Effect of the Composition and Structure of Excipient Emulsion on the

University, 665 Huntington Avenue, Boston, Massachusetts 02115, United States. J. Agric. Food Chem. , 2017, 65 (41), pp 9128–9138. DOI: 10.1021/...
2 downloads 10 Views 13MB Size
Subscriber access provided by UNIV OF CAMBRIDGE

Article

Effect of the composition and structure of excipient emulsion on the bioaccessibility of pesticides residue in agricultural products Ruojie Zhang, Wenhao Wu, Zipei Zhang, Yeonhwa Park, Lili He, Baoshan Xing, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02607 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 18, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 59

Journal of Agricultural and Food Chemistry

1

Effect of the composition and structure of excipient emulsion

2

on the bioaccessibility of pesticides residue in agricultural

3

products

4

Ruojie Zhang1, Wenhao Wu2, Zipei Zhang1, Yeonhwa Park1, Lili

5

He1, Baoshan Xing2, and David Julian McClements1,3

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1

Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA 2 Stockbridge School of Agriculture, University of Massachusetts Amherst, Amherst, MA 01003, USA 3 Laboratory for Environmental Health NanoScience, Center for Nanotechnology and Nanotoxicology, T. H. Chan School of Public Health, Harvard University 665 Huntington Avenue, Boston, MA 02115, USA

Journal: Journal of Agricultural and Food Chemistry Submitted: June 2017

1

David Julian McClements, Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA. 413 545 1019; [email protected] 1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

23

ABSTRACT

24

The influence of co-ingestion of food emulsions with tomatoes on the

25

bioaccessibility of a model pesticide (chlorpyrifos) was studied. Emulsions were

26

fabricated with different oil contents (0 to 8%), lipid compositions (MCT and corn oil)

27

and particle diameters (d32 = 0.17 and 10 µm). The emulsions were then mixed with

28

chlorpyrifos-contaminated tomato puree, and the mixtures were subjected to a

29

simulated gastrointestinal tract (GIT) consisting of mouth, stomach, and small

30

intestine. The particle size, surface charge, and microstructure of the emulsions was

31

measured as they passed through the GIT, and chlorpyrifos bioaccessibility was

32

determined after digestion. The composition and structure of the emulsions had a

33

significant impact on chlorpyrifos bioaccessibility.

34

increasing oil content, and was higher for corn oil than MCT, but did not strongly

35

depend on oil droplet size. These results suggest that co-ingestion of emulsions with

36

fruits or vegetables could increase pesticide bioaccessibility.

37

Keywords: nanoemulsions; pesticides; bioaccessibility; toxicity; bioavailability

Bioaccessibility increased with

38

2 ACS Paragon Plus Environment

Page 2 of 59

Page 3 of 59

Journal of Agricultural and Food Chemistry

39 40

INTRODUCTION Oil-in-water emulsions, such as dressings, dips, sauces, and creams, are

41

commonly co-ingested with fruits and vegetables. 1.

42

and dips are consumed with raw vegetables (such as lettuce, tomatoes, radishes, celery,

43

and broccoli), hot sauces are consumed with cooked vegetables (such as cabbage,

44

carrots, broccoli, kale, beans, or peas), and creams or ice-creams are consumed with

45

hot or cold fruits (such as apples, blueberries, strawberries, or raspberries).

46

studies have shown that co-ingestion of emulsions with fruits and vegetables may

47

substantially increase the oral bioavailability of lipophilic nutraceuticals present

48

within these natural products.

49

increase the bioaccessibility of carotenoids in carrots,

50

and yellow peppers. 14. The efficacy of emulsions at enhancing the bioaccessibility of

51

lipophilic nutraceutical depends on their composition and structure, such as lipid

52

content, lipid phase composition, particle size, and interfacial properties.

53

Emulsions may enhance oral bioavailability through a number of mechanisms related

54

to their impact on the bioaccessibility, absorption, and/or transformation of

55

nutraceuticals in the gastrointestinal tract (GIT). 2.

56

development of excipient emulsions whose compositions and structures are

57

specifically designed to enhance the bioavailability of nutraceuticals in foods. 1.

1-4

For example, salad dressings

Previous

. For instance, emulsions have been shown to 5-8

, tomatoes,

9-12

, mangoes, 13,

5-7, 15-17

.

