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Food Hydrocolloids 25 (2011) 122e130

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Food Hydrocolloids

journal homepage: www.elsevier .com/locate/ foodhyd

Control of lipase digestibility of emulsified lipids by encapsulation withincalcium alginate beads

Yan Li a,b, Min Hu b, Yumin Du a, Hang Xiao b, David Julian McClements b,*

aDepartment of Environmental Science, Wuhan University, Wuhan 430079, ChinabDepartment of Food Science, University of Massachusetts, Amherst, MA 01003, USA

a r t i c l e i n f o

Article history:Received 17 March 2010Accepted 2 June 2010

Keywords:Lipid digestionEmulsionCalciumAlginateBeadsDelivery systemHydrogel particlesSatietyObesity

* Corresponding author. Tel.: +1 413 545 1019.E-mail address: mcclements@foodsci.umass.edu (D

0268-005X/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.foodhyd.2010.06.003

a b s t r a c t

Structured delivery systems, fabricated from natural lipids and polymers, are finding increasing use toimprove the oral bioavailability of poorly water-soluble drugs and nutraceuticals, as well as to control therelease of lipophilic bioactive molecules within the human gastrointestinal tract. This study focused onthe development of filled hydrogel particles to control the digestion and release of encapsulated lipids.These filled hydrogel particles were fabricated by trapping sub-micron lipid droplets within calciumalginate beads. These particles remained intact when the pH was varied from 1 to 7, but exhibited someshrinkage at pH 1 and 2. The free fatty acids released from the filled hydrogel particles after addition ofpancreatic lipase were monitored using a pH-stat in vitro digestion model. Encapsulation of lipid dropletswithin calcium alginate beads (d ¼ 2.4 mm) reduced the free fatty acids released from around 100% toless than 12% after 120 min. The rate and extent of lipid digestion increased with decreasing bead size(from 3.4 to 0.8 mm), decreasing degree of cross-linking (i.e., lower calcium or alginate concentrations),and decreasing triglyceride molecular weight (i.e., tributyrin > MCT z corn oil). These results haveimportant implications for the design of delivery systems to protect and release lipophilic bioactivecomponents within the human body, as well as to modulate satiety/satiation by controlling the rate oflipid digestibility.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

There is growing interest in the development of delivery systemsto encapsulate, protect and deliver bioactive components, such asnutraceuticals, pharmaceuticals, antioxidants and antimicrobials(Acosta, 2009; Augustin, Sanguansri, Margetts, & Young, 2001;McClements, Decker, Park, & Weiss, 2009b; Muller & Keck, 2004;Porter & Wasan, 2008; Velikov & Pelan, 2008). This knowledge isbeing used by the pharmaceutical industry to design emulsion-based delivery systems to carry active agents to specific locationswithin the GI tract and release them at a controlled rate (Porter &Wasan, 2008; Pouton, 2006). Similar systems are being developedby the food industry to encapsulate, protect and deliver bioactivefood components, such as antimicrobials, antioxidants, nutraceut-icals and flavors (Lundin & Golding, 2009; Lundin, Golding, &Wooster, 2008; McClements, Decker, & Park, 2009a; McClements,Decker, Park, & Weiss, 2007; Patten, Augustin, Sanguansri, Head, &Abeywardena, 2009; Singh, Ye, & Horne, 2009). Emulsion-based

.J. McClements).

All rights reserved.

delivery systems with controlled stability and digestibility withinthe GI tract have also been proposed as an effective means ofcontrolling appetite and therefore combating obesity (Beglinger &Degen, 2004; Cummings & Overduin, 2007; Karra & Batterham,2010; Langhans & Geary, 2010; Strader & Woods, 2005). A deliverysystem that retards lipid digestion and increases the amount ofdigestion products (e.g., free fatty acids) reaching the ileum, canstimulate the ileal brakemechanism that modulates hunger, satietyand satiation (Camilleri & Sweetser, 2007; Cummings & Overduin,2007; Little et al., 2007; Maljaars, Haddeman, Peters, & Masclee,2009; Maljaars, Peters, Mela, & Masclee, 2008).

In this manuscript, we focus on the utilization of hydrogelparticles to control the digestibility of emulsified lipids. Hydrogelparticles can be prepared entirely from edible biopolymers, such asproteins and polysaccharides, and are therefore particularly suit-able for use within the food industry (Burey, Bhandari, Howes, &Gidley, 2008; Malone & Appelqvist, 2003; McClements, Decker, &Weiss, 2007; Norton & Frith, 2001). Food-grade hydrogel particlescan be constructed using a number of different assembly principles,including aggregation/gelation methods, segregation/gelationmethods, injection/gelation methods, and macro-gel disruptionmethods (Burey et al., 2008; McClements et al., 2009b). In the

Y. Li et al. / Food Hydrocolloids 25 (2011) 122e130 123

present study, we used an injection/gelation method to fabricatefilled hydrogel beads consisting of lipid droplets dispersed withincalcium alginate beads. Filled hydrogel beads were formed byinjecting aqueous solutions containing protein-coated lipid drop-lets and alginate molecules into a calcium chloride solution (Fig. 1).The calcium ions promoted gelation of the alginate molecules,which trapped the emulsified lipids within the hydrogel beads.

