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Effect of ellagic acid incorporation on the oxidative stability of sodium caseinate-polysaccharide-stabilized emulsions
Accepted Manuscript Effect of ellagic acid incorporation on the oxidative stability of sodium caseinatepolysaccharide-stabilized emulsions Hyunnho Cho, Ki Seon Yu, Keum Taek Hwang PII:
S0023-6438(17)30433-4
DOI:
10.1016/j.lwt.2017.06.029
Reference:
YFSTL 6324
To appear in:
LWT - Food Science and Technology
Received Date: 8 December 2016 Revised Date:
11 June 2017
Accepted Date: 12 June 2017
Please cite this article as: Cho, H., Yu, K.S., Hwang, K.T., Effect of ellagic acid incorporation on the oxidative stability of sodium caseinate-polysaccharide-stabilized emulsions, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.06.029. 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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Effect of ellagic acid incorporation on the oxidative stability of sodium caseinate-
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polysaccharide-stabilized emulsions
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Hyunnho Cho, Ki Seon Yu, Keum Taek Hwang *
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Department of Food and Nutrition, and Research Institute of Human Ecology, Seoul National
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University, Seoul, 08826, Korea
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*Corresponding author: K. T. Hwang
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Department of Food and Nutrition (Bldg. 222, Rm 508)
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College of Human Ecology
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Seoul National University
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Seoul 08826 Korea
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TEL: +82 2 880 2531; FAX: +82 2 884 0305; EMAIL: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Although ellagic acid (EA) has been known to have various functional activities including
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antioxidant and anticancer, application of EA is limited due to its low solubility. In this study,
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EA was incorporated into sodium caseinate (NaCas) using pH cycle method, a method
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involving higher solubility of EA and dissociation of NaCas in alkaline media. Fluorescence
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spectra showed involvement of hydrophobic interaction. FT-IR showed incorporation of EA
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into NaCas. EA-incorporated NaCas was used as an emulsifier to evaluate effect of EA on the
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oxidative stability of emulsion. High-methoxyl pectin (HMP) or carboxymethyl cellulose
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(CMC) was added to increase stability of emulsion at acidic pH. A stable emulsion was
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formed when the ratio of NaCas to the polysaccharide was 1:1 at pH 4. EA did not affect
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creaming index of the emulsion. However, formation of lipid hydroperoxides in the NaCas-
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HMP and NaCas-CMC-stabilized emulsions with EA was reduced by 22.5% and 24.0%,
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respectively. Volatile lipid oxidation products were also produced less in the emulsions with
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EA than without it. These results suggest that the pH cycle method may be used to
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incorporate EA into NaCas, which could be used as an emulsifier to increase oxidative
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stability of emulsion.
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Keywords: Sodium caseinate; Lipid oxidation; Oil in water emulsion; Protein-polysaccharide
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interaction; Antioxidant
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1. Introduction Sodium caseinate (NaCas) has been used to encapsulate bioactive compounds (Luo, Pan, & Zhong, 2015; Pan, Luo, Gan, Baek, & Zhong, 2014). NaCas is known to dissociate in alkaline conditions
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(Vaia, Smiddy, Kelly, & Huppertz, 2006). Certain polyphenols including rutin and curcumin are
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insoluble in aqueous media, but readily soluble in alkaline media due to their deprotonation (Leung,
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Colangelo, & Kee, 2008; Luo et al., 2015). Dissociation of NaCas and higher solubility of certain
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polyphenols in alkaline condition could be applied to incorporate polyphenols into NaCas, and a pH
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cycle method (Pan & Zhong, 2016) was used to incorporate rutin and curcumin into NaCas without
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using organic solvents (Luo et al., 2015; Pan et al., 2014).
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Ellagic acid (EA) is a phenolic compound commonly found in berries and nuts (Daniel et al., 1989)
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with antioxidant and anticancer activities (Bala, Bhardwaj, Hariharn, & Kumar, 2006). EA has low
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water solubility of about 9.7 µg/mL, but due to its acidic nature, it is more soluble in a basic solvent
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(Bala et al., 2006). Also, electrostatic complexes between NaCas and an anionic polysaccharide, high-
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methoxyl pectin (HMP) or carboxymethyl cellulose (CMC), could increase stability of NaCas against
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pH change and ionic strength to expand the application of NaCas in food matrixes (Cho, Jung, Lee,
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Kwak, & Hwang, 2016). Thus it was hypothesized that EA could be incorporated into NaCas using a
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pH cycle method and EA-incorporated NaCas could be used to form electrostatic complexes with
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HMP or CMC. However, incorporation of EA using a pH cycle method and formation of an
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electrostatic complex using pH-cycled NaCas have not been tested.
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Many health benefits are reported for long chain n-3 polyunsaturated fatty acids (PUFA) including
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eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are abundant in fish oil
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(Abeywardena & Head, 2001). However, PUFA are prone to oxidation during processing and storage,
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which results in rancidity and loss of nutritional values (Sun, Wang, Chen, & Li, 2011). In food
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system, lipid usually exists as an emulsion. However, due to increased surface area, oils in emulsion
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are more susceptible to oxidation (Waraho, McClements, & Decker, 2011). Thus antioxidants are
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commonly added to emulsions to prevent oxidation. Natural antioxidants such as resveratrol, rutin and
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green tea extract have been used to increase oxidative stability of emulsion (Cui, Kong, Chen, Zhang, 3
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2014). However, these studies were conducted at pH 7, thus study involving pH close to that of
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general food systems is needed. Also, efficacy of EA-incorporated NaCas on oxidative stability of an
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emulsion has not been tested. NaCas has been used as a food emulsifier and encapsulant. Thus EA-
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incorporated NaCas could be used as an emulsifier and incorporated EA will accumulate on oil-water
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interface with NaCas and improve oxidative stability of oil. Moreover, it was reported that
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antioxidants in O/W emulsions would be more effective if they are accumulated on oil-water interface
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(Lucas et al., 2010). Finally, HMP or CMC could be added to increase the stability in a low pH range
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(Liu et al., 2012; Surh, Decker, & McClements, 2006).
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In this study, EA was incorporated into NaCas using a pH cycle method, and effect of EA
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incorporation on the oxidative stability of menhaden oil emulsions stabilized by NaCas-
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polysaccharide was studied.
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2. Materials and methods
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2.1 Materials
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Food grade NaCas with 91 g protein/100 g (wet basis) and 5.6 g water/100 g was a kind gift from Samik Dairy & Food Co. Ltd (Seoul, Korea). Food grade HMP (degree of esterification: 71%) and
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sodium benzoate were from Esfood Co. Ltd. (Pocheon, Korea). CMC (degree of substitution: 1.0-1.2)
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was from Ashland Chemical Co. (Changzhou, Jiangsu, China). HCl, NaOH, ethanol (EtOH), hexane,
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isooctane, 2-propanol, methanol, 1-butanol, ammonium thiocyanate, BaCl2 and FeSO4 were
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purchased from Samchun Chemicals (Seoul, Korea). EA, 2-pentenal, 2-hexenal, 2,4-heptadienal, fish
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oil from menhaden, 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt
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(ABTS), potassium persulfate, 14 mL/100 mL of BF3 in methanol and cumene hydroperoxide were
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from Sigma Chemical Co. (St. Louis, MO, USA). A standard fatty acid methyl ester (FAME) mixture
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was from Supelco (Bellefonte, PA, USA).
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2.2 Fatty acid composition analysis 4
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Fish oil was transesterified using a method from Park and Goins (1994) (AOCS 1980). Fish oil (5
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µL), methylene chloride (100 µL) and 0.5 mol/L NaOH in methanol (1 mL) were placed in a test tube,
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followed by flushing with nitrogen gas and heating at 90 °C for 10 min. The tube was cooled to room
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temperature, mixed with 14 mL/100 mL of BF3 in methanol (1 mL), flushed with nitrogen gas and heated at 90 °C for 10 min. After cooling, distilled water (DW; 1 mL) and hexane (2 mL) were added
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and vigorously mixed. Top hexane layer with FAME was taken and analyzed by gas chromatography
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(GC; Agilent 6890, Agilent Technologies, Paolo Alto, CA, USA) using a modified method from Lee
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et al. (2010). A DB-23 column (30 m × 0.25 mm × 0.25 µm; J & W Scientific, Folsom, CA, USA)
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was used and the oven was programmed from 50 °C to 160 °C at 25 °C/ min, to 220 °C at 4 °C/min,
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held for 8 min, and to 250 °C at 25 °C/min, held for 5 min. Temperatures of injector and flame
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ionization detector were 200 °C and 250 °C, respectively. Carrier gas was helium and split ratio was
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30:1 (mL:mL). Identification and quantification of peaks were done by comparing reference retention
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times and peak areas as weight percent of the standard FAME mixture. Fatty acid composition of
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menhaden oil is shown in Table 1, and is in accordance with the previously reported data (Stansby,
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1981).
