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Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion: Impact of protein and calcium concentrations
Accepted Manuscript Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion: Impact of protein and calcium concentrations Qi Fan, Lei Wang, Yuanda Song, Zheng Fang, Muriel Subirade, Li Liang PII:
S0958-6946(17)30135-8
DOI:
10.1016/j.idairyj.2017.06.002
Reference:
INDA 4191
To appear in:
International Dairy Journal
Received Date: 19 January 2017 Revised Date:
8 June 2017
Accepted Date: 8 June 2017
Please cite this article as: Fan, Q., Wang, L., Song, Y., Fang, Z., Subirade, M., Liang, L., Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion: Impact of protein and calcium concentrations, International Dairy Journal (2017), doi: 10.1016/j.idairyj.2017.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion:
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Impact of protein and calcium concentrations
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Qi Fana,b, Lei Wanga,b, Yuanda Song c, Zheng Fanga,b, Muriel Subiraded,e, Li Lianga,b*
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a
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China
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b
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
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c
Colin Ratledge Centre for Microbial Lipids, School of Agriculture Engineering and
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Food Sciences, Shandong University of Technology, Zibo, Shandong, China
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Institute of Nutrition and Functional Foods (INAF), Canada
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e
Department of Food Science, Laval University, Quebec city, Quebec, Canada
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State Key Lab of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu,
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*Corresponding author. Tel.: +86 510 85197367
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E-mail address: [email protected] (L. Liang)
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ABSTRACT
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Whey protein isolate (WPI) is often used in food emulsions and can also interact with
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resveratrol, a natural amphiphilic polyphenol, this interaction being improved by
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heat-denaturation. In this study, oil-in-water emulsions stabilised by heat-denatured
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WPI in the absence and presence of CaCl2 were characterised in terms of size,
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ζ-potential and protein partition. Partition and stability of resveratrol were also
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studied as a function of WPI and calcium concentrations. Size of WPI emulsions was
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dependent on the protein content at the oil-water interface. Partition of resveratrol and
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WPI was positively proportional at the oil-water interface and in the continuous phase.
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The stability of resveratrol increased as the concentration of WPI increased, but
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decreased when the concentration of calcium exceeded 0.20 mM. These data should
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be useful for simultaneous encapsulation of hydrophobic and amphiphilic bioactive
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components in a single emulsion and the protection of the inner oil by combination of
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antioxidant addition.
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1.
Introduction
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Food proteins have been widely used as carrier materials for the encapsulation
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and protection of bioactive molecules, since they have multiple functional properties,
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such as emulsification, gelation, and interaction with bioactive molecules (Chen,
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Remondetto, & Subirade, 2006). Protein stabilised oil-in-water (O/W) emulsions
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consist of three essential components: the dispersed oil phase (in the form of droplets),
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the continuous aqueous phase, and the oil-water interface (stabilised by proteins).
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Whey protein isolate (WPI) generally protected ω-3 oils, essential oils and
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hydrophobic bioactive molecules dissolved in the oil phase against oxidation (Kuhn,
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& Cunha, 2012; Mehyar, Al-lsamil, Al-Ghizzawi, & Holley, 2014; Ozturk, Argin,
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Ozilgen, & McClements, 2015). However, amphiphilic and hydrophilic components
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may not be dissolved in the inner oil phase of protein emulsions. It is possible to
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encapsulate them at the oil-water interface by interacting these bioactive components
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with food proteins (Wang et al., 2016).
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Antioxidant addition could provide a protective effect on the inner oil or
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bioactive component that dissolved in the inner oil phase of O/W emulsion (Paradiso
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et al., 2016; Staszewski, Ruiz-Henestrosa, & Pilosof, 2014; Wang et al., 2016).
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According to the polar paradox, O/W emulsions are better protected from oxidation
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by hydrophobic or amphiphilic antioxidants than by hydrophilic ones, which is
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primarily attributed to the greater affinity of the former to the oil-water interface
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(Zhong & Shahidi, 2011). It has been widely reported that the antioxidants with 3
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non-covalent interactions (Gorji et al., 2015; Liang, Tremblay-Hébert, & Subirade,
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2011; Zorilla, Liang, Remondetto, & Subirade, 2011). Heat-induced denaturation
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exposed more hydrophobic residues of proteins to aqueous media and could
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strengthen hydrophobic interactions and hydrogen binding with polyphenolic
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antioxidants (Liang, & Subirade, 2012; Shpigelman, Israeli, & Livney, 2010). It is
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thus possible to improve the partition of polyphenols at the oil-water interface of
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emulsions based on the sensitivity of polyphenol-protein interactions to
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environmental factors.
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Resveratrol (trans-3,5,4’-trihydroxystilbene) is a natural amphiphilic polyphenol and has antioxidant, anti-inflammatory, antiplatelet aggregation, and
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antibacterial activities (Pangeni, Sahni, Ali, Sharma, & Baboota, 2014; Yang, Wang,
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Zhu, Zhang, & Yan, 2015). However, the low solubility of resveratrol both in water
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and oil and sensitivity to environmental factors (Freitas, Lopes, & Gaspar, 2015;
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Kuršvietienė, Stanevičienė, Mongirdienė, & Bernatonienė, 2016; Zupančič, Lavrič, &
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Kristl, 2015) limited its application in functional foods (Matos, Gutiérrez, Iglesias,
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Coca, & Pazos, 2015; Pando, Beltrán, Gerone, Matos, & Pazos, 2015; Wani et al.,
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2015). Resveratrol could interact with various proteins, including soy and whey
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proteins, caseins and collagen, to form complexes (Bourassa, Bariyanga, &
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Tajmir-Riahi, 2013; Hemar, Gerbeaud, Oliver, & Augustin, 2011; Liang, Tajmir-Riahi,
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& Subirade, 2008; Wan, Wang, Wang, Yuan, & Yang, 2014; Zhang, Mi, & Shen,
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2012). In soy protein isolate (SPI) stabilised O/W emulsion, water-soluble resveratrol
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interface and SPI-resveratrol complex was also used as an efficient emulsifier to
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improve oxidative stability (Wan, Wang, Wang, Yang, & Yuan, 2013; Wan et al.,
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2014). Recently, resveratrol was encapsulated at the oil-water interface by binding to
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natural WPI before emulsification, but the interfacial polyphenol content was only
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about 50% (Wang et al., 2016).
Whey proteins could form soluble aggregates and microgels upon heating,
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depending on protein concentration, ionic strength and pH (Donato, Kolodziejcyk, &
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Rouvet, 2011; Schmitt, Bovay, Vuilliomenet, Rouvet, & Bovetto, 2011). WPI
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microparticles with a gel-like inner structure were successfully obtained by using
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cold-set gelation method at the protein concentrations below the critical formation
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concentration of self-supporting gel, at the concentrations of CaCl2 between 1.7 and
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10 mM and at pH 7.0 (Ni et al., 2015). The influence of calcium ion on
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physiochemical properties of O/W emulsions stabilised by natural WPI and
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β-lactoglobulin, the major whey protein, has been widely studied. Calcium at 2 mM or
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higher concentrations caused aggregation of the emulsions (Agboola, & Dalgleish,
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1995, 1996; Keowmaneechai, & McClements, 2002). In this study, heat-denatured
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WPI in the absence or presence of CaCl2 was used to prepare O/W emulsions at pH
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7.0 in term of characterisation at WPI concentrations between 0.1% and 2.0% and at
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calcium concentrations between 0.05 and 5.00 mM. Moreover, partition and stability
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of resveratrol in the emulsions were investigated to discuss the possibility of the
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polyphenol accumulation at the oil-water interface. The data gathered here should be
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useful for the protection of the inner oil as well as simultaneous encapsulation of
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bioactive components with different solubility in O/W emulsion.
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Materials and methods
2.1.
Materials
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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.
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(Shanghai, China). Other reagents were analytical grade and obtained from
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SinoPharm CNCM Ltd. (Shanghai, China).
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Emulsion preparation
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2.2.
WPI stabilised emulsions in the absence or presence of CaCl2 were prepared
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according to our previous methods (Ni et al., 2015; Wang et al., 2016). Pre-denatured
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WPI solutions at 0.2, 0.4, 1.0, 2.0 or 4.0% (w/w) were prepared by dispersing the
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powder in distilled water under stirring, followed by adjusting to pH 7.0, then holding
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at 90 °C for 35 min and cooling to room temperature. Pre-denatured WPI solutions
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were mixed with 91 mM CaCl2 under stirring and then added into sunflower oil to
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obtain coarse emulsions by using a high speed blender (ATS Engineering Ltd., 6
ACCEPTED MANUSCRIPT Brampton, Ontario, Canada) operating at 14,000 rpm for 1 min, with final WPI
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concentrations of 0.1, 0.2, 0.5, 1.0 and 2.0%, final CaCl2 concentrations of 0.05, 0.2,
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0.8, 1.6, 3.2 and 5 mM, and a final oil concentration of 10%. Oil droplet size was
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reduced by passing the coarse emulsion twice through an ATSAH2100 high-pressure
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homogeniser (ATS Engineering Ltd., Brampton, Ontario, Canada) at a pressure of 50
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MPa. When resveratrol was added, the polyphenol was dissolved in 70% (v/v) ethanol
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at 25.25 g L-1 and then mixed with WPI solutions and incubated for 35 min before
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WPI/CaCl2 mixture. The final polyphenol concentration was 0.13 g L-1 and equal to
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the molar concentration of WPI at 1.0% when calculated based on the molecular
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weight of β-lactoglobulin. Sodium azide (0.04%, w/w) was then added into the
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emulsions to prevent microbial growth and samples were stored at 25 °C for up to 30
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days. All samples were prepared at least in duplicate for each subsequent analysis.
