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Enhancing nutraceutical bioavailability from raw and cooked vegetables using excipient emulsions: Influence of lipid type on carotenoid bioaccessibility from carrots Ruojie Zhang, Zipei Zhang, Liqiang Zou, Hang Xiao, Guodong Zhang, Eric Andrew Decker, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04691 • Publication Date (Web): 20 Nov 2015 Downloaded from http://pubs.acs.org on November 25, 2015
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Journal of Agricultural and Food Chemistry
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Enhancing nutraceutical bioavailability from raw and
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cooked vegetables using excipient emulsions: Influence of
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lipid type on carotenoid bioaccessibility from carrots
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Ruojie Zhang1 , Zipei Zhang1, Liqiang Zou1, Hang Xiao1, Guodong
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Zhang1, Eric Andrew Decker1,2, and David Julian McClements1,2* 1
Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA 2
Department of Biochemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203 Jeddah 21589 Saudi Arabia
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Journal: Journal of Agricultural and Food Chemistry
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Submitted: September 2015
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*Contact Information for Corresponding Author: David Julian McClements, Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA; Tel 413 545 1019; Fax 413 545 1262; email
[email protected].
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ABSTRACT
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The influence of the nature of the lipid phase in excipient emulsions on the
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bioaccessibility and transformation of carotenoid from carrots was investigated using
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a gastrointestinal tract (GIT) model. Excipient emulsions were fabricated using whey
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protein as an emulsifier and medium chain triglycerides (MCT), fish oil, or corn oil as
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the oil phase. Changes in particle size, charge, and microstructure were measured as
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the carrot-emulsion mixtures were passed through simulated mouth, stomach, and
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small intestine regions.
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used to form the excipient emulsions (corn oil > fish oil >> MCT), which was
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attributed to differences in the solubilization capacity of mixed micelles formed from
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different lipid digestion products. The transformation of carotenoids was greater for
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fish oil and corn oil than for MCT, which may have been due to greater oxidation or
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isomerization.
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raw carrots, which was attributed to greater disruption of the plant tissue facilitating
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carotenoid release. In conclusion, excipient emulsions are highly effective at
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increasing carotenoid bioaccessibility from carrots, but lipid type must be optimized
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to ensure high efficacy.
Carotenoid bioaccessibility depended on the type of lipids
The bioaccessibility of the carotenoids was higher from boiled than
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Key words: Excipient emulsion; bioaccessibility; carotenoids; gastrointestinal;
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digestion
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INTRODUCTION
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Carotenoids are a group of highly hydrophobic molecules with a tetraterpenoid
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structure that consists of a long hydrocarbon chain containing many conjugated
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double bonds 1. Carotenoids contribute to the characteristic colors of many plant and
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animal species due to their ability to absorb electromagnetic radiation in the visible
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region 2. In addition, they have been reported to exert various health benefits when
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consumed orally, such as provitamin A activity 3, 4, reduced risk of certain cancers 5-7,
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reduced risk of cardiovascular diseases 8-10, and ability to control obesity 11.
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number of different types of carotenoids have been identified at appreciable levels in
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certain fruits and vegetables, such as carrot, broccoli, kale, spinach, tomato, red
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pepper, cherry, orange, mango 12-16. Consequently, consumption of these natural
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products may have a beneficial impact on human health and wellbeing due to the
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presence of these bioactive carotenoids.
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from fruits and vegetables has been reported to be relatively low and/or highly
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variable 17, 18, which has been attributed to various causes, including limited release
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from plant tissues and low solubilization in gastrointestinal fluids 16, 19, 20.
A large
However, the bioavailability of carotenoids
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An effective approach to increase the bioaccessibility of carotenoids from fruits
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and vegetables is to consume the carotenoid-rich foods with excipient foods 19, 21-25.
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The composition and structure of excipient foods is specifically designed to enhance
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the bioaccessibility of nutraceuticals in nutraceutical-rich foods that are co-ingested
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with it. For example, excipient foods have recently been shown to increase the
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bioaccessibility of curcumin (another highly hydrophobic nutraceutical) from powders
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21, 22
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carotenoids from fruits and vegetables.
. A similar approach can also be adopted to increase the bioaccessibility of
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Oil-in-water emulsions, which consist of lipid droplets dispersed within an
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aqueous medium, are particularly suitable for utilization as excipient foods. The
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composition, size, and interfacial properties of the lipid droplets can be manipulated,
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and lipophilic, amphiphilic, or hydrophilic ingredients can be added to the oil,
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interfacial, or aqueous phases.
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type on the ability of excipient emulsions to increase the bioaccessibility of
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carotenoids from carrots.
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within carrots and must therefore be released from the plant tissues before they can be
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solubilized and absorbed 16.
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phase composition on the bioaccessibility of lipophilic nutraceuticals encapsulated
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within nanoemulsion- or emulsion-based delivery systems.
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that lipid composition had an appreciable influence on the bioaccessibility of the
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lipophilic nutraceuticals 26-30, which was attributed to differences in the solubilization
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capacities of the mixed micelles formed by different lipid digestion products.
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Digestible lipids (such as corn and olive oil) were shown to improve nutraceutical
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bioaccessibility, while indigestible lipids (such as lemon, orange, or mineral oil) had a
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limited impact 26, 27, 30. Moreover, digestible lipids containing long chain fatty acids
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(LCFAs) were more efficient at increasing bioaccessibility than those containing
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medium chain fatty acids (MCFAs) 28, 31, 32. However, much less is known about the
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influence of lipid composition on the ability of excipient emulsions to increase
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nutraceutical bioaccessibility from fruits and vegetables, where the carotenoids first
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have to be liberated from the plant tissues prior to solubilization.