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2.3. Emulsion preparation and stability measurement To find optimum protein to polysaccharide ratio and pH, various concentrations of polysaccharide
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and pH were tested. Emulsions were formed from the method of Surh et al. (2006) with some
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modification. In brief, NaCas and polysaccharides were separately dissolved in DW to have final
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concentration of 1.11 g/100 g and 1 g/100 g, respectively. The solutions were stirred for 2 h and then
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incubated at 4 °C overnight for full hydration. pH was adjusted to 7 using NaOH and HCl. Ten g fish
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oil was added to 90 g NaCas solution, homogenizing at 10,000 rpm for 2 min to form a crude
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emulsion (Matsushita Electric Co. Ltd; Osaka, Japan). Then the crude emulsion was homogenized at
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50 MPa using a nanodispenser (ISA-NLM100; Ilshin autoclave Co., Ltd, Daejoen, Korea) 2 times.
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Sodium benzoate (0.04 g/100 g) was added as an antimicrobial agent, and the emulsion was mixed
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with DW, HMP or CMC, and then pH was adjusted from 7 to 3. Aliquot (5 mL) was taken at each pH
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and stored in a glass test tube (internal diameter 10 mm, height 100 mm) overnight at room
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temperature before stability measurement. Stability of emulsion was characterized by taking digital
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images and measuring creaming index (CI) using following equation:
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CI = 100 × Hs/He
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where Hs is height of the serum layer and He is height of the emulsion. Also, emulsions were
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visualized using an optical microscope (S16C, MICro Scopes, Inc., St. Louis, MO, USA) magnifying
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100 times. The result showed that the emulsion with 0.5 g NaCas, 0.5 g polysaccharides and 5 g fish
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oil per 100 g at pH 4 was the most stable and this condition was used throughout the study.
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2.4. Incorporation of EA into NaCas
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EA was incorporated into NaCas using pH cycle method as described by Pan et al. (2014) with some modification. NaCas solution (1 g/100 g) was adjusted to pH 12 using 4 mol/L NaOH and
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stirred for 30 min. EA powder (0.01 g/100 g) was added and the mixture was stirred for 10 min.
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Previous paper reported that this condition resulted in the highest encapsulation efficiency (Pan et al.,
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2014). The mixture was adjusted back to pH 7, mixed with HMP or CMC solution and then adjusted
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to pH 4. The final concentrations of NaCas, polysaccharides and EA were 0.5 g/100 g, 0.5 g/100 g and
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0.005 g/100 g, respectively. To produce EA-incorporated emulsion, EA was incorporated into NaCas
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using the method mentioned above, but the initial concentrations of NaCas and EA were 1.11 g/100 g
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and 0.011 g/100 g, respectively, to have final concentrations of 0.5 g/100 g NaCas and 0.005 g/100 g
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EA.
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2.5. Binding of EA to NaCas
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2.5.1. Fluorescence spectroscopy
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To confirm the binding of EA to NaCas, intrinsic fluorescence of NaCas in the presence and
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absence of various concentrations of EA was measured (Pan et al., 2014). NaCas (0.01 g/100 g) was
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prepared in DW, adjusting to pH 12. Then the solutions were mixed with various concentrations of EA
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in EtOH (final concentration of EA was 0-1 µg/mL) and intrinsic fluorescence was measured using 6
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FluoroMate FS-2 (Scinco, Seoul, Korea). Excitation wavelength was 280 nm and emission spectrum
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was recorded from 300 to 500 nm.
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2.5.2. Fourier transform infrared (FT-IR) spectroscopy FT-IR spectra of electrostatic complexes, complex incorporated with EA, physical mixture of freeze
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dried electrostatic complexes and EA powder were measured by a Nicolet 6700 FT-IR spectrometer
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(Thermo Nicolet Corp., Madison, WI, USA). Wavenumber range was from 4000 to 650 cm-1 and
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numbers of scan and resolution were 32 and 8, respectively.
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2.6. Antioxidant activity
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Antioxidant activity of the complexes, complex incorporated with EA and native EA were measured using ABTS radical scavenging method (Brandwilliams, Cuvelier, & Berset, 1995). Briefly,
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ABTS (7 mmol/L) and potassium persulfate (2.45 mmol/L) stock solutions were prepared in DW,
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mixed at 1:1 ratio, and incubated overnight in the dark. The solution was diluted with DW at pH 4 to
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have absorbance of 0.70 ± 0.05 at 734 nm. ABTS solution (0.5 mL) was mixed with the samples (30
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µL) and incubated for 5 min in the dark. Absorbance was measured at 734 nm.
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2.7. Encapsulation efficiency (EE)
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EE of EA incorporated in the complexes or emulsions was measured using the method from Xu et al. (2014) with some modification. In brief, EA-incorporated complexes or emulsions were
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centrifuged at 2580 × g for 30 min at 4 °C. The supernatant was removed and the pellet was mixed
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with EtOH and sonicated for 10 min to extract pelleted free EA. For emulsion, the pellet was washed
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with DW before the addition of EtOH to completely remove remaining emulsion.
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Amount of EA was quantified using high-performance liquid chromatography (HPLC) according to
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the method of Daniel et al. (1989) and Espin et al. (2007). A Waters Alliance 2695 separations module
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equipped with a Waters 2996 variable-wavelength diode array detector (Waters, Milford, MA, USA)
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and an Xbridge C18 column (4 µm, 3.9 mm × 300 mm; Millipore Corp., Milford, MA, USA) was 7
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used. Mobile phases were 5 mL/100 mL formic acid (A) and acetonitrile (B) and the gradient started
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from 99 mL/100 mL A for 5 min, followed by a linear decrease from 99 mL/100 mL A to 40 mL/100
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mL A over 40 min. The flow rate was fixed to 1 mL/min, injection volume was 20 µL and EA was
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detected at 254 nm. The samples were filtered with 0.45 µm syringe filter before HPLC analysis.
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2.8. Measurement of lipid oxidation
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2.8.1. Lipid hydroperoxides
The emulsions were stored at room temperature under dark for 2 weeks and aliquots were
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withdrawn periodically to measure lipid hydroperoxides according to the method from Richards,
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Chaiyasit, McClements, and Decker (2002). Emulsion (0.3 mL) was taken, mixed with 1.5 mL
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isooctane/2-propanol (3:1; mL:mL) and vortexed 3 times for 10 s each. The mixture was centrifuged
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at 1274 × g for 1 min and 0.2 mL of clear upper layer was mixed with 2.8 mL methanol/1-butanol (2:1;
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mL:mL), 15 µL 3.94 mol/L thiocyanate solution and 15 µL 0.072 mol/L Fe2+ solution. After 20 min of
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incubation at room temperature under dark, absorbance was measured at 510 nm (Optizen 2020UV;
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Mecasys, Daejeon, Korea) and the amount of lipid hydroperoxides was calculated using a cumene
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hydroperoxide standard curve.
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2.8.2. Volatile lipid oxidation products
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Fresh emulsions (1 mL) were placed at 10 mL screw cap vials and sealed with screw caps with PTFE/silicone septa (Supleco, Bellefonte, PA, USA). The vials were stored at room temperature under
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dark for 2 weeks and headspace volatiles were measured using gas chromatograph mass spectrometer
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(GCMS; Shimadzu GP2010 Ultra, Shimadzu, Kyoto, Japan), equipped with a DB 5 capillary column
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(30.00 m × 0.25 mm × 0.25 µm, Agilent J & W Scientific, Folsom, CA, USA) and
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divinylbenzene/darboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 µm solid microextraction
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(SPME) fibre (Supleco, Bellefonte, PA, USA). A SPME fibre was injected into vial and exposed to the
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headspace for 10 min at 55 °C. The fibre was desorbed at 250 °C for 10 min in the GC injector using
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splitless mode. Oven temperature was held at 40 °C for 2 min and then elevated from 40 °C to 160 °C
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at 6 °C/min and from 160 °C to 280 °C at 10 °C/min. Carrier gas was helium (25.0 cm/s) and injector
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temperature was 250 °C. Volatile compounds were identified by mass detector at 70 eV and 220 °C
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ionization gauge controller.
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2.9. Statistical analysis
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Statistical analysis was conducted using ANOVA (SPSS 22 software, SPSS Inc., Chicago, IL, USA) with Duncan’s multiple-range test (p < 0.05) to determine significance among the samples.
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3. Results and discussion
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3.1. Effect of polysaccharide concentration and pH on the stability of emulsion
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In this study, the concentration of NaCas was fixed to 1 g/100 g when producing crude emulsion as
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this concentration was reported to be sufficient to cover the oil (10 g/100 g) during emulsification (Liu
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et al., 2012). CI and digital images of the emulsions with various concentrations of polysaccharides
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and pH are shown in Fig. 1 and 2.
