (≈−11 to −14 mV) than those in the emulsions (≈−5 mV), which may have been due to some negative charge associated with the starch granules themselves as reported previously (Wu, Degner, & McClements, 2013). There was little change in the ζ-potential of the particles in the RS-hydrogels after they passed through the mouth phase but there was a large decrease in the magnitude of the electrical charge under acidic gastric conditions. This decrease in negative charge may again be attributed to changes in ionization of charged groups and electrostatic screening effects associated with the low pH and high ionic strength of the gastric fluids as discussed earlier. After exposure to the small intestinal phase, the particles present in the digesta and the mixed micelles collected from all the starch-hydrogel samples were highly negatively charged due to the presence of a complex mixture of anionic species within the intestinal digesta, such as bile salts, phospholipids, and free fatty acids. The particles in the mixed micelle phase had fairly similar high negative charges regardless of initial starch type or level, i.e., ζ = − 38 to − 45 mV (Fig. 4). This observation suggests that the starch granules did not strongly interfere with the formation of micelles and vesicles in the small intestinal fluids. On the other hand, there were some appreciable differences between the magnitudes of the negative charges measured on the whole digesta (Fig. 4). In particular, the samples containing resistant starch appeared to have a lower negative charge than the ones containing similar levels of native starch. This may have been due to the contribution of undigested resistant starch granules (which have a low charge) to the overall signal used to calculate the ζ-potential of the digesta. 3.2. Influence of delivery system composition on lipid digestibility The rate and extent of lipid digestion in the different samples were measured using the pH-stat method (Fig. 5). For all the samples, there was a rapid initial release of FFAs, followed by a more gradual release at longer times, until a relatively constant final value was attained. The initial rate of lipid digestion (calculated from the slope of the FFA-time profiles in the first 5 min) was significantly faster in the emulsions than in the filled hydrogels (Fig. 6), which suggests that the lipase molecules could more easily absorb to the surfaces of the free lipid droplets in this system (Golding & Wooster, 2010; Li & McClements, 2010; McClements, 2013). The slower initial digestion rate of the lipid droplets in the presence of the starch hydrogels suggested that the starch

Please cite this article as: Tangsrianugul, N., et al., Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.11.004

6

N. Tangsrianugul et al. / Food Research International xxx (2015) xxx–xxx

Please cite this article as: Tangsrianugul, N., et al., Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.11.004

N. Tangsrianugul et al. / Food Research International xxx (2015) xxx–xxx

Fig. 4. Influence of simulated gastrointestinal conditions on the electrical charge (ζ-potential) of corn oil-in-water emulsions and filled starch-hydrogels stabilized by Tween 80 (* represents not detected).

granules slowed down the movement of lipase to the lipid droplet surfaces, or the movement of lipid digestion products (such as free fatty acids or monoacylglycerols) from the lipid droplets surfaces. The sample initially containing 35% of native starch gave the slowest initial digestion rate (Fig. 6). There are a number of different molecular, structural, and physicochemical factors that could contribute to the influence of starch type and concentration on the initial rate of lipid digestion. First, the presence of any non-digested starch granules may have increased the diffusion path length of the lipase molecules. The presence of particles within a fluid is known to retard the diffusion of small molecules due to an increase in path length associated with tortuosity (Li, Hu, Du, Xiao, & McClements, 2011). Second, the digestion of starch granules releases amylose and amylopectin molecules that will increase the viscosity of the intestinal fluids and therefore retard the movement of lipase molecules. Studies have shown that suspensions of starch granules initially have a high viscosity under simulated gastrointestinal conditions, but that their viscosity decreases appreciably after digestion of the starch by amylase (Hardacre, Yap, Lentle, & Monro, 2015). Consequently, the starch granules may have inhibited the initial stages of lipid digestion when the viscosity was high, but this effect may have been reduced as starch digestion became more extensive. Third, the amylose molecules may have been able to form complexes with the free fatty acids released by lipid digestion (Ai et al., 2014). The formation of these complexes may have altered the extent of starch hydrolysis (Ai et al., 2014), as well as the movement of long chain free fatty acids from the lipid droplet surfaces and the formation of mixed micelles, which is known to impact lipid digestion (Devraj et al., 2013). Despite the differences in the initial rate of lipid digestion, the lipid phase in all of the samples was fully digested by the end of the small intestine period (Fig. 5). The reason that the final amount of FFAs released exceeded 100% may have been because more than two FFAs were produced per triacylglycerol molecule or because of digestion of components arising from the starch granules (rather than the lipid droplets). Starch granules contain starch, lipids, and proteins that could all be hydrolyzed by the digestive enzymes in pancreatin (amylase, lipase, protease). The hydrolysis of each of these components leads to the production of protons (H+) that could decrease the pH of the small intestinal fluids. The volume of NaOH required to neutralize these additional protons will be included in the calculation of the FFAs released, thereby leading to an overestimation of the actual amount of fatty acids released. Even though we did run controls containing starchhydrogels in the absence of lipid droplets, the digestible material arising from the starch granules may have been digested differently in the