Calcium alginate gels are already widely used in many food,pharmaceutical and medical applications (Wittaya-areekul,Kruenate, & Prahsarn, 2006). Alginates are linear copolymerscomposed of two monomeric units, b-1-4-linked D-mannuronicacid (M) and a-1,4-linked L-guluronic (G) acid (Helgerud, Gaserod,Fjaereide, Andersen, & Laresen, 2010). They form gels in the pres-ence of calcium because of the formation of ‘egg-box junctions’where Ca2þ ions act as cationic bridges between anionic GeG richsequences along the polysaccharide backbone (George & Abraham,2006). Previously, it has been reported that calcium alginate beadsare able to protect bioactive molecules from the harsh acidicenvironment of the stomach (Chen et al., 2004). Studies of thebehavior of alginate beads in the gastrointestinal (GI) tract haveshown that they tend to shrink under gastric conditions but swellunder small intestinal conditions (Hoad et al., 2009; Rayment et al.,2009). This characteristic may have important consequences for thedevelopment of targeted delivery systems, which must protecta bioactive component within the stomach but deliver it furtheralong the GI tract. Furthermore, it has been shown that thechemically inert environment present within alginate beads allowsfor the entrapment of a wide range of bioactive substances, cellsand drug molecules (Griffith, 2000). Finally, the physical andchemical properties of alginates (e.g., porosity, degradability) caneasily be modified using mild processing conditions (Gombotz &Wee, 1998).

In our study, we examined the impact of calcium alginate beadcharacteristics on the digestibility of encapsulated lipid dropletsusing an in vitro digestion model to simulate the small intestine.Our objective was to determine whether calcium alginate beadscould be designed to control the digestion of triacylglycerols withinthe GI tract. Before an ingested triacylglycerol can be absorbed by

Fig. 1. Schematic representation of the method used to form the filled calcium alginatebeads: an alginate solution containing lipid droplets was dripped into a stirredaqueous solution of calcium chloride.

the human body, it must be hydrolyzed into diacylglycerols, mon-oacylglycerols, and free fatty acids, through the action of digestiveenzymes such as gastric and pancreatic lipase (Lowe, 2002). Theseenzymes must adsorb to the lipid droplet surfaces to come intoclose proximity to the lipid substrate and catalyze digestion(Wickham, Garrood, Leney, Wilson, & Fillery-Travis, 1998). Anumber of factors may impact the ability of lipase to adsorb to lipiddroplet surfaces: bile salts and phospholipids e which are neededto displace other surface active materials and to solubilize digestionproducts; calcium e which is needed for enzyme activity and toremove long chain fatty acids from droplet surfaces; co-lipase e

which helps lipase attach to droplet surfaces (Bauer, Jakob, &Mosenthin, 2005; Reis et al., 2009a; Reis, Holmberg, Watzke,Leser, & Miller, 2009b; Wickham et al., 1998).

In principle, the digestion of emulsified lipids can be controlledby altering the composition, structure and/or integrity of thematerials surrounding them to alter the ability of lipase to comeinto close contact with the lipid droplet surfaces. Previously, wehave controlled lipid digestibility by coating individual lipid drop-lets with layers of indigestible dietary fibers (Beysseriat, Decker, &McClements, 2006; Klinkesorn & McClements, 2009; Mun,Decker, Park, Weiss, & McClements, 2006). Nevertheless, thesestudies found that dietary fiber coatings do not strongly inhibitlipolysis, presumably because they were either permeable topancreatic lipase or were easily displaced from the lipid dropletsurfaces, thereby enabling the digestive enzymes to come into closeproximity to the lipid substrates. We hypothesized that embeddinglipid droplets within calcium alginate beads may be a more effec-tive means of controlling lipid digestion, because these beads havepreviously been shown to be highly resistant to disruption under GItract conditions (Chen et al., 2004). Hence, the digestive enzymeswill have to diffuse into and through the hydrogel bead matrixbefore they can access the emulsified lipids trapped inside.

2. Materials and methods

2.1. Materials

Powdered lactoglobulin (b-Lg) was obtained fromDavisco FoodsInternational (Lot # JE 002-8-415, Le Sueur, MN). Medium chaintriglyceride (MIGLYOL 812N) (MCT) was purchased from SasolGermany Gmbh. (Witten, Germany). Glyceryl tributyrate (tribu-tyrin) (TB) (>98%) was obtained from the SigmaeAldrich ChemicalCompany (St. Louis, MO). Corn oil (CO) was purchased from a localsupermarket and was used without further purification. Sodiumalginate (Alginic acid, sodium salt; Product Number 180947;Viscosity of 1% aqueous solution ¼ 20e40 cps), bile extract(porcine, B8613) and lipase from porcine pancreas (activity � 2.0USP units/mg, Type II, L3126) were obtained from SigmaeAldrich.The composition of bile extract (BS, batch# 058K0066, Sigma-eAldrich) has been reported: total bile salt content ¼ 49 wt%; with10e15% glycodeoxycholic acid, 3e9% taurodeoxycholic acid, 0.5e7%deoxycholic acid, 1e5% hyodeoxycholic acid, and 0.5e2% cholicacid; 5 wt% phosphatidyl choline (PC); Ca2þ< 0.06wt%; CMC of bileextract 0.07 � 0.04 mM; the mole ratio of BS to PC being around 15to 1. Type II lipase contains amylase and protease, as well as lipases.It has been reported that lipase activity is 100e400 units/mgprotein (using olive oil) and 30e90 units/mg protein (using tri-acetin) for 30 min incubation. Analytical grade hydrochloric acid(HCl) and sodium hydroxide (NaOH) were also purchased fromSigmaeAldrich. Calcium chloride (CaCl2.2H2O) was obtained fromFisher Scientific. Purified water from a Nanopure water system(Nanopure Infinity, Barnstead International, Dubuque, IA) was usedfor the preparation of all solutions.

