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Food and Beverage Chemistry/Biochemistry

Vitamin E encapsulation within oil-in-water emulsions: Impact of emulsifier type on physicochemical stability and bioaccessibility Shanshan Lv, Yanhua Zhang, Haiyan Tan, Ruojie Zhang, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06347 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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Journal of Agricultural and Food Chemistry

Vitamin E encapsulation within oil-in-water emulsions: Impact of emulsifier type on physicochemical stability and bioaccessibility Shanshan Lv1,2, Yanhua Zhang*1, Haiyan Tan1, Ruojie Zhang2, David Julian McClements*2 1

Key Laboratory of Bio-based Material Science and Technology (Ministry of Education),

College of Material Science and Engineering, Northeast Forestry University, Harbin, 150040, P.R. China 2

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

Corresponding Author: Yanhua Zhang; David Julian McClements Corresponding author emails: [email protected]; [email protected]

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Abstract

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The influence of plant-based (gum arabic and quillaja saponin) and animal-based (whey

3

protein isolate, WPI) emulsifiers on the production and stability of vitamin E-fortified emulsions

4

was investigated. Their impact on lipid digestibility and vitamin bioaccessibility was also

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studied utilizing an in vitro gastrointestinal tract. WPI and saponin produced smaller emulsions

6

than gum arabic. All emulsions had good storage stability at room temperature (4 weeks, pH 7).

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Saponin- and gum arabic-emulsions were resistant to droplet aggregation from pH 2 to 8 because

8

these emulsifiers generated strong electro-steric repulsion. WPI-coated droplets flocculated

9

around pH 5 due to a reduction in charge near their isoelectric point. Lipid digestion was slower

10

in saponin-emulsions, presumably because the high surface-activity of saponins inhibited their

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removal by bile acids and lipase. Vitamin bioaccessibility was higher in WPI- than in saponin-

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or gum arabic-emulsions. This information may facilitate the design of more efficacious

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vitamin-fortified delivery systems.

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Keywords: natural emulsifier; vitamin E; lipid digestion; bioaccessibility; nanoemulsions

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Journal of Agricultural and Food Chemistry

Introduction With the recent improvement in global living standards, more people are paying attention to

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the incorporation of essential nutrients and nutraceuticals into their diets. Consequently, many

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companies are developing a new generation of functional food and beverage products that are

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fortified with these bioactive components. Vitamin E is a group of isomeric micronutrients, with

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α-tocopherol having the strongest biological activity.1-2 Daily intake of α-tocopherol may benefit

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human health due to its antioxidant activity and ability to inhibit various diseases.3-5 However, it

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is a strongly hydrophobic molecule, making it hard to disperse directly into foods and beverages

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that have an aqueous continuous phase. Moreover, exposure to light, heat, and oxygen promotes

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the chemical degradation of α-tocopherol during storage, leading to a reduction in its biological

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activity and nutritional benefits. To overcome these challenges, α-tocopherol can be

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encapsulated and protected using colloidal delivery systems.6-7

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Oil-in-water emulsions are particularly suitable for encapsulating and delivering lipophilic

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vitamins because they can be designed to have good physicochemical stability and to promote

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vitamin bioavailability.8 Emulsifiers play an essential role in the production and stabilization of

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emulsions, as well as in determining their functional performance. Selection of an appropriate

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emulsifier is therefore crucial to the success of any emulsion-based delivery system. Consumers

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are increasingly demanding more ethical and sustainable food products, which has promoted the

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food industry to search for plant-derived ingredients to replace synthetic or animal-derived

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ones.9-10 There has, therefore, been great interest in the identification of natural plant-derived

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emulsifiers that can be used in functional foods and beverages.11

37 38

In the current study, three types of natural emulsifier were tested to establish their impact on the production, stability, and performance of vitamin E-fortified emulsions. Quillaja saponin

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(QS) consists of a group of surface-active substances extracted from the Quillaja saponaria

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Molina tree.12-13 Gum arabic (GA) consists of a blend of amphiphilic glycoproteins and

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polysaccharides extracted from the exudate of two species of acacia tree.14-16 Whey protein

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isolate (WPI) consists of a blend of amphiphilic globular proteins isolated from bovine milk,

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such as -lactoglobulin, -lactalbumin, and bovine serum albumin.17-18 These three emulsifiers

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vary in their molecular weights, structures, polarities, and electrical characteristics, which affect

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their performance in emulsions.