NaCas-stabilized emulsions were stable at pH 7, but phase separation occurred at pH 3, 4 and 5
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since the pH is near the isoelectric point of NaCas. After 2 weeks, severe aggregation occurred in all
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the emulsions except at pH 7. The emulsions with 0.25 g/100 g polysaccharides were not stable to
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aggregation and phase separation, possibly due to bridging flocculation (Liu et al., 2012). The
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emulsions with 0.5 g/100 g polysaccharides were the most stable to aggregation and phase separation
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at pH 4. In the proceeding experiments, emulsions with 0.5 g/100 g NaCas, 0.5 g/100 g
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polysaccharides and 5 g/100 g fish oil at pH 4 were used. From the microscopic images, particle size
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of the emulsions was about 2-4 µm and was not analyzed further as the objective of this study was to
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examine oxidative stability of the emulsion rather than its physical characterization.
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3.2. Incorporation of EA into NaCas
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3.2.1. Fluorescence spectra of EA-incorporated NaCas
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In order to determine if NaCas and EA interact via hydrophobic interaction, intrinsic fluorescence 9
ACCEPTED MANUSCRIPT spectra of NaCas with and without various concentrations of EA were measured (Fig. 3). NaCas has
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maximum intensity at 336 nm when excited at 280 nm and the presence of a quencher resulted in
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lower intensity (Cogan, Kopelman, Mokady, & Shinitzky, 1976). Fluorescence intensity of NaCas
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decreased as the concentration of EA increased, indicating that hydrophobic interaction occurred
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between NaCas and EA. Decrease in fluorescence intensity could be due to dynamic quenching, the
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collision of a quencher and a fluorophore or due to static quenching, complex formation between a
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quencher and fluorophore (Mohammadi, Bordbar, Divsalar, Mohammadi, & Saboury, 2009). To
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determine whether the quenching is dynamic or static, Stern-Volmer equation was used:
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F0/F = 1 + kq τ0 [Q] = 1 + KSV[Q]
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where F0 and F are fluorescence intensities in the absence and presence of EA, respectively. KSV, [Q],
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and kq are Stern-Volmer quenching constant, concentration of EA and the fluorescence quenching
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constant, respectively. τ0 is lifetime of fluorophore fluorescence in the absence of quencher (10-8s;
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Lakowicz & Weber, 1980). kq value was 7.85 × 1011 (mol/L)-1s-1, which is higher than the maximum
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dynamic quenching constant of 2 x 1010 (mol/L)-1s-1 (Lange, Kothari, Patel, & patel, 1998). This result
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indicates that the quenching interaction between NaCas and EA was static and NaCas formed
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complex with EA.
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FT-IR spectra of EA, EA-incorporated complexes, complexes without EA and physical mixture are shown in Fig. 4. The spectrum of EA showed similar characteristic bands as described in previous
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papers (Bulani et al., 2016; Gopalakrishnan et al., 2014). Most of the characteristic peaks of EA were
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not visible in physical mixture of EA and the complex as the concentration of EA was much lower
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than that of the complex. However, some of the characteristic peaks of EA were still visible in the
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physical mixture including a sharp peak around 3471 cm-1 from OH stretching (Bulani et al., 2016).
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Although physical mixture and EA-incorporated complexes have the same concentrations of EA and
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the complex, characteristic peaks of EA were not visible in the EA-incorporated complexes and the
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spectra of the EA-incorporated complexes were almost identical to that of empty complex. These
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results, along with the fluorescence spectra, indicate that EA could be incorporated into the complex.
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3.3. Antioxidant activity of EA and EA-incorporated complexes Antioxidant activity of EA and the electrostatic complexes with and without EA was evaluated using ABTS radical scavenging assay (Table 2). The complexes showed antioxidant activities as
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NaCas, HMP and CMC have antioxidant activities (Kitts, 2005; Moseley, Walker, Waddington, &
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Chen, 2003; Ro et al., 2013). EA incorporated into the complexes and native EA showed similar
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antioxidant activity. EA-incorporated NaCas-HMP showed the highest antioxidant activity (p < 0.05).
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Higher antioxidant activity of the EA-incorporated NaCas-HMP compared to the EA-incorporated
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NaCas-CMC could be due to the higher antioxidant activity of NaCas-HMP compared to NaCas-
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CMC.
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3.4. EE of the complex and emulsion
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EE of EA in the complex and emulsion are shown in Table 3. There was no significant difference
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between NaCas-HMP and NaCas-CMC. However, EE of the complexes was significantly higher than
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that of the emulsions (p < 0.05). One of the possible reasons for the lower EE of the emulsions is loss
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of EA during preparation of emulsion. Also, the high pressure homogenization process during
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emulsion formation or presence of oil could affect structure of NaCas, consequently binding EA to the
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protein.
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3.5. Effect of EA incorporation on the stability of emulsion
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3.5.1. Effect of EA incorporation on the physical stability of emulsion
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To evaluate effect of pH cycle treatment on the stability of emulsion, CI of the emulsions with
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native NaCas, pH-cycled NaCas and EA-incorporated NaCas were measured (Table 4). CI of the
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emulsions prepared using NaCas-HMP was significantly lower than that of NaCas-CMC (p < 0.05),
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which is in accordance with Fig. 2A. However, CI of the emulsions prepared with pH-cycled NaCas
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or EA-incorporated NaCas were not significantly different from those of the emulsions with native 11
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NaCas (p > 0.05). Chen, Zhang, and Tang (2016) also reported that complexation of curcumin to soy
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protein isolate did not affect CI of the emulsions.
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3.5.2. Effect of EA incorporation on the oxidative stability of emulsion Incorporation of EA reduced lipid hydroperoxides in the emulsions compared to the emulsions
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without EA (Fig. 5). After 14 days of storage, lipid hydroperoxides of NaCas-HMP and NaCas-CMC-
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stabilized emulsions with EA were 22.5% and 24.0%, respectively, lower compared to the pH-cycled
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controls. pH-cycle treatment did not affect formation of lipid hydroperoxides.
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Volatile lipid oxidation products in the emulsions stored for 14 days were analyzed by SPME
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(Table 5). Among lipid oxidation products from fish oil or oil rich in n-3 fatty acids, 2-pentenal, 2-
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hexenal and 2,4-heptadienal were identified in this study (Frankel, 1993; Jimenez-Alvarez et al.,
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2008). In accordance with the result of lipid hydroperoxides, there was no significant difference
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between the emulsions with native NaCas and pH-cycled NaCas (p > 0.05) except for 2-hexenal in the
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NaCas-CMC-stabilized emulsion (p < 0.05). For the NaCas-HMP-stabilized emulsion, incorporation
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of EA reduced formation of 2-pentenal, 2-hexenal and 2,4-heptadienal by 70.1%, 39.9% and 34.5%,
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respectively. For the NaCas-CMC-stabilized emulsion, their reductions were 80.9%, 58.3% and
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46.1%, respectively. There was no significant difference between the emulsions with NaCas-HMP and
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NaCas-CMC (p > 0.05). Other papers reported that incorporation of antioxidant resulted in reduced
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amount of hexanal (Cui et al., 2014, Wan et al., 2014). However, in this study, there was no difference
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in the concentration of hexanal in all the samples (data not shown). This may be due to difference in
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the fatty acid composition as previous papers used soybean or corn oil, while menhaden oil, rich in n-
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3 PUFA, was used in this study (Table 1). Jimenez-Alvarez et al. (2008) reported that 2-propenal, 2-
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hexenal and 2,4-heptadienal but not hexanal were formed during oxidation of EPA and DHA.
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4. Conclusion In this study, it was demonstrated that EA could be incorporated into NaCas using pH-cycle method. When applied to emulsions, EA increased oxidative stability of the emulsions without affecting CI. 12
ACCEPTED MANUSCRIPT The results of this study showed that incorporation of EA into NaCas could be a way to increase
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utilization of poorly water soluble EA as an emulsifier with antioxidant activity in food systems.
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However, further study on the effect of EA incorporation on the other properties of emulsion,
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including particle size, zeta potential and flocculation, is needed.
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References
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Abeywardena, M. Y., & Head, R. J. (2001). Longchain n-3 polyunsaturated fatty acids and blood
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fatty-acid composition in foods. Journal of Food Science, 59, 1262-1266. Richards, M. P., Chaiyasit, W., McClements, D. J., & Decker, E. A. (2002). Ability of surfactant
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micelles to alter the partitioning of phenolic antioxidants in oil-in-water emulsions. Journal of
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Sun, Y. E., Wang, W. D., Chen, H. W., & Li, C. (2011). Autoxidation of unsaturated lipids in food
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emulsion. Critical Reviews in Food Science and Nutrition, 51, 453-466.
