Microgel-in-Microgel Biopolymer Delivery Systems: Controlled

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Da Ma, Zongcai Tu, Hui Wang, zipei zhang, and David Julian McClements



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Page 1 of 35

Microgels 1 + Biopolymer 2

Journal of Agricultural and Food Chemistry

Oil Droplets + Biopolymer 1

O/M

ACS 1Paragon Plus Environment

Step 1

O/M1/M2

Step 2


1

Microgel-in-microgel biopolymer delivery systems: Controlled

2

digestion of encapsulated lipid droplets under simulated

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gastrointestinal conditions

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Da Ma a,c, Zong-Cai Tu a,b*, Hui Wang a, Zipei Zhang c, David Julian McClements c*

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a

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Jiangxi, 330047, China

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b

College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi, 330022, China

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c

Department of Food Science, University of Massachusetts, Amherst, MA 01060, USA.

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang,

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Journal: Journal of Agricultural and Food Chemistry

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Submitted: January 2018

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* Corresponding authors:

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Prof. Zongcai Tu

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235 Nanjing Easter Road, Nanchang, Jiangxi, China

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E-mail: [email protected]; Tel: +86-79188121868; Fax: +86-791-88305938;

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Prof. David Julian McClements

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University of Massachusetts Amherst, MA 01003, USA

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E-mail: [email protected]; Fax: +413-545-1262; Tel: +413-545-2275

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ACS Paragon Plus Environment

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Page 3 of 35

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Abstract Structural design principles are increasingly being used to develop colloidal delivery

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systems for bioactive agents.

In this study, oil droplets were encapsulated within

25

microgel-in-microgel systems.

26

protein-coated oil droplets (d43 = 211 nm).

27

carrageenan-in-alginate (O/MC/MA) or alginate-in-carrageenan (O/MA/MC) microgels.

28

vibrating nozzle encapsulation unit was used to form the smaller inner microgels (d43 =170-324

29

µm), while a hand-held syringe was used to form the larger outer microgels (d43 =2200-3400

30

µm). Calcium alginate microgels (O/MA) were more stable to simulated gastrointestinal tract

31

(GIT) conditions than potassium carrageenan microgels (O/MC), which was attributed to the

32

stronger cross-links formed by divalent calcium ions than the monovalent potassium ions. As a

33

result, the microgel-in-microgel systems had different gastrointestinal fates depending on the

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nature of the external microgel phase, i.e., the O/MC/MA system was more resistant to rupture

35

than the O/MA/MC system. The rate of lipid digestion under simulated small intestine

36

conditions decreased in the following order: free oil droplets > O/MC > O/MA > O/MA/MC >

37

O/MC/MA.

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microgels in the small intestine, since a hydrogel network surrounding the oil droplets inhibits

39

lipid hydrolysis by lipase. The structured microgels developed in this study may have interesting

40

applications for the protection or controlled release of bioactive agents.

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Keywords: lipid digestion; microgels; gastrointestinal fate; nanoemulsions; structural design

Initially, a nanoemulsion was formed that contained small whey These oil droplets were then loaded into either A

This effect was attributed to differences in the integrity and dimensions of the


2


42

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Page 4 of 35

Introduction There is growing interest in the design, fabrication, and application of biopolymer microgels

44

in foods for the encapsulation, protection, and release of bioactive agents, such as vitamins,

45

minerals, nutraceuticals, enzymes, or probiotics 1-5. Biopolymer microgels are small particles

46

that contain a cross-linked network of biopolymer molecules inside. The structure and

47

composition of biopolymer microgels can be controlled by careful selection of the ingredients

48

and manufacturing methods used to produce them, which enables one to tailor their functionality

49

for specific applications 6-7.

50

polarity, and molecular interactions of biopolymer microgels can be manipulated, which gives

51

one great scope in creating a wide range of functional attributes, such as increased lubrication,

52

novel textures, improved stability, or controlled digestibility 3, 8-13. Food-grade biopolymer

53

microgels are usually fabricated from proteins and/or polysaccharides, such as whey protein 14,

54

soy protein 15, egg protein 16, casein 17, alginate 18, carrageenan 19, and pectin 20. Each of these

55

biopolymers has its own unique molecular and physicochemical properties, which enables

56

microgels with different functional attributes to be created.

57

biopolymer microgels can be altered by coating them with biopolymers or colloidal particles so

58

as to change their surface polarity, charge, stability, aggregation state, release characteristics, or

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digestibility 13, 21-22.

60 61

For instance, the composition, dimensions, shape, pore size,

After formation, the properties of

In the current study, an alternative approach was examined for tailoring the functionality of biopolymer microgels, which is based on embedding one kind of microgel in another kind of


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microgel. These “microgel-in-microgel” systems are formed using a two-step process: (i) a

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suspension of relatively small microgels (M1) is formed from a biopolymer gelling agent using

64

an appropriate fabrication method; (ii) then these microgels are mixed with another biopolymer

65

gelling agent and then a suspension of microgels-in-microgels (M1/M2) is formed using another

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fabrication method. This approach has previously been used to encapsulate small whey protein

67

microgels inside larger alginate microgels 23. These structured biopolymer systems can be

68

further extended to encapsulate hydrophobic substances, for example by dispersing oil droplets

69

in one or both of the biopolymer phases.

70

internal biopolymer phase to form oil-in-microgel-in-microgel systems (O/M1/M2) (Figure 1).