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Previous studies have reported that QS and WPI are better at producing emulsions

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containing fine oil droplets than GA because of their higher surface activity and faster adsorption

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rate during homogenization.19 Under neutral pH conditions, the emulsions formed using QS and

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WPI were more stable to creaming than those formed using GA because they contained smaller

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oil droplets. Conversely, the GA-emulsions had better stability to droplet aggregation when

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exposed to alterations in environmental conditions, such as pH variations, high salt levels, or

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elevated temperatures. The WPI-emulsions tended to aggregate around their isoelectric points, at

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high ionic strengths, and when heated due to changes in the colloidal interactions acting amongst

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the oil droplets. The QS-emulsions were shown to have good aggregation stability over most of

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the pH range found in foods but were unstable at pH 2, which was attributed to a reduction in

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their negative surface potential in this strongly acidic environment.20-21

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Most previous studies on these emulsifiers have focused on their influence on the

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physicochemical stability of emulsions. There is a much poorer understanding of the influence

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of these emulsifiers on the behavior of vitamin-fortified emulsions under gastrointestinal

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conditions. The current study was therefore carried out to determine the influence of these

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natural emulsifiers on the gastrointestinal behavior of vitamin-fortified emulsions. We

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hypothesized that it is important to understand this process because encapsulated vitamins should

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be released and solubilized inside the lumen of the human gut prior to being transported and

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absorbed by the intestinal epithelium cells.22 Previous researchers have shown that the

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bioaccessibility and/or bioavailability of oil-soluble vitamins encapsulated within emulsion-

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based delivery systems depends on numerous factors, including droplet size, oil level, carrier oil

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type, and interfacial properties 23-28. Consequently, it is important to optimize the composition,

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structure, and physicochemical properties of these systems to ensure good performance. Some

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previous studies have measured the bioaccessibility of emulsified vitamin E stabilized by

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different kinds of emulsifier 29-31, but they have not focused on a direct comparison of plant- and

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animal-based emulsifiers and the differences in the mechanisms involved. The insights gained

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form the current research should therefore be helpful for improving the nutritional quality of

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functional foods and beverages.

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Materials and Methods

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Materials

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Vitamin E (α-tocopherol, purity 95%) was provided by Fisher Scientific (Waltham, MA).

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Corn oil (Mazola) was obtained from a local commercial supplier. Quillaja saponin (Q-Naturale

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200) was kindly supplied by Ingredion Inc. (Westchester, IL). Whey protein isolate (WPI) was

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bought from Davisco Foods International (Le Sueur, MN). Gum Arabic (GA) was purchased

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from TIC Gums (Belcamp, MD). All these emulsifiers were used without further purification.

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The mucin, pepsin, lipase, and bile extract (from porcine) were purchased from the Sigma-

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Aldrich Chemical Company (St. Louis, MO).

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purchased either from Fisher Scientific or Sigma-Aldrich. All concentrations are reported as

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weight percentages (w/w), unless otherwise stated.

All other reagents and chemicals were

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Emulsion Preparation Aqueous phases were prepared by dissolving the natural emulsifiers (1.5% w/w) in 5 mM

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phosphate buffer (pH 7.0). Oil phases were prepared by mixing vitamin E (20% w/w) and corn

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oil (80% w/w). Emulsions were prepared by blending the oil phase (10% w/w) and aqueous

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phase (90% w/w) together and then passing the resulting coarse emulsion through a

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microfluidizer (M110Y, Microfluidics, Newton, MA) three times at 12,000 psi.

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Emulsion Stability

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The storage stability of the emulsions was investigated by incubating them in the dark at

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room temperature for 4 weeks. The pH stability of the emulsions was determined by preparing a

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series of systems with different pH values (2.0-8.0) using HCl or NaOH solutions.

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In vitro Digestion

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A three-stage simulated GIT, consisting of mouth, stomach, and small intestine, was

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employed to explore the potential gastrointestinal behavior of the emulsions. Briefly, emulsions

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(2 wt.% oil level) were mixed with simulated saliva fluid (containing 3 mg/mL mucin) at a ratio

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of 1:1, the pH was adjusted to 6.8 and then the samples were incubated for 2 min to mimic the

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mouth phase. After 2 min, the mouth phase was mixed with simulated gastric fluid (with 3.2

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mg/mL pepsin) at a ratio of 1:1, the pH was adjusted to 2.5 and the system was incubated for 2 h

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to mimic the stomach phase. Finally, 30 mL of the stomach fluid contents were collected and

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subjected to the simulated small intestinal digestion condition. At this stage, the pH was