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2.3.
Size and ζ-potential measurements
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Size distribution and ζ-potential of WPI emulsions in the absence and presence
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of CaCl2 were measured on a NanoBrooker Omni Particle size Analyzer (Brookhaven
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Instruments Ltd, New York, USA) with a He/Ne laser (λ = 633 nm). Samples were
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diluted with distilled water at pH 7.0 by 100 times before measurement. All
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measurements were conducted at 25 °C and at a scattering angle of 173°. Size
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distribution was obtained by using a NNLS mode analysis.
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2.4.
Determination of WPI at the oil/water interface
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Adsorption of WPI at the oil/water interface was determined according to the method described by Wan et al. (2013) and Wang et al. (2016) with some
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modifications. WPI emulsion was centrifuged twice at 13,000 × g for 30 min at 4 °C
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(Wang et al., 2016), using a 5804 R centrifuge (Eppendorf Co. Ltd, Hamburg,
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Germany). The WPI contents in the subnatant and in the whole emulsion were
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determined by using the Kjeldahl method (Wan et al., 2013; Ye, Srinivasan, & Singh,
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2000). The percentage of WPI at the oil-water interface was calculated from the
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difference between the protein amount in the whole emulsion and that in the subnatant
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divided by the content in the whole emulsion. The content of WPI at the oil-water
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interface was calculated according to the protein content in the whole emulsion
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multiplied by the protein interfacial percentage. Surface WPI concentration was
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calculated from interfacial WPI content and surface area of oil droplets determined by
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size distribution (Chen & Tang, 2016; Zhao et al., 2015).
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2.5.
Quantitation of resveratrol using high performance liquid chromatography
WPI emulsion was centrifuged at 13,000 × g for 30 min at 4 °C, the
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subnantant was collected and centrifuged at 150,000 × g for 30 min using a CP70ME
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ultra-centrifuge (Hitachi Co. Ltd, Tokyo, Japan). The contents of resveratrol in the
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whole emulsion (Cw), in the subnatant obtained from first centrifugation (Csub) and the 8
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liquid extraction and high performance liquid chromatography (HPLC) (Camont et al.,
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2009; Wang et al., 2016). Briefly, methanol exacts were measured on an HPLC
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system equipped with a C18 column (5 µm, 4.6 mm × 250 mm, Waters, Milford, MA,
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USA). Polydatin was used as an internal standard. The content of resveratrol was the
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sum of both trans- and cis-resveratrol contents. The percentage of free resveratrol in
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the continuous phase was calculated from Csup divided by Cw. The percentage of
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resveratrol bound to WPI in the continuous phase was calculated from the difference
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between Csub and Csup divided by Cw. The percentage of resveratrol in the continuous
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phase was calculated from Csub divided by Cw. The percentage of resveratrol at the
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oil-water interface was calculated from the difference between Cw and Csub divided by
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Cw. Stability of resveratrol in the whole emulsion, in the continuous phase and at the
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oil-water interface was expressed as the retention of the polyphenol during storage at
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25 °C and calculated by a percentage relative to the corresponding initial content of
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trans-resveratrol.
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2.6.
Fluorescence measurement
Fluorescence spectra of resveratrol in the emulsions stabilised by WPI at 0.1,
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0.2, 0.5, 1.0, 2.0% and by WPI at 0.5% in the presence of 0, 0.05, 0.2, 0.8 and 1.6 mM
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CaCl2 were measured on a Cary Eclipse spectrophotometer (Agilent Co. Ltd, New
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York, USA) equipped with a front-surface accessory. The incidence angle of the 9
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excitation radiation was set at 15°. The emission spectra were recorded from 330 to
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550 nm with an excitation wavelength of 330 nm. Both the excitation and emission
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slit widths were set at 5 nm.
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Statistical analysis
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Data are presented as mean values ± standard deviations and analysed for
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significant difference based on two-way ANOVA and Pearson’s correlation by using
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the statistical software package SPSS, version 20.0 (IBM Co. Ltd, New York, USA).
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Results and discussion
3.1.
Characterisation of WPI emulsions
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3.1.1. Size and ζ-potential
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Fig. 1 shows size distributions of the emulsions stabilised by heat-denatured
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WPI in the absence and presence of CaCl2. Size distributions of all the emulsions
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were divided into two peaks. In the absence of CaCl2, size distributions were around
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160 and 930 nm at 0.1% WPI and decreased to 115 and 410 nm at 0.2%, then
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remained constant as the concentrations of WPI increased up to 2% (Fig. 1A–E). By
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increasing the concentrations of CaCl2, size distributions of WPI emulsions gradually
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increased. The effect of CaCl2 became less pronounced as the concentrations of WPI 10
ACCEPTED MANUSCRIPT increased. The influence of CaCl2 on size distributions of heat-denatured WPI
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emulsions was similar to that of calcium at concentrations between 2 and 16 mM on
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natural β-lactoglobulin emulsions (Agboola, & Dalgleish, 1995). At 0.1% of WPI
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emulsions, creaming occurred after preparation for about 0.5 h when the
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concentration of CaCl2 exceeded 0.2 mM, size distributions were thus not analysed. At
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0.2% WPI, an abrupt increase in the size distributions was observed at the CaCl2
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concentrations between 0.2 and 0.8 mM. The salt concentrations for the abrupt
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changes increased as WPI concentrations increased and no aggregation was observed
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at 2% WPI. After centrifugation, the particles in all the subnatants had uniform size
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distributions, which were close or similar to the small ones of the whole emulsions.
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The particle size distributions of the centrifuged samples changed from a bit smaller
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at the WPI concentrations between 0.1% and 0.5% to a bit larger at higher protein
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concentrations, the former being more pronounced at higher CaCl2 concentrations
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while the latter being more pronounced at lower CaCl2 concentrations. The effects
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might be due to the impact of centrifugation on the particle with different structures.
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Therefore, in the whole emulsions, the smaller size distributions were for WPI
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particles and the larger ones were for WPI stabilised oil droplets.
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Zeta-potentials of all emulsions were negative at pH 7.0 (Table 1) since whey
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proteins (e.g., β-lactoglobulin and α-lactalbumin) have an isoelectric point of about 5
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(Almécija, Ibáñez, Guadix, & Guadix, 2007). All ζ-potentials showed a uniform
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distribution (data not shown). The ζ-potential was about –32 mV at a WPI
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concentration of 0.1%, decreased to about –27 mV as the protein concentration 11
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being about –38 mV at 2.0% (Table 1). Since Ca2+ neutralised negative charges of
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WPI, the ζ-potential values decreased as the concentrations of CaCl2 increased,
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reaching –24 mV at 0.2 mM when the concentration of WPI was 0.1%. The decreased
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values were similar at higher WPI concentrations, with ζ-potential reaching about –22
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and –15 mV at 3.2 and 5 mM CaCl2 when the protein concentrations were between 0.2%
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and 1.0% and reaching –25 mV at 5 mM CaCl2 and 2% WPI. The emulsion droplets
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was stabilised against aggregation by electrostatic repulsion and against coalescence
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by steric hindrance (Wade & Beattie, 1997; Wu et al., 2015). In the case of the
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emulsions stabilised by caseins, small concentrations of Ca2+ cause a decrease in the
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thickness of the adsorbed layer, reducing steric stabilisation, and high concentrations
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of Ca2+ binds to the protein to form calcium bridges between the emulsion droplets.
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However, Ca2+ did not completely neutralise the ζ-potential, so it may not be
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sufficient to destabilise the emulsion (Dalgleish, 1997; Sjöblom, 2001).
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3.1.2. Adsorption of WPI at the oil-water interface
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In O/W emulsions, WPI existed both at the oil droplet surface and in the
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continuous phase. The percentage of WPI at the oil-water interface was about 66%
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when the protein concentration was 0.1% and decreased as the protein concentrations
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increased, being about 32% at 2% (Fig. 2A). However, the interfacial WPI
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percentages increased gradually as the concentrations of CaCl2 increased, which was
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more pronounced at the lower protein concentrations. The interfacial WPI percentage 12
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WPI percentages were similar and increased to about 79% at 0.2% and 0.5% WPI. At
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5 mM CaCl2, the interfacial WPI percentages were similar and increased to about 82%
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at the protein concentration between 0.2% and 1.0%. The interfacial protein
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percentages were significantly lower and increased only to 43% at 2.0% WPI.
