Improved oxidative stability of fish oil emulsion by grafted ovalbumin-catechin conjugates

Accepted Manuscript Improved Oxidative Stability of Fish Oil Emulsion by Grafted Ovalbumin-Catechin Conjugates Jin Feng, He Cai, Hua Wang, Chunyang Li, Songbai Liu PII: DOI: Reference:

S0308-8146(17)31390-0 http://dx.doi.org/10.1016/j.foodchem.2017.08.055 FOCH 21604

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

14 April 2017 26 July 2017 17 August 2017

Please cite this article as: Feng, J., Cai, H., Wang, H., Li, C., Liu, S., Improved Oxidative Stability of Fish Oil Emulsion by Grafted Ovalbumin-Catechin Conjugates, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/ j.foodchem.2017.08.055

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.

Improved Oxidative Stability of Fish Oil Emulsion by Grafted Ovalbumin-Catechin Conjugates

Jin Feng a,b, He Cai a, Hua Wang c, Chunyang Li b, Songbai Liu a, *

a

Department of Food Science and Nutrition, Fuli Institute of Food Science, Zhejiang

Key Laboratory for Agro-Food Processing, Zhejiang R & D Center for Food Technology and Equipment, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China b

Department of Functional Food and Bio-active compounds, Institute of Agro-food

Processing, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China c

Center of Analysis and Measurement, Zhejiang University, 866 Yuhangtang Road,

Hangzhou 310058, China

* Corresponding Author. E-mail address: [email protected] (S. Liu).

1

ABSTRACT Conjugates of ovalbumin (OVA) with (+)-catechin (C), (-)-epigallocatechin (EGC) or (-)-epigallocatechin gallate (EGCG) were synthesized through a free-radical grafting approach. The covalent binding between OVA and catechins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted

laser

desorption/ionization

time-of-flight

mass

spectrometry

(MALDI-TOF-MS). OVA-catechin conjugates were further characterized by a combination of steady-state fluorescence spectroscopy, differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) analyses. Results suggested that the conjugates contained less α-helix but more β-sheet secondary structure than OVA. The catechin-grafting reaction led to improved surface hydrophobicity and decreased tertiary conformation stability of OVA. Compared with that emulsified by OVA, fish oil emulsion coated by conjugates demonstrated smaller droplet size, better storage stability and less viscosity. Besides, conjugates inhibited the lipid oxidation in fish oil emulsion more effectively than OVA because of their higher antioxidant activity and interfacial accumulation.

Keywords: Ovalbumin; Catechins; Conjugates; Fish oil emulsion; Lipid oxidation

2

1. Introduction Fish oils contain high level of long-chain ω-3 polyunsaturated fatty acids (Lω-3 PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are beneficial to human health by reducing the risk of cardiovascular diseases (Su et al., 2015). However, the application of fish oils as functional food ingredient is limited because of their high hydrophobicity. Fish oils therefore usually have to be incorporated into emulsion-based delivery systems before they are introduced into aqueous food products (von Staszewski, Ruiz-Henestrosa, & Pilosof, 2014). The utilization of fish oils in food products can also bring about a challenge since PUFAs’ degradation through auto-oxidation during processing and storage, leading to losses in stability and safety as well as the sensory and nutritional quality (von Staszewski et al., 2014; Waraho, McClements, & Decker, 2011). Many food proteins, such as milk protein, can be utilized as emulsifiers because they are amphiphilic molecules that adsorbed to oil droplet surface preventing them from aggregation (Liu, Wang, Sun, & Gao, 2016; Liu, Wang, Xu, Sun, & Gao, 2016; Wei, Yang, Fan, Yuan, & Gao, 2015; Yi, Fan, Zhang, & Zhao, 2016; Yi, Zhang, Liang, Zhong, & Ma, 2015). The physicochemical properties of emulsions are dependent on the properties of the interfacial coating around lipid droplets, such as polarity, thickness, charge and chemical composition (Mao, Dubot, Xiao, & McClements, 2013). In oil-in-water emulsions, the antioxidant capacity of interfacial emulsifiers can have a significant influence on the rate of lipid oxidation by affecting the location and reactivity of pro-oxidative transition metals, lipid hydroperoxide and free radicals 3

(Chityala, Khouryieh, Williams, & Conte, 2016; von Staszewski et al., 2014). Covalent conjugation of proteins with polyphenols would enhance their antioxidant activities by introducing a large amount of phenolic hydroxyl groups. Besides, it is reported to be a promising way to improve the emulsifying activity and emulsifying stability of proteins (Liu, Sun, Yang, Yuan, & Gao, 2015; Wei et al., 2015). Therefore, compared with protein alone, protein-polyphenol conjugate may provide

better

protection

for

easily

oxidized

lipophilic

nutraceuticals

in

emulsion-based systems. Protein-polyphenol conjugates could be conveniently synthesized via a non-enzymatic free-radical grafting approach, which employs hydrogen peroxide (H2O2)-ascorbic acid (Vc) redox pair as radical initiator system (Spizzirri et al., 2009). This approach is preferable for the synthesis of conjugates in mild conditions without the use or generation of toxic chemicals. Conjugates of gelatin-gallic acid/catechin (Spizzirri et al., 2009), β-lactoglobulin-catechin (Yi et al., 2015), α-lactalbumin-catechin (Yi et al., 2016) and lactoferrin-chlorogenic acid/epigallocatechin gallate/gallic acid (Liu et al., 2015) were successfully prepared via this method and the effect of polyphenol modification on the structural and functional properties of these proteins has been well evaluated. Non-covalent protein-polyphenol complexes, such as gelation-grape seed proanthocyanidin (Su et al., 2015) and β-lactoglobulin-green tea polyphenols mixture (von Staszewski et al., 2014), have been proved to be more effective in inhibiting lipid oxidation in fish oil emulsions than their pure protein counterparts. Complexes formed by physical interactions such as electrostatic, hydrophobic, van der Waals and 4

hydrogen bonding readily undergo dissociation when the environmental or solution conditions are altered (Wei et al., 2015). The strong hydrophilicity of these polyphenols prevents them from partitioning into the oil phase or residing at the droplet surface and hence reduces their antioxidant activity towards PUFAs. On the contrary, conjugates held together by covalent bonds are more stable and display greater potential in inhibiting lipid oxidation because the grafted polyphenols are mostly accumulated at the lipid-water interfacial (Wei et al., 2015). Ovalbumin (OVA) is a global phosphoglycoprotein of 42-47 kDa molecular weight and it is composed by 385 amino acids, with the hydrophobic amino acids (about half of total) mainly buried in protein interior whereas the charged ones (about one third of total) mainly located on protein surface (Sponton, Perez, Carrara, & Santiago, 2015). As the most abundant component in egg white protein (~ 65 %), OVA is an important food ingredient with good emulsifying, foaming and gelling properties (Sponton et al., 2015). Catechins such as (+)-catechin (C), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) are the major antioxidants in green tea (Seeram et al., 2006). Previous studies suggested that protein-polyphenol conjugates fabricated via radical copolymerization could effectively inhibit β-carotene degradation by acting as interfacial antioxidants (Yi et al., 2016; Yi et al., 2015). Therefore, we speculate that the grafting of catechins onto OVA endows OVA with higher antioxidant activity which allows OVA to act as not only stabilizers but also antioxidants to control lipid oxidation in fish oil emulsions. In this work, catechins such as C, EGC and EGCG were grafted onto OVA 5

molecules respectively via the aforementioned radical-mediated copolymerization method. OVA-catechin conjugates were characterized by a combination of SDS-PAGE, MALDI-TOF-MS, steady-state fluorescence spectroscopy, DSC, FTIR spectroscopy and CD analyses. The influence of catechin grafting on the antioxidant activity of OVA was evaluated by DPPH, ABTS and ORAC assay, respectively. Finally, the storage stability and rheological behavior of fish oil emulsions coated by OVA or OVA-catechin conjugates were investigated and the lipid oxidation was monitored by determining the content of lipid hydroperoxide and thiobarbituric acid reactive substances (TBARS). This study provides valuable information for the designation of emulsion-based systems for healthy lipids, such as ω-3 PUFAs, using novel protein-polyphenol conjugate emulsifiers. 2. Materials and methods