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adjusted to 7.0, then 1.5 mL simulated small intestinal fluid (containing 3.75 M NaCl and 0.25 M

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CaCl2) and 3.5 mL bile extract solution was added. Afterward, the pH was adjusted back to 7.0

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and 2.5 mL lipase solution (24 mg/mL) was added to mimic the small intestine digestion. The

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temperature of the entire digestion process was controlled at 37 °C. A pH-stat method was used

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to monitor lipolysis in the small intestine phase. The free fatty acids (FFA) released were

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calculated as follows 32:

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𝐹𝐹𝐴(%) =

𝑉𝑁𝑎𝑂𝐻 × 𝐶𝑁𝑎𝑂𝐻 × 𝑀𝑙𝑖𝑝𝑖𝑑 2𝑊𝑙𝑖𝑝𝑖𝑑

× 100

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Here, VNaOH is the NaOH consumption during the small intestinal digestion process, CNaOH is the

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NaOH concentration (0.25 M), Mlipid is the molar mass of digestible lipid (824 g·mol-1), and

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Wlipid is the digestible lipid weight in the initial digestion system. The GIT model used in our

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study is closely related to the standardized INFOGEST international consensus procedure

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developed for in vitro digestion studies33, but was optimized for application to emulsions by our

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group some years ago 32. This method was employed so that the results of this study could be

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directly compared to our previous studies on related systems.

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Particle Characterization

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The characteristics of the particles in the various systems were measured using static light

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scattering and electrophoresis. A Mastersizer 3000 and Zetasizer NanoZS (Malvern,

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Worcestershire, UK) were used to measure the particle size and charge, respectively. Before

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analysis, the samples from the stomach phase were diluted with pH 2.5 phosphate buffer, while

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the other samples were diluted with pH 7.0 phosphate buffer to avoid multiple scattering effects.

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Confocal fluorescence microscopy (Nikon D-Eclipse C1 80i, USA) was performed to observe

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the samples’ microstructures. Prior to observation, Nile Red (1 mg mL-1 ethanol) was added to

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stain the lipid phase.

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Vitamin Bioaccessibility

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The mixed micelle phase was obtained by centrifuging (4°C, 41 657 g, 50 min) the digest

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that remained after digestion of the samples within the small intestine. Vitamin bioaccessibility

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was determined by measuring the level of vitamin E in the mixed micelle and digest phases 7 ACS Paragon Plus Environment

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utilizing high-performance liquid chromatography (HPLC, Agilent 1100, Agilent Technologies,

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USA) with a C18 column (250 × 4.6 mm, 5 μm). Before HPLC analysis, the oil-soluble vitamin

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was extracted from the samples using a hexane/ethanol mixture (1/1, v/v). Briefly, 3 mL

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samples were mixed with the mixed organic solvent, and then centrifuged at 2500 g for 2 min to

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obtain a supernatant layer. This extraction process was repeated three times. Afterward, the

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supernatant layers were combined together and dried under nitrogen. Finally, the dried samples

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were dissolved in methanol and filtered through a 0.45 μm filter before carrying out HPLC

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analysis. The details of the HPLC analysis conditions have been reported in our previous

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work.34 Briefly, a mixture of 95% methanol and 5% double distilled water was used as the

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mobile phase. An isocratic elution running at 1.0 mL/min was carried out to separate the vitamin

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E at a wavelength of 295 nm. Finally, the vitamin E bioaccessibility was calculated as follows:

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Bioaccessibility(%) =

𝐶𝑚𝑖𝑐𝑒𝑙𝑙𝑒 𝐶𝐷𝑖𝑔𝑒𝑠𝑡𝑎

× 100

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Here, Cmicelle and CDigesta represent the vitamin concentrations in the mixed micelle fraction and

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total digesta collected after the small intestine phase.

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Statistical Analysis

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All experiments were repeated at least two or three times and the mean and standard

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deviation values were obtained. ANOVA analysis was employed to analyze the significant

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difference at a significance level of 0.05.

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Result and Discussion

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Impact of emulsifier type on emulsion formation

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In these experiments, the influence of emulsifier type on the characteristics of the oil droplets produced using standardized homogenization conditions was measured (Figure 1). The 8 ACS Paragon Plus Environment

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emulsions formed using QS and WPI had much smaller droplets than those formed using GA.