Y. Li et al. / Food Hydrocolloids 25 (2011) 122e130124

2.2. Solution preparation

An emulsifier solution was prepared by dispersing 1.0 wt%powdered b-Lg into buffer solution (5 mM phosphate, pH 7.0) andstirring for at least 2 h. The protein solution was then stored at 4 �Covernight to ensure complete hydration. An alginate solution(4.0 wt%) was made by dispersing alginate powder into buffersolution (5 mM phosphate, pH 7.0) and stirring overnight. Calciumsolution was prepared by dissolving a certain amount of CaCl2powder into double distilled water.

2.3. Emulsion preparation

A primary emulsion was prepared by homogenizing 10 wt% oilphase with 90 wt% aqueous phase (1.0 wt% b-Lg, pH 7.0) witha high-speed blender for 2 min (M133/1281-0, Biospec Products,Inc., ESGC, Switzerland) followed by five passes at 9000 psi througha high pressure homogenizer (Microfluidics M-110Y, F20Y 75 mminteraction chamber, Newton, MA). Most of the experiments werecarried out usingMCTas the oil phase, but in some experiments theoil phase consisted of either 100% CO or 50 wt% CO/50 wt% TB.These emulsions were referred to as stock emulsions.

2.4. Unfilled bead preparation

Unfilled calcium alginate beads were formed using a syringe todrip 6 mL of 2 w/v% sodium alginate solution into 10 mL of 10 w/v%CaCl2 solution with continuous stirring (Fig. 1). The beads wereallowed to crosslink with Ca2þ for 30min at room temperature, andthen kept overnight in CaCl2 gelling solution to obtain stronglygelled beads. Before experiments, beads were collected by filtrationand washed with distilled water to remove excess Ca2þ on thesurface. The beads were then either stored and used in their wetform or lyophilized and kept in a desiccator prior to beingrehydrated.

2.5. Filled bead preparation

Filled calcium alginate beads were prepared using a similarmethod as that for unfilled beads. A droplet-alginate mixture wasprepared by mixing stock emulsion and alginate solution and thenstirring for at least 30 min to ensure complete mixing. This mixturewas then left to stand for a further 30 min to allow any air bubblesto rise to the surface before preparing the beads. Filled beads wereprepared using a syringe to drip 6 mL emulsion-alginate solution(containing 300 mg or 5 w/v% oil) into 10 mL of CaCl2 solution withcontinuous stirring (Fig. 1). The beads were allowed to crosslinkwith Ca2þ for 30min at room temperature, and then kept overnightin the same CaCl2 gelling solution to form strongly gelled beads.Beads were then collected by filtration and washed with distilledwater to remove excess Ca2þ on the surface. In some experiments,the concentrations of the alginate and calcium solutions werevaried to study their effects on lipid digestion. Beads with differentsizes were prepared by controlling the dripping rate and needlesize. The rate and extent of lipid digestionwas determined on these“wet” calcium alginate beads, i.e., beads were not dried and storedprior to use (which may have altered their properties).

2.6. Physicochemical characteristics of the beads

The overall appearance of the calcium alginate beads wasobserved using a digital camera (PowerShot SX110 IS, Canon, USA).The dimensions (diameter) of the beads was determined usinga digital micrometer (0e300 mm, EC10, High Precision DigitalCaliper, Tresna Instruments, Guilin, China). The diameter of at least

10 individual beads was measured and the mean and standarddeviation was calculated. When sliced open with a sharp knife thebeads had a uniform milky appearance, which suggested that thelipid droplets were evenly distributed throughout them. In futurestudies, it would be useful to examine the internal microstructureand pore size of the beads using microscopy methods.

2.7. Dynamic in vitro digestion model

The in vitro digestion model used in this study was a modifica-tion of those described previously (Mun et al., 2006; Zangenberg,Mullertz, Kristensen, & Hovgaard, 2001). Beads containing300 mg oil were placed in a glass beaker containing 30 mL buffersolution (5 mM phosphate, pH 7.0) and then incubated in a waterbath at 37.0 �C for 10 min. The system was then adjusted to pH 7using NaOH or HCl solutions. Then 5.0 mL of bile extract solution(187.5 mg bile extract dissolved in phosphate buffer, pH 7.0, 37.0 �C)and 1.0 mL of CaCl2 solution (187.5 mM CaCl2 in double distilledwater, 37.0 �C) were added to the samples with continuous stirringand the system was adjusted back to pH 7 if required.

1.5 mL of freshly prepared pancreatic lipase suspension (60 mgpancreatic lipase powder dispersed in phosphate buffer, pH 7,37.0 �C) was added to the above mixture. The final compositionwithin the reaction vessel was 300 mg lipid, 5 mg/mL bile extract,1.6 mg/ml pancreatic lipase, and 5 mM CaCl2. A pH-stat automatictitration unit (Metrohm, USA Inc.) was then used to automaticallymonitor the pH and maintain it at pH 7.0 by titrating appropriateconcentrations of NaOH solution to neutralize any free fatty acids(FFA) formed. The volume of NaOH (0.25 M NaOH) added to thesamples was recorded, and used to calculate the concentration offree fatty acids generated by lipolysis.