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Surh, J., Decker, E. A., & McClements, D. J. (2006). Influence of pH and pectin type on properties
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and stability of sodium-caseinate stabilized oil-in-water emulsions. Food Hydrocolloids, 20,
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607-618.
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Vaia, B., Smiddy, M. A., Kelly, A. L., & Huppertz, T. (2006). Solvent-mediated disruption of bovine
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casein micelles at alkaline pH. Journal of Agricultural and Food Chemistry, 54, 8288-8293. von Staszewski, M., Ruiz-Henestrosa, V. M. P., & Pilosof, A. M. R. (2014). Green tea polyphenolsbeta-lactoglobulin nanocomplexes: Interfacial behavior, emulsification and oxidation stability
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of fish oil. Food Hydrocolloids, 35, 505-511.
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Wan, Z. L., Wang, J. M., Wang, L. Y., Yuan, Y., & Yang, X. Q. (2014). Complexation of resveratrol
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with soy protein and its improvement on oxidative stability of corn oil/water emulsions. Food
414 415 416 417
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Chemistry, 161, 324-331.
Waraho, T., McClements, D. J., & Decker, E. A. (2011). Mechanisms of lipid oxidation in food dispersions. Trends in Food Science & Technology, 22, 3-13. Xu, W., Jin, W. P., Zhang, C. L., Li, Z. S., Lin, L. F., Huang, Q.,… Li, B. (2014). Curcumin loaded
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and protective system based on complex of kappa-carrageenan and lysozyme. Food Research
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International, 59, 61-66. 16
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Fig. 1. Microscopic images of the emulsions prepared with NaCas (0.5 g/100 g NaCas and 5 g/100 g
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oil), NaCas-HMP (0.5 g/100 g NaCas, 0.25-0.5 g/100 g HMP and 5 g/100 g oil) and NaCas-CMC (0.5
423
g/100 g NaCas, 0.25-0.5 g/100 g CMC and 5 g/100 g oil) at pH 3-7 after stored for 14 days (n = 2).
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The scale bars represent a length of 100 µm.
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NaCas, sodium caseinate; HMP, high-methoxyl pectin; and CMC, carboxymethyl cellulose.
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Fig. 2. Effect of polysaccharide concentration and pH on creaming index (A) and visual appearance
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(B) of the emulsions prepared with NaCas (0.5 g/100 g NaCas and 5 g/100 g oil, ), NaCas-HMP (0.5
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g/100 g NaCas, 5 g/100 g oil and 0.25 g/100 g HMP,
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g/100 g NaCas, 5 g/100 g oil and 0.25 g/100 g CMC,
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stored for 14 days.
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Values are means ± standard deviations (n = 2).
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NaCas, sodium caseinate; HMP, high-methoxyl pectin; and CMC, carboxymethyl cellulose.
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or 0.5 g/100 g HMP, ×) and NaCas-CMC (0.5
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or 0.5 g/100 g CMC, ▲) at pH 3-7 after
Fig. 3. Fluorescence spectra of sodium caseinate (0.01 g/100 g) in the presence and absence of various
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concentrations (0-1µg/mL) of ellagic acid (EA). Excitation and emission wavelengths were 280 nm
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and from 300 to 450 nm, respectively.
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Fig. 4. FT-IR spectra of ellagic acid (EA), electrostatic complexes (0.5 g/100 g NaCas and 0.5 g/100 g
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polysaccharides), complex incorporated with EA (0.5 g/100 g NaCas, 0.5 g/100 g polysaccharides and
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0.005 g /100 g EA), physical mixture of freeze dried electrostatic complexes prepared with HMP (A)
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and CMC (B).
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NaCas, sodium caseinate; HMP, high-methoxyl pectin; CMC, carboxymethyl cellulose, and FT-IR,
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Fourier transform infrared.
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Fig. 5. Effect of ellagic acid (EA) on the formation of lipid hydroperoxides. Emulsions with and 17
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without EA were stored for 14 days under dark at room temperature and concentrations of lipid
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hydroperoxides were measured. NaCas-HMP (dashed line,
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EA_NaCas-HMP (dashed line, ▲); NaCas-CMC (solid line,
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EA_NaCas-CMC (solid line, ▲).
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Values are means ± standard deviations (n = 3).
452
Different letters represent significant difference (p < 0.05).
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NaCas, sodium caseinate; NaCasP, pH cycled NaCas; HMP, high-methoxyl pectin; and CMC,
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carboxymethyl cellulose.
); NaCasP-HMP (dashed line, );
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); NaCasP-CMC (solid line, ); and
18
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0.28 ± 0.07
14:0
8.40 ± 0.05
15:0
0.78 ± 0.01
16:0
18.2 ± 0.1
17:0
0.93 ± 0.01
18:0
3.6 ± 0.1
SFA
32.2 ± 0.3
16:1
11.01 ± 0.08
18:1n-9
5.67 ± 0.03
20:1
0.85 ± 0.25
MUFA
17.5 ± 0.2
18:2n-6
1.57 ± 0.02
18:3n-3
1.42 ± 0.01
20:4n-6
0.62 ± 0.58
20:5n-3
13.9 ± 0.1
22:6n-3
12.6 ± 0.1
PUFA
28.7 ± 0.5
Total identified
79.8 ± 0.2
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Menhaden fish oil
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Fatty acid composition of menhaden fish oil (g/100 g).
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Values are means ± standard deviations (n=3).
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SFA: saturated fatty acid; MUFA: mono unsaturated fatty acid; and PUFA: poly unsaturated fatty acid.
ACCEPTED MANUSCRIPT Table 2 ABTS radical scavenging rate of ellagic acid (EA) and electrostatic complexes (0.5 g/100 g NaCas and 0.5 g/100 g polysaccharides) with and without incorporated EA.
56 ± 2b
NaCas-HMP
36 ± 2d
NaCas-CMC
22.8 ± 0.3e
NaCasP-HMP
38 ± 2d
NaCasP-CMC
25 ± 4e
EA_NaCasP-HMP
61 ± 2a
EA_NaCasP-CMC
46 ± 1c
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ABTS radical scavenging rate (%)
Different letters represent significant difference (p < 0.05).
ABTS, 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt; NaCas, sodium caseinate; NaCasP, pH cycled NaCas; HMP, high-methoxyl pectin; and CMC, carboxymethyl
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cellulose.
ACCEPTED MANUSCRIPT Table 3 Encapsulation efficiency of ellagic acid (EA) incorporated in the complexes (0.5 g/100 g NaCas and 0.5 g/100 g polysaccharides) and the emulsions (0.5 g/100 g NaCas, 0.5 g/100 g polysaccharides and
Complex
Emulsion
NaCas-HMP
99.8 ± 0.0
a
68 ± 9b
NaCas-CMC
99.1 ± 0.6a
67 ± 2b
Values are means ± standard deviations (n = 3). Different letters represent significant difference (p < 0.05).
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Encapsulation efficiency (%)
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5 g/100 g oil).
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NaCas, sodium caseinate; HMP, high-methoxyl pectin; and CMC, carboxymethyl cellulose.
ACCEPTED MANUSCRIPT Table 4 Effect of pH cycle treatment and ellagic acid (EA) incorporation on creaming index of the emulsions (0.5 g/100 g NaCas, 0.5 g/100 g polysaccharides and 5 g/100 g oil) after 14 days of storage.
5.4 ± 0.8a
NaCasP-HMP
3.9 ± 0.9a
EA_NaCasP-HMP
4.57 ± 0.08a
NaCas-CMC
7.4 ± 0.3b
NaCasP-CMC
8 ± 2b
EA_NaCasP-CMC
8.1 ± 0.9b
Values are means ± standard deviations (n = 3).
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NaCas-HMP
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Creaming index (%)
NaCas, sodium caseinate; NaCasP, pH cycled NaCas; HMP, high-methoxyl pectin; and CMC,
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carboxymethyl cellulose.
ACCEPTED MANUSCRIPT Table 5 Effect of ellagic acid (EA) on the formation of volatile lipid oxidation products of the emulsions stored for 14 days (peak area, × 105). 2-Hexenal
2,4-Heptadienal
NaCas-HMP
723 ± 199a
519 ± 96ab
534 ± 75a
NaCasP-HMP
878 ± 9.6a
577 ± 18ab
EA_NaCasP-HMP
263 ± 30b
347 ± 28cd
NaCas-CMC
647 ± 192a
456 ± 36bc
NaCasP-CMC
931 ± 359a
607 ± 138a
606 ± 139a
EA_NaCasP-CMC
178 ± 93b
253 ± 23d
327 ± 34c
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2-Pentenal
580 ± 87a
380 ± 34bc
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481 ± 53ab
Values are means ± standard deviations (n = 3).
Different letters represent significant difference (p < 0.05).
NaCas, sodium caseinate; NaCasP, pH cycled NaCas; HMP, high-methoxyl pectin; and CMC,
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carboxymethyl cellulose.