71

The impact of using this approach on the rate and extent of lipid digestion under simulated

72

gastrointestinal tract (GIT) conditions was then determined. We hypothesized that

73

encapsulation of the oil droplets within these structured microgels would be an effective way of

74

controlling their gastrointestinal fate, which may be useful for a number of food and medical

75

applications.

76

to protect bioactives from degradation, to modulate blood triglyceride levels, to deliver

77

bioactives to specific regions of the GIT, or to control satiety/satiation responses 3. This might

78

be achieved due to the fact that the encapsulated lipid droplets are more effectively isolated from

79

the surrounding aqueous phase when they are dispersed within the internal microgel phase of

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Materials and Methods

In this study, we embedded oil droplets within the

For instance, they could be used to develop foods with controlled flavor profiles,


4


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Page 6 of 35

Materials Whey protein isolate (WPI) powder (BIPro JE-099-2-420) was supplied by Davisco Foods

84

International (Eden Prairie, MN, USA), which was stated to contain 97.7 % protein, 0.3% fat,

85

and 1.8% ash. Powdered κ-carrageenan (Ticaloid® 710 H, Lot No.34695) with a degree of

86

esterification > 99% and alcohol content < 0.1% (ethanol, isopropanol, or methanol) was

87

obtained from TIC Gums, Inc. (White Marsh, MD).

88

food supplier (Mazola, ACH Food Companies, Inc., Memphis, TN). Nile Red (N3013-100MG)

89

and acetic acid (ACS reagent, ≥99.7%) were purchased from the Sigma Chemical Company (St.

90

Louis, MO).

91

(Pittsburgh, PA). Double distilled water from a laboratory purification system (Nanopure Infinity,

92

Barnstead International) was used for all experiments.

93

Sample preparation

94

Nanoemulsion preparation

95

Corn oil was purchased from a commercial

All other analytical-grade reagents were purchased from Fisher Scientific

An oil-in-water nanoemulsion was prepared using a method described previously with some

96

slight modifications 24. Initially, an aqueous phase was prepared by dispersing 1.00 g of

97

powdered whey protein isolate (WPI) into 89.00 g of 5 mM phosphate buffer solution (pH 7.0),

98

and then stirring overnight at ambient temperature to disperse and dissolve the proteins. Then,

99

90.0 g of the aqueous phase was blended with 10.0 g of the oil phase (corn oil) using a

100

high-shear mixing device (M133/1281-0, Biospec Products, Inc., ESGC, Switzerland) for 2 min

101

at ambient temperature.

102

homogenizer (Microfluidizer, M110A, Microfluidics, Newton, MA, USA) at a pressure of

The coarse emulsion formed was then passed through a mechanical


5

Page 7 of 35


103

13,000 psi (around 900 bar) for 3 passes. The nanoemulsion formed was then stored in a

104

refrigerator at 4 oC to inhibit any microbial growth prior to use.

105

Microgel Preparation

106

A number of lipid-loaded biopolymer microgels with different compositions and structures

107

were formed by altering the ingredients and fabrication procedures used (Figure 1).

108

sake of clarity, the oil phase is referred to as O, the microgel phase formed from carrageenan is

109

referred to as MC, and the microgel phase formed from alginate is referred to as MA. Relatively

110

large microgels were formed using a simple hand-held syringe, while relatively small microgels

111

were formed using a vibrating nozzle encapsulation unit (Encapsulator B-390, BUCHI,

112

Switzerland). A schematic representation of the procedure used to prepare the oil droplet-loaded

113

microgel-in-microgel systems is shown in Figure 1.

114

For the

O/MC systems: Relatively small oil loaded-carrageenan microgels (O/MC) were formed

115

using an injection-gelation approach described previously 25, with some slight modifications.

116

First, 2.5 g of powdered κ-carrageenan was dispersed in 100 ml of pH 7-adjusted double distilled

117

water by stirring at 60 oC for 3 hours to ensure complete dissolution, and then the system was

118

cooled to 35 oC. The resulting carrageenan solution was then mixed with the nanoemulsion (1:4

119

v/v) at 35 oC for 2 hours to form a final system that contained 0.5% w/v carrageenan and 0.8 %

120

w/v oil droplets.

121

potassium chloride solution using a vibrating nozzle encapsulation unit (Encapsulator B-390,

122

BUCHI, Switzerland).

The oil droplet/carrageenan mixture was then injected into a 10% (w/v)

The oil droplet-loaded carrageenan microgels formed were then held in


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the potassium chloride solution for 1 hour at ambient temperature to harden them.

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microgels were then placed on filter paper and washed with pH 4-adjusted double distilled water

125

to remove any excess potassium chloride from their surfaces.

126

The

O/MA systems: Relatively small oil droplet-loaded alginate microgels (O/MA) were formed

127

using a similar approach. First, 2.5 g of powdered alginate was dispersed in 100 mL of pH

128

7-adjusted double distilled water by stirring at 60 oC for 3 hours, and then cooling to 35 oC.

129

The alginate solution and nanoemulsion were then mixed together (1:4 v/v) at 35 oC for 2 hours,

130

and then injected into a 10% (w/v) calcium chloride solution using the encapsulation unit.

131

oil droplet-loaded alginate microgels formed were then incubated in the calcium chloride

132

solution for 1 hour at ambient temperature to harden them.

133

placed on filter paper and washed with pH 7-adjusted double distilled water to remove any

134

excess calcium chloride form their surfaces.

