The effect of pectin on in vitro β-carotene bioaccessibility and lipid digestion in low fat emulsions

Food Hydrocolloids 49 (2015) 73e81

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The effect of pectin on in vitro b-carotene bioaccessibility and lipid digestion in low fat emulsions Tina A.J. Verrijssen*, Sarah H.E. Verkempinck, Stefanie Christiaens, Ann M. Van Loey, Marc E. Hendrickx Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Department of Microbial and Molecular Systems (M2S), KU Leuven, Kasteelpark Arenberg 22, PB 2457, 3001, Leuven, Belgium

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 August 2014 Received in revised form 11 January 2015 Accepted 14 February 2015 Available online 21 March 2015

In this work, we investigated how pectin with different DM, with or without the presence of an additional emulsifier (L-a-phosphatidylcholine), influences on the one hand the in vitro bioaccessibility of bcarotene, loaded in the oil phase of an oil-in-water emulsion, and on the other hand the lipid digestion. As a consequence, the relation between the b-carotene bioaccessibility and the lipid digestion was investigated as well. For this research, two types of oil-in-water emulsions have been investigated. The first type contained 5% olive oil enriched with b-carotene and water in which only 2% citrus pectin (CP) (with a DM of 99%, 66% or 14%) was dissolved. In this type, only pectin is present that can function as emulsifier. The second type contained 5% enriched oil and water in which 1% L-a-phosphatidylcholine and 0 or 2% CP (with a DM of 99%, 66% or 14%) were dissolved. Results show that the influence of pectin DM on the in vitro b-carotene bioaccessibility (incorporation of b-carotene in the micelles) and the lipid digestion (incorporation of free fatty acids (FFAs) and monoacylglycerols (MAGs) in the micelles) was dependent on the presence of phosphatidylcholine but was less dependent on the particle size (distributions) or the viscosity. In the emulsions with phosphatidylcholine, an increase of on the one hand the incorporation of b-carotene and on the other hand the incorporation of FFAs and MAGs in the micelles was seen by decreasing the DM of the citrus pectin from 99% to 66%, whereas both incorporations decreased again by decreasing the DM further to 14%. In the emulsions without phosphatidylcholine, an increase of the incorporation of b-carotene into the micelles was seen by decreasing the DM. On the contrary, the incorporation of FFAs and MAGs into the micelles remained. This means that there was a clear relation between the incorporation of b-carotene and the incorporation of FFAs and MAGs in the micelles for the emulsions without phosphatidylcholine, whereas this was not the case for the emulsions containing phosphatidylcholine. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Pectin Degree of methyl-esterification In vitro b-carotene bioaccessibility Lipid digestion Emulsion L-a-phosphatidylcholine

1. Introduction Soups and sauces, full of vegetables like tomatoes and carrots, are good sources of water, fibres (e.g. pectin), micronutrients such as vitamins and/or pro-vitamins (e.g. carotenoids) and lipids. These lipids are often added to increase the palatability of the food. Besides the risk of obesity when too much lipids are taken, lipids are also important macronutrients, providing energy, essential fatty acids and lipid soluble nutrients (e.g. carotenoids) which are needed in a human diet. A balance should be found between too

* Corresponding author. Tel.: þ32 16 37 67 67. E-mail address: [email protected] (T.A.J. Verrijssen). http://dx.doi.org/10.1016/j.foodhyd.2015.02.040 0268-005X/© 2015 Elsevier Ltd. All rights reserved.

large intake and uptake of lipids and not taking up essential fatty acids and lipid soluble nutrients like carotenoids (Armand et al., 1996; Bauer, Jakob, & Mosenthin, 2005; Lowe, 1994). Carotenoids are lipid soluble nutrients, present in fruits and vegetables, which have benefical health effects. Besides their antioxidant properties, they seem to be of interest due to their influence on the modulation of immune responses and the regulation of cell growth (Rock, 1997). Some carotenoids like b-carotene also have a provitamin A-activity. Vitamin A is an important micronutrient for preventing night blindness, good immune function, growth, development and gastrointestinal functioning of the body (Grune et al., 2010; Haskell, 2012). Only a certain amount of the dietary carotenoids will be absorbed and used by the human body due to several factors, e.g. the presence of lipids or fibres (e.g. pectin)

