Improving Resveratrol Bioaccessibility Using Biopolymer

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Improving Resveratrol Bioaccessibility using Biopolymer Nanoparticles and Complexes: Impact of Protein-Carbohydrate Maillard Conjugation Gabriel Davidov-Pardo, Sonia Pérez-Ciordia, Maria Remedios Marín-Arroyo, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00777 • Publication Date (Web): 06 Apr 2015 Downloaded from http://pubs.acs.org on April 13, 2015

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Journal of Agricultural and Food Chemistry

Improving Resveratrol Bioaccessibility using Biopolymer Nanoparticles and Complexes: Impact of Protein-Carbohydrate Maillard Conjugation

Gabriel Davidov-Pardo1,2, Sonia Pérez-Ciordia2, María R. Marín-Arroyo2 and David Julian McClements1,3, *

1

Department of Food Science, University of Massachusetts, Amherst, MA, USA Department of Food Technology, Ænoltec research group, Public University of Navarre, Campus Arrosadia s/n, Pamplona 31006, Spain. 3 Department of Biochemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203 Jeddah 21589 Saudi Arabia 2

*Corresponding author: [email protected] Department of Food Science – University of Massachusetts 102 Holdsworth Way - Chenoweth Lab, Room 228 Amherst, MA 01002 Tel.: +1-413-545-7157

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Abstract The impact of encapsulating resveratrol in biopolymer nanoparticles or biopolymer

3

complexes on its physicochemical stability and bioaccessibility was determined.

The

4

biopolymer nanoparticles consisted of a zein core surrounded by a caseinate or caseinate-

5

dextran shell. The biopolymer complexes consisted of resveratrol bound to caseinate or

6

caseinate-dextran.

7

reaction. Both the biopolymer nanoparticles and complexes protected trans-resveratrol

8

from isomerization when exposed to UV-light, with the nanoparticles being more effective.

9

Nanoparticles coated by caseinate-dextran were more stable to aggregation under simulated

10

gastrointestinal conditions than those coated by caseinate, presumably due to greater steric

11

repulsion.

12

both biopolymer nanoparticles and biopolymer complexes. These results have important

13

implications for the development of effective delivery systems for incorporating lipophilic

14

nutraceuticals into functional foods and beverages.

15

Keywords: Nanoparticles; resveratrol; encapsulation; antisolvent precipitation; Maillard;

16

bioaccessibility; biopolymers

The caseinate-dextran conjugates were formed using the Maillard

The bioaccessibility of resveratrol was enhanced when it was encapsulated in


2

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Introduction

18

Resveratrol (trans-3,5,4'-trihydroxy-stilbene) is a polyphenolic compound that can

19

be extracted from wine industry by-products, especially grape skins 1. This compound has

20

been shown to have several potential benefits for human health

21

utilization as a nutraceutical ingredient within the food industry is currently limited due to

22

its poor water-solubility, low oral bioavailability, and chemical instability

23

problems can be overcome by encapsulating resveratrol within well-designed colloidal

24

delivery systems, thereby facilitating its utilization as a functional ingredient in the food

25

and other industries. In previous studies, we demonstrated that the fabrication of

26

biopolymer nanoparticles by antisolvent precipitation of zein is a simple and effective

27

means of creating colloidal delivery systems for resveratrol 8. In this case, the bioactive

28

compound and hydrophobic protein are dissolved in an ethanol solution, which is then

29

injected into water (the antisolvent), leading to the formation of nutraceutical-fortified zein

30

nanoparticles. Lipophilic bioactive compounds can also be encapsulated using other

31

approaches based on the formation of biopolymer nanoparticles, e.g., polyphenol-protein

32

complexes 9. Indeed, resveratrol has previously been shown to form complexes with

33

sodium caseinate, ß-lactoglobulin, lactoferrin, and whole buttermilk, which is driven by a

34

combination of hydrophobic, hydrogen bonding, and van der Waals interactions 10-13. In all

35

these cases, the solubility of resveratrol in aqueous solutions was increased.

2-4

. Nevertheless, its

5-7

. These

36

The stability of protein-based colloidal delivery systems is often governed by the

37

electrostatic repulsive interactions operating between the nanoparticles or complexes. A

38

major disadvantage of this kind of stabilization mechanism is that the particles tend to

39

aggregate when the surface charge is not larger enough to generate a strong repulsion,

40

which may occur near the proteins isoelectric point or at high salt concentrations ACS Paragon Plus Environment

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The aggregation of colloidal particles often leads to the formation of precipitates that

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cannot simply be redispersed. These destabilizing conditions often occur in the food

43

industry, and so there is a need to create more stable protein-based colloidal delivery

44

systems.

45

Improvements in the stability of protein-based colloidal delivery systems have been

46

achieved by creating Maillard conjugates between the proteins and high molecular weight

47

polysaccharides

48

Maillard conjugates to stabilize zein nanoparticles against changes in pH, ionic strength,

49

and temperature

50

that prevents the formation of aggregates when the charge of the proteins is close to zero.

51

The Maillard reaction is a non-enzymatic reaction that is greatly accelerated by heat and

52

alkaline conditions

53

carbohydrate with a free amino group of a protein (either a lysine residue or an N-terminal

54

amino group). The initial stages of the reaction involved the interaction of an amino group

55

on a protein with a carbonyl group on a polysaccharide, leading to the formation of an N-

56

substituted glycosylamine and then to an aldimine (Schiff base), which undergoes a a

57

rearrangement that results in an Amadori rearrangement product

58

stages, a series of complex chemical reactions occurs, which eventually leads to

59

degradation of the Amadori products and the formation of a wide variety of reaction

60

products

61

undesirable to food quality. Therefore, it is often important to prevent the later stages of the

62

Maillard reaction from occurring when using it to stabilize proteins.

22

16-19

20

. For example, in a previous study we demonstrated the ability of

. The hydrophilic polysaccharide chains provide strong steric repulsion

16

. It involves the condensation of the carbonyl group of a reducing

21

. After these initial

. Some of these products have color and aroma characteristics that are

63

As mentioned earlier, one of the main limitations to using resveratrol as a nutraceutical

64

ingredient is its low oral bioavailability. Nanoparticles can often be designed to increase the


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bioavailability of the compounds encapsulated within them. The small size of the particles

66

confers colloidal delivery systems with unique physicochemical characteristics that may

67

enhance the bioavailability of lipophilic nutraceuticals

68

may occur for a number of different reasons: enhanced solubility in intestinal fluids;

69

improved protection against chemical or metabolic transformations within the

70

gastrointestinal tract; prolonged residence time due to mucoadhesion of the particles to the

71

intestinal wall; increased absorption by epithelium cells

72

hydrolysis of Maillard conjugates by the digestive enzymes found in the gastrointestinal

73

tract is lower than the hydrolysis of native proteins

74

the bioaccessibility of compounds encapsulated in conjugated or non-conjugated protein-

75

based delivery systems.

