- Email: [email protected]
Impact of gum Arabic on the partition and stability of resveratrol in sunflower oil emulsions stabilized by whey protein isolate
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Protocols
Impact of gum Arabic on the partition and stability of resveratrol in sunflower oil emulsions stabilized by whey protein isolate Haixia Zhanga,b,1, Qi Fana,b,c,1, Di Lia,b, Xing Chena,b, Li Lianga,b,
T
⁎
a
State Key Lab of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China c Jinan Fruit Research Institute, China Supply and Marketing Cooperatives, Jinan, Shandong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Whey protein isolate Gum Arabic Resveratrol Emulsion
In protein-stabilized oil-in-water emulsions, a co-emulsifier may also be an antioxidant, increasing the oxidative stability of the oil and adding nutritional value to the formulation. We investigated the impact of gum Arabic on the partition and stability of resveratrol in sunflower oil emulsions produced using whey protein isolate in the absence and presence of calcium. Gum Arabic increased the protein and resveratrol contents at the oil-water interface and the stability of resveratrol, which was enhanced by calcium. Resveratrol increased the oxidative stability of the oil. These results indicate that resveratrol is stable in the interfacial membrane of emulsions made with whey protein isolate, calcium and gum Arabic and suggest that oil-in-water emulsions could be used as potential carriers of co-encapsulated functional oils and polyphenolic antioxidants.
1. Introduction
particles [8,9]. Soy protein isolate (SPI) complexes with resveratrol are efficient emulsifiers for improving oxidative stability of corn oil/water emulsions [10]. About 50% of the resveratrol bound by whey protein isolate (WPI) is adsorbed at the interface in O/W emulsions of sunflower oil [11]. Adding calcium into emulsions stabilized with heatdenatured WPI appears to improve the polyphenol content of the surface but decrease storage stability [12]. However, the stability of resveratrol bound by β-lactoglobulin, a major whey protein, has been improved through the formation of protein-pectin complex particles [13]. Complexation of proteins with polysaccharides may improve functional properties of individual polymers [14,15]. Secreted by Acacia senegal, gum Arabic (GA) is an anionic polysaccharide with a high molecular weight and 2% covalently bound proteinaceous material. At a pH below isoelectric point (pI ˜ 5) of WPI, WPI-GA complexes have been used for the encapsulation and protection of single components such as conjugated linoleic acid, ω-3 rich tuna oil and D-limonene [16–18]. The viability of probiotic bacteria and the oxidative stability of tuna oil have been improved by co-encapsulating them in WPI-GA complex coacervate matrix at pH 3.75 [19]. At pH ranged from 5 to 7, positively charged domains remaining on WPI interact with negatively charged GA to form weak complexes [20]. At pH 7, 7.5% WPI and 2.5% GA is a better stabilizer of 10% canola oil emulsions than either WPI or
Food proteins have been widely used as carrier materials for the encapsulation and protection of bioactive components, since they possess functional properties including emulsification and gelation and interact with low-molecular-weight ligands and polysaccharides [1,2]. Proteins usually exert compact and random coil structures, or disperse in the form of colloidal particles at the oil-water interface [3]. In general, hydrophobic components are dissolved in the inner oil phase of protein-stabilized emulsions. According to the polar paradox, non-polar and amphiphilic antioxidants provide better protection of oxidationlabile substances in oil-in-water (O/W) emulsions than polar antioxidants do, due to their greater affinity to the oil-water interface [4]. Some antioxidants are known to co-adsorb to droplet surfaces, where they may function as co-emulsifiers, although they could not form physically-stable emulsions when used in isolation [5]. It is thus necessary to improve the content of interfacial antioxidants not only for oxidative stability of O/W emulsions but also for co-encapsulation of bioactive components in a single emulsion. Resveratrol (trans-3,5,4′-trihydroxystibene) is a natural polyphenol with antioxidant, anti-inflammatory, antibacterial, anti-apoptopic and anti-depressant effects [6,7]. It forms molecular complexes with proteins and can thus be encapsulated in protein-based nano/micro-
⁎
Corresponding author at: State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China. E-mail address: [email protected] (L. Liang). 1 These authors contributed equally to the work. https://doi.org/10.1016/j.colsurfb.2019.06.034 Received 14 March 2019; Received in revised form 23 May 2019; Accepted 15 June 2019 Available online 17 June 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
2.5. Fluorescence measurement
GA alone [21]. In this study, the impact of gum Arabic on the encapsulation and protection of resveratrol in WPI-stabilized O/W emulsions without and with CaCl2 was investigated. At neutral condition, metal ions can improve biomacromolecular interaction and assembly by neutralizing negative charges, electrostatic shielding and inducing conformational changes [22,23]. Emulsions were characterized in terms of size distribution, ζ-potential and interfacial protein content. Partition and stability of resveratrol were analyzed to discuss the feasibility of its encapsulation and protection at the oil-water interface. Moreover, the oxidative stability of the oil phase in the absence and presence of resveratrol was measured. The results obtained are applicable to improving the oxidative stability of oils and co-encapsulated components in single emulsions.
Fluorescence spectra of resveratrol in the emulsion were obtained using a Cary Eclipse spectrophotometer (Agilent Co. Ltd, New York, USA) equipped with a front-surface accessory. The wavelength of excitation was set at 320 nm and the incidence angle of the excitation radiation was 15°. Spectra were recorded from 350 to 550 nm. Spectral resolution was 2.5 nm for both excitation and emission. 2.6. Quantitation of resveratrol using HPLC Emulsions were centrifuged twice at 13,000–20,000 × g for 30 min at 4 °C. The aqueous phase was then clarified at 150,000 × g for 30 min at 4 °C. A sample of whole emulsion or of non-clarified or clarified aqueous phase was blended with an equal volume of polydatin solution (150 μM or 15 μM in methanol), vortex-mixed with 4 volumes of methanol. After centrifugation at 3500 × g at 4 °C, the supernatant was passed through a 0.45-μm syringe filter and injected into an HPLC system equipped with a C18 column (5 μm, 4.6 mm × 250 mm, Waters, Milford, MA, USA) at 35 °C. The mobile phase was a mixture of methanol and distilled water (50:50, v/v) at 1 mL min−1. UV detection at 306 nm and 285 nm was used to quantitate trans-resveratrol and cisresveratrol, respectively. The reported resveratrol content was the sum of both isomers. The percentage of the resveratrol bound to free WPI (Rb) was calculated from the difference between the total amount in the aqueous phase (Raq) and the amount in the clarified aqueous phase (Rc) divided by the amount in the whole emulsion (Rw) using the expression Rb = (Raq − Rc)/Rw × 100. The percentage of free resveratrol (Rf) in the aqueous phase was calculated as Rf = Rc/Rw × 100. Resveratrol at the oil-water interface (Ri) was calculated as Ri = (Rw − Raq)/ Rw × 100. The content of resveratrol remaining in the whole emulsion, in the continuous phase and at the oil-water interface during storage at 45 °C was expressed as a percentage of the corresponding initial contents of trans-resveratrol.
2. Materials and method 2.1. Materials WPI (BioPRO, ˜92%) was obtained from Davisco International Inc. (Le Sueur, MN, USA). Sunflower oil (Brand Duoli) was purchased from a local retailer. Resveratrol (trans-isomer, purity > 98%) was purchased from Sango Biotech Co. (Shanghai, China). Acacia tree gum Arabic, 2thiobarbituric acid (TBA, ≥98%), 1,1,3,3-tetramethoxypropane (TEP, 99%) and cumene hydroperoxide (CHP, technical grade, 80%) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Other reagents were analytical grade and obtained from SinoPharm CNCM Ltd. (Shanghai, China). 2.2. Emulsion preparation Emulsions were prepared without and with CaCl2 according to our method described prevously [12]. A 1% (w/w) solution of WPI adjusted to pH 7.0 was heated at 90 °C for 35 min, then cooled to room temperature and mixed with 50.5 g L−1 resveratrol in 70% (v/v) ethanol at a volume ratio of 20:1 for 35 min. Calcium chloride (0.1%–1%, w/w) was added at this point. Gum Arabic solution (4%, w/w) at pH 7.0 was then stirred in for 35 min. Sunflower oil was blended in by using a high speed mixer (ATS Engineering Ltd., Brampton, Ontario, Canada) operating at 14,000 rpm followed by passing twice through an ATSAH2100 high-pressure homogenizer (ATS Engineering Ltd., Brampton, Ontario, Canada) at 50 MPa and 10 °C to obtain an O/W emulsion with an oil content of 10% (w/w), WPI content of 0.5% (w/w), 0.13 g L−1 resveratrol, 0, 0.2 and 1.6 mmol/L CaCl2, and 0%, 0.05%, 0.10%, 0.50%, and 1.00% (w/w) gum Arabic in the continuous phase. The molar concentration of resveratrol was twice that of WPI. Some of emulsion samples containing sodium azide (0.02%, w/w) were stored at 45 °C for 30 days.
