Chemical stability and bioaccessibility of β-carotene ...

Food Hydrocolloids 57 (2016) 301e310

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Chemical stability and bioaccessibility of b-carotene encapsulated in sodium alginate o/w emulsions: Impact of Ca2þ mediated gelation bastien Cambier a, Lucien Hoffmann a, Torsten Bohn a, b, * Christos Soukoulis a, Se a

Environmental Research and Innovation (ERIN) Department, Luxembourg Institute of Science and Technology (LIST), 41 rue du Brill, L-4422 Belvaux, Luxembourg b Population Health Department, Luxembourg Institute of Health (LIH), rue 1A-B Thomas Edison, L-1445 Strassen, Luxembourg

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 October 2015 Received in revised form 25 January 2016 Accepted 1 February 2016 Available online 20 February 2016

In the present work, 5% (w/w) canola oil-in-water submicron emulsions containing sodium alginate were investigated as b-carotene (0.05% w/w) carriers. O/w emulsions prepared via spontaneous emulsification were Tween-80 (SA, 1 and 1.5% w/w) stabilised. Ionotropic gelation of the o/w emulsions' continuous phase via in situ Ca2þ release, in the presence (sheared o/g emulsions) or absence (quiescent o/g emulsions) of mechanical stirring (1000 rpm, 6 h) was conducted. b-Carotene chemical stability in both o/w and o/g emulsions following 65 day storage periods at 4, 20 and 37 C, as well as its bioaccessibility under in vitro oro-gastro-intestinal digestion conditions were evaluated. Oxidative degradation rates of b-carotene ranged from 0.22 to 2.77%/day. Although Ca2þ-mediated gelation of o/w emulsions’ aqueous phase enhanced b-carotene chemical stability, oxidative degradation rates between quiescent o/g (0.07 e1.42%/day) and sheared o/g (0.19e1.50%/day) emulsions were comparable. Based on the activation energies of b-carotene degradation calculated by the Arrhenius kinetic model, sheared o/g emulsions exerted the lowest storage temperature dependency, followed by the quiescent o/g and SA containing o/ w emulsions. Moderate (ca. 27%) to high (ca. 48%) bioaccessibility of b-carotene was achieved in quiescent and sheared o/g emulsions respectively, showing no dependency on SA content. Contrarily, bcarotene bioaccessibility was reduced from ca. 29e16% for 1 and 1.5% SA containing o/w emulsions. Digesta micellar fractions of o/g emulsions exerted smaller lipid droplet size and lower surface tension values compared to the SA containing o/w emulsions. Therefore, it was postulated that enhanced bcarotene bioaccessibility in o/g emulsions was associated with their higher colloidal stability throughout gastrointestinal passage. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Carotenoids Ionotropic gelation Storage stability Degradation In vitro digestion Bioavailability

1. Introduction Carotenoids are pigments naturally occurring in plants and some bacterial species, which are of paramount importance in photosynthesis and photo-sensitive chemical reactions (Krinsky & Yeum, 2003). According to current knowledge, adoption of a carotenoid rich diet appears to confer health benefits to the human host, such as preserving major body functions and exerting a preventive role against inflammatory associated diseases, such as cardiovascular, ophthalmological, pulmonary and neurodegenerative complications, as well as several types of cancer (Krinsky &

* Corresponding author. Environmental Research and Innovation (ERIN) Department, Luxembourg Institute of Science and Technology (LIST), 41 rue du Brill, L-4422 Belvaux, Luxembourg. E-mail address: [email protected] (T. Bohn). http://dx.doi.org/10.1016/j.foodhyd.2016.02.001 0268-005X/© 2016 Elsevier Ltd. All rights reserved.

Johnson, 2005; Stahl & Sies, 2005). It has been postulated that carotenoid health benefits are primarily associated with their ability to affect cellular signalling cascades such as various transcription factors, influencing the expression of genes related to antioxidant defence, anti-inflammatory and anti-cancer properties (Kaulmann & Bohn, 2014; Stahl & Sies, 2005). Absorption of carotenoids is generally influenced by several physicochemical and physiological parameters, including the release from the food matrix (as the result of mechanical, chemical and enzymatic processes throughout the gastrointestinal passage), incorporation in the co-digested lipid droplets, interaction with endogenous lipid surface active compounds (mainly bile salts and phospholipids), promoting the formation of mixed micelles and eventually, transport of the mixed micelles to the small intestinal epithelium (Yonekura & Nagao, 2007). Therefore, the complexity of the absorptive mechanisms, together with the carotenoids’ poor