The percentage of free fatty acids released was calculated fromthe number of moles of NaOH required to neutralize the FFAdivided by the number of moles of FFA that could be produced fromthe triacylglycerols if they were all digested (assuming 2 FFAproduced per 1 triacylglycerol molecule):

%FFA ¼ 100� VNaOH �mNaOH �MLipid

wLipid � 2

!(1)

Here VNaOH is the volume of sodium hydroxide required toneutralize the FFA produced (in mL), mNaOH is the molarity of thesodium hydroxide solution used (in M), wLipid is the total weight ofoil initially present in the reaction vessel (0.3 g), and MLipid is themolecular weight of the oil (based on their average fatty acidcompositions the molecular weights of CO and MCT were taken tobe 800 and 500 g mol�1, respectively).

It should be noted that the total amount of FFA’s that can bereleased may be higher than that calculated based on the aboveassumption (i.e., 2 FFA per TAG), e.g., if FFA come from other sources(such as phospholipids or bile) or if some of the MAGs are brokendown to FFAs and glycerol.

2.8. Statistical analysis

All experiments were performed at least twice on freshlyprepared samples. The results were then reported as averages andstandard deviations of these measurements.

3. Results and discussion

3.1. Influence of calcium alginate beads on digestion

Initially, we carried out an experiment to determine the influ-ence of encapsulating oil droplets within calcium alginate beads on

20

25

Large Beads

Medium Beads

Y. Li et al. / Food Hydrocolloids 25 (2011) 122e130 125

the rate and extent of lipid digestion. Three samples were preparedcontaining the same total amount of lipid (MCT): (i) non-encap-sulated oil droplets; (ii) non-encapsulated oil droplets mixed withunfilled calcium alginate beads; and, (iii) oil droplets encapsulatedinside calcium alginate beads (Fig. 2). The filled and unfilledcalcium beads were formed using similar calcium and alginateconcentrations, as well as similar preparation conditions so thatthey had the same degree of cross-linking and similar particlediameters (d ¼ 2.4 � 0.2 mm). The non-encapsulated (free) oildroplets underwent complete digestion (z100% FFA released)within the first 25 min of hydrolysis, with the amount of FFAreleased increasing steeply during the first 5 min and then levelingoff at longer digestion times (Fig. 2). On the other hand, the oildroplets encapsulated within the calcium alginate beads weredigested at a much slower rate, with<8% FFA being released withinthe first 25min of digestion. Even after 120min,<12% FFA had beenreleased (data not shown), which suggested that the alginate beadswere highly effective at retarding lipid digestion. A number ofphysicochemical phenomena might account for the decreaseddigestion rate for the encapsulated oil droplets. First, pancreaticlipase has to adsorb to the oil droplet surfaces, so that it can comeinto close proximity to the lipid substrate (emulsified tri-acyglycerols), before any digestion can occur (Reis et al., 2009b). Inthe absence of calcium alginate beads, the pancreatic lipase ispresent in the aqueous medium that surrounds each lipid dropletand can therefore rapidly adsorb to the droplet surfaces (in thepresence of bile and co-lipase), thereby initiating the lipolysisprocess. On the other hand, when oil droplets are trapped withincalcium alginate beads the pancreatic lipase (and other compo-nents) must penetrate into the beads and diffuse through the gelnetwork before it can reach the droplet surfaces. The rate ofdiffusion of the pancreatic lipase will depend on the pore size of thegel network and on any specific interactions of the enzymewith themolecules that comprise the gel network e.g., electrostatic orhydrophobic interactions (Gombotz & Wee, 1998). Second, the freefatty acids generated by the action of pancreatic lipase must beremoved from the droplet surfaces otherwise the lipolysis reactionis retarded (Bauer et al., 2005; Fave, Coste, & Armand, 2004). Themovement of FFAs and protons away from the droplet surfaces may

0

20

40

60

80

100

120

0 5 10 15 20 25Digestion Time (min)

%

A

F

F

Free Droplets

Free Droplets + Beads

Encapsulated Droplets

Fig. 2. Dependence of the percentage of free fatty acids released from emulsified tri-acylglycerol (MCT) droplets due to lipase digestion on the structure of the system: (i)free droplets; (ii) free droplets þ calcium alginate beads; (iii) droplets encapsulatedinside calcium alginate beads. The beads were prepared by dripping either 0 or 5 w/v%lipid and 2 w/v% alginate solution into 10 w/v% CaCl2 solutions.

also be slowed down by the gel network surrounding the oildroplets. Previous studies have shown that if the FFAs are notremoved from the lipid droplet surface they can inhibit the inter-facial lipolysis reaction (Dahan & Hoffman, 2008; Zangenberg et al.,2001). Interestingly, when non-encapsulated (free) oil dropletswere mixed with the same type and concentration of unfilledcalcium alginate beads, there was no inhibition of lipolysis (Fig. 2).This demonstrated that the beads themselves were not responsiblefor retarding the lipid digestion process (e.g., by trapping or bindingthe pancreatic lipase). Instead, the delayed digestion rate appears tobe due to the fact that the droplets are trapped within the gelnetwork, which increases the diffusion path length of the pancre-atic lipase (and possibly other digestive components) to the drop-lets, and of the free fatty acids away from the droplet surfaces.

3.2. Influence of bead dimensions on lipid digestibility

If the retardation of the lipid digestion process is due to the factthat pancreatic lipase must diffuse into the calcium alginate beadsbefore the inner oil droplets can be digested, then onewould expectthe digestion rate to depend on bead dimensions. We thereforeprepared a series of calcium alginate beads with different averagesizes, but containing the same amount of oil droplets. Calciumalginate beads with mean diameters of 0.82 � 0.15, 2.37 � 0.16, and3.37 � 0.21 mm were prepared and are referred to as “small”,“medium” and “large” beads, respectively.