2.1. Materials

Ovalbumin (OVA, from egg while, purity >80%) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Fish oil (containes approximately 20% EPA and 50% DHA) were purchased from Xunda marine biological products Co., Ltd. (Wuxi, China).

2,2-diphenyl-1-picrylhydrazyl

2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic 2,2′-azobis(2-methylpropionamidine)

acid

(DPPH),

ammonium

dihydrochloride

salt)

(ABTS),

(AAPH)

and

(-)-epigallocatechin (EGC) were purchased from Aladdin Co., Ltd. (Shanghai, China). 8-Anilino-1-naphthalenesulfonic

acid 6

(ANS),

6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), (+)-catechin (C), (-)-epigallocatechin gallate (EGCG) and Folin-Ciocalteu’s phenol reagent (2M) were purchased from Sigma-Aldrich Corp. (St. Louis, NJ, USA). All other chemicals used were of analytical grade and used as purchased.

2.2. Preparation of OVA-catechin conjugates

OVA-C conjugate (OVA-C), OVA-EGC conjugate (OVA-EGC) and OVA-EGCG conjugate (OVA-EGCG) were prepared using Vc/H2O2 redox pair as an initiator system (Spizzirri et al., 2009). Briefly, 1 g of OVA was dissolved in 100 mL of ultrapure water in a flask, then 1.0 mL of H2O2 (10 M) and 0.25 g of ascorbic acid were added and incubated at 25 oC under stirring for 2 h. After that, 0.7 mmol of C, EGC or EGCG was introduced to the mixture and the reaction was allowed to proceed for 24 h. The above procedures were carried out under the protection of nitrogen. The unreacted catechin was removed by dialysis (MWCO: 12000-14000 Da) at room temperature for 48 h with eight changes of water until no free polyphenols existed in the dialysis fluid, which was monitored using Folin-Ciocalteu method described later. The resulting solution was frozen and dried in a FD-1A freeze-drier (Boyikang laboratory instruments Co., Ltd, China) to afford powdered solid. The control OVA was prepared under the same condition but in the absence of catechins.

2.3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was carried out according to the method of Laemmli (Laemmli, 1970) 7

with an 8% acrylamide separating gel and a 5% stacking gel. 5 μL of conjugation solution (5 mg mL-1) was mixed with 5 μL of ultrapure water and 10 μL of 2×SDS sample buffer (100 mM Tris, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol and 200 mM 2-mercaptoethanol) and then heated for 5 min at 100 oC. 15 μL of each sample was loaded to designated well for the electrophoresis at 100 mV with a Tris-HEPES-SDS running buffer (100 mM Tris, 100 mM HEPES, 3 mM SDS, pH 8.0). The gels were stained with Coomassie brilliant blue R-250 for protein visualization.

2.4. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis

MALDI-TOF-MS were carried out on AUTOFLEX Ⅱ

TOF/TOF mass

spectrometer (Bruker Daltonics, Billerica, MA, USA). The lyophilized samples were dissolved in ultrapure water to reach a concentration of 1 mg/mL and then 0.5 μL of these solutions were brought onto the target and covered with 0.5 μL of matrix (saturated sinapinic acid in 50 % acetonitrile with 0.1 % trifluoroacetic acid). The samples were dried in air for 10 min prior to analysis by MALDI-TOF-MS. The mass spectra were recorded in the reflector mode with an acceleration voltage of 20 kV and an effective flight path of 200 cm.

2.5. Steady-state fluorescence spectroscopy

The intrinsic fluorescence spectra of OVA and OVA-catechin conjugates were 8

recorded by a F4600 fluorescence spectrophotomer (Hitachi Co., Japan) at 25 oC using 1.0 cm path length quartz cells. Sample solutions (2 mg mL -1) were prepared in 10 mM phosphate buffered saline (PBS, pH 7.0). Excitation was done at 295 nm and the emission spectra were scanned over the range of 300-450 nm. The excitation and emission slit width were set at 2.5 and 5.0 nm, respectively. The surface hydrophobicity (H0) was determined with the fluorescence probe ANS according to the method of Alizadeh-Pasdar and Li-Chan (Alizadeh-Pasdar & Li-Chan, 2000) with modifications. Series dilutions in 10 mM PBS (pH 7.0) were prepared with the stock solutions of samples (10 mg/mL) to reach a final concentration of 0.05-0.5 mg mL-1. Twenty microliters of ANS solution (8.0 mM) was added to 4 mL of each dilution. Excitation was done at 390 nm and the emission was measured at 470 nm. The initial slope of florescence intensity versus sample concentration (mg mL -1) (calculated by linear regression analysis) was used as an index of H0.

2.6. Differential scanning calorimetry (DSC)

The thermal transition profiles of powdered OVA and OVA-catechin conjugates were characterized using a model Q2000 calorimeter (TA Instrument, New Castle, DE, USA). About 5-10 mg of powder was sealed in a hermetic aluminum pan and heated from 20 to 200 oC at a rate of 10 oC/min. Nitrogen was used as the transfer gas at a flow rate of 25 mL/min. The instrument was calibrated using Indium under the same condition as samples.

2.7. Fourier transform infrared (FTIR) spectroscopy 9

The FTIR spectra of OVA and conjugates were recorded on a FTIR Bruker Tensor 27 spectroscopy (Bruker Optik Gmbh, Ettlingen, Germany) using a KBr disk containing 1% finely ground samples. The spectra were scanned ranging from 500 to 4000 cm-1 at a resolution of 4 cm-1. Fourier self-deconvolution and secondary derivative were applied in the range of 1600-1700 cm-1 which was assigned to the amideⅠin protein FTIR spectra using Peakfit 4.0. Area of peaks corresponding to α-helix (1650-1660 cm-1), β-sheet (1610-1642), β-turn (1660-1680), β-antiparallel (1680-1700) and random coil (1642-1650) was measured with Gaussian function (Beauchemin et al., 2007). The area of all component bands assigned to a given secondary conformation were summed up and then divided by the total area to obtain its content.

