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Food Safety and Toxicology

Impact of Food Emulsions on the Bioaccessibility of Hydrophobic Pesticide Residues in Co-ingested Natural Products: Influence of Emulsifier and Dietary Fiber Type Ruojie Zhang, Wenhao Wu, zipei zhang, Shanshan Lv, Baoshan Xing, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06930 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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Impact of Food Emulsions on the Bioaccessibility of Hydrophobic

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Pesticide Residues in Co-ingested Natural Products: Influence of

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Emulsifier and Dietary Fiber Type

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Ruojie Zhang1, Wenhao Wu2, Zipei Zhang1, Shanshan Lv1,3, Baoshan Xing2, and

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David Julian McClements1,4

7 8 9 10 11 12 13 14 15 16 17 18 19

1

Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA 2

Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003 3 Key Laboratory of Bio-based Material Science and Technology (Ministry of Education), College of Material Science and Engineering, Northeast Forestry University, Harbin, 150040, People’s Republic of China 4 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: Dec.6

1

David Julian McClements, Department of Food Science, University of

Massachusetts Amherst, Amherst, MA 01003, USA. 413 545 1019; [email protected]

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ABSTRACT

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In the typical Western diet, fruits and vegetables are often consumed with food

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products that exist as oil-in-water emulsions, such as creams, dressings, and sauces.

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Studies have shown that co-ingestion of fruits and vegetables with emulsions can

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increase the bioavailability of beneficial lipophilic bioactive agents, such as

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nutraceuticals or vitamins.

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with low levels of detrimental lipophilic agents, such as hydrophobic pesticides.

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therefore examined the impact of co-ingesting a common agricultural product

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(tomatoes) with model food emulsions on the bioaccessibility of a hydrophobic

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pesticide (chlorpyrifos).

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Tween 80) and dietary fiber types (xanthan, chitosan, β-glucan) on the bioaccessibility

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of the pesticide was measured using a simulated gastrointestinal model.

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bioaccessibility depended on the type of emulsifier used to formulate the emulsions:

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phospholipids > Tween 80 > whey protein.

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pesticide bioaccessibility by an amount that depended on the nature of the emulsifier

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used. Overall, our results suggest that the bioaccessibility of undesirable pesticides on

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fruits and vegetables will depend on the nature of the emulsions they are consumed

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with.

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Keywords:

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emulsifier; dietary fiber

Agricultural produce, however, may also be contaminated We

The impact of emulsifier types (phospholipids, whey protein,

Chlorpyrifos

Dietary fiber type also influenced

excipient emulsions; nanoemulsions; pesticides; bioaccessibility;

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2


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INTRODUCTION

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In a typical American diet, it is common to co-consume fruits and vegetables with

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food products consisting of oil-in-water emulsions, such as salad dressings, cooking

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sauces, dessert creams, and fruit smoothies.

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oil-in-water emulsions could be beneficial for human health because this leads to an

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increase in the bioavailability of lipophilic nutraceuticals

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lipids in the emulsions leads to the formation of mixed micelles in the gastrointestinal

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tract (GIT), which can solubilize and transport the hydrophobic bioactive agents 4.

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the other hand, fruits and vegetables may also contain undesirable hydrophobic

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substances, such as pesticides, which are introduced during crop growth and/or

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postharvest processing 5. Co-ingestion of emulsions with fruits and vegetables may

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therefore increase the bioavailability of undesirable pesticides, as well as any desirable

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nutraceuticals or vitamins. Indeed, in a previous study, we showed that oil-in-water

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emulsions could increase the bioaccessibility of hydrophobic pesticides on tomatoes 6.

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Moreover, we found that the efficacy of these emulsions at enhancing the

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bioaccessibility of the pesticides depended on their composition and structure. For

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instance, pesticide bioaccessibility increased with increasing fat content in the

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emulsions, and was higher when corn oil was used as the fat phase rather than medium

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chain triglycerides (MCT).

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micelles formed by corn oil have larger hydrophobic domains than those formed by

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MCT 7.

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within their hydrophobic interiors.

The co-ingestion of natural foods with

1-3

.

The digestion of the

On

This latter effect was attributed to the fact that the mixed

As a result, they are capable for solubilizing more of the non-polar pesticides

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Another important factor that is known to impact lipid digestion and the

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bioaccessibility of hydrophobic substances is the surface properties of the droplets in

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the emulsions

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known to impact the gastrointestinal fate of oil droplets

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would expect that the nature of the droplet surfaces in oil-in-water emulsions would

8-9

.

Surface properties, such as charge, thickness and polarity, are

3


10-12

.

