Improvement of Î²-Carotene Bioaccessibility from Dietary Supplements

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Improvement of #-carotene bioaccessibility from dietary supplements using excipient nanoemulsions Laura Salvia-Trujillo, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00804 • Publication Date (Web): 20 May 2016 Downloaded from http://pubs.acs.org on May 25, 2016

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Journal of Agricultural and Food Chemistry

Improvement of β-carotene bioaccessibility from dietary supplements using excipient nanoemulsions Laura Salvia-Trujillo and David Julian McClements* Department of Food Science, University of Massachusetts, Amherst, MA, 01003, USA.

* Corresponding author (Tel: 413 545 1019; Fax: 413 545 1262; E-mail: [email protected]).

1 ACS Paragon Plus Environment


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ABSTRACT

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The influence of excipient nanoemulsions on β-carotene bioaccessibility from

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commercial dietary supplements (tablets or soft gels) was studied employing an in vitro

4

gastrointestinal tract (GIT) model. Excipient nanoemulsions were formulated from long or

5

medium chain triglycerides (LCT or MCT) to determine the impact of lipid type on

6

carotenoid bioaccessibility. Dietary supplements were tested using the GIT model in the

7

absence or presence of excipient nanoemulsions. β-carotene bioaccessibility from tablets

8

(0.3%) or soft gels (2.4%) was low when tested in isolation. LCT nanoemulsions greatly

9

improved β-carotene bioaccessibility from tablets (20%) and slightly improved it from soft

10

gels (5%), whereas MCT nanoemulsions only slightly improved bioaccessibility. These

11

results were attributed to the ability of large carotenoid molecules to be incorporated into

12

large mixed micelles formed by LCT digestion but not by small ones formed by MCT

13

digestion. Our results indicate that excipient nanoemulsions have considerable potential for

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improving nutraceutical bioavailability from dietary supplements.

15 16 17

Keywords: β-carotene; nutraceuticals; nanoemulsions; excipient foods; bioaccessibility; lipid digestion

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INTRODUCTION

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Nutraceuticals are food components isolated from natural sources that are claimed to

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provide health benefits beyond basic nutrition 1. Carotenoids are an important category of

22

nutraceuticals that have received particular interest due to their potential health benefits 2.

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Carotenoids are found at appreciable levels in certain highly colored fruits, vegetables, and

24

algae, and have been reported to have pro-vitamin A and antioxidant activity, as well as the

25

ability to combat certain types of cancer, heart disease, and eye disease 3-5. Carotenoids may

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be incorporated into functional foods or they may be delivered in the form of dietary

27

supplements 2. The consumption of dietary supplements (such as tablets, capsules, and soft

28

gels) containing carotenoids has recently increased due to consumer’s growing interest in

29

their health benefits 2. However, carotenoid bioavailability from dietary supplements is

30

generally very low 6, which may be one reason why clinical trials with carotenoid

31

supplements have often shown little or no significant benefit 7, 8.

32

interest in elucidating the main factors that determine carotenoid bioavailability, and in using

33

this knowledge to develop more efficacious nutraceutical supplements.

34

Consequently, there is

The oral bioavailability of carotenoids from functional foods or supplements depends on

35

numerous physiochemical and biochemical processes, including release from the food or

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supplement matrix, solubilization in the gastrointestinal fluids, transformation by chemical or

37

biochemical reactions, and absorption by intestinal epithelial cells 9-12. Carotenoid

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bioaccessibility can be considered to be the fraction released from a food or supplement and

39

then solubilized by the mixed micelles present in the GIT 13, 14. These mixed micelles are

40

colloidal particles (micelles and vesicles) that are formed from bile salts and phospholipids

41

arising from gastrointestinal secretions and free fatty acids and monoacylglycerols arising

42

from any digested lipids 15-17. The mixed micelles contain hydrophobic domains capable of



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incorporating lipophilic bioactives, such as carotenoids, that would normally have poor

44

aqueous solubility. Once incorporated into the micelles the lipophilic bioactives are

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transported to the epithelium cells where they can be absorbed. Therefore, ingestion of a lipid

46

source that favors the formation of mixed micelles tends to enhance the bioaccessibility of co-

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ingested lipophilic nutraceuticals 14, 18, 19.

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The bioaccessibility of carotenoids in some dietary supplements has been reported to be

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very low because they are delivered in the form of tablets that contain no lipid source capable

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of forming mixed micelles 20. Carotenoid bioaccessibility can be increased by incorporating

51

them into soft gels that consist of an edible biodegradable shell (such as gelatin) and an oily

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inner core containing the carotenoids. However, the bioaccessibility of carotenoids has been

53

reported to be much lower when they are dispersed in bulk oils than in emulsified oils 21.

