Vitamin E Encapsulation in Plant-Based Nanoemulsions Fabricated

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Food and Beverage Chemistry/Biochemistry

Vitamin E encapsulation in plant-based nanoemulsions fabricated using dual-channel microfluidization: Formation, stability and bioaccessibility Shanshan Lv, Jiyou Gu, Ruojie Zhang, Yanhua Zhang, Haiyan Tan, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 21 Sep 2018 Downloaded from http://pubs.acs.org on September 21, 2018

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

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Journal of Agricultural and Food Chemistry

ACS Paragon Plus Environment


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Vitamin E encapsulation in plant-based nanoemulsions fabricated using dual-

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channel microfluidization: Formation, stability and bioaccessibility

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Shanshan Lv1,2, Jiyou Gu1, Ruojie Zhang2, Yanhua Zhang*1, Haiyan Tan1, David Julian

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McClements*2

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1

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College of Material Science and Engineering, Northeast Forestry University, Harbin, 150040,

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P.R. China

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2

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

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

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Corresponding authors: Yanhua Zhang; David Julian McClements

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Corresponding author emails: [email protected]; [email protected]

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Abstract In this study, vitamin E was encapsulated in oil-in-water nanoemulsions fabricated using a

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dual-channel microfluidizer. A long chain triacylglycerol (corn oil) was used as a carrier oil and

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a biosurfactant (quillaja saponin) was used as a natural emulsifier. The impact of vitamin-to-

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carrier oil ratio on the formation, storage stability, and bioaccessibility of the nanoemulsions was

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determined. The lipid droplet size formed during homogenization increased with increasing

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vitamin content, which was attributed to a large increase in lipid phase viscosity. The storage

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stability of the nanoemulsions decreased as the vitamin content increased because the larger lipid

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droplets creamed faster. The rate and extent of lipid hydrolysis in the small intestine decreased as

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the vitamin content increased, probably because the vitamin molecules inhibited the ability of

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lipase to reach the triacylglycerols inside the lipid droplets. Vitamin bioaccessibility decreased as

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the vitamin level in the lipid phase increased, which was attributed to the reduced level of mixed

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micelles available to solubilize the tocopherols. The optimized nanoemulsion-based delivery

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system led to a relatively high vitamin bioaccessibility (53.9%). This research provides valuable

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information for optimizing delivery systems to increase the bioaccessibility of oil-soluble

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vitamins.

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Keywords: emulsion; nanoemulsion; vitamin E; digestion; bioaccessibility

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35 36

Introduction Vitamin E is a term that actually refers to a family of lipophilic bioactive substances which includes

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α, β, γ, and δ forms of tocopherol and tocotrienol.1, 2 These substances are commonly incorporated into

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functional foods and beverages, nutritional supplements, and pharmaceutical preparations because of their

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benefits on human health and wellbeing.3, 4 α-tocopherol is reported to be the most bioactive form of this

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vitamin group and is therefore widely used in commercial products.1, 5, 6 It acts as an oil-soluble

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antioxidant that can inhibit lipid oxidation by scavenging free radicals.

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The addition of vitamin E to food products often confronts various challenges because of its poor

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water-solubility and strong susceptibility to chemical degradations, especially at elevated temperatures,

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oxygen levels, and light exposures.7-9 Furthermore, the lipophilic nature of vitamin E reduces its solubility

45

in the small intestinal fluids, which limits its bioaccessibility and bioavailability. An effective method to

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tackle these problems is to encapsulate α-tocopherol in nanoemulsion-based delivery systems. This type

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of colloidal delivery system can be specifically designed to enhance the inclusion of vitamin E into a wide

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range of commercial products in a form that is chemically stable and has high bioaccessibility.

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The utilization of oil-in-water nanoemulsions for the encapsulation and delivery of hydrophobic

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functional components in foods and pharmaceuticals is attracting growing interest.10-13 Nanoemulsions

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can be produced by many methods, including microfluidization, spontaneous emulsification, and phase

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inversion methods.14-16 Microfluidization is one of the most efficient methods because it can produce

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extremely fine droplets with a narrow particle size distribution.17 Recently, a new homogenization

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method, known as dual-channel microfluidization, has been introduced for efficiently producing

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nanoemulsions.18 This type of homogenizer can produce nanoemulsions directly from separate oil and

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water phases using a single pass, thereby reducing energy costs and production time. Based on these

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advantages, dual-channel microfluidization was used to produce the nanoemulsion-based delivery

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systems for Vitamin E in this study.

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On its own, vitamin E is a highly viscous oily material that is challenging to homogenize directly


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using dual-channel microfluidization. A carrier oil must therefore be mixed with the vitamin E prior to

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homogenization. In general, the type and level of carrier oil used play important roles in the successful

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formation of nanoemulsion delivery systems.19-21 Saberi et. al.20 reported that the lipid droplet size first

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decreased and then increased as the ratio of vitamin-to-carrier oil used to prepare the nanoemulsions

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increased. It has also been reported that bigger lipid droplets were created when a higher level of viscous

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oils were used,7, 19 while other researchers reported the opposite.14 These complex effects depend on the

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homogenization mechanism used, the oil phase viscosity,5, 19 and the interfacial tension.22

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Highly hydrophobic bioactive agents, like vitamin E, normally possess poor oral bioaccessibility and

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bioavailability due to their low water-solubility. However, its bioaccessibility can be increased

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appreciably by co-ingesting it with a digestible lipid that forms mixed micelles in the gastrointestinal

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fluids.6, 23, 24 Previous research has shown that a variety of factors impact the bioaccessibility of

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hydrophobic bioactives, including lipid type,6, 24-27 lipid concentration,28, 29 and emulsifier type.30 These

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effects could be attributed to the digestion and absorption behavior of the lipid and bioactives. During

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digestion in the small intestinal phase, the triacylglycerols (TAGs) in the small oil droplets are hydrolyzed

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into free fatty acids (FFAs) and monoacylglycerols (MAGs) due to the action of lipase. Then, these FFAs

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and MAGs interact with phospholipids and bile salts in the gastrointestinal fluids to form mixed micelles.