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This may have been because of differences in the surface activity and adsorption kinetics of the

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different emulsifiers. The ability of emulsifiers to reduce the interfacial tension is a critical

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factor in the formation of fine droplets during microfluidization, because the breakup of oil

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droplets is facilitated when the interfacial tension is reduced.35 As reported in our previous

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study, QS and WPI reduced the interfacial tension more effectively than GA.19 Consequently,

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they should generate finer oil droplets inside the homogenizer by promoting a higher degree of

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droplet disruption.35 Moreover, the relatively small QS and WPI emulsifiers are likely to adsorb

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to the lipid droplet surfaces more quickly than the relatively large GA emulsifiers, thereby

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inhibiting droplet coalescence inside the homogenizer.

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Under neutral pH conditions, the ζ-potential values for the droplets in all three emulsions

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were strongly negative (Figure 1b), thereby creating a strong electrostatic repulsion between

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them.34 As a result, all three emulsions were relatively stable to aggregation after they were

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prepared.

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Effect of pH on emulsion stability

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The pH-stability of emulsions plays an important role in determining their application in

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many foods and beverages. Consequently, the influence of pH on the electrical properties and

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aggregation stability of the different emulsifier-coated oil droplets was studied. The mean

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particle diameter of QS- and GA-emulsions did not change appreciably from pH 2 to 8 (Figure

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2a), indicating they had relatively good pH-stability. Even so, there was a small increase in

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particle size for the QS-emulsions at the most acidic condition used (pH 2), which suggests that

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some particle aggregation occurred due to the reduction in droplet charge (Figure 2b).

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Consequently, the electrostatic repulsive forces acting amongst the QS-coated droplets was

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reduced, thereby leading to flocculation 29. Interestingly, the GA-emulsions did not exhibit any

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change in particle size across the full pH range used, even though the droplet charge also

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decreased notably under acidic conditions. This is because the droplets in GA-emulsions are

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mainly prevented from aggregating by steric forces, rather than electrostatic ones.36 Unlike the

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other two emulsions, the size of the particles in the WPI-emulsions was highly sensitive to pH,

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being relatively large at pH 5 but small at lower (pH 2~4) and higher (pH 6~8) values. This

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phenomenon is a result of changes in the electrostatic forces acting between the droplets as the

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pH was varied 9. At relatively low and high pH values, there is a high net surface potential

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associated with the droplets, leading to intense electrostatic repulsive forces. Conversely, around

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the isoelectric point of the adsorbed proteins, the net surface potential on the droplets is fairly

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low, thereby generating only a weak repulsion.

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After storage, the appearance of the emulsions was consistent with the results of the light

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scattering analysis. Briefly, no changes in appearance were observed in the QS- or GA-

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emulsions over the pH range used, whereas droplet creaming was observed at pH 5 in the WPI-

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emulsions (Figure 3). In particular, a droplet-enriched layer was seen at the top of the test tubes

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and a droplet-depleted layer was observed at the bottom.

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The QS- and GA-emulsions had fairly similar ζ-potential versus pH patterns, with the

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surface potential decreasing from highly negative at pH 8 to slightly negative at pH 2 (Figure

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2b). A fairly similar pH-dependence of the surface potential has previously been reported for

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these two emulsifiers.20, 37 The ζ-potential of these systems became less negative below 4, which

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is due to carboxylic acid protonation (-COOH) of the emulsifiers around their pKa values.20 QS

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and GA have been reported to have pKa values around pH 3.2 and 2.2, respectively.38-39 As

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mentioned earlier, the ζ-potential of the GA-coated lipid droplets was near zero at pH 2 but the

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droplets were stable to aggregation and gravitational separation (Figures 2a and 3). This is

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because the large GA molecules create a thick interfacial coating around the droplets.15-16 Under

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neutral conditions, the GA is highly charged and so the droplets are stabilized by a blend of steric

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and electrostatic repulsive forces. Under highly acidic conditions, the GA loses most of its

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charge but is still able to stabilize the droplets through a strong steric repulsion. This is not the

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case for the QS-coated droplets, because the interfacial layer is too thin to generate a strong long-

205

range steric repulsion.