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(McClements & Decker, 2009; Xu et al., 2014). The terms bioavailability or bioaccessibility of a nutrient are used to describe the fraction of this nutrient that is respectively available for utilization in physiological functions and for storage or available for absorption into the small intestinal mucosa (Castenmiller & West, 1998). For lipid soluble nutrients like carotenoids, the conversion into a watersoluble form, like micelles, is important before they can be absorbed into the mucosa. Therefore, the knowledge of lipid digestion is important to understand the absorption of carotenoids. Lipid digestion can be divided into two steps: hydrolysis of the dietary lipids and formation of micelles. Lipase hydrolyses the lipids, mostly triacylglycerols (TAGs), into the hydrolysed products diacylglycerols (DAGs), monoacylglycerols (MAGs) and free fatty acids (FFAs). The efficiency and rate of this step are depending on several factors including the properties of the surrounding medium of the lipid droplets (e.g. viscosity or presence of interacting compounds) and properties of the oil-water-interface (e.g. droplet size, droplet composition, oil type, surface active compounds) (Hu, Li, Decker, & McClements, 2010; McClements & Decker, 2009). McClements, Decker, Park, & Weiss, 2008; McClements, Decker, & Park, 2008 described a number of factors why fibres might interact with this step. Fibres may (i) directly interact with lipase or with co-lipase, reducing the enzyme activity, (ii) form a protective membrane around the lipid droplets, preventing lipase or co-lipase to interact, (iii) increase the viscosity which can increase the duration in the stomach and small intestinal phase but also decrease the transport between substrate and enzymes. Besides these possibilities, some fibres can also function as emulsifiers depending on their structure and properties (Akhtar, Dickinson, Mazoyer, & Langendorff, 2002; Leroux, Langendorff, Schick, Vaishnav, & Mazoyer, 2003; Leroux et al., 2003). An emulsifier can form smaller oil droplets by adsorbing at the oil-water interface and decreasing the surface tension, thereby enlarging the surface area available for lipase to interact, resulting in an increase of lipase binding and activity. On the other hand, competitive adsorption processes can occur between the surface active compounds (emulsifiers) and lipase, which can interfere with the binding of lipase to the droplet surface or with the lipase activity (McClements & Decker, 2009; Singh & Ye, 2013). The lipase hydrolysis products can form micelles together with bile acids, phospholipids and lipid-soluble compounds, like carotenoids. The efficiency and micelle formation rate depend on the properties of the surrounding medium and of the interface as well. Also for this step, McClements et al. (2008) described the reasons why fibres might interact. On the one hand, fibres may increase the system viscosity increasing the residence time in the small intestinal phase and decreasing the transport between the different compounds thereby influencing micelle formation. On the other hand, some fibres can bind bile salts thereby preventing them from incorporation into the micelles or emulsifying the lipids. The role of the fibre structure on lipid digestion is although not fully understood and seems important because different fibre properties (e.g. length, embranchment, hydrophobicity, or charge) result in differences in e.g. binding properties, pH and viscosity which might be important for lipid digestion (Eastwood & Mowbray, 1976; Falk & Nagyvary, 1982; McClements & Decker, 2009; Verrijssen, Balduyck, et al., 2014). Pectin is a fibre located in the cell wall and middle lamellae of dicotyledonous plants, so present in fruits and vegetables together with carotenoids. Pectin structure largely depends on the plant source, the ripening stage, the processing and storage (Sila et al., 2009). The degree of methylesterification (DM) of pectin is of interest because it determines the pectin functionality, such as its hydrophobicity and its charge density which might influence the interactions with all compounds present in the sample or digestion juices (e.g. ions, lipids, lipase, bile acids or micelles).

It has to be noted that besides the influence fibres are assumed to have on lipid digestion, intake of dietary fibre can also protect against diseases like coronary artery diseases, hypertension, colon cancer and diabetes (Mehta & Kaur, 1992; Reiser, 1987). The aim of this work was to investigate the relation between the in vitro b-carotene bioaccessibility and lipid digestion in model systems which might represent (simplified) soups or sauces and allow to study the interactions between different compounds. To better understand the effect of pectin (DM) and phosphatidylcholine on the b-carotene bioaccessibility and the lipid digestion, also structural characteristics, such as the particle size distributions and the viscosity of the different emulsions, were investigated at different stages of digestion. The model systems contain water, enriched olive oil (with b-carotene from carrots), 0 or 1% emulsifier (L-a-phosphatidylcholine, PHC) (from egg yolk) and (0 or 2%) citrus pectin with different structures in terms of DM. In plant-based food products, extra emulsifiers are often added, which makes it of interest to investigate the effect of its addition. Phosphatidylcholine is choosen as extra emulsifier because more and more consumers prefer foods with natural ingredients (like phosphatidylcholine from plant sources) instead of chemically prepared additives. In addition, phospholipids play a role in the micelle formation (McClements & Decker, 2009) and Marisiddaiah, Rangaswamy, and Vallikannan (2011) found improvement of b-carotene bioavailibality in rats by phospolipids. Besides the fact that the tested emulsions represent simplified soups and sauces, it is known that carotenoids can be isolated from natural sources and can be used as nutraceutical ingredients. Because of the hydrophobicity of carotenoids, emulsions are suitable for successfully incorporating the carotenoids into a wide range of food and beverage products. It is therefore interesting to investigate emulsions as study object. 2. Material and methods 2.1. Materials Citrus pectin (CP) (Sigma Aldrich) was used for the preparation of pectin with different degree of methyl-esterification (DM). Carrots (Daucus carota cv. Nerac) were purchased in a local shop and stored at 4  C before used. Olive oil (extra virgin) was kindly donated by Vandemoortele (Ghent, Belgium). All chemicals and reagents were from Sigma Aldrich, except for NaCl, HCl, urea, anhydrous sodium sulphate and ethanol (from VWR); CaCl2.2H20, NH4Cl and MgCl2 (from Merck); hexane, sulphuric acid and acetone (from Chem Lab); glucose and NaHCO3 (from Fisher Scientific); heptane (from Fluka); KCl (from MP Biomedicals) and diethylether €n). All chemicals and reagents were of (from Riedel-De Hae analytical grade. 2.2. Preparation of citrus pectin with different DM Citrus pectin (CP) with different DM was prepared by incubating high methyl-esterified CP (DM of 98.6%) (Sigma Aldrich) with purified carrot pectin-methyl-esterase (PME) for 4 min or 30 h, as described by Verrijssen, Balduyck, et al. (2014). The DM values of the pectin samples were measured by using Fourier transforminfrared (FT-IR) spectroscopy (IRAffinity-1, Shimadzu) and were 98.6% (±1.5), 65.6% (±5.8) and 14.1% (±1.1). Therefore, the different pectin samples will be further called “CP99”, “CP66” and “CP14”. 2.3. Preparation of oil-in-water emulsions enriched with bcarotene The procedure to prepare olive oil enriched with b-carotene from carrots is described by Verrijssen, Vanierschot, et al. (2014).