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study, MCT, corn oil, and fish oil were used to prepare excipient emulsions because
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they are food-grade digestible lipids with appreciably different fatty acid
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compositions 26: MCT mainly contains medium chain saturated fatty acids (C8 and
In this study, we focus on the influence of lipid phase
Carotenoids are initially present in a crystalline form
Previous studies have examined the influence of lipid
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This research has shown
In the present
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C10); Corn oil mainly contains long chain unsaturated fatty acids (C16 and C18); fish
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oil mainly contains long chain monounsaturated and polyunsaturated fatty acids (C18
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to C22).
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Carrots are typically consumed in either a raw or cooked formed.
It has been
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reported that thermal treatments can increase carotenoid bioaccessibility from many
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carotenoid-rich plants, which has been attributed to plant wall disruption, swelling,
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and separation, as well as to pectin dissolution and de-polymerization 20, 33-37.
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However, some studies have reported that heat treatment may have a limited or even
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negative effect on carotenoid bioaccessibility 38-41. These differences in the effects of
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thermal treatments may be due to a number of factors: variations in mechanical
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treatments used to prepare plant tissues (such as peeling, chopping, or slicing) leading
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to different plant tissue shapes and sizes; variations in thermal treatments (such as
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time, intensity, and nature) leading to different levels of plant tissue disruption; and,
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the presence of other food components (such as lipids, proteins, carbohydrates, or
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minerals) leading to differences in gastrointestinal fate; variations in the sophistication
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of the gastrointestinal model used to determine the bioaccessibility. For this reason,
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we also examined the influence of thermal processing (raw versus boiled) on
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carotenoid bioaccessibility from carrots using a simulated gastrointestinal model.
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Mixtures of carrot and excipient emulsion were passed through a simulated
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gastrointestinal tract (GIT), which included mouth, stomach, and small intestine
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phases, and then the bioaccessibility of the carotenoids was measured.
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the size, charge, organization, and digestion of the lipid particles were also measured
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throughout the GIT model to provide some insight into the origin of any differences in
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gastrointestinal fate for these systems.
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MATERIALS AND METHODS
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Materials
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Raw carrots were purchased from a local supermarket. Whey protein isolate (WPI)
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was obtained from Davisco Foods International Inc. (Le Sueur, MN). As stated by the
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manufacturer, the protein content was 97.6% (dry basis). Medium chain triglyceride
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(MCT) oil was obtained from Coletica (Northport, NY). Corn oil was purchased from
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a commercial food supplier (Mazola, ACH Food Companies, Memphis, TN). The
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manufacturer reported that the saturated, monounsaturated, and polyunsaturated fat
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content of this product were approximately 14, 29, and 57%, respectively. Fish oil
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was purchased from DSM Nutritional Products Ltd. (Basel, Switzerland). The
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manufacturer reported that DHA, EPA and total omega-3 levels in this product were
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14.8, 10.1, and 31.2%, respectively. Mucin from porcine stomach, pepsin from
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porcine gastric mucosa (250 units/mg), porcine lipase (100-400 units/mg), and
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porcine bile extract were purchased from Sigma-Aldrich (Sigma Chemical Co., St.
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Louis, MO).
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St. Louis, MO) and α-carotene was purchased from Sinostandards Bio-Tech Co., Ltd.
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( Chendu, China). HPLC grade methanol and MTBE were purchased from Fisher
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Scientific (Pittsburgh, PA). All other chemicals used in this paper were purchased
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from either Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO) or Fisher Scientific
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(Pittsburgh, PA). All solvents and reagents were of analytical grade. Double distilled
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water from a water purification system (Nanopure Infinity, Barnstaeas International,
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Dubuque, IA) was used for preparation of all solutions.
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Carrot preparation
β-carotene was purchased from Sigma-Aldrich (Sigma Chemical Co.,
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Fresh carrots were cut into disks (approximately 10 mm high and 15 mm wide).
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Individual disks were either left raw or boiled for 10 min at 100 ºC. Raw or boiled
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carrot were then mixed with an equal mass of excipient emulsion and then placed in a
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household blender for 1 min to breakdown the carrot structure.
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Emulsion preparation
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A stock emulsion was prepared by homogenizing an oil phase (8 wt%) with an
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aqueous phase (92 wt%) using a high-speed blender for 2 min (M133/1281-0, Biospec
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Products, Inc., ESGC, Switzerland) followed by passing through a microfluidizer
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(M110Y, Microfluidics, Newton, MA) with a 75 µm interaction chamber (F20Y) at
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11,000 psi for 5 passes.
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the aqueous phase consisted of WPI emulsifier solution (emulsifier-to-oil: 1:10, pH
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7.0).
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stock emulsion with buffer solution.
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In vitro digestion model
The oil phase consisted of MCT, fish oil or corn oil, while
Emulsions containing 4 wt% oil contents were then prepared by diluting the
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The mixture of carrot and emulsion was passed through a GIT model designed to
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mimic the mouth, stomach, and small intestine phases. This model was a slight
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modification of that described in detail in our previous study 42, and so is only briefly
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summarized below.