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water solubility, high crystallinity (of especially some apolar carotenes) and their chemically labile character leads to rather low bioavailability, e.g. 10e20% for carotenes and up to 40% for xanthophylls (Bohn, 2008). Incorporation of carotenoids in liquideliquid carriers (LLCs) has been reported as being an efficient strategy to improve their solubility and micellisation properties during gastrointestinal passage (Li & McClements, 2011; Matalanis, Jones, & McClements, 2011; McClements, Decker, & Park, 2008; McClements & Li, 2010; McClements, Decker, & Weiss, 2007; Soukoulis & Bohn, 2015). In general, LLCs can be fabricated via either high energy homogenisation, using specific mechanical devices (e.g. high shear mixers, high pressure homogenisers, colloidal mills, sonicators and microfluidisers), or low energy approaches such as spontaneous emulsification or emulsion phase transition (Ostertag, Weiss, & McClements, 2012). In principle, spontaneous emulsification relies on the formation of ultrafine droplets at the boundary between an aqueous and organic phase when the latter are brought into contact (Komaiko & McClements, 2015). A large number of LLCs for the oral delivery of bioactive compounds such as lipophilic vitamins and polyphenols have been successfully produced via spontaneous emulsification (McClements et al., in press). The bioavailability of carotenoids in food emulsions is structurally, colloidally and physicochemically related to factors such as the physical state and structural conformation of the lipid phase, the type of surface active compounds, the colloidal changes of the emulsion elements during GI passage, and the modulating effect of the aqueous phase (as primarily driven by the presence of soluble dietary fibre and the presence of amphiphilic compounds) on micelle formation (micelle hydrodynamic volume) and transport (microviscosity, diffusion rate) phenomena (Liu, Hou, Lei, Chang, & Gao, 2012; Rao, Decker, Xiao, & McClements, 2013b; Salvia-Trujillo, Qian, Martín-Belloso, & McClements, 2013a, 2013b; Soukoulis & Bohn, 2015; Yonekura & Nagao, 2009). On the other hand, carotenoids encapsulated in LLCs exert a rather limited chemical stability compared to their dehydrated analogues (e.g. spray or freezedried glassy matrices). This is especially evident throughout exposure to ambient or higher temperatures, pro-oxidants, light and oxygen, and moderately acidic (pH 4e6) conditions (Boon, McClements, Weiss, & Decker, 2009; Qian, Decker, Xiao, & McClements, 2012a, 2012c; Xu, Yuan, Gao, McClements, & Decker, 2013). Interfacial engineering approaches based on hydrocolloids, such as electrostatic layered lipid droplets, gel-in-oil-gel structured emulsions, self-structuring or co-structuring of the bulk aqueous phase etc., all implemented for bioactive compound encapsulation, have drawn attention over the last years (Liu, Wang, Sun, & Gao, 2016a; Liu, Wang, Xu, Sun, & Gao, 2016b; Matalanis et al., 2011; Mun, Kim, Shin, & McClements, 2015; Soukoulis & Bohn, 2015; Xu et al., 2014). Sodium alginate is one of the most interesting anionic biopolymers, exerting both a self-structuring (under acidic conditions) or co-structuring (complexation with cationic polymers) ability (Draget, 2009). It is well established that sodium alginate can interact with Ca2þ (preferentially with homoguluronic sequences), leading to the formation of complexes that induce the spatial molecular rearrangement of the backbone chain known as egg-box structure conformation (Draget, 2009). Calcium mediated gelation of sodium alginate has found extensive technological applications, such as for food restructuring and texturing, food coating and packaging, as well as encapsulation of bioactive compounds, enzymes and living cells (Banerjee & Bhattacharya, 2012; Comaposada, Gou, Marcos, & Arnau, 2015; De Prisco & Mauriello, 2016; Dickinson, 2015; Draget, 2009; Li & Nie, 2016). Regarding carotenoids, divalent ion crosslinking of sodium alginate has been primarily focused on microbead formation e

extrusion methodologies (Donhowe, Flores, Kerr, Wicker, & Kong, 2014; Leach, Oliveira, & Morais, 1998). Recently, sodium alginate covalent structuring via energy dissipation (shear-induced breakup of gel clusters), promoting the formation of a continuous aqueous phase containing gelled microparticulates, with promising techs, Douaire, & nological aspects, has been demonstrated (Farre ndez Farre s, Moakes, & Norton, 2014; Norton, 2013; Ferna s & Norton, 2014). However, potential applicaFern andez Farre tions of both quiescent and fluid gels based on covalent ion-active biopolymers for lipophilic bioactive compound encapsulation purposes, including carotenoids, are yet non-existing. Therefore, the aim of the present investigation was to evaluate the impact of sodium alginate Ca2þ structured sub-micron emulsions, fabricated via spontaneous emulsification, on the chemical stability of b-carotene during storage, under light-temperature controlled conditions. In addition, the bioaccessibility, i.e. the fraction of a compound that is released during digestion and potentially available for further uptake, of b-carotene following invitro gastro-intestinal digestion as affected by the emulsion structuring approach, was also investigated. 2. Materials and methods 2.1. Materials Low viscosity sodium alginate (12 cP, 1% in water at 25 C), bcarotene standard powder (purity >95%), d-glucono-lactone (GDL), Tween 80, porcine pepsin (250 IU/mg), porcine pancreatin (8xUSP specifications) and porcine bile extract were purchased from Sigma Aldrich (Leuven, Belgium). Sodium alginate was used for the preparation of the hydrocolloid solutions without further purification. Calcium carbonate was purchased from Merck GmbH (Darmstadt, Germany), whilst canola oil (Mazola, Bekkevoort, Belgium) was obtained from the local market. 2.2. Preparation of the lipid and aqueous phases For the preparation of the lipid phase, b-carotene (0.05% w/w, SigmaeAldrich, Leuven, Belgium) was dispersed into canola oil (Mazola), sonicated for 10 min at an amplitude of 40% (1 s pulse mode) and then left to fully solubilise at ambient temperature for 2 h under constant magnetic stirring (dark, vial was flushed with nitrogen). Then, the b-carotene enriched canola oil was blended at a ratio of 3:2 with Tween 80 (SigmaeAldrich) and kept under agitation until the preparation of the sub-micron emulsions. The aqueous phase was prepared by dispersing sodium alginate (2 and 3%, w/w) in deionised water (Millipore, USA) and left to fully dissolve and hydrate overnight. Calcium carbonate (Merck GmbH) was mixed with the 1 and 1.5% (w/w) sodium alginate solutions to achieve final concentrations of 15 mM and 22.5 mM, respectively. The sodium alginate (SA) solutions were successively sonicated for 5 min at 90% amplitude (Hielscher UP200S, GmbH, Teltow, Germany) to ensure complete dispersion of CaCO3. 2.3. Preparation of the oil-in-gel (o/g) emulsions Sub-micron o/w emulsions (10% w/w oil) were prepared via the spontaneous emulsification method at ambient temperature (20 ± 2 C), as recently described by Komaiko and McClements (2015). Specifically, 5 g of the lipid phase were added drop-wise (ca. 0.5 g/min) into 45 g of Millipore (18 MU) water under constant magnetic stirring (750 rpm). The formed emulsions were further agitated under the same conditions for 10 min. The obtained o/w emulsions were 1:1 blended with the sodium alginate solutions in order to obtain 5% o/w emulsions containing either 1 or