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During digestion, lipophilic vitamins are liberated from inside the oil droplets and then solubilized inside

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the hydrophobic domains of the mixed micelles, which can then transverse the mucus layer and reach the

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epithelial intestinal cells to be absorbed and then packed into chylomicrons.6, 31 The co-ingestion of

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lipophilic vitamins with digestible lipids is therefore a critical factor impacting their bioaccessibility.24

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The solubilization capacity of the mixed micelles formed depends on types and levels of FFAs and MAGs

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released from the digested lipid phase. Typically, the bioaccessibility of hydrophobic molecules tends to

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increase as the initial lipid content increases, as well as when the dimensions of the non-polar core inside

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the mixed micelles increases.32, 33

84 85

In the current study, we fabricated vitamin E-loaded nanoemulsions using dual-channel microfluidization and investigated the impact of vitamin-to-carrier oil ratio and emulsifier level on their



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fabrication and stability. We also evaluated the influence of lipid phase composition on the

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gastrointestinal fate of the nanoemulsions and on vitamin E bioaccessibility using a static in vitro

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digestion model. This research provides valuable knowledge that could be used to improve the design and

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fabrication of vitamin E delivery systems for application in commercial 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 purchased from Fisher Scientific Inc. (Waltham, MA).

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Quillaja saponin, which is sold under the trade name Q-Naturale 200, was kindly donated by Ingredion

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Inc. (Westchester, IL). Corn oil produced by a commercial supplier (Mazola, ACH Food Company,

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Memphis, TN) was used as the carrier oil to formulate the nanoemulsions. Gastrointestinal components,

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including mucin (from porcine stomach), pepsin (from porcine gastric mucosa), lipase (from porcine

97

pancreas), and bile extract (porcine), were all purchased from the Sigma-Aldrich Chemical Co. (St. Louis,

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MO). The other chemical reagents utilized in this study were of analytical grade and mainly purchased

99

from Sigma-Aldrich (St. Louis, MO).

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Preparation of Vitamin-loaded Nanoemulsions Aqueous emulsifier phases were made by dispersing quillaja saponin into phosphate buffer (5 mM,

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pH 7.0) to obtain a range of final emulsifier levels (0.5% - 3.0% w/w). Lipid phases with different

103

compositions were made by mixing vitamin E and corn oil in different ratios. These mixtures were then

104

stirred at room temperature for 120 minutes to fully dissolve the two components. Nanoemulsions were

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not produced using 100% vitamin E because it was too viscous to pass through the microfluidizer. In

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particular, it tended to trap air bubbles inside that caused extensive foaming and poor homogenization

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performance.

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Nanoemulsions were created using a dual-channel microfluidizer (Microfluidics PureNano, Newton,

109

MA, USA) operating at a pressure of 14,000 psi, as described previously 18. The final oil level in the

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nanoemulsions was fixed at 10% (w/w) by controlling the flow rate of the lipid and aqueous phases, as


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described previously.18

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Storage stability vitamin-loaded nanoemulsions

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Newly created nanoemulsions were poured into glass test tubes and then stored in the dark at room

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temperature for 4 weeks. Characteristics of stored samples were tested periodically as described later.

115

In vitro digestion of vitamin-loaded nanoemulsions

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The nanoemulsions were passed through a three-stage simulated gastrointestinal tract (GIT)

117

consisting of mouth, stomach, and small intestinal phases, to evaluate the impact of lipid phase

118

composition on their behavior. The details of this method have been described in our previous

119

publications6. Briefly, the samples were sequentially passed through a mouth phase (salts, mucin, pH 6.8,

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2 min), a stomach phase (salts, pepsin, pH 2.5, 2 hours), and a small intestine phase (salts, bile extract,

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lipase, pH 7.0, 2 hours). The digestion of the lipids in the small intestine was monitored by measuring the

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free fatty acids released over time using a pH-stat method. The control used in these experiments was a

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solution containing quillaja saponin (but no oil) that was analyzed under the same conditions. The results

124

for the control were subtracted from that of the samples.

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Particle characterization

126

Particle dimensions were determined using laser diffraction (Mastersizer 3000, Malvern Instruments

127

Ltd., Malvern, Worcestershire, UK), while particle charge (ζ-potential) was assessed using electrophoresis

128

(Zetasizer Nano ZA series, Malvern Instruments Ltd. Worcestershire, UK). Prior to analysis, the samples

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collected from the initial, stomach, and small intestine phases were diluted with phosphate buffer solution

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(5 mM, pH 7.0), while the samples collected from the stomach phase were diluted with acidified double

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distilled water (pH 2.5) to avoid multiple scattering effects.

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Microstructure analysis

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Microstructures were acquired using a confocal fluorescence microscope with a 40× objective lens

134

attached (Nikon D-Eclipse C1 80i, Nikon, Melville, NY, USA). Lipids were stained with Nile Red (1

135

mg/mL ethanol) prior to observation and wavelengths of 543 and 605 nm were used to stimulate



136

excitation and emission of this dye.

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Bioaccessibility of vitamin E

138

After passing through the whole GIT model, the raw digesta resulting from the small intestinal phase

139

was centrifuged (Thermo Scientific, Waltham, MA) at 18,000 rpm and 4 °C for 50 min. The resulting

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supernatant was considered as the “micelle” phase in which the vitamin E was solubilized. The digesta

141

and micelle phase were extracted three times with hexane/ethanol (1/1, v/v) to obtain the vitamin E. Then,

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3 mL of the same solvent was added to 3 mL of samples and vortex-mixed vigorously, then the mixture

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was centrifuged for 2 min at 4000 rpm (Sorvall ST8, Thermo Fisher Scientific, Inc.). The supernatant

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layer was transferred into another tube for further extraction. The final supernatant was collected and

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dried under nitrogen to obtain vitamin E. Afterwards, the dried samples were dissolved in methanol and

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filtered through a filter membrane (0.45 µm) for HPLC analysis (Agilent 1100 series, Agilent

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Technologies, USA). A C18 column (250 × 4.6 mm, 5 µm, Beckman Coulter) was used for the

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chromatography analysis. Separation was carried out with an isocratic elution (methanol / double distilled

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water; 95/5 (v/v)) using a fluid flow rate of 1.0 ml/min, a column temperature of 30 °C and an injection

150

volume of 20 µL. A wavelength of 295 nm was used for the UV detector. Vitamin bioaccessibility was

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calculated using the expression:

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Bioaccessibility(%) =

𝐶1234554 × 100 𝐶627489:

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Here, Cmicelle is the vitamin E concentration in the micelle fraction and CDigesta is the total vitamin E

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concentration in the digesta collected at the end of the small intestine phase.