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Initial system: An aliquot (20 mL) of carrot/emulsion mixture was placed into a
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glass beaker in an incubated shaker (Innova Incubator Shaker, Model 4080, New
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Brunswick Scientific, New Jersey, USA) at 37 ºC.
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Mouth phase: 20 mL of simulated saliva fluid (SSF) containing 0.03 g/mL mucin
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was preheated to 37 ºC and then mixed with the initial systems. After being adjusted
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to pH 6.8, the mixture was incubated in the incubator shaker for 10 min at 37 ºC to
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mimic agitation in the mouth.
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Stomach phase: 20 mL of the “bolus” sample resulting from the mouth phase was
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mixed with 20 mL of simulated gastric fluid (containing 0.0032 g/mL pepsin) that had
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been preheated to 37 ºC, and then the pH was adjusted to 2.5. This mixture was
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incubated in the incubator shaker for 2 h at 37 ºC to mimic stomach conditions.
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Small intestine phase: 30 g of “chyme” sample from the stomach phase was
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poured into a 100 mL glass beaker that was placed into a water bath at 37 ºC and then
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adjusted to pH 7.00. 1.5 mL of simulated intestinal fluid was added to the reaction
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vessel, followed by 3.5 mL of bile salt solution with constant stirring. The pH of the
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reaction system was adjusted back to 7.00. 2.5 mL of lipase solution was then added
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to the sample and an automatic titration unit (Metrohm,USA Inc.) was used to monitor
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the pH and maintain it at pH 7.0 by titrating 0.25 M NaOH solution into the reaction
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vessel for 2 h at 37 ºC. The amount of free fatty acids released was calculated from
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the titration curves as described previously 43.
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Particle size and charge measurements
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The particle size distribution and ζ-potential of the particles in the samples were
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measured as they passed through the various stages of the GIT model. It was not
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possible to obtain information about the lipid droplets in the presence of the
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homogenized carrot tissue because the large plant tissue fragments (around 200 µm)
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dominated the light scattering signal. For this reason, the plant tissue fragments were
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removed prior to particle size and charge analysis.
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samples with buffer solution, allowing the plant tissue fragments to sediment to the
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bottom of the test tubes due to gravity, and then collecting the upper layer of emulsion.
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It should be noted that dilution and sedimentation may have altered the size and
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charge of the particles originally present in the system.
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measured the microstructure of the samples using microscopy (next section).
This was achieved by diluting the
For this reason, we also
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The particle size distribution of the emulsions was determined using static light
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scattering (Mastersizer 2000, Malvern Instruments Ltd., Malvern, Worcestershire,
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UK), which utilizes measurements of the angular scattering pattern of emulsions.
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Samples were diluted in aqueous solutions and stirred in the dispersion unit with a
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speed of 1200 rpm to ensure homogeneity. Phosphate buffer (5 mM, pH 7.0) was used
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to dilute initial, mouth, and small intestine samples, while pH 2.5 adjusted distilled
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water was used to dilute stomach samples. Average particle sizes are reported as the
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surface-weighted mean diameter (d32).
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The ζ-potential of emulsions was measured using an electrophoresis instrument
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(Zetasizer Nano ZS series, Malvern Instruments Ltd. Worcestershire, UK). Prior to
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analysis, initial, mouth, and small intestine samples were diluted with 5 mM
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phosphate buffer (pH 7.0), whereas stomach samples were diluted with pH
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2.5-adjusted distilled water.
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Microstructure measurements
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The microstructures of samples were measured after exposure to the various
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stages of the GIT model using either optical or confocal scanning laser microscopy
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with a 20× objective lens or a 60× oil immersion objective lens (Nikon D-Eclipse C1
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80i, Nikon, Melville, NY, US.). Before analysis 2 mL samples were mixed with 0.1
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mL Nile Red solution (1 mg/mL ethanol) to dye the oil phase. The excitation and
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emission spectrum for Nile red were 543 nm and 605 nm, respectively. An aliquot of
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sample was placed on a microscope slide, covered by a cover slip, and then
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microstructure images were acquired using image analysis software (NIS-Elements,
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Nikon, Melville, NY).
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Bioaccessibility and Transformation
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The overall bioavailability of a carotenoid depends on its bioaccessibility, 44-46
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absorption, and transformation within the gastrointestinal tract
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study, we only focused on the bioaccessibility and transformation of the carotenoids.
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The bioaccessibility was taken to be the fraction of carotenoids present within the
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digesta resulting from the small intestine phase that were solubilized within the mixed
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micelle phase, whereas the transformation was taken to be the fraction of carotenoids
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present within the digesta compared to the initial amount.
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The bioaccessibility of the carotenoids was determined after each sample had
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been subjected to the full in vitro digestion process using a method described
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previously
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and centrifuged at 18,000 rpm (41657 × g), 4 ºC for 50 min, which resulted in
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samples that contained a sediment at the bottom with a clear supernatant above. The
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supernatant was collected and assumed to be the “micelle” fraction, in which the
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carotenoids were solubilized. The bioaccessibility was calculated from the
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concentrations of total carotenoids determined in the micelle fraction and supernatant
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using the procedure described previously. The bioaccessibility of carotenoids was then
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calculated using the following equation:
26, 47
. After the small intestinal stage, raw digesta samples were collected
= 100 ×
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Where, Cmicelle and CDigesta are the concentrations of carotenoids in the mixed micelle
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phase and in the overall digesta after the simulated intestinal digestion, respectively.