1.5% w/w sodium alginate. Finally, the o/w emulsions were mixed with d-glucono-lactone (at a 2:1 GDL to CaCO3 ratio), to trigger the slow in situ release of Ca2þ ions. Sheared o/g emulsions were fabricated under constant paddle stirring (IKA GmbH, Staufen, Germany) at 1000 rpm for 6 h. Quiescent o/g emulsions were prepared by rapid mixing of the CaCO3 containing o/w emulsions with d-glucono-lactone, and pouring the obtained system into amber glass vials, allowing ionotropic gelation to occur. Additionally, 5% o/w emulsions comprising 1 or 1.5% w/w SA in the absence of Ca2þ were also prepared and used for the purposes of the present work as the reference b-carotene LLCs. 2.4. Rheological characterisation of the o/w and o/g emulsions All emulsion systems were agitated for 30 min using a paddle mechanical stirrer (IKA GmbH, Staufen, Germany) at 1000 rpm and successively conditioned for 18 h at 4 C prior to rheological characterisation. Steady state shear flow measurements applying an upwarddownward ramp shear stress range from 0.1 to 500 s1 with a 60 s maintenance shear rate step (at 5001) were carried out. Upward ramp shear stress (t) e shear rate data ( g_ ) data were fitted to the Herschel-Bulkley model:

t ¼ t0 þ Κ g_ n

(1)

where t0 equals the yield stress (Pa), K the consistency coefficient (mPa*s-n) and n the rheological behaviour index (dimensionless). Strain-sweep measurements were performed on the sodium alginate containing o/w emulsions at 1 Hz to determine the linear viscoelastic region (LVR). The viscoelastic properties of the SA containing o/w emulsions were measured by small frequency amplitude sweeps (0.1e10 Hz) at a constant strain of 0.1%. 2.5. Light microscopy A light microscope (Zeiss, Jena, Germany) was used to visualise the structural aspects of the structured emulsions. Systems were diluted with Millipore deionised water at the ratio of 1:1000. 2.6. Chemical stability of b-carotene during storage For testing the chemical stability of b-carotene, approximately 20 mL of the o/w or o/g emulsion were transferred into screw capped amber coloured glass vials. Samples were stored in the dark in temperature controlled incubators set at 4, 20 and 37 C, for a period of 65 days. Periodically, aliquots of 500 mg were taken and extracted and analysed for all-trans b-carotene, following the method of Corte-Real, Richling, Hoffmann, and Bohn (2014), with minor modifications. Briefly, 5 mL of an extraction solvent comprised of n-hexane, acetone and ethanol (50:25:25, v/v) were mixed with 500 mg of the emulsion, vortexed for 1 min and subsequently centrifuged for 3 min at 4000 g and 4 C to allow complete phase separation. A second extraction step, adding 4 mL of pure n-hexane, and 1 mL of saturated sodium chloride solution was applied to facilitate complete extraction of b-carotene. The collected organic phases were combined and evaporated to dryness under a nitrogen stream (TurboVapLV, Biotage, Eke, Belgium), whilst the obtained dry residues were flushed with argon and stored at 80 C prior to analyses. The dry b-carotene containing residues were dissolved in 1 mL of pure n-hexane and the resulting organic phase was spectrophotometrically measured at 450 nm. The residual b-carotene (mg per g of emulsion) was calculated according to the formula:

amount of b carotene ¼

303

1% E1cm

A $m$d

(2)

1% where A equals the measured absorbance at 450 nm, E1cm is the 1 extinction coefficient of b-carotene in n-hexane (2590 g L cm1), m the mass (g) of the emulsion and d the length (cm) of the cuvette.