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The ζ-potential of the WPI-emulsions went from strongly negative at neutral pH to strongly

207

positive at acidic pH, with a zero charge around pH 5 (Figure 2b). The instability of these

208

emulsions around pH 5 is because the protein layer is too thin to generate a strong steric

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repulsion and the electrostatic repulsion is not strong enough to outweigh the van der Waals and

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hydrophobic attractive forces. 40

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Impact of emulsifier type on the storage stability

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The impact of the three emulsifiers on the stability of the emulsions during storage at room

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temperature was also studied (pH 7.0). The particle size of the QS- and WPI-emulsions did not

214

exhibit any significant changes throughout 28-days storage (Figure 4a). Moreover, no creaming

215

or flocculation was observed by visual inspection (Figure S1) or microscopy analysis (data not

216

shown), respectively. The good storage stability of emulsions formed using these emulsifiers has

217

also been reported previously.19, 34 This phenomenon is mainly due to the relatively small

218

dimensions of the oil droplets present in these emulsions, which made them more stable to

219

aggregation and creaming.41 Moreover, the ζ-potential of both these emulsions remained fairly

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constant and highly negative throughout storage (Figure S2), suggesting there was little change

221

in interfacial composition during this period.

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Interestingly, the GA-emulsions exhibited a small but noticeable rise in particle size during

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storage (Figure 4a), even though the surface charge on the droplets did not change appreciably

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(Figure S2). Moreover, some creaming was observed in these emulsions after 7 days storage

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(Figure S1). Finally, confocal microscopy indicated that the extent of droplet aggregation in

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these emulsions increased progressively during storage (Figure 4c). The level of GA used in our

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study (1.5 wt.%) was relatively low and may therefore not have been sufficient to saturate the

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surfaces of the lipid droplets. As a result, some coalescence may have occurred when two

229

partially covered oil droplets collided. Alternatively, a single GA molecule may have desorbed

230

from the surface of one droplet and then reattached itself to the surface of a different droplet,

231

leading to bridging flocculation.42 A schematic representation of this process is shown in Figure

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4b.

233

In summary, the emulsions stabilized by the saponin and protein appeared to have good

234

storage stability, whereas those stabilized by the polysaccharide were prone to flocculation. In

235

practice, this problem would be overcome by increasing the GA level employed to produce the

236

original emulsions. However, in this study we wanted to compare emulsifiers at similar usage

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levels.

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Influence of emulsifier type on simulated gastrointestinal behavior

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A static three-stage GIT model was utilized to investigate the influence of emulsifier type on

240

the gastrointestinal behavior of the emulsions. Each emulsion was subjected to a simulated

241

human gut by exposing it sequentially to artificial oral, gastric, and small intestinal conditions.

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Alterations in the structural and physicochemical properties of the emulsions were determined

243

after being incubated in each GIT phase.

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Journal of Agricultural and Food Chemistry

Fresh emulsions

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Emulsions stabilized by the three emulsifiers were produced by microfluidization as

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described earlier. The QS- and WPI-emulsions contained smaller droplets than the GA-

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emulsions (Figure 5a). All three systems had particle size distributions (PSDs) that were

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monomodal (Figure S3) and contained oil droplets were uniformly distributed throughout them

249

(Figure 6). The fresh emulsions all had a relatively strong negative charge (Figure 5b),

250

accounting for their good stability. The origin of these differences was discussed in an earlier

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section and so will not be repeated here.

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Mouth stage

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After being incubated in simulated saliva, the mean particle size of any of the emulsions did

254

not change significantly as determined by static light scattering (Figure 5a). The PSDs of all the

255

emulsions remained monomodal and similar to those of the initial emulsions (Figure S3).

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Confocal microscopy analysis showed that widespread droplet aggregation did not occur under

257

artificial mouth conditions (Figure 6). Taken together, our data suggests that all emulsions

258

remained relatively resistant to flocculation and coalescence in the mouth, which is in agreement

259

with previous studies on emulsions stabilized by these emulsifiers.34, 43 Other studies, however,

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have reported that appreciable levels of bridging and/or depletion flocculation can be induced in

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artificial saliva for other types of emulsifiers.44-45 The relatively good stability of the emulsions

262

prepared in our study may have been due to various reasons. First, the electrostatic and steric

263

repulsive forces acting amongst the lipid droplets are sufficiently strong to inhibit their

264

aggregation. Second, the incubation time in the oral phase (2 min) used in our work was

265

relatively short compared to that used in many previous studies (10 min), so the mucin did not

266

have sufficient time to act on the lipid droplets.