18, 25

23

. This increase in bioavailability

24

. Studies have shown that the

. This could result in differences in

76

The aim of the current study was to determine the effect of encapsulation on the

77

physicochemical stability and bioaccessibility of resveratrol. Resveratrol was encapsulated

78

using two different approaches: (i) within biopolymer complexes; (ii) within biopolymer

79

nanoparticles. The biopolymer complexes were formed by binding the resveratrol to either

80

caseinate or caseinate-dextran. The biopolymer nanoparticles were formed by coating a

81

zein core with either a caseinate or a caseinate-dextran shell. The caseinate-dextran

82

complexes were formed by heating the protein and carbohydrate together under appropriate

83

conditions to induce the Maillard reaction.

84

bioaccessibility of the encapsulated resveratrol were determined to establish the potential

85

efficacy of these two delivery systems. The results of this study will facilitate the rational

86

design of effective encapsulation technologies that can enhance both the stability and

87

effectiveness of hydrophobic nutraceuticals.

The physicochemical stability and


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Material and methods

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Materials

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Resveratrol standard (purity ≥ 99%) was purchased from Extrasynthese (Lyon,

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France). Sodium dodecylsufate (SDS), o-phthalaldehyde (OPA, ≥ 99%), leucin, and 2-

92

mercaptoethanol were purchased from Merck (Madrid, Spain). Dextran (40 kDa), pepsin,

93

pancreatin and bile extract were purchased from Sigma-Aldrich Co. (Madrid, Spain).

94

Ethanol 96% was purchased from OPPAC (Navarra, Spain). Sodium tetraborate analytical

95

grade was purchased from Probus (Barcelona, Spain). Soy lecithin was purchased from

96

Guinama (Valencia, Spain). Ammonium carbonate analytical grade was purchased from

97

Alfa Aesar (Ward Hill, MA, USA). All other chemicals, reagents, and solvents were

98

purchased from Panreac (Barcelona, Spain) and were of analytical grade unless otherwise

99

stated. Resveratrol (99%) from grape skin extract was purchased from Changsha Organic

100

Herb Inc. (Changsha, China). Food grade zein was purchased from Kobayashi Perfumery

101

Co. (Tokio, Japan). Sodium caseinate was kindly provided by VicorQuimia (Barcelona,

102

Spain).

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Maillard conjugate formation

104

Sodium caseinate (2% w/v) and dextran (3.5% w/v) were solubilized overnight at 5 ºC

105

in double distilled water. The completely solubilized and hydrated samples were

106

subsequently spray-dried (Mini Spray Drier B-191, Büchi, Flawil, Switzerland) using an

107

inlet temperature of 180 ºC, a feed rate of 7.5 mL/min, a compressed air pressure of 600

108

kPa, and air flow rate of 35 m3/h. Maillard conjugation reactions were performed by

109

incubating the spray-dried mixture at 60 ºC and 76% relative humidity for up to 48 h

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The relative humidity was maintained using a desiccator containing a saturated potassium

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bromide solution. After conjugation, the samples were allowed to cool to room temperature ACS Paragon Plus Environment

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.

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and ground using a mortar and pestle, then stored in a desiccator prior to use.

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Maillard conjugate characterization

114

Conjugation efficiency

115

The conjugation efficiency was determined by measuring reduction in free amino

116

groups using the OPA assay. OPA reagent was prepared freshly prior to use according to

117

Pan, Mu, Hu, Yao and Jiang

118

v/v), 25 mL 0.100 M sodium tetraborate buffer (pH 9.5), 2.5 mL 20% SDS solution (w/v),

119

and 0.10 mL 2-mercaptoethanol were mixed together and brought to a final volume of 50

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mL. After solubilizing the conjugates 5 mg mL-1, 0.10 mL of the dispersion was mixed with

121

2.70 mL of OPA reagent and incubated for 1 min at room temperature. The absorbance at

122

340 nm was then measured immediately using a UV-VIS spectrophotometer (Thermo

123

Scientific Multiskan GO spectrophotometer, ThermoFisher Scientific, Vartaa, Finland).

124

Calibration curves were constructed using L-leucine (0,2-5 mM) as a standard compound

125

containing an amino group 17, 26. The conjugation efficiency was defined as follows:

26

. In short, 40 mg OPA (dissolved in 1.0 mL 95% ethanol

126 127

(1) Stabilizing effect of the conjugation

128

The stabilizing effect of conjugation on casein was assessed by measuring the

129

reduction in turbidity of conjugated samples around the isoelectric point of the protein (pH

130

4.6). Samples were dissolved in double distilled water at concentrations of 5 mg mL-1, and

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subsequently acidified to pH 4.6 with HCl. Nephelometric Turbidity Units (NTU) of the

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samples were measured in a turbidimeter (Turbiquant 3000 IR, Merck, Darmstadt

133

Germany).

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Browning

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To assess browning caused by the Maillard reaction the absorbance of conjugates was ACS Paragon Plus Environment

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measured at 420 nm. Conjugates were dissolved in double distilled water (5 mg/mL), and

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then diluted 5 times. The absorbance at 420 nm was measured using the UV-VIS

138

spectrophotometer with 1 cm polystyrene cuvettes.

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Resveratrol solubility

140

The solubility of resveratrol in the solvent was assessed by quantifying the amount of

141

resveratrol dispersed in the antisolvent phase (1% (w/v) sodium caseinate solution, pH 7)

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after 120 h storage at room temperature

143

1500 µg mL-1) were dispersed in the antisolvent phase following the procedure described in

144

section 2.5 without zein. After this storage period, samples were centrifuged 10 min at 1000

145

rpm using an eppendorf centrifuge (Centrifuge 5415R, Eppendorf, Hamburg, Germany).

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An aliquot of supernatant was collected, diluted in DMSO, and measured as described in

147

section 2.7. The diluted antisolvent phase without grape skin extract was used as a blank.

148

Fabrication of resveratrol-loaded core-shell nanoparticles

27

. Different amounts of grape skin extract (25-

149

Zein (3% w/v) was dissolved in 85% (v/v) ethanol by stirring for 45 min followed by

150

filtration using a filter paper. The filtered zein solution was subsequently added to the

151

antisolvent at a 1:5 ratio under continuous stirring (440 rpm) at room temperature. The

152

antisolvent phase consisted of aqueous sodium caseinate or Maillard conjugate solutions

153

(pH 7.0, final protein concentration of 1% w/v). After addition, the particle suspensions

154

were stirred for an additional 2 min. Ethanol was removed from the mixtures using a

155

vacuum oven (Binder, Binder GmbH, Tuttligen, Germany) at 30 ºC for 1.5 h. For the

156

nanoparticles loaded with resveratrol, 0.15% (w/v) of grape skin extract was dissolved in

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the solvent phase prior to particle formation. The final particle suspensions contained 0.5%

158

(w/v) zein and 0.025% (w/v) resveratrol.


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Fabrication of resveratrol-loaded biopolymer complexes

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Resveratrol-loaded biopolymer complexes were prepared by mixing resveratrol (0.15%

161

(w/v) of grape skin extract in 85% (v/v) ethanol) with an aqueous solution containing

162

sodium caseinate or Maillard conjugates (Section 2.4). The final particle suspensions

163

contained 1% (w/v) of sodium caseinate and 0.025% (w/v) resveratrol.