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This may be because emulsified oils are usually digested more rapidly and extensively than

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bulk oils and therefore quickly form mixed micelles that can incorporate carotenoids 22. In

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addition, the bioaccessibility of carotenoids is highly dependent on the type of oil used to

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deliver them 23, 24, and so any oil used in a soft gel must be carefully selected. In general, it

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has been noted that the biological activity of many nutraceuticals is reduced when they are

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isolated from their original location in foods and then delivered as supplements 25.

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Consequently, there is interest in finding strategies to enhance the bioavailability of

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nutraceuticals from supplements.

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Excipient emulsions are an effective strategy for improving the bioavailability of

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lipophilic nutrients and nutraceuticals 21, 26, 27. Typically, an excipient emulsion does not

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contain any bioactive components itself, but it can enhance the bioavailability of

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nutraceuticals in foods or supplements consumed with it. An excipient emulsion may achieve

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this goal by modulating the bioaccessibility, absorption, and/or stability of the co-ingested


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nutraceuticals in the GIT. Indeed, numerous recent studies indicate that the oral

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bioavailability of hydrophobic bioactives (such as carotenoids or curcuminoids) in foods can

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be improved by co-administrating them with excipient emulsions 28-30. In the current work,

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the focus was on the utilization of oil-in-water nanoemulsions as excipients for carotenoids

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delivered in the form of dietary supplements. These excipient nanoemulsions consist of small

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lipid droplets (diameter < 200 nm) dispersed within an aqueous phase 31. An advantage of

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using nanoemulsions as excipients is their rapid digestion within the GIT due to their small

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droplet dimensions (high surface area), which can lead to rapid solubilization of any lipophilic

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bioactives into the mixed micelle phase 22.

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The main objective of this research was therefore to determine the impact of excipient

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nanoemulsions on the bioaccessibility of β-carotene present within dietary supplements

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(tablets and soft gels) using a simulated GIT consisting of mouth, stomach, and small

79

intestine phases. Moreover, the impact of lipid phase type (medium or long chain

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triacylglycerides) on the efficacy of the excipient nanoemulsions was investigated, as

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previous studies have reported this factor has a strong influence on β-carotene bioaccessibility

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from foods 29, 32. The knowledge gained from our study could lead to the development of

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more efficacious dietary supplements that provide improved health benefits to consumers.

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MATERIALS AND METHODS

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Materials. The dietary supplements were purchased in a local commercial store. The

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manufacturer of the β-carotene tablets (Whole Foods Market, Austin, TX, USA) reported that

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they contained 25,000 IU of vitamin A (100% as β-carotene), as well as rice flour, vegetable

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capsule (hydroxypropyl methylcellulose; HPMC), silicon dioxide, and magnesium stearate

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(from a vegetable source) as other ingredients. The manufacturer of the β-carotene soft gels



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(Country life, Hauppauge, NY, USA) reported that each soft gel contained 25,000 IU of

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vitamin A (100% as β-carotene), 0.5 g of medium chain triglycerides, 10 mg of soy lecithin,

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and 4 IU of vitamin E (as d-α-tocopherol). Both β-carotene dietary supplements claimed to

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provide 500% of the recommended daily intake of carotenoids. Corn oil (long chain

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triacylglycerides, LCT) was obtained from Mazola ACH Food Companies (Memphis, TN,

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USA) and Miglyol 812 (medium chain triacylglycerides, MCT) was purchased from SASOL

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(Houston, TX, USA). Tween 80, monobasic and dibasic phosphates, pepsin, bile salts and

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lipase were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). Chloroform

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was purchased from Fisher Scientific (Waltham, MA). Aqueous solutions and nanoemulsions

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were prepared using purified water obtained from a Milli-Q filtration system (EMD Millipore,

100 101

Merck KGaA, Darmstadt, Germany). Excipient nanoemulsion formation. Excipient nanoemulsions were prepared from an

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oil phase that consisted of either pure corn oil (LCT) or pure Miglyol 812 (MCT), an an

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aqueous phase that consisted of a 5 mM phosphate buffer solution (pH 7.0). A course

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emulsion was obtained by blending 10% (w/w) oil phase (LCT or MCT), 1% (w/w) surfactant

105

(Tween 80), and 89% (w/w) aqueous phase using a high-shear blender at 20,000 rpm for 2

106

min. Nanoemulsions were formed by passing coarse emulsions through a high pressure

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homogenizer (Microfluidizer, Model M110-P, Microfluidics, Newton, MA) at 20,000 psi for

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3 passes.

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Simulated Gastrointestinal Tract. An in vitro GIT model consisting of mouth,

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stomach, and small intestine stages was used to mimic the potential gastrointestinal fate of

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dietary supplements alone or mixed with excipient nanoemulsions. This model is based on

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previous attempts to develop standardized GIT models to compare samples under similar

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conditions 33, 34.