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The electrical properties of the emulsifier-coated oil droplets were altered appreciably after

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encountering the simulated oral fluids (Figure 5b). The absolute value of the -potential

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decreased appreciably on the QS-coated droplets, but only slightly on the WPI- and GA-coated

270

droplets. This reduction in negative charge could have occurred because of ion binding or

271

electrostatic screening effects linked to the existence of mucin or inorganic salts in the artificial

272

saliva.45-46

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Gastric stage

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There were obvious alterations in the dimensions and charge of the particles within the

275

emulsions after they had been incubated in the artificial gastric fluids for 2h (Figure 5). The

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particle size of the GA-emulsions stayed fairly constant, that of the QS-emulsions increased

277

somewhat, and that of the WPI-emulsions increased appreciably (Figure 5a). Similarly, the PSD

278

of the GA-emulsions remained relatively unchanged, while those of the QS- and WPI-emulsions

279

shifted upward (Figure S3). These results indicate that the GA-coated droplets were highly

280

resistant to aggregation under simulated gastric conditions, the GS-coated droplets were

281

moderately resistant, and the WPI-coated droplets were strongly prone to aggregation. The light

282

scattering data were supported by the microscopy images, which indicated the QS- and GA-

283

emulsions had relatively good stability to droplet aggregation but the WPI-emulsions exhibited

284

severe droplet flocculation (Figure 6). Our results agree with prior work that has also shown

285

that QS-emulsions are relatively stable to aggregation in simulated stomach conditions, whereas

286

protein-stabilized emulsions are unstable.34, 43

287 288

The tendency for protein-coated droplets to aggregate under stomach conditions has been attributed to several mechanisms: (i) reduced electrostatic repulsion caused by pH changes; (ii)

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increased electrostatic screening caused by salts; (iii) proteolysis of absorbed proteins by pepsin;

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and, (iv) bridging flocculation caused by mucin.46-49

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After incubation within the gastric fluids, the ζ-potential values became slightly positive for

292

all the emulsions (Figure 5b). The strong acidity and high ionic strength of gastric fluids largely

293

account for this effect.34, 44 The low pH means the adsorbed emulsifiers should have a positive

294

or slightly negative charge (Figure 1b). The high salt concentration means the surface potential

295

will be reduced as a result of electrostatic screening. Moreover, the existence of anionic mucin

296

within the gastric fluids may have altered the surface potential by adsorbing to any positively

297

charged regions on the droplet surfaces, particularly for the protein-coated droplets.49 Moreover,

298

the partial digestion of the proteins adsorbed to the lipid droplet interfaces by pepsin may also

299

have altered the surface potential.6, 46, 50

300

Small intestine stage

301

After being incubated in the artificial small intestinal fluids, all of the digested emulsions

302

contained particles with fairly similar mean particle diameters and surface potentials (Figure 5)

303

as well as broad particle size distributions (Figure S2). Moreover, the confocal microscopy

304

images showed that they all contained relatively large lipid-rich particles (Figure 6). After

305

digestion, there are various kinds of molecular species in the gastrointestinal fluids that can

306

assemble into numerous types of colloidal particles. For instance, triacylglycerols, free fatty

307

acids, monoglycerides, bile salts, phospholipids, peptides, and undigested emulsifiers may be

308

present as lipid droplets, micelles, vesicles, liquid crystals, or calcium soaps.34, 44 This diversity

309

of particles accounts for the wide range of dimensions seen in the PSDs (Figure S2).

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Influence of emulsifier on lipid digestion We hypothesize that the lipid digestion rate would impact the speed of mixed micelle

312

generation, as well as the kinetics of vitamin release from the lipid droplets, which would be

313

expected to affect the final vitamin bioaccessibility. The influence of emulsifier type on lipid

314

digestion was thus determined using the pH-stat method to establish FFA-time profiles for the

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various emulsions (Figure 7). Generally, FFA generation occurred rapidly throughout the first

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10 minutes of lipolysis but then more gradually at later times. The rapid generation of FFAs in

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the initial period suggests that lipase quickly attached itself to the surfaces of the oil droplets and

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then hydrolyzed the triacylglycerols.51 The slower release of FFAs at longer times was probably

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because most of the triacylglycerols had already been hydrolyzed thereby making it harder for

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the lipase to access the few remaining undigested triacylglycerols inside the droplet interiors.

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The WPI- and GA-emulsions released very similar levels of FFAs throughout the entire

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digestion process. But there were some distinct differences in the FFA-time relationships for the

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QS-emulsions. First, a short-lag phase was observed before rapid lipid digestion occurred.