164

Resveratrol quantification

165

The resveratrol level contained in the nanoparticles was measured using a UV-visible

166

spectrophotometer (Thermo Scientific Multiskan GO spectrophotometer, ThermoFisher

167

Scientific, Vartaa, Finland) at a wavelength of 307 nm. Pure resveratrol was dissolved in

168

dimethyl sulfoxide (DMSO) at 100 µg/mL and then diluted to create a calibration curve

169

with concentrations ranging from 0.2 to 6.0 µg/mL.

170

Characterization of nanoparticles

171

Particle size and -potential

172

Mean particle diameter, polydispersity index, and particle size distribution based on

173

number were obtained using a dynamic light scattering instrument (Zetasizer 3000,

174

Malvern Instruments, Malvern, UK). The ζ-potential was determined by particle

175

electrophoresis using the same instrument. Samples were diluted 20 times to avoid multiple

176

scattering effects and equilibrated at 25 ˚C prior to analysis.

177

Retention efficiency (RE)

178 179

The capacity of the delivery systems to retain resveratrol was determined using the following equation:

180

(2)

181

Here, CT is the resveratrol contained in the suspension and CF is the amount of free

182

resveratrol. The free resveratrol was determined as the amount of resveratrol that was ACS Paragon Plus Environment

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collected in the filtrate receiver after being centrifuged at 4020 g for 30 min using a using a

184

bench top centrifuge (Sigma 3K30, Sigma GmbH, Osterode am Harz, Germany). The

185

sample was placed in a centrifugal filter (Amicon® Ultracel-10 kDa 2 mL Millipore, Cork

186

Ireland)

187

taken into account to correct the actual amount of resveratrol in the filtrate. The resveratrol

188

contained in the nanoparticles and the filtrate was assessed by diluting the suspension in

189

DMSO

190

resveratrol were used as blank samples.

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UV-light stability

28

29

. The recovery yield of resveratrol after passing through the filter (92%) was

prior to quantification as described in Section 2.7. The suspensions without

192

The stability of trans-resveratrol to light induced isomerization was assessed using a

193

UV-light lamp and cabinet. An aliquot of 5 mL of each sample was poured into a Petri dish

194

(60 mm diameter) and exposed to UV-light at 365 nm for 1h 7. The remaining trans-

195

resveratrol was measured as described in Section 2.7 after the sample was diluted in

196

DMSO. The suspensions without resveratrol were used as blanks.

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In vitro digestion model

198

A dynamic in vitro gastrointestinal model was used to study the influence of the

199

biopolymer particles composition on resveratrol bioaccessibility. The gastrointestinal

200

model was based on the work by Minekus, et al.

201

was not included in this work since most liquids do not require an oral phase, mainly due to

202

the very short residence times in the oral cavity 30.

203

Gastric phase

30

with modifications. The mouth phase

204

The suspension (10 mL) was mixed with simulated gastric fluid (SGF, 10 mL) that had

205

a composition shown in Table 1. The resulting mixture was readjusted to pH 3.0 using

206

NaOH or HCl solutions, and then shaken continuously at 150 rpm and 37 ºC in an incubator


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(Incubator Mini Shaker, VWR, Radnor, PA, USA) for 2 h to mimic the temperature and

208

motility of the stomach. The samples were then transferred to the intestinal phase. An

209

aliquot of the gastric phase was diluted 10 times in the simulated gastric buffer and the

210

particle size and ζ-potential were analyzed as described in Section 2.8.1.

211

Intestinal phase

212

The “chyme” sample collected from the gastric phase (15 mL) was mixed with

213

simulated intestinal fluids (SIF, 15 mL) with a composition shown in Table 1. The resulting

214

mixture was then readjusted to pH 7.0 using NaOH or HCl solutions, and then shaken

215

continuously at 150 rpm and 37 ºC in an incubator (Incubator Mini Shaker, VWR, Radnor,

216

PA, USA) for 2 h to mimic the temperature and movement of the intestine. The samples

217

were then transferred to centrifugal tubes to determine the bioaccessibility of resveratrol.

218

An aliquot of the intestinal phase was diluted 5 times in the simulated intestinal buffer and

219

the particle size and ζ-potential were analyzed as described in Section 2.8.1.

220

Bioaccessibility determination

221

The bioaccessibility of resveratrol was evaluated after the samples had passed through

222

the simulated small intestine phase of the gastrointestinal model. Aliquots of 10 mL of the

223

samples were centrifuged (10000 g for 40 min at 10 ºC) using a bench top centrifuge

224

(Sigma 3K30, Sigma GmbH, Osterode am Harz, Germany). After centrifugation, the

225

samples separated into a sediment phase at the bottom and a clear micelle phase at the top.

226

Aliquots (5 mL) were collected from the supernatant (“micelle phase”) and filtered using a

227

syringe filter (17 mm, 0.45 µm, PVDF, ThermoFisher Scientific, Rockwood, TN USA).

228

After filtration the samples were diluted in DMSO and the resveratrol was measured

229

according to Section 2.7. The bioaccessibility (BA) was determined using the following

230

equation:


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(3)

232

where, CM is the resveratrol concentration in the micelle phase and CI is the initial

233

concentration of resveratrol in the suspension 27. The suspensions without resveratrol were

234

used as blanks.

235

Data analysis

236

All experiments were performed in triplicate and the results are given as mean values ±

237

standard deviation. Differences among the treatments were determined using an analysis of

238

variance (ANOVA) and a post-hoc Tukey test with a confidence level of 95%. The

239

analyses were made using SPSS software (IBM Corporation., Armonk NY, USA).

240

Results and Discussion

241

Conjugation characterization

242

Initially, we carried out a series of experiments to assess the time-dependence of the

243

formation of protein-carbohydrate conjugates through the Maillard reaction (Figure 1).

244

The conjugation efficiency of the reaction of sodium caseinate with dextran was analyzed

245

using the OPA test, which quantifies the amount of unreacted amino groups remaining on

246

the protein after conjugation (Figure 1A). The effect of conjugation on the aggregation

247

stability of the sodium caseinate was assessed by measuring the turbidity of the conjugate

248

solutions at the isoelectric point of the protein, i.e., pH 4.6 (Figure 1B). The formation of

249

brown colors in the samples as a result of the later stages of the Maillard reaction was

250

determined by measuring the solution absorbance at 420 nm (Figure 1C).

251

The conjugation efficiency increased with increasing incubation time during the first

252

24 h, and then reached a relatively constant level. The average number of amino groups per

253

caseinate molecule available to react with reducing sugars of the polysaccharides has been

254

reported to be 13.6

17, 26

. This suggests that the number of dextran molecules attached per ACS Paragon Plus Environment

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caseinate molecule was about 5.0 after 24 h incubation (37% efficiency), and about 5.5

256

after 48 h incubation (41% efficiency). The reduction in the conjugation rate with

257

incubation time can be attributed to two effects: (i) a reduction in the number of amino

258

groups available to react; (ii) steric hindrance caused by already attached dextran molecules

259

17

260

near the isoelectric point of sodium caseinate. The absence of a net charge on the protein

261

molecules reduces the electrostatic repulsion, which leads to the formation of protein

262

aggregates

263

protein aggregates were of a size that scattered light strongly (Figure 1B Insert)

264

observed reduction in turbidity of the caseinate solutions after conjugation with dextran

265

(Figure 1B), suggested that the carbohydrate chains inhibited protein aggregation near the

266

isoelectric point. This stabilization can be attributed to an increase in the steric repulsion

267

between the molecules after conjugation. The turbidity decreased sharply from 0 to 8 h

268

incubation, which indicated that this was sufficient time to form stable protein-carbohydrate

269

conjugates. Finally, the presence of a brown color in the solutions due to the formation of

270

secondary Maillard reaction products was determined by measuring changes in their

271

absorbance at 420 nm

272

quality perspective, and should therefore be minimized. In addition the appearance of color

273

is the result of the degradation of the Amadori product

274

Maillard reaction can lead to dissociation of the protein-carbohydrate conjugate. Therefore,

275

it is important to keep the browning as low as possible. The absorbance of the samples

276

continued to increase throughout the incubation period (Figure 1C), and therefore it is

277

advantageous to use as short a conjugation time as possible.