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Emulsions were prepared by mixing for 10 min (ultra turax, Waring Commercial, Torrington, CT, USA) 5% (w/w) the enriched oil with demineralized water, in which 0 or 1% (w/w) L-a-phosphatidylcholine (PHC) and 0 or 2% (w/w) citrus pectin (CP99, CP66 or CP14) were dissolved. After mixing, the emulsions were high pressure homogenized at 100 MPa (Stansted Fluid Power, Pressure cell homogeniser, U.K.) using a single cycle. The pH was adjusted to 6.0 using a (1 M) sodium hydroxide solution. The different emulsions are further indicated as “0e1%PHC 0e2%CPdm emulsion”, depending on the PHC concentration (%), the CP concentration (%) and the CP DM (dm%). Emulsions were prepared in duplicate to take into account the variability due to the preparation procedure. Each emulsion was independently submitted to the in vitro digestion procedure. 2.4. In vitro digestion The digestion of the oil-in-water emulsions was simulated by using stomach juice, small intestinal juice, bile extract and a (1M) bicarbonate solution as described by Versantvoort, Oomen, Van de Kamp, Rompelberg, and Sips (2005). The validation of the composition of these digestion juices was published in Versantvoort, Van de Kamp, and Rompelberg (2004). Stomach digestion was mimicked by adding 12 ml stomach juice (mainly containing ions, glucose, urea, pepsin and mucin; pH 1.3) to 6 g emulsion followed by incubation for 2 h at 37  C (end-over-end rotation, 40 rpm). The small intestinal digestion was simulated by adding 12 ml duodenal juice (mainly containing ions, urea, pancreatin and lipase; pH 8.1), 6 ml bile extract (mainly containing ions, urea and bile; pH 8.2) and 2 ml 1 M bicarbonate to the sample. These samples were incubated for 2 h (at 37  C) while rotating end-over-end (40 rpm). The headspace of the tubes was flushed with nitrogen before each incubation step (10 s) and the samples were kept in the dark during the digestion procedure to reduce the influence of oxygen and light (Verrijssen, Balduyck, et al., 2014). 2.5. Particle size distribution Particle size distributions of the initial emulsions and the digested emulsions (after the stomach phase and after the small intestinal phase) were measured by laser diffraction (Beckman Coulter Inc., LS 13 320, Miami, Florida). A few droplets of each sample were poured into a stirring tank, filled with deionized water. The sample was pumped into the measurement cell wherein the laser light (wavelength main illumination source: 750 nm; wavelengths halogen light for Polarization Intensity Differential Scattering (PIDS): 450 nm, 600 nm, 900 nm) is scattered by the particles. The parameters D(v,0.10), D(v,0.25), D(v,0.50), D(v,0.75) and D(v,0.90) were calculated from the intensity profile of the scattered light using the instrument's software (Mie theory) and reported accordingly. All analyses were carried out in duplicate. Besides laser diffraction, the microstructure of the samples was visualized by microscopy pictures, using a light microscope (Olympus BX-41) equipped with an Olympus XC-50 digital camera (Olympus, Opticel Co. Ltd., Tokyo, Japan). 2.6. Viscosity The viscosity of the initial and the digested (after stomach phase and after small intestinal phase) emulsions was measured using a stress-controlled rheometer (MCR 501, Anton Paar, Graz, Austria) at 25  C. As geometry, a concentric cylinder (double wall couette cell) was used. First, a constant shear rate of 100 s1 was applied for 60 s, followed by a rest-period (shear rate of 0 s1) of 300 s to neglect loading history of the emulsion. The viscosity was measured by