2.7. Simulation of the gastro-intestinal digestion The digestion of SA o/w and o/g emulsions was carried out adopting the standardised protocol described by Minekus et al. (2014). In brief, for the oral phase preparation, 5 g of the o/w emulsions were mixed with 4.975 mL simulated saliva fluid and 25 mL CaCl2 (0.3 M). The obtained oral phase was preconditioned at 37 C and successively mixed with 9.995 mL simulated gastric fluid (containing 25000 U mL1 pepsin from porcine mucosa) and 5 mL CaCl2 0.3 M, adjusted at pH ¼ 3 with HCl 1 M and incubated at 37 C for 1 h under constant shearing at 100 rpm, mimicking physiological stomach antral forces in a shaking water bath (model 1083, GFL GmbH, Germany). The incubated gastric chyme was mixed with 19.96 mL of simulated intestinal fluid (containing 4 mg mL1 pancreatin from porcine pancreas and 24 mg mL1 bile extract porcine) and 40 mL CaCl2 (0.3 M), adjusted to pH ¼ 7 using NaOH (1 M) and finally incubated at 37 C for 2 h under constant shaking. The obtained gastric digesta samples were quenched in liquid nitrogen and stored at 80 C prior to further physicochemical examination. Rheological characterisation of the oral, gastric and intestinal phases was carried out on the day of digestion trials, preparing separate samples. 2.7.1. Rheological characterisation of oral, gastric and intestine phases The simulated oral, gastric and intestine chyme systems were rheologically characterised at 25 ± 0.03 C. Steady state shear flow measurements applying an upward-downward ramp shear stress range from 0.1 to 200 s1 with a 60 s maintenance shear rate step (at 5001) were carried out. Upward ramp shear stress (t) e shear rate data ( g_ ) data were fitted to the Herschel-Bulkley model. 2.7.2. Physicochemical characterisation of micellar fraction of digesta samples Lipid droplet size distribution and zeta-potential of micellar fraction samples were determined using Nanoparticle Tracking Analysis (NTA, NanoSight, Malvern Instruments Ltd., Malvern, UK). Prior to analysis, samples were diluted with deionised water (Millipore Inc., USA) to obtain a mean particle concentration of 108109 particles mL1. The airewater interfacial properties of the digesta samples, preconditioned at 25 ± 0.1 C, were determined via the weight-drop method as previously described by Permprasert and Devahastin (2005). Surface tension of the micellar fraction of the digesta samples was calculated according to the equation:

sdigesta ¼

mdigesta sH2O mH2O

(3)

where sH2O denotes the surface tension of pure water, i.e. 71.99 dyn/cm (Pallas & Harrison, 1990). 2.7.3. Bioaccessibility of b-carotene In order to determine the in-vitro bioaccessibility of b-carotene, digesta aliquots of 12 mL were transferred to 15 mL plastic centrifuge tubes and centrifuged at 4500 g for 1 h at 4 C. After centrifugation, aliquots of 4 mL, corresponding to the micellar

304


Table 1 Steady state rheological properties (at 25 C) of b-carotene loaded o/w and o/g emulsions containing sodium alginate, calculated using the Herschel-Bulkley model. Sample

Yield stress t0 (mPa)

Consistency coefficient K (mPa s-n)

O/w emulsion 1% O/w emulsion 1.5% Sheared o/g emulsion 1% Sheared o/g emulsion 1.5% Quiescent o/g emulsion 1% Quiescent o/g emulsion 1.5%

ns ns 1.13 3.84 1.06 1.47

0.01 0.17 0.44 0.92 1.87 6.66

± ± ± ±

0.06a 0.12c 0.15ab 0.24b

± ± ± ± ± ±

0.00a 0.01b 0.03c 0.21d 0.08e 0.43f

Rheological behaviour index n 0.95 0.79 0.61 0.64 0.48 0.41

± ± ± ± ± ±

0.05e 0.04d 0.02c 0.02c 0.03b 0.01a

Thixotropic index (%) 0.13 0.68 1.32 1.42 6.03 5.13

± ± ± ± ± ±

0.01a 0.04b 0.15c 0.18c 0.33e 0.32d

R2 0.99 0.99 0.99 0.99 0.98 0.99

ns ¼ non-significant. a Values in a column not sharing the same superscripts are significantly different (p < 0.05) according to Tukey's post-hoc test.

fraction, were carefully collected from the intermediate phase using a syringe and passed through a 0.2 mm Nylon membrane filter (Acrodisc® 13 mm Syringe Filters, PALL Life Sciences, Ann Harbor, MI, USA) into a 15 mL falcon tube. b-Carotene transferred into the mixed micelles was extracted by adding 4 mL of an extraction solvent consisting of n-hexane, acetone and ethanol (50:25:25, v/ v). A second solvent step using 2 mL of pure n-hexane was applied when necessary. The b-carotene solvent extracts were evaporated to dryness under a stream of nitrogen and eventually the obtained b-carotene residues were re-dissolved in 1 mL of n-hexane and passed through a 0.45 mm syringe filter (Acrodisc, CR 4 mm, PALL Life Sciences, Ann Harbor, MI, USA), prior to spectrophotometric analysis. b-Carotene bioaccessibility was calculated according to the formula:

Bioaccessibilityð%Þ ¼ 100

amount of bcaroteneinthemicellar fraction amount of bcaroteneintheinitialemulsion (4)

2.8. Statistical analyses Normal distribution of data was verified by quantileequantile (QeQ) plots whilst box plots were used to verify the equality of variance. Two way analysis of variance (ANOVA) was performed to evaluate the significance of the effect of emulsion structuring method (dilute state, sheared or quiescent ion mediated gels) and sodium alginate concentration (1 and 1.5% w/w). Following significant F-values, Tukey's post hoc comparison tests were carried out for all group-wise comparisons. A p < 0.05 (2-sided) was considered as statistically significant. All statistical analyses were performed with SPSS vs. 19.0 (IBM, Inc., Chicago, IL). All values in the text are expressed as mean ± SD. 3. Results and discussion 3.1. Rheological characterisation of the sodium alginate structured emulsions

Fig. 1. Dynamic rheological spectra (open symbols ¼ 1% SA, closed symbols ¼ 1.5% SA, △ ¼ G0 , , ¼ G00 and B ¼ tand) of o/w and o/g emulsions containing b-carotene at 25 C. a: quiescent o/g emulsion, b: sheared o/g emulsion and c: o/w emulsion.

