Increasing Carotenoid Bioaccessibility from Yellow Peppers Using

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Increasing carotenoid bioaccessibility from yellow peppers using excipient emulsions: Impact of lipid type and thermal processing Xuan Liu, Jinfeng Bi, Hang Xiao, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04217 • Publication Date (Web): 11 Sep 2015 Downloaded from http://pubs.acs.org on September 16, 2015

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Journal of Agricultural and Food Chemistry

Increasing carotenoid bioaccessibility from yellow peppers using excipient emulsions: Impact of lipid type and thermal processing

Xuan Liu1, 2, Jinfeng Bi1, Hang Xiao2,3, and David Julian McClements2,3,4*

1

Institute of Food Science and Technology CAAS, Key Laboratory of Agro-Products Processing,

Ministry of Agriculture, Beijing 100193, China 2

Department of Food Science, University of Massachusetts, Amherst, MA 01003

3

Center for Bioactive Delivery, Institute of Applied Life Science, University of Massachusetts,

Amherst, MA 01003 4

Department of Biochemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203

Jeddah 21589 Saudi Arabia

•

Corresponding author: (Tel: 413 545-1019; Fax: 413 545-1262; E-mail: [email protected]).

1

ACS Paragon Plus Environment


1 2

ABSTRACT Many phytochemicals from fruits and vegetables exert biological activities that

3

may be beneficial to human health, but these benefits are not fully realized because of

4

their poor oral bioavailability. The objective of this research was to establish the

5

potential of excipient emulsions to increase carotenoid bioaccessibility from raw and

6

cooked yellow peppers using a gastrointestinal model that included oral, gastric, and

7

intestine phases. The influence of oil type (medium chain triglycerides, MCT; long

8

chain triglycerides, LCT; and, indigestible orange oil, OO) on microstructural changes,

9

particle properties, lipid digestibility, and carotenoid bioaccessibility was investigated.

10

Oil type had a major impact, with carotenoid bioaccessibility decreasing in the

11

following order: LCT > MCT > OO > control (no oil). Conversely, thermal treatment

12

(raw versus boiled) had little influence on carotenoid bioaccessibility. These results

13

will facilitate the rational design of excipient emulsions that boost the bioavailability

14

of phytochemicals in fruits and vegetables.

15 16 17

Keywords: excipient emulsions; carotenoids; yellow pepper; oil type; thermal processing; bioaccessibility

18 19

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INTRODUCTION Bioactive phytochemicals, often referred to as nutraceuticals, are naturally

22

present in a wide variety of fruits and vegetables 1, 2. In vitro and in vivo studies

23

suggest that many of these phytochemicals have biological activities potentially

24

beneficial to human health, including antioxidant, antimicrobial, anti-inflammatory,

25

anticancer and anti-hypertension activity, as well as the ability to promote reductions

26

in blood serum lipids, cholesterol, and glucose 2-7. Consumption of diets containing

27

high levels of these nutraceuticals might therefore be an effective means of combating

28

or treating chronic diseases, such as cancer, diabetes, heart disease, eye disease, and

29

hypertension 8.

30

In general, the level of bioactive phytochemicals present within the human diet

31

may be increased using two approaches. First, the nutraceuticals may be extracted

32

from fruits and vegetables, purified, and then introduced into other foods as

33

nutraceutical ingredients in either a pure or encapsulated form 9-11. Second,

34

nutraceuticals may be consumed as an integral part of fresh or processed fruits and

35

vegetables 5, 12. The consumption of whole fruits and vegetables is often

36

recommended because it is believed that phytochemicals exhibit their beneficial

37

health effects more effectively in this form 13, 14. One limitation of obtaining

38

nutraceuticals directly from fruits and vegetables is that they often have a relatively

39

poor oral bioavailability 15, 16. In addition, there may be large variations in

3



40

bioavailability depending on nutraceutical type, fruit and vegetable type, the nature of

41

processing used, and the nature of the food matrix 17, 18. Numerous physicochemical

42

mechanisms may be responsible for the low and variable bioavailability of

43

phytochemicals, including poor release from plant tissues, low solubility in GIT fluids,

44

limited absorption by epithelium cells, and/or transformation within the GIT 19-22.

45

Various studies have shown that the bioavailability of nutraceuticals in fruits and

46

vegetables may be enhanced by co-ingesting them with certain types of food matrices

47

22

48

sources can be increased when they are mixed with digestible lipids prior to

49

introduction into the gastrointestinal tract, including carrots 23, mangoes 21, 24, peppers

50

17

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of digestible lipids is associated with the formation of colloidal structures (mixed

52

micelles) in the GIT that are capable of solubilizing hydrophobic nutraceuticals and

53

transporting them to the epithelium cells where they can be absorbed 17, 27.

54

Carotenoid bioaccessibility may also be increased by reducing their chemical

55

degradation within foods or within the GIT by incorporating natural antioxidants 28. It

56

has been reported that the bioaccessibility of carotenoids may vary widely (from

57

around 1 to 100%) depending on the plant origin, thermal and mechanical processing

58

conditions, and food matrix effects 17, 29, 30. Consequently, there is a need to

59

understand the impact of these factors on the bioavailability of nutraceuticals so as to

60

develop efficacious functional foods that are more beneficial to human health.