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ACCEPTED MANUSCRIPT Highlights Emulsion was formed using sodium caseinate and polysaccharide. Ellagic acid was incorporated into sodium caseinate using pH cycle method. Incorporation of ellagic acid was confirmed by fluorescence spectra and FT-IR.
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Ellagic acid reduced formation of volatile lipid oxidation products.
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Incorporation of ellagic acid reduced formation of lipid hydroperoxides.
S0023-6438(17)30433-4
DOI:
10.1016/j.lwt.2017.06.029
Reference:
YFSTL 6324
To appear in:
LWT - Food Science and Technology
Received Date: 8 December 2016 Revised Date:
11 June 2017
Accepted Date: 12 June 2017
Please cite this article as: Cho, H., Yu, K.S., Hwang, K.T., Effect of ellagic acid incorporation on the oxidative stability of sodium caseinate-polysaccharide-stabilized emulsions, LWT - Food Science and Technology (2017), doi: 10.1016/j.lwt.2017.06.029. 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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Effect of ellagic acid incorporation on the oxidative stability of sodium caseinate-
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polysaccharide-stabilized emulsions
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Hyunnho Cho, Ki Seon Yu, Keum Taek Hwang *
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Department of Food and Nutrition, and Research Institute of Human Ecology, Seoul National
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University, Seoul, 08826, Korea
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*Corresponding author: K. T. Hwang
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Department of Food and Nutrition (Bldg. 222, Rm 508)
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College of Human Ecology
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Seoul National University
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Seoul 08826 Korea
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TEL: +82 2 880 2531; FAX: +82 2 884 0305; EMAIL: [email protected]
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ACCEPTED MANUSCRIPT Abstract
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Although ellagic acid (EA) has been known to have various functional activities including
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antioxidant and anticancer, application of EA is limited due to its low solubility. In this study,
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EA was incorporated into sodium caseinate (NaCas) using pH cycle method, a method
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involving higher solubility of EA and dissociation of NaCas in alkaline media. Fluorescence
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spectra showed involvement of hydrophobic interaction. FT-IR showed incorporation of EA
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into NaCas. EA-incorporated NaCas was used as an emulsifier to evaluate effect of EA on the
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oxidative stability of emulsion. High-methoxyl pectin (HMP) or carboxymethyl cellulose
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(CMC) was added to increase stability of emulsion at acidic pH. A stable emulsion was
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formed when the ratio of NaCas to the polysaccharide was 1:1 at pH 4. EA did not affect
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creaming index of the emulsion. However, formation of lipid hydroperoxides in the NaCas-
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HMP and NaCas-CMC-stabilized emulsions with EA was reduced by 22.5% and 24.0%,
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respectively. Volatile lipid oxidation products were also produced less in the emulsions with
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EA than without it. These results suggest that the pH cycle method may be used to
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incorporate EA into NaCas, which could be used as an emulsifier to increase oxidative
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stability of emulsion.
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Keywords: Sodium caseinate; Lipid oxidation; Oil in water emulsion; Protein-polysaccharide
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interaction; Antioxidant
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2
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1. Introduction Sodium caseinate (NaCas) has been used to encapsulate bioactive compounds (Luo, Pan, & Zhong, 2015; Pan, Luo, Gan, Baek, & Zhong, 2014). NaCas is known to dissociate in alkaline conditions
45
(Vaia, Smiddy, Kelly, & Huppertz, 2006). Certain polyphenols including rutin and curcumin are
46
insoluble in aqueous media, but readily soluble in alkaline media due to their deprotonation (Leung,
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Colangelo, & Kee, 2008; Luo et al., 2015). Dissociation of NaCas and higher solubility of certain
48
polyphenols in alkaline condition could be applied to incorporate polyphenols into NaCas, and a pH
49
cycle method (Pan & Zhong, 2016) was used to incorporate rutin and curcumin into NaCas without
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using organic solvents (Luo et al., 2015; Pan et al., 2014).
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Ellagic acid (EA) is a phenolic compound commonly found in berries and nuts (Daniel et al., 1989)
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with antioxidant and anticancer activities (Bala, Bhardwaj, Hariharn, & Kumar, 2006). EA has low
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water solubility of about 9.7 µg/mL, but due to its acidic nature, it is more soluble in a basic solvent
54
(Bala et al., 2006). Also, electrostatic complexes between NaCas and an anionic polysaccharide, high-
55
methoxyl pectin (HMP) or carboxymethyl cellulose (CMC), could increase stability of NaCas against
56
pH change and ionic strength to expand the application of NaCas in food matrixes (Cho, Jung, Lee,
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Kwak, & Hwang, 2016). Thus it was hypothesized that EA could be incorporated into NaCas using a
58
pH cycle method and EA-incorporated NaCas could be used to form electrostatic complexes with
59
HMP or CMC. However, incorporation of EA using a pH cycle method and formation of an
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electrostatic complex using pH-cycled NaCas have not been tested.
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Many health benefits are reported for long chain n-3 polyunsaturated fatty acids (PUFA) including
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eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are abundant in fish oil
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(Abeywardena & Head, 2001). However, PUFA are prone to oxidation during processing and storage,
64
which results in rancidity and loss of nutritional values (Sun, Wang, Chen, & Li, 2011). In food
65
system, lipid usually exists as an emulsion. However, due to increased surface area, oils in emulsion
66
are more susceptible to oxidation (Waraho, McClements, & Decker, 2011). Thus antioxidants are
67
commonly added to emulsions to prevent oxidation. Natural antioxidants such as resveratrol, rutin and
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green tea extract have been used to increase oxidative stability of emulsion (Cui, Kong, Chen, Zhang, 3
ACCEPTED MANUSCRIPT & Hua, 2014; Wan, Wang, Wang, Yuan, & Yang, 2014; von Staszewski, Ruiz-Henestrosa, & Pilosof,
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2014). However, these studies were conducted at pH 7, thus study involving pH close to that of
71
general food systems is needed. Also, efficacy of EA-incorporated NaCas on oxidative stability of an
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emulsion has not been tested. NaCas has been used as a food emulsifier and encapsulant. Thus EA-
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incorporated NaCas could be used as an emulsifier and incorporated EA will accumulate on oil-water
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interface with NaCas and improve oxidative stability of oil. Moreover, it was reported that
75
antioxidants in O/W emulsions would be more effective if they are accumulated on oil-water interface
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(Lucas et al., 2010). Finally, HMP or CMC could be added to increase the stability in a low pH range
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(Liu et al., 2012; Surh, Decker, & McClements, 2006).
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In this study, EA was incorporated into NaCas using a pH cycle method, and effect of EA
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incorporation on the oxidative stability of menhaden oil emulsions stabilized by NaCas-
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polysaccharide was studied.
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2. Materials and methods
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2.1 Materials
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Food grade NaCas with 91 g protein/100 g (wet basis) and 5.6 g water/100 g was a kind gift from Samik Dairy & Food Co. Ltd (Seoul, Korea). Food grade HMP (degree of esterification: 71%) and
86
sodium benzoate were from Esfood Co. Ltd. (Pocheon, Korea). CMC (degree of substitution: 1.0-1.2)
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was from Ashland Chemical Co. (Changzhou, Jiangsu, China). HCl, NaOH, ethanol (EtOH), hexane,
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isooctane, 2-propanol, methanol, 1-butanol, ammonium thiocyanate, BaCl2 and FeSO4 were
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purchased from Samchun Chemicals (Seoul, Korea). EA, 2-pentenal, 2-hexenal, 2,4-heptadienal, fish
90
oil from menhaden, 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt
91
(ABTS), potassium persulfate, 14 mL/100 mL of BF3 in methanol and cumene hydroperoxide were
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from Sigma Chemical Co. (St. Louis, MO, USA). A standard fatty acid methyl ester (FAME) mixture
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was from Supelco (Bellefonte, PA, USA).
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2.2 Fatty acid composition analysis 4
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Fish oil was transesterified using a method from Park and Goins (1994) (AOCS 1980). Fish oil (5
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µL), methylene chloride (100 µL) and 0.5 mol/L NaOH in methanol (1 mL) were placed in a test tube,
98
followed by flushing with nitrogen gas and heating at 90 °C for 10 min. The tube was cooled to room
99
temperature, mixed with 14 mL/100 mL of BF3 in methanol (1 mL), flushed with nitrogen gas and heated at 90 °C for 10 min. After cooling, distilled water (DW; 1 mL) and hexane (2 mL) were added
101
and vigorously mixed. Top hexane layer with FAME was taken and analyzed by gas chromatography
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(GC; Agilent 6890, Agilent Technologies, Paolo Alto, CA, USA) using a modified method from Lee
103
et al. (2010). A DB-23 column (30 m × 0.25 mm × 0.25 µm; J & W Scientific, Folsom, CA, USA)
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was used and the oven was programmed from 50 °C to 160 °C at 25 °C/ min, to 220 °C at 4 °C/min,
105
held for 8 min, and to 250 °C at 25 °C/min, held for 5 min. Temperatures of injector and flame
106
ionization detector were 200 °C and 250 °C, respectively. Carrier gas was helium and split ratio was
107
30:1 (mL:mL). Identification and quantification of peaks were done by comparing reference retention
108
times and peak areas as weight percent of the standard FAME mixture. Fatty acid composition of
109
menhaden oil is shown in Table 1, and is in accordance with the previously reported data (Stansby,
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1981).