. The turbidity of the samples at pH 4.6 is related to the formation of protein aggregates

31

. Solutions containing aggregated caseinate appeared turbid because the

22, 33

32

. The

. This type of brown color is usually undesirable for a food

22

, which in advance stages of the


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Overall, our results suggest that a 24 h incubation period was an optimum compromise

279

between increasing conjugation efficiency and decreasing browning reactions. Although

280

there is a sharp increase in absorbance between 16 and 24 h, it is not until 24 that the

281

conjugation efficiency and turbidity reached plateau values with no changes observed

282

between 24 and 36 h. We therefore used this time to fabricate the conjugates for the rest of

283

the experiments described in this study.

284

Resveratrol solubility

285

One of the factors that determine the efficacy of a delivery system is the maximum

286

amount of the functional ingredient that can be incorporated into it. The amount of

287

resveratrol that could be incorporated into aqueous solutions was therefore determined.

288

Increasing amounts of grape skin extract were dispersed in a 1% (w/v) sodium caseinate

289

solution (Figure 2). The dispersions were prepared using the same procedure as used to

290

produce the nanoparticles by antisolvent precipitation (Section 2.5), and then the mixtures

291

were stored for 120 h at room temperature to allow the crystallization of any undissolved

292

resveratrol 34. The mixtures were then centrifuged, which caused the resveratrol crystals to

293

form a sediment at the bottom of the tubes, leaving a supernatant containing the dissolved

294

resveratrol. The amount of dissolved resveratrol was then measured using a

295

spectrophotometer. Adding up to 250 µg mL-1 of grape skin extract to the aqueous solution

296

resulted in complete solubility of the resveratrol, indicated by the similarity of the added

297

and measured resveratrol amounts. In this region the solution was either saturated or super-

298

saturated with resveratrol. When 500 µg mL-1 of grape skin extract was added the amount

299

of measured resveratrol was only about 83% of the added resveratrol, which indicates that

300

part of the resveratrol crystalized and precipitated. With a further increase in the amount of

301

grade skin extract added, the measured resveratrol concentration actually decreased and the


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302

variability of the data became larger. This effect can be attributed to the fact that some of

303

the supersaturated resveratrol crystallized, so that the amount of resveratrol remaining

304

dissolved in the aqueous solution was equal to the equilibrium solubility.

305

The relatively high supersaturation of resveratrol in the aqueous solutions can be

306

attributed to the presence of the sodium caseinate. It is known that proteins can act as

307

nucleation and crystallization inhibitors by forming complexes with hydrophobic bioactive

308

compounds, thus decreasing the nucleation rate

309

caseinate are known to form complexes through hydrophobic interactions and hydrogen

310

bonding

311

aqueous solution was insufficient to bind all the resveratrol leading to the formation of

312

nuclei and crystals during the storage period 34, 35. From Figure 2 it can be deduced that the

313

nucleation rate at grape skin extract concentrations above 500 µg mL-1 was faster than the

314

ability of the proteins to bind resveratrol leading to sudden precipitation and lower amounts

315

of resveratrol in the supernatant.

10

34

. In particular resveratrol and sodium

. At 500 µg mL-1 of added grape skin extract, the amount of protein in the

316

If a hydrophobic crystalline bioactive compound is going to be incorporated into an

317

aqueous based system, then it is important to ensure that it is present at a level below the

318

loading capacity of the delivery system.

319

concentration will not be exceeded and so bioactive precipitation during production,

320

storage, or utilization will be avoided. For this reason, the grape skin extract concentration

321

used in the remainder of this study was 250 µg mL-1, i.e. below the saturation level (Figure

322

2). Another important consideration when working with bioactive compounds, is to reach

323

the minimum concentration to promote a health benefit. In this case 250 µg mL-1 of grape

324

skin extract in the colloidal dispersions represents 25 times the concentration typically

325

found in wine (5 mg/l 36).

Under these conditions, the saturation


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Retention efficiency

327

In this study, the retention efficiency means the amount of resveratrol associated within

328

the biopolymer nanoparticles or complexes. The retention efficiency is important because it

329

determines the fraction of the bioactive compound within a delivery system that is

330

protected from degradation by external conditions, such as heat, light, or oxygen. The

331

delivery systems could be classified into two groups according to their measured retention

332

efficiencies (Figure 3), with the biopolymer nanoparticles (RE ≈ 83%) having a

333

significantly higher retention efficiency (p < 0.05) than the biopolymer complexes (RE ≈

334

68%). The retention efficiency of the biopolymer nanoparticles measured in this work was

335

similar to that obtained in our previous study where resveratrol was also encapsulated in

336

zein nanoparticles formed by antisolvent precipitation 8. All the biopolymer nanoparticles

337

formed in the current study had an inner core of zein surrounded by an outer layer of

338

caseinate molecules. However, in some cases the caseinate molecules were covalently

339

attached to dextran molecules through the Maillard reaction.

340

The retention efficiency was similar for protein nanoparticles, protein nanoparticles

341

coated by Maillard conjugates, and a physical mixture of dextran and protein nanoparticles

342

(p > 0.05). These results suggest that it was the protein core (zein and caseinate) that was

343

primarily responsible for encapsulating the resveratrol, rather than the carbohydrate shell.

344

Presumably, the non-polar resveratrol molecules were trapped within the hydrophobic core

345

formed by zein and caseinate. The retention efficiency of the biopolymer complexes was

346

about 15% lower than that of biopolymer nanoparticles (Figure 3). For these systems, the

347

retention efficiency was similar for caseinate alone, caseinate conjugated to dextran, and a

348

physical mixture of dextran and caseinate (p > 0.05), which suggested that it was the

349

caseinate that was primarily responsible for resveratrol binding. Hydrophobic forces are


16

Page 17 of 43


10

350

known to play an important role in the binding of polyphenols to proteins

351

account for the fact that dextran did not play an important role in determining the binding

352

of the resveratrol to the caseinate molecules.

353

efficiency was higher for the biopolymer nanoparticles than the biopolymer complexes may

354

have been because zein has a higher hydrophobicity than caseinate.