Consequently, one


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modulate the bioaccessibility of pesticides on co-ingested fruits and vegetables.

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emulsions, such as dressings, sauces, and creams, may be stabilized by a variety of

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different types of emulsifier, including proteins, polysaccharides, phospholipids, and

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surfactants

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lipophilic bioactive compounds was significantly impacted by the nature of the

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emulsifiers used to formulate the emulsions 9. In addition, it was reported that some

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emulsifiers could alter the permeability of intestinal cell walls, thereby altering the

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absorption of certain bioactive components

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type of emulsifier used to form an emulsion would impact the bioaccessibility of any

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hydrophobic pesticides co-ingested with it.

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13-14

.

Food

Previous studies have reported that the bioaccessibility of some

15

.

We therefore hypothesized that the

Dietary fibers are often used in food emulsions as stabilizers and thickeners to

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improve their functional and sensory attributes 16-18.

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to improve the physical stability of emulsions by inhibiting droplet aggregation and

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gravitational separation

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gastrointestinal fate of emulsions through a variety of mechanisms, including altering

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the rheology of the gastrointestinal fluids, forming coatings around the fat droplets,

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altering fat droplet aggregation, and binding GIT components such as lipase, calcium,

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bile salts, and fatty acids 10.

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in emulsions would also alter the bioaccessibility of hydrophobic pesticides.

19-20

Dietary fibers have been shown

. In addition, they have been shown to impact the

We therefore hypothesized that the type of dietary fiber

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In the current study, we therefore examined the influence of emulsifier and dietary

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fiber type in emulsions on the bioaccessibility of pesticides on a commonly consumed

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fresh produce.

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pesticide-contaminated agriculture produce.

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(LogP = 5.2) organophosphorus pesticide that is commonly used for insect and pest

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control on tomatoes

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whey protein, and phospholipids, were used to fabricate the emulsions because they

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have different molecular characteristics. Three dietary fibers, xanthan (anionic),

Chlorpyrifos contaminated-tomato was used as a model for a

21-22

.

Chlorpyrifos is a highly hydrophobic

Four food-grade emulsifiers, Tween 80, quillaja saponin,

4


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chitosan (cationic) and β-glucan (neutral), were incorporated into the emulsions

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because they have different electrical properties. The impact of emulsion composition

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on the bioaccessibility of the chlorpyrifos from the tomatoes was studied using a

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simulated GIT, consisting of mouth, stomach and small intestinal phases. The results

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from this study will help us to understand the influence of food matrix effects on the

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bioavailability of pesticides in the human diet.

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MATERIALS AND METHODS

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Materials

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Fresh tomatoes (organic) and corn oil (Mazola) were purchased from a local

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supermarket. Tween 80 was purchased from Sigma-Aldrich (St. Louis, MO). Quillaja

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saponins (Q-Naturale) was provided by Ingredion Inc. (Westchester, IL). Whey protein

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isolate (WPI) was provided by Davisco Foods International Inc. (Le Sueur, MN).

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Sunflower phospholipids (Sunlipon 65) was kindly donated by Perimondo (New York,

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NY, USA).

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(Belcamp, Md., USA), which was reported to have an average molecular weight around

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60 × 106 g/mol. Chitosan (ChitoClear) was obtained from Primex (Iceland). It was

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reported to have a molecular weight of about 3.2 × 106 g/mol and a degree of

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deacetylation of about 75%. β-glucan was donated by Zhangjiakou Yikang

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Biotechnology Co., Ltd. (Zhangjiakou, China). It was reported to have an average

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molecular weight of around 0.6-0.7 × 106 g/mol.

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porcine lipase (100-400 units/mg), porcine bile extract, and unlabeled chlorpyrifos

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were purchased from the Sigma-Aldrich Company (St. Louis, MO). Radioactive-

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labeled [14C] chlorpyrifos with specific activity of 26.8 mCi/mmol was obtained from

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Dow AgroSciences LLC (Indianapolis, IN). A liquid scintillation cocktail (Ultima Gold

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XR) was obtained from PerkinElmer (PerkinElmer, Inc., Walthanm, MA). Double

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distilled water was prepared using a Nanopure Infinity system (Barnstaeas

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Internationals, Dubuque, IA). All solvents and reagents used in this study were of

Xanthan gum (Ticanxan® Xanthan EC) was provided by TIC Gums

Mucin, pepsin (250 units/mg),

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analytical grade.