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The transformation of the carotenoids calculated using the following expression:
= 100 ×
!"
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Here, CInitial is the initial concentration of carotenoids in the samples prior to passage
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through the simulated gastrointestinal tract.
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methods are described in the following sections.
The carotenoid extraction and analysis
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Carotenoid extraction and HPLC procedure
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The extraction method was adapted from
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with some modification. In brief, 3
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mL digesta or micelle aqueous phase was extracted using a hexane:acetone (1:1, v/v)
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mixture, vigorously shaken, and then centrifuged for 2 min at 4000 rpm (1788.8 ×g).
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The supernatant layer was collected in a second tube. The extraction process was
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repeated three times. The combined organic fractions were mixed with saturated
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sodium chloride solution and the mixture was shaken vigorously. After the
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supernatant hexane layer was collected, the lower phase was extracted again with
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hexane. Combined supernatant hexane phases were then diluted with hexane to an
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appreciate concentration and filtered through 0.45 µm filter (VWR International,
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Philadephia, PA, USA) to be analyzed by HPLC. All procedures were carried out on
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ice and with low light exposure.
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The HPLC system (Agilent 1100 series, Agilent Technologies, Santa Clara, CA,
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USA) consisted of a binary solvent delivery system, an on-line degasser, an
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auto-sampler, a column temperature controller, a diode array detector (DAD), and a
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variable wavelength detector (VWD). System control and data analysis were
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processed using instrument software (Agilent ChemStation). A C-30 reversed phase
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column (250 mm × 4.6 mm id, 5 µm, YMC Carotenoid, YMC Inc., Wilmington, NC)
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was used as the stationary phase. The injection volume was 20 µL and the flow rate
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was 1 mL/min. The detection wavelength was set at 450 nm. The mobile phase was
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composed of (A) methanol/MTBE/1M ammomium acetate (95:3:2 v/v/v) and (B)
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methanol/MTBE/1M ammonium acetate (25:75:2 v/v/v). A linear gradient program
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was performed as follows: initial condition of mobile phase A: B was 85:15; followed
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70:30 for 10 min, 52:48 for 12 min, 52:48 for 18 min, 35:65 for 26 min and then back
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to the initial condition for 30 min to allow re-equilibration. The content of α-carotene
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and β-carotene in the samples were calculated from carotenoid standard curves.
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Statistical analysis
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Experiments were carried out using two or three freshly prepared samples. The
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results are reported as averages and standard deviations, and the differences among
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treatments were calculated based on an analysis of variance (ANOVA) and a post-hoc
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Duncan test with a confidence level of 95 %. These analyses were carried out using
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statistical analysis software (SPSS, IBM Corporation, Armonk, NY, USA).
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RESULTS AND DISCUSSIONS
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Carotenoid type in carrots
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The type of carotenoids in the carrots was initially determined by HPLC
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chromatography, which indicated that the dominant carotenoids were β-carotene and
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α-carotene (Figure 1). This finding is in agreement with previous studies of the
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carotenoid content of carrots
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bioaccessibility of β-carotene and α-carotene in this study because the levels of the
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other carotenoids were so low.
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Physical and structural properties of excipient emulsions in GIT model
49-51
. For this reason, we only investigated the
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Information about changes in the physical and structural properties of emulsified
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food systems (such as droplet size, aggregation state, and charge) as they pass through
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the GIT is important for a number of reasons. First, the size of the individual lipid
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droplets in an emulsion determines the surface area of the lipid phase exposed to the
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digestive enzymes and other components in the surrounding aqueous phase.
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the aggregation state of the lipid droplets (e.g., free versus flocculated) will influence
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the ability of digestive enzymes to reach the surfaces of the lipid droplets. It will be
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harder for lipases to adsorb to the surfaces of the lipid droplets in the interior of a floc,
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than those on the edges.
Second,
Third, the electrical charge of the colloidal particles in the
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GIT provides indirect information about their interfacial composition, as well as
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determining their interactions with any charged components in the gastrointestinal
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fluids.
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Before exposure to the GIT model, excipient emulsions prepared using MCT, fish
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oil, or corn oil all had fairly similar mean particle diameters (d32 ≈ 0.14 µm) and
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monomodal particle size distributions (Figures 2 and 3). On the other hand, there
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were some differences in the electrical properties (ζ-potential) of the lipid droplets
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made using different oils: -54.7, -67.3 and -66.3 mV for MCT, fish oil and corn oil,
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respectively. The highly negative charge on the lipid droplets can be attributed to the
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fact that the adsorbed proteins are well above their isoelectric point (pI = 5.1) under
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neutral pH conditions. The slight differences in the magnitude of the electric charges
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on the different excipient emulsions can be attributed to impurities in the lipid phases,
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such as free fatty acids or phospholipids. The influence of lipid type on droplet charge
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has also been reported in previous studies 52.
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Initial: After blending with carrot (and then removing by sedimentation), the
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initial mean particle diameter (d32) of all the excipient emulsions increased
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significantly: 1.91, 2.22, and 1.66 µm for MCT, fish oil and corn oil, respectively
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(Figure 2a). However, all emulsions still maintained a monomodal particle size
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distribution (Figure 3a) and appeared fairly uniform in the confocal images (Figure
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4a).
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droplet flocculation or coalescence had occurred.