7

Fig. 5. The calculated FFA released from corn oil-in-water emulsions and filled starchhydrogels stabilized by Tween 80 measured in a pH-stat in vitro digestion model.

presence of lipid droplets. Consequently, simply subtracting the NaOH volume measurements for the controls (starch granules only) from the samples (starch granules plus fat droplets) may not be fully accounted for this effect.

4. Conclusions The objective of this study was to investigate the influence of encapsulation of lipid droplets within starch-hydrogels on their potential GIT fate. Initially, we hypothesize that lipid droplets encapsulated within resistant starch hydrogels would be digested more slowly than those in native starch hydrogels. Our results showed that the starch-hydrogels altered the behavior of the lipid droplets within the simulated GIT by an amount that depended on starch type, i.e., native starch (NS) versus resistant starch (RS). The lipid droplets in the filled starch hydrogels were mainly located in the regions between the starch granules. The starch granules in the NS-hydrogels were progressively digested as they moved through the GIT due to the action of amylase in the mouth and small intestine phases. Conversely, the starch granules in the RS-hydrogels remained intact throughout the entire GIT due to their resistance to hydrolysis by digestive enzymes in the upper GIT. Encapsulation of the lipid droplets within the starch-hydrogels decreased the initial rate of lipid digestion, which suggests that the presence of both digested and non-digested starch granules inhibited the ability of lipase to interact with the droplet surfaces. However, the lipid phase was fully digested within all of the samples at the end of the small intestine phase, which suggests that the starch granules only slowed down lipid digestion, rather than preventing it. The main reason for this phenomenon can be attributed to the fact that the lipid droplets were mainly located between the starch granules, and so the resistant starch did not have a major impact on lipid digestion since the lipase could still easily access the lipid droplet surfaces. This information may be useful for designing functional foods that can slow down the rate of lipid digestion, and therefore control blood serum triacylglycerol levels. Nevertheless, further work is required using animal or human trials to verify the efficacy of these systems. In addition, it should be stressed that there are numerous kinds of native and resistant starch ingredients that may be utilized in foods, which may behave differently from each other. Consequently, it is not possible to generalize the results of this study to different types of starches. Instead, further studies will be required to compare different starch types under simulated and actual gastrointestinal conditions.

Fig. 3. Influence of simulated gastrointestinal conditions on microstructure of: (a) corn oil-in-water emulsions; (b) RS-hydrogels; and (c) NS-hydrogels stabilized by Tween 80 measured using confocal and optical microscopy. The scale bars represent a length of 20 μm, and the red regions represent lipids.

Please cite this article as: Tangsrianugul, N., et al., Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.11.004

8

N. Tangsrianugul et al. / Food Research International xxx (2015) xxx–xxx

Fig. 6. Initial rate of free fatty acid release from corn oil-in-water emulsions and filled starch- hydrogels measured using a pH-stat during small intestine digestion. Different capital letters mean significant differences (p b 0.05) between samples.