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decreasing the shear rate linearly from 100 to 0.1 s1. Each shear rate was applied for 40 s and it was verified that steady state viscosities were obtained in this way. Evaporation was considered negligible due to the short duration of the tests. All analyses were carried out in duplicate. 2.7. In vitro b-carotene bioaccessibility The in vitro b-carotene bioaccessibility was measured after digesting the emulsions by the in vitro digestion model described above. After the small intestinal phase, the micelle fraction was collected by ultracentrifugation (165 000 g, 1 h and 5 min, 4  C) and the amount of b-carotene in this fraction was determined according to the procedure described by Verrijssen, Balduyck, et al. (2014). In addition, the initial amount of b-carotene in each emulsion was measured. The in vitro b-carotene bioaccessibility (B /C) is defined as the amount of b-carotene in the micelles after digestion per g initial emulsion (B) relatively to the initial amount of b-carotene in the emulsion per g initial emulsion (C). All analyses were carried out in triplicate. 2.8. In vitro lipid digestion The amount of lipids was measured before digestion (in the initial emulsions), after the small intestinal phase and in the micelle fraction after digestion to measure on the one hand the release of free fatty acids (FFAs) and monoacylglycerols (MAGs) from triacylglycerol (TAG) and on the other hand the micellar incorporation of FFAs and MAGs. The micelle fraction was collected by (ultra) centrifuging (165 000 g, 1 h and 5 min, 4  C) the digested sample (after the small intestinal phase). A procedure to identify and quantify the amount of TAG, DAG (diacylglycerol), MAG and FFA in the different samples was implemented based on the procedure of Helbig, Silletti, Timmerman, Hamer, and Gruppen (2012). The optimization of this procedure resulted in some modifications in the extraction step and the quantification step. In the extraction step, 2 ml sample was taken and poured into an extraction mix, containing 2 ml ethanol, 3 ml diethylether-heptane (DEE-Hep, 1:1 v/v) and 0.2 ml 2.5 M sulphuric acid. This mixture was vortexed for 2 min after which it was rotated (end-over-end) for 30 min and centrifuged for 5 min at 500 g (20  C). The upper layer was collected in a new tube. 1 ml of DEE-Hep was added to the lower layer and the mixture was vortexed (1 min), mixed (end-over-end rotation, 15 min) and centrifuged again. The upper layer was again collected and added to the previous upper layer. 100 mg anhydrous sodium sulphate and an internal standard (50 ml trilaurin (0.2 g/5 ml DEEHep) for the quantification of TAGs and 100 ml methyl heptadecanoate (0.04 g/5 ml DEE-Hep) for the quantification of MAGs and FFAs) were added to the collection of the two upper layers, after which the whole volume was brought to 5 ml with DEE-Hep. The type and concentration of internal standard were chosen so to not interfere with the compounds present in the sample and to have an abundance comparable to the lipids in the sample. The second step was the identification and quantification of the compounds with a GCeMS. In the GC (Agilent Technologies, 6890N Network GC System, United States) with column VF-ht, UltiMetal (Varian BV, Middelburg, Netherlands), 0.2 ml sample was injected splitless. The oven temperature was programmed at 80  C (1 min) and increased with 6  C/min to 400  C (2 min). For the detection of the compounds, a MS-system (Agilent Technologies, 5973 inert Mass Selective Detector, United States), was used. Standard curves were used to quantify the different compounds based on the amount internal standard added (Verrijssen et al., 2015). The LM/LSIP e value represents the incorporation of FFAs and MAGs in the micelle fraction and is defined as the amount of FFAs

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and MAGs in the micelles (after digestion) per g initial emulsion (LM) relative to the amount of FFAs and MAGs after the small intestinal phase (SIP) per g initial emulsion (LSIP). 2.9. Statistical analysis Differences in mean relative b-carotene bioaccessibility and LM/ LSIP e values were analysed using one-way anova and the Tukey's Studentized Range Post-hoc Test (Statistical Software Package SAS, version 9.2., Cary, N.C., U.S.A.). The level of significance was 95% (P < 0.05). 3. Results and discussions 3.1. 0%PHC emulsions 3.1.1. Particle size distributions during in vitro digestion The 0%PHC emulsions were first studied before digestion to investigate the influence of pectin DM on the particle size distribution (Table 1). The pectin DM seems to have little influence because the initial particle size distributions are similar (diameters around 1.07e1.32 mm). The 0%PHC 2%CP66 emulsion however contains smaller particles than the other emulsions which lead to the idea that CP66 is a better emulsifier (D(v; 0.9) of 2.62 compared to 3.72e5.57). This can be explained by the available, block-wise oriented, hydrophilic and hydrophobic groups of the galacturonic acid (GalA)-molecules in CP66 at pH 6 (Fraeye, Duvetter, Doungla, Van Loey, & Hendrickx, 2010). The carboxylic groups of GalA of CP99 are esterified so this pectin is more hydrophobic and CP14 has more negative charges at pH 6 so is more hydrophilic than CP66. Nevertheless, all samples remained stable so CP99 and CP14 could also act as emulsifier. Besides the action of CP as an emulsifier due to changes at the droplet interface, another possible explanation can be that CP alters the properties of the continuous phase and works as a stabilizer or thickner (Chen, McClements, & Decker, 2010). The small differences in particle size distributions before digestion can be due to the high pressure homogenization-step at 100 MPa. The results of the particle size distribution analysis of the 0%PHC emulsions during digestion show that the 0%PHC 2%CP99 emulsion has a similar particle size distribution before digestion compared to the one after the stomach phase, while 0%PHC 2%CP66 and CP14 emulsions both show larger particles after the stomach phase (Table 1 and Fig. 1). These trends were also seen in the microscopy pictures (results not shown). The emulsion where CP99 was used as emulsifier, seems to retain the structural properties (Fig. 1 A), although oil droplet flocculation was expected due to addition of mucin, a highly glycosylated protein, present in the stomach juice (Vingerhoeds, Blijdenstein, Zoet, & van Aken, 2005; Vingerhoeds, Silletti, de Groot, Schipper, & van Aken, 2009). This oil droplet flocculation showed up in the 1%PHC emulsion (see further, Fig. 4). This means that the droplet flocculation due to mucin dependson the type of