. For example, the bioaccessibility of carotenoids from various types of plant

, salads 25 and sweet potatoes 26. This increase in oral bioavailability in the presence

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It has recently been proposed that excipient foods can be designed to boost the

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bioavailability of nutraceuticals in fruits and vegetables 31, 32. The type, concentration,

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and structural organization of the ingredients in an excipient food are specifically

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selected to enhance nutraceutical bioavailability by altering their bioaccessibility,

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absorption, and/or transformation in the GIT 22. The first step in the design of an

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excipient food involves identifying the major factors that normally limit the

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bioavailability of the desired nutraceuticals 22 An excipient food is then designed to

68

overcome these factors by incorporating specific ingredients or structures within it 31.

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The objective of the present study was to investigate the utilization of excipient

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emulsions to increase the bioaccessibility of carotenoids from yellow peppers.

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Peppers are known to be a good source of carotenoids 33-35, and so their consumption

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may be beneficial to human health. The overall bioavailability of carotenoids is often

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limited due to their relatively low bioaccessibility, which may be due to poor release

74

from the plant cell tissues and/or due to poor solubilization within the GIT fluids 22.

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In this study, we examined the effectiveness of oil-in-water excipient emulsions

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containing either digestible or indigestible lipids on the bioaccessibility of carotenoids

77

from yellow peppers 36. Our hypothesis was that digestible lipids would be rapidly

78

digested within the small intestine phase of the GIT and form mixed micelles capable

79

of solubilizing any carotenoids released from the yellow peppers. In addition, we

80

examined the influence of lipid phase type, long chain triglycerides (LCT), medium

81

chain triglycerides (MCT) and indigestible oil (orange oil), since this factor has 5



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previously been shown to impact the bioaccessibility of carotenoids encapsulated in

83

colloidal delivery systems 37, 38. The information obtained from this study would

84

facilitate the rational design of excipient foods that can boost the bioavailability of

85

health promoting nutrients and nutraceuticals from natural sources.

86

MATERIAL AND METHODS

87

Chemicals. Fresh yellow peppers and corn oil (a source of LCTs) were obtained

88

from a local supermarket. Miglyol 812 (a source of MCTs) was obtained from

89

SASOL (Houston, TX). Orange oil (OO，10× decolorized) was obtained from the

90

Chemistry Store (Cayce, SC). β-carotene (≥ 93%), gallic acid (≥ 99%), lipase (porcine

91

pancreas, type II), bile extract, and calcium chloride were purchased from Sigma-

92

Aldrich (St. Louis, MO). All other chemicals were of analytical grade. All aqueous

93

solutions were prepared using double-distilled water from a water purification system.

94

Preparation of Excipient Emulsion and Yellow Pepper. An aqueous phase was

95

prepared by dispersing 1.5% non-ionic surfactant (Tween 20) in buffer solution (10

96

mM phosphate, pH7.0). An organic phase was prepared that contained LCT, MCT, or

97

orange oil. A coarse emulsion was then prepared by blending the organic phase (8%,

98

w/w) with the aqueous phase (92%, w/w) using a high-shear mixer (10,000 rpm, 2

99

min). Excipient emulsions were then formed by passing the coarse emulsion through a

100

microfluidizer (Model 101, Microfluidics, Newton, MA) three times at a

101

homogenization pressure of 9,000 psi.

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Yellow peppers were cut into small pieces that were used directly (“raw”) or

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were subjected to a thermal treatment (“boiled”) by heating them in a pan of boiling

104

water for 5 minutes. These two treatments were used to take into account that peppers

105

may be consumed raw (e.g., in salads) or they may be cooked (e.g., in sauces).

106

Simulated gastrointestinal tract. The yellow peppers were converted into a

107

puree by grinding with a mortar and pestle to simulate tissue breakdown in the mouth

108

due to mastication. The pureed yellow pepper (6 g) was then mixed with excipient

109

food (6 g of buffer or emulsion), and the resulting mixture was passed through a static

110

simulated GIT 37, 38:

111

Mouth phase: Simulated saliva fluid (SSF) was prepared by dispersing mucin

112

and various salts in water and then stirring overnight, as described previously 39. The

113

pepper/excipient sample (12 g) was then mixed with SSF (12 g) and the system was

114

adjusted to pH 6.8 with sodium hydroxide solution. The resulting mixture was

115

incubated at 37 °C for 10 min with continuous agitation (100 rpm).

116

Stomach phase: Simulated gastric fluid (SGF) was prepared by dissolving 2 g of

117

sodium chloride and 7 mL of hydrogen chloride (37%) in 1 L of water, and then

118

dispersing 0.064 g of pepsin into20 mL of this solution followed by stirring for 45

119

min.The sample resulting from the mouth phase (20 g) was then mixed with SGF (20

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g), and adjusted to pH 2.5 with sodium hydroxide solution. The stomach sample was

121

placed in an incubator at 37 °C for 2 hours with continuous swirling at 100 rpm to

122

simulate gastric digestion.

123

Small intestine: A sample from the stomach phase (30 g) was placed in a

124

temperature-controlled (37 °C) water bath and the pH value was adjusted to pH 7.0.