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Statistical analysis

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All measurements were carried out on two or more samples and the data obtained is expressed as

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means and standard deviations. ANOVA analysis was applied to determine if the data was significantly

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different (p < 0.05).


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Result and Discussion

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Impact of VE-to-carrier oil ratio on formation of emulsions

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The influence of vitamin E-to-carrier oil ratio on the formation of emulsions fabricated using the

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dual-channel microfluidizer was evaluated. A lipid phase containing pure vitamin E was not used because

163

it was so viscous that it could not be pumped through the microfluidizer chamber. The mean particle

164

diameter of the emulsions increased slightly when the vitamin E level in the lipid phase was raised from 0

165

to 60%, followed by a steep increase when it was increased from 60% to 80% (Figure 1). The form of the

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particle size distributions (PSDs) also depended on the composition of the oil phase. In particular, the

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PSDs became broader as the vitamin E concentration increased. This effect was mainly attributed to the

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increase in lipid phase viscosity as the fraction of vitamin E it contained increased.5, 19-21 Lipid phase

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viscosity is known to determine the disruption of oil droplets inside high-pressure homogenizers – since it

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takes longer for the oil droplets to become deformed and disrupted as the disperse phase viscosity rises.21

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Consequently, droplets containing high viscosity oils may pass through the disruption zone inside the

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microfluidizer before they can breakup.

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174 175

Figure 1. Influence of oil phase composition (wt.% of Vitamin E in VE/corn oil mixtures) on (a) mean

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particle diameter; and (b) particle size distribution of emulsion (1.0 wt.% quillaja saponin)

177



178

The ζ-potential values were fairly similar for emulsions with different vitamin E levels in the lipid

179

phase, ranging from around -63 to -67 mV (Figure 2). This suggests that the electrical characteristics of

180

the lipid droplets were primarily a result of the emulsifier coating. The relatively high anionic nature of

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the lipid droplets is mainly due to the presence of anionic groups on the biosurfactant used. Quillaja

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saponin molecules have carboxyl functional groups that are negatively charged over the pH range in most

183

foods. The highly charged lipid droplets in the initial emulsions are prevented from aggregating due to the

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strong electrostatic repulsion that acts between them.

185 186

Figure 2. ζ-Potential of nanoemulsions with different vitamin E concentrations in oil phase

187 188

Impact of emulsifier concentration on emulsion formation

189

A series of emulsions was prepared containing relatively high levels of vitamin E (60% or 80%) in

190

the lipid phase, as a high vitamin loading capacity is often required in commercial products. The impact

191

of emulsifier level on the particle dimensions in the emulsions formed was then measured (Figure 3). The

192

mean particle diameter (d4,3) decreased steeply when the emulsifier level was raised from 0.5% to 1.5%.

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This trend is the result of two main factors: (i) a faster interfacial tension reduction promoting more

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extensive breakup of the droplets; (ii) a faster increase in surface coverage opposing droplet

195

coalescence.22, 35, 36 Above 1.5% emulsifier, the mean particle diameter remained fairly constant because


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all the droplet surfaces were saturated and so emulsifier concentration no longer influenced droplet

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breakup or coalescence. At saturation emulsifier levels, the generation of small droplets is regulated by

198

the efficiency of droplet disruption inside the homogenizer instead of the emulsifier level22. As expected,

199

more smaller droplets were observed in the PSDs as the emulsifier concentration increased.

200

201 202

Figure 3. Influence of emulsifier concentration on main particle diameter (a) 60% VE; (c) 80% VE; and

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particle size distribution (b) 60% VE; (d) 80% VE. The PSD data has been shifted up the y-axis to

204

facilitate sample comparisons.

205 206 207

The magnitude of the negative charge on the lipid droplets increased as the emulsifier concentration was increased (Figure S1), which suggests that there was a higher level of the anionic quillaja saponin



208

molecules adsorbed at the surfaces of the lipid droplets. However, in all cases, the lipid droplet surface

209

potential was strongly negative, and should have been sufficient to prevent droplet aggregation in the

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initial emulsions.

211

Storage stability of emulsions

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Impact of VE-to-carrier oil ratio on storage stability of emulsions

213

The storage stability of emulsion-based products determines the shelf life of commercial foods and

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beverages. Emulsion breakdown during storage is often the result of physical instability mechanisms,

215

including coalescence, flocculation, or creaming.38 The influence of lipid phase composition on the

216

physical stability of vitamin E-enriched emulsions was therefore followed when they were stored at room

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temperature for 4 weeks. The mean droplet diameter remained fairly constant during storage, suggesting

218

that had good stability to flocculation and coalescence (Figure 4a). Similarly, the ζ-potential of all the

219

emulsions did not change appreciably (Figure 4b). Additionally, the emulsions exhibited good storage

220

stability, with no evidence of aggregation, creaming, or oiling off seen by eye (Figure S2). The ability of

221

the emulsions to resist creaming is because the lipid droplets were initially small and also stable to

222

aggregation throughout storage.39, 40 Overall, the emulsions containing both vitamin E levels displayed

223

excellent stability when stored at room temperature. The only exception was for the emulsion containing

224

80% vitamin E after 28 days storage, when a thin oil layer was observed at the top, indicating that some

225

phase separation occurred. This phenomenon may have been because these emulsions contained a fraction

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of relatively big droplets, which would have been more prone to coalescence.

227 228


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Figure 4. The influence of storage time on (a) mean droplet diameter and (b) ζ-potential of emulsions

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with different vitamin E concentrations in the oil phase. The nanoemulsions were formulated with 1.5

232

wt.% quillaja saponin in the aqueous phase.