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Mouth Phase: A simulated saliva fluid (SSF) that consisted of mucin, salts, and water

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was prepared as described previously 35. The amounts of SSF, nanoemulsion, and phosphate

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buffer included in each sample to prepare the mouth phase are summarized in Table 1.

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For the tablet alone, 20 mL of phosphate buffer and 20 mL of SSF were mixed together,

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and then the dry tablet ( 0.5 g) was introduced into the same container. For the tablets with

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excipient nanoemulsions, 16 mL of phosphate buffer, 4 mL of nanoemulsion, and 20 mL of

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SSF were mixed together, and then the tablet was added. This led to an oil concentration of 1

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wt% LCT or MCT in the mouth phase. In the case of the soft gels, the volumes of buffer and

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SSF were adjusted so that the total concentration of oil in the mouth was 1%, taking into

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account that each soft gel contained 0.5 g of MCT. Finally, for the soft gel mixed with LCT

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excipient nanoemulsions, the concentration of the components was adjusted to reach a final

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concentration of 1% MCT from the soft gel and 1% of LCT from the excipient nanoemulsion

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in the mouth phase.

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The pH of the resulting mixtures were changed to pH 6.8, and then the samples were held

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at 37 ºC for 10 min with stirring (100 rpm). The period that the samples spent in the mouth

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phase was longer than would be expected in practice when taking supplements (a few

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seconds). This was done so that the capsules had time to disintegrate within the mouth and

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release the encapsulated β-carotene in either powder or liquid form. In this way, a

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representative aliquot could be taken to further continue with the simulation of the stomach

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and intestinal phases.

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Gastric phase: Simulated gastric fluid (pH 1.2) containing NaCl, HCl, and water was

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prepared as previously described 36. The sample resulting from the mouth phase was

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combined with SGF (1:1 v/v) leading to a mixture that contained 0.5% (w/w) oil. The mixture

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was changed to pH 2.5 using 1 M NaOH and stirred for 120 minutes (37º C, 100 rpm).



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Small Intestine Phase: Samples from the gastric phase were exposed to simulated small

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intestine conditions and the amount of free fatty acids (FFAs) released over time was

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measured using a pH-stat as described previously22. Briefly, samples (30 mL) from the

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gastric phase were mixed with solutions (7.5 mL) containing bile extract, calcium chloride

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and lipase (pH 7.0, 37º C, 100 rpm). The amount of alkaline solution required to maintain a

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neutral pH was then measured over time, and converted into the percentage of FFAs released

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37

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. Particle characteristics: The physicochemical properties of the dietary supplements

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(with or without nanoemulsion addition) were determined in the initial, mouth, stomach and

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small intestine stages.

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Particle size was determined by static light scattering (LS 13 320, Beckman Coulter Inc.,

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USA). The instrument used consists of a 5 mW laser diode with a wavelength of 750 nm and

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a secondary tungsten-halogen light source projected through a set of filters, which transmit

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three wavelengths (450 nm, 600 nm, and 900 nm), to measure particles that fall in the

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submicron range. Buffer solutions adjusted to an appropriate pH were used to dilute the

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samples prior to analysis: pH 6.8 (mouth phase): pH 2.5 (stomach phase); pH 7 (small

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intestine phase). The mean particle diameter is given as the volume-weighted mean diameter

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(μm). Particle ζ-potential was determined using phase-analysis light scattering (Zetasizer

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NanoZS, Malvern Instruments, Worcestershire, UK). As for particle size analysis, buffer

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solutions adjusted to an appropriate pH were used to dilute (1:10) the samples prior to

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analysis.

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β-carotene bioaccessibility. Carotenoid bioaccessibility (BA) was quantified by

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measuring the fraction of β-carotene solubilized in the micelle phase at the end of the GIT

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model as described previously22:


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𝐵𝑖𝑜𝑎𝑐𝑐𝑒𝑠𝑠𝑖𝑏𝑖𝑙𝑖𝑡𝑦 =

𝐶𝑚𝑖𝑐𝑒𝑙𝑙𝑒 × 100 𝐶𝐷𝑖𝑔𝑒𝑠𝑡𝑎

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Where Cmicelle and CDigesta are the β-carotene concentrations in the micelle phase and in the

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total digesta after the in vitro digestion, respectively.

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Optical and confocal microscopy. Changes in the microstructure of the samples

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throughout the GIT were determined by optical and confocal fluorescence microscopy as

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described previously22. The general appearance of the samples was determined by optical

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microscopy, whereas the location of the carotenoids after the small intestine phase was

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determined using confocal fluorescence microscopy. Carotenoids naturally fluoresce and so it

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is possible to detect their location in lipid colloidal structures 38, 39. By the same principle, it is

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possible to track the possible migration of carotenoids from the dietary supplements to the oil

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droplets after mixing with the nanoemulsions.