2.8. Circular dichroism (CD) analysis

CD spectra were recorded on a J-815 CD spectrometer (Jasco, Tokyo, Japan). The secondary structure of OVA and conjugates was investigated by far-UV CD spectra in the wavelength range of 190-250 nm using a quartz cuvette with a 1.0 mm path length. Samples were prepared with 10 mM PBS (pH 7.0) to obtain a concentration of 0.2 mg mL-1. Scanning was carried out at 50 nm/min at 25 oC. The collected data were analyzed using the online program DichroWeb. Far-UV CD spectra were utilized to probe the tertiary structure. Each sample with a concentration of 2 mg mL -1 was placed in a 10 mm quartz cuvette and scanned from 250 to 300 nm. The spectrum was an average of eight scans for each measurement. 10

2.9. Measurement of the amount of catechins bound to OVA

The amount of catechins bound to OVA was measured using Folin-Ciocalteu method (Slinkard & Singleton, 1977) with modifications. In brief, 0.5 mL of conjugate solution (1 mg mL-1) was thoroughly mixed with 2.5 mL of fresh Folin-Ciocalteu reagent (0.5 M). After 4 min, 2 mL of Na2CO3 solution (7.5%, w/v) was added, and the mixture was allowed to stand in dark for 2 h. The absorbance at 760 nm was measured with a UV-spectrophotometer against a control prepared using OVA solution (1 mg mL-1) with the same procedure. The amount of catechins bound to OVA was expressed as equivalent content of C, EGC or EGCG using the equations obtained from the calibration curves of each catechin, and the result was expressed as μmol catechin equivalent (CE) per g conjugate.

2.10. Antioxidant activity

The DPPH• scavenging activity was determined based on the method described by Brand-Williams et al. (Brand-Williams, Cuvelier, & Berset, 1995) with slight modification. Briefly, 2 mL of fresh DPPH• solution (0.2 mM, methanol) was thoroughly mixed with 2 mL of diluted sample (0.7 mg mL -1). The mixture was allowed to stand in the dark for 30 min, after which the absorbance was measured at 517 nm. The DPPH• scavenging activity was determined on the basis of the Trolox calibration curve, which was carried out by the method described above, and expressed as μmol Trolox equivalent (TE) per g sample. The ABTS•+ scavenging activities of OVA and conjuages were evaluated according 11

to Miller et al. (Miller, Riceevans, Davies, Gopinathan, & Milner, 1993). The stock solution was prepared by reacting of 10 mg of ABTS with 2.6 mL of potassium persulfate solution (2.45 mM). The mixture was kept in dark at 4 oC overnight before use. The ABTS working solution was prepared by diluting the stock solution with ultrapure water to reach an absorbance of 0.70 ± 0.02 at 734 nm. Then 1 mL of diluted sample and 3 mL of ABTS working solution were mixed and incubated in dark for 30 min, after which the absorbance at 734 nm was measured. The ABTS •+ scavenging activity was determined on the basis of the Trolox calibration curve, which was carried out by the method described above, and expressed as μmol Trolox equivalent (TE) per g sample. Oxygen radical absorbance capacity (ORAC) assay was carried out using fluorescein sodium salt as fluorescence probe. The reaction was carried out at 37 oC in 75 mM PBS (pH 7.4) and the final volume of mixture was 200 μL. Briefly, 100 μL of diluted sample and 50 μL of fluorescein sodium salt (0.4 μM) were mixed in the well of a black 96-well microplate (96F nontreated, Nunc, Denmark) and incubated at 37 o

C for 15 min. Then 50 μL of AAPH solution (60 mM) was added rapidly and the

fluorescence (Ex: 374 nm; Em: 485nm) was recorded every minute for 100 min in a SpectraMax M5 reader (Molecular Devices, California, USA). The ORAC values of OVA and OVA-catechin conjugates were determined on the basis of the Trolox calibration curve, which was carried out by the method described above, and expressed as μmol Trolox equivalent (TE) per g sample.

2.11. Preparation of fish oil emulsions 12

OVA or OVA-conjugates were dissolved in ultrapure water and stirred for 3 h to form a 1 % solution. Fish oil was emulsified with 1 % OVA or conjugate solution at 10000 rpm for 10 min with a T-25 blender (IKA-Werk, Staufen, Germany) to produce coarse emulsions at an oil-to-water ratio of 1:9. Fine emulsions were then formed by passing the coarse emulsions through a high pressure homogenizer at an operational pressure of 60 MPa for three cycles. The pH of emulsions was adjusted to pH 3.5 or 7.0 by HCl (1M) or NaOH (1M) with stirring for at least 1 h for stabilization.

2.12. Droplet size and ζ-potential

The Z-average diameter (DZ), polydispersity index (PDI) and ζ-potential of emulsions were determined using a commercial zeta-sizer (Nano-ZS 90, Malvern Instruments Ltd, United Kingdom) at 25 oC with a He/Ne laser (λ = 633 nm) and 900 scattering angle. Emulsions were diluted by 20 times with ultrapure water and adjusted to pH 3.5 or 7.0 before determination to avoid multiple scattering phenomenon.

2.13. Rheological behavior

The rheological properties of emulsions were measured with a MCR302 rheometer (Aoton paar GmbH, Austria) fitted with a 40 mm diameter of steel parallel plate. The apparent viscosity as a function of the shear rate was determined in the range of 1-100 s-1. Dynamic oscillatory measurements were carried out in the linear viscoelasticity range (LVR). Storage modulus (G') and loss modulus (G") were recorded in the 13

0.1-100 rad/s angular frequency sweep at a strain amplitude of 1 % (in the LVR). Samples were individually loaded on the measuring plateau and allowed to stand for 2 min prior to test. All measurements were carried out at 25 ± 0.1 oC and performed in triplicate.

2.14. Determination of interfacial protein fraction

The fraction of protein adsorbed at oil-water interface at pH 7.0 was determined according to the method described by Wan et al. with slight modification (Wan, Wang, Wang, Yang, & Yuan, 2013). The fresh emulsions were centrifuged at 15,000g at 4 oC for 2 h. After centrifugation, the subnatants were carefully removed using a syringe and filtered through a 0.45 μm filter. The amount of protein remaining in subnatants was determined by the Bradford assay (Bradford, 1976) with a standard calibration curve prepared using emulsifier concentrations ranging from 0.2 to 1.0 mg/mL. The amount of interfacial protein was calculated by the difference between the total amount of protein used to prepare emulsions and that measured in the subnatants after centrifugation. The interfacial protein fraction (Fip) was determined as the ratio of the amount of interfacial protein to the total amount of protein in the emulsion and the continuous phase protein fraction (Fcp) was determined by the equation: Fcp = 1 - Fip.

2.15. Lipid oxidation

Fresh fish oil emulsions were transferred to falcon tubes and kept at 25 oC in dark for 14 days. The DZ, PDI and ζ-potential of emulsions were determined at the end of 14

the storage test. Aliquots of samples were withdrawn every 24 h for the measurement of lipid hydroperoxide and thiobarbituric acid reactive substances (TBARS), respectively. The content of lipid hydroperoxide was measured to evaluate the primary products of lipid oxidation according to the method described in a previous report (Su et al., 2015). Aliquot of fish oil emulsion (0.3 mL) was mixed with 1.5 mL of isooctane/2-propanol mixture (3:1, v:v) and then vortexed for 1 min. After centrifugation at 5000g for 30 min, 0.2 mL of the clear upper layer was mixed with 2.8 mL of methanol/1-butanol (2:1, v:v). The mixture (3 mL) was then reacted with 15 μL of 3.94 M ammonium thiocyanate and 15 μL of ferrous iron solution (prepared by reacting 0.132 M barium chloride with 0.144 M ferrous sulfate). The reaction was allowed to proceed for 20 min at room temperature in dark, after which the absorbance was measured at 510 nm using a spectrophotometer. The content of lipid hydroperoxide was determined based on a standard curve of cumene hydroperoxide (CHP) and expressed as mmol CHP equivalent (CHPE) per kg oil. The content of TBARS was measured to evaluate the secondary products of lipid oxidation. One mL of emulsion was mixed with 2 mL of TBA working solution (prepared by mixing 15 g of trichloroacetic acid, 0.375 g of TBA, 1.76 mL of 12 M HCl and 89.2 mL of H2O) in test tubes and placed in a boiling water bath for 10 min. Then the mixture was cooled down to room temperature and centrifuged at 5000g for 30 min. The absorbance was measured at 532 nm using a spectrophotometer. TBARS content was calculated from a standard curve of 1,1,3,3-tetramethoxypropane, and 15

expressed as mmol malondialdehyde (MDA) equivalent (MDAE) per kg oil.