Rheographs (Supplementary Fig. 1) illustrate the viscosimetric response of the emulsions at shear rate range of 1e200 s1. Steady state rheological data calculated using the HerschelBulkley model (Table 1) revealed that all systems exhibited a shear thinning behaviour (n < 1), which was more pronounced in the case of quiescent oil-in-gel emulsions. Although the increase of sodium alginate concentration was positively associated with increased pseudoplasticity, its impact was more remarkable for SA stabilised o/w emulsions compared to the ionotropically gelled ones. Nevertheless, quiescent o/g emulsions exerted


significantly higher (p < 0.001) consistency coefficients and thixotropic index values compared to the sheared o/g emulsions. Therefore, it can be denoted that although sheared o/g emulsions exhibited a rather comparable deformability (intrinsic elasticity) and particle packing ability than the quiescent gelled systems, they showed a better structure recovery capacity compared to those of quiescent o/g emulsion systems (Ma, Lin, Chen, Zhao, & Zhang, 2014). Due to the fact that the critical molar ratio of CaCO3 to alginate was identical for both o/g emulsion systems, it would be expected that the degree of sodium alginate crosslinking should at least be comparable. It is therefore assumed that the diversified rheological behaviour of the o/g emulsions was associated mainly with the structure conformational changes attained as a function of the shearing step. Specifically, the higher macroviscosity and thixotropicity, as well as the more evident shear thinning behaviour of the quiescent o/g emulsions could be attributed not only to the formation of Ca2þ e SA dimer structures, but also to the inter-clustering of the ordered dimers, leading to the formation of a three-dimensional crosslinked ndez Farre s & Norton, 2014). Under shearing network (Ferna conditions, the supramolecular aggregation of egg-box dimers

305

was less promoted than the quiescent gelled systems, resulting in lower macroviscosity and higher structure recovery upon shearing forces suspension. Oscillatory frequency sweep (0.01e0.1Hz) measurements (Fig. 1) of the linear viscoelastic region (LVR) were carried out on SA containing o/w and o/g emulsions, following a 24 h relaxation period. According to the dynamic oscillatory rheological spectra, emulsions structured via Ca2þ-mediated gelation (Fig. 1a, b) exhibited a dominantly elastic character (G0 > G00 ). In both cases, elastic moduli showed only a slight dependency on frequency, implying that the anisotropic formed alginate microparticles remained entangled independently of the sodium alginate concentration or cross-linking method. On the contrary, o/w emulsions containing sodium alginate in the dilute state exhibited a predominant viscous-like character (G0 > G00 , tand > 1) within the entire frequency ramp range, corroborating the findings of Ma et al. (2014). 3.2. Light microscopy SA containing o/w emulsions conditioned at 4

C

for 24 h

Fig. 2. Light microscope micrographs illustrating lipid droplet distribution of structured emulsions. Left column: 1% w/w sodium alginate, right column: 1.5% w/w sodium alginate. a,b: o/w emulsion, c,d: sheared o/g emulsion, e,f: quiescent o/g emulsion. Bar scale ¼ 10 mm.

306


implied the occurrence of lipid droplet coalescence during the gelation process, which has also been previously reported for emulsion filled gels or gel-in-oil-in-gel systems (Patel et al., 2015). Regarding that no significant differences in the lipid droplet mean size between the quiescent and sheared o/g emulsion were detected, we assume that the lipid coalescence occurring in all o/g emulsions may be associated with the lipid destabilisation due to their high ionic strength (Dickinson & Davies, 1999). 3.3. b-Carotene degradation during storage

Fig. 3. Degradation rates of b-carotene encapsulated in o/w emulsions structured with sodium alginate as influenced by storage temperature. a ¼ 4 C, b ¼ 20 C and c ¼ 37 C. Blue line ¼ o/w emulsion, green ¼ sheared o/g emulsion and blue ¼ quiescent o/g emulsion. Straight line ¼ 1% sodium alginate, dashed line ¼ 1.5% sodium alginate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

exerted significantly smaller lipid droplets compared to the o/g ones (Fig. 2). However, in all cases, the obtained o/w and o/g emulsions were well-residing in the sub-micron region (0.22 < d3,2 < 0.89 mm, data not shown). It should be also noted that the Ca2þ mediated gelation of the o/w emulsions inhibited the occurrence of lipid droplet flocculation phenomena (small lipid droplet flocks were evidenced solely in the case of SA stabilised systems). This may be associated with the increased elasticity of the macroscopic emulsion (as a result of the three-dimensional polymer network formed via the inter-clustering of the Ca2þ e alginate dimers), diminishing the diffusion of lipid droplets in the continuous phase and therefore, inhibiting lipid particle collision due to insufficient repulsive forces between them (Torre & Pinho, 2015). On the contrary, the increased Sauter diameter of the lipid droplets