355

UV-light stability

356

. This would

In addition, the fact that the retention

UV-light triggers the isomerization of trans-resveratrol to cis-resveratrol 7, which 37, 38

357

results in a loss of biological activity

. The stability of the various delivery systems

358

against isomerization was assessed by exposing them to UV-light (365 nm) for 60 min in

359

Petri dishes. Resveratrol dissolved in dimethyl sulfoxide at the same concentration was

360

used as a control. There was a significant difference (p < 0.05) in the amount of trans-

361

resveratrol remaining among the different samples after exposure (Figure 4). Both the

362

biopolymer nanoparticles and complexes were able to protect the resveratrol against

363

isomerization, with the nanoparticles being more effective. Conjugation of the caseinate

364

with dextran, or the addition of free dextran, did not have a significant (p > 0.05) effect on

365

the protection of resveratrol in either type of delivery system. The protection of resveratrol

366

against exposure to light may have occurred through a number of different mechanisms.

367

Proteins absorb UV-light due to the presence of aromatic groups and double bonds

368

which would reduce the intensity of light waves reaching the resveratrol. The suspensions

369

of biopolymer nanoparticles contained 50% (w/w) more protein than the suspensions of

370

biopolymer complexes, which may account for their better ability to inhibit isomerization.

371

On the other hand, the higher protection of the nanoparticle systems might be because they

372

scattered light more effectively, and thereby allowed less light to reach the resveratrol

373

This effect is seen in the insert to Figure 4, which compares the appearance of suspensions


39

32

,

.

17


Page 18 of 43

374

of biopolymer nanoparticles (turbid) and biopolymer complexes (clear).

Finally, the

375

retention efficiency of the systems (Figure 3) is closely related to the protection of

376

resveratrol, being more protective the systems with higher retention efficiency. The

377

treatments with lower retention efficiency have a higher amount of free resveratrol

378

dispersed in the aqueous solution, which results in less protection from exposure to UV-

379

light.

380

In vitro digestion model

381

The biopolymer nanoparticles and complexes were passed through the simulated

382

gastrointestinal tract to assess resveratrol bioaccessibility. Changes in the size and charge

383

of the biopolymer nanoparticles were measured throughout the simulated GIT, and the

384

bioaccessibility of resveratrol for both type of biopolymer delivery systems was measured

385

at the end.

386

Initial biopolymer nanoparticles

387

The non-conjugated biopolymer nanoparticles had similar diameters in the absence

388

(238 nm) and presence (237 nm) of non-adsorbed dextran, which suggested that this

389

carbohydrate did not directly influence their particle size (Figure 5A). In addition, the ζ-

390

potential of the non-conjugated biopolymer nanoparticles was similar in the absence (-50

391

mV) and presence (-50 mV) of non-adsorbed dextran (Figure 5B), which suggested that

392

this carbohydrate did not appreciably modify interfacial properties. The conjugation of the

393

caseinate with dextran did have an effect on particle size and charge, with the mean

394

diameter falling to 224 nm and the ζ-potential falling to -47 mV. Particle size distribution

395

measurements of the suspensions indicated that they had monomodal and narrow

396

distributions (Figure 6). The slight reduction in particle size upon Maillard conjugation

397

suggests that there is a change in the packing of the caseinate molecules at the droplet


18

Page 19 of 43


17

. The slight reduction in the ζ-

398

interfaces due to the presence of the carbohydrate chains

399

potential can be explained by a reduction of the number of ionizable amino groups on the

400

protein after conjugation with dextran. In addition, the presence of the dextran may have

401

increased the thickness of the hydration layer around the protein nanoparticles where the

402

charge is measured, thereby leading to a decrease in surface potential

403

that there were no flocs or aggregates in the initial conjugated or non-conjugated

404

nanoparticle suspensions.

405

Gastric phase

406

18

. Figure 7 shows

Samples were exposed directly to the gastric phase because liquids typically only 30

407

spend a very short time in the oral cavity before swallowing

408

non-conjugated nanoparticle suspensions increased greatly (d > 1000 nm) after exposure to

409

the simulated gastric phase (Figure 5A). Conversely, the increase in particle size of the

410

conjugated nanoparticle suspensions was much less (d ≈ 530 nm). The ζ-potential of the

411

particles in the gastric “chyme” was slightly positive (Figure 5B), which can be attributed

412

to the fact that both caseinate and zein were below their isoelectric point. The particle size

413

distribution

414

nanoparticles, but only moderate aggregation of the conjugated ones after exposure to

415

gastric fluids (Figure 6). The destabilization of protein-based colloidal delivery systems

416

under simulated gastric conditions may occur for a number of reasons, including loss of

417

charge due to pH changes, electrostatic screening due to an increased ionic strength, and

418

proteolysis by pepsin 18. Further information about the relative importance of pepsin on the

419

stability of the nanoparticle systems was obtained by omitting pepsin from the gastric

420

fluids. The particle diameter did not increase for the conjugated nanoparticles and only

measurements

shows

extensive

aggregation


. The mean diameter of the

of

the

non-conjugated

19


Page 20 of 43

421

increased by about 50 nm for the non-conjugated ones (data not shown), suggesting that the

422

hydrolysis of the proteins by pepsin was an important destabilizing factor during this phase.

423

The observed increase in particle size can be attributed to aggregation of the protein

424

nanoparticles (Figure 7). The lower reactivity of digestive enzymes toward the conjugated

425

nanoparticles can be attributed to the steric hindrance exerted by the dextran molecules

426

attached to their exteriors

427

proteolysis of certain amino acids due to glycation 25.

428

Intestinal phase

40

. Another possible explanation is the reduction of the

429

After the gastric phase, the resulting “chyme” was mixed with small intestinal fluids

430

(SIF) and then incubated for 2 h. After exposure to simulated intestinal conditions, all the

431

nanoparticle systems had fairly similar size distributions and mean diameters (d ≈ 750 nm)

432

(Figures 5A and 6). Furthermore the ζ-potential of all the samples became highly negative

433

(Figure 5B), which can be attributed to the fact that the pH was above the isoelectric point

434

of the proteins, and due to the presence of anionic bile salts in the SIF. The fact that all the

435

samples had relatively similar particle sizes and charges suggests that they all contained

436

fairly similar types of particle after digestion

437

mixed micelles and liposome kind of structures formed by bile salts and/or aggregated

438

peptides remaining after digestion of the proteins by proteases. To confirm if the bile salts

439

or the pancreatin were responsible for the changes in size and distribution, a digestion made

440

with only pancreatin or bile extract was performed for the conjugated biopolymer

441

nanoparticles (data non shown). The results indicated that the digestion with only bile salts

442

resulted in the same size and distributions than the ones showed in Figure 5A and 6.