2.16. Statistical analysis

All tests were performed in triplicate, and the data were presented as means ± the standard deviation. The results were subjected to least significant difference (LSD) in one-way analysis of variance (ANOVA) using the PASW statistics 18 software to analyze the difference. Differences with a P value of < 0.05 were considered significant. 3. Results and discussion

3.1. Molecular mass

The molecular mass of OVA, control OVA and OVA-catechin conjugates were investigated by SDS-PAGE and MALDI-TOF-MS, respectively. As depicted in Fig. 1a, the major band corresponding to OVA was found at about 45 kDa and small amount of ovotransferrin was observed at about 78 kDa, which was in accordance with our recent report (Feng, Wu, Wang, & Liu, 2016). The protein band in SDS-PAGE pattern did not change when OVA reacted only with the Vc/H2O2 redox pair system (control OVA), suggesting that there was no conjugated OVA product in the absence of catechins. In contrast, OVA-catechin conjugates migrated up slightly compared with OVA indicating higher molecular mass. These high molecular mass complexes were not separated by SDS or mercaptoethanol, which were used to break non-covalent bindings, revealing that catechins were covalently conjugated onto OVA. 16

This phenomenon was consistent with the apparent increase in the molecular mass of other proteins after their covalent conjugation with polyphenols (Liu et al., 2015; Yi et al., 2016). MALDI-TOF-MS was further applied to determine the accurate molecular mass of OVA and conjugates. As depicted in Fig. 1b, a peak with molecular mass of 44561.32 Da was observed for OVA. The peak position was quite similar for OVA and control OVA indicating that Vc could not be conjugated onto OVA, which was in accordance with the SDS-PAGE result. On the other hand, OVA-catechin conjugates were found to contain one main component with molecular mass of 44988.56, 44855.12 and 45182.77 for OVA-C, OVA-EGC and OVA-EGCG, respectively. The increase in the molecular mass of OVA after the catechin-grafting reaction confirmed the covalent attachment between these two components because they were not separated by the high acceleration voltage and long-distance flight at high speed with this technique (Wei et al., 2015). The reaction of OVA with EGCG (458.4 Da) resulted in the highest increase in molecular mass of 621.45 Da, which accounted for the grafting of one or two molecules of EGCG to one OVA molecule. Similar results were also observed in the reaction of OVA with C or EGC.

3.2. Protein conformation

The intrinsic (or Trp) fluorescence study provides a sensitive mean of monitoring the polarity of the environment around Trp residues and the conformation changes in proteins (Tang, Sun, & Foegeding, 2011). As shown in Fig. 2a, the fluorescence 17

spectrum of OVA displayed an emission maxima at 334.06 nm, which is a typical fluorescence profile of Trp residues in the hydrophobic interior of a global protein (Tang et al., 2011). Compared with OVA, conjugates showed a red-shifted (up to 4.9-21.1 nm) emission maxima with considerably decreased magnitude. Similar result was observed when grafting catechins to ovotransferrin (You, Luo, & Wu, 2014). Commonly, the emission maxima of proteins suffers a red-shift when Trp residues are more exposed to the hydrophilic solvent, and the quantum yield of fluorescence decreases when the chromophores interact with the quenching agent either in solvent or in the protein itself (Feng, Wu, Wang, & Liu, 2017; Tang & Sun, 2011). It could be therefore deduced from Fig 2a that the catechin-grafting reaction resulted in the unfolding and denaturation of OVA, which decreased its tertiary conformation stability. The surface hydrophobicity (H0) of OVA and conjugates was determined at pH 7.0 with the fluorescence probe ANS and the results were summarized in the last column of Table 1. The H0 values for OVA and control OVA were 103.42 and 111.21, respectively, suggesting that the free-radical grafting approach could hardly change the tertiary structure of OVA in the absence of catechins. On the other hand, the covalently attached catechins were supposed to reduce the surface hydrophobicity of conjugates because of their hydrophilic nature (Wei et al., 2015). Nevertheless, the H0 values of conjugates were generally more than two times higher than that of OVA. As evidenced by the intrinsic fluorescence study (Fig. 2a), the catechin-grafting reaction led to the unfolding of protein structure, which caused the exposure of interior 18

hydrophobic clusters onto

protein surface, thereby improving its surface

hydrophobicity. The thermal stability of OVA and conjugates was evaluated using the peak melting temperature (Tp) as an indicator. Tp reflected the disrupting of the hydrogen bond and disulfide bond maintaining the tertiary conformation of proteins (Tang et al., 2011). Therefore, higher Tp is associated with higher thermal stability and more compact tertiary conformation of a global protein (Feng et al., 2016; Tang et al., 2011). Each sample showed only one prominent endothermic peak in the DSC profile (Fig 2b), corresponding to the thermal transition of protein. Tp, as well as the onset melting temperature (To) of OVA decreased considerably after the catechin-grafting reaction (e.g. from 102.4 to 74.8-96.6 oC for Tp), suggesting decreased thermal stability, or tertiary conformation stability. Values of ΔH decreased in the order: OVA > OVA-C > OVA-EGC > OVA-EGCG, which revealed that the conjugates contained less proportion of undenatured or ordered structure than OVA (Tang et al., 2011). The decreased tertiary conformation stability of conjugates has also been evidenced by the aforementioned intrinsic fluorescence study (Fig. 2a). The effect of polyphenol grafting on the thermal stability of global protein has been thoroughly reviewed by Liu et al. (2017). In general, an improvement in thermal stability of global proteins can be achieved by conjugation with polyphenols. For example, the Tp of lactoferrin increased by 5-15.2 oC after grafting with EGCG, chlorogenic acid or gallic acid via radical-mediated copolymerization method (Liu et al., 2015); the alkaline-modification by EGCG improved the Tp of α-lactalbumin, 19