125

Then, 4 mL of bile salt solution (187.5 mg/4 mL) and 1.5 mL of simulated small

126

intestine solution (10 mM of calcium chloride and 150 mM of sodium chloride) were 7



127

added into the mixture and the mixture was adjusted to pH 7.0. Afterwards, 4 mL of

128

freshly prepared lipase suspension (187.5 mg/4 mL) dissolved in phosphate buffer

129

was added to the mixture. The pH of the mixture was monitored and the volume of

130

0.25 M NaOH (mL) necessary to neutralize the free fatty acids (FFA) released from

131

the lipid digestion was recorded over two hours using an automatic titration (pH stat)

132

method (Metrohm USA Inc., Riverview, FL). The percentage of FFA released was

133

calculated using the following equation: 134  VNaOH (t ) ⋅ CNaOH ⋅ M Oil  FFA(= t ) 100 ×  135  2 ⋅ mOil 

136

(1)

137 138

Here, VNaOH(t) is the volume of NaOH solution required to neutralize the FFAs

139

produced at digestion time t (L), CNaOH is the molarity of the NaOH solution used to

140

titrate the sample (mol/L), Moil is the molecular weight of the oil (g/mol), and moil is

141

the total mass of lipid initially present in the incubation cell (g).The molecular

142

weights of the LCT and MCT were taken to be 872 and 492 g/mol, respectively.

143

Particle Characterization in Excipient Emulsions and Digesta. The mean

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particle diameter, particle size distribution, and electrical charge of the samples were

145

monitored after exposure to each stage of the simulated GIT. The mean particle

146

diameter and particle size distribution were measured using static light scattering

147

(Mastersizer 2000, Malvern Instruments Ltd., Worcestershire, UK). The samples were

148

diluted in 10 mM phosphate buffer (pH 7.0) prior to measurement to avoid multiple

149

scattering effects. The particle size was reported as the surface-weighted mean

150

diameter (d32). The electrical charge (ζ-potential) on the particles in the samples was

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measured using a particle electrophoresis method (Nano ZS, Malvern Instruments,

152

Malvern, UK).

153

Bioaccessibility. The bioaccessibility of β-carotene was determined after each

154

sample had been subjected to the full in vitro digestion process using a method

155

described previously

156

collected and centrifuged (4400 rpm for 1 hour), which resulted in samples that

157

contained a sediment at the bottom with a supernatant above. The supernatant was

158

collected and assumed to be the “micelle” fraction, in which the carotenoids were

159

solubilized. The bioaccessibility was calculated from the concentrations of total

160

carotenoids determined in the micelle fraction and supernatant using a procedure

161

described previously

162

using the following equation:

37, 38

. After the small intestine stage, raw digesta samples were

37, 38

. The bioaccessibility of total carotenoids was calculated

163 164

 C Bioaccessibility = 100 ×  Micelle C  RawDigesta

   

165 166

Where, Cmicelle and CRawDigesta are the concentrations of total carotenoids in the

167

micelle fraction and in the overall sample (raw digesta) after the simulated intestinal

168

digestion experiment, respectively. The carotenoid content of each sample was

169

determined according to the method described previously 40.

170

Microstructure Analysis. The microstructure of the samples initially and after

171

exposure to various GIT phases was characterized using optical and confocal

172

scanning fluorescence microscopy (Nikon D-Eclipse C1 80i, Nikon, Melville, NY).

173

The samples analyzed by confocal microscopy were stained with a lipophilic 9



174

fluorescent dye (Nile Red) to highlight the location of the lipid phase. The Nile red

175

was dissolved in absolute ethyl alcohol at a concentration (1 mg/mL). Then, before

176

analysis 2 mL samples were mixed with 20 μL Nile Red solution (1 mg/mL ethanol)

177

to dye the oil phase. All images were captured with a 10× eyepiece and a 60×

178

objective lens (oil immersion).

179

Statistical Analysis. All experiments were conducted in at least duplicate and

180

analyses of each sample were also carried out in duplicate. Results are expressed as

181

the mean and the standard deviation of these measurements. Variance analyses were

182

performed by statistical analysis software (SPSS19.0, IBM). Multiple comparisons

183

were conducted to determine the significant differences at 5% level (p< 0.05).

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RESULTS AND DISCUSSION

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The potential impact of excipient emulsions on the gastrointestinal fate of the

186

nutraceuticals in the yellow peppers was assessed using a static in vitro digestion

187

model: mouth, stomach, and small intestine.

188

Properties of Initial Excipient Emulsions. Initially, the size and charge of the

189

lipid droplets in the excipient emulsions was characterized before mixing them with

190

the raw or boiled pureed yellow pepper samples. The mean particle diameter (d32) of

191

the excipient emulsions containing digestible lipids was relatively small: 0.14 and

192

0.18 µm for the MCT and LCT emulsions, respectively (Figure 1). In addition, these

193

two emulsions had monomodal particle size distributions (Figure 2c and 2d), and

194

exhibited no visible phase separation throughout storage for one week (data not 10


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shown). Optical and confocal microscopy images indicated that the droplets in these

196

emulsions were relatively small and evenly distributed throughout the samples

197

(Figure 3). Thus, microfluidization was an effective means of preparing stable

198

excipient emulsions from these types of triglyceride oils. Conversely, the initial

199

excipient emulsions containing the indigestible lipids (orange oil) had relatively large

200

mean particle diameters (0.64 µm) as measured by light scattering (Figure 1a), and

201

there were some large droplets observed in the particle size distributions (Figure 2b)

202

and microscopy images (Figure 3). After a few hours storage, the orange oil

203

emulsions separated into a cream layer at the top and a serum layer at the bottom (data

204

not shown). The reason for the relatively large droplet size in these emulsions can be

205

attributed to Ostwald ripening: orange oil is a relatively polar lipid and so a

206

substantial amount may diffuse from the small droplets to the large droplets due to a

207

thermodynamic driving force associated with differences in interfacial curvature 41.