Acknowledgments Financial support from the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant no. PHD/0124/2554) to N.T. and M.S. is acknowledged. The authors thank Tate & Lyle for providing the native corn starch used in these experiments. This material was partly based upon work supported by the Cooperative State Research, Extension, Education Service, USDA, Massachusetts Agricultural Experiment Station (Project No. 831) and USDA, NRI Grants (2011-03539, 2013-03795, 2011-67021, and 2014-67021). This project was also partly funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant numbers 330-130-1435-DSR, 299130-1435-DSR, 87-130-35-HiCi. The authors, therefore, acknowledge with thanks DSR technical and financial support. References Ai, Y.F., Zhao, Y.S., Nelson, B., Birt, D.F., Wang, T., & Jane, J.L. (2014). Characterization and in vivo hydrolysis of amylose stearic acid complex. Cereal Chemistry, 91(5), 466–472. Aleixandre, A., & Miguel, M. (2008). Dietary fiber in the prevention and treatment of metabolic syndrome: A review. Critical Reviews in Food Science and Nutrition, 48(10), 905–912. Baixauli, R., Salvador, A., Martinez-Cervera, S., & Fiszman, S.M. (2008). Distinctive sensory features introduced by resistant starch in baked products. LWT- Food Science and Technology, 41(10), 1927–1933. Benmoussa, M., Moldenhauer, K.A.K., & Hamaker, B.R. (2007). Rice amylopectin fine structure variability affects starch digestion properties. Journal of Agricultural and Food Chemistry, 55(4), 1475–1479. Beysseriat, M., Decker, E.A., & McClements, D.J. (2006). Preliminary study of the influence of dietary fiber on the properties of oil-in-water emulsions passing through an in vitro human digestion model. Food Hydrocolloids, 20(6), 800–809. Biliaderis, C.G., & Izydorczyk, M.S. (2007). Functional food carbohydrates (1st ed.). Bocan Raton, FL: CRC Press. Brouns, F., Kettlitz, B., & Arrigoni, E. (2002). Resistant starch and “the butyrate revolution”. Trends in Food Science & Technology, 13(8), 251–261. Brown, I.L., Yotsuzuka, M., Birkett, A., & Henriksson, A. (2006). Prebiotics, synbiotics and resistant starch. Journal of Japanese Association for Dietary Fiber Research, 10, 1–9. Cai, C., Zhao, L., Huang, J., Chen, Y., & Wei, C. (2014). Morphology, structure and gelatinization properties of heterogeneous starch granules from high-amylose maize. Carbohydrate Polymers, 102, 606–614. Charalampopoulos, D., Wang, R., Pandiella, S.S., & Webb, C. (2002). Application of cereals and cereal components in functional foods: A review. International Journal of Food Microbiology, 79(1–2), 131–141. Chung, H. -J., Liu, Q., Lee, L., & Wei, D. (2011). Relationship between the structure, physicochemical properties and in vitro digestibility of rice starches with different amylose contents. Food Hydrocolloids, 25(5), 968–975.