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Second, the rate of lipid digestion in the initial stages was slower than for the WPI- and GA-

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emulsions. Third, the level of FFAs generated by the end of the small intestinal stage was lower

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than for the other two emulsions. This data suggests that the saponins slightly suppressed lipid

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digestion. The hydrolysis of lipids involves a series of physicochemical processes that mainly

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occur at the oil droplet surfaces and so interfacial phenomena are critical.22 The lag-phase,

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slower digestion rate, and lower digestion extent for the QS-emulsions may therefore be

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associated with the behavior of the saponins at the oil/water interface. As mentioned earlier, QS

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reduces the interfacial tension more than WPI or GA, indicating that it is more surface-active. It

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is therefore possible that the saponins formed a strong interfacial film at the surfaces of the oil

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droplets that inhibited the adsorption of the bile salts and/or lipase, thereby suppressing

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digestion. Another possible reason is that the saponins formed electrostatic complexes with the

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calcium ions in the gastrointestinal fluids. The highly anionic saponins could have bound

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strongly to the cationic calcium ions thereby reducing the level of calcium ions available to

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precipitate and remove long-chain FFAs from the lipid droplet surfaces.44

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The faster digestion for the WPI- and GA-coated droplets is probably because the interfacial

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layers formed by these emulsifiers were less effective at preventing the attachment of bile salts

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and lipase to the surfaces of the oil droplets. The whey proteins are likely to have been partially

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digested by pepsin in the stomach, thereby forming a relatively weak interfacial layer. The GA

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molecules have a relatively low surface activity and would therefore be displaced more easily

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from the surfaces of the oil droplets. Previous studies have also demonstrated that lipid digestion

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depends on the characteristics of the emulsifiers used to coat the oil droplets.52-53

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Influence of emulsifier on vitamin bioaccessibility

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Finally, the influence of emulsifier type on the bioaccessibility of the encapsulated vitamin

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was evaluated after the emulsions were incubated in simulated small intestine conditions. The

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WPI-emulsions led to the highest bioaccessibility, whereas the QS- and GA-emulsions had fairly

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similar bioaccessibilities (Figure 8). Nevertheless, in all cases, the measured bioaccessibility

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was relatively high (65-85%).

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In general, the bioaccessibility of lipophilic bioactives depends on the fraction of digested

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triacylglycerols and the absolute amount of FFAs generated during lipid digestion. Lipophilic

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bioactives are typically located in the interior of the lipid droplets and so the surrounding

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triacylglycerols have to be digested before they can be released. Moreover, once they are

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released from the droplets they have to be incorporated into the hydrophobic domains within the

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mixed micelles, otherwise they will simply precipitate or form a separate layer. In our study, we

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did not find a strong correlation between the final level of lipid digestion and vitamin

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bioaccessibility. The highest level of FFAs generated was for both the WPI- and the GA-

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emulsions (Figure 7), but the highest bioaccessibility was only for the WPI-emulsions (Figure

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8).

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This phenomenon cannot be simply explained, as lipid digestion and bioactive solubilization

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are complex processes influenced by multiple factors. It is possible that the lipids were digested

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in the GA-emulsions, but for some reason, the vitamin was not fully solubilized in the mixed

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micelles or the mixed micelles that were formed precipitate. It is known that polysaccharides

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can also interact with various digestive components, including lipase, bile acids, and calcium

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ions.54 Therefore, the GA may interact with bile salts and/or FFAs, thereby reducing the

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incorporation of vitamin E into the mixed micelles or causing the vitamin-enriched mixed

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micelles to precipitate and so not be measured. These results suggest that the bioaccessibility of

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lipophilic bioactive compounds is not only influenced by the final amount of lipid digestion

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products generated but also by other factors. Clearly, further studies are required to better

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understand this complex phenomenon.

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It is interesting to compare the in vitro bioaccessibility of vitamin E determined in this study

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(65-85%) to its reported in vivo bioavailability. A recent feeding study using rats reported that

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the relative bioavailability of vitamin E (tocopherol) delivered in palm oil-in-water emulsions

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was around 82.5% 55, which is in reasonable agreement with our bioaccessibility data.

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In summary, emulsion-based delivery systems were fabricated from two plant-derived

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emulsifiers (quillaja saponin and gum arabic) and one animal-derived emulsifier (whey protein

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isolate). QS and WPI were more effective at producing emulsions containing small oil droplets,

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presumably due to the relatively high surface activity and rapid adsorption kinetics of these

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emulsifiers. The good storage stability of these emulsions was linked to their relatively small

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droplet size and the ability of the emulsifiers to generate strong repulsive interactions between

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the droplets: mainly electrostatic for QS and WPI, and steric for GA. All the emulsions were

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relatively stable to flocculation from pH 2 to 8, with the exception of the WPI-emulsions at pH 5,

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which was linked to the reduction in electrostatic repulsion around the isoelectric point of the

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protein. The WPI- and GA-emulsions had fairly similar digestion profiles, whereas the QS-

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emulsions were digested more slowly. This phenomenon was linked to the ability of the

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saponins to adsorb strongly to the lipid droplet surfaces, thereby inhibiting the attachment of the

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bile salts and/or lipase. Interestingly, we did not find a strong correlation between the final level

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of lipid digestion that had occurred and the vitamin bioaccessibility. The WPI-emulsions gave a

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significantly higher bioaccessibility of the vitamin E than the other two emulsions. Our results

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show that emulsifier type impacts the gastrointestinal fate of emulsions, which may have

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important consequences for designing more effective vitamin-enriched delivery systems.