208

The resulting increase in droplet size due to this process leads to enhanced creaming

209

and coalescence of the droplets, which accounts for the visual appearance of phase

210

separation.

211

The lipid droplets in all the excipient emulsions were moderately anionic, with ζ-

212

potentials of -11, -14 and -19 mV for the MCT, LCT and orange oil emulsions,

213

respectively (Figure 4a). The negative charge on the droplets can be attributed to the

214

presence of anionic impurities (such as free fatty acids) in the nonionic surfactant or

215

lipid ingredients used to fabricate the emulsions 42. 11



216

Influence of Mouth Phase. The pureed yellow peppers (raw or boiled) were

217

mixed with either excipient emulsion (MCT, LCT or orange oil) or buffer solution

218

(control) and then exposed to simulated mouth conditions. Samples were collected at

219

the end of the incubation period and then their particle sizes, microstructures, and

220

electrical characteristics were measured (Figures 1 to 4).

221

In the absence of lipid droplets (control), the light scattering measurements

222

indicated that both the raw and boiled yellow pepper samples contained relatively

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large particles (d32 ≈ 10 µm) after exposure to the mouth phase (Figure 1). These

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large particles were presumably fragments of yellow pepper generated during the

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simulated mastication process. Indeed, the optical microscopy images clearly showed

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the presence of a number of relatively large particles within these samples (Figures

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3b and 3d). There was evidence of bright regions within the confocal microscopy

228

images for the yellow pepper in buffer samples, which suggests that the Nile red dye

229

may have been localized within some hydrophobic regions in the plant tissue (Figures

230

3a and 3c). The fragments in both the raw and boiled yellow pepper control samples

231

had fairly high negative charges (ζ ≈ - 18 mV), which may have been due to the

232

presence of anionic biopolymers (e.g., pectin) at the surfaces of the yellow pepper

233

pieces. Interestingly, the raw and boiled samples behaved very similarly throughout

234

the GIT (see later). These results suggest that boiling did not have a major impact on

235

the initial size or electrical properties of the yellow pepper fragments. Nevertheless,

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there are likely to have been other changes that would not be detected by light

237

scattering or microscopy methods, such as tissue softening.

238

In the presence of the excipient emulsions, the mean particle diameter measured

239

by light scattering was much smaller than that for the yellow pepper alone,

240

particularly for the MCT and LCT samples (Figures 1a and 1b). Conversely, the

241

optical and confocal microscopy images clearly suggested that there were large

242

particles in these samples (Figure 3). These large particles appeared to be tissue

243

fragments from the yellow pepper, as well as some clumps of droplets. These

244

apparent contradictory results may be attributed to the ability of the mucin molecules

245

from the simulated saliva fluids to promote depletion flocculation of the lipid droplets

246

43

247

group (Tween 20), and so one would not expect the mucin molecules to strongly

248

adhere to the droplet surfaces. However, the presence of non-adsorbed biopolymers

249

in the aqueous phase surrounding the droplets generates an osmotic attraction between

250

them that causes them to become flocculated 44. The forces holding the droplets

251

together in aggregates formed by depletion flocculation are usually relatively weak,

252

and reduced when the sample is diluted and stirred for light scattering measurements.

253

Hence, the droplets may have been highly aggregated in the original samples, but

254

appear to be relatively small in the light scattering measurements. The mixtures

255

containing orange oil emulsions and yellow pepper had much larger mean particle

256

diameters than the ones containing digestible lipids. In addition, the particle size

. The lipid droplets were coated by a non-ionic surfactant with a polymeric head-

13



257

distribution data showed that most these samples had a bimodal distribution with a

258

population of small particles around 0.1-0.3 µm and a population of large particles

259

around 10 µm (Figure 2b). The small particles were likely to be the lipid droplets and

260

the large particles the yellow pepper fragments. The large mean particle size in these

261

samples can be attributed to the contribution of large orange oil droplets formed due

262

to Ostwald ripening 41. Nevertheless, the microscopy images also indicated that

263

extensive droplet flocculation still occurred under oral conditions, which can be

264

attributed to the depletion flocculation mechanism discussed earlier.

265

The electrical charges on the particles in the mixed systems were more highly

266

negative than those in the control systems (Figure 4), but they followed a similar

267

trend to those observed in the initial emulsions, with the charge becoming

268

increasingly negative in the following order: control < MCT < LCT < orange oil. The

269

negative charge on the particles in the mixed systems is due to the fact that they

270

contained a mixture of anionic lipid droplets and anionic yellow pepper fragments.