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The content of WPI at the oil-water interface increased as the concentrations
of the protein and CaCl2 increased (Fig. 2B). Surface WPI concentration increased as
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the protein concentration increased in the absence of CaCl2 and as the salt
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concentration increased at a fixed protein concentration (Fig. 2C). The increase in the
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surface concentration was dependent on both WPI and CaCl2 concentrations. At 0.05
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and 0.2 mM CaCl2, surface WPI concentration decreased when the protein
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concentration increased from 0.1% to 0.2% and then increased gradually at higher
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protein concentrations. Surface WPI concentration was the most at the protein
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concentrations of 0.5% and 1.0% when the concentrations of CaCl2 were 3.2 and 5
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mM, respectively. An abrupt increase in the interfacial protein content and surface
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protein concentration was observed at the salt concentrations between 0.2 and 0.8 mM
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at 0.2% WPI, between 1.6 and 3.2 mM at 0.5% WPI and between 3.2 and 5 mM at 1.0%
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WPI (Fig. 2B and C). The concentrations were consistent with that for an abrupt
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increase in the size of WPI particles in the continuous phases and WPI stabilised oil
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droplets (Fig. 1). Therefore, calcium induced aggregation of WPI resulted in a greater
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number of protein molecules adsorb at the surface of oil droplets, leading to an
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increase in the size of oil droplets. In the case that surface charges of WPI particles
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protein particles adsorbed at the oil-water interface than did charged particles, with
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that the former were highly aggregated and formed a continuous network while the
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latter might spread at the interface to form a continuous protein membrane (Destribats,
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Rouvet, Gehin-Delval, Schmitt, & Binks, 2014). As the concentrations of CaCl2
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increased, ζ-potentials of WPI particles decreased (Table 1), thus leading to greater
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protein absorption at the oil-water interface (Fig. 2B). When the concentration of WPI
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was 2.0%, ζ-potential was greater than that of other emulsions (Table 1), the increase
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in interfacial WPI content and surface protein concentration was thus slower as the
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concentration of CaCl2 increased (Fig. 2B,C).
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Partition of resveratrol in WPI emulsions
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3.2.
3.2.1. Percentage of resveratrol in the continuous phase Fig. 3A shows the percentage of free resveratrol in the continuous phase of the
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emulsions stabilised by WPI in the absence and presence of CaCl2. In the absence of
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CaCl2, the percentage of free resveratrol gradually increased from 22% to 29% as the
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concentration of WPI increased from 0.1% to 1.0% and then decreased to 22% at 2%
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WPI. At 0.1% WPI, the percentage of free resveratrol was similar in the absence and
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presence of CaCl2. At 0.2%, the percentage of free resveratrol did not change when
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the concentration of CaCl2 was less than 0.2 mM and then decreased to 11% at 0.8 and
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1.6 mM and to 7% at 3.2 and 5 mM. At 0.5% WPI, the percentage of free resveratrol
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decreased gradually to about 21% at 5 mM. Influence of CaCl2 on the percentage of
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free resveratrol was different at higher protein concentrations. At 1% WPI, the
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percentage of free resveratrol increased to 41% at 0.05 mM CaCl2 and then began to
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gradually decrease with the salt concentration, being about 7% at 5 mM. At 2% WPI,
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the percentage of free resveratrol increased gradually to 31% at 3.2 mM CaCl2 and
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then decreased to 14% at 5 mM.
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Fig. 3B shows the percentage of resveratrol bound to WPI in the continuous
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phase of the emulsions stabilised by WPI in the absence and presence of CaCl2. In the absence of CaCl2, the percentage of bound resveratrol was about 19% at the
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concentrations of WPI being 0.1% and 0.2%, about 25% at 0.5% and 1.0% WPI and
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increased to 47% at 2% WPI. At 0.1% WPI, the percentage of bound resveratrol was
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similar in the absence and presence of CaCl2. At 0.2% WPI, the percentage of bound
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resveratrol decreased slowly to 10% as the concentration CaCl2 increased to 5 mM. At
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0.5% WPI, the percentage of bound resveratrol decreased gradually to 12% as the
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concentration of CaCl2 increased to 1.6 mM and was zero at higher salt concentrations.
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At 1% WPI, the percentage of bound resveratrol was between 19% and 24% at the
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concentration of CaCl2 less than 3.20 mM and then decreased to 6% at 5 mM.
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However, at 2% WPI, the percentage of bound resveratrol decreased gradually to 31%
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as the concentration CaCl2 increased to 3.2 mM and then increased to 42% at 5 mM.
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3.2.2. Percentage of resveratrol at the oil-water interface 15
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Resveratrol is not soluble in bulk oil including sunflower oil and rapeseed oil (Filip et al., 2003). Resveratrol interacts firstly with WPI to form protein-ligand
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complexes and could then adsorb at the oil–water interface (Wang et al., 2016). The
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percentage of resveratrol at the oil-water interface was 59% when the concentration of
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WPI was 0.1% and decreased as the protein concentrations increased, being about 31%
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at 2% WPI (Fig. 3C). The interfacial polyphenol percentages were basically similar to
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that of WPI at the interface (Fig. 2A). When the concentrations of WPI were between
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0.2% and 2%, the interfacial resveratrol percentages increased as the concentration of
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CaCl2 increased. A significant positive correlation between the interfacial resveratrol
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percentage and that of WPI at the interface exists (Table 2). These results indicate that
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the accumulation of resveratrol at the oil-water interface was dependent on the
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adsorption of WPI at the oil droplet surface. However, in the case of 0.1% WPI, the
13
percentages of resveratrol at the oil-water interface kept constant as the concentrations
14
of CaCl2 increased to 0.2 mM. In the mixtures of WPI with resveratrol, there was an
15
equilibrium between free resveratrol and WPI-resveratrol complexes, since their
16
interaction involving noncovalent bonds are reversible (Mohammadi, & Moeeni, 2015;
17
Zhang, Liu, Subirade, Zhou, & Liang, 2014). As the concentrations of WPI increased,
18
the percentages of protein-bound resveratrol increased, thus the percentages of
19
resveratrol and WPI became more similar at the oil-water interface.
20
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As a whole, resveratrol could be free and bound to WPI in the continuous
21
phase and adsorbed together with WPI at the oil-water interface, except that the
22
polyphenol was only free in the continuous phase and bound to WPI at the interface 16
ACCEPTED MANUSCRIPT when the concentrations of CaCl2 were between 3.2 and 5 mM at 0.5% WPI. Addition
2
of CaCl2 at the concentrations less than 0.2 mM had no influence on the partition of
3
resveratrol in the emulsion stabilised by WPI at 0.1%. When the concentrations of
4
WPI were between 0.2% and 2%, except for the case at 2% WPI and 5 mM CaCl2, the
5
ratio of the percentages of resveratrol bound to WPI at the interface and in the
6
continuous phase increased with the salt concentration, indicating that resveratrol tend
7
to be accumulated by complexation with WPI at the oil-water interface more than in
8
continuous phase.
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1
9 10 11
3.2.3. Microenvironment of resveratrol
Fluorescence of resveratrol was sensitive to the polarity of surrounding environment. Fluorescence emission maximum (λmax) of resveratrol was around 404
13
nm in 10 mM phosphate buffer at pH 7.4 and shifted to 383 nm at 70% ethanol with a
14
significant increase in the intensity (Liang & Subirade, 2012; Liang et al., 2008). The
15
λmax of resveratrol was around 380 nm in the emulsion stabilised by WPI in the
16
absence and presence of CaCl2 (Fig. 4), suggesting that resveratrol was encapsulated
17
in a hydrophobic environment of WPI. Fluorescence intensity of resveratrol increased
18
as the concentrations of WPI increased (Fig. 4A) and as the concentrations of Ca2+
19
increased at a fixed WPI concentration of 0.5% (Fig. 4B), indicating that more
20
resveratrol molecules were bound by WPI. Therefore more resveratrol molecules were
21
adsorbed together with WPI at the surface of oil droplets (Fig. 3).
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3.3.
Stability of resveratrol
2 3
Trans-resveratrol is liable to transform to cis-isomer, depending on storage conditions (Trela & Waterhouse, 1996). In this study, cis-resveratrol was only
5
detected after storage for 20 days, with the contents below 10% in the whole
6
emulsions and below 5% in the continuous phases (data not shown). The total
7
contents of both trans- and cis-resveratrol were shown in the following.
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9
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3.3.1. Dependence on WPI concentration
Creaming was observed for the emulsions at the WPI concentration of 0.1%
11
after preparation for about 12 h. The retention of resveratrol in the whole emulsions
12
stabilised by WPI between 0.2% and 2% is shown in Fig. 5A. In the whole emulsion
13
at 0.2% WPI, resveratrol was stable until 5 days and then began to degrade gradually
14
over time, with the percentage of about 57% remaining after storage of 30 days. The
15
polyphenol stability was significantly improved as the concentrations of WPI
16
increased to 0.5%, with about 78% remaining after 30 days. A further increase in the
17
WPI concentration up to 2% led to a slight increase in the percentage of resveratrol
18
remaining after 20 days, but the difference was not quite statistically significant.
EP
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10
In the continuous phases at 0.2% WPI, resveratrol was stable within one day
20
and then degraded gradually over time, with the percentage of about 68% remaining
21
after 30 days (Fig. 5B). The stability of resveratrol was also improved at higher WPI
22
concentrations, with similar retention of resveratrol until 20 days. After storage for 30 18
ACCEPTED MANUSCRIPT days, the percentages of resveratrol remaining at the concentrations of WPI between 1%
2
and 2% became greater than that at 0.5%, with the value being about 87% at 2% WPI.