2.7. Lipid oxidation Lipid hydroperoxides were measured as the primary oxidation products according to the method of Dong [25]. Exactly 0.1 mL of emulsion or corresponding bulk oil was vortex-mixed with 1.5 mL of isooctane/isopropanol mixture (3:1, v:v) for 30 s then centrifuged at 3000 rpm for 5 min. Exactly 0.2 mL of the organic phase was collected and added to 2.8 mL of methanol/n-butanol (2:1, v:v) then reacted with 15 μL of 3.94 mol/L ammonium thiocyanate and 15 μL of ferrous iron solution prepared by reacting 0.132 mol/L barium chloride with 0.144 mol/L ferrous sulfate. The absorbance at 510 nm was measured after reaction for 20 min using a Shimadzu UV-1800 spectrophotometer (Shimadzu Corp, Kyoto, Japan). The hydroperoxide content was determined from a standard curve and expressed as mmol CHP equivalent per kg of oil. Secondary products of lipid oxidation were measured as thiobarbituric acid reactive substances (TBARS) according to the method of Feng [26]. Exactly 2 mL of emulsion was mixed with 2 mL of TBA working solution containing 15% (w/v) trichloroacetic acid, 0.375% (w/v) TBA and 0.25 mol/L HCl and placed in a boiling water bath for 15 min. The mixture was cooled down to room temperature and centrifuged at 10,000 rpm for 10 min and the supernatant was passed through a 0.45μm syringe filter. The absorbance at 532 nm was measured. The TBARS content was calculated from a standard curve of TEP and expressed as μmol malondialdehyde (MDA) equivalent per kg of oil.
2.3. Size and ζ-potential measurements Size distribution and ζ-potential of WPI emulsions in the absence and presence of CaCl2 and/or gum Arabic were measured on a NanoBrooker Omni Particle size Analyzer (Brookhaven Instruments Ltd, New York, USA) with a He/Ne laser (λ = 633 nm). Samples were diluted by 100 times before measurement at 25 °C and at a scattering angle of 173°. 2.4. Interfacial protein determination Emulsions were centrifuged twice at 13,000 × g for 30 min at 4 °C using a 5804 R centrifuge (Eppendorf Co. Ltd, Hamburg, Germany). The amount of protein in the subnatant (As) and in the whole emulsion (Aw) was determined using the Kjeldahl method. Percentage of protein at the oil-water interface was calculated as (Aw − As)/Aw × 100% [12,24].
2.8. Statistical analysis All samples were prepared at least in duplicate for measurements. Values presented are mean ± standard deviation. Data were analyzed 750
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
for significant differences using the online GraphPad QuikCalcs Free ttest calculator (GraphPad Software Inc. San Diego, CA, USA). 3. Results and discussion 3.1. Influence of gum Arabic on WPI-stabilized emulsions 3.1.1. Size distribution In emulsions stabilized by WPI alone, two peaks were observed, at about 130 nm and 550 nm (Fig. 1A), which have been shown to correspond respectively to WPI particles and WPI-stabilized oil droplets [12]. Particles in emulsions stabilized by WPI plus 0.05% GA were smaller and distributed around 120 nm and 375 nm, whereas higher GA concentrations resulted in particles of sizes similar to those in emulsions stabilized by WPI alone. The presence of 0.2 mmol/L CaCl2 shifted the peaks to around 140 and 620 nm in the absence of GA, then to 90 and 320 nm at 0.10% GA and to 270 and 1030 nm at 1.00% GA (Fig. 1B). In the presence of 1.6 mM CaCl2, the peaks were around 190 and 990 nm in the absence of GA, then shifted to 170 nm and 570 nm at 0.05% GA and to 360 nm and 1420 nm at 0.50% GA (Fig. 1C). At a polysaccharide concentration of 1.00%, WPI-Ca-GA emulsions had a single size peak around 2580 nm. 3.1.2. Zeta-potential WPI-stabilized emulsions had a ζ-potential of −27.35 mV at pH 7.0 (Table 1). Whey proteins are isoelectric at about pH 5 and thus negatively charged at higher values. Stabilization of oil droplets by WPI has been attributed primarily to electrostatic repulsion [27]. The ζ-potentials of emulsions stabilized by WPI and GA were about −35 mV, regardless of the polysaccharide concentration. Adding 0.2 mmol/L CaCl2 lowered the ζ-potential at all GA concentrations except for the cases of 0% and 1.00% GA. At 1.6 mmol/L, CaCl2 further lowered the ζ-potential, to −23.60 mV at 0% GA, to −28.15 mV at 1.00% and to intermediate values at GA concentrations in between. These results suggest GA increases electrostatic repulsion between droplets and hence the stability of the emulsion against aggregation. Moreover, hydrophilic part of GA is also believed to provide stability against droplet aggregation through steric repulsion [27]. An absolute ζ-potential of 20 mV may provide sufficient stabilization, due to steric stabilization of biopolymers [28]. 3.1.3. Interfacial protein content At gum Arabic concentrations up to 0.10%, about 40% of the WPI added was incorporated into the emulsion interface (Table 2). This percentage increased to about 49% at higher concentrations of GA. Adding 0.2 mmol/L CaCl2 further increased the amount slightly at 0.10% and 1.00% GA, while at 1.6 mmol/L CaCl2, the amount was about 68–70% at GA concentrations up to 0.50% and then increased to about 76% at 1.00% GA. When the concentration of GA was 1.00%, the increased amount of protein at the oil-water interface was about 0.04–0.05%, while the polysaccharide itself carries 0.02% covalently attached proteinaceous material. Therefore, adding GA to the emulsion formulation increased the adsorption of WPI at the oil-water interface in the absence and presence of CaCl2. The observed effect of GA on droplet size and ζ-potential of WPIstabilized sunflower oil emulsions (Fig. 1 and Table 1) is similar to that of 1–4% GA on 10% soybean oil emulsions stabilized by 1% soy protein concentrate, in which there was competitive adsorption between GA and soy protein at the O/W interface [29]. However, at pH 7.0, WPI was reportedly a much more efficient stabilizer of emulsified orange oil and vitamin E-acetate, being more effective than GA at lower concentrations and producing smaller droplets [27]. Based on the percentages of protein found at the sunflower oil emulsion interface (Table 2), GA did not replace WPI but increased its interfacial adsorption. At low concentrations, GA might also act as an emulsifier, absorbing at the oil droplet surface and reducing interfacial tension and droplet size
Fig. 1. Size distribution of WPI-stabilized emulsions in the absence and presence of 0.05%, 0.10%, 0.50% and 1.00% gum Arabic at 0 (A), 0.2 (B) and 1.6 (C) mmol/L CaCl2.
751
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
Table 1 Effect of gum Arabic on ζ-potential (mV) of O/W emulsions stabilized by 0.5% WPI plus CaCl2 at different concentrations. Concentration of CaCl2 (mM)
Concentration of gum Arabic (%) 0.00
0.0 0.2 1.6
0.05
−27.35 ± 0.99 −25.41 ± 0.77Aa −23.60 ± 0.26Ac Aa
0.10
−35.85 ± 0.73 −30.40 ± 0.84Bb −24.62 ± 1.71ABc Ba
0.50
−35.35 ± 1.08 −32.40 ± 0.72BCb −25.64 ± 0.56Bc Ba
1.00
−36.54 ± 0.70 −34.17 ± 0.45Cb −25.96 ± 0.34Bc Ba
−33.64 ± 1.26Ba −33.25 ± 1.32BCa −28.15 ± 0.25Cc
Values with the different superscript letter (upper case for gum Arabic concentration, lower case for CaCl2 concentration) are significantly different (P < 0.05).
interface (Fig. 3A). Therefore, the addition of GA increased the adsorption of both free and encapsulated resveratrol molecules at the oilwater interface. The percentage of resveratrol associated with the interface was greater than that of WPI (Fig. 3 and Table 2), which is consistent with previous measurements of green tea polyphenol adsorption to the oilwater interface of β-LG-stabilized emulsions [33]. A similar phenomenon was observed without and with 0.05–1.00% GA in the presence of 0.2 mmol/L CaCl2. These results suggest that encapsulation of resveratrol occurs mainly at the O/W interface and is not in association with WPI dispersed in the continuous phase. At 1.6 mM CaCl2 and GA concentrations ≤0.10%, the percentages of resveratrol associated with the interface were lower than those of WPI. However, this difference disappeared at higher GA concentrations, due to almost complete transfer of the protein-bound polyphenol molecules to the oil-water interface (Fig. 3).