271

Influence of Stomach Phase. The samples resulting from the oral phase were

272

incubated in simulated gastric fluids, and then their size, microstructure, and charge

273

were measured (Figures 1 to 4). In the yellow pepper controls (no lipids), the particle

274

size remained relatively large after exposure to gastric conditions, d32 ≈ 10 µm

275

(Figure 1). Hence, the highly acidic environment of the gastric fluids did not appear

276

to breakdown the yellow pepper tissue fragments. On the other hand, the mean

277

particle diameter remained relatively small in the presence of the digestible lipid 14


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droplets (MCT and LCT), which suggests that these droplets were resistant to droplet

279

coalescence under gastric conditions. In addition, the mean particle diameter of the

280

mixtures containing the orange oil emulsions remained relatively high, which can be

281

attributed to droplet growth by Ostwald ripening. The optical and confocal

282

micrographs indicated that the large clumps of droplets observed in the excipient

283

emulsions in the mouth stage, were not present after exposure to the gastric phase

284

(Figure 3). The breakdown of these clumps may have been because the emulsions

285

were diluted (1:1) with simulated gastric fluids, which weakened the attractive

286

osmotic forces acting between the droplets, thereby reducing the tendency for

287

depletion flocculation to occur 44. In addition, the change in solution pH and ionic

288

strength may have altered the relative magnitude of the other attractive and repulsive

289

colloidal interactions operating between the lipid droplets, leading to changes in the

290

tendency for droplets to come together. The particle size distribution data also

291

showed that there was a reduction in the population of large particles after exposure to

292

gastric conditions (Figure 2). This observation suggests that the presence of the lipid

293

droplets somehow promoted the disruption of the yellow pepper fragments under

294

simulated gastric conditions, although the origin of this effect is currently unknown.

295

The ζ-potential of the gastric samples was measured under the same solution

296

conditions (pH 7) as those used for the other stages of the simulated GIT, so that

297

alterations in interfacial properties could be directly compared between different

298

samples. The ζ-potential on the particles in all the samples became slightly more 15



299

negative after they were exposed to stomach conditions (Figure 3), which suggests

300

that there was some alteration in the interfacial composition of the lipid droplets

301

and/or the yellow pepper fragments. This alteration may have occurred due to

302

adsorption of anionic species to their surfaces, such as mucin or pepsin.

303

Influence of Small Intestine Phase. The gastric samples were then exposed to

304

simulated small intestine conditions and their particle size, microstructure, and charge

305

were measured at the end of the incubation period (Figures 1 to 4). In the control

306

yellow pepper samples (no lipid), there was an appreciable decrease in the mean

307

particle size measured by light scattering (Figure 1), which may have been due to

308

some breakdown of the yellow pepper fragments, or due to the contribution of

309

particles originating from the small intestine fluids to the light scattering signal (such

310

as mixed micelles formed by bile salts or insoluble aggregates from the lipase extract).

311

The optical microscopy images of both the raw and boiled yellow pepper samples

312

indicate that there were still some large fragments present (Figures 3b and 3d),

313

which suggests that the small sizes measured by light scattering were probably due to

314

particles arising from the simulated small intestinal fluids. These results highlight the

315

importance of confirming light scattering results using microscopy analysis.

316

In the presence of lipid droplets, there was an appreciable change in the mean

317

particle diameter after exposure to small intestine conditions: the particle size

318

increased in the mixtures containing digestible lipid droplets, but decreased in the

319

mixtures containing indigestible lipid droplets (Figure 1).This change in particle 16


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dimensions can be attributed to the formation of a complex mixture of different

321

colloidal particles that can scatter light, such as undigested lipid droplets, micelles,

322

vesicles, insoluble calcium soaps, and liquid crystals. The number, type, and

323

dimensions of these particles formed is likely to depend strongly on lipid composition.

324

Consequently, it is difficult to interpret the results from light scattering measurements

325

in such complex colloidal mixtures. The optical and confocal microscopy images

326

indicated that various kinds of structures were formed in the small intestinal fluids

327

depending on lipid type (Figure 3). In the orange oil emulsions, there appeared to be

328

a fairly even distribution of relatively small particles spread through the system.

329

Interestingly, both the raw and boiled yellow peppers containing MCT emulsions,

330

contained an interwoven network of hair-like structures after digestion. These long

331

thin structures may have been liquid crystals formed from the medium chain fatty

332

acids and other substances in the digesta. To the authors knowledge this is the first

333

time this kind of structure has been observed, and their origin and nature is currently

334

unknown. The samples that initially contained LCT excipient emulsions contained a

335

number of large lipophilic spheroids after digestion, which may have been undigested

336

fat droplets or large vesicle-like structures. These structures were particularly

337

apparent in the boiled yellow pepper sample. Overall, our microscopy results show

338

that there is a large change in the microstructures formed within the digesta produced

339

in the small intestine phase. Other studies have also indicated that various kinds of

17



340

structures may be formed in simulated small intestine fluids depending on the nature

341

of the lipid phase used 45, 46.