This phenomenon has led to the

58

Previous research in this area has mainly focused on the ability of emulsions to

59

increase the bioavailability of beneficial bioactive agents in fruits and vegetables.

60

However, many natural products also contain potentially detrimental bioactive agents

61

that may be introduced during crop production and storage, such as pesticides.

62

It is therefore possible that co-ingestion of emulsions with these products could

63

increase the bioavailability of these undesirable substances. The purpose of the

64

current study was therefore to examine the potential impact of co-ingestion of

65

emulsions with natural produce on the bioaccessibility of a hydrophobic pesticide.

66

The bioaccessibility of selected pesticides from various types of soil 3 ACS Paragon Plus Environment

21-24

18-20

.

and

Journal of Agricultural and Food Chemistry

25-27

Page 4 of 59

67

food

68

knowledge, there have been no previous studies of the impact of processed foods on

69

the bioaccessibility of pesticides on natural foods co-ingested with them.

70

studies suggest that the bioaccessibility of pesticides is highly dependent on their

71

oil-water partition coefficients (LogP values), with the lipid content of the sample

72

being a critical factor influencing the bioaccessibility of lipophilic pesticides..

73

We therefore postulated that food emulsions, which are a source of readily digestible

74

lipids, would increase the bioavailability of lipophilic pesticides on fruits and

75

vegetables.

samples has been measured in previous studies.

However, to the authors’

Previous

28-31

.

76

In this study, chlorpyrifos was used as a representative pesticide, because it is

77

widely used for controlling agricultural and household insects, and is commonly

78

detected in foods.

79

octanol/water partition coefficient (LogP=5.2),

80

impacted by co-ingestion with emulsions.

81

product because chlorpyrifos is widely used to control insect pests on this commonly

82

consumed food. 34-36.

32

. In addition, it is a strongly hydrophobic molecule with a high 33

, and may therefore be strongly

Tomato was used as a model natural

83

A simulated GIT, consisting of mouth, stomach and small intestine phases, was

84

used to study the potential gastrointestinal fate of the tomato-emulsion mixtures. After

85

passing through the GIT model, the amount of chlorpyrifos solubilized in the mixed

86

micelle phase was used as a measure of its bioaccessibility.

87

composition and structure of the emulsions on the bioaccessibility of chlorpyrifos was

88

also studied, including lipid droplet concentration, composition, and size.

89

results from this study will provide valuable insights into the potential impact of food

90

matrix effects on the bioavailability of pesticides in the human diet.

91

MATERIALS AND METHODS

92

Materials

The impact of the

The

93

Fresh organic tomatoes were purchased from a local market. Whey protein isolate

94

(WPI) was purchased from Davisco Foods International Inc. (Le Sueur, MN), which 4 ACS Paragon Plus Environment

Page 5 of 59

Journal of Agricultural and Food Chemistry

95

was reported to contain 97.6% protein (dry basis).

96

oil was purchased from Coletica (Northport, NY). Corn oil was obtained from a

97

commercial food supplier (Mazola, ACH Food Companies, Memphis, TN). The

98

saturated, monounsaturated, and polyunsaturated fat content of this product were

99

reported to be approximately 14, 29, and 57%, respectively. Gastrointestinal

100

components, including mucin from porcine stomach, pepsin from porcine gastric

101

mucosa (250 units/mg), porcine lipase (100-400 units/mg), and porcine bile extract,

102

were obtained from the Sigma-Aldrich Chemical Co. (St. Louis, MO). Unlabeled

103

chlorpyrifos was purchased from the Sigma-Aldrich Chemical Co. (St. Louis, MO)

104

and radioactive-labeled [14C]chlorpyrifos (specific activity of 26.8 mCi/mmol) was

105

purchased from Dow AgroSciences LLC (Indianapolis, IN). Scintiverse cocktail

106

(Ultima Gold XR) was purchased from PerkinElmer (PerkinElmer, Inc., Walthanm,

107

MA).

108

from a water purification system (Nanopure Infinity, Barnstaeas International,

109

Dubuque, IA) was used for preparation of all solutions and emulsions.