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Statistical analysis. Each experiment were carried out twice, and the results are given as

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the mean and standard deviation calculated from these values. The analysis of variance was

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calculated using commercial statistical software (JMP 12, SAS Institute Inc.). The Student’s t

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test was employed to ascertain statistical differences at a 5% significance level (p < 0.05).

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RESULTS AND DISCUSSION

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The experiments were designed to determine the impact of mixing dietary supplements with LCT or MCT nanoemulsions on their gastrointestinal behavior using a GIT model. Properties of excipient nanoemulsions. In the absence of the dietary supplements, both

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the LCT and MCT nanoemulsions initially had monomodal size distributions and mean

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particle diameters of 0.169 and 0.151 μm, respectively (Figure 1A). Hence, the

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homogenization conditions and emulsifier type used were suitable for fabricating stable

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nanoemulsions from the two lipid phases used. 9 ACS Paragon Plus Environment


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Impact of passage through GIT on particle size. Initially, the impact of GIT conditions on the particle size and microstructure of the dietary supplements was determined. Tablets: The mean diameter of particles resulting from the carotenoid-enriched tablets

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after they were dispersed within the mouth phase was around 75 μm, and remained high after

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exposure to the stomach and small intestine phases (Figure 1B). The particle size

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distributions suggested that these samples contained particles that ranged in diameter from a

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few micrometers to several hundred micrometers (Figure 2A). These particles are likely to be

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components from the tablet formulation that were insoluble and indigestible in the

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gastrointestinal fluids, such as carotenoid crystals and/or silicon dioxide particles. Indeed,

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microscopy measurements showed that large insoluble aggregates were present in these

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systems throughout the GIT model (Figure 3A).

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The impact of mixing the tablets with excipient nanoemulsions prior to passing them

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through the simulated GIT was then determined. The mean particle diameter stayed large for

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the systems consisting of tablets and excipient nanoemulsions throughout the simulated GIT

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(Figure 1B), because large insoluble and indigestible particles from the tablets remained

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(Figure 3) that dominated the light scattering profiles. Nevertheless, the presence of the

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small nanoemulsion droplets could be observed in the mixed samples in certain regions of the

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GIT, with a peak being observed in the particle size distributions around 0.1 μm in the mouth

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and stomach regions (Figures 2B and 2C). This peak moved rightwards after exposure of

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the samples to the small intestine phase (Figure 1B) due to the digestion of the nanoemulsion

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droplets by lipase and the formation of micelles and vesicles that contributed to the overall

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light scattering pattern. The microscopy measurements indicted that a number of the large

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particles observed in the samples containing only tablets were broken down in the presence of

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the nanoemulsions, especially in the stomach and small intestine phases (Figure 3). This


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effect may have been due to solubilization of some of the -carotene crystals by the lipid

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phase in the nanoemulsions.

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Soft Gels: Initially, the soft gels consisted of a gelatin shell and an oily core that

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contained a mixture of carotenoid crystals and MCT. After exposure to mouth conditions, the

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outer shell of the soft gels dissolved and the oily core material was released, which led to the

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formation of some relatively large particles (d  89 μm) in the simulated saliva fluids (Figure

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1B). These particles were probably oil droplets formed when the MCT released from the soft

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gels was sheared under simulated oral conditions. Indeed, optical microscopy images showed

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that these samples contained large droplets (light-colored) with smaller irregular-shaped

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particles (dark-colored) inside them (Figure 3A). Confocal fluorescent microscopy suggested

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that these irregular-shaped particles were carotenoid crystals, as they appeared as bright

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objects in the images (Figure 3B). The particle size distribution remained relatively

220

unchanged after passage through the simulated gastric fluids (Figure 2B) and some large

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carotenoid-containing oil droplets were still observed in the microscopy images (Figure 3A),

222

which indicated that these particles were resistant to disruption in the highly acidic simulated

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gastric fluids. However, an appreciable change in the particle size distribution of the soft gel

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samples occurred after they were exposed to simulated small intestine conditions (Figure

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2B). There was a reduction in the number of larger particles present and an increase in the

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number of smaller particles, which is consistent with digestion of the large lipid droplets by

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lipase and the formation of smaller micelles and vesicles. Moreover, optical microscopy

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suggested that the MCT droplets had been digested, leaving behind clusters of carotenoid

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crystals (Figure 3).