135

The

The microgels formed were then

O/MC/MA systems: Relatively small oil droplet-loaded carrageenan microgels were prepared

136

as described earlier.

137

temperature for 30 minutes, and then injected into a 10% w/v calcium chloride solution using a

138

hand-held syringe (BD Safety-Lok 10 mL Syringe with a 0.6 mm diameter tip, Franklin Lakes,

139

NJ), and then left for 1 hour to harden.

140

filtration using Miracloth (rayon-polyester mesh with a typical pore size of 22-25 µm) and

141

washed with pH 7-adjusted double distilled water to remove residual calcium chloride.

142 143

They were then mixed with 0.5% alginate solution (1:1 v/v) at ambient

The O/MC/MA microgels formed were then collected by

O/MA/MC systems: These systems were formed using a similar approach, but by inverting the biopolymer used in the two microgel fabrication steps.

Relatively small alginate microgels


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144

were prepared as described earlier.

They were then mixed with 0.5% carrageenan solution (1:1

145

v/v) at ambient temperature for 30 minutes and then injected into a 10% (w/v) potassium

146

chloride solution using the hand-held syringe, and left for 1 hour to harden.

147

microgels formed were collected by filtration using Miracloth and then washed with pH

148

7-adjusted double distilled water to remove residual potassium chloride.

149

Simulated gastrointestinal tract

The O/MA/MC

150

An in vitro static gastrointestinal tract (GIT) model was used to study the impact of microgel

151

structure on their behavior under simulated gastrointestinal conditions, which is closely related to

152

the recently developed standardized international consensus method 26. The GIT model used in

153

this study has been described in detail in a recent publication 27, and so only a brief summary is

154

given here. The samples were passed through simulated mouth, stomach, and small intestine

155

phases, and the free fatty acids (FFAs) released within the small intestine phase were continually

156

monitored using a pH stat method.

157

delivery systems studied is was important to ensure that they all had similar initial lipid contents

158

(0.2 g of oil in 7.5 g sample). This was achieved by carrying out a mass balance analysis of the

159

amount of oil droplets in each sample.

160

O/MC/MA and O/MA/MC systems were 2 g, 2.5 g, 2.5 g, 5 g and 5 g, respectively.

To directly compare the rate of FFA release in the different

Hence, the mass of the initial emulsion, O/MC, O/MA,

161

Initially, both the sample and the simulated saliva fluid were incubated to 37 oC.

162

g of the sample (containing 0.2 g oil) was mixed with 7.5 g of the simulated saliva and the

163

sample was adjusted to pH 6.8.

164

using an incubation unit (Innova Incubator Shaker, Model 4080, New Brunswick Scientific,

Then, 7.5

This mixture was then stirred at 100 rpm for 2 min at 37 oC


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Page 10 of 35

165

Edison, NJ). Then, simulated gastric fluid (15 g) containing 0.048 g pepsin was added to the

166

mixture, the system was adjusted to pH 2.5, and then stirred at 100 rpm for 2 h at 37 °C. Small

167

intestine conditions were then modeled by adding simulated intestinal fluids (1.5 mL), lipase (2.5

168

mL), and bile salts (3.5 mL) to the mixture and then adjusting to pH 7.00. The pH of the system

169

was monitored and controlled using an automatic titration unit (Metrohm, USA Inc.).

170

was maintained at 7.00 throughout the small intestine phase by titrating 0.25 mM NaOH into the

171

mixture throughout a 2 h incubation period at 37 oC. The amount of free fatty acids released was

172

calculated from the titration curves using the following equation:

173

%FFA = 100 ×

௏ಿೌೀಹ ×௠ಿೌೀಹ ×ெ೗೔೛೔೏ ௐಽ೔೛೔೏ ×ଶ

The pH

(1)

174

Here, VNaOH and mNaOH are the volume and molarity of the sodium hydroxide solution added, and

175

Mlipid and Wlipid are the molecular weight and weight of the lipids (corn oil) in the small intestine

176

phase. The initial rate of lipid digestion was determined by calculating the linear slope of FFA

177

versus time in the region where the free fatty acids released was < 50%.

178

Microstructure analysis

179

The microstructure of the samples was monitored using optical and confocal fluorescence

180

scanning laser microscopy (Nikon D-Eclipse C1 80i, Nikon, Melville, NY) using 20× and 10×

181

objective lenses.

182

microscope (NIS-Elements, Nikon, Melville, NY).

183

the hand-held injection method, a freezing microtome (Cryostar NX70, Thermo Electron

184

Corporation, MA) was used to cut them into thin slices to observe their internal structure.

185

incubation temperature and section thickness used in the microtome were −20 °C and 10 µm,

The images acquired were analyzed using the software associated with the For the relatively large microgels formed by


The

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186

respectively.

For the confocal fluorescence measurements, the lipid phase of the samples was

187

dyed using 0.1 ml Nile red solution prior to analysis, and excitation and emission wavelengths of

188

543 and 605 nm were used to acquire the images, respectively.

189

Particle size and charge

190

The particle size distributions of the microgels produced using the encapsulation device

191

were evaluated using a static light scattering instrument (Mastersizer 2000, Malvern Instruments,

192

Ltd., Worcestershire, U.K.).

193

water and then stirred to make sure they were homogeneous and to avoid multiple scattering

194

effects.