(Fig. 1). At high concentrations, GA might interact with interfacial WPI [20], resulting in an increase of droplet size (Fig. 1). The negative charges of GA at the interface provide additional stabilization by increasing repulsive forces between droplets (Table 1). Calcium ions are known to cause aggregation of heat-denatured WPI [30]. The emulsifying capacity of WPI appears to decrease as aggregate size increases, possibly because larger aggregates require more time to migrate and are less capable of re-configuring and aligning with the oilwater interface [31]. At lower concentrations, GA therefore might have had more emulsifying action by reducing oil droplet size, especially as the concentration of Ca2+ increased (Fig. 1). Adding Ca2+ also decreases negative charges on WPI particles [30], which would reduce repulsion and favor interaction between local positive charges on the protein and negative charges on the polysaccharide, thus allowing an increase in droplet size (Fig. 1). 3.2. Encapsulation of resveratrol at the oil-water interface
3.3. Storage stability of resveratrol Fluorescence of resveratrol is sensitive to the polarity of surrounding environment. The λmax of resveratrol is around 404 nm in phosphate buffer and shifts to 394 nm by binding to β-LG and to 383 nm in 75% ethanol [32]. The λmax around 380 nm in WPI emulsions was similar in the absence and presence of calcium and/or gum Arabic (Fig. 2), indicating that resveratrol was encapsulated in a hydrophobic environment. At 0.2 and 1.6 mmol/L CaCl2, fluorescence intensity of resveratrol at λmax was about 1.05 and 1.48 times that in its absence. The intensity gradually increased as the GA concentration increased from 0.05% to 1.00%, but less as the concentration of CaCl2 increased. These results suggest that both CaCl2 and GA cause more resveratrol molecules to shift to the hydrophobic phase and thereby improve its encapsulation. Resveratrol was not detected in the oil phase of WPI-stabilized emulsions when measured after oil extraction with isooctane/isopropanol mixture. Resveratrol forms complexes with whey proteins including β-LG, α-LA and BSA [8,32] and is adsorbed at the surface of oil droplets stabilized by soy and whey proteins [10,11]. About 49%, 52% and 61% of the resveratrol was associated with the oil-water interface at 0, 0.2 and 1.6 mM CaCl2 respectively, and this is unchanged by addition of 0.05% gum Arabic (Fig. 3A). At 1.00% GA, these values shift to about 63%, 63% and 74% respectively. The portion of the resveratrol found in the continuous (aqueous) phase generally decreased as the GA concentration increased, which was more pronounced in the presence of CaCl2 than in its absence (Fig. 3B). This decrease was less than the increase in amount of resveratrol adsorbed to the oil-water
The trans-resveratrol contents of the sunflower oil emulsions stored for 30 days at 45 °C are shown in Fig. 4 for the whole emulsion (A), the oil-water interface (B) and the aqueous phase (C). In the absence of gum Arabic, the content dropped below 70% of its initial value, and CaCl2 exacerbated this at the oil-water interface, as has been noted previously [12], but not in the aqueous phase. Gum Arabic appeared to counteract the effect of CaCl2. In the presence of 1.00% GA, CaCl2 appeared to have little if any de-stabilizing effect on resveratrol. In the best cases, about 75% of the initial content remained. A stronger counteraction might have been observed at 1.6 mmol/L CaCl2, but no measurement was performed because of visible creaming of these emulsions at 0.50% and 1.00% GA. At 1.6 mmol/L CaCl2 and 0.10% GA, the final resveratrol contents in the whole emulsion, the oil-water interface and the aqueous phase were respectively 62%, 54% and 66% of the initial values. 3.4. Oxidation stability of emulsified sunflower oil The contents of lipid hydroperoxide in all fresh emulsions were between 1.66 and 1.99 mmol CHPE/kg oil (Fig. 5A). After storage at 45 °C for 30 days, the content of lipid hydroperoxide in WPI emulsions increased to 73.18 mmol CHPE/kg oil. Gum Arabic at 0.05% and 0.10% decreased the content slightly, while gum Arabic at 1.00% increased it to a value even higher than that without the polysaccharide. The effect was similar at 0 and 0.2 mmol/L CaCl2. However, at 1.6 mmol/L CaCl2,
Table 2 Effect of gum Arabic on interfacial protein content (%) of O/W emulsions stabilized by 0.5% WPI plus CaCl2 at various concentrations. Concentration of CaCl2 (mM)
Concentration of gum Arabic (%) 0.00
0.0 0.2 1.6
0.05 Aa
40.43 ± 0.63 40.44 ± 0.24Aa 67.92 ± 2.21Ab
0.10 Aa
39.72 ± 0.33 39.93 ± 0.36Aa 67.67 ± 1.96Ab
0.50 Aa
40.40 ± 0.12 41.89 ± 0.34Bb 68.10 ± 1.53Ac
1.00 Ba
48.05 ± 0.88 48.29 ± 0.55Ca 70.35 ± 0.48Ab
49.21 ± 0.28Ba 50.81 ± 0.26Db 75.94 ± 0.16Bc
Values with the different superscript letter (upper case for gum Arabic concentration, lower case for CaCl2 concentration) are significantly different (P < 0.05). 752
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
Fig. 3. Content of resveratrol adsorbed at the oil-water interface (A) and bound to WPI in the continuous phase (B) of sunflower oil emulsions as a function of the concentration of gum Arabic at 0, 0.2 and 1.6 mmol/L CaCl2.
10 μmol MDA/kg oil in resveratrol-free emulsions containing ≤0.10% GA and to 16.60 μmol MDA/kg oil at 1.00% GA. These changes were indifferent to the CaCl2 concentration. The effect of resveratrol was to decrease these values slightly, especially in association with 1.6 mmol/ L CaCl2. The antioxidant effect of resveratrol on emulsified sunflower oil (Fig. 5) is consistent with previous findings obtained using corn oil emulsified with soy protein isolate [10]. Resveratrol at the oil-water interface could protect the oil simply by reacting with oxidizing agents. It might also scavenge free radicals produced by the decomposition of lipid hydroperoxides accumulating at the oil interface [10]. The fraction of the resveratrol at the interface increased with the concentration of CaCl2. In addition, calcium-induced aggregation might thicken the interfacial membrane in comparison with WPI alone, thus further improving the oxidative stability of emulsified oil. However, calciumcrosslinked aggregates of heat-denatured WPI are believed to have a gel-like porous structure that is relatively permeable to oxygen [30]. This might be one reason for the enhanced protection of the oil (Fig. 5) at the expense of greater losses of resveratrol molecules (Fig. 4). In contrast, gum Arabic at concentrations ≤0.10% decreased the loss of resveratrol at the oil-water interface but not the oxidation of the oil, and even increased oxidation slightly at higher concentrations (Fig. 5), possibly by allowing TBARS to increase in the fresh emulsions (Fig. 5C and D).
Fig. 2. Fluorescence emission spectra of resveratrol in WPI-stabilized emulsions in the absence and presence of 0.05%, 0.10%, 0.50% and 1.00% gum Arabic at 0 (A), 0.2 (B) and 1.6 (C) mmol/L CaCl2.
the lipid hydroperoxide content decreased to about 36 mmol CHPE/kg oil and was independent of the GA concentration. The presence of resveratrol (Fig. 5B) lowered all of these values by 20 mmol or more at 0 or 0.2 mmol/L CaCl2 and by at least 12 mmol at 1.6 mmol/L CaCl2. The TBARS contents of fresh emulsions were about 3.50 μmol MDA/ kg oil and indifferent to the CaCl2 concentration (Fig. 5C) and to resveratrol (Fig. 5D) at GA ≤ 0.10%. However, at higher GA concentrations, the initial TBARS contents were greater, reaching about 7.85 μmol MDA/kg oil at 1.00% GA. After storage, TBARS rose to about
753
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
Fig. 4. Content of resveratrol remaining in the whole WPI emulsions (A), at the oil-water interface (B) and in the continuous phase (C) as a function of the concentration of gum Arabic at 0, 0.2 and 1.6 mM CaCl2 after storage for 30 days.
4. Conclusions Ternary complexes of WPI with calcium and gum Arabic appear to stabilize oil-in-water emulsions. Gum Arabic improved the encapsulation of resveratrol at the oil-water interface and its stability in the emulsions, especially in the presence of CaCl2. The presence of resveratrol improved the oxidative stability of emulsified sunflower oil. These results suggest that WPI-Ca-GA emulsions could be used as effective carriers for the co-encapsulation of functional oils and polyphenols.