3
In the oil-water interface (Fig. 5C), the overall change trend in the retention of
4
resveratrol were basically similar to that in the whole emulsions (Fig. 5A), except that
5
a more significant decrease was observed after 10 days, with the resveratrol
6
percentage of about 50% remaining after 30 days at the WPI concentrations of 0.2%. As a whole, when the concentration of WPI was 0.2%, the percentage of
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1
resveratrol remaining at the oil-water interface was greater than that in the continuous
9
phases during storage until 10 days (Fig. 5B,C). This is due to that resveratrol was
10
accumulated by complexation with WPI at the oil-water interface while resveratrol
11
was both free and complexed by WPI in the continuous phase (Fig. 3). WPI particles
12
adsorbed at the oil-water interface, providing better protective effect against the loss
13
of resveratrol. After then, the stability of resveratrol were better in the continuous
14
phase instead (Fig. 5B,C). It has been reported that addition of resveratrol could
15
improve the stability of α-tocopherol dissolved in the inner oil phase and of the inner
16
oil phase of the emulsions (Medina et al., 2010; Wan et al., 2013; Wang et al., 2015).
17
However, upon lipid oxidation occurred, resveratrol could accept free radicals by
18
donation of phenolic hydrogen atoms, leading to faster oxidation at the oil-water
19
interface (Wan et al., 2014). The phenomena were less pronounced when the
20
concentrations of WPI were 0.5% and 1%. At 2% WPI, the stability of resveratrol at
21
the oil-water interface were better than that in the continuous phase until 20 days and
22
then became similar after storage for 30 days. As the concentrations of WPI increased,
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19
ACCEPTED MANUSCRIPT 1
the stability of the inner oil phase was improved, thus delaying the impact on the loss
2
of resveratrol (Wang et al., 2016).
3
5
3.3.2. Dependence on CaCl2 concentration
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In the whole emulsions at 0.5% WPI, addition of CaCl2 at the concentrations
less than 0.20 mM basically had no significant influence on the retention of resveratrol
7
during storage (Fig. 6A). However, the percentage of resveratrol remaining decreased
8
when the concentrations of CaCl2 further increased, with the value being about 36% at
9
1.6 mM after 30 days. Similar trend was also observed in the percentages of
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resveratrol remaining in the continuous phase and the oil-water interface (Fig. 6B,C).
11
It has been reported that addition of CaCl2 had no influence on the stability of
12
α-tocopherol dissolved in the inner oil phase of emulsion (Wang, 2015).
13
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CaCl2-induced WPI aggregates had more hydrophobic surface exposed to solvents, which might be the reason for the greater loss of resveratrol (Ni et al., 2015).
15
In the absence and presence of CaCl2, the stability of resveratrol was similar, being
16
better at the oil-water interface until 10 days and being better in the continuous phase
17
after then (Fig. 6B,C).
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In O/W emulsion stabilised by heat-denatured WPI, resveratrol partitioned at
19
the oil-water interface and in the continuous phase. The percentage of resveratrol at
20
the oil-water interface decreased as the concentration of WPI increased but increased
21
as the concentration of CaCl2 increased. Opposite was influence of WPI and CaCl2
22
concentrations on the stability of resveratrol. The emulsion stabilised by calcium 20
ACCEPTED MANUSCRIPT 1
induced WPI aggregates was thus not an effective carrier for resveratrol. But the
2
accumulation of resveratrol at the oil-water interface would provide better protection
3
for the inner oil phase against oxidation.
5
4.
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4
Conclusions
6
Oil-in-water emulsions were stabilised by heat-denatured WPI particles and its
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7
calcium aggregate particles. Small WPI particles and large oil droplets co-existed in
9
the emulsions. At the oil-water interface, the accumulation of resveratrol was
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positively proportional to the contents of interfacial WPI, except for the case of 0.1%
11
WPI in the presence of CaCl2. Resveratrol at the oil-water interface was basically
12
more stable than that in the continuous phase at the initial stage of storage and then
13
became less stable at the ending of storage. The stability of resveratrol in the whole
14
emulsions increased as the concentration of WPI increased but decreased when the
15
concentration of calcium exceeded 0.2 mM, especially at 1.6 mM. In the future,
16
WPI-polysaccharide complexation was used to improve the stability of resveratrol in
17
the presence of CaCl2.
19
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Acknowledgements
20 21 22
This work was supported by the National Natural Science Foundation of China (NSFC Project 31571781) and the Fundamental Research Funds for the Central 21
ACCEPTED MANUSCRIPT 1
Universities (JUSRP51711B).
2 3
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Figure captions
2
Fig. 1. Size distributions of O/W emulsions (—–) stabilised by WPI at concentrations
4
of 0.1% (A), 0.2% (B), 0.5% (C), 1.0% (D) and 2.0% (E) and subnatants (– – –) after
5
centrifugation in the absence and presence of CaCl2 at various concentrations.
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6
Fig. 2. Interfacial WPI percentage (A), interfacial WPI content (B) and surface WPI
8
concentration (C) of the emulsions stabilised by the protein at various concentrations
9
(, 0.1%;
, 0.2%;
, 0.5%;
concentration of CaCl2.
11
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, 1.0% and , 2.0%) as a function of the
Fig. 3. Percentage of resveratrol free (A) and bound to WPI (B) in the continuous
13
phase and at oil-water interface (C) of the emulsions stabilised by various
14
concentrations of WPI (, 0.1%;
15
different concentrations of CaCl2.
, 0.5%;
, 1.0% and , 2.0%) with
EP
, 0.2%;
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16
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12
17
Fig. 4. Fluorescence emission spectra of resveratrol in the emulsions stabilised by
18
WPI at various concentrations (A): —–, 0.1%; – · · –, 0.2%; – · – ·, 0.5%; · · · ·, 1.0%; –
19
– –, 2.0% and in the emulsions stabilised by WPI at 0.5% in the presence of CaCl2 at
20
various concentrations (B): —–, 0 mM; – · · –, 0.05 mM; – · – ·, 0.20 mM; · · · ·, 0.80 mM;
21
– – –, 1.60 mM.
22 1
ACCEPTED MANUSCRIPT 1
Fig. 5. Persistence of resveratrol in the whole emulsion (A), in the continuous phase
2
(B) and at the oil-water interface (C) at various concentrations of WPI (, 0.2%;
3
0.5%; , 1.0% and
4
material, Tables S1–S3).
,
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, 2.0%) and stored for up to 30 days (see also Supplementary
5
Fig. 6. Persistence of resveratrol in the whole emulsion (A), in the continuous phase
7
(B) and at the oil-water interface (C) at 0.5% WPI made with different concentrations
8
of CaCl2 (, 0 mM;
9
stored for up to 30 days (see also Supplementary material, Tables S4–S6).
, 0.80 mM and
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, 0.05 mM; , 0.20 mM;
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6
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10
2
, 1.60 mM) and
ACCEPTED MANUSCRIPT Table 1 ζ-Potential of oil-in-water emulsions stabilised by WPI at various concentrations as a
Concentration of
Concentration of WPI (%)
CaCl2 (mM)
0.1
0.2
0.5
0
-32.1±0.1Ba
-28.2±1.0CDa
-27.4±1.0Da
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function of the concentration of CaCl2. a
0.05
-27.6±0.8Bb
-27.5±0.9CDb
-27.1±1.6Db
0.20
-24.6±0.7Bb
-28.0±0.8CDb
-25.4±0.8Db
-30.0±1.5Cb
-35.2±5.6Ab
0.80
−
-27.3±0.7CDc
-23.6±1.7Dc
-23.1±1.9Cc
-30.6±1.1Ac
1.60
−
-25.2±0.0CDc
-23.6±0.3Dc
-24.9±0.2Cc
-29.9±0.2Ac
3.20
−
-22.2±0.8CDd
-22.6±0.2Dd
-23.2±1.9Cd
-29.0±0.8Ad
5.00
−
-16.1±1.0De
-15.0±1.3Ce
-25.3±0.4Ae
-30.6±1.8Ca
-37.8±5.6Aa
-30.1±2.8Cb
-36.0±4.9Ab
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2.0
-15.5±1.2CDe
Values with the different superscript letter (upper case for WPI concentration, lower
EP
a
1.0
AC C
case for CaCl2 concentration) are significantly different (P < 0.05).