233 234

Impact of emulsifier level on the storage stability of emulsions

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Emulsifier concentration affects interfacial packing and charge thereby influencing emulsion stability

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to droplet aggregation during storage. Hence, it is useful to investigate the influence of emulsifier level on

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the storage stability of the emulsions. Again, only emulsions containing 60% or 80% vitamin E within the

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lipid phase were selected for these studies, since for commercial food applications it is often necessary to

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utilize a high vitamin loading capacity. The particle size and charge of emulsions loaded with 60% or

240

80% vitamin E in the lipid phase were therefore measured at different storage times (Figure 5). The mean

241

droplet diameter of both emulsions increased during storage at the lowest emulsifier concentration

242

(0.5%), while it remained almost constant over the 28 days of storage at the higher emulsifier

243

concentrations (1.0% - 3.0%). These results indicated that raising the emulsifier level improved the

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storage stability of systems with high vitamin E levels. This effect can be attributed to the fact that the

245

droplet surfaces were saturated with emulsifier, the droplets have a high surface potential, and the lipid

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droplets were small. Consequently, there were strong electrostatic repulsive forces acting between them

247

that inhibited their aggregation and their small size would have inhibited their creaming.



248

249 250

Figure 5. Influence of storage time on the mean droplet diameter of emulsions containing (a) 60% VE;

251

(b) 80% VE and ζ-potential values of emulsions containing (c) 60% VE; (d) 80% VE

252 253

For the initial emulsions, the negative charge on the lipid droplets increased as the emulsifier level

254

used to create them was increased (Figures 5c and d), which would partially account for their increased

255

aggregation stability. The magnitude of the negative charge on the lipid droplets was reduced after

256

storage, especially for the emulsions containing 80% vitamin E after 28 days. This result suggests that

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there was probably an alteration in the interfacial composition and/or structure of the lipid droplets over

258

time.

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During storage, no evidence of phase separation was visible in most of the emulsions, except for the


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ones at the lowest emulsifier concentration (0.5%) (Figure S3). Immediately after preparation, the

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emulsions all had a uniform white appearance. For all the emulsions containing 60% vitamin E, their

262

appearance remained white and homogeneous after storage for 7 days. However, the emulsion stabilized

263

by 0.5% emulsifier had a thin oil layer at the top after prolonged storage (28 days). For the emulsions

264

containing 80% vitamin E, a thin oil layer was observed at the top of the emulsion at the lowest emulsifier

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concentration (0.5%) at 7 days storage, which became more apparent at 28 days. After this storage time,

266

thin oil layers were also observed at intermediate emulsifier levels (1.0% - 2.0%) but not at the higher

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ones (2.5% - 3.0%). In summary, the emulsions containing lower vitamin E levels and higher emulsifier

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levels were the most stable during storage. The relatively good stability of these emulsions during storage

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may be due to their small droplet sizes 39, 40 and Brownian motion effects.41 Smaller oil droplets have a

270

slower rate of gravitational separation and a greater tendency to be randomly dispersed due to Brownian

271

motion effects.

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Overall, our results indicate that the emulsions were not stable at low emulsifier concentrations

273

(0.5%). The main reason for this instability was that there was not enough emulsifier available to coat all

274

of the lipid droplet surfaces, resulting in incomplete coverage during homogenization.42, 43 This meant that

275

relatively large droplets were produced at his low emulsifier level. According to Freitas et. al.,44

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emulsions become highly unstable when the droplet diameter exceeds about 1µm. From Figure 5a and

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5b, the mean droplet diameters of the emulsions at 0.5% emulsifier were appreciably higher than 1µm,

278

these emulsions were therefore particularly unstable during storage. Thus, plant-based emulsions that are

279

stable at ambient temperature can be formulated provided a sufficiently high level of quillaja saponin is

280

used.

281

Impact of oil phase composition on gastrointestinal fate of VE-loaded nanoemulsions

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Emulsions consisted of different compositions (80% corn oil + 20% VE; 60% corn oil + 40% VE,

283

40% corn oil + 60% VE, and 20% corn oil + 80% VE) stabilized by 1.5% (w/w) quillaja saponin were

284

passed sequentially through the different stages of the simulated GIT. The lipid droplet properties and



285

emulsion microstructure were then characterized after at each stage (Figures 6 to 9).

286

287 288

Figure 6. Mean droplet diameter (d4,3) of vitamin E encapsulated emulsions after exposure to different

289

regions of the simulated GIT model. It should be noted that light scattering data should be treated with

290

caution since samples are diluted prior to analysis.

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Figure 7. Particle size distributions of vitamin E encapsulated emulsions after exposure to different

294

regions of the simulated GIT (a) 20% VE; (b) 40% VE; (c) 60% VE; (d) 80% VE. The PSD data has been

295

shifted up the y-axis to facilitate comparison of different samples.

296 297

Initial phase: As discussed earlier, the d4,3 values of all the initial emulsions increased slightly (from

298

0.62 to 0.97 µm) as the level of vitamin E in the oil phase increased (from 20 to 80%), while the PSD

299

changed from monomodal to bimodal, which was probably due to an increase in lipid phase viscosity.6

300

Confocal and optical microscopy measurements showed that small lipid droplets were uniformly

301

distributed within the initial emulsion samples (Figure 9 and Figure S4). The decreased intensity of the

302

fluorescence signals for the initial emulsions with increasing carrier oil level was because the Nile red dye



303

was originally dispersed in the corn oil. As the vitamin E concentration increased, the number of large oil

304

droplets in the images increased, especially for the 80% vitamin E encapsulated emulsion, which accounts

305

for the bimodal PSDs observed for these systems.