41

. Presumably, these particles consisted of


20

Page 21 of 43


443

Bioaccessibility

444

Finally, the bioaccessibility of resveratrol encapsulated within the biopolymer nanoparticles

445

or bound to the biopolymer complexes was determined by measuring the amount of

446

resveratrol in the micelle phase after the intestinal step of the in vitro digestion model. It is

447

assumed that only resveratrol incorporated within the mixed micelles is available for

448

absorption within the small intestine, i.e., is bioaccessible 23. Figure 8 shows the percentage

449

of resveratrol present in the micelle phase after the small intestine stage. In the absence of

450

lipids, the mixed micelle phase will primarily be composed of micelles and liposomes

451

formed by bile salts and phospholipids in the gastrointestinal fluids, as well as any

452

undigested soluble proteins and peptides. Thus, the resveratrol measured in the mixed

453

micelle phase could be present within micelles or liposomes

454

peptides 10. It can be seen that there was a significant difference (p < 0.05) in the resveratrol

455

contained in the mixed micelle phase between the different delivery systems and the

456

resveratrol dispersed in water at the same concentration. When resveratrol was directly

457

added to water it immediately formed crystals that could be seen by the naked eye (Figure

458

8, Insert). Conversely, the resveratrol encapsulated within the biopolymer nanoparticles or

459

complexes did not form any visible crystals. The crystallization and precipitation of

460

resveratrol could explain the lower bioaccessibility of the free bioactive component

461

compared to the encapsulated ones 27. Nevertheless, the bioaccessibility of resveratrol was

462

still above 50%, it has been demonstrated by fluorescence quenching studies that

463

polyphenols can bind to the enzymes present in the gastric and intestinal fluids increasing

464

their solubility in the micelle phase

465

nanoparticles did not affect the bioaccessibility of resveratrol when compared to the ones

466

bound to sodium caseinate. Neither the conjugation nor the presence of dextran had a

44

42, 43

, or bound to proteins or

. The encapsulation of grape skin extract in the


21


Page 22 of 43

467

significant effect (p > 0.05). Previous studies have shown that other types of caseinate-

468

based and zein-based delivery systems are also able to retain hydrophobic nutraceuticals

469

under simulated gastrointestinal conditions

470

retained within the colloidal particles in the gastrointestinal tract, presumably accounts for

471

the fact that it did not crystallize and was available to be solubilized within mixed micelles

472

or to bind to peptides or proteins after digestion. The fact that all the biopolymer

473

nanoparticle samples had similar characteristics after the small intestine phase (Figures 5A,

474

5B and 6) can account for the lack of differences in the bioaccessibility among the different

475

delivery systems. It is known from in vivo studies that resveratrol undergoes extensive

476

metabolism within the gastrointestinal track and liver mainly due to glucorination and

477

sulfonation, and that only traces reach the circulatory system in an active form 1. One can

478

speculate that these delivery systems may slow down or prevent the extensive metabolism

479

and increase the bioactivity of resveratrol after consumption. Further research is clearly

480

needed to test this hypothesis.

17, 28, 45, 46

. The fact that the resveratrol is

481

Both biopolymer nanoparticles (consisting of a zein core and a caseinate or caseinate-

482

dextran shell) and biopolymer complexes (consisting of caseinate or caseinate-dextran)

483

protected resveratrol against isomerization and increased its bioaccessibility, but the

484

biopolymer particles were more effective. In conclusion both delivery systems have the

485

potential for application in functional food and beverage products.

486

complexes are easier to form and can be incorporated into transparent products, whereas the

487

biopolymer nanoparticles are more difficult to form, can only be used in turbid or opaque

488

products, but are more effective.


The biopolymer

22

Page 23 of 43


References

1.

Walle, T.; Hsieh, F.; DeLegge, M. H.; Oatis Jr, J. E.; Walle, U. K., High absorption but

very low bioavailability of oral resveratrol in humans. Drug Metab. Dispos. 2004, 32, 1377-1382.

2.

Alves, N. E. G., Studies on mechanistic role of natural bioactive compounds in the

management of obesity, an overview. Open Nutraceuticals J 2012, 5, 193.

3.

Catalgol, B.; Batirel, S.; Taga, Y.; Ozer, N. K., Resveratrol: French paradox revisited.

Front. Pharmacol. 2012, 3 JUL.

4.

Neves, A. R.; Lucio, M.; Martins, S.; Lima, J. L. C.; Reis, S., Novel resveratrol

nanodelivery systems based on lipid nanoparticles to enhance its oral bioavailability. Int. J. Nanomedicine 2013, 8, 177-187.

5.

Hung, C.-F.; Chen, J.-K.; Liao, M.-H.; Lo, H.-M.; Fang, J.-Y., Development and

Evaluation of Emulsion-Liposome Blends for Resveratrol Delivery. J. Nanosci. Nanotechnol. 2006, 6, 2950-2958.

6.

Patel, K. R.; Scott, E.; Brown, V. A.; Gescher, A. J.; Steward, W. P.; Brown, K., Clinical

trials of resveratrol. In Resveratrol and Health, Vang, O.; Das, D. K., Eds. 2011; Vol. 1215, pp 161-169.


23


7.

Page 24 of 43

Trela, B. C.; Waterhouse, A. L., Resveratrol: Isomeric molar absorptivities and

stability. J. Agric. Food Chem. 1996, 44, 1253-1257.

8.

Davidov-Pardo, G.; Joye, I. J.; McClements, D. J., Encapsulation of resveratrol in

biopolymer particles produced using liquid antisolvent precipitation. Part 1: Preparation and characterization. Food Hydrocolloids 2015, 45, 309-316.

9.

Joye, I. J.; McClements, D. J., Biopolymer-based nanoparticles and microparticles:

Fabrication, characterization, and application. Curr. Opin. Colloid Interface Sci. 2014, 19, 417-427.

10. Acharya, D. P.; Sanguansri, L.; Augustin, M. A., Binding of resveratrol with sodium caseinate in aqueous solutions. Food Chem. 2013, 141, 1050-1054.

11. Ye, J. H.; Thomas, E.; Sanguansri, L.; Liang, Y. R.; Augustin, M. A., Interaction between Whole Buttermilk and Resveratrol. J. Agric. Food Chem. 2013, 61, 7096-7101.

12. Liang, L.; Tajmir-Riahi, H. A.; Subirade, M., Interaction of beta-Lactoglobulin with resveratrol and its biological implications. Biomacromolecules 2008, 9, 50-56.

13. Hemar, Y.; Gerbeaud, M.; Oliver, C. M.; Augustin, M. A., Investigation into the interaction between resveratrol and whey proteins using fluorescence spectroscopy. Int. J. Food Sci. Technol. 2011, 46, 2137-2144.


24

Page 25 of 43


14. Hu, K.; McClements, D. J., Fabrication of surfactant-stabilized zein nanoparticles: A pH modulated antisolvent precipitation method. Food Res. Int. 2014, 64, 329-335.

15. Zhong, Q.; Jin, M., Zein nanoparticles produced by liquid–liquid dispersion. Food Hydrocolloids 2009, 23, 2380-2387.

16. Oliver, C. M.; Melton, L. D.; Stanley, R. A., Creating proteins with novel functionality via the maillard reaction: A review. Crit. Rev. Food Sci. Nutr. 2006, 46, 337-350.

17. Markman, G.; Livney, Y. D., Maillard-conjugate based core-shell co-assemblies for nanoencapsulation of hydrophobic nutraceuticals in clear beverages. Food Funct. 2012, 3, 262-70.

18. Lesmes, U.; McClements, D. J., Controlling lipid digestibility: Response of lipid droplets coated by β-lactoglobulin-dextran Maillard conjugates to simulated gastrointestinal conditions. Food Hydrocolloids 2012, 26, 221-230.

19. Wooster, T. J.; Augustin, M. A., β-Lactoglobulin-dextran Maillard conjugates: Their effect on interfacial thickness and emulsion stability. J. Colloid Interface Sci. 2006, 303, 564-572.