230 231

As the soft gels already contained MCT, we only mixed them with LCT nanoemulsions in this study. The presence of the small lipid droplets from the LCT nanoemulsions caused a



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significant alteration in the particle size and microstructure of the soft gel samples throughout

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the GIT. In the mouth phase, the number of larger particles in the microscopy images

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decreased, whereas the number of smaller particles increased (Figure 2E), leading to a

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reduction in the mean particle diameter (Figure 1B). These effects are partly due to the

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contribution that the nanoemulsion droplets make to the light scattering pattern, however,

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there also seemed to be a reduction in the number and size of the MCT droplets from the soft

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gels (Figure 3). This phenomenon may have occurred because there was some free surfactant

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arising from the LCT nanoemulsions that helped to facilitate the formation of smaller MCT

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droplets when the soft gel samples were sheared in the mouth phase. In addition, there

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appeared to be less carotenoid crystals present within the large MCT droplets released from

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the soft gels (Figure 3), which may have been because some of the carotenoids were

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solubilized in the small LCT droplets from the nanoemulsions. The particle size distribution

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and microstructure of the samples remained relatively unchanged after exposure to the

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stomach phase, again suggesting that the particles were stable under these conditions.

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However, the particle size and microstructure did change appreciably after the samples were

247

subjected to small intestine conditions (Figures 2E and 3). There was a decrease in the

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number of larger particles (presumably MCT droplets from soft gels) and a decrease in the

249

number of smaller particles (presumably LCT droplets from the nanoemulsions), presumably

250

due to lipid digestion. This led to the formation of a particle size distribution that contained

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particles that varied in diameter from a fraction of a micrometer to several hundred

252

micrometers (Figure 2E). These samples are will contain a mixture of numerous types of

253

colloidal particles after lipid digestion, including micelles, vesicles, undigested oil droplets,

254

carotenoid crystals, calcium soaps, and other insoluble matter 40.


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Impact of passage through GIT on particle charge. The ζ-potential of particles arising

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from the carotenoid-enriched dietary supplements was measured as they passed through the

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GIT (Figure 4). Again, measurements were made in the absence and presence of excipient

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nanoemulsions to establish their potential influence on the GIT fate of the supplements.

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At the end of the mouth phase, the ζ-potential values of all the dietary supplement

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samples were moderately negative (-12 to -16 mV) regardless of supplement type or

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nanoemulsion addition (Figure 4). This negative ζ-potential is due to various types of anionic

262

particles in the simulated saliva that all make a contribution to the electrophoretic signal. For

263

example, the tablets contained silicon dioxide and magnesium stearate, whereas the soft gels

264

contained lecithin and gelatin. In addition, the simulated saliva contained mucin, which is a

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relatively large anionic glycoprotein 41. After exposure to the stomach phase, the ζ-potential of

266

all of the dietary supplements became close to zero (-0.4 to -1.9 mV), with no significant

267

differences between them. The reduction in the magnitude of the electrical potential when the

268

samples moved from the mouth to the stomach fluids might be due to the high acidity and

269

ionic strength of the gastric environment. At low pH, protonation of anionic groups (e.g.,

270

−COOH) would lead to neutralization of negative charges present in the neutral mouth phase

271

(e.g., −COO ). In addition, the high ionic strength of the gastric phase reduces the electrical

272

potential through electrostatic screening.

-

273

At the end of the small intestine phase there was a large increase in the negative charge

274

on the particles in all of the dietary supplements, with the extent of the effect depending on

275

supplement type and nanoemulsion addition (Figure 4). In particular, the negative charge

276

was higher for soft gels than for tablets, and was higher for samples containing LCT than for

277

those containing MCT. In the small intestine, the observed increase in negative charge may

278

have been due to numerous reasons. First, certain components arising from the supplements



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may have become negatively charged under the neutral pH conditions in the small intestinal

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fluids, e.g., gelatin and lecithin from the soft gels. This would account for the higher negative

281

charge observed for the soft gels than for the tablets. Second, anionic bile salts from the small

282

intestinal fluids will have been present at the surfaces of the micelles, vesicles, and oil

283

droplets generated by lipid digestion and therefore contributed to the net negative charge.

284

Third, the hydrolysis of triglycerides by lipase releases anionic free fatty acids (FFAs) that

285

may also have accumulated at the surfaces of the various types of colloidal particles present

286

after lipid digestion 42. Long chain FFAs produced by digestion of LCT are known to be

287

more likely to accumulate at particle surfaces than the medium chain FFAs produced by

288

MCT43, which may be why the ζ-potential was more negative for the samples to which LCT

289

nanoemulsions were added. More highly negatively charged colloidal particles in the small

290

intestinal fluids after lipid digestion have also been reported for LCT than for MCT in other

291

studies, which was associated with the same mechanism 24.

292

Influence of Dietary Supplement Type on Lipid Digestion. The hydrolysis of any

293

digestible lipids in the various samples after addition of lipase to the small intestine phase was

294

monitored using a pH-stat method. The volume of alkaline (0.1 N NaOH) solution needed to

295

maintain neutral pH conditions was measured (Figure 5A), and then this value was converted

296

into the amount of FFAs released by the lipids (Figure 5B).