313
occurred due to the high shear forces involved in the blending process, or due to some
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components released from the carrots that promoted droplet flocculation (such as
315
acids, biopolymers, or minerals).
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have been because some of the smaller droplets present within the original excipient
The increase in particle size after blending with the carrots suggests that some Droplet aggregation may have
Alternatively, the increase in particle size may
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emulsions were preferentially trapped within the plant tissue structure.
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The microstructures of carrot/emulsion mixtures (that had not been separated by
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sedimentation) were examined using a combination of optical and confocal
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microscopy (Figure 4b). The optical microscopy images indicated that the plant cell
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tissue from the carrots remained partly intact, while the confocal microscopy images
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indicated that some of the lipid droplets were distributed around the carrot fragments
323
but that others were internalized within the plant cell tissue.
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observed for excipient emulsions containing all three types of oil phase (images not
325
shown).
326
due to capillary forces generated by the pores in the cellular structure or due to
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specific attractive interactions between the lipid droplets and components within the
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cellular structure (e.g., electrostatic or hydrophobic attraction).
Similar behavior was
The internalization of the lipid droplets by the plant tissue may have been
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There was no appreciable difference in the mean particle diameter of excipient
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emulsions that had been mixed with either raw or boiled carrot (Figures 2b).
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However, there did appear to be a greater accumulation of lipids within the carrot
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tissues (higher fluorescence intensity) for the boiled carrots than for the raw carrots
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(Figure 4b). This difference suggests that boiling promoted the disruption of the plant
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cell tissue, thereby making them more accessible to interaction with the lipid droplets.
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For example, cellular tissue disruption may have led to an increase in pore size, which
336
allowed more lipid droplets to be internalized by the carrot pieces.
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discussed later, this effect may account for the higher bioaccessibility of carotenoids
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from boiled carrot than from raw carrot.
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similarly in the GIT, and therefore only the results for the fish oil are shown.
As will be
All of the different oils behaved fairly
340
Compared to the original excipient emulsions, the magnitude of the negative
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charge on the lipid droplets decreased after mixing with the carrots, e.g., -38.6, -39.5
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and -35.6 mV for the MCT, fish oil, and corn oil, respectively (Figures 5a and 5b).
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This result suggests that there may have been some components released from the
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carrot tissue during the blending process that altered the surface charge or ionic
345
strength of the system, such as acids, minerals, or biopolymers.
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may have altered the ζ-potential through electrostatic screening or ion binding effects
347
53
These components
.
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Mouth: After passing through the oral stage, the mean particle diameter of all the
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emulsions measured using light scattering increased, which was most obvious in the
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samples containing boiled carrots (Figure 2a and 2b). A corresponding rightwards
351
shift in the particle size distribution was observed for each sample. However,
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extensive flocculation of the same emulsions was observed in the confocal
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microscopy images (Figure 4a). Droplet flocculation may be attributed to depletion
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effects resulting from the presence of non-adsorbed mucin molecules within the saliva:
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this type of floc is only held together by weak attractive forces and is easily separated
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by dilution and stirring stir during light scattering measurements
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confocal microscopy images of the carrot/emulsion mixtures suggest that some of the
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lipid droplets were internalized within the carrot tissue (Figure 4b), which again may
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be due to capillary forces or specific interactions.
42
. The optical and
360
The magnitude of the negative charge on all the samples decreased appreciably
361
after incubation in the mouth stage (Figures 5a and 5b). This effect can be attributed
362
to the presence of mineral ions and mucin in the saliva solution, which reduced the
363
surface potential through electrostatic screening and binding effects 53.
364
Stomach: After being exposed to the gastric phase, the particle size of all the
365
excipient emulsions increased further (Figures 2a and 2b), and there was evidence of
366
extensive flocculation in the confocal microscopy images (Figure 4a). In addition,
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367
there appeared to be greater accumulation of the lipid droplets within the carrot tissue
368
for all samples (Figure 4b).
369
droplets decreased to close to zero after incubation in the gastric fluids, which can be
370
attributed to the low pH and high ionic strength of the simulated stomach phase
371
(Figures 5a and 5b).
372
positively charged at a pH below their isoelectric point was probably because of the
373
adsorption of anionic mucin molecules to the cationic-protein coated surfaces, as well
374
as possibly some adsorption of anionic biopolymers released from the carrot tissue
375
(such as pectins).
376
composition and properties due to the action of the gastric proteases in the simulated
377
gastric fluids on the adsorbed protein layer 54.
The magnitude of the electrical potential of the lipid
The fact that the protein-coated droplets did not become
In addition, there may have been some changes in interfacial
378
Small Intestine: After exposure to the small intestine phase, the particle sizes of
379
all the systems became fairly similar and were relatively small (Figures 2a and 2c)
380
and almost no lipid droplets could be seen in the confocal microscopy images (Figure
381
4a). The small irregularly shaped lipid-rich particles observed in the small intestine
382
phase are likely to be mixed micelles formed from lipid digestion products (FFAs and
383
MAGs) , as well as bile salts and phospholipids from the simulated intestinal fluids.
384
Interestingly, we did not observe a strong fluorescence signal within the carrot tissue
385
after exposure to small intestine conditions (Figure 4b), which suggested that any
386
lipid droplets trapped within them had been digested by lipase.