β-lactoglobulin and lactoferrin appreciably (Wei et al., 2015). The discrepancy between the result of present work and that of the references suggested that the physico-chemical properties of polyphenols and proteins and the preparation methods would affect the thermal stability of conjugates appreciably. The FTIR spectra of OVA and conjugates were depicted in Supplementary Fig. 1. OVA showed typical bands at 3289.87 (amide A, associated with N-H stretching coupled with hydrogen bonding), 1641.13 (amideⅠ, associated with C=O stretching vibration of the peptide linkage), 1536.09 (amideⅡ, associated with C-N stretching and N-H bending of amino groups) and 1241.93 cm-1 (amide Ⅲ, associated with N-H in-plane deformation) (Feng et al., 2016; Liu et al., 2015), respectively. It should be noted that both the position and shape of these bands were obviously changed after the catechin-grafting reaction, which implied alterations in the secondary conformation. It was demonstrated that amideⅠband is the most informative part of the FTIR spectrum with regards to the secondary structure of protein (Byler & Susi, 1986). Therefore, the Fourier self-deconvolution was applied to the region of 1600-1700 cm-1 in the origin spectra. Supplementary Fig. 2 showed the amideⅠregion fitted with a Guassian line shape function and a quantitative analysis of the secondary structure of samples. In this work, OVA contained 26.11% α-helix, 33.55% β-sheet, 16.16% β-turn, 9.98% β-antiparallel and 15.00% unordered structure. In general, the catechin-grafting reaction resulted in a decrease in α-helix, β-turn and β-antiparallel fractions with a parallel increase in the content of other structures. These changes were believed to 20

arise from the covalent interactions between OVA and catechins. The secondary structure contents of OVA and conjugates were also investigated by far-UV CD spectroscopy. As depicted in Fig. 2c, a broad negative band ranging from 210-220 nm was observed in the far-UV CD spectrum of OVA and the catechin-grafting reaction resulted in a remarkable decrease in the ellipicity of this negative band, suggesting that the secondary conformation of OVA has been distinctively changed. The secondary structure composition of samples was evaluated with DichroWeb online using SELCON algorithms (Whitmore & Wallace, 2004). In the present study, OVA contained 28.51% α-helix, 21.22% β-sheet, 23.22% β-turn and 29.82% unordered structure. In general, the conjugation with catechins led to an increased content of β-sheet with a parallel decrease in the fraction of α-helix. This trend was also observed in the aforementioned FTIR analysis (Supplementary Fig. 2) though some differences between the results of these two methods should be noted because lyophilized powder was used for FTIR test whereas aqueous solution for far-UV CD spectroscopy. Similar results were observed in C grafted α-lactalbumin (Yi et al., 2016). Nevertheless, it was reported that the conjugation of lactoferrin with chlorogenic acid, gallic acid or EGCG resulted in an increased fraction of highly order α-helix together with decreased fraction of other structures (Liu et al., 2015). This discrepancy revealed that the effect of polyphenol grafting on the secondary conformation of proteins was highly dependent on the physico-chemical properties of polyphenols and proteins utilized. CD spectra in near-UV region were utilized to probe the tertiary/quaternary 21

conformation of OVA and conjugates. As illustrated in Fig. 2d, the near-UV CD spectrum of OVA showed predominant positive bands at 260, 269, 276, 285 and 292 nm, respectively, which was in accordance with previous reports (Naeem, Khan, Muzaffar, Ahmad, & Saleemuddin, 2011; Wong, Kadir, & Tayyab, 2015). Occurrence of these peaks would have been because the presence of disulfide bonds and aromatic chromophores such as Trp, Tyr and Phe, in proteins (Kelly, Jess, & Price, 2005; Wong et al., 2015). Specifically, Trp shows a peak close to 290 nm; Tyr shows a peak between 275 and 282 nm; Phe shows sharper bands with fine structure between 255 and 270 nm; Disulphide bonds usually show weak broad band centered around 260 nm (Kelly et al., 2005). The conjugation of OVA with EGCG caused the highest loss of mean residue ellipicity of these peaks, followed by C and EGC. In general, the decreased magnitude of near-UV ellipicity was associated with a decrease in the tertiary conformation stability of proteins because these hydrophobic chromophores will shift to a more hydrophilic environment (Tang et al., 2011), which was in accordance with the intrinsic fluorescence study (Fig. 2a). On the other hand, the decrease in near-UV ellipicity would be also attributed to the increased flexibility in the quaternary conformation of proteins (Tang et al., 2011).

3.3. Antioxidant activity

The catechin content in conjugates was expressed as equivalent of each catechin by comparing the obtained data with the calibration curves. As shown in Table 1, the catechin content were 65.43, 42.77 and 60.01 CE μmol g -1 for powdered OVA-C, 22

OVA-EGC and OVA-EGCG, respectively. The amounts of bound polyphenols in this work were comparable to those in a previous study regarding the conjugates of lactoferrin with EGCG, chlorogenic aicd and gallic acid (Liu et al., 2015). The effect of catechin grafting on the antioxidant activity of OVA was systematically evaluated by a combination of DPPH, ABTS and ORAC assay. The DPPH• scavenging capacity was 14.65, 13.27, 39.43, 38.39 and 71.93 μg TE g-1 for OVA, control OVA, OVA-C, OVA-EGC and OVA-EGCG, respectively. This result suggested that the DPPH• scavenging capacity of OVA was appreciably improved via its conjugation with catechins due to the introduction of a large amount of phenolic hydroxyl groups. Besides, it was worth noting that that the catechin-grafting enhanced the tertiary conformation flexibility of OVA (Fig. 2a and b) and led to the exposure of buried residues, thereby improving its hydrogen-donating ability. On the contrary, the antioxidant activity of protein changed slightly by the radical copolymerization method in the absence of catechins. Similar results were also observed in ABTS and ORAC assay indicating that the catechin-grafting reaction was a potential method to improve the antioxidant activity of OVA. Data in Table 1 demonstrated that the bound amount of catechins decreased with the trend: OVA-C > OVA-EGCG > OVA-EGC. Nevertheless, OVA-EGCG exhibited the highest antioxidant capacity, followed by OVA-C and OVA-EGC. This paradox would have been because the higher antioxidant activity of EGCG than C (Supplementary Table 1).

3.4. Droplet characterization

23

The droplet size, PDI and ζ-potential of fish oil emulsions coated by OVA, OVA-C, OVA-EGC or OVA-EGCG were summarized in Table 2. All fresh emulsion adopted a droplet size below 400 nm with a PDI value below 0.3, suggesting that a 0.9 wt% emulsifier was suitable for the preparation of fish oil emulsions. It was worth noting that emulsions coated by OVA-catechin conjugates showed an appreciable smaller droplet size than those by OVA. The covalent modification of OVA with catechins resulted in the exposition of deeply buried hydrophobic residues onto protein surface, thereby improving its surface hydrophobicity (evidenced by H0 result) and surface activity. On the other hand, the results of intrinsic fluorescence (Fig. 2a), DSC (Fig. 2b) and near-UV CD analyses (Fig. 2d) revealed the decreased stability or improved flexibility in OVA tertiary structure by the catechin-grafting reaction. Both factors were favorable for the improvement of the emulsifying properties of protein and therefore the production of lipid droplets with smaller size (Tang et al., 2011). As reported, the emulsifying activity and stability of lactoferrin were significantly improved after its covalent conjugation with EGCG or chlorogenic acid (Liu et al., 2015). For all emulsions, the oil droplets were negatively charged at pH 7.0 while adopted a positive ζ-potential at pH 3.5 because the isoelectric point (pI) of OVA was previously determined to be about 4.5 (Feng et al., 2016). The ζ-potential of lipid droplet at pH 3.5 was similar for emulsions stabilized by OVA and OVA-catechin conjugates. Nevertheless, when at pH 7.0, the magnitude of surface charge of lipid droplets decreased with the trend: OVA-EGCG > OVA-EGC ≈ OVA-C > OVA. This 24