195

measurement chamber, which was attributed to leaching of potassium ions (the cross-linking

196

agent) out of the microgels. Consequently, the O/MC samples were also analyzed after the

197

samples had been diluted with 10 mM KCl solution. The refractive indices of the water and

198

particles used in the calculations of the particle size distributions were 1.33 and 1.472,

199

respectively. There are some uncertainties associated with defining a refractive index for the

200

microgel particles because they vary in internal composition.

201

particles, the particle size determined by static light scattering is not strongly dependent on the

202

refractive index 28.

203

(d43) calculated from the particle size distributions. The microgel-in-microgel systems were too

204

large (> 1 mm) to analyze by static light scattering, and so their size was determined manually

205

using digital calipers (0–300 mm, EC10, High Precision Digital Caliper, Tresna Instruments,

Initially, these samples were diluted with pH 7-adjusted distilled

However, the carrageenan-microgels (O/MC) were found to breakdown within the

However, for relatively large

Average particle sizes are reported as the volume-weighted mean diameter


10


206

Guilin, China).

207

and standard deviation were calculated.

208

Page 12 of 35

The diameter of multiple microgel particles was measured, and then the mean

The surface potential (ζ-potential) of the particles in the nanoemulsions and simple

209

microgels was determined using a commercial electrophoresis instrument, which measures the

210

direction and velocity of particle movement in a well-defined electrical field (Zetasizer Nano ZS

211

series, Malvern Instruments Ltd. Worcestershire, UK). Samples were diluted 10- to 20-fold

212

with phosphate buffer (pH 7.0) prior to analysis to avoid multiple scattering effects.

213

carrageenan microgels were also analyzed after they had been diluted with a potassium chloride

214

solution (10 mM) to avoid their disintegration.

215

analyzed using this method because the particles were too large.

216

Statistical analysis

217

The

The microgel-in-microgel systems could not be

The experiments were carried out in triplicate using freshly prepared samples. Results were

218

expressed as the calculated means ± standard deviations (n=3) and data were analyzed using a

219

statistical software package (Version 16.0, SPSS, lnc., Chicago, USA)

220

Results and discussion

221

Initial nanoemulsion and microgel properties

222

Initially, the properties of the nanoemulsions and biopolymer microgels were characterized

223

after their fabrication.

The protein-coated lipid droplets in the oil-in-water nanoemulsions had a

224

relatively small mean diameter (d43 = 211 ± 1 nm), which indicates that the microfluidization

225

method used to produce them was highly effective. The droplets in the nanoemulsions had a


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Page 13 of 35


226

relatively high negative surface potential (ζ = -21.7 ± 2.6 mV), which can be attributed to the

227

fact that the pH was above the isoelectric point of the adsorbed proteins.

228

The simple microgels produced using the vibrating nozzle encapsulation device had roughly

229

spherical shapes with diameters around 100 to 400 µm when observed by confocal microscopy

230

(Figure 2).

Moreover, the lipid droplets were mainly located within the interior of the

231

microgels.

Light scattering measurements indicated that the nanoemulsion-loaded alginate

232

microgels (O/MA) had a monomodal particle size distribution (Figure 3), and a mean particle

233

diameter (d43) of around 170 ± 40 µm.

234

the nanoemulsion-loaded carrageenan microgels (O/MC), however there were some problems

235

with this method. When the O/MC microgels were diluted in distilled water prior to analysis

236

they were found to partially dissociate.

237

cationic potassium ions (K+) holding the anionic carrageenan molecules together in the microgels

238

into the surrounding aqueous phase.

239

after they were diluted with a 10 mM KCl solution, so as to avoid this effect. This level of KCl

240

was selected because it was found not to lead to dissociation of the microgels when they were

241

diluted. The mean particle diameter was much larger for the samples diluted in KCl solution

242

(324 ± 3 µm) than for the samples diluted in water (8.6 ± 0.5 µm).

243

differences in the particle size distributions of the carrageenan microgels depending on the

244

dilution method (Figure 3).

245

uniform distribution than the ones diluted in water.

246

implications for the application and characterization of carrageenan-based microgels cross-linked

Light scattering was also used to characterize the size of

This effect was attributed to movement of some of the

For this reason, the O/MC microgels were also analyzed

Moreover, there were major

The samples diluted in KCl solution were larger and had a more This phenomenon has important


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Page 14 of 35

247

using potassium ions.

248

either beneficial or detrimental to its application as a delivery system, depending on the situation.

249

Moreover, the potential for microgel dissociation during particle characterization using light

250

scattering methods should be taken into account to avoid obtaining unrealistic results. For this

251

reason, light scattering analysis was not used to characterize the microgels in the remainder of

252

the studies.

253

This type of microgel appears to be sensitive to dilution, which may be

Both types of microgels initially had a relatively high negative surface potential (ζ = -42.2 ±

254

0.5 mV for O/MC and = -25.8 ± 2.4 mV for O/MA) when they were diluted with distilled water,

255

which can be attributed to the anionic groups on the polysaccharide chains: carrageenan has

256

sulfate groups, whereas alginate has carboxyl groups 29-30. There are a number of reasons for the

257

higher magnitude of the negative charge on the carrageenan microgels.

258

may have had a higher linear charge density than the alginate.