110

Emulsion preparation

Medium chain triglyceride (MCT)

All solvents and reagents were of analytical grade. Double distilled water

111

Initially, stock emulsions were fabricated by homogenizing 10 wt% oil phase

112

with 90 wt% aqueous phase. Different oil types, which were representative of medium

113

chain triglycerides (MCT) and long chain triglycerides (LCT) were used as the oil

114

phase.

115

aqueous phase was prepared by dispersing WPI in buffer solution (5 mM phosphate

116

buffer, pH 7.0) to a final concentration (1.0 wt%), stirring for at least 3 hours at

117

ambient temperature, and then storing at 4 ºC overnight to completely hydrate the

118

protein. The aqueous phase was filtered using Whatman qualitative filter paper

119

(Fisher scientific) before use to remove any large particles.

120

containing relatively large droplets were prepared by blending the oil-water mixture

121

using a high-shear mixer for 2 min (M133/1281-0, Biospec Products, Inc., ESGC,

122

Switzerland). Fine emulsions containing relatively small droplets were prepared by

Corn oil was used as an example of a widely utilized LCT food oil.

5 ACS Paragon Plus Environment

The

Coarse emulsions

Journal of Agricultural and Food Chemistry

123

passing the coarse emulsions through a high-pressure homogenizer (M110Y,

124

Microfluidics, Newton, MA) with a 75 µm interaction chamber (F20Y) three times at

125

a pressure of 11,000 psi.

126

8 wt.%) were prepared by dilution of the originally prepared emulsions.

127

Preparation of chlorpyrifos standard solution

Fine emulsions with different oil concentrations (2, 4, and

128

Stock pesticide solutions were prepared by dissolving radio-labeled [14C]

129

chlorpyrifos or unlabeled chlorpyrifos in acetonitrile at a level of 50 ppm. The stock

130

solution containing [14C] chlorpyrifos was only used for the bioaccessibility

131

determinations.

132

to avoid unnecessary pollution and cost.

133

Tomato-pesticide sample preparation

All other experiments were performed using unlabeled chlorpyrifos

134

Fresh tomatoes were cut into pieces (approximately 10 mm × 10 mm in height

135

and width) and then blended for 1 min using a household blender to break down the

136

tomato structure. 10 g of the resulting tomato puree were mixed with 100 µL

137

chlorpyrifos standard solution (containing either [14C] chlorpyrifos or unlabeled

138

chlorpyrifos) to obtain final chlorpyrifos concentrations of 0.5 ppm, which is the

139

maximum residue (MRL) level for chlorpyrifos.

140

chlorpyrifos-treated tomato was mixed with either phosphate buffer (control) or

141

emulsion (sample).

142

Gastrointestinal tract model

37

. An equal amount of

143

The mixtures of chlorpyrifos-treated tomatoes and emulsions were passed

144

through a simulated GIT designed to mimic passage of a food through the human

145

mouth, stomach, and small intestine phases. This model followed the one described in

146

detail in our previous study 38 with some slight modifications:

147

Initial system: 20 g of sample (tomato with or without pesticide) was placed into

148

a glass beaker in an incubated shaker (Innova Incubator Shaker, Model 4080, New

149

Brunswick Scientific, New Jersey, USA) at 37 ºC to warm up the samples.

150

Mouth phase: A simulated saliva fluid (SSF) containing 0.03 g/g mucin has been 6 ACS Paragon Plus Environment

Page 6 of 59

Page 7 of 59

Journal of Agricultural and Food Chemistry

151

prepared and preheated to 37 ºC.

152

mixed with an equal amount of the SSF (20 g), and then the mixture was adjusted to

153

pH 6.8. The mixture was then placed in an incubator shaker for 2 min at 37 ºC to

154

mimic the mouth phase.

155

An aliquot of the initial sample (20 g) was then

Stomach phase: A simulated gastric fluid containing 0.0032 g/g pepsin had been

156

prepared and preheated to 37 ºC.

157

from the mouth phase was mixed with an equal amount of the SGF (20 g), and then

158

the mixture was adjusted to pH 2.5.

159

shaker for 2 h at 37 ºC to mimic the stomach phase.