306

Mouth stage: At the end of the oral stage, the d43 values and PSDs of all the emulsions remained

307

fairly similar to those of the original emulsions (Figures 6 and 7). This result suggests that the saponin-

308

coated lipid droplets were relatively stable to aggregation under these conditions due to the relatively

309

strong electrostatic repulsion generated by their negative surface charge. Good stability of saponin-coated

310

oil droplets under simulated oral conditions has also been reported previously.6, 26 The microstructural

311

images (confocal and optical) of the emulsions did not show any noticeable flocculation of the emulsions

312

after the mouth stage (Figure 9 and Figure S4), consistent with the laser diffraction analysis. The

313

magnitude of ζ-potential of all the lipid droplets showed an appreciable decrease after being exposed to

314

the mouth stage (Figure 8). This reduction is probably due to the mucin and mineral ions in the simulated

315

saliva, since these charged entities may have caused electrostatic screening.45

316

Stomach stage: At the end of the stomach stage, the d43 values and PSDs of the emulsions remained

317

relatively small. However, confocal and optical microscopy showed there was some lipid droplet

318

aggregation in the stomach stage (Figure 9 and Figure S4). A potential explanation for this discrepancy is

319

the sample preparation method used for light scattering measurements.23 The emulsions are diluted

320

around 100-fold for the light scattering measurements, which can cause disruption of any weak flocs. The

321

observed aggregation of the emulsions in the stomach may have been caused by either depletion or

322

bridging flocculation caused by mucin from the mouth stage and pepsin from the stomach stage.34, 46

323

Depletion flocculation is caused by non-adsorbed polymers, whereas bridging flocculation is caused by

324

polymers adsorbed to the surfaces of multiple lipid droplets.24, 47 ζ-potential analysis indicated that the

325

strength of the negative charge on the lipid droplets decreased appreciably after the emulsions were

326

incubated within the simulated stomach phase (Figure 8). This decrease is probably the result of the low

327

pH and high ionic strength of the gastric environment, which is known to reduce the charge on adsorbed

328

ionic emulsifiers and cause electrostatic screening.


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Small intestine stage: At the end of the small intestine stage, the mean particle diameters, PSDs, and

330

microstructures of the samples exhibited appreciable changes compared to the stomach stage (Figures 6,

331

7, 9 and Figure S4). The mean particle diameter increased significantly, especially for the samples

332

containing high vitamin E concentrations, while all the samples had broad PSDs. The observed changes

333

may have occurred for numerous reasons, including alterations in the original colloidal particles (lipid

334

droplets) and formation of new colloidal particles (micelles, liposomes, and calcium salts).30, 48 All of

335

these factors could account for the large particle size and broader PSDs observed after the small intestine.

336

The charge on the particles were negative after exposure to the small intestine phase (Figure 8), which

337

can be attributed to the presence of various types of anionic substances, such as quillaja saponins, free

338

fatty acids, phospholipids, and bile salts.

339 340

Figure 8. ζ-potential of vitamin E encapsulated emulsions after exposure to different regions of a

341

simulated GIT.



342 343

Figure 9. Microstructure of vitamin E encapsulated emulsions after exposure to different regions of the

344

simulated GIT (scar bar: 10 µm)


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345


Influence of oil phase composition on lipid digestion

346 347

Figure 10 (a) Volume of NaOH (0.25 M) required to maintain a constant pH (7.00); (b) amount of fatty

348

acids released from emulsions as measured in pH-stat in vitro digestion model.

349 350

The influence of oil phase composition on lipid digestion was evaluated using an automatic titration

351

method. The volume of NaOH solution titrated into the system to keep the pH at 7.0 was recorded over

352

time (Figure 10a) and the percentage of FFAs released was calculated from this information (Figure

353

10b). As expected, the volume of NaOH solution needed to maintain neutral conditions was appreciably

354

different in emulsions containing different lipid phase compositions (Figure 10a). The volume of NaOH

355

consumed decreased with decreasing carrier oil (corn oil) concentration in the lipid phase because then

356

there were less triacylglycerol molecules available to hydrolyze. Only a small amount of NaOH was

357

consumed by the end of digestion for the control samples, which can be attributed to the hydrolysis of

358

other constituents present, such as phospholipids and emulsifier. For this reason, the volume of NaOH

359

consumed for the control samples was subtracted from that of the test samples to calculate the FFA

360

release from the emulsions. The FFAs released was expressed as the percentage of total digestible lipids

361

within each lipid phase i.e., only the corn oil.

362

It was noteworthy that the amount of NaOH consumed for the emulsion containing 80% vitamin E in



363

its lipid phase was very similar to that for the control samples (Figure 10a). The most probable reason for

364

this phenomenon is that the high level of vitamin E inside the droplets prevented the lipase from coming

365

into contact with the triacylglycerol molecules. Moreover, the high viscosity of the lipid phase may have

366

inhibited the movement of the triacylglycerol molecules through the lipid droplets. For this reason, during

367

the first 65 min of digestion, the consumed NaOH volume of the samples containing 80% vitamin E in the

368

lipid phase was almost the same as that for the control sample. After 65 min, the volume of NaOH

369

required increased somewhat above the control sample, because the lipase and triacylglycerols were

370

eventually able to come into contact at the surfaces of the lipid droplets.

371

The lipid digestion profiles of emulsions containing different oil compositions were appreciably

372

different. Most of the emulsions followed a fairly similar trend except for the ones containing 80%

373

vitamin E in the lipid phase. The percentage of FFAs released increased quickly throughout the initial

374

stages of digestion, followed by a relatively slow release until a fairly steady final value was attained

375

(Figure 10b). The lipid digestion rate and extent of the emulsions decreased with increasing vitamin E

376

level, which may have occurred for various reasons. First, there was a decrease in the specific surface area

377

of the lipid phase entering the small intestine with increasing vitamin levels i.e., larger droplets.49 Second,

378

the fraction of triacylglycerol molecules actually present at the oil-water interface would have decreased

379

as the vitamin level increased.

