Effect of the Composition and Structure of Excipient Emulsion on the

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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

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Journal of Agricultural and Food Chemistry

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Effect of the composition and structure of excipient emulsion

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on the bioaccessibility of pesticides residue in agricultural

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products

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

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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

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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

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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


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ABSTRACT

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

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

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

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

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

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

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

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

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

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significant impact on chlorpyrifos bioaccessibility.

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

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

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fruits or vegetables could increase pesticide bioaccessibility.

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Keywords: nanoemulsions; pesticides; bioaccessibility; toxicity; bioavailability

Bioaccessibility increased with

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

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commonly co-ingested with fruits and vegetables. 1.

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

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

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

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

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

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substantially increase the oral bioavailability of lipophilic nutraceuticals present

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within these natural products.

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increase the bioaccessibility of carotenoids in carrots,

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

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

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

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Emulsions may enhance oral bioavailability through a number of mechanisms related

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

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nutraceuticals in the gastrointestinal tract (GIT). 2.

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development of excipient emulsions whose compositions and structures are

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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

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

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

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However, many natural products also contain potentially detrimental bioactive agents

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

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

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

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

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

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

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18-20

.

and


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food

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

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

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

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

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

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

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

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vegetables.

samples has been measured in previous studies.

However, to the authors’

Previous

28-31

.

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

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

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detected in foods.

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octanol/water partition coefficient (LogP=5.2),

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impacted by co-ingestion with emulsions.

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

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consumed food. 34-36.

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

, and may therefore be strongly

Tomato was used as a model natural

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

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

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

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micelle phase was used as a measure of its bioaccessibility.

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

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

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

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

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MATERIALS AND METHODS

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Materials

The impact of the

The

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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MA).

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

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

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Emulsion preparation

Medium chain triglyceride (MCT)

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

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

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

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

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phase.

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

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

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

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

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

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

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

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

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


The

Coarse emulsions


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

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

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a pressure of 11,000 psi.

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

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Preparation of chlorpyrifos standard solution

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

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

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

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

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determinations.

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to avoid unnecessary pollution and cost.

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Tomato-pesticide sample preparation

All other experiments were performed using unlabeled chlorpyrifos

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

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

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

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

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

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maximum residue (MRL) level for chlorpyrifos.

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

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emulsion (sample).

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Gastrointestinal tract model

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. An equal amount of

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The mixtures of chlorpyrifos-treated tomatoes and emulsions were passed

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

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

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detail in our previous study 38 with some slight modifications:

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

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

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

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

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prepared and preheated to 37 ºC.

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

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

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mimic the mouth phase.

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

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

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prepared and preheated to 37 ºC.

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

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the mixture was adjusted to pH 2.5.

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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

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

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

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

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

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

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

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

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

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

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

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

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Particle characterization

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

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

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

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described in our previous study. 5.

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

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

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

The particle size distribution and ζ-potential of



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

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

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

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

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

The

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Confocal microscopy images of the samples were taken to characterize their

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

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

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

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

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

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

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then microstructure images were acquired using image analysis software

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

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Pesticide bioaccessibility

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The bioaccessibility of chlorpyrifos was determined after they had passed through

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the small intestinal phase.

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

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

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following expression:

The bioaccessibility was calculated from measurements

% = 100 ×

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

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

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Chlorpyrifos determination

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

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intensity of the

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LS6500).

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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)

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

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

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calculated from the liquid scintillation counting results.

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Statistical analysis

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

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results are reported as averages and standard deviations calculated from these

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

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Duncan's test and a P-value of

Effect of the Composition and Structure of Excipient Emulsion on the

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