259

cross-linked using monovalent counter-ions (K+), whereas the alginate was cross-linked using

260

divalent counter-ions (Ca2+), and so there may have been more charge neutralization for the

261

alginate. Third, some of the potassium ions may have leached out of the carrageenan microgels

262

when they were diluted in water, which increased the amount of negative charge on the

263

polysaccharide molecules. The magnitude of the ζ-potential of the carrageenan microgels

264

diluted with potassium chloride solution was appreciably lower (-21.3 ± 0.4 mV) than those

265

diluted with water (-42.2 ± 0.5 mV), which can be attributed to two effects.

266

of potassium chloride in the diluent prevented the loss of cationic potassium ions from the

267

microgels, thereby leading to a smaller negative charge on the potassium carrageenan microgels.


First, the carrageenan

Second, the carrageenan was

First, the presence

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268

Second, the increase in ionic strength of the diluent due to the presence of the KCl will have

269

caused electrostatic screening, which is known to reduce the magnitude of the ζ-potential 31.

270

The microgel-in-microgel systems were too large to analyze by light scattering, and to place

271

directly under the microscope. For this reason, their initial internal microstructures were

272

determined using optical and confocal scanning fluorescence microscopy after obtaining slices

273

using a microtome (Figure 4).

274

digital calipers.

275

hydrophobic fluorescence probe (Nile red) so as to ascertain the location of the oil droplets.

276

The microscopy images clearly indicated that oil-in-microgel-in-microgel systems had been

277

successfully fabricated using the two-step approach used (Figure 1). The lipid droplets were

278

clustered together inside the inner microgel phase, which was surrounded by the droplet-free

279

outer microgel phase.

280

were a few millimeters big and had quite irregular shapes.

281

diameters using digital calipers indicated that the O/MC/MA systems had a mean diameter of

282

2240 ± 260 µm, while the O/MA/MC systems had a mean diameter of 3400 ± 580 µm. The much

283

larger size of the microgel-in-microgel systems compared to the microgel systems can be

284

attributed to the differences in the dimensions of the nozzles used to prepare them. The

285

hand-held syringe had a nozzle diameter of about 600 µm, whereas the encapsulation unit had a

286

nozzle diameter of about 160 µm.

287

appeared to be larger than those produced using alginate for both devices.

288

due to differences in the rheology of the initial biopolymer solutions, or the kinetics of

In addition, their average particle size was determined using

For the fluorescence microscopy images, the lipid phase was dyed red using a

The microscopy images suggested that the microgel-in-microgel systems The measurements of their mean

Interestingly, the microgels produced using carrageenan


This may have been

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289

biopolymer gelation within the hardening solutions. One would expect alginate to gel faster

290

than carrageenan because the cross-linking agent is a divalent ion (calcium) compared to a

291

monovalent ion (potassium).

292

spherical shape more rapidly when they were injected into the hardening solution.

293

GIT fate of nanoemulsions and simple microgels

294

Consequently, the alginate microgels may have been locked into a

Before examining the behavior of the microgel-in-microgel systems, the properties of the

295

simple oil droplet-loaded alginate and carrageenan microgels were characterized as they passed

296

through the simulated GIT.

297

more structurally complex microgel-in-microgel systems.

298

(O/MC) microgels were therefore passed through the simulated mouth, stomach, and small

299

intestine phases and changes in their structural properties were monitored using confocal

300

fluorescence microscopy and light scattering (Figure 2, Table 1).

301

non-encapsulated nanoemulsions were also measured for comparison.

302

This information is useful for understanding the behavior of the Alginate (O/MA) and carrageenan

The properties of the

The nanoemulsions appeared to be relatively stable to aggregation after exposure to the

303

simulated mouth conditions, with the majority of the oil droplets being evenly distributed

304

throughout the microscopy images (Figure 2).

305

mean particle diameter compared to the initial sample (Table 1), which may have been because

306

of electrostatic screening, bridging, or depletion flocculation occurring in the simulated oral

307

fluids32.

308

simulated stomach conditions.

309

and enzyme activity of the gastric fluids 32. Whey protein-coated lipid droplets should have a

Nevertheless, there was a slight increase in the

Extensive droplet flocculation occurred when the nanoemulsions were incubated in This effect can be attributed to the high acidity, mucin content,


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310

positive charge in gastric fluids, because the pH is well below the isoelectric point of the

311

adsorbed proteins (pI ≈ 5) 33.

312

anionic mucin molecules arising from the mouth phase 34. In addition, the presence of salts in the

313

simulated gastric fluids may have promoted flocculation by reducing the electrostatic repulsion

314

between the droplets 35. Finally, the whey proteins adsorbed to the droplet surfaces may have

315

been partially hydrolyzed by pepsin in the gastric fluids, which reduced their ability to inhibit

316

droplet flocculation 33. After exposure to the simulated small intestine phase, the large flocs

317

formed in the stomach were broken down and there appeared to be numerous small particles

318

dispersed throughout the samples (Figure 2).

319

nanoemulsions were digested by lipases and proteases in the simulated small intestinal fluids,

320

resulting in the formation of colloidal particles such as mixed micelles, vesicles, and calcium

321

salts 36-37.

322

the mouth (-21.7 ± 2.6 mV), to slightly negative in the stomach (-2.8 ± 0.3 mV), to highly

323

negative in the small intestine (-40.4 , ± 3.2), which can be attributed to changes in the pH, ionic

324

strength, and composition of the system in the different GIT phases, as discussed elsewhere 32.