387
Compared to the samples in the stomach stage, there was an appreciable increase
388
in the magnitude of the negative charge on the particles in all of the samples after
389
exposure to small intestine conditions (Figures 5a and 5b). This relatively high
390
negative charge can be attributed to the presence of various types of anionic particles
391
in the digesta, such as bile salts, phospholipids, free fatty acids, and undigested lipid
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392
droplets. In addition, there was a noticeable difference in the surface charges of the
393
particles formed from different excipient emulsions: -24.4, -32.8 and -44.4 mV for
394
MCT, fish oil, and corn oil with raw carrot, respectively. This result indicated that the
395
composition and properties of the particles formed by various lipids after digestion
396
was quite different. This difference may be related to the nature of the lipid digestion
397
products formed after lipase hydrolysis.
398
more readily that MCFAs , thereby leading to a higher negative charge.
399
Intestinal digestion of excipient emulsions
LCFAs may accumulate at particle surfaces
400
In this section, the influence of lipid type on the rate and extent of excipient
401
emulsion digestion was examined using an automatic titration method (pH stat). The
402
volume of NaOH (0.25M) titrated into the samples to maintain a constant pH (7.0)
403
was measured as a function of digestion time throughout the small intestine phase
404
(Figure 6) and the amount of free fatty acids released was calculated from these
405
profiles (Figure 7).
406
The total amount of NaOH titrated into the samples depended on lipid type
407
(Figures 6a and 6b): a higher amount of alkaline solution was required to neutralize
408
the FFAs produced by MCT emulsions than by fish oil and corn oil emulsions. The
409
greater amount of NaOH required for MCT is due to its lower molecular weight,
410
which means there are a higher number of free fatty acids being produced per gram of
411
lipid. The overall digestion profiles of the samples containing different excipient
412
emulsions were fairly similar: there was a rapid initial production of free fatty acids
413
during the first 10 minutes followed by a more gradual increase at longer incubation
414
times (Figures 7a and 7b). The most noticeable difference between the FFA profiles
415
was that corn oil appeared to produce the least amount of fatty acids at longer
416
digestion times.
This result is consistent with the confocal microscopy images
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Page 18 of 43
417
collected after small intestine digestion, in which there was a fluorescence signal in
418
the samples containing corn oil; but, almost nothing in the samples containing MCT
419
and fish oil.
420
the lipid droplet surfaces, thereby inhibiting further lipid digestion 55.
421
Carotenoid bioaccessibility and transformation
A likely reason for this phenomenon is that the LCFAs accumulate at
422
In this section, we investigated the influence of lipid type on the bioaccessibility
423
and transformation of carotenoids original located within carrots. The bioaccessibility
424
was taken to be the fraction of carotenoids in the digesta that were present within the
425
mixed micelle phase, while the transformation was taken to be the total amount of
426
carotenoids in the digesta relative to the total amount of carotenoids in the initial
427
sample.
428
Previous studies have shown that the bioaccessibility of carotenoids can be
429
increased by incorporating digestible lipids into the system since they form mixed
430
micelles that can solubilize these highly hydrophobic molecules
431
bioaccessibilities of α-carotene and β-carotene were therefore measured because they
432
are the major carotenoids in carrots. A slightly higher bioaccessibility was observed
433
for α-carotenoids than for β-carotenoids for all samples, which is consistent with
434
previous reports 20.
20, 56
. The
435
The bioaccessibility of carotenoids from carrot clearly depended on the lipid type
436
used to prepare the excipient emulsions (Figures 8a and 8b). The highest
437
bioaccessibility was observed for excipient emulsions containing corn oil, followed
438
by those containing fish oil. On the other hand, an extremely low bioaccessibility was
439
observed for the carrots mixed with emulsified MCT. This result is consistent with
440
previous studies that have shown that long chain triacylglycerols enhance carotenoid
441
bioaccessibility more effectively than medium chain triacylglycerols when added to a
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29, 57
442
carotenoid-rich salad or lycopene-rich tomato pulp
. Similar trends were also
443
found in emulsion-based delivery systems containing lipophilic components e.g.,
444
β-carotene or vitamin E 26, 28. This effect can be attributed to the fact that the micelles
445
formed by long chain fatty acids have higher solubilization capacity for relatively
446
long hydrophobic components (such as carotenoids) compared with those formed by
447
medium chain fatty acids 26.
448
by LCFAs are large enough to accommodate carotenoids, whereas those formed by
449
MCFAs are too small 58.
The hydrophobic regions in micelles or vesicles formed
450
The fact that the carotenoid bioaccessibility was lower for fish oil than for corn
451
oil may be due to differences in the degree of unsaturation of the fatty acid chains.
452
Our result is in agreement with previous research that showed a slightly lower
453
lycopene bioaccessibility when mixed with fish oil than with sunflower oil 29.
454
oil contains a relatively high proportion of polyunsaturated fatty acids, which tend to
455
have non-polar tails that adopt a kinked (rather than linear) structure.
456
the dimensions of the hydrophobic domains in the micelles and vesicles formed from
457
fish oil may be smaller than those formed from corn oil.
Fish
Consequently,
458
The bioaccessibility of the carotenoids was appreciably higher for boiled carrots
459
that for raw carrots, e.g., it was around 12 and 25% for raw and boiled carrots mixed
460
with corn oil excipient emulsions (Figure 8).
461
boiling may occur due to a number of reasons: swelling, separation, and disintegration
462
of cell walls, as well as dissolution and depolymerisation of pectin molecules 20, 33-36.