An aliquot of the “bolus” sample (20 g) resulting

The mixture was then placed in an incubator

160

Small intestine phase: An aliquot of the “chyme” sample (30 g) from the stomach

161

phase was placed into a 100-mL glass beaker that was incubated in a water bath at 37

162

ºC, and then the sample was adjusted to pH 7.00 with constant stirring. 1.5 mL of

163

simulated intestinal fluid (SIF) was then added to the reaction vessel, followed by 3.5

164

mL of bile salts solution (final concentration is 5mg/mL in reaction cell). The mixture

165

in the reaction system was then adjusted back to pH 7.00. Finally, 2.5 mL of lipase

166

solution (final concentration is 1.6mg/mL in reaction cell) was added to the mixture

167

and an automatic titration unit (Metrohm, USA Inc.) was activated to monitor the pH

168

and maintain it at a constant value (pH 7.0) by titrating 0.25 N NaOH solution into the

169

reaction vessel for 2 h at 37 ºC. The amount of free fatty acids released due to the

170

lipid digestion was calculated from the titration curves as described previously. 39.

171

Particle characterization

172

The characteristics of the colloidal particles in the tomato-emulsion mixtures

173

were measured as they passed through the simulated GIT. A pre-treatment for samples

174

was required to avoid the interference from large tomato tissue fragments, which was

175

described in our previous study. 5.

176

the samples were determined using a static light scattering device (Mastersizer 2000,

177

Malvern Instruments Ltd., Malvern, Worcestershire, UK) and an electrophoresis

178

instrument (Zetasizer Nano ZS series, Malvern Instruments Ltd. Worcestershire, UK),

The particle size distribution and ζ-potential of

7 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 59

179

respectively. Phosphate buffer (5 mM, pH 7.0) was used to dilute the initial, mouth,

180

and small intestine samples and acidified water (pH 2.5) was used to dilute the

181

stomach samples to avoid multiple scattering effects. The refractive index of the MCT

182

and corn oil used in the calculations were 1.445 and 1.472, respectively.40.

183

particle sizes are reported as the surface-weighted mean diameter. (d32).

The

184

Confocal microscopy images of the samples were taken to characterize their

185

microstructures at various stages in the GIT model. A confocal scanning laser

186

microscope with a 20× objective lens was used to acquire the images (Nikon

187

D-Eclipse C1 80i, Nikon, Melville, NY, US.). 2 mL samples were mixed with 0.1 mL

188

Nile Red solution (1 mg/mL ethanol) to dye the oil phase before analysis. The

189

excitation and emission spectrum for Nile Red were 543 nm and 605 nm, respectively.

190

An aliquot of sample was placed on a microscope slide, covered by a cover slip, and

191

then microstructure images were acquired using image analysis software

192

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

193

Pesticide bioaccessibility

194

The bioaccessibility of chlorpyrifos was determined after they had passed through

195

the small intestinal phase.

196

of the chlorpyrifos concentration in the mixed micelle fraction and in the total digesta,

197

as described previously. 5. The bioaccessibility was then calculated using the

198

following expression:

The bioaccessibility was calculated from measurements

  % = 100 ×

 

199

Where, Cmicelle and CDigesta are the concentrations of chlorpyrifos in the mixed micelle

200

phase and in the overall digesta after the simulated intestinal digestion, respectively.

201

Chlorpyrifos determination

202

The chlorpyrifos concentration in the samples was determined by measuring the 14

203

intensity of the

204

LS6500).

205

was placed in to an 8 mL hinge cap vial (PerkinElmer, Inc., Walthanm, MA) and then 8

C-radioactive signal using liquid scintillation counting (Bechman

Briefly, 2 mL of sample (the mixed micelle phase or the overall digesta)

ACS Paragon Plus Environment

Page 9 of 59

Journal of Agricultural and Food Chemistry

206

4 mL of Scintiverse cocktail (Ultima Gold XR, PerkinElmer, Inc., Walthanm, MA)

207

was added and the system was mixed. The concentration of chlorpyrifos was then

208

calculated from the liquid scintillation counting results.

209

Statistical analysis

210

All experiments were performed on at least three freshly prepared samples. The

211

results are reported as averages and standard deviations calculated from these

212

measurements using a statistical software package (SPSS). Means were subject to

213

Duncan's test and a P-value of