Colonna, P., Leloup, V., & Buléon, A. (1992). Limiting factors of starch hydrolysis. European Journal of Clinical Nutrition, 46, S17–S32. Cui, S.W. (2005). Food carbohydrates: chemistry, physical properties and applications. Boca Raton, FL: Taylor and Francis. Devraj, R., Williams, H.D., Warren, D.B., Mullertz, A., Porter, C.J.H., & Pouton, C.W. (2013). In vitro digestion testing of lipid-based delivery systems: Calcium ions combine with fatty acids liberated from triglyceride rich lipid solutions to form soaps and reduce the solubilization capacity of colloidal digestion products. International Journal of Pharmaceutics, 441(1–2), 323–333. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17(1), 25–39. Elleuch, M., Bedigian, D., Roiseux, O., Besbes, S., Blecker, C., & Attia, H. (2011). Dietary fibre and fibre-rich by-products of food processing: Characterisation, technological functionality and commercial applications: A review. Food Chemistry, 124(2), 411–421. Englyst, H.N., Kingman, S.M., & Cummings, J.H. (1992). Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition, 46, 30–50. Espinal-Ruiz, M., Parada-Alfonso, F., Restrepo-Sanchez, L. -P., Narvaez-Cuenca, C. -E., & McClements, D.J. (2014). Impact of dietary fibers methyl cellulose, chitosan, and pectin on digestion of lipids under simulated gastrointestinal conditions. Food & Function, 5(12), 3083–3095. Fuentes-Zaragoza, E., Riquelme-Navarrete, M.J., Sánchez-Zapata, E., & Pérez-Álvarez, J.A. (2010). Resistant starch as functional ingredient: A review. Food Research International, 43(4), 931–942. Golding, M., & Wooster, T.J. (2010). The influence of emulsion structure and stability on lipid digestion. Current Opinion in Colloid & Interface Science, 15(1–2), 90–101. Hardacre, A.K., Yap, S.Y., Lentle, R.G., & Monro, J.A. (2015). The effect of fibre and gelatinised starch type on amylolysis and apparent viscosity during in vitro digestion at a physiological shear rate. Carbohydrate Polymers, 123, 80–88. Israelachvili, J.N. (2011). Intermolecular and surface forces (3rd ed.). Lodon, UK: Academic Press. Jiang, H.X., Lio, J.Y., Blanco, M., Campbell, M., & Jane, J.L. (2010). Resistant-starch formation in high-amylose maize starch during kernel development. Journal of Agricultural and Food Chemistry, 58(13), 8043–8047. Keogh, J.B., Wooster, T.J., Golding, M., Day, L., Otto, B., & Clifton, P.M. (2011). Slowly and rapidly digested fat emulsions are equally satiating but their triglycerides are differentially absorbed and metabolized in humans. Journal of Nutrition, 141(5), 809–815. Kosaraju, S.L. (2005). Colon targeted delivery systems: Review of polysaccharides for encapsulation and delivery. Critical Reviews in Food Science and Nutrition, 45(4), 251–258. Lehmann, U., & Robin, F. (2007). Slowly digestible starch — Its structure and health implications: A review. Trends in Food Science & Technology, 18(7), 346–355. Li, Y., & McClements, D.J. (2010). New mathematical model for interpreting pH-stat digestion profiles: Impact of lipid droplet characteristics on in vitro digestibility. Journal of Agricultural and Food Chemistry, 58(13), 8085–8092. Li, Y., Hu, M., Du, Y.M., Xiao, H., & McClements, D.J. (2011). Control of lipase digestibility of emulsified lipids by encapsulation within calcium alginate beads. Food Hydrocolloids, 25(1), 122–130. Lundin, L., Golding, M., & Wooster, T.J. (2008). Understanding food structure and function in developing food for appetite control. Nutrition and Dietetics, 65, S79–S85. Mayer, S., Weiss, J., & McClements, D.J. (2013). Behavior of vitamin E acetate delivery systems under simulated gastrointestinal conditions: Lipid digestion and bioaccessibility of low-energy nanoemulsions. Journal of Colloid and Interface Science, 404, 215–222. McClements, D.J. (2005). Food emulsions: Principles, practices and techniques. Boca Raton, FL: CRC Press. McClements, D.J. (2013). Edible lipid nanoparticles: Digestion, absorption, and potential toxicity. Progress in Lipid Research, 52(4), 409–423. McClements, D.J., & Li, Y. (2010). Structured emulsion-based delivery systems: Controlling the digestion and release of lipophilic food components. Advances in Colloid and Interface Science, 159(2), 213–228. McClements, D.J., & Xiao, H. (2014). Excipient foods: designing food matrices that improve the oral bioavailability of pharmaceuticals and nutraceuticals. Food & Function, 5(7), 1320–1333. McClements, D.J., Decker, E.A., & Park, Y. (2009). Controlling lipid bioavailability through physicochemical and structural approaches. Critical Reviews in Food Science and Nutrition, 49(1), 48–67. Mun, S., Kim, Y. -R., & McClements, D.J. (2015). Control of β-carotene bioaccessibility using starch-based filled hydrogels. Food Chemistry, 173(0), 454–461. Nugent, A.P. (2005). Health properties of resistant starch. Nutrition Bulletin, 30(1), 27–54. Pasquier, B., Armand, M., Guillon, F., Castelain, C., Borel, P., Barry, J.L., ... Lairon, D. (1996). Viscous soluble dietary fibers alter emulsification and lipolysis of triacylglycerols in duodenal medium in vitro. Journal of Nutritional Biochemistry, 7(5), 293–302. Sajilata, M.G., Singhal, R.S., & Kulkarni, P.R. (2006). Resistant starch — A review. Comprehensive Reviews in Food Science and Food Safety, 5(1), 1–17. Salvia-Trujillo, L., Qian, C., Martín-Belloso, O., & McClements, D.J. (2013). Influence of particle size on lipid digestion and β-carotene bioaccessibility in emulsions and nanoemulsions. Food Chemistry, 141(2), 1472–1480. Santipanichwong, R., & Suphantharika, M. (2009). Influence of different beta-glucans on the physical and rheological properties of egg yolk stabilized oil-in-water emulsions. Food Hydrocolloids, 23(5), 1279–1287. Sarkar, A., Goh, K.K.T., Singh, R.P., & Singh, H. (2009). Behaviour of an oil-in-water emulsion stabilized by beta-lactoglobulin in an in vitro gastric model. Food Hydrocolloids, 23(6), 1563–1569. Schirmer, M., Höchstötter, A., Jekle, M., Arendt, E., & Becker, T. (2013). Physicochemical and morphological characterization of different starches with variable amylose/amylopectin ratio. Food Hydrocolloids, 32(1), 52–63.

