Enhancing Nutraceutical Bioavailability from Raw and Cooked

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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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ACS Paragon Plus Environment


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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

30

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

38

isomerization.

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raw carrots, which was attributed to greater disruption of the plant tissue facilitating

40

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

43 44

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

50

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


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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Changes in


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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.


.

In the current


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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

283

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

285

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

287

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

289

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,

291

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

296

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

298

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,

300

respectively. The highly negative charge on the lipid droplets can be attributed to the

301

fact that the adsorbed proteins are well above their isoelectric point (pI = 5.1) under

302

neutral pH conditions. The slight differences in the magnitude of the electric charges

303

on the different excipient emulsions can be attributed to impurities in the lipid phases,

304

such as free fatty acids or phospholipids. The influence of lipid type on droplet charge

305

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

308

significantly: 1.91, 2.22, and 1.66 µm for MCT, fish oil and corn oil, respectively

309

(Figure 2a). However, all emulsions still maintained a monomodal particle size

310

distribution (Figure 3a) and appeared fairly uniform in the confocal images (Figure

311

4a).

312

droplet flocculation or coalescence had occurred.

313

occurred due to the high shear forces involved in the blending process, or due to some

314

components released from the carrots that promoted droplet flocculation (such as

315

acids, biopolymers, or minerals).

316

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



317

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emulsions were preferentially trapped within the plant tissue structure.

318

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

321

tissue from the carrots remained partly intact, while the confocal microscopy images

322

indicated that some of the lipid droplets were distributed around the carrot fragments

323

but that others were internalized within the plant cell tissue.

324

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

327

specific attractive interactions between the lipid droplets and components within the

328

cellular structure (e.g., electrostatic or hydrophobic attraction).

Similar behavior was

The internalization of the lipid droplets by the plant tissue may have been

329

There was no appreciable difference in the mean particle diameter of excipient

330

emulsions that had been mixed with either raw or boiled carrot (Figures 2b).

331

However, there did appear to be a greater accumulation of lipids within the carrot

332

tissues (higher fluorescence intensity) for the boiled carrots than for the raw carrots

333

(Figure 4b). This difference suggests that boiling promoted the disruption of the plant

334

cell tissue, thereby making them more accessible to interaction with the lipid droplets.

335

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.

337

discussed later, this effect may account for the higher bioaccessibility of carotenoids

338

from boiled carrot than from raw carrot.

339

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

341

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).

343

This result suggests that there may have been some components released from the

344

carrot tissue during the blending process that altered the surface charge or ionic

345

strength of the system, such as acids, minerals, or biopolymers.

346

may have altered the ζ-potential through electrostatic screening or ion binding effects

347

53

These components

.

348

Mouth: After passing through the oral stage, the mean particle diameter of all the

349

emulsions measured using light scattering increased, which was most obvious in the

350

samples containing boiled carrots (Figure 2a and 2b). A corresponding rightwards

351

shift in the particle size distribution was observed for each sample. However,

352

extensive flocculation of the same emulsions was observed in the confocal

353

microscopy images (Figure 4a). Droplet flocculation may be attributed to depletion

354

effects resulting from the presence of non-adsorbed mucin molecules within the saliva:

355

this type of floc is only held together by weak attractive forces and is easily separated

356

by dilution and stirring stir during light scattering measurements

357

confocal microscopy images of the carrot/emulsion mixtures suggest that some of the

358

lipid droplets were internalized within the carrot tissue (Figure 4b), which again may

359

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,



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


Page 16 of 43

Page 17 of 43


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



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


Page 19 of 43


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%,



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


Page 21 of 43


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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663 664



Page 28 of 43

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


Page 29 of 43


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



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


Page 31 of 43


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


1000 10000


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


Page 32 of 43

Page 33 of 43


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



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


Page 34 of 43

Page 35 of 43


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


Dc


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).


Page 37 of 43


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


100

120


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


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


Page 39 of 43


Raw 120

FFA Released (%)

100 80 60 MCT

40

Fish Oil Corn Oil

20 0 0

20

40

60

80

Digestion Time (min) 722


100

120


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


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


Page 41 of 43


Raw 30% α-carotene

Bioaccessibility (%)

25%

β-carotene

20% A

15%

A B

10%

B

5% 0%

C

C

MCT

Fish Oil

729 730


Corn Oil


Boiled A A

30% α-carotene


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


Page 42 of 43

Page 43 of 43


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


20% 15%

α-carotene β-carotene

Raw Carrots

10% 5% 0% MCT

Fish Oil Corn Oil


Enhancing Nutraceutical Bioavailability from Raw and Cooked

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