Pectin and gastric pH interactively affect DHA-rich emulsion in vitro digestion microstructure, digestibility and bioaccessibility

Accepted Manuscript Pectin and gastric pH interactively affect DHA-rich emulsion in vitro digestion microstructure, digestibility and bioaccessibility Xinjie Lin, Amanda J. Wright PII:

S0268-005X(17)30995-5

DOI:

10.1016/j.foodhyd.2017.06.010

Reference:

FOOHYD 3939

To appear in:

Food Hydrocolloids

Received Date: 26 June 2016 Revised Date:

26 April 2017

Accepted Date: 8 June 2017

Please cite this article as: Lin, X., Wright, A.J., Pectin and gastric pH interactively affect DHA-rich emulsion in vitro digestion microstructure, digestibility and bioaccessibility, Food Hydrocolloids (2017), doi: 10.1016/j.foodhyd.2017.06.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Gastric stage E

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pH 4.8

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Duodenal Stage High DHA bioaccessibility

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Pectin and gastric pH interactively affect DHA-rich emulsion in vitro digestion microstructure, digestibility and bioaccessibility

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Xinjie Lin and Amanda J. Wright*

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Department of Human Health & Nutritional Sciences, University of Guelph, Guelph, ON, Canada

To whom correspondence should be addressed. Department of Human Health & Nutritional

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Sciences, University of Guelph, Guelph, ON, Canada. Email: [email protected]. Phone: 519-824-4120 x54697. Fax: 519-763-5902

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Abstract

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Lipid digestibility and bioaccessibility (i.e. solubilization) may be impacted by various factors.

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For example, dietary fibres may interact with lipids and other digestive molecules during

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gastrointestinal digestion, and gastric pH may impact lipid droplet microstructure. Thus, these

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parameters may have implications for the release and uptake of bioactive fatty acids such as

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DHA. This study investigated how apple pectin (0.00, 5.68, or 100.00 mg/5.0 g emulsion),

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combined with variable gastric pH (2.0, 3.0 or 4.8), impacted the in vitro digestion of a DHA-

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rich algal oil lecithin-stabilized emulsion (10:1.2:88.8 oil:lecithin:water, D3,2 = 0.137 ± 0.001

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nm). Low gastric pH (2.0 and 3.0) induced severe emulsion destabilization. However, the

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addition of a low-level of pectin (5.68 mg/5.0 g emulsion) reduced the destabilization at pH 3.0.

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Small lipid droplets, maintained either by this low-level of pectin at pH 3.0 or by a higher gastric

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pH (4.8), were associated with greater early (p<0.05), but not eventual (p>0.05) duodenal

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lipolysis (pH 7.0) and higher DHA bioaccessibility (>69%, p<0.05). Samples containing 100.00

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mg pectin/5.0 g emulsion had the lowest overall lipolysis and DHA bioaccessibility across all pH

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values (p<0.05). Therefore, pectin content and gastric pH interactively impacted emulsion

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digestion. The presence of applesauce, matched for pectin concentration, further destabilized the

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emulsion and limited lipid digestibility and DHA bioaccessibility, compared to the pectin-only

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samples. Overall, a stable emulsion microstructure during gastric digestion promoted in vitro

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lipid digestibility and DHA bioaccessibility.

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Key Words

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Apple pectin; Emulsification; Food matrix; In vitro digestibility; DHA bioaccessibility

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1. Introduction

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Lipids play essential, but complicated, roles in health and disease. Understanding the factors that

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impact lipid digestion and absorption can offer insights into ways to modify lipemia, with

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implications for cardiovascular disease risk. The digestion of dietary triacylglycerols (TAG) in

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the gastrointestinal (GI) tract is a critical process determining their availability for absorption,

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with the proportion of lipids solubilized within the aqueous phase of the digestate referred to as

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‘bioaccessible’ (Hedrén, Mulokozi, & Svanberg, 2002) and recognized as a critical precursor to

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bioavailability (Gregory, Quinlivan, & Davis, 2005). Various factors, including lipid

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intramolecular and supramolecular structure (Michalski et al., 2013) impact TAG digestion. For

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example, long-chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and

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docosahexaenoic acid (DHA) can be structurally resistant to lipolysis when located in the sn-1

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and sn-3 positions of a TAG (Bottino, Vandenburg, & Reiser, 1967; Brockerhoff, Hoyle, &

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Huang, 1966). In terms of supramolecular structure, emulsification can aid in digestion by

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minimizing the requirement for emulsification within the GI tract, and increasing the oil-water

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interfacial area for lipase adsorption (Michalski et al., 2013; Parada & Aguilera, 2007; Wilde &

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Chu, 2011). Therefore, pre-emulsification can be a strategy to enhance the absorption of TAGs

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containing long-chain polyunsaturated fatty acids, which tend to be lipolysis-resistant DHA and

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EPA (Walker, Decker, & McClements, 2015).

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Dietary ingredients may also impact lipid digestive processes. For example, viscous fibres have

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the potential to alter the microstructure of emulsions and therefore impact lipid digestion. This

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includes by inducing emulsion droplet aggregation, thereby reducing the interfacial area for

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lipases, thickening the intestinal contents, thereby limiting contact between lipases and lipid

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droplets, and binding calcium ions and/or bile salts which act to reduce the presence of

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accumulated FFA at lipid droplet surfaces (Dikeman & Fahey, 2006; Zhang, Zhang, Zhang,

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Andrew, & McClements, 2015). Such interactions and their impacts on lipid digestion and

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bioavailability require further investigation, especially with the intense interest in development

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of so-called functional foods and dietary recommendations related to PUFA and dietary fibre

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consumption. The apple-derived soluble fibre pectin is one of the most widely utilized soluble

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fibres by the food industry (Colin-Henrion, Mehinagic, Renard, Richomme, & Jourjon, 2009).

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Although soluble fibre consumption has widely recognized health benefits, the impact of pectin

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and other dietary fbires on the bioavailability and metabolism of other nutrients, including lipids,

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is not entirely clear. Some animal studies have shown attenuated postprandial lipemia (Lærke,

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Meyer, Kaack, & Larsen, 2007; Leclere, Lairon, Champ, & Cherbut, 1993; Suzuki & Kajuu,

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1983) or fasting blood lipid levels (Trautwein, Kunath-Rau, & Erbersdobler, 1998) with pectin

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consumption, although such lipid-lowering effects in humans are not as widely studied or

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observed (Brouns et al., 2012; Morgan & Tredger, 1993). Some in vitro studies have also shown

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that pectin has the potential to impact lipid digestion processes (Beysseriat, Decker, &

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McClements, 2006; Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, & Narváez-Cuenca, 2014;

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Verrijssen, Verkempinck, Christiaens, Van Loey, & Hendrickx, 2015; Zhang et al., 2015).