380

Influence of oil phase composition on vitamin E bioaccessibility

381

The impact of carrier oil composition on vitamin E bioaccessibility was determined after the small

382

intestine stage (Figure 11). The vitamin bioaccessibility decreased from 53.9% to 12.6% as the vitamin E

383

level in the oil phase increased, which may be due to changes in the solubilization capacity of the mixed

384

micelle phase. Lipophilic bioactives, such as vitamin E, are normally incorporated inside the hydrophobic

385

regions in mixed micelles in the intestinal fluids and then carried through the mucus layer to the

386

epithelium cells 49. Mixed micelles are mainly comprised of phospholipids and bile acids from

387

endogenous intestinal secretions, in addition to monoacylglycerols and free fatty acids produced by


Page 22 of 31

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388

digestion of exogeneous lipids.24 The solubilization of lipophilic components typically increases as the

389

mixed micelles level in the intestinal fluids increases.50 Our FFA release results suggest that an increased

390

level of mixed micelles would be formed when the vitamin content in the oil droplets decreased (more

391

corn oil), and so there should be an increased ability to solubilize the vitamin. Hence, the vitamin

392

bioaccessibility was much higher when the corn oil content in the oil phase was 80% (20% vitamin E)

393

than for the other three delivery systems. In addition, with the decreasing of the lipid content in the

394

emulsions, the lipid digestion extent decreased and led to a decrease in the amount of vitamin E released

395

from the lipid droplets during digestion. Consequently, some of the vitamin E might have remained

396

trapped within the undigested lipid phase, resulting in less available to be incorporated into the mixed

397

micelles. This phenomenon should also account for the decrease of the bioaccessibility as the lipid

398

content decreased.

399

400 401

Figure 11. Influence of oil phase composition on the bioaccessibility of vitamin E.

402 403

The results of this study may have practical implications for the rational design and development of

404

delivery systems for lipophilic vitamins. The vitamin-to-carrier oil ratio has to be optimized to obtain a

405

high initial loading but also a high bioaccessibility. For instance, if the initial vitamin doses in the



406

different emulsions are kept the same, the actual amount of vitamin available will be higher for the 40%

407

and 60% vitamin E-loaded delivery system. However, there will be a significant fraction of the vitamin E

408

that is not bioaccessible in these systems, which will lead to some waste of the vitamin. To ensure a

409

higher fraction of vitamin E is actually absorbed after ingestion, it should be encapsulated within delivery

410

systems containing 20% vitamin E and 80% carrier oil.

411

Influence of storage on the bioaccessibility of vitamin E

412

Finally, we evaluated the influence of storage at 4 °C on the bioaccessibility of vitamin E in the

413

delivery systems. As discussed in the previous section, the emulsion containing 20% vitamin E had the

414

highest bioaccessibility and so this system was selected for study. Before digestion, the particle size and

415

ζ-potential value were determined to evaluate the emulsion stability during storage under refrigeration

416

conditions, i.e., 4 °C (Figure S5). The results showed that the emulsions were stable over the 12 weeks

417

study period with no noticeable increases in particle size occurring. The vitamin bioaccessibility was

418

determined by measuring the levels of vitamin E in the micelle phase and digest using HPLC (Figure 12).

419

There was a decrease in the vitamin level in both the micelle phase and digest after storage, with the

420

vitamin E concentration decreasing from 315 ppm to 266 ppm in the micelle phase and from 584 ppm to

421

504 ppm in the digest. This suggests that about 86% of the vitamin was retained in the system after

422

storage for 12 weeks, indicating that the vitamin had fairly good stability to degradation. As expected, the

423

bioaccessibility of the vitamin E remained almost the same after storage, because it is simply the ratio

424

between the vitamin in the mixed micelle and digest. The bioaccessibility changed from 54% initially to

425

53% after storage for 12 weeks.

426


Page 24 of 31

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427 428

Figure 12. Influence of storage at 4 °C on the concentration of α-Tocopherol within the micelle phase and

429

the total digesta, and the influence on the bioaccessibility of vitamin E in delivery system (20% VE)

430 431

In summary, this study has shown that plant-based vitamin E fortified nanoemulsions can be

432

fabricated using a dual-channel microfluidizer. The formation, stability, and vitamin bioaccessibility of

433

these nanoemulsions depended on lipid phase composition, which therefore had to be optimized. The rate

434

and extent of lipid digestion decreased as the vitamin E level in the oil phase was increased,

435

which was attributed to the reduced opportunity for lipase to interact with the digestible oil

436

inside the droplets. As a result, the bioaccessibility of vitamin E decreased as its level within the

437

oil phase increased. Vitamin bioaccessibility did not decrease after the nanoemulsions were

438

stored at refrigerated temperatures for 12 weeks, suggesting that this type of delivery system may

439

be an effective means of delivering vitamin E. Our results could be helpful in the rational design

440

of more effective emulsion-based delivery systems for hydrophobic vitamins, nutrients, and

441

nutraceuticals. Furthermore, our emulsions were formulated entirely from natural ingredients,

442

which may be beneficial for many food and nutraceutical applications.



443

Acknowledgements

444

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

445

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

446

AFRI Grants (2016-08782). Shanshan Lv would like to thank the Chinese Scholarship Council

447

for support.

448 449

References

450

(1) Cordero, Z.; Drogan, D.; Weikert, C.; Boeing, H., Vitamin E and Risk of Cardiovascular Diseases: A

451

Review of Epidemiologic and Clinical Trial Studies. Critical Reviews in Food Science and Nutrition

452

2010, 50, 420-440.

453

(2) Imola Gabriela, Z.; Carlos Ernesto, A.; Cristina Mirela, S., Nanoparticles with entrapped α-tocopherol:

454

synthesis, characterization, and controlled release. Nanotechnology 2008, 19, 105606.

455

(3) Ozturk, B.; Argin, S.; Ozilgen, M.; McClements, D. J., Formation and stabilization of nanoemulsion-

456

based vitamin E delivery systems using natural biopolymers: Whey protein isolate and gum arabic. Food

457

Chemistry 2015, 188, 256-263.

458

(4) Duhem, N.; Danhier, F.; Préat, V., Vitamin E-based nanomedicines for anti-cancer drug delivery.

459

Journal of Controlled Release 2014, 182, 33-44.

460

(5) Hategekimana, J.; Chamba, M. V. M.; Shoemaker, C. F.; Majeed, H.; Zhong, F., Vitamin E

461

nanoemulsions by emulsion phase inversion: Effect of environmental stress and long-term storage on

462

stability and degradation in different carrier oil types. Colloids and Surfaces A: Physicochemical and

463

Engineering Aspects 2015, 483, 70-80.