Fig. 5. Formation of lipid hydroperoxides (A, B) and TBARS (C, D) in WPIstabilized emulsions without (A, C) and with (B, D) resveratrol as a function of the concentration of gum Arabic at 0, 0.2 and 1.6 mmol/L CaCl2 at 45 °C after 0 (solid symbols) and 30 (open symbols) days.
754
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
Acknowledgments
[16] X. Yao, S. Xiang, K. Nie, Z. Gao, W. Zhang, Y. Fang, K. Nishinari, G.O. Phillips, F. Jiang, Whey protein isolate/gum arabic intramolecular soluble complexes improving the physical and oxidative stabilities of conjugated linoleic acid emulsions, RSC Adv. 6 (2016) 14635–14642. [17] D. Eratte, B. Wang, K. Dowling, C.J. Barrow, B.P. Adhikari, Complex coacervation with whey protein isolate and gum arabic for the microencapsulation of omega-3 rich tuna oil, Food Funct. 5 (2014) 2743–2750. [18] J. Su, Q. Guo, L. Mao, Y. Gao, F. Yuan, Food Hydrocolloids Effect of gum arabic on the storage stability and antibacterial ability of β-lactoglobulin stabilized D -limonene emulsion, Food Hydrocoll. 84 (2018) 75–83. [19] D. Eratte, S. McKnight, T.R. Gengenbach, K. Dowling, C.J. Barrow, B.P. Adhikari, Co-encapsulation and characterisation of omega-3 fatty acids and probiotic bacteria in whey protein isolate-gum Arabic complex coacervates, J. Funct. Foods 19 (2015) 882–892. [20] M. Klein, A. Aserin, P. Ben Ishai, N. Garti, Interactions between whey protein isolate and gum Arabic, Colloids Surf. B: Biointerfaces 79 (2010) 377–383. [21] M. Klein, A. Aserin, I. Svitov, N. Garti, Enhanced stabilization of cloudy emulsions with gum Arabic and whey protein isolate, Colloids Surf. B: Biointerfaces 77 (2010) 75–81. [22] B.T. O’Kennedy, J.S. Mounsey, The dominating effect of ionic strength on the heatinduced denaturation and aggregation of β-lactoglobulin in simulated milk ultrafiltrate, Int. Dairy J. 19 (2009) 123–128. [23] G.E. Remondetto, Molecular mechanisms of Fe2+-induced β-lactoglobulin cold gelation, Biopolymers 69 (2003) 461–469. [24] Z.L. Wan, J.M. Wang, L.Y. Wang, X.Q. Yang, Y. Yuan, Enhanced physical and oxidative stabilities of soy protein-based emulsions by incorporation of a water-soluble stevioside-resveratrol complex, J. Agric. Food Chem. 61 (2013) 4433–4440. [25] S. Dong, B. Wei, B. Chen, D.J. McClements, E.A. Decker, Chemical and antioxidant properties of casein peptide and its glucose Maillard reaction products in fish oil-inwater emulsions, J. Agric. Food Chem. 59 (2011) 13311–13317. [26] J. Feng, H. Cai, H. Wang, C. Li, S. Liu, Improved oxidative stability of fish oil emulsion by grafted ovalbumin-catechin conjugates, Food Chem. 241 (2018) 60–69. [27] B. Ozturk, S. Argin, M. Ozilgen, D.J. McClements, Formation and stabilization of nanoemulsion-based vitamin E delivery systems using natural biopolymers: whey protein isolate and gum Arabic, Food Chem. 188 (2015) 256–263. [28] S. Honary, F. Zahir, Effect of zeta potential on the properties of nano-drug delivery systems—a review (Part 2), Trop. J. Pharm. Res. 12 (2013) 265–273. [29] B. Wang, L.J. Wang, D. Li, B. Adhikari, J. Shi, Effect of gum Arabic on stability of oil-in-water emulsion stabilized by flaxseed and soybean protein, Carbohydr. Polym. 86 (2011) 343–351. [30] Y. Ni, L. Wen, L. Wang, Y. Dang, P. Zhou, L. Liang, Effect of temperature, calcium and protein concentration on aggregation of whey protein isolate: formation of gellike micro-particles, Int. Dairy J. 51 (2015) 8–15. [31] R.S.H. Lam, M.T. Nickerson, The effect of pH and temperature pre-treatments on the physicochemical and emulsifying properties of whey protein isolate, LWT – Food Sci. Technol. 60 (2015) 427–434. [32] L. Liang, H.A. Tajmir-Riahi, M. Subirade, Interaction of β-lactoglobulin with resveratrol and its biological implications, Biomacromolecules 9 (2008) 50–56. [33] M. von Staszewski, V.M. Pizones Ruiz-Henestrosa, A.M.R. Pilosof, Green tea polyphenols-β-lactoglobulin nanocomplexes: interfacial behavior, emulsification and oxidation stability of fish oil, Food Hydrocoll. 35 (2014) 505–511.
This work was supported by the National Natural Science Foundation of China (NSFC Project 31571781) and the Fundamental Research Funds for the Central Universities (JUSRP51711B). References [1] L. Chen, G.E. Remondetto, M. Subirade, Food protein-based materials as nutraceutical delivery systems, Trends Food Sci. Technol. 17 (2006) 272–283. [2] A. Matalanis, O.G. Jones, D.J. McClements, Structured biopolymer-based delivery systems for encapsulation, protection, and release of lipophilic compounds, Food Hydrocoll. 25 (2011) 1865–1880. [3] D.J. McClements, S.M. Jafari, Improving emulsion formation, stability and performance using mixed emulsifiers: a review, Adv. Colloid Interface Sci. 251 (2018) 55–79. [4] F. Shahidi, Y. Zhong, Revisiting the polar paradox theory: a critical overview, J. Agric. Food Chem. 59 (2011) 3499–3504. [5] D.J. McClements, E. Decker, Interfacial antioxidants: a review of natural and synthetic emulsifiers and coemulsifiers that can inhibit lipid oxidation, J. Agric. Food Chem. 66 (2018) 20–25. [6] M.R. de Oliveira, A.L. Chenet, A.R. Duarte, G. Scaini, J. Quevedo, Molecular mechanisms underlying the anti-depressant effects of resveratrol: a review, Mol. Neurobiol. 55 (2018) 4543–4559. [7] T. Yang, L. Wang, M. Zhu, L. Zhang, L. Yan, Properties and molecular mechanisms of resveratrol: a review, Pharmazie 70 (2015) 501–506. [8] H. Cheng, Z. Fang, A.M. Wusigale, Y. Bakry, L. Chen, Liang, Complexation of transand cis-resveratrol with bovine serum albumin, β-lactoglobulin or α-lactalbumin, Food Hydrocoll. 81 (2018) 242–252. [9] G. Davidov-Pardo, I.J. Joye, D.J. McClements, Encapsulation of resveratrol in biopolymer particles produced using liquid antisolvent precipitation. Part 1: preparation and characterization, Food Hydrocoll. 45 (2015) 309–316. [10] Z.L. Wan, J.M. Wang, L.Y. Wang, Y. Yuan, X.Q. Yang, Complexation of resveratrol with soy protein and its improvement on oxidative stability of corn oil/water emulsions, Food Chem. 161 (2014) 324–331. [11] L. Wang, Y. Gao, J. Li, M. Subirade, Y. Song, L. Liang, Effect of resveratrol or ascorbic acid on the stability of α-tocopherol in O/W emulsions stabilized by whey protein isolate: simultaneous encapsulation of the vitamin and the protective antioxidant, Food Chem. 196 (2016) 466–474. [12] Q. Fan, L. Wang, Y. Song, Z. Fang, M. Subirade, L. Liang, Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion: impact of protein and calcium concentrations, Int. Dairy J. 73 (2017) 128–135. [13] H. Cheng, Z. Fang, T. Liu, Y. Gao, L. Liang, A study on β-lactoglobulin-triligandpectin complex particle: formation, characterization and protection, Food Hydrocoll. 84 (2018) 93–103. [14] C. Schmitt, C. Sanchez, S. Desobry-Banon, J. Hardy, Structure and technofunctional properties of protein-polysaccharide complexes: a review, Crit. Rev. Food Sci. Nutr. 38 (1998) 689–753. [15] B.L.H.M. Sperber, H.A. Schols, M.A. Cohen Stuart, W. Norde, A.G.J. Voragen, Influence of the overall charge and local charge density of pectin on the complex formation between pectin and β-lactoglobulin, Food Hydrocoll. 23 (2009) 765–772.