emulsifier, what was also found by Verrijssen, Balduyck, et al. (2014). The type of molecules at the oil-water-interface surrounding the lipid droplets, in this case an emulsifier, influences their stability and the exposed lipid surface area (McClements, Decker, & Park, 2008). In summary, it can be hypothesized that CP99 binds the hydrophobic oil droplets strongly due to its hydrophobic, methoxylated GalA-groups and mucin cannot interact. CP66 and CP14 on the other hand can form small gel-like pectin clusters with the available calcium-ions in the stomach juice, namely pectin-Caþ2-crosslinkings, probably stabilized by other ions available in the juice (Grant, Morris, Rees, Smith, & Thom, 1973; mazong et al., 2012; Walter, 1991). These crosslinkings can Ngoue occur due to the free carboxyl-groups on the GalA of CP66 and CP14 and may cluster the oil droplets. These gel-like pectin clusters embedding oil droplets are visualized by microscopy (Fig. 2). After the small intestinal phase, smaller particle sizes are found for the 0%PHC 2%CP66 emulsion compared to the particle sizes after the stomach phase, whereas the particle distribution of the 0%PHC 2%CP14 emulsion became bimodal. On the one hand, oil droplets are assumed to be digested by lipase after this phase, which lead to CP66 and CP14 in the continuous phase instead of surrounding the oil droplets, which might change properties such as binding capacity. On the other hand, the pH, the ion concentration and the amount of proteins is different in the small intestinal phase compared to the conditions in the stomach phase, leading to different or new pectin-Caþ2-crosslinkings (Endress, Mattes, & Norz, 2006; Grant et al., 1973; Thibault & Ralet, 2003; ). 3.1.2. In vitro b-carotene bioaccessibility and lipid digestion The in vitro b-carotene bioaccessibility (Fig. 3 A) and the incorporation of FFAs and MAGs in the micelle fraction (Fig. 3 B) follow a similar trend. No significant differences are noticed between the 0% PHC 2%CP99 and the 0%PHC 2%CP14 emulsion, whereas the incorporation of b-carotene and lipids into micelles is higher for the 0% PHC 2%CP66 emulsion. From the particle size distributions, it could be expected that the smaller oil droplets in the stomach phase and the small intestinal phase of the 0%PHC 2%CP99 emulsion would increase the b-carotene bioaccessibility and the lipid incorporation, but it might be that CP99 binds the oil droplets that much that lipase acts slower. The 0%PHC 2%CP14 emulsion on the other hand showed large pectin-clusters which means that steric hindering or high viscosity (results not shown) may inhibit the lipase activity and/or micelle formation. The LSIP-value was the same for all emulsions and contained only MAGs and FFAs which means that for all samples, all TAGs were hydrolysed to MAGs and FFAs after the 2 h-small intestinal phase (results not shown). For this reason, it seems that not the hydrolysis but the incorporation of MAGs and FFAs into the micelles is different for the 0%PHC 2%CP66 emulsion compared to the others. Another possibility is that the lipase conversion is the same after 2 h but that the lipase activity was higher in the 0%PHC 2%CP66 emulsion so that micelle formation occurred longer.