The rate and extent of lipid digestibility increased withdecreasing bead diameter (Fig. 3), e.g., 11.3, 11.8 and 20.5% FFAswere released after 2 h digestion for large, medium and smallbeads. The most likely reason for this observation is that the frac-tion of oil droplets close to the bead surface increases as the meanbead diameter decreases:

0

5

10

15

0 10 20 30 40 50 60 70 80 90 100 110 120

%

A

F

F

Digestion Time (min)

Small Beads

Fig. 3. Dependence of the percentage of free fatty acids released from filled calciumalginate beads due to lipase digestion on bead diameter: small beads,d ¼ 0.82 � 0.15 mm, medium beads, d ¼ 2.37 � 0.16 mm, and large beadsd ¼ 3.37 � 0.21 mm. Filled hydrogel particles were prepared by dripping 5 w/v% lipidand 2 w/v% alginate solution into 10 w/v% CaCl2 solutions. The inset shows photo-graphs of samples taken before and after digestion.

100% 0.5 mm

Y. Li et al. / Food Hydrocolloids 25 (2011) 122e130126

Fshell ¼ Vshell ¼ 1��1� 2d

�3

(2)

0%

20%

40%

60%

80%

0 20 40 60 80 100 120

d e b r o s b A

t n u o

m

A

Time (min)

4 mm

2 mm

1 mm

Fig. 4. Predicted dependence of the amount of lipase (¼m(t)/m(N)) that has diffusedinto filled calcium alginate beads with time for beads with different diameters(0.5e4 mm, shown next to curves). It was assumed that the alginate beads initiallycontained no lipase, and that they had pore diameters of 20 nm.

0

5

10

15

20

25

0 20 40 60 80 100 120

%

A

F

F

Digestion Time (min)

100%SB: 0% MB

75% MB: 25% MB

50% SB: 50% MB

25% SB: 75% MB

0% SB: 100% MB

Fig. 5. Dependence of the amount of free fatty acids released from filled calciumalginate beads due to lipase digestion on the ratio of small beads (d ¼ 0.82 mm) tomedium beads (d¼ 2.37 mm). Filled hydrogel particles were prepared by dripping 5 w/v% lipid and 2 w/v% alginate solution into 10 w/v% CaCl2 solutions.

Vbead d

Here, Fshell is the fraction of oil droplets within a shell ofthickness d from the bead surface, Vbead and Vshell are the volumesof the total bead and the shell layer respectively, and d is thediameter of the overall bead. If we assume that the pancreatic lipasecan diffuse a distance d into the alginate beads in a certain time,which is largely independent of oil droplet size, then Equation (2)shows that the fraction of oil droplets accessible to digestionincreases with decreasing bead size. An estimate of the time takenfor pancreatic lipase to diffuse into the lipid droplets is given by thefollowing expression (Crank, 1975):

F ¼ MðtÞMðNÞ ¼ 1� 6

p2

XNn¼1

1n2

exp

� Dgeln

2p2t

a2

!(3)

Here, F is the fraction of protein that has diffused into thehydrogel particle at time t,M(t) andM(N) are the concentrations ofprotein present within the hydrogel particle at time t and at equi-librium, n is an integer, a is the radius of the hydrogel beads, andDgel is the diffusion coefficient of protein (pancreatic lipase)through the hydrogel matrix. This equation is based on diffusion ofprotein into an empty spherical hydrogel particle that is submergedin a liquid maintaining a constant protein concentration (Crank,1975). The following equation can be used to predict the diffusioncoefficient of a protein through a polymer gel network (Chan &Neufeld, 2009):

Dgel ¼ Dwexp

� p

rH � rfzþ 2rf

!!(4)

Here, Dw is the diffusion of the protein through pure water, rH isthe hydrodynamic radius of the protein molecule (pancreaticlipase), rf is the cross-sectional radius of the polymer chains in thegel network (alginate), and z is the mesh pore diameter of thenetwork. The following values have been reported for alginate gels:pore diameter (z)¼ 4e400 nm; alginate chain radius (rf)¼ 0.83 nm(Chan & Neufeld, 2009). The radius of gyration (rH) of pancreaticlipase inwater has been estimated to be 1.67 nm (Peters, vanAalten,Edholm, Toxvaerd, & Bywater, 1996). The diffusion coefficient ofprotein in water is given by: Dw ¼ kBT/6phrH, where kB is Boltz-man’s constant, T is absolute temperature, and h is solvent viscosity.We have used these equations to predict the impact of alginatebead dimensions on the time for pancreatic lipase molecules todiffuse into the beads (Fig. 4). For these calculations, we assumedthat the pore diameter (z) was 20 nm. These predictions indicatethat the amount of protein present within the hydrogel particlesshould increase with time more rapidly for smaller particles thanlarger particles, which supports the general trends observed in ourexperimental data. Nevertheless, the calculated diffusion profilesfor protein into the hydrogel beads appear to be appreciably fasterthan the rate of FFA release (Fig. 3). There may be a number ofpossible reasons to account for this: (i) the actual pore size of thegel network in the alginate beadsmay have been less than that usedin the theoretical calculations; (ii) the actual size of the pancreaticlipase molecules may have been bigger than the one used in thecalculations, e.g., if pancreatic lipase moved as a molecular complexwith co-lipase; (iii) the pancreatic lipase molecules may havephysically interacted with molecules in the gel network so thattheir movement was hindered, e.g., electrostatic interactionsbetween positive patches on pancreatic lipase and negative patcheson alginate; (iv) the slow rate of FFA movement away from the oildroplet surfaces may have caused the pancreatic lipase reaction tobe retarded; (v) the slow diffusion of protons (Hþ) from the droplet

surface may have reduced the expected rate of pH reduction; (vi)encapsulation of the lipid droplets within hydrogel beads may haveprevented bile salts from getting to the lipid droplet surfacesthereby altering the normal digestion process.