phenomenon might arise from the deprotonation of catechins because the pKa1 values were reported to be 8.97, 7.73 and 7.68 for C, EGC and EGCG, respectively (Muzolf, Szymusiak, Gliszczynska-Swiglo, Rietjens, & Tyrakowska, 2008). Another possible explanation would be the polyphenol-grafting reaction shifted the pI of protein to lower pH values (Rawel, Rohn, Kruse, & Kroll, 2002). The particle diameter and PDI of oil droplets coated by OVA increased appreciably (more than doubled) after 14 days of storage at either pH 3.5 or 7.0, which would be attributed to the droplet aggregation or coalescence because emulsions were thermodynamically unstable. By contrast, the diameter of droplets coated by conjugates was increased by only 3.6-35.9 nm and the PDI values were still below 0.3 at the end of storage test, indicating their better stability against droplet aggregation or coalescence. Catechins resided at the oil-water interface acted as cross-linkers between OVA molecules and enabled a more compact coating around the lipid droplets, thereby improving their storage stability by enhancing the steric repulsion between them (Liu, Wang, Xu, et al., 2016). At pH 7.0, the storage stability of conjugates-coated emulsions would also benefit from the higher magnitude of electrical charge on lipid droplets. For each case, the magnitude of ζ-potential of lipid droplets decreased slightly after 14 days, which was most likely initiated by the rearrangement of the emulsifiers on the surface of lipid droplet (Tamm, Herbst, Brodkorb, & Drusch, 2016).

3.5. Rheological properties

25

The rheological behavior is one of the most significant properties of food emulsions. Emulsion products with good textures, sensory and shelf life often require careful manipulation of emulsion rheology (Liu, Wang, Xu, et al., 2016). Fig. 3a and b showed the viscosity of fresh emulsions as a function of shear rate. It was found that all emulsions exhibited a remarkable shear-thinning behavior at tested range of shear rate at both pH 3.5 and 7.0. As the shear rate sufficiently to overcome the Brownian motion, the emulsion droplet became more ordered along the flow field and offered less resistance to the flow, thereby showing lower viscosity (Sun, Gunasekaran, & Richards, 2007). The flow curves were fitted with the Ostwald de Waale model: , where the ƞ is the apparent viscosity (Pa·s), γ is the shear rate (s -1), k is the consistence index (Pa·sn) and n is the flow behavior index. The corresponding parameters were summarized in the insets in Fig. 3a and b. The correlation coefficient R2 was above 0.96 for all emulsions suggesting that the Ostwald de Waale model fitted the flow curves adequately. All emulsions exhibited typical non-Newtonian behaviors as suggested by the low n values (0.4196-0.7603). In this work, emulsions coated by OVA adopted a higher viscosity with a more obvious pseudoplastic behavior compared with those stabilized by OVA-catechin conjugates, as evidenced by their higher k values but lower n values. This result would have been because that OVA-coated emulsions were more prone to flocculate, which was in agreement with their larger particle size (Table 2). Small-amplitude oscillatory shear tests were carried out to provide information 26

about the fluid-like and solid-like characteristics of fish oil emulsions. As depicted in Fig. 3c and d, both G' and G" were angular frequency (ω)-dependent and increased with ω. At low frequency, G' was lower than G" for all emulsions, indicating that liquid-like vicious behavior dominated the emulsions over the solid-like one. As the frequency increased, G' and G" approached each other and a cross-over was detected, beyond which the elastic contribution predominated. It could be found that the G' curves of all emulsions were almost coincided with each other. On the other hand, the G" for emulsions stabilized by OVA was higher than those by conjugate. In addition, the intersection point (where G' = G") typically moved to lower frequency values for conjugate-stabilized emulsions. These results revealed that conjugate-stabilized emulsions were less vicious (or more elastic) compared with those coated by OVA (Liu, Wang, Sun, et al., 2016), which was consistent with the steady-state flow tests (Fig. 3a and b). Similar results have been observed in medium-chain triglyceride oil emulsions coated by lactoferrin and lactoferrin-chlorogenic acid conjugate by Liu et al. (Liu, Wang, Xu, et al., 2016).

3.6. Lipid oxidation

Fish oil naturally contains high levels of ω-3 PUFAs, which renders it highly susceptible to lipid oxidation. Lipid hydroperoxide generated from the reaction between oxygen and unsaturated fatty acids are the primary products of this process while TBARS are the secondary lipid oxidation products formed by the degradation of lipid hydroperoxide (Su et al., 2015). As depicted in Fig. 4a and b, the content of 27

lipid hydroperoxide in fresh OVA-stabilized emulsion was 4.43 mmol CHPE/kg oil, and increased gradually with time to reach 37.44 and 31.23 mmol CHPE/kg oil after 14 days of storage at pH 7.0 and 3.5, respectively. By contrast, significantly less amount of lipid hydroperoxide was formed in emulsions stabilized by OVA-catechin conjugates, suggesting that they were oxidized more slowly. Similar result was also observed in terms of secondary oxidation products TBARS (Fig. 4c and d). For instance, the TBARS content in OVA-coated emulsion was about 0.75 mmol MDAE/kg oil at the end of the storage test at pH 7.0, but never exceed 0.50 mmol MDAE/kg oil for emulsions stabilized by OVA-catechin conjugates. The droplet-water interfacial is the contact region between the dispersed lipids and aqueous phase where the lipid oxidation is supposed to be most prevalent (Laguerre et al., 2009). Emulsions stabilized by conjugates would be beneficial because catechins bound to OVA could scavenge the free radicals and/or inactivate the prooxidants such as transition metals at lipid-water interfacial (Waraho et al., 2011), thereby blocking the decomposition of lipid hydroperoxide (LOOH) into alkoxyl (LO •) and peroxyl (LOO• •) radicals. In addition, the activity of these highly reactive free radicals abstracting a hydrogen from unsaturated fatty acid (LH) to form new radicals was inhibited, which in turn retarded the lipid oxidation (Chityala et al., 2016). The content of lipid hydroperoxide and TBARS in emulsions during storage decreased in the order of emulsifiers: OVA > OVA-EGC > OVA-C > OVA-EGCG, suggesting that there was a good correlation between the inhibition of lipid oxidation and the antioxidant capacity of emulsifiers (Table 1). On the other hand, data in 28

Supplementary Fig. 3 indicated that fish oil emulsions stabilized by OVA-conjugates exhibited significantly higher interfacial protein fraction (Fip) while lower continuous phase protein fraction (Fcp) than that stabilized by OVA. This could result from the reduced interfacial tension of proteins after the catechin-grafting reaction, which made the conjugates bind more strongly to the lipid-water interfacial once adsorbed (Wei et al., 2015). Therefore, the lipid droplet of emulsions stabilized by conjugates might adopt a more compact and defectless interfacial film, which could efficiently block the penetration and diffusion of oxidation initiators to react with ω-3 PUFAs. In this work, the lipid oxidation was pH-dependent, being more pronounced at pH 7.0 than at pH 3.5 for all emulsions. We presumed that the lipid oxidized faster at pH 7.0 because of the electrostatic attraction of ionic oil droplet (Table 2) with cationic transition metals (Waraho et al., 2011). By contrast, when at pH 3.5, positively charged fish oil droplet tended to repel irons from the droplet surface, thereby retarding lipid oxidation (Hu, McClements, & Decker, 2004). Besides, catechins especially EGC and EGCG readily undergo oxidative degradation under neutral or alkaline conditions, which would compromise their antioxidant activity.