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2.3. Emulsion preparation and stability measurement To find optimum protein to polysaccharide ratio and pH, various concentrations of polysaccharide
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and pH were tested. Emulsions were formed from the method of Surh et al. (2006) with some
115
modification. In brief, NaCas and polysaccharides were separately dissolved in DW to have final
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concentration of 1.11 g/100 g and 1 g/100 g, respectively. The solutions were stirred for 2 h and then
117
incubated at 4 °C overnight for full hydration. pH was adjusted to 7 using NaOH and HCl. Ten g fish
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oil was added to 90 g NaCas solution, homogenizing at 10,000 rpm for 2 min to form a crude
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emulsion (Matsushita Electric Co. Ltd; Osaka, Japan). Then the crude emulsion was homogenized at
120
50 MPa using a nanodispenser (ISA-NLM100; Ilshin autoclave Co., Ltd, Daejoen, Korea) 2 times.
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Sodium benzoate (0.04 g/100 g) was added as an antimicrobial agent, and the emulsion was mixed
122
with DW, HMP or CMC, and then pH was adjusted from 7 to 3. Aliquot (5 mL) was taken at each pH
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and stored in a glass test tube (internal diameter 10 mm, height 100 mm) overnight at room
124
temperature before stability measurement. Stability of emulsion was characterized by taking digital
125
images and measuring creaming index (CI) using following equation:
126
CI = 100 × Hs/He
127
where Hs is height of the serum layer and He is height of the emulsion. Also, emulsions were
128
visualized using an optical microscope (S16C, MICro Scopes, Inc., St. Louis, MO, USA) magnifying
129
100 times. The result showed that the emulsion with 0.5 g NaCas, 0.5 g polysaccharides and 5 g fish
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oil per 100 g at pH 4 was the most stable and this condition was used throughout the study.
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2.4. Incorporation of EA into NaCas
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EA was incorporated into NaCas using pH cycle method as described by Pan et al. (2014) with some modification. NaCas solution (1 g/100 g) was adjusted to pH 12 using 4 mol/L NaOH and
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stirred for 30 min. EA powder (0.01 g/100 g) was added and the mixture was stirred for 10 min.
136
Previous paper reported that this condition resulted in the highest encapsulation efficiency (Pan et al.,
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2014). The mixture was adjusted back to pH 7, mixed with HMP or CMC solution and then adjusted
138
to pH 4. The final concentrations of NaCas, polysaccharides and EA were 0.5 g/100 g, 0.5 g/100 g and
139
0.005 g/100 g, respectively. To produce EA-incorporated emulsion, EA was incorporated into NaCas
140
using the method mentioned above, but the initial concentrations of NaCas and EA were 1.11 g/100 g
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and 0.011 g/100 g, respectively, to have final concentrations of 0.5 g/100 g NaCas and 0.005 g/100 g
142
EA.
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2.5. Binding of EA to NaCas
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2.5.1. Fluorescence spectroscopy
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To confirm the binding of EA to NaCas, intrinsic fluorescence of NaCas in the presence and
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absence of various concentrations of EA was measured (Pan et al., 2014). NaCas (0.01 g/100 g) was
148
prepared in DW, adjusting to pH 12. Then the solutions were mixed with various concentrations of EA
149
in EtOH (final concentration of EA was 0-1 µg/mL) and intrinsic fluorescence was measured using 6
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FluoroMate FS-2 (Scinco, Seoul, Korea). Excitation wavelength was 280 nm and emission spectrum
151
was recorded from 300 to 500 nm.
152
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2.5.2. Fourier transform infrared (FT-IR) spectroscopy FT-IR spectra of electrostatic complexes, complex incorporated with EA, physical mixture of freeze
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dried electrostatic complexes and EA powder were measured by a Nicolet 6700 FT-IR spectrometer
156
(Thermo Nicolet Corp., Madison, WI, USA). Wavenumber range was from 4000 to 650 cm-1 and
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numbers of scan and resolution were 32 and 8, respectively.
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2.6. Antioxidant activity
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Antioxidant activity of the complexes, complex incorporated with EA and native EA were measured using ABTS radical scavenging method (Brandwilliams, Cuvelier, & Berset, 1995). Briefly,
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ABTS (7 mmol/L) and potassium persulfate (2.45 mmol/L) stock solutions were prepared in DW,
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mixed at 1:1 ratio, and incubated overnight in the dark. The solution was diluted with DW at pH 4 to
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have absorbance of 0.70 ± 0.05 at 734 nm. ABTS solution (0.5 mL) was mixed with the samples (30
165
µL) and incubated for 5 min in the dark. Absorbance was measured at 734 nm.
167 168
2.7. Encapsulation efficiency (EE)
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EE of EA incorporated in the complexes or emulsions was measured using the method from Xu et al. (2014) with some modification. In brief, EA-incorporated complexes or emulsions were
170
centrifuged at 2580 × g for 30 min at 4 °C. The supernatant was removed and the pellet was mixed
171
with EtOH and sonicated for 10 min to extract pelleted free EA. For emulsion, the pellet was washed
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with DW before the addition of EtOH to completely remove remaining emulsion.
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Amount of EA was quantified using high-performance liquid chromatography (HPLC) according to
174
the method of Daniel et al. (1989) and Espin et al. (2007). A Waters Alliance 2695 separations module
175
equipped with a Waters 2996 variable-wavelength diode array detector (Waters, Milford, MA, USA)
176
and an Xbridge C18 column (4 µm, 3.9 mm × 300 mm; Millipore Corp., Milford, MA, USA) was 7
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used. Mobile phases were 5 mL/100 mL formic acid (A) and acetonitrile (B) and the gradient started
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from 99 mL/100 mL A for 5 min, followed by a linear decrease from 99 mL/100 mL A to 40 mL/100
179
mL A over 40 min. The flow rate was fixed to 1 mL/min, injection volume was 20 µL and EA was
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detected at 254 nm. The samples were filtered with 0.45 µm syringe filter before HPLC analysis.
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2.8. Measurement of lipid oxidation
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2.8.1. Lipid hydroperoxides
The emulsions were stored at room temperature under dark for 2 weeks and aliquots were
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withdrawn periodically to measure lipid hydroperoxides according to the method from Richards,
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Chaiyasit, McClements, and Decker (2002). Emulsion (0.3 mL) was taken, mixed with 1.5 mL
187
isooctane/2-propanol (3:1; mL:mL) and vortexed 3 times for 10 s each. The mixture was centrifuged
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at 1274 × g for 1 min and 0.2 mL of clear upper layer was mixed with 2.8 mL methanol/1-butanol (2:1;
189
mL:mL), 15 µL 3.94 mol/L thiocyanate solution and 15 µL 0.072 mol/L Fe2+ solution. After 20 min of
190
incubation at room temperature under dark, absorbance was measured at 510 nm (Optizen 2020UV;
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Mecasys, Daejeon, Korea) and the amount of lipid hydroperoxides was calculated using a cumene
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hydroperoxide standard curve.
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2.8.2. Volatile lipid oxidation products
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Fresh emulsions (1 mL) were placed at 10 mL screw cap vials and sealed with screw caps with PTFE/silicone septa (Supleco, Bellefonte, PA, USA). The vials were stored at room temperature under
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dark for 2 weeks and headspace volatiles were measured using gas chromatograph mass spectrometer
198
(GCMS; Shimadzu GP2010 Ultra, Shimadzu, Kyoto, Japan), equipped with a DB 5 capillary column
199
(30.00 m × 0.25 mm × 0.25 µm, Agilent J & W Scientific, Folsom, CA, USA) and
200
divinylbenzene/darboxen/polydimethylsiloxane (DVB/CAR/PDMS) 50/30 µm solid microextraction
201
(SPME) fibre (Supleco, Bellefonte, PA, USA). A SPME fibre was injected into vial and exposed to the
202
headspace for 10 min at 55 °C. The fibre was desorbed at 250 °C for 10 min in the GC injector using
203
splitless mode. Oven temperature was held at 40 °C for 2 min and then elevated from 40 °C to 160 °C
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at 6 °C/min and from 160 °C to 280 °C at 10 °C/min. Carrier gas was helium (25.0 cm/s) and injector
205
temperature was 250 °C. Volatile compounds were identified by mass detector at 70 eV and 220 °C
206
ionization gauge controller.