755
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Protocols
Impact of gum Arabic on the partition and stability of resveratrol in sunflower oil emulsions stabilized by whey protein isolate Haixia Zhanga,b,1, Qi Fana,b,c,1, Di Lia,b, Xing Chena,b, Li Lianga,b,
T
⁎
a
State Key Lab of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China c Jinan Fruit Research Institute, China Supply and Marketing Cooperatives, Jinan, Shandong, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Whey protein isolate Gum Arabic Resveratrol Emulsion
In protein-stabilized oil-in-water emulsions, a co-emulsifier may also be an antioxidant, increasing the oxidative stability of the oil and adding nutritional value to the formulation. We investigated the impact of gum Arabic on the partition and stability of resveratrol in sunflower oil emulsions produced using whey protein isolate in the absence and presence of calcium. Gum Arabic increased the protein and resveratrol contents at the oil-water interface and the stability of resveratrol, which was enhanced by calcium. Resveratrol increased the oxidative stability of the oil. These results indicate that resveratrol is stable in the interfacial membrane of emulsions made with whey protein isolate, calcium and gum Arabic and suggest that oil-in-water emulsions could be used as potential carriers of co-encapsulated functional oils and polyphenolic antioxidants.
1. Introduction
particles [8,9]. Soy protein isolate (SPI) complexes with resveratrol are efficient emulsifiers for improving oxidative stability of corn oil/water emulsions [10]. About 50% of the resveratrol bound by whey protein isolate (WPI) is adsorbed at the interface in O/W emulsions of sunflower oil [11]. Adding calcium into emulsions stabilized with heatdenatured WPI appears to improve the polyphenol content of the surface but decrease storage stability [12]. However, the stability of resveratrol bound by β-lactoglobulin, a major whey protein, has been improved through the formation of protein-pectin complex particles [13]. Complexation of proteins with polysaccharides may improve functional properties of individual polymers [14,15]. Secreted by Acacia senegal, gum Arabic (GA) is an anionic polysaccharide with a high molecular weight and 2% covalently bound proteinaceous material. At a pH below isoelectric point (pI ˜ 5) of WPI, WPI-GA complexes have been used for the encapsulation and protection of single components such as conjugated linoleic acid, ω-3 rich tuna oil and D-limonene [16–18]. The viability of probiotic bacteria and the oxidative stability of tuna oil have been improved by co-encapsulating them in WPI-GA complex coacervate matrix at pH 3.75 [19]. At pH ranged from 5 to 7, positively charged domains remaining on WPI interact with negatively charged GA to form weak complexes [20]. At pH 7, 7.5% WPI and 2.5% GA is a better stabilizer of 10% canola oil emulsions than either WPI or
Food proteins have been widely used as carrier materials for the encapsulation and protection of bioactive components, since they possess functional properties including emulsification and gelation and interact with low-molecular-weight ligands and polysaccharides [1,2]. Proteins usually exert compact and random coil structures, or disperse in the form of colloidal particles at the oil-water interface [3]. In general, hydrophobic components are dissolved in the inner oil phase of protein-stabilized emulsions. According to the polar paradox, non-polar and amphiphilic antioxidants provide better protection of oxidationlabile substances in oil-in-water (O/W) emulsions than polar antioxidants do, due to their greater affinity to the oil-water interface [4]. Some antioxidants are known to co-adsorb to droplet surfaces, where they may function as co-emulsifiers, although they could not form physically-stable emulsions when used in isolation [5]. It is thus necessary to improve the content of interfacial antioxidants not only for oxidative stability of O/W emulsions but also for co-encapsulation of bioactive components in a single emulsion. Resveratrol (trans-3,5,4′-trihydroxystibene) is a natural polyphenol with antioxidant, anti-inflammatory, antibacterial, anti-apoptopic and anti-depressant effects [6,7]. It forms molecular complexes with proteins and can thus be encapsulated in protein-based nano/micro-
⁎
Corresponding author at: State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China. E-mail address: [email protected] (L. Liang). 1 These authors contributed equally to the work. https://doi.org/10.1016/j.colsurfb.2019.06.034 Received 14 March 2019; Received in revised form 23 May 2019; Accepted 15 June 2019 Available online 17 June 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
2.5. Fluorescence measurement
GA alone [21]. In this study, the impact of gum Arabic on the encapsulation and protection of resveratrol in WPI-stabilized O/W emulsions without and with CaCl2 was investigated. At neutral condition, metal ions can improve biomacromolecular interaction and assembly by neutralizing negative charges, electrostatic shielding and inducing conformational changes [22,23]. Emulsions were characterized in terms of size distribution, ζ-potential and interfacial protein content. Partition and stability of resveratrol were analyzed to discuss the feasibility of its encapsulation and protection at the oil-water interface. Moreover, the oxidative stability of the oil phase in the absence and presence of resveratrol was measured. The results obtained are applicable to improving the oxidative stability of oils and co-encapsulated components in single emulsions.
Fluorescence spectra of resveratrol in the emulsion were obtained using a Cary Eclipse spectrophotometer (Agilent Co. Ltd, New York, USA) equipped with a front-surface accessory. The wavelength of excitation was set at 320 nm and the incidence angle of the excitation radiation was 15°. Spectra were recorded from 350 to 550 nm. Spectral resolution was 2.5 nm for both excitation and emission. 2.6. Quantitation of resveratrol using HPLC Emulsions were centrifuged twice at 13,000–20,000 × g for 30 min at 4 °C. The aqueous phase was then clarified at 150,000 × g for 30 min at 4 °C. A sample of whole emulsion or of non-clarified or clarified aqueous phase was blended with an equal volume of polydatin solution (150 μM or 15 μM in methanol), vortex-mixed with 4 volumes of methanol. After centrifugation at 3500 × g at 4 °C, the supernatant was passed through a 0.45-μm syringe filter and injected into an HPLC system equipped with a C18 column (5 μm, 4.6 mm × 250 mm, Waters, Milford, MA, USA) at 35 °C. The mobile phase was a mixture of methanol and distilled water (50:50, v/v) at 1 mL min−1. UV detection at 306 nm and 285 nm was used to quantitate trans-resveratrol and cisresveratrol, respectively. The reported resveratrol content was the sum of both isomers. The percentage of the resveratrol bound to free WPI (Rb) was calculated from the difference between the total amount in the aqueous phase (Raq) and the amount in the clarified aqueous phase (Rc) divided by the amount in the whole emulsion (Rw) using the expression Rb = (Raq − Rc)/Rw × 100. The percentage of free resveratrol (Rf) in the aqueous phase was calculated as Rf = Rc/Rw × 100. Resveratrol at the oil-water interface (Ri) was calculated as Ri = (Rw − Raq)/ Rw × 100. The content of resveratrol remaining in the whole emulsion, in the continuous phase and at the oil-water interface during storage at 45 °C was expressed as a percentage of the corresponding initial contents of trans-resveratrol.
2. Materials and method 2.1. Materials WPI (BioPRO, ˜92%) was obtained from Davisco International Inc. (Le Sueur, MN, USA). Sunflower oil (Brand Duoli) was purchased from a local retailer. Resveratrol (trans-isomer, purity > 98%) was purchased from Sango Biotech Co. (Shanghai, China). Acacia tree gum Arabic, 2thiobarbituric acid (TBA, ≥98%), 1,1,3,3-tetramethoxypropane (TEP, 99%) and cumene hydroperoxide (CHP, technical grade, 80%) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Other reagents were analytical grade and obtained from SinoPharm CNCM Ltd. (Shanghai, China). 2.2. Emulsion preparation Emulsions were prepared without and with CaCl2 according to our method described prevously [12]. A 1% (w/w) solution of WPI adjusted to pH 7.0 was heated at 90 °C for 35 min, then cooled to room temperature and mixed with 50.5 g L−1 resveratrol in 70% (v/v) ethanol at a volume ratio of 20:1 for 35 min. Calcium chloride (0.1%–1%, w/w) was added at this point. Gum Arabic solution (4%, w/w) at pH 7.0 was then stirred in for 35 min. Sunflower oil was blended in by using a high speed mixer (ATS Engineering Ltd., Brampton, Ontario, Canada) operating at 14,000 rpm followed by passing twice through an ATSAH2100 high-pressure homogenizer (ATS Engineering Ltd., Brampton, Ontario, Canada) at 50 MPa and 10 °C to obtain an O/W emulsion with an oil content of 10% (w/w), WPI content of 0.5% (w/w), 0.13 g L−1 resveratrol, 0, 0.2 and 1.6 mmol/L CaCl2, and 0%, 0.05%, 0.10%, 0.50%, and 1.00% (w/w) gum Arabic in the continuous phase. The molar concentration of resveratrol was twice that of WPI. Some of emulsion samples containing sodium azide (0.02%, w/w) were stored at 45 °C for 30 days.