297

There were appreciable differences in the volume of alkaline solution titrated into the

298

different samples to neutralize the FFAs formed (Figure 5A). These differences are due to

299

differences in the total type and amount of lipids present in the various samples. For the

300

tablets, the volume of alkaline solution added was relatively small because they contained no

301

digestible lipids. For the soft gels, the rise in the volume of alkaline solution added during

302

digestion was relatively low, which can be attributed to digestion of the oily core material


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(MCT). For all the samples containing nanoemulsions, there was a rapid initial rise in the

304

volume of alkaline solution added followed by a slower rise at longer times (Figure 5A). The

305

total volume of alkaline solution added to the systems containing MCT nanoemulsions was

306

much greater than that for the systems containing LCT nanoemulsions, which can be

307

attributed to the fact that MCT has a lower molecular weight than LCT and so there are more

308

FFAs per gram of oil. To make a better comparison, the %FFAs released over time was

309

therefore calculated assuming that 2 free fatty acids were released per triglyceride molecule

310

(Figure 5B). Major differences in the FFA release profiles were observed for the different

311

dietary supplements depending on the nature of any excipient nanoemulsions used.

312

Tablets: For the tablet + nanoemulsions, the majority of FFAs were liberated during the

313

first 10 min followed by a slight increase throughout the remainder of the small intestine

314

phase. Interestingly, the total level of FFAs liberated by the end of lipid digestion exceeded

315

100%, which may be because more than 2 free fatty acids were released per triglyceride

316

molecule or that there was some other components within the tablets that were digested in the

317

presence of the nanoemulsions. The rapid digestion of the triglycerides in these systems is

318

because the nanoemulsions contained small digestible lipid droplets (d < 200 nm) that had a

319

high surface area. Consequently, lipase easily adsorbed to the droplet surfaces and promoted

320

digestion of the emulsified lipids. For the sake of comparison the fraction of FFAs released

321

from the nanoemulsions in the absence of dietary supplements was also measured (Figure

322

5C). In this case, the %FFAs released also increased sharply in the first 10min, followed by a

323

slower rise at later times. However, the total level of FFAs released by the finish of the small

324

intestine phase was much closer to 100%. Again, this suggests that the tablets contained

325

some constituents that were only digested in the presence of the nanoemulsions or that

326

promoted further digestion of the triglycerides.



327

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Soft gels: For the soft gels, the release of FFAs was initially much slower than for the

328

dietary supplements mixed with the excipient nanoemulsions, and by the completion of the

329

small intestine stage a smaller percentage of triglycerides (92%) had been digested (Figure

330

5B). This may have been because the oily phase released from the soft gels formed large

331

MCT droplets with corresponding low interfacial areas (Figure 3). Consequently, they would

332

be digested more slowly by the lipase molecules 37. For the soft gel + LCT nanoemulsion,

333

there was a fast rate of lipid digestion initially due to the small size (high surface area) of the

334

lipid droplets in the nanoemulsions. However, in this case the fraction of FFAs released on

335

completion of the small intestine phase (67%) was appreciably lower than for the other

336

samples. This effect might be due to the presence of a higher total amount of digestible lipids

337

in this system (2%) compared to the other systems (1%). Indeed, other studies have reported

338

that lipid digestion is suppressed when the total lipid content is too high because the level of

339

lipase per unit mass of lipids is reduced and because the amount of bile salts and calcium ions

340

available to remove FFAs from the lipid droplet interfaces is reduced 33.

341

Influence of Dietary Supplement Type on β-carotene Bioaccessibility. Finally, the

342

impact of adding excipient nanoemulsions to the tablets and soft gels on β-carotene

343

bioaccessibility was assessed (Figure 6).

344

Tablets: The bioaccessibility of β-carotene was found to be extremely low for the tablets

345

alone (0.3%), which is consistent with other studies on the bioaccessibility of carotenoids in

346

health supplements20. This result can be attributed to the absence of any digestible lipids

347

within the tablets, so that no mixed micelles were formed in the small intestinal fluids upon

348

completion of digestion. The presence of mixed micelles is important for the solubilization

349

and transport of highly hydrophobic bioactive molecules, such as carotenoids 44. Mixing the

350

tablets with MCT nanoemulsions only caused a moderate improvement in β-carotene


Page 17 of 39


351

bioaccessibility (2.0%), but mixing them with LCT nanoemulsions caused a substantial

352

improvement (19.4%). The fact that LCT is much more effective than MCT at promoting

353

carotenoid bioaccessibility has also been reported in earlier studies 23, 24. This effect is because

354

the mixed micelles formed by LCT have large non-polar domains that can easily

355

accommodate large non-polar carotenoid molecules, whereas those formed by MCT do not.