20. Davidov-Pardo, G.; Joye, I. J.; Espinal-Ruiz, M.; McClements, D. J., Encapsulation of resveratrol in biopolymer nanoparticles stabilized by Maillard conjugation. Food Res. Int. 2015, Under review.


25


Page 26 of 43

21. Ames, J. M., Control of the Maillard reaction in food systems. Trends in Food Science &Technology 1990, 1, 150-154.

22. Van Lancker, F.; Adams, A.; De Kimpe, N., Chemical modifications of peptides and their impact on food properties. Chem Rev 2011, 111, 7876-903.

23. Joye, I. J.; Davidov-Pardo, G.; McClements, D. J., Nanotechnology for increased micronutrient bioavailability. Trends Food Sci. Technol. 2014, 40, 168-182.

24. Li, Z.; Jiang, H.; Xu, C.; Gu, L., A review: Using nanoparticles to enhance absorption and bioavailability of phenolic phytochemicals. Food Hydrocolloids 2015, 43, 153-164.

25. Yi, J.; Lam, T. I.; Yokoyama, W.; Cheng, L. W.; Zhong, F., Controlled Release of βCarotene in β-Lactoglobulin–Dextran-Conjugated Nanoparticles’ in Vitro Digestion and Transport with Caco-2 Monolayers. J. Agric. Food Chem. 2014, 62, 8900-8907.

26. Pan, X.; Mu, M.; Hu, B.; Yao, P.; Jiang, M., Micellization of casein-graft-dextran copolymer prepared through Maillard reaction. Biopolymers 2006, 81, 29-38.

27. Pool, H.; Mendoza, S.; Xiao, H.; McClements, D. J., Encapsulation and release of hydrophobic bioactive components in nanoemulsion-based delivery systems: impact of physical form on quercetin bioaccessibility. Food Funct. 2013, 4, 162-174.


26

Page 27 of 43


28. Luo, Y. C.; Zhang, B. C.; Whent, M.; Yu, L. L.; Wang, Q., Preparation and characterization of zein/chitosan complex for encapsulation of alpha-tocopherol, and its in vitro controlled release study. Colloids Surf., B 2011, 85, 145-152.

29. Podaralla, S.; Perumal, O., Influence of Formulation Factors on the Preparation of Zein Nanoparticles. Aaps Pharmscitech 2012, 13, 919-927.

30. Minekus, M.; Alminger, M.; Alvito, P.; Ballance, S.; Bohn, T.; Bourlieu, C.; Carriere, F.; Boutrou, R.; Corredig, M.; Dupont, D.; Dufour, C.; Egger, L.; Golding, M.; Karakaya, S.; Kirkhus, B.; Le Feunteun, S.; Lesmes, U.; Macierzanka, A.; Mackie, A.; Marze, S.; McClements, D. J.; Menard, O.; Recio, I.; Santos, C. N.; Singh, R. P.; Vegarud, G. E.; Wickham, M. S. J.; Weitschies, W.; Brodkorb, A., A standardised static in vitro digestion method suitable for food - an international consensus. Food Funct. 2014, 5, 11131124.

31. Uskokovic, V., Dynamic light scattering based microelectrophoresis: main prospects and limitations. J. Dispersion Sci. Technol. 2012, 33, 1762-1786.

32. McClements, D. J., Food Emulsions: Principles, Practice, And Techniques. CRC PressINC: 2005; p 374.

33. Moreno, F. J.; Molina, E.; Olano, A.; López-Fandiño, R., High-Pressure Effects on Maillard Reaction between Glucose and Lysine. J. Agric. Food Chem. 2002, 51, 394-400.


27


Page 28 of 43

34. McClements, D. J., Crystals and crystallization in oil-in-water emulsions: Implications for emulsion-based delivery systems. Adv. Colloid Interface Sci. 2012, 174, 1-30.

35. Joye, I. J.; McClements, D. J., Production of nanoparticles by anti-solvent precipitation for use in food systems. Trends Food Sci. Technol. 2013, 34, 109-123.

36. Baur, J. A.; Sinclair, D. A., Therapeutic potential of resveratrol: The in vivo evidence. Nat. Rev. Drug Discovery 2006, 5, 493-506.

37. Fauconneau, B.; WaffoTeguo, P.; Huguet, F.; Barrier, L.; Decendit, A.; Merillon, J. M., Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests. Life Sci. 1997, 61, 2103-2110.

38. Rius, C.; Abu-Taha, M.; Hermenegildo, C.; Piqueras, L.; Cerda-Nicolas, J. M.; Issekutz, A. C.; Estan, L.; Cortijo, J.; Morcillo, E. J.; Orallo, F.; Sanz, M. J., Trans- but Not Cis-Resveratrol Impairs Angiotensin-II-Mediated Vascular Inflammation through Inhibition of NF-kappa B Activation and Peroxisome Proliferator-Activated Receptorgamma Upregulation. J. Immunol. 2010, 185, 3718-3727.

39. Luo, Y. C.; Wang, T. T. Y.; Teng, Z.; Chen, P.; Sun, J. H.; Wang, Q., Encapsulation of indole-3-carbinol and 3,3 '-diindolylmethane in zein/carboxymethyl chitosan nanoparticles with controlled release property and improved stability. Food Chem. 2013, 139, 224-230. ACS Paragon Plus Environment

28

Page 29 of 43


40. Corzo-Martínez, M.; Soria, A. C.; Belloque, J.; Villamiel, M.; Moreno, F. J., Effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine βlactoglobulin. Int. Dairy J. 2010, 20, 742-752.

41. Sarkar, A.; Horne, D. S.; Singh, H., Interactions of milk protein-stabilized oil-inwater emulsions with bile salts in a simulated upper intestinal model. Food Hydrocolloids 2010, 24, 142-151.

42. Wan, Z.-L.; Wang, J.-M.; Wang, L.-Y.; Yang, X.-Q.; Yuan, Y., Enhanced physical and oxidative stabilities of soy protein-based emulsions by incorporation of a watersoluble stevioside-resveratrol complex. J. Agric. Food Chem. 2013, 61, 4433-4440.

43. Bonechi, C.; Martini, S.; Ciani, L.; Lamponi, S.; Rebmann, H.; Rossi, C.; Ristori, S., Using liposomes as carriers for polyphenolic compounds: The case of transresveratrol. PLoS One 2012, 7.

44. Moser, S.; Chegeni, M.; Jones, O. G.; Liceaga, A.; Ferruzzi, M. G., The effect of milk proteins on the bioaccessibility of green tea flavan-3-ols. Food Res. Int. 2014, 66, 297305.

45. Patel, A. R.; Heussen, P. C. M.; Hazekamp, J.; Drost, E.; Velikov, K. P., Quercetin loaded biopolymeric colloidal particles prepared by simultaneous precipitation of quercetin with hydrophobic protein in aqueous medium. Food Chem. 2012, 133, 423429.


29


Page 30 of 43

46. Pan, X.; Yao, P.; Jiang, M., Simultaneous nanoparticle formation and encapsulation driven by hydrophobic interaction of casein-graft-dextran and beta-carotene. J. Colloid Interface Sci. 2007, 315, 456-63.

Note Dr. Gabriel Davidov-Pardo is recipient of a post-doctoral fellowship by the Secretaría de Ciencia Tecnología e Innovación del Distrito Federal (SECITI, Mexico City).