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However, experiment conditions and models have varied widely and discrepant results may

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relate to this and the fact that pectin’s physiological properties can be affected by parameters

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such as concentration (Flourie, Vidon, Chayvialle, Palma, & Bernier, 1985; Verrijssen et al.,

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2014), degree of methylation (Brouns et al., 2012; Dongowski, 1995; Trautwein et al., 1998),

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degree of amidation (Hagesaether & Sande, 2007), and the presence of other molecules such as

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emulsifiers in the digestate (Zhang et al., 2015). More research is needed to identify the

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physiological mechanisms involved in pectin’s impacts on lipid digestion.

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Indeed interactions between dietary lipids and fibres should be considered when formulating

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foods or making dietary and supplement recommendations. Interactions between ingested food

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molecules and digestive parameters are additionally important to consider in terms of possible

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impacts on GI function and nutrient absorption. For example, gastric pH can vary depending on

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stomach contents. While the pH of an empty stomach may be below 2.0, the ingestion of food

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can buffer the pH to as high as 6.7, with the pH gradually returning to fasted state levels

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(Dressman et al., 1990; Minekus et al., 2014). We recently reported that a soy lecithin-stabilized

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emulsion was extensively destabilized at an in vitro gastric pH of 1.6, and that these changes

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resulted in a significantly lower DHA in vitro bioaccessibility (Lin, Wang, Li, & Wright, 2014).

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Therefore, the purpose of this research was to investigate how the presence of apple pectin (5.68

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or 100.00 mg in 5.0 g emulsion), as purified powder or contained in applesauce, and gastric

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acidity (pH 2.0, 3.0 or 4.8) influenced the in vitro GI fate of an algal oil emulsion stabilized by

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soy lecithin, in order to support the design of emulsions for enhanced bioavailability. It was

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hypothesized that apple pectin and gastric pH would interactively change the emulsion

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microstructure and, in turn, influence the subsequent in vitro lipolysis and DHA bioaccessibility.

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2. Material and Methods

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2.1. Materials

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All chemicals and reagents were purchased from Sigma Chemical Co. (MO, USA), including

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pectin from apple (76282), D-galacturonic acid monohydrate (48280), porcine bile extract

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(B8631), pancreatin (from porcine pancreas, P1750), pancreatic lipase (from porcine pancreas,

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L3126), and Nile Red (72485). Life's DHA S35-O300 algal oil was supplied by DSM (MD,

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USA), and liquid soy lecithin was from NOW Foods (IL, USA). Applesauce (Mott’s Fruitsations

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unsweetened apple sauce, Canada Dry Mott’s Inc, Mississauga, Ontario, Canada) was purchased

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from a local supermarket.

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2.2. Characterization of applesauce and pectin

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The total solids content of the applesauce was determined by drying to constant weight using a

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105°C oven method (Sluiter et al., 2008). The alcohol insoluble solids (AIS) fraction of the

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applesauce was also determined, as described by Le Bourvellec et al. (2011). Briefly, the

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applesauce was extracted by ethanol at room temperature, then filtered and washed with acetone

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solutions until the supernatant was colorless. The residue was then dried at 40 °C for 24 h and

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weighed as AIS. The soluble pectin content of the applesauce was determined by colorimetric

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assay (Melton & Smith, 2001). The polysaccharide was first hydrolyzed by sulphuric acid to

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galacturonic acid, which react with m-hydroxydiphenyl and absorption at 525 nm was

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measured using a UV–vis spectrophotometer (Hewlett Packard 8451A Diode Array

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Spectrophotometer) and with a standard curve based on D-galacturonic acid (0 – 80 mg/mL).

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Accordingly, the pectin content of the applesauce was determined to be 5.68 ± 0.50 mg/g. Total

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polyphenols in the applesauce were determined by an adapted Folin–Ciocalteu method (Almeida

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et al., 2011). Briefly, applesauce was extracted by acidified methanol for 2 h at 85 °C to

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eliminate vitamin C. After cooling, the sample was centrifuged at 2,500 xg for 15 min, and the

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aliquot diluted and then mixed with the Folin–Ciocalteu reagent and saturated sodium carbonate

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solution. The absorbance at 750 nm was determined after standing at room temperature for 30

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min. Polyphenol concentration was calculated based on a gallic acid (0 – 0.5 mg/mL) calibration

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curve. Accordingly, the total polyphenols per gram (wet weight basis) of the applesauce was

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0.778 ± 0.016 milligram of gallic acid equivalents (GAE). The protein content in the purified

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apple pectin was determined using the Bradford method as described (Chee & Williams, 2008)

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and found to be 0.74 ± 0.03 wt% protein. All the above determinations were made in triplicate.

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2.3. Preparation of soy lecithin-stabilized emulsions

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A soy lecithin-stabilized emulsion (10.0 wt% algal oil, 1.2 wt% soy lecithin and 88.8 wt% Milli-

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Q water) was prepared as previously described (Lin et al., 2014). Soy lecithin was first dispersed

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in the oil phase and the mixture pre-homogenized for 30-60 seconds with water using a handheld

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mixer at speed 2 (10,000 rpm) (Ultra-Turrax, IKa T18 Basic, Germany) and then passed at

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15,000 psi (103.42 MPa) through a microfluidizer (M110-EH Microfluidizer Processor,

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Microfluidics, MA, USA). The freshly prepared emulsion was collected in amber glass jars and

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stored under nitrogen at 4 °C for up to one week.

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2.4. Particle size and ζ-potential measurements

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Mean droplet diameters (D3,2: the surface area-weighted mean, D4,3: the volume-weighted mean)

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before and after gastric digestion were determined by laser diffraction (Mastersizer S, Malvern

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Southborough, MA, USA) using a refractive index of 1.488 for the algal oil. Samples analyzed

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during the in vitro digestion experiments were drawn 0.5 cm from the bottom of vials. Control

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solutions of purified pectin (0, 0.568 and 10 mg/mL, equal to those in the gastric digestate) and

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applesauce (0.1 g/mL) were also subjected to analysis, although those based on purified pectin

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were too diluted to reach the necessary opacity for the light scattering measurement.

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ζ-potential was measured using a particle electrophoresis instrument (NanoZS, Malvern

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Instruments Ltd, UK). Prior to analysis, in order to minimize multiple scattering effects, meal

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samples and gastric digestates were dispersed in 5 mM sodium phosphate buffer solutions at the

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pH of samples to achieve an oil concentration of 0.01 wt%.

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2.5. In vitro digestion experiments

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Firstly, 0.00, 5.68, or 100.00 mg purified apple pectin was added to 5.0 g of the aforementioned

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emulsion to study the effects of pectin content on the emulsion digestion. In another set of

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samples, 1.0 g applesauce (containing 5.68 mg pectin) was added to the emulsion, instead of

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pectin, to compare the effects of the food matrix versus isolated pectin. The pectin in applesauce

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has a high degree of methylation (Le Bourvellec et al., 2011), similar to the purified apple pectin

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used in this study. To achieve the highest pectin concentration (100.00 mg/5.0 g emulsion), an

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additional applesauce-containing meal sample was prepared with 5.0 g emulsion, 1.0 g

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applesauce and 94.32 mg purified pectin. The five meal samples studied are listed in Table 1.