356

Moreover, it has been reported that highly non-polar bioactive molecules delivered with LCT

357

are mainly solubilized in small micelles after lipid digestion, whereas those delivered with

358

MCT are mainly located within large vesicles 45. The β-carotene trapped within these large

359

vesicles may therefore sediment during the centrifugation step used to separate the micelle

360

fraction, which would lead to a lower measured bioaccessibility. One would expect that small

361

micelles easily move through the layer of mucus coating the epithelium cells, whereas large

362

vesicles cannot. Consequently, the bioaccessibility measured using the in vitro method would

363

be reflective of that expected in vivo.

364

Soft gels: The bioaccessibility of the β-carotene in the soft gels was also relatively low

365

after in vitro digestion (2.4%), which again may be due to the fact that the large non-polar

366

carotenoid molecules were not easily incorporated into small micelles. Indeed, the optical

367

microscopy images show numerous clusters of crystalline β-carotene present in the soft gel

368

samples after incubation in small intestine conditions (Figure 3). Mixing the soft gels with

369

the LCT nanoemulsions only led to a modest increase in β-carotene bioaccessibility (4.8%),

370

which was much less than was observed when the LCT nanoemulsions were mixed with the

371

tablets (19.4%). This difference is probably because the lipid phase was not completed

372

digested for the soft gels containing LCT nanoemulsions due to the relatively high total lipid

373

content in these samples (Figure 5B). Consequently, not all of the carotenoids were released

374

from the oil phase. This hypothesis is consistent with the microscopy measurements showing



Page 18 of 39

375

there were undigested oil droplets containing carotenoid crystals in the samples after

376

incubation in small intestine fluids (Figure 3). This result shows the importance of carefully

377

selecting both the type and amount of lipid phase used in excipient nanoemulsions (at least for

378

in vitro experiments).

379

In summary, this in vitro study has shown that the bioaccessibility of β-carotene from

380

commercial dietary supplements (tablets or soft gels) is relatively low when they are taken

381

alone. The low bioaccessibility may be caused by the lack of digestible lipids (tablets), or by

382

the fact that the digestible lipids present do not form micelles capable of solubilizing large

383

non-polar β-carotene molecules (soft gels). The co-ingestion of excipient nanoemulsions

384

formulated using LCT as a lipid phase significantly increased β-carotene bioaccessibility in

385

both types of dietary supplements. Nevertheless, the observed improvement in

386

bioaccessibility was much more substantial for the tablets than for the soft gels, which was

387

attributed to a limitation of the in vitro digestion model for samples containing high lipid

388

levels. Moreover, excipient nanoemulsions formulated with LCT were much more effective

389

than those formulated with MCT at improving bioaccessibility, which was attributed to the

390

fact that they could form a mixed micelle phase containing hydrophobic domains large

391

enough to trap the large hydrophobic carotenoid molecules. Overall, our results may be useful

392

in formulating dietary supplements with improved bioavailability characteristics. For

393

example, soft gels could be formulated to contain an LCT nanoemulsion (rather than a bulk

394

oil) inside the capsules, or tablets could be formulated to contain a spray-dried LCT

395

nanoemulsion within them. Finally, we note that a relatively simple in vitro GIT model was

396

used here, and that it will be important to carry our further studies employing animal or

397

human feeding trials to confirm the results in practice.


Page 19 of 39


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supplements: differential health impact (Part 2). Agro Food Industry Hi-Tech 2015, 26, 20-22.

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Effects To Enhance Nutraceutical Bioavailability: Increase of Curcumin Bioaccessibility

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Salvia-Trujillo, L.; Qian, C.; Martín-Belloso, O.; McClements, D. J., Influence of

Qian, C.; Decker, E. A.; Xiao, H.; McClements, D. J., Nanoemulsion delivery

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Fardet, A., Complex foods versus functional foods, nutraceuticals and dietary

McClements, D. J.; Xiao, H., Excipient foods: designing food matrices that improve

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McClements, D. J., Enhancing Nutraceutical Bioavailability from Raw and Cooked

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Vegetables Using Excipient Emulsions: Influence of Lipid Type on Carotenoid

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Bioaccessibility from Carrots. Journal of Agricultural and Food Chemistry 2015, 63, 10508-

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516

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625-637.

520

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521

Nutrition & Food Research 2007, 51, 107-115.

522

45.

523

characterization of the colloidal phases produced on digestion of common formulation lipids

524

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525

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526 527


Page 25 of 39

528 529


FUNDING This material is based upon work supported by the Cooperative State Research,

530

Extension, Education Service, United State Department of Agriculture, Massachusetts

531

Agricultural Experiment Station (MAS00491) and by the United States Department of

532

Agriculture, NRI Grants (2013-03795 and 2014-67021).