325

Consequently, the cationic oil droplets may be linked together by

It is likely that the lipids and proteins within the

The ζ-potential of the protein-coated oil droplets went from moderately negative in

The oil droplet-loaded alginate microgels (O/MA) remained intact in the mouth and stomach

326

phase, with the oil droplets (stained red) still trapped inside.

However, there appeared to be

327

some fragmentation of their structure after exposure to the small intestine phase.

328

were still present as relatively large particles, with evidence of lipid droplets trapped inside. The

329

microscopy observations were supported by the data from the light scattering measurements

330

(Table 1).

Even so, they

In contrast, the microscopy images indicated that the oil droplet-loaded carrageenan


16


Page 18 of 35

331

microgels (O/MC) exhibited some slight structural changes in the mouth phase, but considerable

332

fragmentation and disintegration after exposure to both stomach and small intestine conditions

333

(Figure 2).

334

light scattering for this system when it was diluted with either water or KCl solution (Table 1),

335

which highlighted the importance of the potassium ions in maintaining microgel structure.

336

Moreover, there were appreciable differences between the results obtained using microscopy and

337

light scattering. This effect can be attributed to the fact that the carrageenan microgels

338

fragmented into irregular shaped particles, and were therefore no longer spherical as assumed by

339

the theory used to interpret the light scattering data. In addition, the carrageenan microgels were

340

more fragile than the alginate ones, which may have led to some changes in their structure within

341

the light scattering instrument (where the samples are diluted and stirred).

342

values of the alginate microgels (O/MΑ) were measured in the different GIT stages because of

343

the problems arising from dissociation of the carrageenan microgels when diluted and stirred

344

with water. The ζ-potential on the O/MΑ microgels went from moderately negative in the

345

mouth (-9.7 ± 3.6 mV), to slightly negative in the stomach (-2.9 ± 0.5 mV), to moderately

346

negative in the small intestine (-24.1 ± 0.9 mV), which can again be attributed to alterations in

347

the pH, ionic strength, and composition of the system in different GIT phases, as discussed in our

348

earlier study 27. Measurements of the ζ-potential of carrageenan microgels diluted with water

349

followed a similar trend to those for the alginate microgels, but they were not used because the

350

microgels had become partially dissociated.

Very different results were obtained for the mean particle diameters determined by

Only, the ζ-potential

Measurements were not made using KCl solution


17

Page 19 of 35


351

for these samples because it may have changed their composition or structure, and therefore led

352

to misleading results.

353

These results show that alginate and carrageenan microgels behaved quite differently under

354

simulated GIT conditions, which can be attributed to differences in their cross-linking.

The

355

alginate molecules were held together by divalent calcium ions (Ca2+), whereas the carrageenan

356

molecules were held together by monovalent potassium ions (K+). Compared to the calcium ions,

357

the potassium ions are more likely to be released from the gel network by dilution, or in the

358

presence of other types of monovalent cation (such as the sodium ions in the simulated GIT

359

fluids). As a result, potassium carrageenan microgels are more susceptible to breakdown under

360

GIT conditions than calcium alginate microgels, which may be important for designing delivery

361

systems that will release bioactive components in different regions of the GIT. These results

362

are consistent with earlier studies of the GIT fate of curcumin-loaded biopolymer microgels,

363

which also showed that alginate microgels were more stable to GIT conditions than carrageenan

364

ones 27.

365

GIT fate of microgel-in-microgel systems

366

Changes in the properties of the two types of oil droplet-loaded microgel-in-microgel

367

systems (O/MC/MA and O/MA/MC) were measured as they were passed through the simulated

368

GIT (Figure 5).

369

organization of the microgel phases.

370

(O/MC/MA) remained intact in the mouth and stomach regions of the simulated GIT, but partly

371

broke down in the small intestine region.

The two systems behaved differently depending on the initial structural The systems with alginate as the outer microgel phase

Indeed, white spherical beads, with dimensions fairly


18


Page 20 of 35

372

similar to those in the initial samples, could be seen in the mouth and stomach regions. However,

373

in the small intestine phase these beads had largely broken down, but there were still some fairly

374

large microgel fragments remaining, as seen in the microscopy images and by the fact that a

375

white sediment settled to the bottom of the containers (Figure 5).

376

carrageenan as the outer microgel phase (O/MA/MC) exhibited discernable fragmentation and

377

disintegration in the mouth, stomach, and small intestine.

378

showed that they contained larger more irregular shaped particles that broke down into many

379

smaller fragments in the mouth and stomach, and then were almost completely disintegrated in

380

the small intestine (Figure 5). These effects can be attributed to the different sensitivities of the

381

alginate and carrageenan hydrogels to GIT conditions (see previous section). The calcium

382

alginate hydrogel is more resistant to breakdown than the potassium carrageenan hydrogel 27.

383

Consequently, the O/MC/MA system, which has an outer alginate layer, is more resistant to

384

disintegration than the O/MA/MC system, which has an outer carrageenan layer. Different kinds

385

of microgel-in-microgel system may be useful for the release of encapsulated components in

386

different regions of the GIT.

387

outer microgel phase so that it is released in the stomach, whereas another bioactive component

388

could be dispersed in the inner microgel phase so that it can be released in the small intestine or

389

colon. Alternatively, the two different microgel phases may be used to encapsulate two

390

different bioactive components that might normally adversely interact with each other.

391

there are some potentially interesting applications for this kind of structured microgel system.