These experiments indicate that an effective way of controllinglipid digestion and release within the GI tract is to control the sizeof the calcium alginate beads. An alternative means of controllingthe digestion and release rate is to use a mixture of different sizedbeads. We therefore prepared a series of samples with the sameoverall oil droplet content, but with the droplets encapsulated indifferent ratios of small (d ¼ 0.82 � 0.15 mm) and medium(d ¼ 2.37 � 0.16 mm) size alginate beads. The rate of lipid digestionprogressively increased as the fraction of smaller beads within themixture increased from 0 to 100% (Fig. 5). This experiment suggeststhat the rate of lipid digestion can be controlled quite precisely byvarying the fraction of different sized beads used to encapsulate theoil droplets.

Y. Li et al. / Food Hydrocolloids 25 (2011) 122e130 127

3.3. Influence of bead composition on lipid digestibility

The composition of calcium alginate beads can be varied byaltering either the alginate concentration within the syringe or thecalcium concentration in the hardening solution used to preparethem (Fig. 1). Altering the alginate or calcium concentration wouldbe expected to alter the internal structure of the calcium alginatebeads, particularly the pore size of the gel network of aggregatedalginate molecules (Chan & Neufeld, 2009; Gombotz & Wee, 1998).A decrease in pore size should slow down the diffusion of pancre-atic lipase into the calcium alginate beads, and therefore decreasethe rate of lipid digestion (Chan & Neufeld, 2009). We thereforeexamined the influence of both alginate and calcium concentrationon the lipid digestion rate. The oil type used to prepare the initialemulsions in these experiments was MCT, and only medium-sizedcalcium alginate beads were formed.

3.3.1. The effect of alginate concentrationA series of filled medium-sized alginate beads were prepared by

using different alginate concentrations in the syringe (0.25e2.0 wt%) but the same amount of CaCl2 concentration in the hardeningsolution (10 wt%). Stable spherical beads could not be formed atlower alginate concentrations. The rate and extent of lipid digestiondecreased as the alginate concentrationwithin the beads increased(Fig. 6). For example, after 2 h of digestion the percentage of freefatty acids released were 84.1 � 9.2%, 60.6 � 2.2%, 30.6 � 1.1%,17.5 � 1.8% for 0.25%, 0.5% 1.0%, 2.0% alginate, respectively. Thisdecrease can at least partly be attributed to the formation ofa denser gel network within the beads with increasing alginatecontent, which would slow down the movement of the pancreaticlipase molecules into the beads (Chan & Neufeld, 2009; Gombotz &Wee, 1998). Visual comparison of the appearance of the beadsbefore and after 2 h digestion indicated that they maintained theiroriginal shape when the alginate concentration was �1.0%, butwere broken down to smaller particles at lower alginate concen-trations. Matrix erosion/degradation may therefore also partlyaccount for the observed increase in the rate and extent of lipiddigestion when the alginate concentration was decreased.

3.3.2. The effect of Ca2þ concentrationA series of filled medium-sized alginate beads was prepared

using the same alginate concentration in the syringe (2.0 wt%), but

0

20

40

60

80

100

120

0 20 40 60 80 100 120Digestion Time (min)

%

A

F

F

Free Droplets 0.25% alginate 0.5% alginate 1.0% alginate 2.0% al inate

Fig. 6. Amount of free fatty acids released from free droplets and from calcium alginatebeads due to lipase digestion for beads prepared using different alginate concentra-tions. Filled hydrogel particles were prepared by dripping 5 w/v% lipid and 0e2 w/v%alginate solutions into 10 w/v% CaCl2 solutions.

different amounts of CaCl2 concentration in the hardening solution(0.5e10 wt%). The rate and extent of lipid digestion decreased asthe calcium concentration used to harden the beads increased(Fig. 7). For example, after 2 h of digestion the percentage of freefatty acids released were 50.0 � 0.8%, 45.4 � 2.0%, 26.4 � 8.4%,19.1 � 3.5%, 16.9 � 1.2 for 0.5%, 1.0%, 2.0% 5.0%, 10.0% Ca2þ,respectively. The decrease in the rate of FFA production withincreasing calcium concentration can again be attributed to theformation of a denser gel network, which would retard the diffu-sion of the pancreatic lipase into the beads, and thereby decreasethe digestion rate (Chan & Neufeld, 2009; Gombotz &Wee,1998). Inthis case, the shape of the beads did not change appreciably afterdigestion for all calcium concentrations used, suggesting thatdegradation/erosion was not an important mechanism promotinglipid digestion. These results imply that the digestion rate ofencapsulated lipids can also be controlled by varying the compo-sition of the calcium alginate beads.