ACCEPTED MANUSCRIPT Table 2 Results of the Pearson correlation coefficient (r) between the percentages of
Parameter
0.5
1.0
r
0.988*
0.987*
0.967*
p
0.000
0.000
2.0
0.934*
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0.002
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WPI concentration (%)
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S0958-6946(17)30135-8
DOI:
10.1016/j.idairyj.2017.06.002
Reference:
INDA 4191
To appear in:
International Dairy Journal
Received Date: 19 January 2017 Revised Date:
8 June 2017
Accepted Date: 8 June 2017
Please cite this article as: Fan, Q., Wang, L., Song, Y., Fang, Z., Subirade, M., Liang, L., Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion: Impact of protein and calcium concentrations, International Dairy Journal (2017), doi: 10.1016/j.idairyj.2017.06.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Partition and stability of resveratrol in whey protein isolate oil-in-water emulsion:
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Impact of protein and calcium concentrations
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Qi Fana,b, Lei Wanga,b, Yuanda Song c, Zheng Fanga,b, Muriel Subiraded,e, Li Lianga,b*
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a
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China
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b
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
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c
Colin Ratledge Centre for Microbial Lipids, School of Agriculture Engineering and
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Food Sciences, Shandong University of Technology, Zibo, Shandong, China
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d
Institute of Nutrition and Functional Foods (INAF), Canada
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e
Department of Food Science, Laval University, Quebec city, Quebec, Canada
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State Key Lab of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu,
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*Corresponding author. Tel.: +86 510 85197367
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E-mail address: [email protected] (L. Liang)
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____________________________________________________________________
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ABSTRACT
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Whey protein isolate (WPI) is often used in food emulsions and can also interact with
5
resveratrol, a natural amphiphilic polyphenol, this interaction being improved by
6
heat-denaturation. In this study, oil-in-water emulsions stabilised by heat-denatured
7
WPI in the absence and presence of CaCl2 were characterised in terms of size,
8
ζ-potential and protein partition. Partition and stability of resveratrol were also
9
studied as a function of WPI and calcium concentrations. Size of WPI emulsions was
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dependent on the protein content at the oil-water interface. Partition of resveratrol and
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WPI was positively proportional at the oil-water interface and in the continuous phase.
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The stability of resveratrol increased as the concentration of WPI increased, but
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decreased when the concentration of calcium exceeded 0.20 mM. These data should
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be useful for simultaneous encapsulation of hydrophobic and amphiphilic bioactive
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components in a single emulsion and the protection of the inner oil by combination of
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antioxidant addition.
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_____________________________________________________________________
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1.
Introduction
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Food proteins have been widely used as carrier materials for the encapsulation
4
and protection of bioactive molecules, since they have multiple functional properties,
5
such as emulsification, gelation, and interaction with bioactive molecules (Chen,
6
Remondetto, & Subirade, 2006). Protein stabilised oil-in-water (O/W) emulsions
7
consist of three essential components: the dispersed oil phase (in the form of droplets),
8
the continuous aqueous phase, and the oil-water interface (stabilised by proteins).
9
Whey protein isolate (WPI) generally protected ω-3 oils, essential oils and
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hydrophobic bioactive molecules dissolved in the oil phase against oxidation (Kuhn,
11
& Cunha, 2012; Mehyar, Al-lsamil, Al-Ghizzawi, & Holley, 2014; Ozturk, Argin,
12
Ozilgen, & McClements, 2015). However, amphiphilic and hydrophilic components
13
may not be dissolved in the inner oil phase of protein emulsions. It is possible to
14
encapsulate them at the oil-water interface by interacting these bioactive components
15
with food proteins (Wang et al., 2016).
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bioactive component that dissolved in the inner oil phase of O/W emulsion (Paradiso
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et al., 2016; Staszewski, Ruiz-Henestrosa, & Pilosof, 2014; Wang et al., 2016).
19
According to the polar paradox, O/W emulsions are better protected from oxidation
20
by hydrophobic or amphiphilic antioxidants than by hydrophilic ones, which is
21
primarily attributed to the greater affinity of the former to the oil-water interface
22
(Zhong & Shahidi, 2011). It has been widely reported that the antioxidants with 3
ACCEPTED MANUSCRIPT different hydro-solubility could bind to natural and denatured whey proteins by
2
non-covalent interactions (Gorji et al., 2015; Liang, Tremblay-Hébert, & Subirade,
3
2011; Zorilla, Liang, Remondetto, & Subirade, 2011). Heat-induced denaturation
4
exposed more hydrophobic residues of proteins to aqueous media and could
5
strengthen hydrophobic interactions and hydrogen binding with polyphenolic
6
antioxidants (Liang, & Subirade, 2012; Shpigelman, Israeli, & Livney, 2010). It is
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thus possible to improve the partition of polyphenols at the oil-water interface of
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emulsions based on the sensitivity of polyphenol-protein interactions to
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environmental factors.
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Resveratrol (trans-3,5,4’-trihydroxystilbene) is a natural amphiphilic polyphenol and has antioxidant, anti-inflammatory, antiplatelet aggregation, and
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antibacterial activities (Pangeni, Sahni, Ali, Sharma, & Baboota, 2014; Yang, Wang,
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Zhu, Zhang, & Yan, 2015). However, the low solubility of resveratrol both in water
14
and oil and sensitivity to environmental factors (Freitas, Lopes, & Gaspar, 2015;
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Kuršvietienė, Stanevičienė, Mongirdienė, & Bernatonienė, 2016; Zupančič, Lavrič, &
16
Kristl, 2015) limited its application in functional foods (Matos, Gutiérrez, Iglesias,
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Coca, & Pazos, 2015; Pando, Beltrán, Gerone, Matos, & Pazos, 2015; Wani et al.,
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2015). Resveratrol could interact with various proteins, including soy and whey
19
proteins, caseins and collagen, to form complexes (Bourassa, Bariyanga, &
20
Tajmir-Riahi, 2013; Hemar, Gerbeaud, Oliver, & Augustin, 2011; Liang, Tajmir-Riahi,
21
& Subirade, 2008; Wan, Wang, Wang, Yuan, & Yang, 2014; Zhang, Mi, & Shen,
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2012). In soy protein isolate (SPI) stabilised O/W emulsion, water-soluble resveratrol
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interface and SPI-resveratrol complex was also used as an efficient emulsifier to
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improve oxidative stability (Wan, Wang, Wang, Yang, & Yuan, 2013; Wan et al.,
4
2014). Recently, resveratrol was encapsulated at the oil-water interface by binding to
5
natural WPI before emulsification, but the interfacial polyphenol content was only
6
about 50% (Wang et al., 2016).
Whey proteins could form soluble aggregates and microgels upon heating,
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depending on protein concentration, ionic strength and pH (Donato, Kolodziejcyk, &
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Rouvet, 2011; Schmitt, Bovay, Vuilliomenet, Rouvet, & Bovetto, 2011). WPI
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microparticles with a gel-like inner structure were successfully obtained by using
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cold-set gelation method at the protein concentrations below the critical formation
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concentration of self-supporting gel, at the concentrations of CaCl2 between 1.7 and
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10 mM and at pH 7.0 (Ni et al., 2015). The influence of calcium ion on
14
physiochemical properties of O/W emulsions stabilised by natural WPI and
15
β-lactoglobulin, the major whey protein, has been widely studied. Calcium at 2 mM or
16
higher concentrations caused aggregation of the emulsions (Agboola, & Dalgleish,
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1995, 1996; Keowmaneechai, & McClements, 2002). In this study, heat-denatured
18
WPI in the absence or presence of CaCl2 was used to prepare O/W emulsions at pH
19
7.0 in term of characterisation at WPI concentrations between 0.1% and 2.0% and at
20
calcium concentrations between 0.05 and 5.00 mM. Moreover, partition and stability
21
of resveratrol in the emulsions were investigated to discuss the possibility of the
22
polyphenol accumulation at the oil-water interface. The data gathered here should be
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useful for the protection of the inner oil as well as simultaneous encapsulation of
2
bioactive components with different solubility in O/W emulsion.
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2.
Materials and methods
2.1.
Materials
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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.
11
(Shanghai, China). Other reagents were analytical grade and obtained from
12
SinoPharm CNCM Ltd. (Shanghai, China).
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Emulsion preparation
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2.2.
WPI stabilised emulsions in the absence or presence of CaCl2 were prepared
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according to our previous methods (Ni et al., 2015; Wang et al., 2016). Pre-denatured
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WPI solutions at 0.2, 0.4, 1.0, 2.0 or 4.0% (w/w) were prepared by dispersing the
19
powder in distilled water under stirring, followed by adjusting to pH 7.0, then holding
20
at 90 °C for 35 min and cooling to room temperature. Pre-denatured WPI solutions
21
were mixed with 91 mM CaCl2 under stirring and then added into sunflower oil to
22
obtain coarse emulsions by using a high speed blender (ATS Engineering Ltd., 6
ACCEPTED MANUSCRIPT Brampton, Ontario, Canada) operating at 14,000 rpm for 1 min, with final WPI
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concentrations of 0.1, 0.2, 0.5, 1.0 and 2.0%, final CaCl2 concentrations of 0.05, 0.2,
3
0.8, 1.6, 3.2 and 5 mM, and a final oil concentration of 10%. Oil droplet size was
4
reduced by passing the coarse emulsion twice through an ATSAH2100 high-pressure
5
homogeniser (ATS Engineering Ltd., Brampton, Ontario, Canada) at a pressure of 50
6
MPa. When resveratrol was added, the polyphenol was dissolved in 70% (v/v) ethanol
7
at 25.25 g L-1 and then mixed with WPI solutions and incubated for 35 min before
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WPI/CaCl2 mixture. The final polyphenol concentration was 0.13 g L-1 and equal to
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the molar concentration of WPI at 1.0% when calculated based on the molecular
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weight of β-lactoglobulin. Sodium azide (0.04%, w/w) was then added into the
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emulsions to prevent microbial growth and samples were stored at 25 °C for up to 30
12
days. All samples were prepared at least in duplicate for each subsequent analysis.