Conversely, the systems with

Visual observation of these samples

For instance, one bioactive component could be dispersed in the


Clearly,

19

Page 21 of 35

392


Comparison of lipid digestion of different microgel systems

393

Previous studies have shown that the external dimensions and pore size of microgels are

394

important factors for inhibiting lipid digestion 13, 18, because they determine the ability of the

395

lipase molecules to reach the lipid droplet surfaces.

396

developed in this study may therefore be a particularly effective means of controlling the rate

397

and extent of lipid digestion of emulsified fats by restricting the ability of the lipase molecules to

398

reach the oil droplet surfaces.

399

The microgel-in-microgel systems

We therefore compared the ability of the different kinds of microgels to alter the lipid

400

digestion profiles of the encapsulated oil droplets.

Each of the delivery systems was passed

401

through the mouth and stomach phases, and then the release of free fatty acids from the samples

402

was measured throughout the small intestine phase using an automatic titration method (pH-stat).

403

There were clear differences in the lipid digestion profiles of the samples depending on their

404

initial microstructures (Figure 6).

405

lipid digestion (Figure 7), which can be attributed to the fact that the lipase molecules could

406

easily access the surface of the free oil droplets.

407

systems had a rate of lipid digestion that was between that of the nanoemulsions and

408


409

nanoemulsions because the lipid droplets were trapped inside hydrogel networks that inhibited

410

the ability of the lipase molecules to reach the oil droplet surfaces 18.

411

lipid digestion depends on how fast the lipase molecules can penetrate through the hydrogel

412

networks or how rapidly the biopolymers disintegrate and release the oil droplets. The rate of

The oil-in-water nanoemulsions had the fastest initial rate of

The simple microgel (O/MA and O/MC)

The rate of lipid digestion was slower than that of the


In this case, the rate of

20


Page 22 of 35

413

lipid digestion was slower for the oil droplet-loaded alginate microgels than for the oil

414

droplet-loaded carrageenan microgels, which can be attributed to the greater resistance of the

415

alginate hydrogels to disruption under GIT conditions 27. Presumably, the rate of lipid digestion

416

was faster for the simple microgels than for the microgel-in-microgel systems because of their

417

smaller dimensions. The slowest rate of lipid digestion was observed for the two oil

418

droplet-loaded microgel-in-microgel systems (O/MC/MA and O/MA/MC) (Figure 7), which can

419

be attributed to their relatively large size when entering the small intestine, and the ability of the

420

biopolymer network to inhibit access of lipase to the oil droplet surfaces.

421

studies have shown that the ability of microgels to inhibit lipid digestion increases as their

422

diameter increases, which was attributed to the smaller surface area and greater distance that the

423

lipase molecules have to travel into the interior of the microgels to reach the oil droplets 18. The

424

O/MC/MA systems were digested more slowly than the O/MA/MC systems because they had an

425

outer microgel phase consisting of alginate, which is more resistant to degradation.

426

Consequently, these microgels are likely to have had larger dimensions throughout the lipid

427

digestion process, thereby being more effective at inhibiting the access of the lipase to the oil

428

droplets. These results clearly show that the structure of microgels can be manipulated to create

429

different kinds of lipid digestion profiles, which may be important for some practical

430

applications.

431

Conclusions


Indeed, previous

21

Page 23 of 35

432


This study has shown that microgel-in-microgel systems can be fabricated using relatively

433

simple sequential injection-gelation methods.

The gastrointestinal fate of these systems

434

depends on the type of biopolymers and cross-linking agent used to construct them, as well as

435

their relative location within the system.

436

gastrointestinal conditions than potassium carrageenan hydrogels.

437

to construct microgels that will breakdown in different regions of the GIT, thereby releasing any

438

encapsulated components at a desired location.

439

some interesting applications within functional foods and other products intended for oral

440

ingestion.

441

microgel phase, and another bioactive agent in the external microgel phase.

442

if the two bioactive components would normally adversely interact with each other and lose their

443

activity, or if it was desirable to release one of the bioactive agents in one part of the GIT (such

444

as the mouth or stomach) and the other bioactive agent in another part of the GIT (such as the

445

small intestine or colon).

446

control the rate and extent of lipid digestion in delivery systems, which may be useful for

447

sustained delivery of bioactive components or for controlling satiety/satiation responses.

Calcium alginate hydrogels are more resistant to Consequently, it is possible

This type of structured microgel may have

For instance, it may be possible to encapsulate one bioactive agent in the internal This may be useful

In this study, we showed that structured microgels could be used to

448

The microgel-in-microgel systems developed in this study were relatively large (a few

449

millimeters), which would limit their application in many commercial products. These large

450

particles could be easily discerned by the eye, would be detected as individual entities in the

451

mouth, and would rapidly sediment in low viscosity products.

452

be partially broken down in the mouth prior to swallowing due to mastication processes. Some


In addition, they would tend to

22


Page 24 of 35

453

of these problems may be overcome by incorporating them into highly viscous or semi-solid

454

foods, such as puddings, sauces, soups, or comminuted meat products.

455

will be important to identify commercially viable methods of creating smaller

456


457

adversely affect the desirable quality attributes of food products, and to establish that they can

458

exhibit their beneficial effects in actual gastrointestinal tracts, which will require animal or

459

human feeding studies.

460

Acknowledgments

461

For wider application, it

In addition, it will be important to ensure that they do not

This work was supported by China Agriculture Research System (CARS-45). Nanchang

462

university graduate innovation special fund project (cx2015108).