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Meal samples were subjected to in vitro digestion experiments simulating gastric and duodenal

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conditions using a method slightly modified from Minekus et al. (Minekus et al., 2014).

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Simulated digestion fluids were prepared as described (Minekus et al., 2014) with the exception

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that additional phospholipids (76 mg) were included in the 10 mL simulated duodenal fluid

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(SDF) (Mansbach II, Tso, & Kuksis, 2001). The digestions were performed in amber glass jars

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placed in a 37 °C 250 rpm shaking water bath (New Brunswick Scientific Co. Inc., NJ, USA).

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Meal samples warmed at 37 °C for 15 min were then mixed with 5 mL simulated gastric fluids

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(SGF). The mixture contained 2,000 U/mL pepsin and 12.6 mg/mL pyrogallol (as an antioxidant)

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and was incubated for 2 h at pH 2.0, 3.0 or 4.8, i.e. representing a range of gastric pH values

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observed depending on food buffering capacity (Minekus et al., 2014; Simonian, Vo, Doma,

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Fisher, & Parkman, 2005). Ten mL of SDF were then added to start the duodenal phase of

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digestion. The final digestion mixture contained 550 U/ml pancreatic lipase, 10 mg/mL bile

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extract (~10 mM bile salts), and 3.8 mg/mL (~8 mM) phospholipids. The duodenal digestate was

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adjusted to pH 7.0 by addition of 1 N sodium hydroxide and the digestion was carried out for 2

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h. For the particle size measurements, sample and digestive fluid volumes were reduced to 1/5 of

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those stated above. To assess the potential for PUFA oxidation during digestion, the

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concentration of thiobarbituric acid reactive substances (TBARS) were determined in the final

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digestate samples using a colorimetric assay (Larsson, Cavonius, Alminger, & Undeland, 2012).

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TBARS concentrations in the digestate samples were not significantly different from DHA-lipid-

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free controls containing only water, emulsifier, digestive fluids, and applesauce and/or pectin

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(p>0.05, data not shown), indicating a high degree of n-3 PUFA preservation during the

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digestion experiments.

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2.6. Measurement of digestate viscosity

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Digestate viscosity during the gastric and duodenal phases was measured using a stress-

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controlled rheometer (AR 2000, TA Instruments, New Castle, DE, USA) equipped with a

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concentric cylinder (double wall Couette cell) geometry at 37 °C. A constant shear of 10 torque

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(N•m) was applied for 10 s to eliminate any loading history and apparent viscosity determined by

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increasing the shear rate logarithmically from 10 to 120 s-1 at 10 points for 60 s each. All

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analyses were performed in triplicate.

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2.7. Determination of FFA release during in vitro digestion

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0.1 mL aliquots of duodenal digestate were collected at 2, 5, 10, 15, 30, 60, 90, and 120 min for

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determination of FFA using a non-esterified fatty acid (NEFA) kit (Wako Diagnostics, VA,

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USA) as per the manufacturer’s instructions (Nik, Corredig, & Wright, 2010). FFA from 100 uL

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of sample were extracted into hexane (1000 uL) under acidic conditions (100 uL 1 M HCl) and

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the mixture vortexed for 10 s before centrifugation at 16,873 g for 30 min (5418 Laboratory

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Centrifuge, Eppendorf Hamburg, Germany). The hexane extract was then diluted to 3 mL and 5

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uL was added to 225 uL of Reagent A in a 96-well plate (in duplicate) and the plate incubated at

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37 °C for 10 min. 75 uL of Reagent B was then added with an additional 15 min incubation at

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37 °C. FFA were determined using a UV-VIS micro-plate spectrophotometer (Spectramax plus,

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Molecular Devices Corporation, CA, USA) by measuring the absorbance at λmax of 550 nm and a

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standard curve prepared using oleic acid ranging from 0.1 to 1.97 mM. The percentage of lipid

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hydrolysis was calculated based on the moles of FFA present at each time point with respect to

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the total moles of FA present initially, correcting for background fluid contributions.

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2.8. Isolation of aqueous phase containing bile salt micelles

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Digestate samples at the end of digestion were immediately centrifuged at 144,000 g at 7 °C for 1

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h using a Sorvall WX Ultra 80 Ultracentrifuge (Mandel Scientific, ON, Canada) to separate any

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undigested oil from the aqueous phase and pellet. The aqueous fraction was collected with a fine

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tip disposable pipette, quantified using a graduated cylinder, and stored in glass vials under

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nitrogen at -80 °C until lipid extraction and analysis.

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2.9. Analysis of DHA bioaccessibility by gas chromatography

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Lipid extractions were performed based on the Folch method, as described (Martin, Nieto-

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Fuentes, Señoráns, Reglero, & Soler-Rivas, 2010) with slight modifications. Afterwards, the

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samples were saponified, methyled and FAME quantified, as previously described (Merino et al.,

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2011). FAMEs were separated by gas chromatography using an Agilent 7890A Network GC

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System (Agilent Technologies, CA, USA) with a Supelco SP 2560 fused-silica capillary column

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(100m × 0.25mm i.d., 0.2 µm film thickness; Sigma-Aldrich, St Louis, MO). The internal

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standard (C17:0) method was used to determine the concentration of DHA in the digestate

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aqueous phase samples. DHA bioaccessibility was defined as the weight percentage of DHA

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from the original algal oil that was incorporated into the aqueous phase by the end of the

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digestion.

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2.10. Confocal microscopy

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Confocal laser microscopy imaging was used to investigate differences in emulsion droplet

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microstructure before and after in vitro digestion. Undiluted emulsion and digestate samples

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were placed on concave glass slides, covered and sealed tight to prevent sample dryness. Nile

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Red (added to the algal oil prior to emulsification at 0.01% (w/w) per gram) was excited at 543

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nm. The resulting images (1024×1024 pixels) were obtained using a 63 x oil immersion

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objective lens.

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2.11. Statistical analyses

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All measurements were completed in triplicate, unless otherwise stated. Results are expressed as

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mean ± standard deviation. ANOVA (with Tukey’s post hoc testing) and Pearson correlation

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analyses were performed using GraphPad Prism version 6.00 for Mac OS X (GraphPad Software,

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San Diego California USA, www.graphpad.com) with a significance level of p<0.05.