The increase of bioaccessibility by
463
The transformation of the carotenoids was also determined for the raw carrots by
464
measuring the amount present within the digesta compared to the original amount
465
present. The percentage of carotenoids remaining after transformation depended on
466
lipid type, e.g., for α-carotene the values were 82 ± 7%, 80 ± 8% and 102 ± 13%,
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Page 20 of 43
467
while for β-carotene the values were 70 ± 14%, 79 ± 7% and 91 ± 19% for fish oil,
468
corn oil, and MCT, respectively. The decrease in the amount of carotenoids present
469
may have occurred due to their isomerization and/or oxidation within the simulated
470
gastrointestinal conditions, particularly within the highly acidic environment of the
471
stomach
472
number of ways.
473
non-polar and polar environments during their passage throughout the GIT.
474
Carotenoid degradation would be expected to occur more rapidly when the
475
carotenoids are present within an aqueous phase than within an organic phase.
476
Second, different types of oils may naturally contain different types of antioxidants
477
and pro-oxidants, which would affect carotenoid stability to oxidation reactions.
478
both carotenoids there was less transformation when MCT was used as an oil phase
479
than when fish or corn oil was used, which may be because MCT consists mainly of
480
saturated medium chain fatty acids that are more stable to oxidation.
481
bioaccessibility (B*) and transformation (T*) data for the raw carrots we estimated
482
that the fraction of the original carotenoids available for absorption (B*×T*) was
483
around 6.8%, 11.1% and 0.2% for α-carotene and around 4.6%, 9.2% and 0.1 % for
484
β-carotene when fish oil, corn oil, or MCT were used as the oil phase, respectively.
485
These results clearly show that oil type has an important influence on the ability of
486
excipient emulsions to increase the bioavailability of carotenoids from natural
487
sources.
44
. Oil type may influence the degree of transformation of carotenoids in a First, oil type may affect the partitioning of carotenoids between
For
Based on the
488
In conclusion, the bioaccessibility of carotenoids from many fruits and vegetables
489
is relatively low due to restricted release from the plant tissue matrix and low
490
solubilization in the gastrointestinal fluids. This study has shown that an effective
491
approach to increase the bioaccessibility of carotenoids from raw and cooked carrots
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492
is to consume them with excipient emulsions. The bioaccessibility of carotenoids
493
from carrot depended on the lipid type of the excipient emulsions: corn oil > fish oil >>
494
MCT, which was attributed to the different solubilization capacities of the mixed
495
micelle phases formed after lipid digestion.
496
optimizing the nature of the oil phase used to design excipient emulsions.
497
bioaccessibility of the carotenoids was higher for boiled than for raw carrots, which
498
may due to swelling and disintegration of the cell wall matrix at high temperatures.
499
In conclusion, excipient emulsions may be used to increase the bioaccessibility of
500
hydrophobic nutraceuticals from fruits and vegetables.
501
useful for designing diets for improved health and wellness.
502
desirable to encourage people to consume fruits and vegetables with specially
503
designed creams, sauces, or dressings.
504
ACKNOWLEDGEMENTS
505
This result highlights the importance of The
This knowledge may be For example, it may be
This material is based upon work supported by the United States Department of
506
Agriculture, NRI grants (2013-0379 and 2013-03795).
This project was also partly
507
supported by the National Natural Science Foundation of China (NSFC31428017).
508 509
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30000 2
Absorbance (mAU)
25000
20000 1 15000
10000
5000
0
0 665
5
10
15
20
25
30
Retention time (min)
666
Figure 1. HPLC profile (absorbance versus retention time) of carotenoids isolated
667
from carrots. Peaks: 1. α-carotene;2. β-carotene.
668
ACS Paragon Plus Environment
Page 29 of 43
Journal of Agricultural and Food Chemistry
Raw
MCT
Fish Oil
Corn Oil
Mean Particle Diameter (µm)
Aa Ab
10
Ac
Ba Bb
Bb
Ca
Ba
Cb
1
Da Cb
Db
0.1 Initial
Mouth
Stomach Intestine
669 670
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Mean Particle Diameter (µm)
Boiled
MCT
Fish Oil
10 Ab Ba Ba Bb
Ca Cb
Page 30 of 43
Corn Oil
Aa
Ac
Bb
1
Ca
Da Da
0.1 Initial
Mouth
Stomach Intestine
671 672
Figure 2. Surface-weighted mean particle diameter (d32) of excipient emulsions
673
containing different lipid types after mixing with (a) raw or (b) boiled carrots and
674
exposing to simulated GIT conditions. Samples designated with different capital
675
letters (A, B, C) were significantly different (Duncan, p < 0.05) when compared
676
between different GIT regions (same lipid type). Samples designated with different
677
lower case letters (a, b, c) were significantly different (Duncan, p < 0.05) when
678
compared between different lipid types (same GIT region).
679 680
ACS Paragon Plus Environment
Page 31 of 43
Journal of Agricultural and Food Chemistry
681 682
60 Raw Initial
Volume Fraction (%)
50 40
Mouth
30 Stomach
20 10 0 0.01
Intestine
0.1
1
10
100
Particle Diameter (µm) 683 684
ACS Paragon Plus Environment
1000 10000
Journal of Agricultural and Food Chemistry
685
Volume Fraction (%)
60
Boiled
50
Initial
40 Mouth
30 20
Stomach
10 Intestine
0 0.01
0.1
1
10
100
1000 10000
Particle Diameter (µm) 686 687
Figure. 3. Particle size distribution of excipient emulsions containing fish oil after
688
incubation with (a) raw or (b) boiled carrots and exposure to different GIT regions.