207
209 210
2.9. Statistical analysis
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Statistical analysis was conducted using ANOVA (SPSS 22 software, SPSS Inc., Chicago, IL, USA) with Duncan’s multiple-range test (p < 0.05) to determine significance among the samples.
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3. Results and discussion
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3.1. Effect of polysaccharide concentration and pH on the stability of emulsion
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In this study, the concentration of NaCas was fixed to 1 g/100 g when producing crude emulsion as
215
this concentration was reported to be sufficient to cover the oil (10 g/100 g) during emulsification (Liu
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et al., 2012). CI and digital images of the emulsions with various concentrations of polysaccharides
217
and pH are shown in Fig. 1 and 2.
NaCas-stabilized emulsions were stable at pH 7, but phase separation occurred at pH 3, 4 and 5
219
since the pH is near the isoelectric point of NaCas. After 2 weeks, severe aggregation occurred in all
220
the emulsions except at pH 7. The emulsions with 0.25 g/100 g polysaccharides were not stable to
221
aggregation and phase separation, possibly due to bridging flocculation (Liu et al., 2012). The
222
emulsions with 0.5 g/100 g polysaccharides were the most stable to aggregation and phase separation
223
at pH 4. In the proceeding experiments, emulsions with 0.5 g/100 g NaCas, 0.5 g/100 g
224
polysaccharides and 5 g/100 g fish oil at pH 4 were used. From the microscopic images, particle size
225
of the emulsions was about 2-4 µm and was not analyzed further as the objective of this study was to
226
examine oxidative stability of the emulsion rather than its physical characterization.
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227 228
3.2. Incorporation of EA into NaCas
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3.2.1. Fluorescence spectra of EA-incorporated NaCas
230
In order to determine if NaCas and EA interact via hydrophobic interaction, intrinsic fluorescence 9
ACCEPTED MANUSCRIPT spectra of NaCas with and without various concentrations of EA were measured (Fig. 3). NaCas has
232
maximum intensity at 336 nm when excited at 280 nm and the presence of a quencher resulted in
233
lower intensity (Cogan, Kopelman, Mokady, & Shinitzky, 1976). Fluorescence intensity of NaCas
234
decreased as the concentration of EA increased, indicating that hydrophobic interaction occurred
235
between NaCas and EA. Decrease in fluorescence intensity could be due to dynamic quenching, the
236
collision of a quencher and a fluorophore or due to static quenching, complex formation between a
237
quencher and fluorophore (Mohammadi, Bordbar, Divsalar, Mohammadi, & Saboury, 2009). To
238
determine whether the quenching is dynamic or static, Stern-Volmer equation was used:
239
F0/F = 1 + kq τ0 [Q] = 1 + KSV[Q]
240
where F0 and F are fluorescence intensities in the absence and presence of EA, respectively. KSV, [Q],
241
and kq are Stern-Volmer quenching constant, concentration of EA and the fluorescence quenching
242
constant, respectively. τ0 is lifetime of fluorophore fluorescence in the absence of quencher (10-8s;
243
Lakowicz & Weber, 1980). kq value was 7.85 × 1011 (mol/L)-1s-1, which is higher than the maximum
244
dynamic quenching constant of 2 x 1010 (mol/L)-1s-1 (Lange, Kothari, Patel, & patel, 1998). This result
245
indicates that the quenching interaction between NaCas and EA was static and NaCas formed
246
complex with EA.
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FT-IR spectra of EA, EA-incorporated complexes, complexes without EA and physical mixture are shown in Fig. 4. The spectrum of EA showed similar characteristic bands as described in previous
251
papers (Bulani et al., 2016; Gopalakrishnan et al., 2014). Most of the characteristic peaks of EA were
252
not visible in physical mixture of EA and the complex as the concentration of EA was much lower
253
than that of the complex. However, some of the characteristic peaks of EA were still visible in the
254
physical mixture including a sharp peak around 3471 cm-1 from OH stretching (Bulani et al., 2016).
255
Although physical mixture and EA-incorporated complexes have the same concentrations of EA and
256
the complex, characteristic peaks of EA were not visible in the EA-incorporated complexes and the
257
spectra of the EA-incorporated complexes were almost identical to that of empty complex. These
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results, along with the fluorescence spectra, indicate that EA could be incorporated into the complex.
259 260 261
3.3. Antioxidant activity of EA and EA-incorporated complexes Antioxidant activity of EA and the electrostatic complexes with and without EA was evaluated using ABTS radical scavenging assay (Table 2). The complexes showed antioxidant activities as
263
NaCas, HMP and CMC have antioxidant activities (Kitts, 2005; Moseley, Walker, Waddington, &
264
Chen, 2003; Ro et al., 2013). EA incorporated into the complexes and native EA showed similar
265
antioxidant activity. EA-incorporated NaCas-HMP showed the highest antioxidant activity (p < 0.05).
266
Higher antioxidant activity of the EA-incorporated NaCas-HMP compared to the EA-incorporated
267
NaCas-CMC could be due to the higher antioxidant activity of NaCas-HMP compared to NaCas-
268
CMC.
269 270
3.4. EE of the complex and emulsion
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EE of EA in the complex and emulsion are shown in Table 3. There was no significant difference
272
between NaCas-HMP and NaCas-CMC. However, EE of the complexes was significantly higher than
273
that of the emulsions (p < 0.05). One of the possible reasons for the lower EE of the emulsions is loss
274
of EA during preparation of emulsion. Also, the high pressure homogenization process during
275
emulsion formation or presence of oil could affect structure of NaCas, consequently binding EA to the
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protein.
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3.5. Effect of EA incorporation on the stability of emulsion
279
3.5.1. Effect of EA incorporation on the physical stability of emulsion
280
To evaluate effect of pH cycle treatment on the stability of emulsion, CI of the emulsions with
281
native NaCas, pH-cycled NaCas and EA-incorporated NaCas were measured (Table 4). CI of the
282
emulsions prepared using NaCas-HMP was significantly lower than that of NaCas-CMC (p < 0.05),
283
which is in accordance with Fig. 2A. However, CI of the emulsions prepared with pH-cycled NaCas
284
or EA-incorporated NaCas were not significantly different from those of the emulsions with native 11
ACCEPTED MANUSCRIPT 285
NaCas (p > 0.05). Chen, Zhang, and Tang (2016) also reported that complexation of curcumin to soy
286
protein isolate did not affect CI of the emulsions.
287
289
3.5.2. Effect of EA incorporation on the oxidative stability of emulsion Incorporation of EA reduced lipid hydroperoxides in the emulsions compared to the emulsions
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without EA (Fig. 5). After 14 days of storage, lipid hydroperoxides of NaCas-HMP and NaCas-CMC-
291
stabilized emulsions with EA were 22.5% and 24.0%, respectively, lower compared to the pH-cycled
292
controls. pH-cycle treatment did not affect formation of lipid hydroperoxides.
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Volatile lipid oxidation products in the emulsions stored for 14 days were analyzed by SPME
294
(Table 5). Among lipid oxidation products from fish oil or oil rich in n-3 fatty acids, 2-pentenal, 2-
295
hexenal and 2,4-heptadienal were identified in this study (Frankel, 1993; Jimenez-Alvarez et al.,
296
2008). In accordance with the result of lipid hydroperoxides, there was no significant difference
297
between the emulsions with native NaCas and pH-cycled NaCas (p > 0.05) except for 2-hexenal in the
298
NaCas-CMC-stabilized emulsion (p < 0.05). For the NaCas-HMP-stabilized emulsion, incorporation
299
of EA reduced formation of 2-pentenal, 2-hexenal and 2,4-heptadienal by 70.1%, 39.9% and 34.5%,
300
respectively. For the NaCas-CMC-stabilized emulsion, their reductions were 80.9%, 58.3% and
301
46.1%, respectively. There was no significant difference between the emulsions with NaCas-HMP and
302
NaCas-CMC (p > 0.05). Other papers reported that incorporation of antioxidant resulted in reduced
303
amount of hexanal (Cui et al., 2014, Wan et al., 2014). However, in this study, there was no difference
304
in the concentration of hexanal in all the samples (data not shown). This may be due to difference in
305
the fatty acid composition as previous papers used soybean or corn oil, while menhaden oil, rich in n-
306
3 PUFA, was used in this study (Table 1). Jimenez-Alvarez et al. (2008) reported that 2-propenal, 2-
307
hexenal and 2,4-heptadienal but not hexanal were formed during oxidation of EPA and DHA.
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308 309 310 311
4. Conclusion In this study, it was demonstrated that EA could be incorporated into NaCas using pH-cycle method. When applied to emulsions, EA increased oxidative stability of the emulsions without affecting CI. 12
ACCEPTED MANUSCRIPT The results of this study showed that incorporation of EA into NaCas could be a way to increase
313
utilization of poorly water soluble EA as an emulsifier with antioxidant activity in food systems.