In order to assess the shelf-life of the fabricated emulsions, and therefore their feasibility as carriers for retaining the biological activity of b-carotene, the chemical stability of b-carotene was monitored for a period of 65 days at three storage temperatures (4, 20 and 37 C). The emulsions were stored in the dark but were exposed to oxygen (ambient air) conditions. As illustrated in Fig. 3, the temporal evolution of b-carotene retained in all emulsions exhibited a fairly linear pattern, suggesting zero order kinetics. Our findings corroborate the observations of Yi, Li, Zhong, and Yokoyama (2014), who reported that the degradation of b-carotene in submicron sodium caseinate stabilised o/w emulsions followed zero order kinetics. As expected, the b-carotene degradation rates increased with higher storage temperature (Table 2). Storage of emulsions at 18 and 37 C was associated with a ca. two-fold and an approx. 7- to 12-fold increase of b-carotene degradation rates when compared to systems stored under chilling conditions (Table 3). Regarding the emulsion structuring method, we have pointed out that Ca2þ mediated gelled emulsions (sheared and quiescent) exerted an enhanced stability against b-carotene degradation compared to the sodium alginate containing o/w ones. Generally, parameters such as the exposure to heat, light and oxygen conditions, the presence of pro-oxidants such as enzymes or heavy metals, and/or co-oxidants such as lipid hydroperoxides may significantly impact the oxidative stability of carotenoids (Soukoulis & Bohn, 2015). Given the isotropic character of the emulsions, as supported by the rheological measurements, and due to the absence of light exposure, it is hypothesised that the induction of lipid oxidation initiated by oxygen-free radicals is the governing factor affecting the chemical stability of b-carotene (Boon et al., 2009; Krinsky & Yeum, 2003). Therefore, it can be assumed that mass transport phenomena at the air e water (oxygen transfer to the bulk aqueous phase) and watereoil interfaces (diffusion of oxygen from the continuous water phase to the lipid droplet surface) as effected by the presence of the biopolymers in the bulk aqueous phase, are associated with the oxidative stability of b-carotene. In this context, the ionotropically induced crosslinking of sodium alginate in the continuous aqueous phase resulted in the reduction of the oxygen molecular diffusivity via two main pathways: first, the increased macroviscosity of the bulk aqueous phase due to the supramolecular junction of the egg-box ordered structures and second, the decreased specific surface area of the lipid droplets (due to the larger Sauter diameter). According to Yi et al., (2014) the oxidative stability of b-carotene loaded o/w emulsions showed a linear proportional dependence on lipid droplet mean size. However, it should be pointed out that increasing the concentration of sodium alginate in the continuous aqueous phase did not confer any significant enhancement of the oxidative stability of b-carotene. Similarly, Toniazzo et al. (2014) reported that increased guar and xanthan gum concentrations in b-carotene loaded liposomes did not improve the chemical stability of b-carotene throughout storage at chilling conditions, despite increased macroviscosity.


307

Table 2 Degradation rates and half-life (t1/2) of b-carotene encapsulated in sodium alginate o/w and o/g emulsions stored at 4, 20 and 37 C for 65 days, calculated using zero-order kinetic models. 4 C

Sample

20 C

k-rate (% day1) O/w emulsion 1% O/w emulsion 1.5% Sheared o/g emulsion 1% Sheared o/g emulsion 1.5% Quiescent o/g emulsion 1% Quiescent o/g emulsion 1.5% a

0.22 0.23 0.19 0.19 0.07 0.15

± ± ± ± ± ±

0.02d 0.02d 0.02c 0.01c 0.00a 0.01b

37 C

t1/2 (days)

R2

k-rate (% day1)

225 217 262 267 677 344

0.92 0.91 0.92 0.82 0.86 0.82

0.52 0.67 0.45 0.43 0.28 0.27

± ± ± ± ± ±

0.04c 0.05d 0.02b 0.03b 0.02a 0.03a

t1/2 (days)

R2

k-rate (% day1)

95 74 111 116 178 187

0.89 0.97 0.95 0.93 0.81 0.91

2.77 2.47 1.50 1.30 1.42 1.28

± ± ± ± ± ±

0.22c 0.17c 0.12b 0.10ab 0.11ab 0.09a

t1/2 (days)

R2

18 20 33 39 35 39

0.97 0.99 0.98 0.96 0.97 0.99

Values in a column not sharing the same superscripts are significantly different (p < 0.05) according to Tukey's post-hoc test.

Table 3 Activation energy values (kJ mol1) of oxidative degradation of b-carotene encapsulated in sodium alginate containing o/w and o/g emulsions, calculated according to Arrhenius kinetics model. Sample

Activation energy of b-carotene degradation Ea (kJ/mol)


55.1 51.5 44.8 42.0 63.9 47.6

a

± ± ± ± ± ±

0.2d 0.4c 0.5b 0.3a 0.9f 0.1e

R2 0.996 0.997 0.999 0.999 0.997 0.981


Fig. 4. Comparison of experimental and Arrhenius model predicted degradation rates of b-carotene encapsulated in sodium alginate o/w and o/g emulsions containing bcarotene.