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3. Results and Discussion

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3.1 Physicochemical properties of the meal and gastric digestate samples

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The D3,2 and D4,3 of 10% algal oil emulsion droplets were 0.137 ± 0.001 and 0.215 ± 0.001 µm,

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respectively (pH = 5.1). The emulsion had a monomodal size distribution with the peak centred

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near 0.1 µm (Fig. 1). All digestions and analyses were done using emulsions within 1 week of

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preparation, over which period of time particle size and ζ-potential did not significantly change

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(p>0.05, data not shown). The ζ-potential measurement (−72.0 ± 0.7 mV) demonstrated that the

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emulsion stabilized by soy lecithin was strongly negatively charged, agreeing with a previous

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study on soy lecithin-stabilized emulsion (Lin et al., 2014; Mantovani, Cavallieri, Netto, &

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Cunha, 2013). The negative charge is attributed to the anionic components in soy lecithin

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molecules, such as phosphatidylinositol and phosphatidic acids (Rydhag & Wilton, 1981; Xu,

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Nakajima, Liu, & Shiina, 2010).

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The addition of 5.68 mg purified pectin (i.e. EP) slightly increased the particle size (D3,2 = 0.141

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± 0.002 µm and D4,3 = 0.251 ± 0.005 µm; p<0.05) and slightly decreased the ζ-potential (− 69.1 ±

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1.3 mV, p<0.05). Greater increases in droplet size were observed with the addition of 100.00 mg

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pectin (i.e. EPP, D3,2 = 1.872 ± 0.027 and D4,3 = 2.304 ± 0.035 µm) and the negative charge on

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the lipid droplets was significantly reduced (−35.1 ± 1.1 mV, p<0.05). According to confocal

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microscopy (Fig. 2), droplet aggregation occurred in both EP and EPP (Fig. 2). However, the

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small particle size increase with EP suggests the aggregation in this sample was reversible with

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the shear applied during the laser diffraction measurements. The decrease in charge of the

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emulsion droplets in the presence of 100.00 mg (2.0 wt%) pectin may be attributed to depletion

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flocculation induced by the presence of excessive anionic pectin molecules, as observed at

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concentrations above 0.2 wt% pectin for a Tween-80-stabilized emulsion (Beysseriat et al. 2006).

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The meal samples were exposed to three different gastric pH conditions prior to simulated

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duodenal digestion at pH 7.0. After gastric digestion at pH 2.0, large increases in emulsion

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droplet size were observed for all samples (Figs. 1D and 2) and the surface charge was

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significantly reduced (Fig. 3). Increased droplet size was also observed at gastric pH 3.0,

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although D3,2 for EP was less impacted compared to E and EPP (Figs. 1E and 2). Based on the

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main peak for EP, most of the droplets at pH 3.0 were much smaller than at pH 2.0, although

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there was still a small peak at ~200 µm (Fig. 1E), indicating the presence of some large particles.

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Corresponding to the small droplet size, EP also had the highest surface charge among the three

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meal samples (Fig. 3). With the highest gastric pH investigated (i.e. 4.8), the lipid droplets in E

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and EP remained relatively small compared to those at lower gastric pH values (Fig. 2).

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Similarly, the surface charge of those meal samples was higher than that at lower gastric pH

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values (Fig. 3). The main peaks for E and EP were both left skewed and located near 1 µm,

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although the small peak near 200 µm was still observed for EP (Fig. 1F). In terms of EPP at pH

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4.8, the main peak was located further right compared to E and EP, i.e. near 2 µm, indicating the

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formation of large particles with the higher amount of pectin present (Fig. 1F). The confocal

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image of EPP at pH 4.8 shows the flocculation and coalescence of small lipid droplets (Fig. 2),

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supporting the observations based on size distribution and D3,2 and D4,3 (i.e. 2.629 ± 0.567 and

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13.398 ± 3.253 µm, respectively). This could be related to the limited surface charge of EPP

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compared to the other samples (Fig. 3).

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Factors affecting emulsion microstructure may include the following. Firstly, an acidic gastric

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pH was found to destabilize the lipid droplets emulsified by soy lecithin. Without the addition of

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any pectin, the D3,2 and D4,3 of the emulsion increased to 10.300 ± 0.902 and 14.252 ± 1.535 µm,

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respectively (p<0.05) when the pH was adjusted to 2.0 (from 5.1). At pH 3.0, the droplet size

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was relatively less impacted (D3,2 = 2.319 ± 0.012 µm and D4,3 = 3.952 ± 0.022 µm). At pH 4.8,

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the particle size was relatively very minimally impacted (i.e. D3,2 of 0.196 ± 0.000 and D4,3 of

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0.322 ± 0.001 µm, p<0.05). This observation that particle size of a soy lecithin-stabilized

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emulsion increases with decreasing pH agrees with previous reports (Comas, Wagner, & Tomás,

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2006; Lin et al., 2014), and is likely related to shielding of the emulsifier’s more negatively

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charged components, such as phosphatidylinositol and phosphatidic acids, by hydrogen ions at

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pH values closer to their pKa (~1.5) (Mantovani et al., 2013).

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In addition, the physiochemical properties of pectin could impact the stability of the emulsion

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system. Pectin is an anionic polysaccharide. Thus, at neutral pH, it can lead to the depletion of

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negatively charged emulsifier molecules on the surface of emulsion droplets and result in

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flocculation (Roudsari, Nakamura, Smith, & Corredig, 2006). This may explain the reversible

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flocculation observed when pectin was added to emulsion before digestion (Fig. 2). Other than

291

destabilizing soy lecithin-covered emulsion droplets, the extreme acidity (pH 2.0) during gastric

292

digestion would also protonate free carboxyl groups, resulting in limited negative charge on the

293

pectin molecules. Thus, no electrostatic interactions between pectin molecules and soy lecithin

294

are expected at pH 2.0. At a pH of 3.0, the meal samples were also destabilized, except EP,

295

which showed a bridging network of relatively small lipid droplets, according to Fig. 2.

296

The pectin used in this study contained 0.74 ± 0.03 wt% protein which may contribute to its

297

emulsifying ability (Ngouémazong, Christiaens, Shpigelman, Van Loey, & Hendrickx, 2015)

298

and impact electrostatic interactions. To investigate the charge of the pectin at pH 3.0, an algal

299

oil-in-water emulsion was prepared using 0.1 wt% pectin (same pectin content as EP) with no

300

additional emulsifier. After adjusting the pH to 3.0, this emulsion was found to be negatively

301

charged (−43.3 ± 0.5 mV), indicating that the pectin overall carries a negative charge at this pH.

302

Thus, the negatively charged pectin may not have electrostatic interactions with the anionic

303

components of soy lecithin molecules. However, there are cationic functional groups on soy

304

lecithin, such as choline and ethanolamine (Rosenberg, 1990), and electrostatic interactions

305

between these cationic groups and anionic pectin molecules may allow pectin interfacial

306

adsorption. Despite this, at low pectin concentration individual pectin molecules may adsorb

307

onto multiple lecithin-stabilized emulsion droplets, leading to bridging flocculation (Fig. 2). This

308

was observed in another study with a β-lactoglobulin-stabilized emulsion in the presence of a

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low concentration of pectin (Cho & McClements, 2009). In terms of EPP, the instability of this

310

emulsion in the presence of the highest pectin concentration could be attributed to depletion

311

flocculation induced by non-adsorbed pectin in the aqueous phase of emulsion (Cho &

312

McClements, 2009).