This

material is partly based upon work supported by United States Department of Agriculture, NRI Grants (2011-03539, 2013-03795, 2011-67021, and 2014-67021).


30

Page 31 of 43


Figure captions Figure 1. A) Conjugation efficiency; B) Turbidity at pH 4.6; C) Absorbance at 420 nm of the conjugates of sodium caseinate to dextran 40 kDa. Insert: appearance of the solutions (5 mg mL-1) at pH 4.6, from left to right 0, 8, 16, 24, 32, 40, 48 h of conjugation. The conjugation was induced at 60 ºC with 76% relative humidity for 48 h. The sampling was in intervals of 8 h. The ratio of dextran to sodium caseinate was 1.75 to 1.0.

Figure 2. Dependence of the added and the dissolved grape skin extract in a 1% (w/v) of sodium caseinate solution. The dissolved resveratrol measurements were made at 307 nm after centrifuging the samples to remove any crystals.

Figure 3. Retention efficiency of resveratrol in biopolymer nanoparticles (BnP) and biopolymer complexes (BC) loaded with grape skin extract prepared with conjugated (Con) and non-conjugated (No-con) sodium caseinate in the antisolvent [1.0% (w/v)]. The ratio of dextran to sodium caseinate was 1.75 to 1.0. Zein and grape skin extract concentration in the particle suspensions were 0.5 and 0.025% (w/v), respectively.

a-b

Different letters are

significantly different for each extract separately (p < 0.05)

Figure 4. UV-light stability of trans-resveratrol encapsulated in biopolymer nanoparticles (BnP) and biopolymer complexes (BC) loaded with grape skin extract prepared with conjugated (Con) and non-conjugated (No-con) sodium caseinate in the antisolvent [1.0% (w/v)]. The ratio of dextran to sodium caseinate was 1.75 to 1.0. Zein and grape skin extract concentration in the particle suspensions were 0.5 and 0.025% (w/v), respectively. Insert:


31


left to right appearance of one treatment containing zein and one without it.

Page 32 of 43

a-c

Different

letters are significantly different for each extract separately (p < 0.05)

Figure 5. Effect of the in vitro digestion phase on the mean diameter (A) and zeta-potential (B) of grape skin extract loaded biopolymer nanoparticles (BnP) prepared with conjugated and non-conjugated sodium caseinate in the antisolvent [1.0% (w/v)]. The ratio of dextran to sodium caseinate was 1.75 to 1.0. Zein and grape skin extract concentration in the particle suspensions were 0.5 and 0.025% (w/v), respectively.

Figure 6. Effect of the in vitro digestion phase on the particle size distribution based on number of grape skin extract loaded biopolymer nanoparticles (BnP) prepared with conjugated and non-conjugated sodium caseinate in the antisolvent [1.0% (w/v)]. The ratio of dextran to sodium caseinate was 1.75 to 1.0. Zein and GSE concentration in the particle suspensions were 0.5 and 0.025% (w/v), respectively.

The distributions shown are

representative ones for each treatment.

Figure 7. Microscopic images (40X) of grape skin extract loaded biopolymer nanoparticles (BnP) prepared with conjugated and non-conjugated sodium caseinate in the antisolvent [1.0% (w/v)] after each phase of the in vitro digestion. The ratio of dextran to sodium caseinate was 1.75 to 1.0. Zein and grape skin extract concentration in the particle suspensions were 0.5 and 0.025% (w/v), respectively. Scale bar 20 µm.

Figure 8. In vitro bioaccesability of resveratrol in biopolymer nanoparticles (BnP) and biopolymer complexes (BC) loaded with grape skin extract prepared with conjugated and


32

Page 33 of 43


non-conjugated sodium caseinate in the antisolvent [1.0% (w/v)]. The ratio of dextran to sodium caseinate was 1.75 to 1.0. Zein and grape skin extract concentration in the particle suspensions were 0.5 and 0.025% (w/v), respectively. Insert: Appearance of grape skin extract dispersed in water at 0.025% (w/v). a-b Different letters are significantly different for each extract separately (p < 0.05).


33


Page 34 of 43

Table 1. Final Concentrations of Constituents Before Addition to the Sample of the Simulated Gastric and Intestinal Fluids. Simulated gastric fluid Simulated intestinal fluid pH 3 pH 7 Constituent mmol L-1 KCl 5.52 5.44 KH2PO4 0.72 0.64 NaHCO3 20.00 68.00 NaCl 37.76 30.72 MgCl2(H2O)6 0.08 0.26 (NH4)2CO3 0.40 --NaOH --3.73 HCl 22.48 6.72 CaCl2(H2O)2 0.07 0.30 Lecithina 0.17 --U mL-1 Pepsinb 2000.00 --c Pancreatin --100.00 mg mL-1 Bile extract --6.25 a -1 b Based on the average molecular weight of lecithin (787 g mol ) Made up in SGF. c Made up in SIF and based on the protolithic activity


34

Page 35 of 43


Conjugation efficiency (%)

FIGURE 1

100 80 60 40 20 0

A

0

10

20

30

40

50

Conjugation time (h) B

10000

NTU

1000 100 10

1 0

10

20

30

40

50

Conjugation time (h) C

Absorbance 420 nm

0.08 0.06 0.04 0.02 0.00 0

10

20

30

40

50

Conjugation time (h) ACS Paragon Plus Environment

35


Page 36 of 43

FIGURE 2

450

Measured GSE (µg/ml)

400 350 300

250 200 150 100 50 0 0

250

500

750 1000 1250 1500

Added GSE (µg/ml)


36

Page 37 of 43


FIGURE 3

100

Retention efficiency (%)

90 80

a

a

a

b

b

b

70 60 50 40 30 20

10 0

Treatment


37


Page 38 of 43

Remaining trans-resveratrol (%)

FIGURE 4

80 70

60

a

a

a b

b

b c

50 40 30 20 10 0

Treatment


38

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Mean Particle Diameter (nm)

FIGURE 5

1200 1000

A

Inital Gastric phase Intestinal phase

800 600 400 200 0

Treatment Zeta potential (mV)

B 5 -5 -15 -25 -35 -45

-55 Initial Gastric phase

Treatment ACS Paragon Plus Environment

Intestinal phase 39


Page 40 of 43

FIGURE 6

160

Initial Gastric Phase

140

Intestinal phase

Relative Number (%)

120

No-con BnP+Dex

100 80 60

Con BnP

40

20 No-con BnP

0 10

100

1000

Size (nm)

10000


40

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FIGURE 7

DIGESTION PHASE

TREATMENT Non-con BnP

Con BnP

Initial

Gastric phase

Intestinal phase


41


Page 42 of 43

FIGURE 8

Remaining resveratrol in the micelle phase (%)

100 90 80

a

a

a

a

a

a

70 b

60 50 40 30 20 10 0

Treatment


42

Page 43 of 43


TOC

Simulated Gastric Fluid

Simulated Intestinal Fluid

Centrifugation

Mixed Micelle phase

Concentration Analysis

BIOACCESSIBILITY

Sediment phase

Zein Core

Sodium caseinate

Dextran

Resveratrol


43

Improving Resveratrol Bioaccessibility Using Biopolymer

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