In order to further investigate the impact of the storage temperature, as well as the structuring method on the oxidative stability of b-carotene, the degradation rate constants were fitted to the Arrhenius equation as follows (Dermesonluoglu, Katsaros, Tsevdou, Giannakourou, & Taoukis, 2015):

Ea

lnkbcar ¼ lnkref ;bcar R

1 T

!

(5)

T1ref

where kref,bcar equals the degradation rate of b-carotene at reference temperature (25 C), Ea denotes the activation energy of bcarotene degradation, and R is the universal gas constant. As illustrated in Fig. 4, the predicted b-carotene degradation rate constants were well matched. According to the estimated

activation energies (Table 3), it is fairly depicted that the sheared o/ g emulsions exhibited the lowest Ea values, and therefore these systems were less responsive to storage temperature changes. This is of particular importance as in many cases processed food is subjected to temperature fluctuations throughout postmanufacturing steps, e.g. shipment and storage, leading to shortened shelf-life. It should also be pointed out that the increased sodium alginate concentration induced a significant (p < 0.001) decrease of the activation energies, and consequently the temperature dependency of b-carotene degradation rate constants. It is well established that increased storage temperature may induce lipid droplet aggregative phenomena such as bridging flocculation or coalescence (Qian, Decker, Xiao, & McClements, 2012c), as well as structure-conformational changes due to increased intermolecular thermal expansion of the biopolymer dimers (Ma et al., 2014). This may promote further oxidative degradation of lipophilic compounds, including carotenoids. Thus, increasing sodium alginate concentrations appears to enhance the oxidative stability of bcarotene occurring due to temperature associated physicochemical phenomena (Suppl. Fig. 2), see Fig. 5. Finally, based on the predicted values of b-carotene degradation constant rates at ambient temperature conditions (25 C), the shelflife of the o/w emulsions calculated on the basis of half-time (t1/2) for b-carotene, was ca. 48.2 ± 0.4 days for sodium alginate stabilised systems, and 75.3 ± 3.3 and 99.1 ± 2.2 days for sheared and quiescent o/g emulsion systems, respectively. Qian et al. (2014b) reported a ca. 16 day shelf-life of b-lactoglobulin stabilised b-carotene loaded o/w emulsions stored at 20 C, while Yi et al. (2014) reported a shelf-life ranging from 40 to 125 days for sodium caseinate o/w emulsions with varying lipid droplet mean size stored at 25 C. 3.4. Bioaccessibility of b-carotene Structural design of o/w emulsions purposed as lipophilic compound carriers should attest a high stability against oxidative degradation and promote bioaccessibility and absorption

308


Fig. 5. Flow curves of the simulated oral, gastric and intestinal chymes, obtained by the in vitro digestion of the sodium alginate o/w and o/g emulsions containing bcarotene. Rheological measurements were carried out at 25 C.

(bioavailability) of the bioactive compounds throughout gastrointestinal passage (Soukoulis & Bohn, 2015). Parameters such as the physical state (crystalline or liquid), and colloidal properties of the

lipid phase (Qian, Decker, Xiao, & McClements, 2012; Rao, Decker, Xiao, & McClements, 2013; Salvia-Trujillo et al., 2013a, 2013b), as well as the structure conformation of the continuous aqueous phase as influenced by the presence of food matrix constituents, e.g. proteins or soluble dietary fibre (Verrijssen et al., 2014; Verrijssen, Verkempinck, Christiaens, VanLoey, & Hendrickx, 2015; Yonekura & Nagao, 2009), are known to influence the fractional bioaccessibility of carotenoids. In addition, other food microconstituents such as minerals (Biehler, Kaulmann, Hoffmann, Krause, & Bohn, 2011) or emulsifiers such as free fatty acids (Corte-Real et al., 2014) may also impact the carotenoid bioaccessibility. In the present study, fractional bioaccessibility of b-carotene ranged from 16 to 29% for the SA containing o/w emulsions and 26e49% for the ionotropically gelled ones (Table 4). In the latter case, sheared o/g emulsions resulted in prominently enhanced bioaccessibility of b-carotene (48 ± 2%) compared to the quiescent o/g emulsions (25 ± 4%). The increased sodium alginate concentration resulted in a significant reduction of the percentage of bcarotene transferred to the mixed micelles, accounting for ca. 44% in the case of the sodium alginate stabilised emulsions and 5e18% for the sheared and quiescent o/g emulsions. Fractional bioaccessibility of carotenes in fruit and vegetable matrices is considerably low, that is, 10e20% (Bohn, 2008; Biehler et al. 2011), whereas higher bioaccessibility values up to 50% have been reported for structurally engineered food emulsions (Soukoulis & Bohn, 2015; Verrijssen et al., 2014, 2015; Xu et al., 2014), possibly due to strong matrix binding effects within natural matrices, as well as high contents of dietary fibre. In this context, the obtained results for SA containing o/w and o/ g emulsions appear to be well-residing into the expected bioaccessibility range of b-carotene. Nevertheless, the adverse effect of increased soluble dietary fibre (including sodium alginate) content on the bioaccessibility of carotenes has been previously highlighted (Yonekura & Nagao, 2009). However, it is not well understood how the structure-conformational state of sodium alginate may impact the transfer of carotenoids to the mixed micelles. Hereby, we hypothesise that the enhanced bioaccessibility of b-carotene obtained in the case of o/g emulsions may be associated with the physicochemical colloidal changes occurring during the gastrointestinal passage. It is well established, that sodium alginate, depending on its chemical structure (that is, the glucuronic to mannuronic unit ratio), can undergo significant self-assembly in acidic environments, e.g. pH 1e3 (Draget, Skjåk-Bræk, & Stokke, 2006). Acid self-structuring of sodium alginate in the gastric chyme can result in remarkable changes of the colloidal aspects of the emulsions, such as increased lipid droplet mean size, due to coalescence. On the contrary, in a recent study (Soukoulis, Fisk, Bohn, & Hoffmann, 2016) we have shown that ionotropically structured sodium alginate o/w emulsions undergo a significant decrease of the complex viscosity when exposed to simulated gastric chyme due to the dialysis of Ca2þ e sodium alginate dimers in the presence of monovalent ions, e.g. Naþ and Kþ, found at high