313

3.2. Viscosity of gastric and duodenal digestates

314

The digestate viscosity of each meal sample determined after 1 h of gastric digestion at pH 2.0,

315

3.0 or 4.8 is shown in Fig. 4. Accordingly, at all gastric pH levels, the viscosity of the E and EP

316

digestates was low and independent of shear rate (p>0.05, Fig. 4A, B, and C). The viscosity of

317

the EPP gastric digestates was slightly higher than those of E and EP, but still less than 0.1 Pa.s

318

at all shear rates. At pH 2.0 and 3.0, but not at pH 4.8, the viscosity of EPP digestate decreased

319

with increasing shear rate (Fig. 4A, B, and C). The viscosity of meal samples after 5 min

320

duodenal digestion was also measured (Fig. 4D, E, and F). Shear-thinning behavior was only

321

observed in EP and EPP at gastric pH 3.0, while all other treatments showed Newtonian type

322

behaviour regardless of pH (p>0.05). The pseudoplastic behaviour of EP at gastric pH 4.8 and

323

EPP at all gastric pH values studied may be attributed to the entanglement of pectin molecules

324

formed at low shear rates (Fabek, 2011) and/or weak flocs formed by depletion flocculation

325

(Surh, Decker, & McClements, 2006) being interrupted at the higher shear rates. Comparing at a

326

shear rate of 56.23 s-1, i.e. within a range (10 - 100 s-1) with physiological relevance for in vitro

327

studies (Fabek, Messerschmidt, Brulport, & Goff, 2014), the EPP gastric digestate exhibited the

328

highest apparent viscosity values at all gastric pH values (p<0.05), whereas E and EP

329

consistently had similarly low apparent viscosity (p>0.05, Fig. 5). Similar trends were observed

330

when comparing apparent viscosities at a shear rate of 100 s-1 (data not shown).

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At the start of duodenal digestion, the digestate of each meal sample was adjusted to the same pH

332

(i.e. 7.0). As expected, the duodenal digestates were less viscous than the gastric samples (Figs. 4

333

and 5) because of dilution (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, Narváez-Cuenca,

334

& McClements, 2014; Fabek et al., 2014; Verrijssen et al., 2014). Despite the low values, the

335

trends in duodenal apparent viscosity were similar as those observed for the gastric phase (Fig. 5).

336

Therefore, the presence of pectin at the highest concentration studied was associated with higher

337

digestate viscosity, which may restrict the movability of droplets and so further impact the

338

digestion process (Ngouémazong et al., 2015; Zhang et al., 2015).

339

3.3. In vitro digestibility of the algal oil emulsion

340

Following gastric digestion, the meal samples were exposed to simulated duodenal digestive

341

conditions at pH 7.0 for 2 h. The effects of sample composition on duodenal lipolysis will be

342

discussed first. With a gastric pH of 2.0, there were no significant differences in the early

343

lipolysis observed between E, EP and EPP (i.e. within the first 5 min of duodenal digestion)

344

(p>0.05, Fig. 6A). By 2 h, the lipids in E and EP were hydrolyzed to a similar extent (p>0.05,

345

Fig. 6B) and EPP showed significantly less lipolysis (p<0.05, Fig. 6B). Following gastric pH of

346

3.0, the early lipolysis of EP was higher than E (p<0.05) and was the highest in this group after 2

347

h (p<0.05, Fig. 6). As with gastric pH 2.0, EPP achieved the lowest 2 h lipolysis (p<0.05, Fig. 6).

348

At gastric pH 4.8, E had the highest 5 min lipolysis, followed by EP and then EPP (p<0.05, Fig.

349

6A). However, the difference between E and EP disappeared by 2 h (p>0.05, Fig. 6B).

350

Within meal type, gastric pH impacted duodenal lipolysis. For example, higher gastric pH was

351

generally associated with higher 5 min and 2 h lipolysis (p<0.05, Fig. 6). Of all the samples and

352

conditions investigated, the emulsion (E) treated with gastric pH 4.8 achieved the highest 5 min

353

and 2 h lipolysis (p<0.05), i.e. 22.64 ± 0.81 and 51.34 ± 2.53 %, respectively. EPP consistently

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had the lowest 2 h lipolysis compared (p<0.05, Fig. 6). Therefore, both gastric pH and the

355

inclusion of pectin impacted emulsion lipid digestibility. This seems to relate to differences in

356

microstructure, in that E and EP tended to have smaller emulsion droplets at higher gastric pH

357

(3.0 and 4.8). The early lipolysis achieved was negatively correlated with the D3,2 of lipid

358

droplets at the beginning of duodenal digestion, i.e. the smaller the D3,2 (indicating an intact

359

emulsion microstructure), the greater the early lipolysis (p<0.05, R2 = 0.4377). This is consistent

360

with previous reports that smaller emulsion droplets have relatively higher oil-water interfacial

361

area available for the pancreatic lipase activity and facilitate lipolysis (Golding et al., 2011; Lin

362

et al., 2014; McClements, Decker, & Weiss, 2007). However, in this study, the ultimate lipolysis

363

achieved (2 h) was not favored by an intact emulsion structure (small droplets) before the start of

364

duodenal digestion (p>0.05). Of note, in the current study, FFA released were not removed

365

during digestion. Their interfacial accumulation may have impeded lipolysis (McClements & Li,

366

2010) and eliminated any advantage of smaller emulsion droplets at the beginning of the

367

duodenal stage, albeit the presence of bile salts regulates triacylglycerol hydrolysis by

368

solubilizing FFA to reduce the interfacial accumulation (McClements & Li, 2010).

369

Interestingly, the 2 h lipolysis was correlated with the apparent viscosity of gastric and duodenal

370

digestates at 56.23 s-1, i.e. the higher the viscosity, the lower the lipolysis (p<0.05). Indeed,

371

higher digestate viscosity may restrict contact between lipid droplets and digestive components

372

such as lipases and bile salts (Dikeman & Fahey, 2006). However, this correlation (i.e. between

373

lipolysis and gastric/duodenal stage apparent viscosity) was not observed at the first 5 min of

374

duodenal digestion (p>0.05). Overall, these results suggest that an intact emulsion structure at the

375

beginning of duodenal digestion is important for promoting initial lipolysis, while the digestate

376

viscosity is critical to the overall lipolysis. Other studies have also concluded that pectin can

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have suppressive effects on in vitro lipolysis. For example, an enzyme kinetics study showed that

378

pectic polysaccharides are non-competitive inhibitors of digestive enzymes, especially lipase, in

379

model solutions (Espinal-Ruiz, Parada-Alfonso, Restrepo-Sánchez, & Narváez-Cuenca, 2014).

380

As little as 0.18% pectin was able to decrease the lipolysis of a Tween-80 stabilized emulsion,

381

which was further reduced at higher pectin concentrations (up to 0.90%) (Espinal-Ruiz, Parada-

382

Alfonso, Restrepo-Sánchez, Narváez-Cuenca, et al., 2014). The inclusion of 1.19 mg/mL pectin

383

in a simulated duodenal digestate was also found to suppress lipolysis from egg yolk and trans

384

fatty acid enriched fats (Hur, Kim, Chun, Lee, & Keum, 2013; Hur, Kim, Choi, & Lee, 2013).

385

Similarly, in this study, EPP (100.00 mg pectin/20 mL final digestate = 5.00 mg/mL) had the

386

lowest lipolysis, regardless of gastric pH. Importantly, however, pectin’s effects on lipid

387

digestion depend on many factors, including the concentration of pectin and the interfacial

388

composition of lipid droplets. In another study (Zhang et al., 2015), 0.5 wt% pectin reduced (i.e.

389

by more than 35%) lipolysis of nonionic Tween-80- and lactoferrin-stabilized emulsions,

390

potentially by bridging or depletion mechanisms. In contrast, 0.025 wt% pectin doubled the

391

lipolysis of caseinate-stabilized emulsions by adsorbing to the surfaces of cationic casein-coated

392

droplets and promoting electrostatic and steric repulsion (Zhang et al., 2015). This highlights the

393

importance of rational emulsion formulation to achieve the desired physiological effects of

394

pectin.

395

3.4. DHA bioaccessibility

396

DHA bioaccessibility at the end of duodenal digestion is compared in Fig. 6. Similar as for

397

lipolysis, the DHA bioaccessibility results for EPP were the lowest at every gastric pH

398

investigated (p<0.05). In contrast, E treated with gastric pH 4.8 had the highest DHA

399

bioaccessibility (87.67 ± 2.36%) among all samples, followed by EP after gastric pH 4.8 (75.84

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± 2.91%). Interestingly, a higher DHA bioaccessibility was observed for EP compared with the

401

other samples with gastric pH 3.0 (69.87 ± 3.28%) (p<0.05). Thus, at this pH, the small amount

402

of pectin present was associated with increases in emulsion DHA bioaccessibility, even though

403

the emulsion microstructure was compromised by gastric pH 3.0 (as evidenced by E at pH 3.0 in

404

Figs. 1 and 2). Other than the aforementioned three samples (E at pH 4.8 and E & EP at pH 3.0),

405

none of the samples exceeded a DHA bioaccessibility of 30%. This may be attributed to the fact

406

that micellar solubilization of long-chain PUFA can be less efficient than short- or medium-chain

407

fatty acids, as their chain length leads to lower solubility and adversely impacts their partitioning

408

between oil and aqueous phases (Carlier, Bernard, & Caselli, 1991; Sek, Porter, Kaukonen, &

409

Charman, 2002).

410

A positive correlation was observed between emulsion lipolysis and DHA bioaccessibility

411

(p<0.05, R2 = 0.8211), despite the fact there were large differences in gastric structural stability

412

based on meal sample type and pH differences. However, correlations were not observed

413

comparing across different gastric pH values or pectin concentrations (p>0.05), with the

414

exception that a positive correlation between lipolysis and DHA bioaccesibility was observed for

415

samples treated at gastric pH of 4.8 (p<0.05, R2 = 0.9528). A negative correlation was observed

416

between the gastric digestate apparent viscosity at 56.23 s-1 and meal sample DHA

417

bioaccessibility (p<0.05, R2 = 0.3426), although the duodenal digestate apparent viscosity at

418

56.23 s-1 was not correlated with DHA bioaccessibility (p>0.05). No correlation was found either

419

between particle size (D3,2 or D4,3) before duodenal digestion and DHA bioaccessibility. Instead,

420

based on the confocal microscopy (Fig. 2), EP had smaller droplets than E and EPP at gastric pH

421

of 3.0 and this may be related to its relatively high DHA bioaccessibility. The smaller droplets of

422

EP could provide larger surface area for pancreatic lipase absorption, facilitating lipolysis (Fig.

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5). Nevertheless, the extent of lipolysis in EP was not as pronounced as its DHA bioaccessibility.

424

Thus the low concentration of pectin present may have also influenced the transfer of DHA to

425

the aqueous phase bile salt micelles and vesicles.

426

Overall, in the current study, the bioaccessibility of DHA (i.e. a TAG digestion product) was

427

influenced by the presence of pectin. In contrast, when 2% pectin was present with

428

phosphatidylcholine (i.e. the main component of the soy lecithin used in the current study) for

429

the formation of a beta-carotene-rich olive oil emulsion, the eventual incorporation of released

430

free fatty acids and monoacylglycerols in the micelle fraction did not significantly differ

431

compared to the same sample without pectin (Verrijssen et al., 2015), although only about one

432

quarter of the lipids were incorporated within the micelles in that study. However, beta-carotene

433

bioaccessibility was reduced by the presence of both high and medium methoxyl pectins,

434

potentially attributable to the formation of gel-like clusters between these molecules and

435

phosphatidylcholine, leading to the entrapment of oil droplets, and ultimately attenuated beta

436

carotene transport. The same results were not observed in the presence of low methoxyl pectin or

437

in the absence of phosphatidylcholine (Verrijssen et al., 2015). Although it is understood that

438

dietary fibres interfere with the micellization of carotenoids through the entrapment of oil

439

droplets and bile salts (Palafox-Carlos, Ayala-Zavala, & González-Aguilar, 2011), Verrijssen et

440

al. (2015) and the current study with DHA highlight the complexity of pectin’s interaction with

441

other GI components and resulting impacts on lipid digestion and bioaccessibility of lipophilic

442

molecules.

443

3.5. Digestion of emulsion with applesauce

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The overall aim of this work was to understand ingredient interactions as they apply to lipid

445

digestibility and bioaccessibility. Therefore, digestions of the algal oil emulsion in the presence of

446

applesauce, i.e. a complex food matrix rich in pectin, were also performed. This was done at

447

concentrations of pectin matching the EP and EPP meal samples. This involved adding 1.0 g

448

applesauce (containing 5.68 mg pectin) to 5.0 g of the emulsion in the case of EA and supplementing

449

with additional 94.32 mg purified apple pectin in the case of EAP.

450

The D3,2 and D4,3 of EA were 0.901 ± 0.079 µm and 267.337 ± 54.691 µm, respectively, i.e.

451

significantly higher than those of E and EP (p<0.05, Fig. 1C) owing to the presence of

452

applesauce which, alone, had D3,2 and D4,3 values of 365.339 ± 6.675 and 716.573 ± 12.611 µm,

453

respectively. The addition of applesauce slightly decreased the negative charge of the original

454

emulsion droplets (−63.7 ± 1.1 mV, p<0.05, Fig. 3). This could be attributed to the acidity of

455

applesauce (pH of EA = 3.8 versus pH of E = 5.1), which may reduce the emulsion-stabilizing

456

ability of soy lecithin, as discussed previously. For EAP the ζ-potential was reduced significantly

457

to −36.7 ± 0.8 mV (p<0.05, Fig. 3) and further increases in droplet size were observed, i.e. to

458

7.978 ± 0.367 and 469.108 ± 66.966 µm, for D3,2 and D4,3 respectively (p<0.05, Fig. 1C).

459

Although the large particles present in the applesauce contribute to a high degree of

460

polydispersity in the laser diffraction analysis, the confocal microscopy confirms that the lipid

461

droplets of meal sample before digestion was similar in size (Fig. 2). Similarly, after gastric

462

digestion at pH 4.8, EA and EAP had more significant increases in droplet size than EP and EPP

463

(Fig. 2), likely contributed by large non-lipid particles from the applesauce, although potentially

464

also reflective of lipid droplet destabilization caused by the acidity of applesauce and/or the

465

presence of pectin, as observed in Fig. 2.

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In terms of viscosity, the EA and EAP gastric digestates were higher than EP and EPP (p<0.05)

467

and exhibited obvious shear-thinning behavior at all pH values. Similar observations were found

468

for all EA duodenal digestates, but not EAP for which the duodenal digestate viscosity was

469

independent of shear rate following gastric pH 3.0 and 4.8 (p>0.05, Fig. 3). The apparent

470

viscosity of the EAP digestate at both the gastric and duodenal phases was the highest of all

471

gastric pH treatments, potentially because of the presence of both applesauce and the highest

472

concentration of pectin (Fig. 4). The EA digestates had significantly higher apparent viscosity

473

values than EP (p<0.05) and similar to EPP (p>0.05), except during gastric digestion at a pH of

474

2.0 (EA < EPP, p<0.05, Fig. 4A). The total solids in applesauce (10.45 ± 0.03 wt%) may

475

contribute to the higher viscosities of applesauce-containing samples.

476

In terms of lipolysis, EA was similar to EP and EAP was similar to EPP, despite the fact that the

477

applesauce-containing samples had lower early stage lipolysis when gastric pH was 3.0 and 4.8

478

(p<0.05, Fig. 5A), and lower overall lipolysis with gastric pH 4.8 (p<0.05, Fig. 5B). Therefore,

479

when the emulsion was treated with a relatively higher gastric pH and was able to maintain a

480

stable microstructure (with the help of a low level pectin at pH 3.0), the presence of applesauce

481

limited the lipolysis, potentially because of emulsion-destabilizing effects (pH 3.0 and 4.8 in Fig.

482

2). Applesauce is a complex food matrix which includes potentially anti-lipolytic components

483

such as polyphenols (Sugiyama et al., 2007). For example, 10 µg/mL apple polyphenols have

484

been shown to inhibit ~65% of pancreatic lipase activity in vitro (Sugiyama et al., 2007). The

485

total polyphenol content of the applesauce used in this study was 0.778 ± 0.016 mg GAE/g wet

486

weight basis, which led to a polyphenol concentration of 39 µg/mL GAE in duodenal digestates

487

of EA and EAP. Thus, polyphenols were present in the applesauce at levels which may have

488

contributed to lipid digestion. However, the lack of difference between meal samples with or

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without applesauce when treated with low gastric pH indicates that emulsion destabilization

490

overlapped with any lipase-inhibiting effects of applesauce as a complicated food matrix, and the

491

lipolysis was not limited further by the presence of polyphenols or additional pectin.

492

4. Conclusions

493

This research demonstrated that pectin content and gastric pH had interactive impacts on

494

emulsion digestion. The possibility exists for other digestive conditions to play a role in these

495

processes and also to be influenced by they chemical and physical properties of ingested foods.

496

A low level of pectin (5.68 mg/5.0 g emulsion) protected the emulsion from severe acidic

497

destabilization at gastric pH 3.0, but not 2.0, and contributed to a higher duodenal DHA

498

bioaccessibility. In contrast, at 100.00 mg pectin/5.0 g emulsion, even a relatively high gastric

499

pH (i.e. pH 4.8) could not maintain the emulsion microstructure to allow efficient duodenal

500

digestion and DHA transfer. The experiments with applesauce revealed more extensive

501

destabilization and lower digestibility and DHA bioaccessibility at equivalent levels of pectin,

502

highlighting the complex interactions between molecules from whole food matrices. Lipid

503

digestibility and DHA bioaccessibility were closely related to the stabilization of emulsion

504

droplets during gastric digestion. This contributes to an understanding of how in vitro digestion

505

methods (i.e. gastric pH) can impact results. It can also be useful for the design of DHA-rich

506

lipid based products by suggesting that the addition of apple pectin and consumption with

507

products such as applesauce could compromise the availability of DHA. Thus, extra caution

508

should be addressed when formulating fibre- and DHA-containing products.

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Acknowledgements

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This study was supported by the Natural Sciences and Engineering Research Council of Canada.

512

Thank you to DSM and NOW Foods for providing the algal oil and soy lecithin, respectively.

513

Thank you also to Prof. David Ma and the Department of Food Science for use of their

514

equipment and to Surangi Thilakarathna, Lyn Hillyer, and Dr. Michaela Strüder-Kypke for their

515

technical support.

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516 List of table titles and figure titles

518

Table 1. Composition of meal samples (E, EP, EPP, EA, and EPP) studied and the corresponding

519

duodenal digestate pectin concentration.

520

Table 2. Mean droplet diameters (µm) of meal samples before and after gastric digestion at pH

521

2.0, 3.0 or 4.8.

522

Fig. 1. Size distribution of the emulsion exposed to different acidic pH conditions (A), meal

523

samples EP, EPP, EA, EAP, applesauce and pectin before digestion (B and C), and meal

524

samples EP, EPP, EA, and EAP after 2 h of gastric digestion at pH 2.0 (D), 3.0 (E) and 4.8 (F).

525

Fig. 2. Microstructure of the meal samples before and after 2 h of gastric digestion at pH 2.0, 3.0,

526

and 4.8. G0 = before gastric digestion; G120 = after 2 h (120 min) of gastric digestion; E = 5.0 g

527

emulsion only; EP = 5.0 g emulsion + 5.68 mg pectin; EPP = 5.0 g emulsion + 100.00 mg pectin;

528

EA = 5.0 g emulsion + 1.0 g applesauce; EAP = 5.0 g emulsion + 1.0 g applesauce + 94.32 mg

529

pectin. Scale bars represent 50 µm.

530

Fig. 3. ζ-potential of digestate of different meal samples after gastric digestion. Data are the

531

mean ± SD. Within each figure, different uppercase letters (A, B, C) indicate significant differences

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between meal sample type and different lowercase letters (a, b, c) indicate significant differences

533

between gastric pH values (p<0.05).

534

Fig. 4. Flow behavior of gastric (A, B, C) and duodenal (D, E, F) digestates of meal samples at

535

gastric pH 2.0 (A and D), 3.0 (B and E), and 4.8 (C and F).

536

Fig. 5. Apparent viscosity (Pa.s) at 56.23 s-1 of digestates for meal samples E, EP, EPP, EA, and

537

EAP after 1 h gastric (A) and 5 min duodenal (B) digestion. Data are mean ± SD. Within A or B,

538

different uppercase letters (A, B, C) indicate significant differences between sample type and

539

different lowercase letters (a, b, c) indicate significant differences between gastric pH levels (p<0.05).

540

Fig. 6. Duodenal lipolysis (% FFA release, maximum = 66.7%) of meal samples treated with

541

different gastric pH values. Lipolysis at 5 min (start of duodenal digestion) (A) and 2 h (end of

542

duodenal digestion) (B). Data are the mean ± SD. Different uppercase letters (A, B, C, D) indicate

543

significant differences between samples (E, AP, EP, EAP, EPP). Different lowercase letters (a, b, c)

544

indicate significant differences between gastric pH levels (p<0.05).

545

Fig. 7. DHA bioaccessibility for meal samples treated with gastric pH 2.0, 3.0 or 4.8. Data are

546

mean ± SD of triplicate lipid extractions from digestions performed in triplicate. Different

547

uppercase letters (A, B, C, D) indicate significant differences between samples. Different lower case

548

letters (a, b, c) indicate significant differences between gastric pH conditions.

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Table 1. Composition of meal samples (E, EP, EPP, EA, and EPP) studied and the corresponding duodenal digestate pectin concentration.

Purified Pectin (mg) 5.68

94.32

100.00

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Duodenal Digestate Pectin Content (mg/mL) 0 0.284 5 0.284 5

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1.0 g Applesauce (5.68 mg Pectin) -

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Emulsion (5.0 g)

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Table 2. Mean droplet diameters (µm) of meal samples before and after gastric digestion at pH 2.0, 3.0 or 4.8.

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Meal Before Digestion1 After Gastric Digestion2 Samples Gastric pH = 2.0 Gastric pH = 3.0 Gastric pH = 4.8 E - D3,2 0.137 ± 0.001 34.216 ± 1.491 25.701 ± 1.147 0.519 ± 0.002 E - D4,3 0.215 ± 0.00 38.161 ± 2.352 43.932 ± 4.792 0.956 ± 0.002 0.141 ± 0.002 23.516 ± 1.157 0.468 ± 0.075 0.552 ± 0.208 EP - D3,2 0.251 ± 0.005 37.131 ± 3.917 39.759 ± 42.231 40.267 ± 40.643 EP - D4,3 1.872 ± 0.027 18.540 ± 2.523 7.832 ± 0.223 2.629 ± 0.567 EPP - D3,2 2.304 ± 0.035 39.845 ± 2.511 20.127 ± 0.690 13.398 ± 3.253 EPP - D4,3 EA - D3,2 0.901 ± 0.079 74.180 ± 3.052 104.835 ± 3.468 17.275 ± 0.401 EA - D4,3 267.337 ± 54.691 188.243 ± 4.305 427.138 ± 24.993 347.200 ± 34.049 EAP - D3,2 7.978 ± 0.367 85.608 ± 5.247 70.760 ± 0.979 42.104 ± 2.677 EAP - D4,3 469.108 ± 66.966 409.911 ± 49.598 452.550 ± 43.742 538.052 ± 78.400 1 Samples were taken after addition of pectin and/or applesauce and being mixed without any digestive fluid for 15 min in the 37°C 250 rpm shaking water bath. 2 Samples were taken after 120 min of gastric digestion in the 37°C 250 rpm shaking water bath.

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corresponding duodenal digestate pectin concentration. Table 2. Mean droplet diameters (µm) of meal samples before and after gastric digestion at pH 2.0, 3.0 or 4.8.

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Fig. 1. Size distribution of the emulsion exposed to different acidic pH conditions (A), meal samples EP, EPP, EA, EAP, applesauce and pectin before digestion (B and C), and meal

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samples EP, EPP, EA, and EAP after 2 h of gastric digestion at pH 2.0 (D), 3.0 (E) and 4.8 (F). Fig. 2. Microstructure of the meal samples before and after 2 h of gastric digestion at pH 2.0, 3.0, and 4.8. G0 = before gastric digestion; G120 = after 2 h (120 min) of gastric digestion; E = 5.0 g emulsion only; EP = 5.0 g emulsion + 5.68 mg pectin; EPP = 5.0 g emulsion + 100.00 mg pectin;

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EA = 5.0 g emulsion + 1.0 g applesauce; EAP = 5.0 g emulsion + 1.0 g applesauce + 94.32 mg pectin. Scale bars represent 50 µm.

Fig. 3. ζ-potential of digestate of different meal samples after gastric digestion. Data are the

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Fig. 4. Flow behavior of gastric (A, B, C) and duodenal (D, E, F) digestates of meal samples at gastric pH 2.0 (A and D), 3.0 (B and E), and 4.8 (C and F). Fig. 5. Apparent viscosity (Pa.s) at 56.23 s-1 of digestates for meal samples E, EP, EPP, EA, and EAP after 1 h gastric (A) and 5 min duodenal (B) digestion. Data are mean ± SD. Within A or B,

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Fig. 6. Duodenal lipolysis (% FFA release, maximum = 66.7%) of meal samples treated with different gastric pH values. Lipolysis at 5 min (start of duodenal digestion) (A) and 2 h (end of duodenal digestion) (B). Data are the mean ± SD. Different uppercase letters (A, B, C, D) indicate significant differences between samples (E, AP, EP, EAP, EPP). Different lowercase letters (a, b, c)

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indicate significant differences between gastric pH levels (p<0.05).

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Fig. 7. DHA bioaccessibility for meal samples treated with gastric pH 2.0, 3.0 or 4.8. Data are mean ± SD of triplicate lipid extractions from digestions performed in triplicate. Different uppercase letters (A, B, C, D) indicate significant differences between samples. Different lower case

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Highlights

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Highly acidic gastric pH destabilized a soy lecithin-stabilized DHA-rich emulsion Low pectin concentration retained emulsion microstructure at gastric pH 3.0 A high level of pectin reduced lipolysis and DHA bioaccessibility, regardless of gastric pH Smaller droplets were associated with duodenal lipolysis and DHA bioaccessibility Pectin-containing applesauce further limited lipid digestion and bioaccessibility

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• • • • •