314
However, further study on the effect of EA incorporation on the other properties of emulsion,
315
including particle size, zeta potential and flocculation, is needed.
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Chemistry, 161, 324-331.
Waraho, T., McClements, D. J., & Decker, E. A. (2011). Mechanisms of lipid oxidation in food dispersions. Trends in Food Science & Technology, 22, 3-13. Xu, W., Jin, W. P., Zhang, C. L., Li, Z. S., Lin, L. F., Huang, Q.,… Li, B. (2014). Curcumin loaded
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and protective system based on complex of kappa-carrageenan and lysozyme. Food Research
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International, 59, 61-66. 16
ACCEPTED MANUSCRIPT Legends of Figures
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Fig. 1. Microscopic images of the emulsions prepared with NaCas (0.5 g/100 g NaCas and 5 g/100 g
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oil), NaCas-HMP (0.5 g/100 g NaCas, 0.25-0.5 g/100 g HMP and 5 g/100 g oil) and NaCas-CMC (0.5
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g/100 g NaCas, 0.25-0.5 g/100 g CMC and 5 g/100 g oil) at pH 3-7 after stored for 14 days (n = 2).
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The scale bars represent a length of 100 µm.
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NaCas, sodium caseinate; HMP, high-methoxyl pectin; and CMC, carboxymethyl cellulose.
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Fig. 2. Effect of polysaccharide concentration and pH on creaming index (A) and visual appearance
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(B) of the emulsions prepared with NaCas (0.5 g/100 g NaCas and 5 g/100 g oil, ), NaCas-HMP (0.5
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g/100 g NaCas, 5 g/100 g oil and 0.25 g/100 g HMP,
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g/100 g NaCas, 5 g/100 g oil and 0.25 g/100 g CMC,
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stored for 14 days.
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Values are means ± standard deviations (n = 2).
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NaCas, sodium caseinate; HMP, high-methoxyl pectin; and CMC, carboxymethyl cellulose.
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or 0.5 g/100 g HMP, ×) and NaCas-CMC (0.5
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or 0.5 g/100 g CMC, ▲) at pH 3-7 after
Fig. 3. Fluorescence spectra of sodium caseinate (0.01 g/100 g) in the presence and absence of various
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concentrations (0-1µg/mL) of ellagic acid (EA). Excitation and emission wavelengths were 280 nm
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and from 300 to 450 nm, respectively.
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Fig. 4. FT-IR spectra of ellagic acid (EA), electrostatic complexes (0.5 g/100 g NaCas and 0.5 g/100 g
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polysaccharides), complex incorporated with EA (0.5 g/100 g NaCas, 0.5 g/100 g polysaccharides and
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0.005 g /100 g EA), physical mixture of freeze dried electrostatic complexes prepared with HMP (A)
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and CMC (B).
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NaCas, sodium caseinate; HMP, high-methoxyl pectin; CMC, carboxymethyl cellulose, and FT-IR,
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Fourier transform infrared.
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Fig. 5. Effect of ellagic acid (EA) on the formation of lipid hydroperoxides. Emulsions with and 17
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without EA were stored for 14 days under dark at room temperature and concentrations of lipid
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hydroperoxides were measured. NaCas-HMP (dashed line,
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EA_NaCas-HMP (dashed line, ▲); NaCas-CMC (solid line,
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EA_NaCas-CMC (solid line, ▲).
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Values are means ± standard deviations (n = 3).
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Different letters represent significant difference (p < 0.05).
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NaCas, sodium caseinate; NaCasP, pH cycled NaCas; HMP, high-methoxyl pectin; and CMC,
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carboxymethyl cellulose.
); NaCasP-HMP (dashed line, );
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); NaCasP-CMC (solid line, ); and
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0.28 ± 0.07
14:0
8.40 ± 0.05
15:0
0.78 ± 0.01
16:0
18.2 ± 0.1
17:0
0.93 ± 0.01
18:0
3.6 ± 0.1
SFA
32.2 ± 0.3
16:1
11.01 ± 0.08
18:1n-9
5.67 ± 0.03
20:1
0.85 ± 0.25
MUFA
17.5 ± 0.2
18:2n-6
1.57 ± 0.02
18:3n-3
1.42 ± 0.01
20:4n-6
0.62 ± 0.58
20:5n-3
13.9 ± 0.1
22:6n-3
12.6 ± 0.1
PUFA
28.7 ± 0.5
Total identified
79.8 ± 0.2
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12:0
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Menhaden fish oil
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Fatty acid
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Fatty acid composition of menhaden fish oil (g/100 g).
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Values are means ± standard deviations (n=3).
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SFA: saturated fatty acid; MUFA: mono unsaturated fatty acid; and PUFA: poly unsaturated fatty acid.
ACCEPTED MANUSCRIPT Table 2 ABTS radical scavenging rate of ellagic acid (EA) and electrostatic complexes (0.5 g/100 g NaCas and 0.5 g/100 g polysaccharides) with and without incorporated EA.
56 ± 2b
NaCas-HMP
36 ± 2d
NaCas-CMC
22.8 ± 0.3e
NaCasP-HMP
38 ± 2d
NaCasP-CMC
25 ± 4e
EA_NaCasP-HMP
61 ± 2a
EA_NaCasP-CMC
46 ± 1c
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ABTS radical scavenging rate (%)
Different letters represent significant difference (p < 0.05).
ABTS, 2,2’-azino-bis(3-ethylbenzo-thiazoline-6-sulphonic acid) diammonium salt; NaCas, sodium caseinate; NaCasP, pH cycled NaCas; HMP, high-methoxyl pectin; and CMC, carboxymethyl
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cellulose.
ACCEPTED MANUSCRIPT Table 3 Encapsulation efficiency of ellagic acid (EA) incorporated in the complexes (0.5 g/100 g NaCas and 0.5 g/100 g polysaccharides) and the emulsions (0.5 g/100 g NaCas, 0.5 g/100 g polysaccharides and
Complex
Emulsion
NaCas-HMP
99.8 ± 0.0
a
68 ± 9b
NaCas-CMC
99.1 ± 0.6a
67 ± 2b
Values are means ± standard deviations (n = 3). Different letters represent significant difference (p < 0.05).
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Encapsulation efficiency (%)
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5 g/100 g oil).
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NaCas, sodium caseinate; HMP, high-methoxyl pectin; and CMC, carboxymethyl cellulose.
ACCEPTED MANUSCRIPT Table 4 Effect of pH cycle treatment and ellagic acid (EA) incorporation on creaming index of the emulsions (0.5 g/100 g NaCas, 0.5 g/100 g polysaccharides and 5 g/100 g oil) after 14 days of storage.
5.4 ± 0.8a
NaCasP-HMP
3.9 ± 0.9a
EA_NaCasP-HMP
4.57 ± 0.08a
NaCas-CMC
7.4 ± 0.3b
NaCasP-CMC
8 ± 2b
EA_NaCasP-CMC
8.1 ± 0.9b
Values are means ± standard deviations (n = 3).
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Different letters represent significant difference (p < 0.05).
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NaCas-HMP
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Creaming index (%)
NaCas, sodium caseinate; NaCasP, pH cycled NaCas; HMP, high-methoxyl pectin; and CMC,
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carboxymethyl cellulose.
ACCEPTED MANUSCRIPT Table 5 Effect of ellagic acid (EA) on the formation of volatile lipid oxidation products of the emulsions stored for 14 days (peak area, × 105). 2-Hexenal
2,4-Heptadienal
NaCas-HMP
723 ± 199a
519 ± 96ab
534 ± 75a
NaCasP-HMP
878 ± 9.6a
577 ± 18ab
EA_NaCasP-HMP
263 ± 30b
347 ± 28cd
NaCas-CMC
647 ± 192a
456 ± 36bc
NaCasP-CMC
931 ± 359a
607 ± 138a
606 ± 139a
EA_NaCasP-CMC
178 ± 93b
253 ± 23d
327 ± 34c
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2-Pentenal
580 ± 87a
380 ± 34bc
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481 ± 53ab
Values are means ± standard deviations (n = 3).
Different letters represent significant difference (p < 0.05).
NaCas, sodium caseinate; NaCasP, pH cycled NaCas; HMP, high-methoxyl pectin; and CMC,
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carboxymethyl cellulose.
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ACCEPTED MANUSCRIPT Highlights Emulsion was formed using sodium caseinate and polysaccharide. Ellagic acid was incorporated into sodium caseinate using pH cycle method. Incorporation of ellagic acid was confirmed by fluorescence spectra and FT-IR.
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Ellagic acid reduced formation of volatile lipid oxidation products.
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Incorporation of ellagic acid reduced formation of lipid hydroperoxides.