2.7. Lipid oxidation Lipid hydroperoxides were measured as the primary oxidation products according to the method of Dong [25]. Exactly 0.1 mL of emulsion or corresponding bulk oil was vortex-mixed with 1.5 mL of isooctane/isopropanol mixture (3:1, v:v) for 30 s then centrifuged at 3000 rpm for 5 min. Exactly 0.2 mL of the organic phase was collected and added to 2.8 mL of methanol/n-butanol (2:1, v:v) then reacted with 15 μL of 3.94 mol/L ammonium thiocyanate and 15 μL of ferrous iron solution prepared by reacting 0.132 mol/L barium chloride with 0.144 mol/L ferrous sulfate. The absorbance at 510 nm was measured after reaction for 20 min using a Shimadzu UV-1800 spectrophotometer (Shimadzu Corp, Kyoto, Japan). The hydroperoxide content was determined from a standard curve and expressed as mmol CHP equivalent per kg of oil. Secondary products of lipid oxidation were measured as thiobarbituric acid reactive substances (TBARS) according to the method of Feng [26]. Exactly 2 mL of emulsion was mixed with 2 mL of TBA working solution containing 15% (w/v) trichloroacetic acid, 0.375% (w/v) TBA and 0.25 mol/L HCl and placed in a boiling water bath for 15 min. The mixture was cooled down to room temperature and centrifuged at 10,000 rpm for 10 min and the supernatant was passed through a 0.45μm syringe filter. The absorbance at 532 nm was measured. The TBARS content was calculated from a standard curve of TEP and expressed as μmol malondialdehyde (MDA) equivalent per kg of oil.
2.3. Size and ζ-potential measurements Size distribution and ζ-potential of WPI emulsions in the absence and presence of CaCl2 and/or gum Arabic were measured on a NanoBrooker Omni Particle size Analyzer (Brookhaven Instruments Ltd, New York, USA) with a He/Ne laser (λ = 633 nm). Samples were diluted by 100 times before measurement at 25 °C and at a scattering angle of 173°. 2.4. Interfacial protein determination Emulsions were centrifuged twice at 13,000 × g for 30 min at 4 °C using a 5804 R centrifuge (Eppendorf Co. Ltd, Hamburg, Germany). The amount of protein in the subnatant (As) and in the whole emulsion (Aw) was determined using the Kjeldahl method. Percentage of protein at the oil-water interface was calculated as (Aw − As)/Aw × 100% [12,24].
2.8. Statistical analysis All samples were prepared at least in duplicate for measurements. Values presented are mean ± standard deviation. Data were analyzed 750
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
for significant differences using the online GraphPad QuikCalcs Free ttest calculator (GraphPad Software Inc. San Diego, CA, USA). 3. Results and discussion 3.1. Influence of gum Arabic on WPI-stabilized emulsions 3.1.1. Size distribution In emulsions stabilized by WPI alone, two peaks were observed, at about 130 nm and 550 nm (Fig. 1A), which have been shown to correspond respectively to WPI particles and WPI-stabilized oil droplets [12]. Particles in emulsions stabilized by WPI plus 0.05% GA were smaller and distributed around 120 nm and 375 nm, whereas higher GA concentrations resulted in particles of sizes similar to those in emulsions stabilized by WPI alone. The presence of 0.2 mmol/L CaCl2 shifted the peaks to around 140 and 620 nm in the absence of GA, then to 90 and 320 nm at 0.10% GA and to 270 and 1030 nm at 1.00% GA (Fig. 1B). In the presence of 1.6 mM CaCl2, the peaks were around 190 and 990 nm in the absence of GA, then shifted to 170 nm and 570 nm at 0.05% GA and to 360 nm and 1420 nm at 0.50% GA (Fig. 1C). At a polysaccharide concentration of 1.00%, WPI-Ca-GA emulsions had a single size peak around 2580 nm. 3.1.2. Zeta-potential WPI-stabilized emulsions had a ζ-potential of −27.35 mV at pH 7.0 (Table 1). Whey proteins are isoelectric at about pH 5 and thus negatively charged at higher values. Stabilization of oil droplets by WPI has been attributed primarily to electrostatic repulsion [27]. The ζ-potentials of emulsions stabilized by WPI and GA were about −35 mV, regardless of the polysaccharide concentration. Adding 0.2 mmol/L CaCl2 lowered the ζ-potential at all GA concentrations except for the cases of 0% and 1.00% GA. At 1.6 mmol/L, CaCl2 further lowered the ζ-potential, to −23.60 mV at 0% GA, to −28.15 mV at 1.00% and to intermediate values at GA concentrations in between. These results suggest GA increases electrostatic repulsion between droplets and hence the stability of the emulsion against aggregation. Moreover, hydrophilic part of GA is also believed to provide stability against droplet aggregation through steric repulsion [27]. An absolute ζ-potential of 20 mV may provide sufficient stabilization, due to steric stabilization of biopolymers [28]. 3.1.3. Interfacial protein content At gum Arabic concentrations up to 0.10%, about 40% of the WPI added was incorporated into the emulsion interface (Table 2). This percentage increased to about 49% at higher concentrations of GA. Adding 0.2 mmol/L CaCl2 further increased the amount slightly at 0.10% and 1.00% GA, while at 1.6 mmol/L CaCl2, the amount was about 68–70% at GA concentrations up to 0.50% and then increased to about 76% at 1.00% GA. When the concentration of GA was 1.00%, the increased amount of protein at the oil-water interface was about 0.04–0.05%, while the polysaccharide itself carries 0.02% covalently attached proteinaceous material. Therefore, adding GA to the emulsion formulation increased the adsorption of WPI at the oil-water interface in the absence and presence of CaCl2. The observed effect of GA on droplet size and ζ-potential of WPIstabilized sunflower oil emulsions (Fig. 1 and Table 1) is similar to that of 1–4% GA on 10% soybean oil emulsions stabilized by 1% soy protein concentrate, in which there was competitive adsorption between GA and soy protein at the O/W interface [29]. However, at pH 7.0, WPI was reportedly a much more efficient stabilizer of emulsified orange oil and vitamin E-acetate, being more effective than GA at lower concentrations and producing smaller droplets [27]. Based on the percentages of protein found at the sunflower oil emulsion interface (Table 2), GA did not replace WPI but increased its interfacial adsorption. At low concentrations, GA might also act as an emulsifier, absorbing at the oil droplet surface and reducing interfacial tension and droplet size
Fig. 1. Size distribution of WPI-stabilized emulsions in the absence and presence of 0.05%, 0.10%, 0.50% and 1.00% gum Arabic at 0 (A), 0.2 (B) and 1.6 (C) mmol/L CaCl2.
751
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
Table 1 Effect of gum Arabic on ζ-potential (mV) of O/W emulsions stabilized by 0.5% WPI plus CaCl2 at different concentrations. Concentration of CaCl2 (mM)
Concentration of gum Arabic (%) 0.00
0.0 0.2 1.6
0.05
−27.35 ± 0.99 −25.41 ± 0.77Aa −23.60 ± 0.26Ac Aa
0.10
−35.85 ± 0.73 −30.40 ± 0.84Bb −24.62 ± 1.71ABc Ba
0.50
−35.35 ± 1.08 −32.40 ± 0.72BCb −25.64 ± 0.56Bc Ba
1.00
−36.54 ± 0.70 −34.17 ± 0.45Cb −25.96 ± 0.34Bc Ba
−33.64 ± 1.26Ba −33.25 ± 1.32BCa −28.15 ± 0.25Cc
Values with the different superscript letter (upper case for gum Arabic concentration, lower case for CaCl2 concentration) are significantly different (P < 0.05).
interface (Fig. 3A). Therefore, the addition of GA increased the adsorption of both free and encapsulated resveratrol molecules at the oilwater interface. The percentage of resveratrol associated with the interface was greater than that of WPI (Fig. 3 and Table 2), which is consistent with previous measurements of green tea polyphenol adsorption to the oilwater interface of β-LG-stabilized emulsions [33]. A similar phenomenon was observed without and with 0.05–1.00% GA in the presence of 0.2 mmol/L CaCl2. These results suggest that encapsulation of resveratrol occurs mainly at the O/W interface and is not in association with WPI dispersed in the continuous phase. At 1.6 mM CaCl2 and GA concentrations ≤0.10%, the percentages of resveratrol associated with the interface were lower than those of WPI. However, this difference disappeared at higher GA concentrations, due to almost complete transfer of the protein-bound polyphenol molecules to the oil-water interface (Fig. 3).