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2.3.
Size and ζ-potential measurements
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Size distribution and ζ-potential of WPI emulsions in the absence and presence
17
of CaCl2 were measured on a NanoBrooker Omni Particle size Analyzer (Brookhaven
18
Instruments Ltd, New York, USA) with a He/Ne laser (λ = 633 nm). Samples were
19
diluted with distilled water at pH 7.0 by 100 times before measurement. All
20
measurements were conducted at 25 °C and at a scattering angle of 173°. Size
21
distribution was obtained by using a NNLS mode analysis.
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2.4.
Determination of WPI at the oil/water interface
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Adsorption of WPI at the oil/water interface was determined according to the method described by Wan et al. (2013) and Wang et al. (2016) with some
5
modifications. WPI emulsion was centrifuged twice at 13,000 × g for 30 min at 4 °C
6
(Wang et al., 2016), using a 5804 R centrifuge (Eppendorf Co. Ltd, Hamburg,
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Germany). The WPI contents in the subnatant and in the whole emulsion were
8
determined by using the Kjeldahl method (Wan et al., 2013; Ye, Srinivasan, & Singh,
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2000). The percentage of WPI at the oil-water interface was calculated from the
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difference between the protein amount in the whole emulsion and that in the subnatant
11
divided by the content in the whole emulsion. The content of WPI at the oil-water
12
interface was calculated according to the protein content in the whole emulsion
13
multiplied by the protein interfacial percentage. Surface WPI concentration was
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calculated from interfacial WPI content and surface area of oil droplets determined by
15
size distribution (Chen & Tang, 2016; Zhao et al., 2015).
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2.5.
Quantitation of resveratrol using high performance liquid chromatography
WPI emulsion was centrifuged at 13,000 × g for 30 min at 4 °C, the
20
subnantant was collected and centrifuged at 150,000 × g for 30 min using a CP70ME
21
ultra-centrifuge (Hitachi Co. Ltd, Tokyo, Japan). The contents of resveratrol in the
22
whole emulsion (Cw), in the subnatant obtained from first centrifugation (Csub) and the 8
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2
liquid extraction and high performance liquid chromatography (HPLC) (Camont et al.,
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2009; Wang et al., 2016). Briefly, methanol exacts were measured on an HPLC
4
system equipped with a C18 column (5 µm, 4.6 mm × 250 mm, Waters, Milford, MA,
5
USA). Polydatin was used as an internal standard. The content of resveratrol was the
6
sum of both trans- and cis-resveratrol contents. The percentage of free resveratrol in
7
the continuous phase was calculated from Csup divided by Cw. The percentage of
8
resveratrol bound to WPI in the continuous phase was calculated from the difference
9
between Csub and Csup divided by Cw. The percentage of resveratrol in the continuous
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phase was calculated from Csub divided by Cw. The percentage of resveratrol at the
11
oil-water interface was calculated from the difference between Cw and Csub divided by
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Cw. Stability of resveratrol in the whole emulsion, in the continuous phase and at the
13
oil-water interface was expressed as the retention of the polyphenol during storage at
14
25 °C and calculated by a percentage relative to the corresponding initial content of
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trans-resveratrol.
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2.6.
Fluorescence measurement
Fluorescence spectra of resveratrol in the emulsions stabilised by WPI at 0.1,
20
0.2, 0.5, 1.0, 2.0% and by WPI at 0.5% in the presence of 0, 0.05, 0.2, 0.8 and 1.6 mM
21
CaCl2 were measured on a Cary Eclipse spectrophotometer (Agilent Co. Ltd, New
22
York, USA) equipped with a front-surface accessory. The incidence angle of the 9
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excitation radiation was set at 15°. The emission spectra were recorded from 330 to
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550 nm with an excitation wavelength of 330 nm. Both the excitation and emission
3
slit widths were set at 5 nm.
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2.7.
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Statistical analysis
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Data are presented as mean values ± standard deviations and analysed for
8
significant difference based on two-way ANOVA and Pearson’s correlation by using
9
the statistical software package SPSS, version 20.0 (IBM Co. Ltd, New York, USA).
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3.
Results and discussion
3.1.
Characterisation of WPI emulsions
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3.1.1. Size and ζ-potential
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Fig. 1 shows size distributions of the emulsions stabilised by heat-denatured
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WPI in the absence and presence of CaCl2. Size distributions of all the emulsions
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were divided into two peaks. In the absence of CaCl2, size distributions were around
19
160 and 930 nm at 0.1% WPI and decreased to 115 and 410 nm at 0.2%, then
20
remained constant as the concentrations of WPI increased up to 2% (Fig. 1A–E). By
21
increasing the concentrations of CaCl2, size distributions of WPI emulsions gradually
22
increased. The effect of CaCl2 became less pronounced as the concentrations of WPI 10
ACCEPTED MANUSCRIPT increased. The influence of CaCl2 on size distributions of heat-denatured WPI
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emulsions was similar to that of calcium at concentrations between 2 and 16 mM on
3
natural β-lactoglobulin emulsions (Agboola, & Dalgleish, 1995). At 0.1% of WPI
4
emulsions, creaming occurred after preparation for about 0.5 h when the
5
concentration of CaCl2 exceeded 0.2 mM, size distributions were thus not analysed. At
6
0.2% WPI, an abrupt increase in the size distributions was observed at the CaCl2
7
concentrations between 0.2 and 0.8 mM. The salt concentrations for the abrupt
8
changes increased as WPI concentrations increased and no aggregation was observed
9
at 2% WPI. After centrifugation, the particles in all the subnatants had uniform size
10
distributions, which were close or similar to the small ones of the whole emulsions.
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The particle size distributions of the centrifuged samples changed from a bit smaller
12
at the WPI concentrations between 0.1% and 0.5% to a bit larger at higher protein
13
concentrations, the former being more pronounced at higher CaCl2 concentrations
14
while the latter being more pronounced at lower CaCl2 concentrations. The effects
15
might be due to the impact of centrifugation on the particle with different structures.
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Therefore, in the whole emulsions, the smaller size distributions were for WPI
17
particles and the larger ones were for WPI stabilised oil droplets.
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Zeta-potentials of all emulsions were negative at pH 7.0 (Table 1) since whey
19
proteins (e.g., β-lactoglobulin and α-lactalbumin) have an isoelectric point of about 5
20
(Almécija, Ibáñez, Guadix, & Guadix, 2007). All ζ-potentials showed a uniform
21
distribution (data not shown). The ζ-potential was about –32 mV at a WPI
22
concentration of 0.1%, decreased to about –27 mV as the protein concentration 11
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being about –38 mV at 2.0% (Table 1). Since Ca2+ neutralised negative charges of
3
WPI, the ζ-potential values decreased as the concentrations of CaCl2 increased,
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reaching –24 mV at 0.2 mM when the concentration of WPI was 0.1%. The decreased
5
values were similar at higher WPI concentrations, with ζ-potential reaching about –22
6
and –15 mV at 3.2 and 5 mM CaCl2 when the protein concentrations were between 0.2%
7
and 1.0% and reaching –25 mV at 5 mM CaCl2 and 2% WPI. The emulsion droplets
8
was stabilised against aggregation by electrostatic repulsion and against coalescence
9
by steric hindrance (Wade & Beattie, 1997; Wu et al., 2015). In the case of the
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emulsions stabilised by caseins, small concentrations of Ca2+ cause a decrease in the
11
thickness of the adsorbed layer, reducing steric stabilisation, and high concentrations
12
of Ca2+ binds to the protein to form calcium bridges between the emulsion droplets.
13
However, Ca2+ did not completely neutralise the ζ-potential, so it may not be
14
sufficient to destabilise the emulsion (Dalgleish, 1997; Sjöblom, 2001).
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3.1.2. Adsorption of WPI at the oil-water interface
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In O/W emulsions, WPI existed both at the oil droplet surface and in the
18
continuous phase. The percentage of WPI at the oil-water interface was about 66%
19
when the protein concentration was 0.1% and decreased as the protein concentrations
20
increased, being about 32% at 2% (Fig. 2A). However, the interfacial WPI
21
percentages increased gradually as the concentrations of CaCl2 increased, which was
22
more pronounced at the lower protein concentrations. The interfacial WPI percentage 12
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2
WPI percentages were similar and increased to about 79% at 0.2% and 0.5% WPI. At
3
5 mM CaCl2, the interfacial WPI percentages were similar and increased to about 82%
4
at the protein concentration between 0.2% and 1.0%. The interfacial protein
5
percentages were significantly lower and increased only to 43% at 2.0% WPI.