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Solution preparation

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Emulsifier solutions were prepared by dissolving 1.0 wt% emulsifier (Tween 80,

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Q-Naturale, WPI or sunflower phospholipids) into phosphate buffer (5 mM, pH 7) and

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stirring for at least 2 h. For the small-molecule surfactants (Tween 80 and Q-Naturale)

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this concentration is well-above their critical micelle concentration (CMC). The WPI

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and phospholipids solutions were stored at 4 ºC overnight to ensure complete hydration

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and then filtered to remove any insoluble matter. Xanthan, chitosan and β-glucan

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solutions (1.0 wt%) were prepared by dispersing weighed amounts of powder into

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phosphate buffer (5 mM, pH 7), 1% acetic acid and double distilled water (pH 7),

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respectively, and then stirring overnight to ensure fully dissolution.

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Emulsion preparation

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Emulsions with different emulsifiers were fabricated by mixing 10 wt% corn oil

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with 90 wt% emulsifier solution using a high-speed mixer for 2 min (M133/1281-0,

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Biospec Products, Inc., ESGC, Switzerland) and then passing them through a

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microfluidizer (M110L, Microfluidics, Newton, MA) with a 75 µm interaction chamber

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(F20Y) for three times at an operational pressure of 11,000 psi. Emulsions were then

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diluted to 8 wt% oil phase before mixing with dietary fiber.

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The mixtures of emulsions and dietary fibers were prepared by mixing a certain

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amount of emulsions and dietary fiber solutions to obtain a final concentration of 4 wt%

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oil and 0.5 wt% dietary fiber. The mixtures were then stirred for 30 min before use.

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Chlorpyrifos standard solution preparation

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Unlabeled or radioactive-labeled [14C] chlorpyrifos was dissolved in acetonitrile to

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form a 50 ppm stock pesticide solution. The radioactive labeled [14C] chlorpyrifos was

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only used for the determination of pesticide bioaccessibility, whereas the unlabeled

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chlorpyrifos was used for all other measurements to avoid unnecessary radioactive

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contamination and reduce costs. 6


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Preparation of pesticide-contaminated tomatoes

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Fresh organic tomatoes were diced into small cubes (approximately 10 mm × 10

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mm in height and width) and then blended using a household blender for 1 min to

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disrupt the tomato structure. The resulting tomato puree were then mixed with

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chlorpyrifos standard solution to produce a pesticide-contaminated sample. The final

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concentration of chlorpyrifos was fixed at 0.5 ppm, which is the maximum residue

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(MRL) level for chlorpyrifos . The initial samples were then prepared by mixing equal

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amounts of chlorpyrifos-contaminated tomato with either pure emulsions or dietary

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fiber containing emulsions.

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Gastrointestinal tract model

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The initial samples were passed through an in vitro simulated GIT model

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containing mouth, stomach, and small intestine phases, which has been described in

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detail in our previous study 6.

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Particle characterization

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The particle size and z-potential of the emulsions were determined using a static

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light scattering instrument (Mastersizer 2000, Malvern Instruments Ltd., Malvern,

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Worcestershire, UK) and an electrophoresis device (Zetasizer Nano ZS series, Malvern

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Instruments Ltd. Worcestershire, UK), respectively. To avoid multiple scattering effects,

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the samples were diluted with phosphate buffer (5 mM) at the same pH as them. For

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emulsions with chitosan, phosphate buffer with pH 4.0 was used. For pure emulsions

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and emulsions with xanthan and β-glucan, phosphate buffer with pH 7.0 was used. The

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refractive index of 1.472 was used for corn oil in the calculations 24. The particle sizes

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of the emulsions are reported as the surface-weighted mean diameter (d32).

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The microstructures of the emulsions were acquired using a confocal microscope

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equipped with a 40× objective lens and a digital camera. The samples were dyed using

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Nile Red solution (1mg/mL ethanol) at a ratio of 20:1 and then placed on a microscope

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slide and covered by a cover slip. The microstructure images of the samples were 7



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acquired using image analysis software (NIS-Elements, Nikon, Melville, NY). The

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

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Chlorpyrifos bioaccessibility

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The bioaccessibility was determined using a method adopted from our previous

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study 6. Briefly, the overall digest and mixed micelles were collected after each sample

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had passed through the full GIT model. The bioaccessibility of chlorpyrifos was

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calculated from the chlorpyrifos concentrations determined in the supernatant mixed

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micelle fraction (Cmicelle) and the overall digest (CDigesta) using the following equation: 𝐵𝑖𝑜𝑎𝑐𝑐𝑒𝑠𝑠𝑖𝑏𝑖𝑙𝑖𝑡𝑦 (%) = 100 ×

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𝐶5678998 𝐶:6;8

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