464

(6) Yang, Y.; McClements, D. J., Vitamin E bioaccessibility: Influence of carrier oil type on digestion and

465

release of emulsified α-tocopherol acetate. Food Chemistry 2013, 141, 473-481.

466

(7) Ozturk, B.; Argin, S.; Ozilgen, M.; McClements, D. J., Formation and stabilization of nanoemulsion-


Page 26 of 31

Page 27 of 31


467

based vitamin E delivery systems using natural surfactants: Quillaja saponin and lecithin. Journal of Food

468

Engineering 2014, 142, 57-63.

469

(8) Raikos, V., Encapsulation of vitamin E in edible orange oil-in-water emulsion beverages: Influence of

470

heating temperature on physicochemical stability during chilled storage. Food Hydrocolloids 2017, 72,

471

155-162.

472

(9) Sagalowicz, L.; Leser, M. E., Delivery systems for liquid food products. Current Opinion in Colloid &

473

Interface Science 2010, 15, 61-72.

474

(10) Arora, D.; Jaglan, S., Nanocarriers based delivery of nutraceuticals for cancer prevention and

475

treatment: A review of recent research developments. Trends in Food Science & Technology 2016, 54,

476

114-126.

477

(11) McClements, D. J.; Rao, J., Food-Grade Nanoemulsions: Formulation, Fabrication, Properties,

478

Performance, Biological Fate, and Potential Toxicity. Critical Reviews in Food Science and Nutrition

479

2011, 51, 285-330.

480

(12) Weigel, F.; Weiss, J.; Decker, E. A.; McClements, D. J., Lutein-enriched emulsion-based delivery

481

systems: Influence of emulsifiers and antioxidants on physical and chemical stability. Food Chemistry

482

2018, 242, 395-403.

483

(13) Salvia-Trujillo, L.; Rojas-Graü, A.; Soliva-Fortuny, R.; Martín-Belloso, O., Physicochemical

484

characterization and antimicrobial activity of food-grade emulsions and nanoemulsions incorporating

485

essential oils. Food Hydrocolloids 2015, 43, 547-556.

486

(14) Bouchemal, K.; Briançon, S.; Perrier, E.; Fessi, H., Nano-emulsion formulation using spontaneous

487

emulsification: solvent, oil and surfactant optimisation. International Journal of Pharmaceutics 2004,

488

280, 241-251.

489

(15) Anton, N.; Vandamme, T. F., The universality of low-energy nano-emulsification. International

490

Journal of Pharmaceutics 2009, 377, 142-147.

491

(16) Tadros, T.; Izquierdo, P.; Esquena, J.; Solans, C., Formation and stability of nano-emulsions.

492

Advances in Colloid and Interface Science 2004, 108-109, 303-318.



493

(17) Lee, L.; Norton, I. T., Comparing droplet breakup for a high-pressure valve homogeniser and a

494

Microfluidizer for the potential production of food-grade nanoemulsions. Journal of Food Engineering

495

2013, 114, 158-163.

496

(18) Bai, L.; McClements, D. J., Development of microfluidization methods for efficient production of

497

concentrated nanoemulsions: Comparison of single- and dual-channel microfluidizers. Journal of Colloid

498

and Interface Science 2016, 466, 206-212.

499

(19) Yang, Y.; McClements, D. J., Encapsulation of vitamin E in edible emulsions fabricated using a

500

natural surfactant. Food Hydrocolloids 2013, 30, 712-720.

501

(20) Saberi, A. H.; Fang, Y.; McClements, D. J., Fabrication of vitamin E-enriched nanoemulsions:

502

Factors affecting particle size using spontaneous emulsification. Journal of Colloid and Interface Science

503

2013, 391, 95-102.

504

(21) Alayoubi, A.; Abu-Fayyad, A.; Rawas-Qalaji, M. M.; Sylvester, P. W.; Nazzal, S., Effect of lipid

505

viscosity and high-pressure homogenization on the physical stability of “Vitamin E” enriched emulsion.

506

Pharmaceutical Development and Technology 2015, 20, 555-561.

507

(22) Qian, C.; McClements, D. J., Formation of nanoemulsions stabilized by model food-grade

508

emulsifiers using high-pressure homogenization: Factors affecting particle size. Food Hydrocolloids

509

2011, 25, 1000-1008.

510

(23) Zou, L.; Zheng, B.; Zhang, R.; Zhang, Z.; Liu, W.; Liu, C.; Zhang, G.; Xiao, H.; McClements, D. J.,

511

Influence of Lipid Phase Composition of Excipient Emulsions on Curcumin Solubility, Stability, and

512

Bioaccessibility. Food Biophysics 2016, 11, 213-225.

513

(24) Zhang, R.; Zhang, Z.; Zhang, H.; Decker, E. A.; McClements, D. J., Influence of lipid type on

514

gastrointestinal fate of oil-in-water emulsions: In vitro digestion study. Food Research International 2015,

515

75, 71-78.

516

(25) Qian, C.; Decker, E. A.; Xiao, H.; McClements, D. J., Nanoemulsion delivery systems: Influence of

517

carrier oil on β-carotene bioaccessibility. Food Chemistry 2012, 135, 1440-1447.

518

(26) Ozturk, B.; Argin, S.; Ozilgen, M.; McClements, D. J., Nanoemulsion delivery systems for oil-


Page 28 of 31

Page 29 of 31


519

soluble vitamins: Influence of carrier oil type on lipid digestion and vitamin D3 bioaccessibility. Food

520

Chemistry 2015, 187, 499-506.

521

(27) Meroni, E.; Raikos, V., Physicochemical stability, antioxidant properties and bioaccessibility of

522

[small beta]-carotene in orange oil-in-water beverage emulsions: influence of carrier oil types. Food &

523

Function 2018, 9, 320-330.

524

(28) McClements, D. J., Enhanced delivery of lipophilic bioactives using emulsions: a review of major

525

factors affecting vitamin, nutraceutical, and lipid bioaccessibility. Food & Function 2018, 9, 22-41.