342

In the presence of lipid droplets, there was evidence that the population of large

343

particles observed in the particle size distribution was reduced, which also suggested

344

that there was some breakdown of the yellow pepper fragments under simulated small

345

intestine conditions. Cellular disruption may have occurred because the surface-active

346

bile salts, free fatty acids, and monoacylglycerols present in the intestinal fluids

347

penetrated into the plant tissues and disrupted the bonds between them. After

348

exposure to the small intestine phase the system containing MCT emulsions exhibited

349

two major peaks around 0.27 and 46 µm for raw yellow pepper and 0.21 and 35 µm

350

for boiled, respectively. LCT emulsions exhibited two major peaks around 0.16 and

351

2.2 µm for raw but three major peaks around 0.18, 2.2 and 180 µm for boiled,

352

respectively, which suggests that different kinds of colloidal structures were formed

353

after digestion. These larger particles may have been undigested lipid droplets or

354

liposomes formed from the long chain fatty acids released by lipid digestion or yellow

355

pepper fragments.

356

The electrical charge on the particles in the small intestine phase was negative for

357

all of the samples studied (Figure 3), which can be attributed to the presence of

358

anionic species in the small intestinal fluids, such as bile salts, free fatty acids,

359

peptides, and mucin. Nevertheless, the negative charge was much higher for the

360

samples containing LCT, which may have been because lipid digestion generated long 18


Page 18 of 52

Page 19 of 52


361

chain fatty acids that accumulated at the surfaces of the lipid droplets or were

362

incorporated into micelle micelles. Again, the results were very similar for the

363

samples containing raw and boiled yellow pepper.

364

Lipid Digestion. The digestion of the lipid phase in the mixtures containing

365

excipient emulsions was followed using an automatic titration (pH-stat) method. The

366

volume of alkaline solution that had to be titrated into the reaction vessel to neutralize

367

the free fatty acids (and any other acids) released during digestion of the raw and

368

cooked yellow pepper was measured (Figures 5a and 5b). This data was then used to

369

calculate the percentage of free fatty acids released from the lipid phase for the two

370

digestible oils, i.e., LCT and MCT (Figures 6a and 6b). The volume of alkaline

371

determined for the control sample (no lipid) was subtracted from the volume

372

measured for the samples containing digestible lipids prior to making these

373

calculations so as to account for any changes of pH due to the reactants employed.

374

In the absence of digestible lipids (control and orange oil sample), there was only

375

a slight increase in the volume of alkaline solution added to the reaction chamber

376

during the course of the experiment (Figure 5), which may be due to the digestion of

377

some components in the reagents (such as bile salts or lipase). The volume of alkaline

378

solution titrated into the mixture containing LCT was appreciably lower than that

379

titrated into the one containing MCT, which is at least partly due to the higher

380

molecular weight of LCT 47.

19



381

For both digestible lipids, and for both raw and boiled samples, there was a rapid

382

increase in free fatty acids released during the first few minutes, followed by a much

383

slower increase at later times (Figures 6a and 6b). The rate of increase in FFAs

384

released at later times appeared to be higher for the MCT system than for the LCT

385

system. Consequently, by the end of the two hour digestion period only about 80% of

386

the LCT had been digested, but over 100% of the MCT had been digested. These

387

calculations are based on the assumption that every triglyceride molecule produces

388

two free fatty acids, so that the high value determined for MCT may be because more

389

than two FFAs were released per triglyceride molecule 48. In addition, previous

390

studies have shown that lipid digestion may be inhibited in LCT emulsions because of

391

accumulation of long chain fatty acids at the lipid droplet surfaces 49. These long

392

chain FFAs form a liquid crystalline shell around the lipid droplets that restricts the

393

access of lipase to the remaining triglycerides, thereby inhibiting further digestion.

394

The lipase can continue to function properly once the long chain free fatty acids are

395

removed from the droplet surfaces by solubilization within mixed micelles or by

396

precipitation by calcium ions. It is therefore possible that there was insufficient bile

397

salts or calcium present in the simulated small intestinal fluids used in our study to

398

completely remove all of the long chain FFAs from the LCT droplet surfaces.

399

Finally, at neutral pH not all of the carboxylic acid groups on the fatty acids may be

400

ionized, and therefore do not release protons 48.

20


Page 20 of 52

Page 21 of 52


401

Bioaccessibility. Finally, we measured the bioaccessibility of the carotenoids

402

released from the yellow pepper tissue after their passage through the simulated GIT.

403

The bioaccessibility was calculated by measuring the carotenoid concentrations in the

404

mixed micelle phase and the total digesta. The bioaccessibility of the carotenoids in

405

the control samples (no lipids) and in the samples containing indigestible oil (orange

406

oil) were relatively low (< 30%). This phenomenon can be attributed to the fact that

407

there were no free fatty acids produced that could form mixed micelles with the bile

408

salts and increase the solubilization capacity of the small intestinal fluids. In this case,

409

the carotenoids released from the yellow pepper tissue were probably solubilized in

410

simple micelles that only consisted of bile salts. There was a significant increase in

411

the bioaccessibility of the carotenoids for the yellow peppers (raw and boiled) that

412

had been mixed with the excipient emulsions containing digestible lipids (MCT and

413

LCT). This effect can be attributed to the fact that the free fatty acids produced by

414

lipid digestion were incorporated into mixed micelles with bile salts, which increases

