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