4. Conclusions

In summary, OVA-C, OVA-EGC and OVA-EGCG conjugates were synthesized using a radical copolymerization method. OVA-catechin conjugates contained less α-helix but more β-sheet secondary structure than OVA. Catechin-grafting reaction led to improved surface hydrophobicity and antioxidant activity but decreased tertiary 29

conformation stability of OVA. Fish oil emulsions coated by OVA-catechin conjugates displayed smaller droplet size, less viscosity and better storage and oxidative stability compared with the OVA emulsified counterparts. Our study indicated

the

potential

applications

of

protein-polyphenol

conjugates

in

emulsion-based delivery systems for healthy lipids.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the National Key Research and Development Program (2016YFD0400805), Qinghai Science and Technology Program (2016-NK-C22, 2015-NK-502), Foundation of Fuli Institute of Food Science, Zhejiang University, National Natural Science Foundation of China (No. 31101226), Young Scientist Interdisciplinary Research Foundation of Zhejiang University and National Postdoctoral Program for Innovative Talents (BX201700101).

References

Alizadeh-Pasdar, N., & Li-Chan, E. C. Y. (2000). Comparison of protein surface hydrophobicity measured at various pH values using three different fluorescent probes. J. Agric. Food Chem., 48(2), 328-334. 30

Beauchemin, R., N'Soukpoe-Kossi, C. N., Thomas, T. J., Thomas, T., Carpentier, R., & Tajmir-Riahi, H. A. (2007). Polyamine analogues bind human serum albumin. Biomacromolecules, 8(10), 3177-3183. Bradford, M. M. (1976). Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal. Biochem., 72(1-2), 248-254. Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci. Technol., 28(1), 25-30. Byler, D. M., & Susi, H. (1986). Examination of the secondary structure of proteins by deconvolved FTIR spectra. Biopolymers, 25(3), 469-487. Chityala, P. K., Khouryieh, H., Williams, K., & Conte, E. (2016). Effect of xanthan/enzyme-modified guar gum mixtures on the stability of whey protein isolate stabilized fish oil-in-water emulsions. Food Chem., 212, 332-340. Feng, J., Wu, S., Wang, H., & Liu, S. (2017). Gliadin nanoparticles stabilized by a combination of thermally denatured ovalbumin with gemini dodecyl O-glucoside: The modulating effect of cosurfactant. Colloids Surf. A: Physicochem. Eng. Asp., 516, 94-105. Feng, J., Wu, S. S., Wang, H., & Liu, S. B. (2016). Improved bioavailability of curcumin in ovalbumin-dextran nanogels prepared by Maillard reaction. J. Funct. Foods, 27, 55-68. Hu, M., McClements, D. J., & Decker, E. A. (2004). Impact of chelators on the oxidative stability of whey protein isolate-stabilized oil-in-water emulsions 31

containing omega-3 fatty acids. Food Chem., 88(1), 57-62. Kelly, S. M., Jess, T. J., & Price, N. C. (2005). How to study proteins by circular dichroism. BBA-Proteins Proteom., 1751(2), 119-139. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685. Laguerre, M., Giraldo, L. J. L., Lecomte, J., Figueroa-Espinoza, M.-C., Barea, B., Weiss, J., Decker, E. A., & Villeneuve, P. (2009). Chain length affects antioxidant properties of chlorogenate esters in emulsion: the cutoff theory behind the polar paradox. J. Agric. Food Chem., 57(23), 11335-11342. Liu, F., Ma, C., Gao, Y., & McClements, D. J. (2017). Food-Grade Covalent Complexes and Their Application as Nutraceutical Delivery Systems: A Review. Compr. Rev. Food Sci. F., 16(1), 76-95. Liu, F. G., Sun, C. X., Yang, W., Yuan, F., & Gao, Y. X. (2015). Structural characterization and functional evaluation of lactoferrin-polyphenol conjugates formed by free-radical graft copolymerization. RSC Adv., 5(20), 15641-15651. Liu, F. G., Wang, D., Sun, C. X., & Gao, Y. X. (2016). Influence of polysaccharides on the physicochemical properties of lactoferrin-polyphenol conjugates coated beta-carotene emulsions. Food Hydrocolloid., 52, 661-669. Liu, F. G., Wang, D., Xu, H. G., Sun, C. X., & Gao, Y. X. (2016). Physicochemical properties

of

beta-carotene

emulsions

stabilized

by

chlorogenic

acid-lactoferrin-glucose/polydextrose conjugates. Food Chem., 196, 338-346. Mao, Y. Y., Dubot, M., Xiao, H., & McClements, D. J. (2013). Interfacial engineering 32

using mixed protein systems: emulsion-based delivery systems for encapsulation and stabilization of beta-carotene. J. Agric. Food Chem., 61(21), 5163-5169. Miller, N. J., Riceevans, C., Davies, M. J., Gopinathan, V., & Milner, A. (1993). A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin. Sci., 84(4), 407-412. Muzolf, M., Szymusiak, H., Gliszczynska-Swiglo, A., Rietjens, I., & Tyrakowska, B. E. (2008). pH-dependent radical scavenging capacity of green tea catechins. J. Agric. Food Chem., 56(3), 816-823. Naeem, A., Khan, T. A., Muzaffar, M., Ahmad, S., & Saleemuddin, M. (2011). A partially folded state of ovalbumin at low pH tends to aggregate. Cell Biochem. Biophys., 59(1), 29-38. Rawel, H. M., Rohn, S., Kruse, H. P., & Kroll, J. (2002). Structural changes induced in bovine serum albumin by covalent attachment of chlorogenic acid. Food Chem., 78(4), 443-455. Seeram, N. P., Henning, S. M., Niu, Y. T., Lee, R., Scheuller, H. S., & Heber, D. (2006). Catechin and caffeine content of green tea dietary supplements and correlation with antioxidant capacity. J. Agric. Food Chem., 54(5), 1599-1603. Slinkard, K., & Singleton, V. L. (1977). Total phenol analysis - automation and comparison with manual methods. Am. J. Enol. Vitic., 28(1), 49-55. Spizzirri, U. G., Iemma, F., Puoci, F., Cirillo, G., Curcio, M., Parisi, O. I., & Picci, N. (2009). Synthesis of antioxidant polymers by grafting of gallic acid and catechin on gelatin. Biomacromolecules, 10(7), 1923-1930. 33