415

the solubilization capacity of the small intestinal fluids 50. Moreover, the

416

bioaccessibility of the carotenoids was significantly higher for the samples containing

417

LCT droplets than for those containing MCT droplets, which agrees with earlier

418

studies that have reported that the bioaccessibility of carotenoids encapsulated in

419

emulsion-based delivery systems is higher for LCT than MCT 37, 38. This effect can be

420

attributed to the ability of the long chain free fatty acids arising from LCT digestion to

421

form mixed micelles that have hydrophobic regions big enough to accommodate long 21



422

non-polar molecules45, 46. Conversely, the hydrophobic regions in the mixed micelles

423

formed by MCT are insufficiently large to incorporate the carotenoid molecules. This

424

result has important implications for the design of excipient emulsions to boost the

425

bioavailability of large hydrophobic nutraceuticals, such as carotenoids. Boiling the

426

yellow peppers appeared to have little impact on their overall bioaccessibility. In

427

previous studies, it has been reported that certain types of domestic cooking (such as

428

stir-frying) lead to a major improvement in carotenoid bioaccessibility, whereas

429

others (such as boiling) only cause a modest increase 30. Presumably, the oil used in

430

stir-frying helps to solubilize the carotenoids and to form mixed micelles in the

431

gastrointestinal tract.

432

In conclusion, this study has shown that excipient emulsions can boost the

433

bioaccessibility of carotenoids in yellow pepper, with the magnitude of the effect

434

depending strongly on lipid phase type. Excipient emulsions fabricated from LCT

435

gave a significantly higher bioaccessibility than those fabricated from MCT, which

436

can be attributed to the greater solubilizing power of mixed micelles formed from

437

long chain fatty acids. Boiling the yellow peppers had little influence on their

438

gastrointestinal fate or on carotenoid bioaccessibility. Overall, these results highlight

439

the potential of designing excipient emulsions to boost the fraction of beneficial

440

nutraceuticals present in natural fruits and vegetables. For example, it may be possible

441

to design an excipient dressing to pour on raw peppers in salads, or an excipient

442

creamy sauce to pour on cooked peppers. 22


Page 22 of 52

Page 23 of 52

443


ACKNOWLEDGEMENTS

444

This work was partly supported by the China Scholarship Council (No.

445

201303250031). This material was also partly based upon work supported by the

446

Cooperative State Research, Extension, Education Service, United State Department

447

of Agriculture, Massachusetts Agricultural Experiment Station (Project No. 831) and

448

United States Department of Agriculture, NRI Grants (2011-03539, 2013-03795,

449

2011-67021, and 2014-67021). This project was also partly funded by the Deanship of

450

Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant numbers

451

330-130-1435-DSR, 299-130-1435-DSR, 87-130-35-HiCi). The authors, therefore,

452

acknowledge with thanks DSR technical and financial support.

453 454

23



455

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31



FIGURE CAPTIONS Figure 1a. Influence of passage through a simulated GIT on the mean particle diameter (d32) of samples containing raw yellow pepper and either buffer solution (control) or excipient emulsions with different oil types: long chain triglycerides (LCT), medium chain triglycerides (MCT) or orange oil (OO). Figure 1b. Influence of passage through a simulated GIT on the mean particle diameter (d32) of samples containing boiled yellow pepper and either buffer solution (control) or excipient emulsions with different oil types: long chain triglycerides (LCT), medium chain triglycerides (MCT) or orange oil (OO). Figure 2a. Influence of gastrointestinal stage on the particle size distribution of mixed systems containing raw yellow pepper and buffer solution (control). Figure 2b. Influence of gastrointestinal stage on the particle size distribution of mixed systems containing raw yellow pepper and orange oil excipient emulsions. Figure 2c. Influence of gastrointestinal stage on the particle size distribution of mixed systems containing raw yellow pepper and MCT excipient emulsions. Figure 2d. Influence of gastrointestinal stage on the particle size distribution of mixed systems containing raw yellow pepper and LCT excipient emulsions. Figure 3a. Change in microstructure of raw yellow pepper samples as they passed through the simulated GIT as observed by confocal fluorescent microscopy using a lipid dye.

32


Page 32 of 52

Page 33 of 52


Figure 3b. Change in microstructure of raw yellow pepper samples as they passed through the simulated GIT as observed by optical microscopy. Figure 3c. Change in microstructure of boiled yellow pepper samples as they passed through the simulated GIT as observed by confocal fluorescent microscopy using a lipid dye. Figure 3d. Change in microstructure of boiled yellow pepper samples as they passed through the simulated GIT as observed by optical microscopy. Figure 4a. Influence of passage through a simulated GIT on the electrical characteristics of the particles (ζ-potential) of samples containing raw yellow pepper and either buffer solution (control) or excipient emulsions with different oil types: long chain triglycerides (LCT), medium chain triglycerides (MCT) or orange oil (OO). Figure 4b. Influence of passage through a simulated GIT on the electrical characteristics of the particles (ζ-potential) of samples containing boiled yellow pepper and either buffer solution (control) or excipient emulsions with different oil types: long chain triglycerides (LCT), medium chain triglycerides (MCT) or orange oil (OO). Figure 5a.Volume of alkaline solution (0.25 M NaOH) required to maintain the pH at neutral under simulated small intestine conditions: The samples analyzed originally contained raw yellow pepper and either buffer solution (control) or excipient emulsions with different oil types: LCT, MCT, or orange oil. Figure 5b. Volume of alkaline solution (0.25 M NaOH) required to maintain the pH at neutral under simulated small intestine conditions: The samples analyzed originally 33