Table 4 Physicochemical properties of the micellar fraction of digesta samples and b-carotene bioaccessibility. Sample

Surface tension (dyn/cm)


38.1 37.5 37.6 36.1 37.5 37.1

a

± ± ± ± ± ±

0.10d 0.12c 0.24c 0.20a 0.08c 0.09b

z-potential (mV) 27.7 27.1 24.2 24.7 21.1 20.8

± ± ± ± ± ±

2.8a 2.6a 1.5a 2.7a 0.8b 1.0b

d3,2 (nm) 138 111 97 89 79 90

± ± ± ± ± ±

Apparent viscosity (mPa*s) 4b 20a 4a 7a 7a 8a

1.24 1.62 1.66 2.05 1.72 2.30

± ± ± ± ± ±

0.03a 0.02b 0.04b 0.01c 0.05b 0.04d


b-Carotene bioaccessibility (%) 28.6 16.0 49.3 46.7 27.1 26.3

± ± ± ± ± ±

1.1b 0.3a 0.6d 1.8d 1.8b 4.4b


concentrations in both gastric and intestinal fluids. Therefore, for the o/g emulsion systems, it is assumed that only minor changes of the colloidal aspects of the lipid phase occurred. Following exposure of the gastric chyme systems to the intestinal fluids, lipolysis is taking place, promoting the transport of carotenoids to the mixed micelles. It is well established that the larger the lipid droplets, the lower b-carotene bioaccessibility (Yi et al., 2014). Indeed, according to the physical characteristics of the micellar digesta fraction (Table 4), SA containing o/w emulsions exerted a significantly larger lipid droplet size than the oilin-gel ones, partially explaining the observed bioaccessibility. In addition, micellar digestion fractions of quiescent o/g emulsions exhibited significantly higher (p < 0.001) z-potential (no impact of the dietary fibre concentration), compared to the sheared o/g emulsions. Considering that the formation of the SA e Ca2þ dimers is a rapid phenomenon, the absence of constant stirring may not have allowed the complete arrangement of the free Ca2þ ions in the egg-box, e.g. interclusters compared to the sheared o/g emulsions, resulting in an increased z-potential of the interfacial double layers. Recently, we have shown that bioaccessibility of carotenes is significantly lowered in the presence of divalent minerals such as calcium, magnesium and zinc, due to their ability to induce precipitation of the surface active bile salts and fatty acids (Corte-Real et al., 2016). Therefore, it is postulated that in the case of quiescent o/g emulsion systems, the presence of unbound calcium may also trigger bile salt precipitation, resulting in a reduced proportion of bioaccessible b-carotene compared to sheared o/g emulsions. In summary, the development of sodium alginate o/g emulsions appears to be a viable strategy to ensure competitive chemical stability during storage and good bioavailability aspects compared to that of carotene rich food matrices such as fruits and vegetables. It is assumed that ion mediated structuring of emulsions can preventively impact oxidative degradation of b-carotene via sterical hindering the mass transfer rate of pro-oxidants from the air e water and watereoil interfaces. In addition, b-carotene degradation in SA o/g emulsions was less responsive to temperature fluctuation compared to the stabilised systems. Inasmuch as their feasibility as carrier systems for carotenes, fractional bioaccessibility was maximised for sheared o/g emulsions, while except for SA stabilised o/w systems, the concentration of dietary fibre had only a minor impact on b-carotene bioaccessibility. Taking into consideration the major physicochemical and colloidal changes occurring during gastrointestinal passage, it is postulated that the enhanced fractional bioaccessibility of b-carotene in the case of o/g emulsion systems is associated with their restrained responsiveness to gastrointestinal fluids (particularly the gastric), maintaining the original colloidal state of the emulsions and therefore favouring the accessibility of lipolytic enzymes to the lipid droplets in the simulated small intestinal phase. In addition, the differences observed between the quiescent and sheared o/g emulsions appeared to be related to the amount of unbound calcium that can alter the bioaccessibility of bcarotene via the precipitation of bile salts and free fatty acids. In order to further provide insight into the understanding of the aforementioned mechanistic background, the impact of structure mediating ions (e.g. Naþ, Kþ, Ca2þ) and additional biopolymer types, e.g. kappa-carrageenan, are going to be illustrated in forthcoming studies.

Acknowledgements We are very much indebted to Boris Untereiner for technical assistance with carotenoid extractions.

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