3.3.3. Modeling the impact of gel pore sizeThe impact of pore dimensions on the rate of pancreatic lipase

diffusion into the hydrogel beads was examined by makingpredictions using Equation (3). An alginate bead diameter of2.4 mm was used in the calculations to mimic the medium-sizedbeads used, and a range of z-values was chosen to reflect thosereported in the literature i.e., 4 to 400 nm (Chan & Neufeld, 2009).The predictions indicate that the rate of pancreatic lipase diffusioninto the alginate beads should decrease with decreasing pore size(Fig. 8), which supports the general trends observed in our exper-imental observations (Figs. 6 and 7). Nevertheless, the amount ofpancreatic lipase present within the alginate beads appears toincrease much faster than the FFA produced, which again suggeststhat there may be another mechanism determining the rate of FFAproduction, such as the accumulation of FFA at the droplet surfacesretarding the pancreatic lipase reaction.

In future studies, it would be useful to quantify the pore size andinternal structure of the calcium alginate beads, as well as tomonitor the time-dependence of the penetration of enzyme andbile salts into the beads and themovement of reaction products out.This information could then be used to provide a more mechanisticunderstanding of the influence of bead pore size on their functionalperformance.

0

20

40

60

80

100

120

0 20 40 60 80 100 120

Digestion Time (min)

%

A

F

F

Free Droplets

0.5% Ca

1.0% Ca

2.0% Ca

5.0% Ca

10.0% Ca

Fig. 7. Amount of free fatty acids released from free droplets and from calcium alginatebeads due to lipase digestion for beads prepared using different calcium concentra-tions. Filled hydrogel particles were prepared by dripping 5 w/v% lipid and 2 w/v%alginate solution into 0.5e10 w/v% CaCl2 solutions.

0%

20%

40%

60%

80%

100%

0 20 40 60 80 100 120

d e b r o s b A

t n u o

m

A

Time (min)

4 nm

10 nm 40 nm

400 nm

Fig. 8. Predicted dependence of the amount of lipase (¼m(t)/m(N)) that has diffusedinto filled calcium alginate beads with time for beads with different pore diameters(4e400 nm shown next to lines). It was assumed that the alginate beads hada diameter of 2.4 mm.

Y. Li et al. / Food Hydrocolloids 25 (2011) 122e130128

3.4. Influence of encapsulated lipid type on lipid digestion

For certain applications, it may be desirable to encapsulatedifferent kinds of triacylglycerol oils within calcium alginate beads.Hence, we examined the impact of oil type on the lipid digestionprocess. We prepared filled calcium alginate beads from emulsionscontaining low (tributyrin, TB), medium (MCT) or high (corn oil,CO) molecular weight triglycerides as the oil phase. It was notpossible to prepare stable emulsions when tributyrin was usedsolely as the oil phase because of rapid droplet growth caused byOstwald ripening (OR). Rapid OR occurs in this system becausetributyrin has a relatively high-water-solubility, and therefore itrapidly diffuses from small to large droplets across the interveningaqueous phase (Kabalnov & Shchukin, 1992; Wooster, Golding, &Sanguansri, 2008).

As described previously, a lipid phase consisting of 50% tribu-tyrin and 50% corn oil was therefore used to inhibit OR in theseemulsions (Li, Le Maux, Xiao, & McClements, 2009). Medium-sized

0

20

40

60

80

100

0 10 20 30 40 50 60

%AF

F

Digestion Time (min)

MCT

Corn Oil/Tributyrin

Corn Oil

a b

Fig. 9. (a) Amount of free fatty acids released from free droplets (non-encapsulated) due toAmount of free fatty acids released from filled calcium alginate beads due to lipase digestio5 w/v% lipid and 2 w/v% alginate solution into 10 w/v% CaCl2 solutions.

filled calcium alginate beads were used in this study, which wereprepared using 2% alginate in the syringe and 10% calcium chloridein the hardening solution (Fig. 1). Visual observation of the beadsbefore and after digestion indicated that they remained intact.

Initially, we examined the impact of oil type on lipid digestionfor non-encapsulated oil droplets (Fig. 9a). The rate and extent ofFFA release was greater for MCT than for corn oil, which haspreviously been attributed to the fact that long chain fatty acids areless water-soluble than either medium or short chain fatty acids(Sek, Porter, Kaukonen, & Charman, 2002). Consequently, longchain fatty acids tend to accumulate at the oil droplet surface andinhibit pancreatic lipase activity, unless they are removed throughprecipitation by calcium or incorporation into bile salt micelles. Thefact that not all of the corn oil was digested may therefore beattributed to the relatively low calcium (5 mM) and bile (5 mg/mL)levels used in this study. On the other hand, the relatively highwater-solubility of the medium chain fatty acids generated whenMCT is digested are able to move into the surrounding aqueousphase and therefore do not inhibit pancreatic lipase (Armand et al.,1992). The conclusions of the pH-Stat experiments were supportedby visual observation of the samples after the digestion had beencompleted: a distinct layer of undigested oil was observed on top ofthe emulsions containing CO, but not on the emulsions containingMCT. The emulsion containing CO/TB droplets was digested at a ratethat was between the MCT and corn oil (Fig. 9a). For example, after50 min of digestion, the percentages of free fatty acids releasedwere approximately 40%, 100%, and 88% for CO, MCT, and CO/TB,respectively. This suggests that the majority of the TB had beendigested, while some of the CO remained undigested.