533



534

FIGURE CAPTIONS

535

Figure 1. Droplet size distribution of the initial LCT or MCT excipient nanoemulsions

536

(A) and volume-weighed mean particle size (μm) of carotenoid dietary supplements with or

537

without excipient nanoemulsions during the in vitro digestion. Different capital letters

538

represent significant differences (p < 0.05) between in vitro digestion phases for the same

539

sample. Different lower case letters represent significant differences (p < 0.05) between

540

different samples in a given in vitro digestion phase (mouth, stomach or small intestine).

541

Page 26 of 39

Figure 2. Particle size distributions measured after exposure of the samples to the

542

simulated GIT regions (mouth, stomach and small intestine). The samples initially contained

543

the tablet alone (A), soft gel alone (B), tablet with LCT nanoemulsion (C), tablet with MCT

544

nanoemulsion (D), or soft gel with LCT nanoemulsion (E).

545

Figure 3. (A) Optical images (20) of dietary supplements with or without mixing with

546

excipient nanoemulsions (LCT or MCT) under simulated gastrointestinal conditions. Scale

547

bar is 100 μm; (B) Confocal images (60) of soft gels and soft gels mixed with excipient

548

nanoemulsions (LCT) after the small intestinal phase. Scale bar is 50 μm.

549

Figure 4. ζ-potential (mV) of carotenoid dietary supplements with or without excipient

550

nanoemulsions during the in vitro digestion. Different capital letters represent significant

551

differences (p < 0.05) between in vitro digestion phases for the same sample. Different lower

552

case letters represent significant differences (p < 0.05) between different samples in a given in

553

vitro digestion phase (mouth, stomach, or small intestine).

554

Figure 5. (A) Volume of NaOH (0.1N) solution used to maintain the small intestine

555

phase at pH 7.0: (B) Calculated free fatty acids released (%) from the dietary supplements

556

with or without excipient nanoemulsions; (C) Calculated free fatty acids released (%) from

557

the excipient nanoemulsions on their own.


Page 27 of 39

558


Figure 6. β-carotene bioaccessibility (BA; %) of dietary supplements with or without

559

excipient nanoemulsions (LCT or MCT). Different capital letters represent significant

560

differences (p < 0.05) between samples.

561



Page 28 of 39

TABLES & FIGURES

Table 1. Levels of Different Constituents Included in the Mouth Phase for Each of the Samples Tested. The Nanoemulsion Consisted of 10 wt% Oil Phase (LCT or MCT) and 90 wt% Aqueous Phase. Key: SSF = Simulated Saliva Fluid.

Sample

Phosphate

Nanoemulsion

SSF

Oil from

Oil from

Buffer

(mL)

(mL)

Supplement

Nanoemulsion

(w/v)

(w/v)

(mL) Tablet

20

0

20

-

-

Tablet + LCT nE

16

4

20

-

1% LCT

Tablet + MCT nE

16

4

20

-

1% MCT

Soft Gel

25

0

25

1% MCT

-

Soft Gel + LCT nE

20

5

25

1% MCT

1% LCT


Page 29 of 39


30

A

Relative Volume (%)

151.3 +/- 0.4 nm

20 MCT

10 168.8 +/- 1.4 nm

LCT

0 0.04

0.4 Particle Diameter (nm)

4

Figure 1a



Mean Particle Diameter (μm)

1000

100

Tablet Tablet + LCT nE Soft gel + LCT nE

AaAa AaAa

Page 30 of 39

Soft gel Tablet + MCT nE

B

Aa Aa Aa ABab BabBab Bbc Bb

Ab Bc

Bb

10

1

Mouth

Stomach Small Intestine

Figure 1b


Page 31 of 39


Figure 2.



Page 32 of 39

Figure 3a.


Page 33 of 39


Figure 3b.



Page 34 of 39

Gastrointestinal phase Mouth

Stomach

Small intestine

0 AaAaAaAaAa

-10

Zeta-potential (mV)

-20

Ba Ba Bb

Ba Ba

-30 Ca Cb

Cab

-40 -50 -60 -70

Tablet Soft gel Tablet + LCT nE Tablet + MCT nE Soft gel + LCT nE

Cc

Cd

-80

Figure 4.


Page 35 of 39


Figure 5a.



Page 36 of 39

Figure 5b.


Page 37 of 39


Figure 5c.



Tablet

20

Page 38 of 39

A

Soft gel

β-carotene BA (%)

Tablet + LCT nE Tablet + MCT nE

15

Soft gel + LCT nE

10

B

5

BC

BC

C 0

Figure 6.


Page 39 of 39


Table of Contents Graphic


Improvement of Î²-Carotene Bioaccessibility from Dietary Supplements

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