526

(29) Zhang, R.; Zhang, Z.; Zou, L.; Xiao, H.; Zhang, G.; Decker, E. A.; McClements, D. J., Impact of

527

Lipid Content on the Ability of Excipient Emulsions to Increase Carotenoid Bioaccessibility from Natural

528

Sources (Raw and Cooked Carrots). Food Biophysics 2016, 11, 71-80.

529

(30) Zhang, R.; Zhang, Z.; Zhang, H.; Decker, E. A.; McClements, D. J., Influence of emulsifier type on

530

gastrointestinal fate of oil-in-water emulsions containing anionic dietary fiber (pectin). Food

531

Hydrocolloids 2015, 45, 175-185.

532

(31) Yonekura, L.; Nagao, A., Intestinal absorption of dietary carotenoids. Molecular Nutrition & Food

533

Research 2007, 51, 107-115.

534

(32) Brown, M. J.; Ferruzzi, M. G.; Nguyen, M. L.; Cooper, D. A.; Eldridge, A. L.; Schwartz, S. J.;

535

White, W. S., Carotenoid bioavailability is higher from salads ingested with full-fat than with fat-reduced

536

salad dressings as measured with electrochemical detection. The American Journal of Clinical Nutrition

537

2004, 80, 396-403.

538

(33) Porter, C. J. H.; Trevaskis, N. L.; Charman, W. N., Lipids and lipid-based formulations: optimizing

539

the oral delivery of lipophilic drugs. Nature Reviews Drug Discovery 2007, 6, 231.

540

(34) Sarkar, A.; Goh, K. K. T.; Singh, H., Colloidal stability and interactions of milk-protein-stabilized

541

emulsions in an artificial saliva. Food Hydrocolloids 2009, 23, 1270-1278.

542

(35) Jafari, S. M.; Assadpoor, E.; He, Y.; Bhandari, B., Re-coalescence of emulsion droplets during high-

543

energy emulsification. Food Hydrocolloids 2008, 22, 1191-1202.

544

(36) Wang, M.; Feng, M.-q.; Jia, K.; Sun, J.; Xu, X.-l.; Zhou, G.-h., Effects of flaxseed gum



545

concentrations and pH values on the stability of oil-in-water emulsions. Food Hydrocolloids 2017, 67, 54-

546

62.

547

(37) Wang, Y.; Li, D.; Wang, L.-J.; Adhikari, B., The effect of addition of flaxseed gum on the emulsion

548

properties of soybean protein isolate (SPI). Journal of Food Engineering 2011, 104, 56-62.

549

(38) Domian, E.; Brynda-Kopytowska, A.; Oleksza, K., Rheological properties and physical stability of

550

o/w emulsions stabilized by OSA starch with trehalose. Food Hydrocolloids 2015, 44, 49-58.

551

(39) Xu, J.; Mukherjee, D.; Chang, S. K. C., Physicochemical properties and storage stability of soybean

552

protein nanoemulsions prepared by ultra-high pressure homogenization. Food Chemistry 2018, 240,

553

1005-1013.

554

(40) Luo, X.; Zhou, Y.; Bai, L.; Liu, F.; Deng, Y.; McClements, D. J., Fabrication of β-carotene

555

nanoemulsion-based delivery systems using dual-channel microfluidization: Physical and chemical

556

stability. Journal of Colloid and Interface Science 2017, 490, 328-335.

557

(41) McClements, D. J., Nanoemulsions versus microemulsions: terminology, differences, and

558

similarities. Soft Matter 2012, 8, 1719-1729.

559

(42) Witayaudom, P.; Klinkesorn, U., Effect of surfactant concentration and solidification temperature on

560

the characteristics and stability of nanostructured lipid carrier (NLC) prepared from rambutan (Nephelium

561

lappaceum L.) kernel fat. Journal of Colloid and Interface Science 2017, 505, 1082-1092.

562

(43) Helgason, T.; Awad, T. S.; Kristbergsson, K.; Decker, E. A.; McClements, D. J.; Weiss, J., Impact of

563

Surfactant Properties on Oxidative Stability of β-Carotene Encapsulated within Solid Lipid Nanoparticles.

564

Journal of Agricultural and Food Chemistry 2009, 57, 8033-8040.

565

(44) Freitas, C.; Müller, R. H., Effect of light and temperature on zeta potential and physical stability in

566

solid lipid nanoparticle (SLN™) dispersions. International Journal of Pharmaceutics 1998, 168, 221-229.

567

(45) Singh, H.; Sarkar, A., Behaviour of protein-stabilised emulsions under various physiological

568

conditions. Advances in Colloid and Interface Science 2011, 165, 47-57.

569

(46) Ritzoulis, C.; Siasios, S.; Melikidou, K. D.; Koukiotis, C.; Vasiliadou, C.; Lolakos, S., Interactions

570

between pig gastric mucin and sodium caseinate in solutions and in emulsions. Food Hydrocolloids 2012,


Page 30 of 31

Page 31 of 31


571

29, 382-388.

572

(47) Vingerhoeds, M. H.; Blijdenstein, T. B. J.; Zoet, F. D.; van Aken, G. A., Emulsion flocculation

573

induced by saliva and mucin. Food Hydrocolloids 2005, 19, 915-922.

574

(48) McClements, D. J.; Decker, E. A.; Park, Y.; Weiss, J., Structural Design Principles for Delivery of

575

Bioactive Components in Nutraceuticals and Functional Foods. Critical Reviews in Food Science and

576

Nutrition 2009, 49, 577-606.

577

(49) Zhang, R.; Zhang, Z.; Zou, L.; Xiao, H.; Zhang, G.; Decker, E. A.; McClements, D. J., Enhancement

578

of carotenoid bioaccessibility from carrots using excipient emulsions: influence of particle size of

579

digestible lipid droplets. Food & Function 2016, 7, 93-103.

580

(50) Yang, Y.; Decker, E. A.; Xiao, H.; McClements, D. J., Enhancing vitamin E bioaccessibility: factors

581

impacting solubilization and hydrolysis of α-tocopherol acetate encapsulated in emulsion-based delivery

582

systems. Food & Function 2015, 6, 83-96.


Vitamin E Encapsulation in Plant-Based Nanoemulsions Fabricated

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