We also compared the impact of oil type on lipid digestion offilled calcium alginate beads (Fig. 9b). The relatively high finalcalcium ion concentrations in these systems should be sufficient toprecipitate any long chain FFAs formed, so that pancreatic lipaseactivity should not be inhibited in the CO system. The rate andextent of lipid digestion was relatively slow for beads loaded witheither CO or MCT droplets, e.g., <10% FFAs were released afternearly 2 h of digestion. This effect can be attributed to the fact thatboth these oils have a relatively low water-solubility, and thereforewould be expected to remain within the calcium alginate beads.Consequently, the time taken for pancreatic lipase to diffuse intothe beads would be expected to limit the digestion process. On theother hand, there was an appreciable increase in the amount of FFA

0

5

10

15

20

25

30

35

40

45

0 20 40 60 80 100 120

%AF

F

Digestion Time (min)

MCT

Corn Oil/Tributyrin

Corn Oil

lipase digestion for oil-in-water emulsions prepared using different types of lipid. (b)n for different types of oil phases. Filled hydrogel particles were prepared by dripping

Y. Li et al. / Food Hydrocolloids 25 (2011) 122e130 129

released with time for the filled beads containing CO/TB, with over35% being released after nearly 2 h digestion (Fig. 9b). A possibleexplanation for the increased rate of digestion in this system is thefact that tributyrin has a relatively high-water-solubility (Woosteret al., 2008) and so some of it may have diffused out of the beadsduring the experiment, where it became more accessible todegradation by pancreatic lipase. Besides, butyric acid producedfrom digestion of TB is more soluble than the longer chain fattyacids of CO, which could be easier to diffuse out of the beads duringlipolysis.

3.5. pH stability of filled calcium alginate beads

In reality, delivery systems experience a range of pH and ionicstrength values as they pass from the mouth, to the stomach, to thesmall intestine, and eventually to the colon. Hence, it is useful toinvestigate the influence of pH and ionic strength on the propertiesof the calcium alginate beads. Medium-sized filled calcium alginatebeads were prepared at neutral pH using 2% alginate in the syringeand 10% calcium chloride in the hardening solution (Fig. 1). Thebeads formed were then placed into a series of continuously stirredbuffer solutions with different pH values (1e7) and ionic strengths(0 or 150 mM NaCl) and stored for 37 �C for 24 h. Bead dimensionswere measured after they had been washed with distilled water,freeze-dried, and rehydrated prior to use.

The size of the beads was strongly dependent on pH, but not onionic strength (Fig. 10). In general, the bead diameter decreasedwith decreasing pH, with the biggest reduction in dimensionsoccurring when the pH was reduced from 3 to 2. Recent studiesusing simulated gastrointestinal fluids have also shown that algi-nate beads tend to shrink under highly acid gastric conditions andswell in neutral intestinal conditions (Rayment et al., 2009). Thischange in bead dimensions can be attributed to the electricalcharacteristics of the alginate molecules, which have a pKa around3.5 and therefore tend to lose their negative charge at lower pHvalues. The pH-dependence of bead dimensions has importantconsequences for the development of delivery systems to protectactive components within the stomach, but deliver them in thesmall or large intestines. When calcium alginate beads shrink thereis a reduction in the pore size of the alginate network (Chan &Neufeld, 2009), which should retard the diffusion of digestiveenzymes into the beads, thereby inhibiting the digestion processes.In addition, the diffusion of any encapsulated components out of

1

1.5

2

2.5

3

3.5

1

) m

m

(

r e t e m

a i

D

d a e B

pH

no NaCl

150 mM NaCl

2 3 4 5 6 7

Fig. 10. Effect of pH on the diameter of filled calcium alginate beads in the presence of0 or 150 mM NaCl. The beads tend to shrink when incubated at low pH values (pH 1and 2).

the alginate beads decreases when they shrink (Chan & Neufeld,2009). On the other hand, when they expand at higher pH valuesthere should be an increase in pore size, which will facilitate thediffusion of molecules into and out of the beads.

4. Conclusions

In this study, we have used a pH-stat in vitro digestion model toshow that the rate and extent of lipid digestion can be greatlydecreased by encapsulating lipid droplets within calcium alginatebeads. This effect can be attributed to the ability of the bead matrixto restrict the access of the digestive enzymes (and possibly othercomponents, such as bile acids and co-lipase) to the encapsulatedlipid droplets, as well as to the retardation of the movement ofdigestion products (free fatty acids) away from the droplet surfaces.Visual observation of the beads indicated that their structure wasdisrupted during the digestion process at low alginate or Ca2þ

concentrations, but remained intact at higher levels. The overalllipid digestion rate could be manipulated by controlling bead size,bead composition (calcium/alginate), or lipid type. The digestionrate increased with decreasing bead size, calcium concentration,alginate concentration, and lipid molecular weight. These effectswere interpreted in terms of the influence of the alginate gel poresize and the fraction of lipid droplets near the bead surfaces. Finally,we showed that beads tend to shrink at low pH (1 and 2) and swellat high pH (3e7), but maintain their overall integrity (providedthere was sufficient calcium and alginate present). This researchmay be useful for the rational design of delivery systems toencapsulate, protect and release bioactive components in the food,pharmaceutical, and cosmetic industries.

For future research, it is important to test the digestion of lipiddroplets encapsulated within filled calcium alginate beads usingmore realistic conditions, e.g., using in vitro digestion models thatinclude the mouth, stomach and small intestine or by using animalor human feeding studies. In addition, it would be informative toestablish the physicochemical mechanisms responsible for thedelayed lipid digestion by characterizing the initial internal struc-ture of the beads (e.g., pore size, droplet location), by monitoringthe change in bead properties during digestion, and by tracking thetransport of enzymes, reactants and products throughout thedigestion process.

Acknowledgements

This material is partly based upon work supported by UnitedStates Department of Agriculture, CREES, NRI Grants, and Massa-chusetts Department of Agricultural Resources. We also thank theChina Scholarship Council (China) for financial support of Yan Li.

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