Please cite this article as: Tangsrianugul, N., et al., Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.11.004

N. Tangsrianugul et al. / Food Research International xxx (2015) xxx–xxx Sharma, A., Yadav, B.S., & Ritika (2008). Resistant starch: Physiological roles and food applications. Food Reviews International, 24(2), 193–234. Singh, H., & Ye, A. (2013). Structural and biochemical factors affecting the digestion of protein-stabilized emulsions. Current Opinion in Colloid & Interface Science, 18(4), 360–370. Singh, J., Dartois, A., & Kaur, L. (2010). Starch digestibility in food matrix: A review. Trends in Food Science & Technology, 21(4), 168–180. Steingoetter, A., Radovic, T., Buetikofer, S., Curcic, J., Menne, D., Fried, M., ... Wooster, T.J. (2015). Imaging gastric structuring of lipid emulsions and its effect on gastrointestinal function: A randomized trial in healthy subjects. American Journal of Clinical Nutrition, 101(4), 714–724. Thompson, L.U., Maningat, C.C., Woo, K., & Seib, P.A. (2011). In vitro digestion of RS4-type resistant wheat and potato starches, and fermentation of indigestible fractions. Cereal Chemistry, 88(1), 72–79. Torcello-Gomez, A., & Foster, T.J. (2014). Interactions between cellulose ethers and a bile salt in the control of lipid digestion of lipid-based systems. Carbohydrate Polymers, 113, 53–61. Vingerhoeds, M.H., Blijdenstein, T.B.J., Zoet, F.D., & van Aken, G.A. (2005). Emulsion flocculation induced by saliva and mucin. Food Hydrocolloids, 19(5), 915–922. Viuda-Martos, M., Lopez-Marcos, M.C., Fernandez-Lopez, J., Sendra, E., Lopez-Vargas, J.H., & Perez-Alvarez, J.A. (2010). Role of fiber in cardiovascular diseases: A review. Comprehensive Reviews in Food Science and Food Safety, 9(2), 240–258. Wang, S., & Copeland, L. (2013). Molecular disassembly of starch granules during gelatinization and its effect on starch digestibility: A review. Food & Function, 4, 1564–1580.

9

Wang, S.J., Wang, J.R., Yu, J.L., & Wang, S. (2014). A comparative study of annealing of waxy, normal and high-amylose maize starches: The role of amylose molecules. Food Chemistry, 164, 332–338. Warrand, J. (2006). Healthy polysaccharides —The next chapter in food products. Food Technology and Biotechnology, 44(3), 355–370. Winuprasith, T., & Suphantharika, M. (2015). Properties and stability of oil-in-water emulsions stabilized by microfibrillated cellulose from mangosteen rind. Food Hydrocolloids, 43, 690–699. Wu, B.C., Degner, B., & McClements, D.J. (2013). Creation of reduced fat foods: Influence of calcium-induced droplet aggregation on microstructure and rheology of mixed food dispersions. Food Chemistry, 141(4), 3393–3401. Yamaguchi, M., Kanemori, T., Kanemaru, M., Takai, N., Mizuno, Y., & Yoshida, H. (2004). Performance evaluation of salivary amylase activity monitor. Biosensors and Bioelectronics, 20(3), 491–497. Yang, Y., & McClements, D.J. (2013). Vitamin E bioaccessibility: Influence of carrier oil type on digestion and release of emulsified alpha-tocopherol acetate. Food Chemistry, 141(1), 473–481. Zhang, R., Zhang, Z., Zhang, H., Decker, E.A., & McClements, D.J. (2015). Influence of emulsifier type on gastrointestinal fate of oil-in-water emulsions containing anionic dietary fiber (pectin). Food Hydrocolloids, 45, 175–185. Zou, L.Q., Zheng, B.J., Liu, W., Liu, C.M., Xiao, H., & McClements, D.J. (2015). Enhancing nutraceutical bioavailability using excipient emulsions: Influence of lipid droplet size on solubility and bioaccessibility of powdered curcumin. Journal of Functional Foods, 15, 72–83.

Please cite this article as: Tangsrianugul, N., et al., Simulated gastrointestinal fate of lipids encapsulated in starch hydrogels: Impact of normal and high amylose corn starch, Food Research International (2015), http://dx.doi.org/10.1016/j.foodres.2015.11.004