(Fig. 1). At high concentrations, GA might interact with interfacial WPI [20], resulting in an increase of droplet size (Fig. 1). The negative charges of GA at the interface provide additional stabilization by increasing repulsive forces between droplets (Table 1). Calcium ions are known to cause aggregation of heat-denatured WPI [30]. The emulsifying capacity of WPI appears to decrease as aggregate size increases, possibly because larger aggregates require more time to migrate and are less capable of re-configuring and aligning with the oilwater interface [31]. At lower concentrations, GA therefore might have had more emulsifying action by reducing oil droplet size, especially as the concentration of Ca2+ increased (Fig. 1). Adding Ca2+ also decreases negative charges on WPI particles [30], which would reduce repulsion and favor interaction between local positive charges on the protein and negative charges on the polysaccharide, thus allowing an increase in droplet size (Fig. 1). 3.2. Encapsulation of resveratrol at the oil-water interface
3.3. Storage stability of resveratrol Fluorescence of resveratrol is sensitive to the polarity of surrounding environment. The λmax of resveratrol is around 404 nm in phosphate buffer and shifts to 394 nm by binding to β-LG and to 383 nm in 75% ethanol [32]. The λmax around 380 nm in WPI emulsions was similar in the absence and presence of calcium and/or gum Arabic (Fig. 2), indicating that resveratrol was encapsulated in a hydrophobic environment. At 0.2 and 1.6 mmol/L CaCl2, fluorescence intensity of resveratrol at λmax was about 1.05 and 1.48 times that in its absence. The intensity gradually increased as the GA concentration increased from 0.05% to 1.00%, but less as the concentration of CaCl2 increased. These results suggest that both CaCl2 and GA cause more resveratrol molecules to shift to the hydrophobic phase and thereby improve its encapsulation. Resveratrol was not detected in the oil phase of WPI-stabilized emulsions when measured after oil extraction with isooctane/isopropanol mixture. Resveratrol forms complexes with whey proteins including β-LG, α-LA and BSA [8,32] and is adsorbed at the surface of oil droplets stabilized by soy and whey proteins [10,11]. About 49%, 52% and 61% of the resveratrol was associated with the oil-water interface at 0, 0.2 and 1.6 mM CaCl2 respectively, and this is unchanged by addition of 0.05% gum Arabic (Fig. 3A). At 1.00% GA, these values shift to about 63%, 63% and 74% respectively. The portion of the resveratrol found in the continuous (aqueous) phase generally decreased as the GA concentration increased, which was more pronounced in the presence of CaCl2 than in its absence (Fig. 3B). This decrease was less than the increase in amount of resveratrol adsorbed to the oil-water
The trans-resveratrol contents of the sunflower oil emulsions stored for 30 days at 45 °C are shown in Fig. 4 for the whole emulsion (A), the oil-water interface (B) and the aqueous phase (C). In the absence of gum Arabic, the content dropped below 70% of its initial value, and CaCl2 exacerbated this at the oil-water interface, as has been noted previously [12], but not in the aqueous phase. Gum Arabic appeared to counteract the effect of CaCl2. In the presence of 1.00% GA, CaCl2 appeared to have little if any de-stabilizing effect on resveratrol. In the best cases, about 75% of the initial content remained. A stronger counteraction might have been observed at 1.6 mmol/L CaCl2, but no measurement was performed because of visible creaming of these emulsions at 0.50% and 1.00% GA. At 1.6 mmol/L CaCl2 and 0.10% GA, the final resveratrol contents in the whole emulsion, the oil-water interface and the aqueous phase were respectively 62%, 54% and 66% of the initial values. 3.4. Oxidation stability of emulsified sunflower oil The contents of lipid hydroperoxide in all fresh emulsions were between 1.66 and 1.99 mmol CHPE/kg oil (Fig. 5A). After storage at 45 °C for 30 days, the content of lipid hydroperoxide in WPI emulsions increased to 73.18 mmol CHPE/kg oil. Gum Arabic at 0.05% and 0.10% decreased the content slightly, while gum Arabic at 1.00% increased it to a value even higher than that without the polysaccharide. The effect was similar at 0 and 0.2 mmol/L CaCl2. However, at 1.6 mmol/L CaCl2,
Table 2 Effect of gum Arabic on interfacial protein content (%) of O/W emulsions stabilized by 0.5% WPI plus CaCl2 at various concentrations. Concentration of CaCl2 (mM)
Concentration of gum Arabic (%) 0.00
0.0 0.2 1.6
0.05 Aa
40.43 ± 0.63 40.44 ± 0.24Aa 67.92 ± 2.21Ab
0.10 Aa
39.72 ± 0.33 39.93 ± 0.36Aa 67.67 ± 1.96Ab
0.50 Aa
40.40 ± 0.12 41.89 ± 0.34Bb 68.10 ± 1.53Ac
1.00 Ba
48.05 ± 0.88 48.29 ± 0.55Ca 70.35 ± 0.48Ab
49.21 ± 0.28Ba 50.81 ± 0.26Db 75.94 ± 0.16Bc
Values with the different superscript letter (upper case for gum Arabic concentration, lower case for CaCl2 concentration) are significantly different (P < 0.05). 752
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
Fig. 3. Content of resveratrol adsorbed at the oil-water interface (A) and bound to WPI in the continuous phase (B) of sunflower oil emulsions as a function of the concentration of gum Arabic at 0, 0.2 and 1.6 mmol/L CaCl2.
10 μmol MDA/kg oil in resveratrol-free emulsions containing ≤0.10% GA and to 16.60 μmol MDA/kg oil at 1.00% GA. These changes were indifferent to the CaCl2 concentration. The effect of resveratrol was to decrease these values slightly, especially in association with 1.6 mmol/ L CaCl2. The antioxidant effect of resveratrol on emulsified sunflower oil (Fig. 5) is consistent with previous findings obtained using corn oil emulsified with soy protein isolate [10]. Resveratrol at the oil-water interface could protect the oil simply by reacting with oxidizing agents. It might also scavenge free radicals produced by the decomposition of lipid hydroperoxides accumulating at the oil interface [10]. The fraction of the resveratrol at the interface increased with the concentration of CaCl2. In addition, calcium-induced aggregation might thicken the interfacial membrane in comparison with WPI alone, thus further improving the oxidative stability of emulsified oil. However, calciumcrosslinked aggregates of heat-denatured WPI are believed to have a gel-like porous structure that is relatively permeable to oxygen [30]. This might be one reason for the enhanced protection of the oil (Fig. 5) at the expense of greater losses of resveratrol molecules (Fig. 4). In contrast, gum Arabic at concentrations ≤0.10% decreased the loss of resveratrol at the oil-water interface but not the oxidation of the oil, and even increased oxidation slightly at higher concentrations (Fig. 5), possibly by allowing TBARS to increase in the fresh emulsions (Fig. 5C and D).
Fig. 2. Fluorescence emission spectra of resveratrol in WPI-stabilized emulsions in the absence and presence of 0.05%, 0.10%, 0.50% and 1.00% gum Arabic at 0 (A), 0.2 (B) and 1.6 (C) mmol/L CaCl2.
the lipid hydroperoxide content decreased to about 36 mmol CHPE/kg oil and was independent of the GA concentration. The presence of resveratrol (Fig. 5B) lowered all of these values by 20 mmol or more at 0 or 0.2 mmol/L CaCl2 and by at least 12 mmol at 1.6 mmol/L CaCl2. The TBARS contents of fresh emulsions were about 3.50 μmol MDA/ kg oil and indifferent to the CaCl2 concentration (Fig. 5C) and to resveratrol (Fig. 5D) at GA ≤ 0.10%. However, at higher GA concentrations, the initial TBARS contents were greater, reaching about 7.85 μmol MDA/kg oil at 1.00% GA. After storage, TBARS rose to about
753
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
Fig. 4. Content of resveratrol remaining in the whole WPI emulsions (A), at the oil-water interface (B) and in the continuous phase (C) as a function of the concentration of gum Arabic at 0, 0.2 and 1.6 mM CaCl2 after storage for 30 days.
4. Conclusions Ternary complexes of WPI with calcium and gum Arabic appear to stabilize oil-in-water emulsions. Gum Arabic improved the encapsulation of resveratrol at the oil-water interface and its stability in the emulsions, especially in the presence of CaCl2. The presence of resveratrol improved the oxidative stability of emulsified sunflower oil. These results suggest that WPI-Ca-GA emulsions could be used as effective carriers for the co-encapsulation of functional oils and polyphenols.