contained boiled yellow pepper and either buffer solution (control) or excipient emulsions with different oil types: LCT, MCT, or orange oil. Figure 6a. Percentage of free fatty acids released under simulated small intestine conditions for samples initially containing raw yellow pepper and excipient emulsions with different digestible oil types: LCT or MCT. Figure 6b. Percentage of free fatty acids released under simulated small intestine conditions for samples initially containing boiled yellow pepper and excipient emulsions with different digestible oil types: LCT or MCT. Figure 7. Bioavailability of carotenoids released under simulated GIT conditions from samples initially containing boiled yellow pepper and either buffer solutions or excipient emulsions with different oil types: LCT, MCT, and orange oil.

34


Page 34 of 52

Page 35 of 52


FIGURES

Buffer-Boiled

10

MCT-Boiled

D[3,2] (µm)

LCT-Boiled OO-Boiled

1

0.1

N/A

Initial

Mouth

Stomach

Figure 1a.

35


Small Intestine


Page 36 of 52

Buffer-Boiled

10

MCT-Boiled

D[3,2] (µm)

LCT-Boiled OO-Boiled

1

0.1

N/A

Initial

Mouth

Stomach

Figure 1b.

36


Small Intestine

Page 37 of 52


30 Buffer-RawIntestine

Volume (%)

25

20 Buffer-RawStomach

15

10 Buffer-RawMouth

5

0 0.01

0.1

1 10 100 1000 10000 Particle Diameter (μm)

Figure 2a.

37



Page 38 of 52

30 OO-RawIntestine

25

20

Volume (%)

OO-RawStomach

15 OO-RawMouth

10

5 OO-Initial

0 0.01

1

100

Particle Diameter (μm) Figure 2b.

38


10000

Page 39 of 52


30 MCT-RawIntestine

Volume (%)

25 MCT-RawStomach

20 15

MCT-RawMouth

10

MCT-Initial

5 0 0.01

0.1

1

10

100

1000 10000

Particle Diameter (μm) Figure 2c.

39



30 LCT-RawIntestine

Volume (%)

25

20 LCT-RawStomach

15 LCT-RawMouth

10

5

0 0.01

LCT-Initial

0.1

1

10

100

1000 10000

Particle Diameter (μm) Figure 2d.

40


Page 40 of 52

Page 41 of 52


Control (Raw)

OO (Raw)

MCT (Raw)

Initial Emulsions N/A

Mouth

Stomach

Intestine

Figure 3a.

41


LCT (Raw)


Control (Raw)

OO (Raw)

MCT (Raw)

Initial Emulsions

N/A

Mouth

Stomach

Intestine

Figure 3b.

42


Page 42 of 52

LCT (Raw)

Page 43 of 52


Control (Boiled)

OO (Boiled)

MCT (Boiled)

Initial Emulsions N/A

Mouth

Stomach

Intestine

Figure 3c

43


LCT (Boiled)


Control (Boiled)

OO (Boiled)

MCT (Boiled)

Initial Emulsions

N/A

Mouth

Stomach

Intestine

Figure 3d.

44


Page 44 of 52

LCT (Boiled)

Page 45 of 52


Initial

Mouth

Stomach

N/A

-5

ζ-potential (mV)

-15

-25

-35

-45

Buffer-Raw MCT-Raw LCT-Raw OO-Raw

-55

Figure 4a.

45


Small Intestine


Initial

Mouth

Stomach

N/A

ζ-potential (mV)

-5

-15

-25

-35 Buffer-Boiled

-45

MCT-Boiled LCT-Boiled OO-Boiled

-55

Figure 4b

46


Page 46 of 52

Small Intestine

Page 47 of 52


6

Volume of NaOH (mL)

5

MCT-Raw LCT-Raw

4

OO-Raw Buffer-Raw

3 2 1 0 0

20

40 60 80 100 Digestion Time (min)

Figure 5a.

47


120


Page 48 of 52

7

Volume of NaOH (mL)

6 MCT-Boiled LCT-Boiled OO-Boiled Buffer-Boiled

5 4 3 2 1 0 0

20


Figure 5b.

48


120

Page 49 of 52


FFA Released (%)

100 80 60 40 MCT-Raw

20

LCT-Raw

0 0

20


Figure 6a.

49


120


Page 50 of 52

FFA Released (%)

100 80 60 40 20

MCT-Boiled LCT-Boiled

0 0

20

40

60

80

100

Digestion Time (min) Figure 6b.

50


120

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100

a

90

a

Bioaccessibility (%)

80 70 60

b

50

c

40 30

d e

d

e

20 10

Figure 7.

51


LCT -Boiled

LCT -Raw

MCT Boiled

MCT -Raw

OO - Boiled

OO - Raw

Buffer Boiled

Buffer -Raw

0


Table of Contents Graphic

52


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Increasing Carotenoid Bioaccessibility from Yellow Peppers Using

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