Table 1 Particle size distributions of the initial emulsions. Significant differences (Tukey test: P < 0.05) are indicated with different letters. Emulsion

0%CP 2%CP99 2%CP66 2%CP14

1%PHC 0%PHC 1%PHC 0%PHC 1%PHC 0%PHC 1%PHC

D(v;0.1)

D(v;0.5)

(mm)

(mm)

0.59 0.60 1.30 0.59 0.56 0.61 0.87

± ± ± ± ± ± ±

0.02C 0.00C 0.22A 0.04C 0.07C 0.01C 0.00B

1.15 1.32 2.29 1.07 0.95 1.27 1.77

D(v;0.9) (mm)

± ± ± ± ± ± ±

0.07g 0.04 g 0.36a 0.09 g 0.02 g 0.04 g 0.05b

2.33 5.57 3.30 2.62 1.79 3.72 3.38

± ± ± ± ± ± ±

0.29cd 0.68a 0.37b 0.49c 0.10d 0.20b 0.34b

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Fig. 1. Particle size distribution after stomach phase (A) and after small intestinal phase (B) of the 0%PHC emulsions (0%PHC 2%CP99 emulsion ( ), 0%PHC 2%CP66 emulsion ( ) en 0%PHC 2%CP14 emulsion ( )). (PHC: phosphatidylcholine; CPdm: citrus pectin with degree of methyl-esterification of dm%).

Fig. 2. Representative microscopic image of oil droplet clustering in the 0%PHC 2%CP14 emulsion after the stomach phase.

When samples were incubated for 1.0 h, 1.5 h and 2.0 h in the small intestinal phase, it was seen that not all lipids were already hydrolysed after 1.0 h small intestinal phase, whereas all lipids were hydrolysed after 1.5 h small intestinal phase (results not shown). Probably, pectin delays this hydrolysis (McClements, Decker, & Park, 2008; McClements, Decker, Park et al., 2008). On

the other hand, the incorporation of MAGs and FFAs into the micelles (LSIP-value) was the same for all tested incubation times (results not shown). This means that the maximal incorporation of lipids was already reached after 1.0 h small intestinal phase, even when the lipids were not completely hydrolysed. Three hypotheses can be stated. First, the properties of the formed micelles, like the pH, the electric charge and the type of surface active compounds present, are important for maximal incorporation of lipids (Goncalves et al., 2013; McClements, Decker, Park et al., 2008; McClements, Decker, & Park, 2008). If pectin can interact with the micelles, it might change the surface properties and these changes will be dependent on the DM. Two possible mechanisms can occur so that pectin interacts with micelles. (i) If CP66, with hydrophobic groups (COOCH3) and hydrophilic groups (COO) at the pH of the small intestinal phase, can be incorporated into the micelles, it might influence the incorporation of other compounds by changing the interface properties. It is less likely that CP99, with more hydrophobic groups, or CP14, with more hydrophilic groups, would interact as a surface active compound in the small intestinal phase because competition can occur with other surface active compounds like bile acids, so it might be that these pectins are not present in the micelle fraction. (ii) Eastwood and Mowbray (1976) found that mixed micelles can bind to dietary fibres by molecular interactions. If this would be the case, it is possible that pectin change the interface properties by binding on to the micelles, instead of acting as a surfactant when more surface active compounds are present. A second hypothesis might be that pectin molecules decrease the formation of micelles by binding bile acids

Fig. 3. (A) Percentage in vitro b-carotene bioaccessibility (calculated as the absolute b-carotene bioaccessibility of a particular fraction divided by the initial b-carotene concentration, B/C-value) (mean ± standard deviation) and (B) the incorporation of lipids in the micelle fraction as the LM/LSIP-value (calculated as the amount lipids in the micelle fraction divided by the amount lipids after the small intestinal phase) (mean ± standard deviation) in the 0%PHC emulsions (0%PHC 2%CP99 emulsion ( ), 0%PHC 2%CP99 emulsion ( ) and 0%PHC 2%CP14 emulsion ( )). Significant differences (Tukey test: P < 0.05) are indicated with different letters.

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Fig. 4. Particle size distribution after stomach phase (A) and after small intestinal phase (B) of the 1%PHC emulsions emulsions (1%PHC 0%CP emulsion ( ), 1%PHC 2%CP99 emulsion ( ), 1%PHC 2%CP66 emulsion ( ) en 1%PHC 2%CP14 emulsion ( )).