Fig. 5. Formation of lipid hydroperoxides (A, B) and TBARS (C, D) in WPIstabilized emulsions without (A, C) and with (B, D) resveratrol as a function of the concentration of gum Arabic at 0, 0.2 and 1.6 mmol/L CaCl2 at 45 °C after 0 (solid symbols) and 30 (open symbols) days.
754
Colloids and Surfaces B: Biointerfaces 181 (2019) 749–755
H. Zhang, et al.
Acknowledgments
[16] X. Yao, S. Xiang, K. Nie, Z. Gao, W. Zhang, Y. Fang, K. Nishinari, G.O. Phillips, F. Jiang, Whey protein isolate/gum arabic intramolecular soluble complexes improving the physical and oxidative stabilities of conjugated linoleic acid emulsions, RSC Adv. 6 (2016) 14635–14642. [17] D. Eratte, B. Wang, K. Dowling, C.J. Barrow, B.P. Adhikari, Complex coacervation with whey protein isolate and gum arabic for the microencapsulation of omega-3 rich tuna oil, Food Funct. 5 (2014) 2743–2750. [18] J. Su, Q. Guo, L. Mao, Y. Gao, F. Yuan, Food Hydrocolloids Effect of gum arabic on the storage stability and antibacterial ability of β-lactoglobulin stabilized D -limonene emulsion, Food Hydrocoll. 84 (2018) 75–83. [19] D. Eratte, S. McKnight, T.R. Gengenbach, K. Dowling, C.J. Barrow, B.P. Adhikari, Co-encapsulation and characterisation of omega-3 fatty acids and probiotic bacteria in whey protein isolate-gum Arabic complex coacervates, J. Funct. Foods 19 (2015) 882–892. [20] M. Klein, A. Aserin, P. Ben Ishai, N. Garti, Interactions between whey protein isolate and gum Arabic, Colloids Surf. B: Biointerfaces 79 (2010) 377–383. [21] M. Klein, A. Aserin, I. Svitov, N. Garti, Enhanced stabilization of cloudy emulsions with gum Arabic and whey protein isolate, Colloids Surf. B: Biointerfaces 77 (2010) 75–81. [22] B.T. O’Kennedy, J.S. Mounsey, The dominating effect of ionic strength on the heatinduced denaturation and aggregation of β-lactoglobulin in simulated milk ultrafiltrate, Int. Dairy J. 19 (2009) 123–128. [23] G.E. Remondetto, Molecular mechanisms of Fe2+-induced β-lactoglobulin cold gelation, Biopolymers 69 (2003) 461–469. [24] Z.L. Wan, J.M. Wang, L.Y. Wang, X.Q. Yang, Y. Yuan, Enhanced physical and oxidative stabilities of soy protein-based emulsions by incorporation of a water-soluble stevioside-resveratrol complex, J. Agric. Food Chem. 61 (2013) 4433–4440. [25] S. Dong, B. Wei, B. Chen, D.J. McClements, E.A. Decker, Chemical and antioxidant properties of casein peptide and its glucose Maillard reaction products in fish oil-inwater emulsions, J. Agric. Food Chem. 59 (2011) 13311–13317. [26] J. Feng, H. Cai, H. Wang, C. Li, S. Liu, Improved oxidative stability of fish oil emulsion by grafted ovalbumin-catechin conjugates, Food Chem. 241 (2018) 60–69. [27] B. Ozturk, S. Argin, M. Ozilgen, D.J. McClements, Formation and stabilization of nanoemulsion-based vitamin E delivery systems using natural biopolymers: whey protein isolate and gum Arabic, Food Chem. 188 (2015) 256–263. [28] S. Honary, F. Zahir, Effect of zeta potential on the properties of nano-drug delivery systems—a review (Part 2), Trop. J. Pharm. Res. 12 (2013) 265–273. [29] B. Wang, L.J. Wang, D. Li, B. Adhikari, J. Shi, Effect of gum Arabic on stability of oil-in-water emulsion stabilized by flaxseed and soybean protein, Carbohydr. Polym. 86 (2011) 343–351. [30] Y. Ni, L. Wen, L. Wang, Y. Dang, P. Zhou, L. Liang, Effect of temperature, calcium and protein concentration on aggregation of whey protein isolate: formation of gellike micro-particles, Int. Dairy J. 51 (2015) 8–15. [31] R.S.H. Lam, M.T. Nickerson, The effect of pH and temperature pre-treatments on the physicochemical and emulsifying properties of whey protein isolate, LWT – Food Sci. Technol. 60 (2015) 427–434. [32] L. Liang, H.A. Tajmir-Riahi, M. Subirade, Interaction of β-lactoglobulin with resveratrol and its biological implications, Biomacromolecules 9 (2008) 50–56. [33] M. von Staszewski, V.M. Pizones Ruiz-Henestrosa, A.M.R. Pilosof, Green tea polyphenols-β-lactoglobulin nanocomplexes: interfacial behavior, emulsification and oxidation stability of fish oil, Food Hydrocoll. 35 (2014) 505–511.
This work was supported by the National Natural Science Foundation of China (NSFC Project 31571781) and the Fundamental Research Funds for the Central Universities (JUSRP51711B). References [1] L. Chen, G.E. Remondetto, M. Subirade, Food protein-based materials as nutraceutical delivery systems, Trends Food Sci. Technol. 17 (2006) 272–283. [2] A. Matalanis, O.G. Jones, D.J. McClements, Structured biopolymer-based delivery systems for encapsulation, protection, and release of lipophilic compounds, Food Hydrocoll. 25 (2011) 1865–1880. [3] D.J. McClements, S.M. Jafari, Improving emulsion formation, stability and performance using mixed emulsifiers: a review, Adv. Colloid Interface Sci. 251 (2018) 55–79. [4] F. Shahidi, Y. Zhong, Revisiting the polar paradox theory: a critical overview, J. Agric. Food Chem. 59 (2011) 3499–3504. [5] D.J. McClements, E. Decker, Interfacial antioxidants: a review of natural and synthetic emulsifiers and coemulsifiers that can inhibit lipid oxidation, J. Agric. Food Chem. 66 (2018) 20–25. [6] M.R. de Oliveira, A.L. Chenet, A.R. Duarte, G. Scaini, J. Quevedo, Molecular mechanisms underlying the anti-depressant effects of resveratrol: a review, Mol. Neurobiol. 55 (2018) 4543–4559. [7] T. Yang, L. Wang, M. Zhu, L. Zhang, L. Yan, Properties and molecular mechanisms of resveratrol: a review, Pharmazie 70 (2015) 501–506. [8] H. Cheng, Z. Fang, A.M. Wusigale, Y. Bakry, L. Chen, Liang, Complexation of transand cis-resveratrol with bovine serum albumin, β-lactoglobulin or α-lactalbumin, Food Hydrocoll. 81 (2018) 242–252. [9] G. Davidov-Pardo, I.J. Joye, D.J. McClements, Encapsulation of resveratrol in biopolymer particles produced using liquid antisolvent precipitation. Part 1: preparation and characterization, Food Hydrocoll. 45 (2015) 309–316. [10] Z.L. Wan, J.M. Wang, L.Y. Wang, Y. Yuan, X.Q. Yang, Complexation of resveratrol with soy protein and its improvement on oxidative stability of corn oil/water emulsions, Food Chem. 161 (2014) 324–331. [11] L. Wang, Y. Gao, J. Li, M. Subirade, Y. Song, L. Liang, Effect of resveratrol or ascorbic acid on the stability of α-tocopherol in O/W emulsions stabilized by whey protein isolate: simultaneous encapsulation of the vitamin and the protective antioxidant, Food Chem. 196 (2016) 466–474. [12] Q. Fan, L. Wang, Y. Song, Z. Fang, M. Subirade, L. Liang, Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion: impact of protein and calcium concentrations, Int. Dairy J. 73 (2017) 128–135. [13] H. Cheng, Z. Fang, T. Liu, Y. Gao, L. Liang, A study on β-lactoglobulin-triligandpectin complex particle: formation, characterization and protection, Food Hydrocoll. 84 (2018) 93–103. [14] C. Schmitt, C. Sanchez, S. Desobry-Banon, J. Hardy, Structure and technofunctional properties of protein-polysaccharide complexes: a review, Crit. Rev. Food Sci. Nutr. 38 (1998) 689–753. [15] B.L.H.M. Sperber, H.A. Schols, M.A. Cohen Stuart, W. Norde, A.G.J. Voragen, Influence of the overall charge and local charge density of pectin on the complex formation between pectin and β-lactoglobulin, Food Hydrocoll. 23 (2009) 765–772.
755