Sponton, O. E., Perez, A. A., Carrara, C. R., & Santiago, L. G. (2015). Impact of environment conditions on physicochemical characteristics of ovalbumin heat-induced nanoparticles and on their ability to bind PUFAs. Food Hydrocolloid., 48, 165-173. Su, Y. R., Tsai, Y. C., Hsu, C. H., Chao, A. C., Lin, C. W., Tsai, M. L., & Mi, F. L. (2015). Effect of grape seed proanthocyanidin-gelatin colloidal complexes on stability and in vitro digestion of fish oil emulsions. J. Agric. Food Chem., 63(46), 10200-10208. Sun, C. H., Gunasekaran, S., & Richards, M. P. (2007). Effect of xanthan gum on physicochemical properties of whey protein isolate stabilized oil-in-water emulsions. Food Hydrocolloid., 21(4), 555-564. Tamm, F., Herbst, S., Brodkorb, A., & Drusch, S. (2016). Functional properties of pea protein hydrolysates in emulsions and spray-dried microcapsules. Food Hydrocolloid., 58, 204-214. Tang, C.-H., Sun, X., & Foegeding, E. A. (2011). Modulation of Physicochemical and Conformational Properties of Kidney Bean Vicilin (Phaseolin) by Glycation with Glucose: Implications for Structure-Function Relationships of Legume Vicilins. J. Agric. Food Chem., 59(18), 10114-10123. Tang, C. H., & Sun, X. (2011). A comparative study of physicochemical and conformational properties in three vicilins from phaseolus legumes: implications for the structure-function relationship. Food Hydrocolloid., 25(3), 315-324. von Staszewski, M., Ruiz-Henestrosa, V. M. P., & Pilosof, A. M. R. (2014). Green tea 34

polyphenols-beta-lactoglobulin

nanocomplexes:

interfacial

behavior,

emulsification and oxidation stability of fish oil. Food Hydrocolloid., 35, 505-511. Wan, Z.-L., Wang, J.-M., Wang, L.-Y., Yang, X.-Q., & Yuan, Y. (2013). Enhanced Physical and Oxidative Stabilities of Soy Protein-Based Emulsions by Incorporation of a Water-Soluble Stevioside-Resveratrol Complex. J. Agric. Food Chem., 61(18), 4433-4440. Waraho, T., McClements, D. J., & Decker, E. A. (2011). Mechanisms of lipid oxidation in food dispersions. Trends Food Sci. Tech., 22(1), 3-13. Wei, Z. H., Yang, W., Fan, R., Yuan, F., & Gao, Y. X. (2015). Evaluation of structural and functional properties of protein-EGCG complexes and their ability of stabilizing a model beta-carotene emulsion. Food Hydrocolloid., 45, 337-350. Whitmore, L., & Wallace, B. A. (2004). DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res., 32, W668-W673. Wong, Y. H., Kadir, H. A., & Tayyab, S. (2015). Targeting chemical and thermal stability of ovalbumin by simulated honey sugar cocktail. Int. J. Biol. Macromol., 73, 207-214. Yi, J., Fan, Y. T., Zhang, Y. Z., & Zhao, L. Q. (2016). Characterization of catechin-alpha-lactalbumin conjugates and the improvement in beta-carotene retention in an oil-in-water nanoemulsion. Food Chem., 205, 73-80. Yi, J., Zhang, Y. Z., Liang, R., Zhong, F., & Ma, J. G. (2015). Beta-carotene chemical stability

in

nanoemulsions

was 35

improved

by

stabilized

with

beta-lactoglobulin-catechin conjugates through free radical method. J. Agric. Food Chem., 63(1), 297-303. You, J., Luo, Y. K., & Wu, J. P. (2014). Conjugation of Ovotransferrin with Catechin Shows Improved Antioxidant Activity. J. Agric. Food Chem., 62(12), 2581-2587.

Fig. 1. SDS-PAGE (a) and MOLDI-TOF-MS (b) spectra of OVA, control OVA and OVA-catechin conjugates. Fig. 2. Steady-state fluorescence (a), DSC (b), far-UV (c) and near-UV (d) spectra of OVA and OVA-catechin conjugates. The related thermal parameters were summarized in the inset in b. The secondary structure contents analyzed by DichroWeb were summarized in the inset in c. Different lowercase letters in the same column represent a significant difference (P < 0.05). Fig. 3. The apparent viscosity (a, b) and dynamic storage modulus (G') and loss modulus (G") (c, d) of fish oil emulsions stabilized by OVA or OVA-conjugates (solid symbols for G' and open symbols for G"). Fig. 4. Formation of lipid oxidation markers (a, b: lipid hydroperoxide; c, d: TBARS) 36

in fish oil emulsions stabilized by OVA or OVA-conjugates during storage at 25 oC in dark. Table 1. Catechin content, antioxidant activity and surface hydrophobicity of OVA, control OVA and OVA-catechin conjugates. Table 2. Changes in droplet size, PDI and ζ-potential of fish oil emulsions stabilized by OVA or OVA-catechin conjugates when stored at 25 oC in dark.

37

Figure 1

38

Figure 2

39

Figure 3

40

Figure 4

41

Table 1

Catechin content

DPPH assay

ABTS assay

ORAC assay

-1

-1

-1

Sample

H0 -1

(μmol CE g )

(μmol TE g )

(μmol TE g )

(μmol TE g )

OVA

-

14.65 ± 0.12a

20.17 ± 0.90a

4.43 ± 0.76a

103.42

Control OVA

-

13.27 ± 1.23a

24.22 ± 2.19a

3.96 ± 0.30a

111.21

OVA-C

65.43 ± 1.23c

39.43 ± 1.87b

70.34 ± 1.54c

30.12 ± 1.10c

272.32

OVA-EGC

42.77 ± 0.52a

38.39 ± 2.76b

55.98 ± 2.41b

22.87 ± 0.87b

214.51

OVA-EGCG

60.01 ± 1.02b

71.93 ± 2.12c

108.23 ± 4.77d

56.33 ± 2.19d

268.17

Different lowercase letters in the same column indicate a significant difference (P < 0.05).

42

Table 2

0 day

Emulsifiers

14 days ζ-potential

pH DZ (nm)

PDI

ζ-potential PDI

DZ (nm) (mV)

7.0 386.7 ± 11.2f 0.178 ± 0.043

(mV)

-23.3 ± 0.9

821.2 ± 23.1d 0.376 ± 0.171 -20.2 ± 1.6

OVA 3.5

366.5 ± 5.7e

0.156 ± 0.012

18.2 ± 3.8

778.1 ± 11.3c 0.321 ± 0.063 17.5 ± 1.1

7.0

234.7 ± 5.4a

0.217 ± 0.016

-32.2 ± 1.3

249.3 ± 7.4a

0.211 ± 0.045 -29.4 ± 2.7

3.5

243.3 ± 3.1b

0.189 ± 0.023

20.1 ± 3.3

255.5 ± 7.9a

0.167 ± 0.021 19.7 ± 0.3

7.0

266.1 ± 5.4d

0.125 ± 0.011

-31.2 ± 2.1

283.2 ± 17.3b 0.105 ± 0.009 -30.7 ± 2.1

3.5

241.2 ± 4.3b

0.233 ± 0.017

19.4 ± 1.2

277.1 ± 12.6b 0.187 ± 0.015 18.8 ± 2.9

7.0

253.5 ± 4.2c

0.212 ± 0.028

-40.2 ± 2.1

257.1 ± 12.8a 0.217 ± 0.059 -33.3 ± 4.4

3.5

261.7 ± 2.9d

0.211 ± 0.051

18.4 ± 2.3

282.4 ± 14.8b 0.189 ± 0.030 16.9 ± 0.8

OVA-C

OVA-EGC

OVA-EGCG

Different lowercase letters in the same column indicate a significant difference (P < 0.05).

43

Highlights  Structural and oxidative activity modulation of ovalbumin by catechin grafting  Improved performance of fish oil emulsion derived from grafted ovalbumin  Enhanced oxidative stability of fish oil by ovalbum-catechin conjugates

44