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However, further studies are still needed to verify that the results obtained using in vitro

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screening methods are translatable to industrial practice. In particular, the vitamin-fortified

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delivery systems will have to be robust enough to survive during food production, storage, and

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utilization and animal/human feeding studies are required to confirm the results of the simulated

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GIT studies.

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Acknowledgments

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Shanshan Lv would like to thank the Chinese Scholarship Council for support.

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Supporting Information: Additional Figures

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Notes: The authors declare no competing financial interest. 19 ACS Paragon Plus Environment

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52. Verkempinck, S. H. E.; Salvia-Trujillo, L.; Moens, L. G.; Charleer, L.; Van Loey, A. M.; Hendrickx, M. E.; Grauwet, T., Emulsion stability during gastrointestinal conditions effects lipid digestion kinetics. Food Chemistry 2018, 246, 179-191. 53. Speranza, A.; Corradini, M. G.; Hartman, T. G.; Ribnicky, D.; Oren, A.; Rogers, M. A., Influence of Emulsifier Structure on Lipid Bioaccessibility in Oil–Water Nanoemulsions. Journal of Agricultural and Food Chemistry 2013, 61 (26), 6505-6515. 54. Verkempinck, S. H. E.; Salvia-Trujillo, L.; Denis, S.; Van Loey, A. M.; Hendrickx, M. E.; Grauwet, T., Pectin influences the kinetics of in vitro lipid digestion in oil-in-water emulsions. Food Chemistry 2018, 262, 150-161. 55. Harlen, W. C.; Muchtadi, T.; Palupi, N. S., Bioavailability of alpha-Tocopherol in Palm Oil Emulsion Drink on Rats (Rattus norvegicus) Blood Plasma and Liver. Agritech-Jurnal Teknologi Pertanian 2017, 37 (3), 352-361.

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Funding

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This material was partly based upon work supported by the National Institute of Food and

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Agriculture, USDA, Massachusetts Agricultural Experiment Station (MAS00491) and USDA,

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AFRI Grants (2016-25147, and 2016-08782).

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Figure Captions Figure 1. Impact of emulsifier type on the (a) mean particle diameter (d3,2) and (b) surface potential of oil-in-water emulsions (pH 7.0). Samples denoted with low case letter (a, b, c) were significantly different (p < 0.05). All emulsions were produced by microfluidization at 12,000 psi for 3 passes. Figure 2. Impact of pH on the (a) mean particle diameter (d3,2), and (b) particle charge of emulsions stabilized by different emulsifiers. Figure 3. Impact of pH on the visual appearances of emulsions stabilized by different emulsifiers: (a) QS, (b) WPI, and (d) GA. A schematic representation of the aggregation state of the droplets in the WPI-emulsions at different pH values is shown in (c). Figure 4. Storage stability of emulsions (pH 7): (a) particle size versus time; (b) schematic representation of changes in flocculation during storage; (c) confocal fluorescence microscopy images of GA-emulsions during storage. Figure 5. Impact of emulsifier type on (a) mean particle diameter (d3,2), (b) particle charge of emulsions after exposure to different simulated GIT stages. Samples denoted with different capital letters (A, B, C, D) were significantly different (p < 0.05) when compared with different digestion phase; samples denoted with different low case letters (a, b, c) were significantly different (p < 0.05) when compared at the same digestion phase. Figure 6. Microstructure of emulsions stabilized by different emulsifiers after digested in different simulated GIT stages (a) QS; (b) WPI; (c) GA. Figure 7. Free fatty acids (FFAs) released from emulsions stabilized by different emulsifiers during in vitro small intestinal digestion.

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Figure 8. Impact of emulsifier type on the bioaccessibility of vitamin E determined by measuring the fraction of the vitamin solubilized in the micelle phase after digestion. Samples denoted with low case letters (a, b) were significantly different (p < 0.05).

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