(McClements, Decker, Park et al., 2008; McClements, Decker, & Park, 2008). Falk and Nagyvary (1982) however found that the binding of low DM pectin with bile acids was difficult because of the eletrocstatic repulsions, unless multivalent cationic ions are present (like Mgþ2 and Caþ2). Because the digestion juices used in these tests contain a high concentration of multivalent ions, the polyvalent cation bridging seems possible. In this way, it is possible that the charge or hydrophobicity of pectin, depending on the DM, is determing if pectin can interact with bile and/or how strong they can interact, leading to differences in micelle formation. If less micelles are formed, it is expected that the incorporation of bcarotene into micelles (b-carotene bioaccessibility) and the incorporation of lipids into micelles is lower for those samples. In this case, more hydrophobic pectin (CP99) or more hydrophilic pectin (CP14) would hinder bile acids more then CP66, containing hydrophobic and hydrophilic groups, because the incorporation of bcarotene and lipids into micelles are lower in those samples. The third hypothesis can be that molecular interactions take place between lipids and pectins (Falk & Nagyvary, 1982; Mokady, 1973). Falk and Nagyvary (1982) found hydrogen bondings between methoxycarbonyl groups of low methylesterified pectin with oleic acid. In this way, they can hinder the incorporation of lipids into the micelles. This hydrogen bondings will be also dependent on the pectin DM. 3.2. 1%PHC emulsions 3.2.1. Particle size distributions during in vitro digestion To evaluate the influence of on the one hand phosphatidylcholine and on the other hand the influence of DM in presence of phosphatidylcholine on the b-carotene bioaccessibility and the lipid digestion, the 1%PHC emulsions were first characterised (Table 1). Also for those samples, the initial particle size distributions are similar between the different initial emulsions, although the 1%PHC 0%CP and 1%PHC 2%CP66 show slightly smaller particle sizes. The high pressure homogenisation-step can explain these small differences. Compared to the 0%PHC emulsions, the particle size distribution is different for the 2%CP99 and 2%CP14 emulsions, probably because phosphatidylcholine can also absorb at the droplet surface which affects the particle size distribution (Singh, Ye, & Horne, 2009). The 1%PHC emulsions (Fig. 4) behave different during digestion compared to the 0%PHC emulsions (Fig. 1). After the stomach phase, the 1%PHC 0%CP and 1%PHC 2%CP99 emulsion also contain larger particles compared to particles before digestion, due to oil droplet flocculation (Fig. 5) caused by the influence of mucin (Verrijssen, Vanierschot, et al., 2014; Vingerhoeds et al., 2005). Larger particles after the stomach phase are observed for the 1%PHC 2%CP66

(Fig. 4 A) compared to the initial emulsion (Table 1), although less large particles were found compared to the 0%PHC 2%CP66 emulsion. It seems that in the presence of phosphatidylcholine, phosphatidylcholine is acting as an emulsifier rather than CP66. If CP66 is more available in the continuous phase, it can form more gel-like pectin clusters. The 1%PHC 2%CP14 emulsion showed, like the 0% PHC 2%CP14 emulsion, large particles after the stomach phase, due to gel cluster formation (confirmed by microscopy observation). After the small intestinal phase, more comparable particle size distributions for the emulsions were found by laser diffraction (Fig. 4 B). Pectin clusters were although found in microscopic pictures of the 1%PHC 2%CP66 and 1%PHC 2%CP14 emulsions (results not shown) what means that the formed structures were broken down by the laser diffraction-pump and could not be measured. 3.2.2. Viscosity during in vitro digestion Fig. 6 show the rheological behaviour of the 1% PHC emulsions before digestion (Fig. 6 A), after stomach phase (Fig. 6 B) and after small intestinal phase (Fig. 6 C). It is clear that adding pectin to a 1%PHC emulsion results in an increased viscosity depending on the pectin DM (Fig. 6 A). Before digestion, the 1%PHC 0%CP shows a viscosity of approximately 1.1 mPa s and behaves Newtonian, so this emulsion is rheologically similar as water. Adding 2%CP99 to this emulsion resulted in an increase of viscosity to approximately 17.5 mPa s and the behaviour is still Newtonian. The viscosity increased with lowering the DM and the 1%PHC 2%CP66 and CP14 emulsions are both pseudoplastic because shear thinning-behaviour is noticed. This shear thinning behaviour is probably a consequence of the formation of small

Fig. 5. Representative microscopic image of oil droplet clustering in the 1%PHC 0%CP emulsion after the stomach phase.

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Fig. 6. Viscosity of the 1%PHC emulsions (1%PHC 0%CP emulsion ( ), 1%PHC 2%CP99 emulsion ( ), 1%PHC 2%CP66 emulsion ( ) en 1%PHC 2%CP14 emulsion ( )) before digestion (A), after stomach phase (B) and after small intestinal phase (C).

structures between phosphatidylcholine, pectin and oil droplets. These structures can be broken down due to high shear rates, so the viscosity decreases in function of the shear rate (Steffe, 1996). After the stomach phase, the viscosity of all emulsions decreased except for the viscosity of the 1%PHC 2%CP14 emulsion (Fig. 6 B). The decrease in viscosity probably results from the addition of aqueous stomach juice. The ions and proteins added seem to have a negligible effect on those emulsions compared to the dilution effect. The increase in viscosity of the 1%PHC 2%CP14 is a consequence of the formation of gel-like pectin structures by adding ions and proteins, present in the stomach juice (Lofgren, Guillotin, Evenbratt, Schols, & Hermansson, 2005). After the small intestinal phase, the viscosity decreases for all emulsions compared to the viscosity of the emulsions after the stomach phase (Fig. 6 C). The small intestinal phase juice and bile extract diluted the system and it seems that the additional ions or proteins, did not increased the viscosity. 3.2.3. In vitro b-carotene bioaccessibility and lipid digestion Fig. 7 A shows that the B/C-value of the 1% PHC emulsion (without CP) was approximately 41.9% which means that 41.9% of the b-carotene present in the emulsion, was incorporated into the micelle fraction and is available for absorption. By adding (2%) CP99 to this emulsion, the b-carotene bioaccessibility significantly decreased to 24.7%. Comparing the 0%PHC 2%CP99 emulsion (Fig. 3 A) with the 1%PHC 2%CP99 emulsion (Fig. 7 A), shows that the b-