689
Similar results were obtained with other oil types (not shown).
690 691
ACS Paragon Plus Environment
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Page 33 of 43
Journal of Agricultural and Food Chemistry
692 693
Figure 4a. Microstructure of excipient emulsions containing different lipid types after
694
they were exposed to different regions of a simulated GIT. The emulsions were
695
isolated from the carrot tissue fragments by gravitational separation prior to analysis.
696
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
697 698
Figure 4b. Microstructure of carrot/emulsion mixtures (containing fish oil) after they
699
were exposed to different regions of a simulated GIT.
700
show the microstructure of the carrot tissue, where confocal microscopy images show
701
the location of the lipid phase.
702
(data not shown).
Optical microscopy images
Similar results were obtained for the other oil types
703
ACS Paragon Plus Environment
Page 34 of 43
Page 35 of 43
Journal of Agricultural and Food Chemistry
Raw Initial
Mouth
Stomach
Small Intestinal
0 Aa Aa Aa
ζ-potential (mV )
-10
-20 Ba
-30
Ba Cb
Bb
Cab
-40
Bb
Dc Cbc
MCT
Fish Oil
Corn Oil
-50 704 705
ACS Paragon Plus Environment
Dc
Journal of Agricultural and Food Chemistry
Boiled Initial
Mouth
Stomach
Page 36 of 43
Small Intestinal
0 Ac Ab Aa
ζ-potential (mV )
-10
-20 Ba
-30
Ba
Ba Cc
-40
Da Ca
Bbc Cb
Ca
MCT
Fish Oil
Corn Oil
-50 706 707
Figure 5. Electrical characteristics (ζ-potentials) of excipient emulsions containing
708
different lipid types after mixing with (a) raw or (b) boiled carrots and exposure to
709
different regions of the GIT. Samples designated with different capital letters (A, B, C)
710
were significantly different (Duncan, p < 0.05) when compared between different GIT
711
regions (same lipid types). Samples designated with different lower case letters (a, b,
712
c) were significantly different (Duncan, p < 0.05) when compared between different
713
lipid types (same GIT region).
ACS Paragon Plus Environment
Page 37 of 43
Journal of Agricultural and Food Chemistry
714
Raw 3.5 3.0
NaOH Volume (mL)
2.5 2.0 1.5 MCT
1.0
FIsh Oil Corn Oil
0.5 0.0 0
20
40
60
80
Digestion Time (min) 715 716
ACS Paragon Plus Environment
100
120
Journal of Agricultural and Food Chemistry
Page 38 of 43
Boiled 3.5 3.0
NaOH Volume (mL)
2.5 2.0 1.5 MCT
1.0
FIsh Oil Corn Oil
0.5 0.0 0
20
40
60
80
100
120
Digestion Time (min) 717 718
Figure 6. Volume of NaOH (0.25 M) required to maintain a constant pH (7.00) in
719
carrot/emulsion mixtures containing different lipid types as measured using a pH-stat
720
method: (a) raw carrot; (b) boiled carrot.
721
ACS Paragon Plus Environment
Page 39 of 43
Journal of Agricultural and Food Chemistry
Raw 120
FFA Released (%)
100 80 60 MCT
40
Fish Oil Corn Oil
20 0 0
20
40
60
80
Digestion Time (min) 722
ACS Paragon Plus Environment
100
120
Journal of Agricultural and Food Chemistry
Page 40 of 43
Boiled 120
FFA Released (%)
100 80 60 MCT
40
FIsh Oil Corn Oil
20 0 0
20
40
60
80
100
120
Digestion Time (min) 723 724
Figure 7. Amount of fatty acids released from carrot/emulsion mixtures containing
725
different lipid types as measured using a pH-stat method: (a) raw carrot; (b) boiled
726
carrot.
727 728
ACS Paragon Plus Environment
Page 41 of 43
Journal of Agricultural and Food Chemistry
Raw 30% α-carotene
Bioaccessibility (%)
25%
β-carotene
20% A
15%
A B
10%
B
5% 0%
C
C
MCT
Fish Oil
729 730
ACS Paragon Plus Environment
Corn Oil
Journal of Agricultural and Food Chemistry
Boiled A A
30% α-carotene
Bioaccessibility (%)
25%
β-carotene
20% 15%
B B
10% 5% C C 0% MCT
Fish Oil
Corn Oil
731 732
Figure 8.
Influence of oil type on bioaccessibility of carotenoids from (a) raw or (b)
733
boiled carrots mixed with excipient emulsions. Samples designated with different
734
capital letters (A, B, C) were significantly different (Duncan, p < 0.05) when
735
compared between different lipid types.
736
ACS Paragon Plus Environment
Page 42 of 43
Page 43 of 43
Journal of Agricultural and Food Chemistry
Enhancing nutraceutical bioavailability from raw and cooked vegetables using excipient emulsions: Influence of lipid type on carotenoid bioaccessibility from carrots Zhang et al Graphic for Abstract
Bioaccessibility (%)
20% 15%
α-carotene β-carotene
Raw Carrots
10% 5% 0% MCT
Fish Oil Corn Oil
ACS Paragon Plus Environment