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The content of WPI at the oil-water interface increased as the concentrations
of the protein and CaCl2 increased (Fig. 2B). Surface WPI concentration increased as
8
the protein concentration increased in the absence of CaCl2 and as the salt
9
concentration increased at a fixed protein concentration (Fig. 2C). The increase in the
10
surface concentration was dependent on both WPI and CaCl2 concentrations. At 0.05
11
and 0.2 mM CaCl2, surface WPI concentration decreased when the protein
12
concentration increased from 0.1% to 0.2% and then increased gradually at higher
13
protein concentrations. Surface WPI concentration was the most at the protein
14
concentrations of 0.5% and 1.0% when the concentrations of CaCl2 were 3.2 and 5
15
mM, respectively. An abrupt increase in the interfacial protein content and surface
16
protein concentration was observed at the salt concentrations between 0.2 and 0.8 mM
17
at 0.2% WPI, between 1.6 and 3.2 mM at 0.5% WPI and between 3.2 and 5 mM at 1.0%
18
WPI (Fig. 2B and C). The concentrations were consistent with that for an abrupt
19
increase in the size of WPI particles in the continuous phases and WPI stabilised oil
20
droplets (Fig. 1). Therefore, calcium induced aggregation of WPI resulted in a greater
21
number of protein molecules adsorb at the surface of oil droplets, leading to an
22
increase in the size of oil droplets. In the case that surface charges of WPI particles
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2
protein particles adsorbed at the oil-water interface than did charged particles, with
3
that the former were highly aggregated and formed a continuous network while the
4
latter might spread at the interface to form a continuous protein membrane (Destribats,
5
Rouvet, Gehin-Delval, Schmitt, & Binks, 2014). As the concentrations of CaCl2
6
increased, ζ-potentials of WPI particles decreased (Table 1), thus leading to greater
7
protein absorption at the oil-water interface (Fig. 2B). When the concentration of WPI
8
was 2.0%, ζ-potential was greater than that of other emulsions (Table 1), the increase
9
in interfacial WPI content and surface protein concentration was thus slower as the
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concentration of CaCl2 increased (Fig. 2B,C).
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Partition of resveratrol in WPI emulsions
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3.2.1. Percentage of resveratrol in the continuous phase Fig. 3A shows the percentage of free resveratrol in the continuous phase of the
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emulsions stabilised by WPI in the absence and presence of CaCl2. In the absence of
17
CaCl2, the percentage of free resveratrol gradually increased from 22% to 29% as the
18
concentration of WPI increased from 0.1% to 1.0% and then decreased to 22% at 2%
19
WPI. At 0.1% WPI, the percentage of free resveratrol was similar in the absence and
20
presence of CaCl2. At 0.2%, the percentage of free resveratrol did not change when
21
the concentration of CaCl2 was less than 0.2 mM and then decreased to 11% at 0.8 and
22
1.6 mM and to 7% at 3.2 and 5 mM. At 0.5% WPI, the percentage of free resveratrol
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2
decreased gradually to about 21% at 5 mM. Influence of CaCl2 on the percentage of
3
free resveratrol was different at higher protein concentrations. At 1% WPI, the
4
percentage of free resveratrol increased to 41% at 0.05 mM CaCl2 and then began to
5
gradually decrease with the salt concentration, being about 7% at 5 mM. At 2% WPI,
6
the percentage of free resveratrol increased gradually to 31% at 3.2 mM CaCl2 and
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then decreased to 14% at 5 mM.
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Fig. 3B shows the percentage of resveratrol bound to WPI in the continuous
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phase of the emulsions stabilised by WPI in the absence and presence of CaCl2. In the absence of CaCl2, the percentage of bound resveratrol was about 19% at the
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concentrations of WPI being 0.1% and 0.2%, about 25% at 0.5% and 1.0% WPI and
12
increased to 47% at 2% WPI. At 0.1% WPI, the percentage of bound resveratrol was
13
similar in the absence and presence of CaCl2. At 0.2% WPI, the percentage of bound
14
resveratrol decreased slowly to 10% as the concentration CaCl2 increased to 5 mM. At
15
0.5% WPI, the percentage of bound resveratrol decreased gradually to 12% as the
16
concentration of CaCl2 increased to 1.6 mM and was zero at higher salt concentrations.
17
At 1% WPI, the percentage of bound resveratrol was between 19% and 24% at the
18
concentration of CaCl2 less than 3.20 mM and then decreased to 6% at 5 mM.
19
However, at 2% WPI, the percentage of bound resveratrol decreased gradually to 31%
20
as the concentration CaCl2 increased to 3.2 mM and then increased to 42% at 5 mM.
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3.2.2. Percentage of resveratrol at the oil-water interface 15
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Resveratrol is not soluble in bulk oil including sunflower oil and rapeseed oil (Filip et al., 2003). Resveratrol interacts firstly with WPI to form protein-ligand
3
complexes and could then adsorb at the oil–water interface (Wang et al., 2016). The
4
percentage of resveratrol at the oil-water interface was 59% when the concentration of
5
WPI was 0.1% and decreased as the protein concentrations increased, being about 31%
6
at 2% WPI (Fig. 3C). The interfacial polyphenol percentages were basically similar to
7
that of WPI at the interface (Fig. 2A). When the concentrations of WPI were between
8
0.2% and 2%, the interfacial resveratrol percentages increased as the concentration of
9
CaCl2 increased. A significant positive correlation between the interfacial resveratrol
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2
percentage and that of WPI at the interface exists (Table 2). These results indicate that
11
the accumulation of resveratrol at the oil-water interface was dependent on the
12
adsorption of WPI at the oil droplet surface. However, in the case of 0.1% WPI, the
13
percentages of resveratrol at the oil-water interface kept constant as the concentrations
14
of CaCl2 increased to 0.2 mM. In the mixtures of WPI with resveratrol, there was an
15
equilibrium between free resveratrol and WPI-resveratrol complexes, since their
16
interaction involving noncovalent bonds are reversible (Mohammadi, & Moeeni, 2015;
17
Zhang, Liu, Subirade, Zhou, & Liang, 2014). As the concentrations of WPI increased,
18
the percentages of protein-bound resveratrol increased, thus the percentages of
19
resveratrol and WPI became more similar at the oil-water interface.
20
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As a whole, resveratrol could be free and bound to WPI in the continuous
21
phase and adsorbed together with WPI at the oil-water interface, except that the
22
polyphenol was only free in the continuous phase and bound to WPI at the interface 16
ACCEPTED MANUSCRIPT when the concentrations of CaCl2 were between 3.2 and 5 mM at 0.5% WPI. Addition
2
of CaCl2 at the concentrations less than 0.2 mM had no influence on the partition of
3
resveratrol in the emulsion stabilised by WPI at 0.1%. When the concentrations of
4
WPI were between 0.2% and 2%, except for the case at 2% WPI and 5 mM CaCl2, the
5
ratio of the percentages of resveratrol bound to WPI at the interface and in the
6
continuous phase increased with the salt concentration, indicating that resveratrol tend
7
to be accumulated by complexation with WPI at the oil-water interface more than in
8
continuous phase.
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1
9 10 11
3.2.3. Microenvironment of resveratrol
Fluorescence of resveratrol was sensitive to the polarity of surrounding environment. Fluorescence emission maximum (λmax) of resveratrol was around 404
13
nm in 10 mM phosphate buffer at pH 7.4 and shifted to 383 nm at 70% ethanol with a
14
significant increase in the intensity (Liang & Subirade, 2012; Liang et al., 2008). The
15
λmax of resveratrol was around 380 nm in the emulsion stabilised by WPI in the
16
absence and presence of CaCl2 (Fig. 4), suggesting that resveratrol was encapsulated
17
in a hydrophobic environment of WPI. Fluorescence intensity of resveratrol increased
18
as the concentrations of WPI increased (Fig. 4A) and as the concentrations of Ca2+
19
increased at a fixed WPI concentration of 0.5% (Fig. 4B), indicating that more
20
resveratrol molecules were bound by WPI. Therefore more resveratrol molecules were
21
adsorbed together with WPI at the surface of oil droplets (Fig. 3).
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22 17
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3.3.
Stability of resveratrol
2 3
Trans-resveratrol is liable to transform to cis-isomer, depending on storage conditions (Trela & Waterhouse, 1996). In this study, cis-resveratrol was only
5
detected after storage for 20 days, with the contents below 10% in the whole
6
emulsions and below 5% in the continuous phases (data not shown). The total
7
contents of both trans- and cis-resveratrol were shown in the following.
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9
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3.3.1. Dependence on WPI concentration
Creaming was observed for the emulsions at the WPI concentration of 0.1%
11
after preparation for about 12 h. The retention of resveratrol in the whole emulsions
12
stabilised by WPI between 0.2% and 2% is shown in Fig. 5A. In the whole emulsion
13
at 0.2% WPI, resveratrol was stable until 5 days and then began to degrade gradually
14
over time, with the percentage of about 57% remaining after storage of 30 days. The
15
polyphenol stability was significantly improved as the concentrations of WPI
16
increased to 0.5%, with about 78% remaining after 30 days. A further increase in the
17
WPI concentration up to 2% led to a slight increase in the percentage of resveratrol
18
remaining after 20 days, but the difference was not quite statistically significant.