This material was also partly

463

based upon work supported by the National Institute of Food and Agriculture, USDA,

464

Massachusetts Agricultural Experiment Station (MAS00491) and USDA, AFRI Grants

465

(2014-67021 and 2016-08782).

466


23

Page 25 of 35


467

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beta-lactoglobulin in an in vitro gastric model. Food Hydrocolloids 2009, 23 (6), 1563-1569. 34. Chang, Y. G.; McClements, D. J., Characterization of mucin - lipid droplet interactions: Influence on potential fate of fish oil-in-water emulsions under simulated gastrointestinal conditions. Food Hydrocolloids 2016, 56, 425-433. 35. Sarkar, A.; Goh, K. K. T.; Singh, H., Properties of oil-in-water emulsions stabilized by beta-lactoglobulin in simulated gastric fluid as influenced by ionic strength and presence of mucin. Food Hydrocolloids 2010, 24 (5), 534-541. 36. Marze, S.; Gaillard, C.; Roblin, P., In vitro digestion of emulsions: high spatiotemporal resolution using synchrotron SAXS. Soft Matter 2015, 11 (26), 5365-5373. 37. Yao, M. F.; Xiao, H.; McClements, D. J., Delivery of Lipophilic Bioactives: Assembly, Disassembly, and Reassembly of Lipid Nanoparticles. In Annual Review of Food Science and Technology, Vol 5, Doyle, M. P.; Klaenhammer, T. R., Eds. 2014; Vol. 5, pp 53-81.

559


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Page 28 of 35

Figures

Microgels 1 + Biopolymer 2

Oil Droplets + Biopolymer 1

O/M1

O/M1/M2

Step 1

Step 2

Figure 1. Schematic representation of the two-step process utilized to form oil-in-microgel-inmicrogel systems (O/M1/M2): (i) an O/M1 system is formed by injection of oil droplets and a gelling biopolymer into a hardening solution; (ii) an O/M1/M2 system is formed by injection of microgels and another gelling biopolymer into a hardening solution.


Page 29 of 35


Figure 2. Confocal microscopy images of oil-loaded alginate (O/MA) microgels and oil-loaded carrageenan (O/MC) microgels after exposure to different stages of a simulate GIT.



Page 30 of 35

60 O/MC (With KCl)

Volume Fraction (%)

50 40

O/MC (No KCl)

30 O/MA

20 10

Nanoemulsion

0 0.01

0.1

1

10

100

1000

10000

Particle Diameter (µm) Figure 3. Initial particle size distributions of the nanoemulsions (O), oil-loaded alginate (O/MA) microgels and oil-loaded carrageenan (O/MC) microgels.


Page 31 of 35


Figure 4. Initial microstructures of the oil-alginate-in-carrageenan (O/MA/MC) microgels and oil-loaded carrageenan-in-alginate (O/MC/MA) microgels. Images were obtained by conventional optical (left) and confocal fluorescence microscopy (right) after sectioning the samples using a microtome.



Figure 5. Changes in the appearance of the oil-loaded alginate-in-carrageenan (O/MA/MC) microgels and oil-loaded carrageenan-inalginate (O/MC/MA) microgels as they were passed through the simulated GIT. The confocal fluorescence microscopy images show the microstructure of the systems after the small intestine phase.


Page 32 of 35

Page 33 of 35


90 80

FFA Released (%)

70 60 50 O

40

O/MC

30

O/MA

20

O/MA/MC

10

O/MC/MA

0 0

20

40

60

80

100

120

Digestion Time (min) Figure 6. Release of free fatty acids (FFA) during digestion of the lipids in different kinds of delivery systems: oil-in-water nanoemulsions (O); oil-loaded alginate (O/MA) microgels; oilloaded carrageenan (O/MC) microgels; oil-loaded alginate-in-carrageenan (O/MA/MC) microgels; and, oil-loaded carrageenan-in-alginate (O/MC/MA) microgels.



Initial Digestion Rate (FFA/min)

20

15

10

5

0

O

O/MC

O/MA

O/MA/MC O/MC/MA

Sample Type Figure 7. Initial rate of lipid digestion in different kinds of delivery systems: oil-in-water nanoemulsions (O); oil-loaded alginate (O/MA) microgels; oil-loaded carrageenan (O/MC) microgels; oil-loaded alginate-in-carrageenan (O/MA/MC) microgels; and, oil-loaded carrageenan-in-alginate (O/MC/MA) microgels.


Page 34 of 35

Page 35 of 35


Table 1. The mean particle diameters of oil-loaded delivery systems after exposure to different stages of a simulated GIT: oil-in-water nanoemulsion (O); oil-loaded carrageenan beads (O/MC) analyzed with or without KCl; oil-loaded alginate beads (O/MA); and oil-loaded microgel-inmicrogel systems (O/MC/MA and O/MA/MC). Mean Particle Diameter (µm) Samples

Initial

Mouth

Stomach

Intestine

O

0.21±0.001

4.23±0.81

58.8±6.5

9.1±4.7

O/MC (No KCl)

8.59±0.47

420±16

270±13

2.4±1.9

O/MC (With KCl)

324.4±2.8

315.8±5.2

283±4.4

273±2.9

275±15

275±20

188±3.4

242±9.4

O/MC/MA

2240±260

2080±310

1920±260

619±50

O/MA/MC

3400±580

2590±590

2170±420

280±19

O/MA


Microgel-in-Microgel Biopolymer Delivery Systems: Controlled

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