carotene bioaccessibility decreased by adding phosphatidylcholine. It can be hypothesized that CP99 strongly binds to the oil droplets (as indicated in the particle size distribution) and together with phosphatidylcholine hinders lipolysis by lipase and/or micelle formation. When (2%) CP66 was added to the 1%PHC emulsion, the bcarotene bioaccessibility decreased as well (to 32.2%) but less compared to (2%) CP99. Comparing the 0%PHC 2%CP66 emulsion (Fig. 3 A) with the 1%PHC 2%CP66 emulsion (Fig. 7 A) shows that the b-carotene bioaccessibility decreased as well by adding phosphatidylcholine. For CP66 together with 1%PHC, larger pectin clusters were found which lead to the idea that part of the oil droplets are surrounded by phosphatidylcholine and less by CP66 because CP66 is available for making gel-like pectin clusters. In this way, the bcarotene bioaccessibility is higher than the b-carotene bioaccessibility of the 1%PHC 2%CP99 emulsion. On the other hand, it is clear that the combination of CP66 with phosphatidylcholine leads to a decrease of b-carotene bioaccessibility. Adding (2%) CP14 to the 1%PHC emulsion does not significant influence the b-carotene bioaccessibility (Fig. 7 A). For the 1%PHC 2%CP14 emulsion, it can be assumed that phosphatidylcholine will act as an emulsifier and CP14 will be in the surrounding phase. Although this pectin increases the viscosity (Fig. 6), oil droplets are not embedded in gellike clusters and cannot be hindered or the duration in the small intestinal phase is long enough to overcome this higher viscosity, normally leading to a slower transport (McClements, Decker, Park

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Fig. 7. (A) Percentage in vitro b-carotene bioaccessibility (mean ± standard deviation) and (B) the incorporation of lipids in the micelle fraction as the LM/LSIP-value (mean ± standard deviation) in the 1%PHC emulsions (1%PHC 0%CP emulsion ( ), the 1%PHC 2%CP99 emulsion ( ), the 1%PHC 2%CP66 emulsion ( ) and the 1%PHC 2 %CP14 emulsion ( )). Significant differences (Tukey test: P < 0.05) are indicated with different letters.

et al., 2008; McClements, Decker, & Park, 2008). This can explain that the b-carotene bioaccessibility of the 1%PHC 0%CP emulsion and the 1%PHC 2%CP14 emulsion are not significant different. Based on the results of the 0%PHC emulsions (Fig. 3) and knowledge from literature where they describe a correlation between b-carotene bioaccessibility and lipid digestion (SalviaTrujillo, Qian, Martin-Belloso, & McClements, 2013), also differences in incorporation of lipids in the micelles were expected. The results (Fig. 7 B) show however no significant differences between the MAG and FFA-incorporation into micelles for the different 1% PHC emulsions which means that this incorporation is not influenced by the presence of pectin nor the DM of pectin in combination with the presence of phosphatidylcholine. The lipid incorporation in the emulsions with phosphatidylcholine is however higher than for the emulsions without phosphatidylcholine (Fig. 3 B), although it seems that no further increase is possible. The reason that the incorporation of lipids in the micelles is limited (approximately 1/4th of the MAGs and FFAs), might be that a maximum incorporation into the micelles is reached which was also seen in Verrijssen et al. (2015). Increasing the bile acid concentration did not lead to an increase of micellar incorporation of lipids (results not shown), which means that the maximum lipid incorporation is due to properties of the micelles (Goncalves et al., 2013; McClements, Decker, Park, et al., 2008; Wilde & Chu, 2011) (Goncalves et al., 2013; McClements, Decker, Park et al., 2008; McClements, Decker, & Park, 2008; Wilde & Chu, 2011), although differences were expected because of the presence of pectin with different DM in different emulsions. The incorporation of b-carotene on the other hand, maybe did not reach his maximum yet so it can be influenced by the presence of pectin and/or the DM of pectin. It can be concluded that the presence of phosphatidylcholine, the presence of pectin and the DM of pectin influence the incorporation of b-carotene into the micelles by changing the interfacial properties. 4. Conclusions In this work, we have shown that the influence of the DM of citrus pectin in emulsions on the in vitro b-carotene bioaccessibility and lipid digestion depends on the presence of (L-a-)phosphatidylcholine. For the emulsions without phosphatidylcholine, the pectin DM influences the incorporation of b-carotene and lipids into the micelles and there is a clear relation between both. On the other hand, the influence of the DM of pectin is completely different in emulsions containing phosphatidylcholine. In those

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