EP
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10
In the continuous phases at 0.2% WPI, resveratrol was stable within one day
20
and then degraded gradually over time, with the percentage of about 68% remaining
21
after 30 days (Fig. 5B). The stability of resveratrol was also improved at higher WPI
22
concentrations, with similar retention of resveratrol until 20 days. After storage for 30 18
ACCEPTED MANUSCRIPT days, the percentages of resveratrol remaining at the concentrations of WPI between 1%
2
and 2% became greater than that at 0.5%, with the value being about 87% at 2% WPI.
3
In the oil-water interface (Fig. 5C), the overall change trend in the retention of
4
resveratrol were basically similar to that in the whole emulsions (Fig. 5A), except that
5
a more significant decrease was observed after 10 days, with the resveratrol
6
percentage of about 50% remaining after 30 days at the WPI concentrations of 0.2%. As a whole, when the concentration of WPI was 0.2%, the percentage of
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1
resveratrol remaining at the oil-water interface was greater than that in the continuous
9
phases during storage until 10 days (Fig. 5B,C). This is due to that resveratrol was
10
accumulated by complexation with WPI at the oil-water interface while resveratrol
11
was both free and complexed by WPI in the continuous phase (Fig. 3). WPI particles
12
adsorbed at the oil-water interface, providing better protective effect against the loss
13
of resveratrol. After then, the stability of resveratrol were better in the continuous
14
phase instead (Fig. 5B,C). It has been reported that addition of resveratrol could
15
improve the stability of α-tocopherol dissolved in the inner oil phase and of the inner
16
oil phase of the emulsions (Medina et al., 2010; Wan et al., 2013; Wang et al., 2015).
17
However, upon lipid oxidation occurred, resveratrol could accept free radicals by
18
donation of phenolic hydrogen atoms, leading to faster oxidation at the oil-water
19
interface (Wan et al., 2014). The phenomena were less pronounced when the
20
concentrations of WPI were 0.5% and 1%. At 2% WPI, the stability of resveratrol at
21
the oil-water interface were better than that in the continuous phase until 20 days and
22
then became similar after storage for 30 days. As the concentrations of WPI increased,
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19
ACCEPTED MANUSCRIPT 1
the stability of the inner oil phase was improved, thus delaying the impact on the loss
2
of resveratrol (Wang et al., 2016).
3
5
3.3.2. Dependence on CaCl2 concentration
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In the whole emulsions at 0.5% WPI, addition of CaCl2 at the concentrations
less than 0.20 mM basically had no significant influence on the retention of resveratrol
7
during storage (Fig. 6A). However, the percentage of resveratrol remaining decreased
8
when the concentrations of CaCl2 further increased, with the value being about 36% at
9
1.6 mM after 30 days. Similar trend was also observed in the percentages of
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resveratrol remaining in the continuous phase and the oil-water interface (Fig. 6B,C).
11
It has been reported that addition of CaCl2 had no influence on the stability of
12
α-tocopherol dissolved in the inner oil phase of emulsion (Wang, 2015).
13
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CaCl2-induced WPI aggregates had more hydrophobic surface exposed to solvents, which might be the reason for the greater loss of resveratrol (Ni et al., 2015).
15
In the absence and presence of CaCl2, the stability of resveratrol was similar, being
16
better at the oil-water interface until 10 days and being better in the continuous phase
17
after then (Fig. 6B,C).
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In O/W emulsion stabilised by heat-denatured WPI, resveratrol partitioned at
19
the oil-water interface and in the continuous phase. The percentage of resveratrol at
20
the oil-water interface decreased as the concentration of WPI increased but increased
21
as the concentration of CaCl2 increased. Opposite was influence of WPI and CaCl2
22
concentrations on the stability of resveratrol. The emulsion stabilised by calcium 20
ACCEPTED MANUSCRIPT 1
induced WPI aggregates was thus not an effective carrier for resveratrol. But the
2
accumulation of resveratrol at the oil-water interface would provide better protection
3
for the inner oil phase against oxidation.
5
4.
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4
Conclusions
6
Oil-in-water emulsions were stabilised by heat-denatured WPI particles and its
SC
7
calcium aggregate particles. Small WPI particles and large oil droplets co-existed in
9
the emulsions. At the oil-water interface, the accumulation of resveratrol was
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positively proportional to the contents of interfacial WPI, except for the case of 0.1%
11
WPI in the presence of CaCl2. Resveratrol at the oil-water interface was basically
12
more stable than that in the continuous phase at the initial stage of storage and then
13
became less stable at the ending of storage. The stability of resveratrol in the whole
14
emulsions increased as the concentration of WPI increased but decreased when the
15
concentration of calcium exceeded 0.2 mM, especially at 1.6 mM. In the future,
16
WPI-polysaccharide complexation was used to improve the stability of resveratrol in
17
the presence of CaCl2.
19
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Acknowledgements
20 21 22
This work was supported by the National Natural Science Foundation of China (NSFC Project 31571781) and the Fundamental Research Funds for the Central 21
ACCEPTED MANUSCRIPT 1
Universities (JUSRP51711B).
2 3
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Figure captions
2
Fig. 1. Size distributions of O/W emulsions (—–) stabilised by WPI at concentrations
4
of 0.1% (A), 0.2% (B), 0.5% (C), 1.0% (D) and 2.0% (E) and subnatants (– – –) after
5
centrifugation in the absence and presence of CaCl2 at various concentrations.
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6
Fig. 2. Interfacial WPI percentage (A), interfacial WPI content (B) and surface WPI
8
concentration (C) of the emulsions stabilised by the protein at various concentrations
9
(, 0.1%;
, 0.2%;
, 0.5%;
concentration of CaCl2.
11
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, 1.0% and , 2.0%) as a function of the
Fig. 3. Percentage of resveratrol free (A) and bound to WPI (B) in the continuous
13
phase and at oil-water interface (C) of the emulsions stabilised by various
14
concentrations of WPI (, 0.1%;
15
different concentrations of CaCl2.
, 0.5%;
, 1.0% and , 2.0%) with
EP
, 0.2%;
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12
17
Fig. 4. Fluorescence emission spectra of resveratrol in the emulsions stabilised by
18
WPI at various concentrations (A): —–, 0.1%; – · · –, 0.2%; – · – ·, 0.5%; · · · ·, 1.0%; –
19
– –, 2.0% and in the emulsions stabilised by WPI at 0.5% in the presence of CaCl2 at
20
various concentrations (B): —–, 0 mM; – · · –, 0.05 mM; – · – ·, 0.20 mM; · · · ·, 0.80 mM;
21
– – –, 1.60 mM.
22 1
ACCEPTED MANUSCRIPT 1
Fig. 5. Persistence of resveratrol in the whole emulsion (A), in the continuous phase
2
(B) and at the oil-water interface (C) at various concentrations of WPI (, 0.2%;
3
0.5%; , 1.0% and
4
material, Tables S1–S3).
,
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, 2.0%) and stored for up to 30 days (see also Supplementary
5
Fig. 6. Persistence of resveratrol in the whole emulsion (A), in the continuous phase
7
(B) and at the oil-water interface (C) at 0.5% WPI made with different concentrations
8
of CaCl2 (, 0 mM;
9
stored for up to 30 days (see also Supplementary material, Tables S4–S6).
, 0.80 mM and
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, 0.05 mM; , 0.20 mM;
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6
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2
, 1.60 mM) and
ACCEPTED MANUSCRIPT Table 1 ζ-Potential of oil-in-water emulsions stabilised by WPI at various concentrations as a
Concentration of
Concentration of WPI (%)
CaCl2 (mM)
0.1
0.2
0.5
0
-32.1±0.1Ba
-28.2±1.0CDa
-27.4±1.0Da
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function of the concentration of CaCl2. a
0.05
-27.6±0.8Bb
-27.5±0.9CDb
-27.1±1.6Db
0.20
-24.6±0.7Bb
-28.0±0.8CDb
-25.4±0.8Db
-30.0±1.5Cb
-35.2±5.6Ab
0.80
−
-27.3±0.7CDc
-23.6±1.7Dc
-23.1±1.9Cc
-30.6±1.1Ac
1.60
−
-25.2±0.0CDc
-23.6±0.3Dc
-24.9±0.2Cc
-29.9±0.2Ac
3.20
−
-22.2±0.8CDd
-22.6±0.2Dd
-23.2±1.9Cd
-29.0±0.8Ad
5.00
−
-16.1±1.0De
-15.0±1.3Ce
-25.3±0.4Ae
-30.6±1.8Ca
-37.8±5.6Aa
-30.1±2.8Cb
-36.0±4.9Ab
TE D
M AN U
SC
2.0
-15.5±1.2CDe
Values with the different superscript letter (upper case for WPI concentration, lower
EP
a
1.0
AC C
case for CaCl2 concentration) are significantly different (P < 0.05).
ACCEPTED MANUSCRIPT Table 2 Results of the Pearson correlation coefficient (r) between the percentages of
Parameter
0.5
1.0
r
0.988*
0.987*
0.967*
p
0.000
0.000
2.0
0.934*
SC
0.2
M AN U
0.000
0.002
EP
TE D
An asterisk indicates p < 0.01 (Significant difference at 99% confidence interval).
AC C
a
WPI concentration (%)
RI PT
resveratrol and WPI at the oil-water interface. a
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 1
Figure 2
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
Figure 3
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 4
Figure 5
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
Figure 6
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT