- Email: [email protected]
Water soluble chitosan-caffeic acid conjugates as a dual functional polymeric surfactant
Accepted Manuscript Water soluble chitosan-caffeic acid conjugates as a dual functional polymeric surfactant Huri İlyasoğlu, Zheng Guo PII:
S2212-4292(18)30936-2
DOI:
https://doi.org/10.1016/j.fbio.2019.04.007
Reference:
FBIO 406
To appear in:
Food Bioscience
Received Date: 24 September 2018 Revised Date:
14 April 2019
Accepted Date: 14 April 2019
Please cite this article as: İlyasoğlu H. & Guo Z., Water soluble chitosan-caffeic acid conjugates as a dual functional polymeric surfactant, Food Bioscience (2019), doi: https://doi.org/10.1016/ j.fbio.2019.04.007. 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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Water soluble chitosan-caffeic acid conjugates as a dual functional polymeric surfactant Running title: Chitosan-caffeic acid conjugates as a polymeric surfactant Huri İlyasoğlu 1,2, Zheng Guo 1, a*
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Department of Engineering, Aarhus University, DK-8000 Aarhus C, DENMARK
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Department of Nutrition and Dietetics, Gümüşhane University, 29100 Gümüşhane,
TURKEY
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a*
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Zheng Guo
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Department of Engineering, Aarhus University, Gustav Wieds Vej 10,
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DK-8000 Aarhus C, DENMARK
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E-mail: [email protected]
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Tel.: +45 8715 5528; fax: +45 8715 0201.
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Corresponding Author:
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ACCEPTED MANUSCRIPT ABSTRACT
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A water soluble chitosan-caffeic acid conjugate (C-CAC) was prepared by chemical
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modification of chitosan using activated caffeoyl with dimethyl aminopyridine as a catalyst.
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The C-CAC was characterized using UV-visible spectrometry, Fourier transform infrared,
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nuclear magnetic resonance spectroscopies and differential scanning calorimetry. The
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caffeoyl content in the C-CAC was 65 mg/g. To study the introduction of a new function into
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chitosan, the antioxidant activities of the synthesized C-CAC was determined using three in
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vitro
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ethylbenzothiazoline-6-sulfonic acid)), and ferric reducing antioxidant power. The half
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inhibition concentration values for DPPH and ABTS radicals were 370 and 172 µg/ml,
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respectively; showing its good radical scavenging activity. The emulsifying properties of the
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C-CAC were investigated by preparing 10% fish oil-in-water emulsions. C-CAC not only
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showed a good ability to stabilize oil-in-water emulsions, but also was more effective in
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retarding lipid oxidation compared to chitosan; implying that the synthesized C-CAC was
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functioning in an oil-in-water emulsion system as both emulsifier and antioxidant. Chitosan-
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phenolic conjugates, as a biopolymer-based surfactant, may represent a promising ingredient
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for enabling stable oxidation-sensitive oil-in-water emulsions.
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Keywords: Caffeic acid, chitosan, chitosan-caffeic acid conjugate, fish oil
(2,2-diphenyl-1-picrylhydrazyl),
ABTS
(2,2′-azino-bis(3-
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DPPH
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assays:
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ACCEPTED MANUSCRIPT INTRODUCTION
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Marine biopolymers have interesting intrinsic properties (Manivasagan et al., 2017), such as
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easily available, flexible, biodegradable, biocompatible, and non-toxic. Chitin is one of the
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most abundant biopolymers, comprised of N-acetylglucosamine monomers. Chitosan, the
45
most important derivative of chitin, is obtained by deacetylation of chitin using alkali
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treatment (concentrated sodium hydroxide) or enzyme treatment (chitin deacetylase)
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(Rinaudo,
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biodegradability, and non-toxicity, which makes it a good material for a wide range of
49
industrial applications including biomedical, cosmetics and food (Ngo and Kim, 2010; Yang
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et al., 2016; Wu et al., 2016). Ceylan et al. (2017a, b) reported some innovative applications
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of chitosan with electrospun chitosan nanofibers to coat sea bass fillets to delay
52
microbiological spoilage and to coat sea bream fillets to limit their chemical deterioration.
53
However, chitosan is only soluble in dilute acids below pH 6.0, which limits its usage in
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industrial applications (Pillai et al., 2009). Therefore, the chemical structure of chitosan has
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been modified to improve its solubility to expand the range of utilization. Chitosan has amino
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functional groups that provide the possibility of enabling many chemical reactions such as
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acetylation, alkylation, grafting, etc. N-acetylation of chitosan using carboxylic acid
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anhydrides (Choi et al., 2007; Lee et al., 2005), or carboxylic acid chlorides (Rodrigues, 2005;
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Shelma and Sharma, 2010), and N-alkylation of chitosan with various disaccharides (Lin and
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Chou, 2004) has been done.
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Chitosan has antioxidant activity (Ngo and Kim, 2010). However, its poor solubility and poor
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H-bond donating ability limits its antioxidant efficiency (Xie et al., 2014). To overcome these
63
drawbacks, phenolic acids have been introduced into the chitosan structure using several
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methods, including carbodiimide based chemical coupling (Liu et al., 2017; Pasanphan and
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Chirachanchi, 2008; Woranuch and Yoksan, 2013), free radical mediated grafting (Lee at al.,
Chitosan
has
important
properties
such
as
biocompatibility,
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ACCEPTED MANUSCRIPT 2014; Liu et al., 2014; Liu et al., 2015), and enzyme catalyzed grafting (Bozic et al., 2015).
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The introduction of phenolic acids onto the chitosan structure provides better water-solubility
68
and stronger antioxidant activity. A covalent bond is formed between the phenolic acid and
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the amino group of chitosan, leading to the breaking of some intermolecular and
70
intramolecular hydrogen bonds. The crystallinity is reduced, and the solubility properties are
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improved. Moreover, the phenolic hydroxyl groups in phenolic acids may enhance the
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antioxidant properties of the chitosan (Huo and Luo, 2016).
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The synthesis of polyphenol-chitosan conjugates added phenolic groups onto chitosan
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improved both physicochemical and biological properties, which may help open up new
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applications in the food industry. These polyphenol-conjugates can be used as food coating
76
agents (Yang et al., 2016; Wu et al., 2016), food packaging materials (Nunes et al., 2013;
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Schreiber et al., 2013), and encapsulation agents of functional dietary ingredients (Vishnu et
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al., 2017). Emulsion systems can be used for the encapsulation of functional food ingredients.
79
An emulsion is a thermodynamically unstable system. To prepare kinetically stable
80
emulsions, emulsifiers must be added before homogenization. There is no single emulsifier
81
that can be used in all food emulsion systems (Ogawa et al., 2003). Therefore, the
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development of each food emulsion system depends on the selection of appropriate
83
emulsifiers. Oil-in-water emulsions as delivery systems can enable incorporation of
84
polyunsaturated fatty acids into foods. Polyunsaturated fatty acids are prone to oxidation.
85
Lipid oxidation in emulsions is related to interfacial area, which enables contacts between the
86
oil and pro-oxidation compounds in the aqueous phase. Antioxidants at the interfacial area
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can retard lipid oxidation. Emulsifiers with antioxidant properties would help protect against
88
lipid oxidation (Falkeborg and Guo, 2015). Caffeic acid (3,4-hydroxycinnamic acid), a
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naturally phenolic compound, shows good radical scavenging activity owing to its structure
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including multiple phenolic hydroxyl groups and a -CH=CH-COOH group (Mathew et al.,
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with antioxidant properties. Chitosan-caffeic acid conjugate (C-CAC) may act as both
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emulsifier and antioxidant in food emulsion systems. However, their applications in food
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emulsion systems have not been fully investigated. Therefore, this study focused on designing
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a dual functional emulsifier from the chitosan conjugate. The preparation of caffeic acid
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grafted chitosan was reported (Aytekin et al, 2011; Nunes et al., 2013). However, the grafting
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methods are complicated. For the synthesis of chitosan-caffeic acid conjugate, a simpler and
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faster grafting method was developed. The water-soluble C-CAC was synthesized from
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chitosan with caffeic acid chloride using dimethyl aminopyridine (DMAP) as the catalyst. The
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C-CAC was characterized using spectroscopic techniques (UV-Vis, FTIR and NMR) and
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DSC. The antioxidant activity of the C-CAC was determined using three in vitro assays:
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DPPH, ABTS and FRAP (ferric reducing antioxidant power). The emulsifying properties
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(droplet size, zeta potential, and creaming stability) of the C-CAC were evaluated in 10% fish
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oil-in-water emulsion. The oxidative stability of the emulsion was also investigated using the
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thiobarbutiric acid reactive substances (TBARS) test.
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MATERIALS AND METHODS
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Materials
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Chitosan (Aldrich 448869, low molecular weight: 50-190 kDa, degree of deacetylation: 75-
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85%), caffeic acid, DMAP, thionyl chloride, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS
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(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), TPTZ (2,4,6-
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Tris(2-pyridyl)-s-triazine), sodium triacetate, sodium azide and solvents were obtained from
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Sigma-Aldrich (Søborg, Denmark). Fish oil (refined and bleached sardine oil) was provided
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in dry ice by Marine Bioproducts A/S (Bergen, Norway), and it was immediately stored at -40
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°C and used within 6 months. Its fatty acid composition was 35.2% saturated fatty acids,
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fatty acids. The free fatty acids were <0.05% according to the manufacturer.
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Preparation of caffeic acid chloride
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Caffeic acid was converted to caffeic acid chloride using thionyl chloride. Caffeic acid (20
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mmol) was placed in a two-necked round bottom flask. The flask was fitted with a condenser,
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and then placed in an oil bath at 70 °C. Thionyl chloride (120 mmol) was gradually added
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using a funnel over 10 min. The reaction was maintained at 90 °C with agitation at 400 rpm
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for 2 hr, the excess thionyl chloride was distilled off.
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Preparation of chitosan-caffeic acid conjugate
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Chitosan (0.4 g) was dissolved in 1% acetic acid solution (50 ml) with overnight stirring at
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room temperature (20 °C). Caffeic acid chloride (0.8 g) and DMAP (1.8 g) were then added to
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the chitosan solution. The reaction was done at room temperature for 6 hr. The reaction
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mixture was precipitated with acetone, and centrifugated at 12,500 g (Sigma 2-16 Centrifuge,
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Buch & Holm A/S, Herlev, Denmark) for 10 min. The precipitate was washed with methanol
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a few times to remove excess caffeic acid chloride. The product C-CAC was dried for 24 hr.
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Solubility test
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Approximately 5 mg of C-CAC was mixed with 1 ml of different solvents. They were stirred
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using an ultrasonic bath at full power (M1800 Ultrasonic Cleaner, Branson, Mentor, OH,
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USA) for 2 hr. Water, acetic acid (1%) methanol, 100% ethanol, acetone, and chloroform
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were used as solvents. Ultraviolet-visible (UV-Vis) spectra analysis of chitosan, caffeic acid
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and chitosan-caffeic conjugate were determined (Cary 50 Bio, Varian, Walnut Creek, CA,
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USA) in the range of 200-500 nm.
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Fourier transform infrared (FTIR) analysis
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FTIR spectra were obtained in absorbance mode in the 4000-600 cm-1 region at a resolution 4
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cm-1 with 16 scans, using a spectrophotometer (Q-Interline, Tollese, Denmark) equipped with
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directly. Spectra were analyzed using GRAMS/AI software (purchased in 2001 from Alfasoft
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Ltd., Luton, Bedfordshire, UK).
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Nuclear magnetic resonance (NMR) analysis
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Chitosan and chitosan-caffeic acid conjugate were dissolved in D2O with 1% CD3COOD
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(Sigma-Aldrich, St. Louis, MO, USA) and caffeic acid was solved in CH3OD. 1H NMR
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spectra were obtained using a Bruker Ascend 400 NMR equipment (Bruker Daltonics Inc.,
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Billerica, MA, USA) at 400 MHz with tetramethylsilane as an internal standard.
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Differential scanning calorimetry (DSC) analysis
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The thermal properties were measured on a Pyris 6 DSC system (Perkin-Elmer Inc., San Jose,
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CA, USA). Approximately 5 mg of each sample was put into an aluminum pan and placed in
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the equipment under a purging atmosphere of nitrogen (20 ml min−1), with an empty pan as an
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inert reference. The heating and cooling profile were: (1) Isothermal heating at 25 °C for 3
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min; (2) Heating to 450 °C at 10 °C min−1 (3) Cooling to 25 °C at 10 °C min−1. The DSC
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scans were evaluated using Micro Cal Origin 8.6 software (purchased in 2000 from OriginLab
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Corp., Northampton, MA, USA).
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Determination of caffeic acid content in chitosan-caffeic acid conjugate.
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The caffeic acid content of the C-CAC was determined using a Folin-Ciocalteu method. The
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C-CAC was dissolved in water. A 100 µl sample was mixed with 0.50 ml of diluted Folin-
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Ciocalteu reagent, 0.4 ml of 1 M sodium carbonate and 4 ml of distilled water. The
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absorbance of the mixture was measured at 760 nm after 1 hr. The calibration curve was
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prepared with caffeic acid from 0 to 100 mg ml-1. The caffeic acid content was expressed as
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mg caffeic acid equivalents (CAE)/g of C-CAC.
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Antioxidant activity
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assays).
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For the DPPH radical scavenging assay, a 50 µl sample was mixed with 950 µl of DPPH
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radical solution (100 µM). The absorbance was measured at 515 nm after 60 min at room
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temperature. The DPPH radical scavenging activity was calculated as:
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Scavenging activity (%) = (Ac-As)*100/Ac
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(1)
Where Ac is the absorbance of control, and As is the absorbance of sample.
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For the ABTS radical scavenging assay; an ABTS stock solution was prepared by reacting 7
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mM ABTS (ABTS•+) radical cation solution with 2.45 mM potassium persulfate solution. The
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stock solution was left in the dark at room temperature for 16 hr. The stock solution was
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diluted with 100% ethanol to obtain an absorbance of 0.70 (± 0.02) AU at 734 nm. A 50 µl
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sample was mixed with 950 µl of ABTS•+ solution and the absorbance was measured at 734
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nm after 10 min. The ABTS radical scavenging activity was calculated as:
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Where Ac is the absorbance of control, and As is the absorbance of sample.
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For the FRAP assay, fresh FRAP reagent was prepared by mixing the following solutions
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(10:1:1): acetate buffer solution (pH = 3.6), 10 mM TPTZ solution in 40 mM HCl and 20 mM
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FeCl3 solution, respectively. A 50 µl of the sample solution was mixed with 950 µl of FRAP
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reagent and the absorbance was measured at 595 nm after 20 min. The results were expressed
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as µm ascorbic acid equivalents (AAE) using an ascorbic acid (AA) calibration curve.
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Emulsion preparation
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The C-CAC and chitosan (used as stabilizer) were dispersed in the buffer (100 mM acetic
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acid, pH = 3), and stirred overnight. Emulsions consisting of 1% stabilizer, 0.02%
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antimicrobial agent sodium azide, 88.9% buffer, and 10% fish oil were prepared. Fish oil
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(10%) was added to the aqueous phase during stirring at 6000 rpm with a homogenizer
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min at 10,000 rpm.
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Emulsion properties (droplet size and zeta potential)
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The oil droplet size distribution and zeta potential were determined in a Zetasizer Nano ZS
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(Malvern Instruments, Malvern, UK) at 25 °C. The emulsions were diluted with buffer (1:10).
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The size distribution of oil droplets was determined using DLS using non-invasive backscatter
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optics. Mean values of three measurements were calculated using Archimedes software
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(purchased in 2001 from Particle Metrology System, Malvern, UK).
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Confocal laser scanning microscopy (CLSM)
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The droplet size distribution of the emulsions and the distribution of stabilizer in the
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emulsions were visualized using a confocal laser scanning microscopy (Zeiss LSM 510, Jena,
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Germany). Nile red solution (10 µl, 1 mg ml-1 in acetone) was added to the emulsion (1 ml) to
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observe the lipid phase, and then the emulsion was gently stirred. Nile red molecules were
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excited at a wavelength of 514 nm, and fluorescence emission intensity was collected over
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539-753 nm. Rhodamine 6G (10 µl, 1 mg ml-1 in water) was added to the emulsion (1 ml) to
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observe the lipid phase, and then the emulsion was gently stirred. Rhodamine 6G molecules
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were excited at a wavelength of 514 nm, and fluorescence emission intensity was collected
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over 517-696 nm. The images were analysed using Zeiss LSM Image Browser software.
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Creaming stability
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Emulsions were transferred to clear vials (15 ml, r: 1.8 cm), and stored at 4 °C for two wk.
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The emulsions separated into two layers: a) an optically opaque “cream layer” at the top and
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b) turbid “serum layer” at the bottom. Creaming index (CI) was calculated using Equation 3.
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CI = 100* (Hc/HE)
(3)
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Where, Hc is the height of cream layer, and HE is the height of emulsion using a meter stick
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with 1 mm markings. 9
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TBA (thiobarbutiric acid) solution including 15 g trichloroacetic acid, 375 mg TBA, 1.76 ml
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of 12 N HCl, and 82.9 ml of deionized water (generated from Milli-Q® CLX 7000 water
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purification systems, Merck KGaA, Darmstadt, Germany) was prepared. A 20 µl sample of
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the emulsions was diluted with water to 1 ml. To accelerate oxidation, 250 µl of 25 mM
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FeSO4 was added to the emulsions, and then the mixture was stirred at 200 rpm for 15 min.
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TBA solution (2 ml) was added to the mixture, and then incubated at boiling water bath for 15
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min. The TBA colored complexes were cooled to room temperature with tap water. The
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absorbance of the colored complex was measured after centrifugation at 12,500 g for 5 min.
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The degree of oxidation was calculated as:
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Oxidation (%) = As*100/Ac
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Where As is the absorbance of the sample including stabilizer, Ac is the absorbance of the
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control emulsion prepared with Tween 20 (Mol. Wt. ∼1228; Sigma-Aldrich, St. Louis).
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RESULTS AND DISCUSSION
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Preparation of chitosan-caffeic acid conjugate
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The chitosan-caffeic acid derivative (C-CAC) was synthesized using DMAP as the catalyst
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(Scheme 1). The caffeic acid content of the C-CAC was determined as 65±2 mg CAE/g by the
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Folin-Ciocalteu method. This value was close to the value (73.4 mg CAE/g) reported by Liu
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et al. (2014), who used a free radical mediated grafting method. It was also in the range (13-
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150 mg CAE/g) of the chitosan-caffeic acid conjugate synthesized using a grafting method
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with EDC as the coupling reagent (Aytekin et al., 2011); therefore, it can be concluded that
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the proposed method can give similar yields as more complex methods, and provide sufficient
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synthesis of chitosan-caffeic acid conjugate including similar caffeic acid content obtained
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using the grafting methods.
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The C-CAC was soluble in water and 1% acetic acid solution. The C-CAC was soluble in
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water at a concentration of up to 10 mg ml-1. The reduction of the chitosan crystallinity and
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hydrophilicity of caffeic acid hydroxyl groups is the driving force for improved water
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solubility of C-CAC.
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solvents such as methanol, ethanol, acetone and chloroform.
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UV-Vis spectra
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UV-Vis spectra of the chitosan, caffeic acid and chitosan-caffeic acid conjugate are shown in
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Figure 1. Chitosan had no absorption peak in the range of 200-500 nm. Caffeic acid had two
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absorption peaks at 298 and 326 nm. Chitosan-caffeic acid conjugate showed two absorption
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peaks at 283 and 322 nm, respectively. The UV-Vis absorption peaks of the C-CAC shifted to
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shorter wavelengths, indicating a blue shift. Molecular structure changes could lead to the
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blue shift due to the incorporation of caffeic acid onto chitosan with amide bonds.
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FTIR spectra
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The FTIR spectra of the chitosan, caffeic acid, and chitosan-caffeic acid conjugate are shown
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in Figure 2. The chitosan-caffeic acid conjugate showed characteristic amide bands (amide I:
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1640 cm-1, amide II: 1550 cm-1 and amide III: 1300 cm-1), as for chitosan (Liu et al., 2015).
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The main difference between chitosan and C-CAC was observed at the amide bands. The
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peak height of the amide bands was higher in the C-CAC than that in the chitosan, indicating
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that higher amide stretching could occur due to amide bond formation between chitosan and
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caffeic acid. Moreover, the amide I and II bands shifted to lower wavenumber with the
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incorporation of caffeic acid moieties into chitosan, supporting the formation of NH-CO
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groups.
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The reduction in the absorption at 1305 and 1415 cm-1 was observed with the incorporation of
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caffeic acid onto chitosan, which was previously attributed to the formation of a covalent
265
linkage between caffeic acid and chitosan (Liu et al., 2014)
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The synthesized conjugates were not soluble in common organic
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The 1H NMR spectrum of the chitosan, caffeic acid and C-CAC are shown in Figure 3. In the
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chitosan, a peak representing protons of N-acetylglucosamine was observed at δ 1.9 ppm, and
269
chitinous backbone gave a single peak at δ 3.1 ppm, multiple peaks between δ 3.3 and δ 4.1
270
ppm, and a small peak at δ 4.5 ppm. In addition to these peaks, the C-CAC showed new
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peaks between δ 6.3 and δ 7.6 ppm, corresponding to methine protons of caffeic acid,
272
confirming the coupling of caffeic acid moieties into the chitosan. These findings were
273
consistent with the literature (Aytekin et al., 2011; Liu et al., 2014). In addition to the
274
characteristic peaks of chitosan, the chitosan-grafted-caffeic acid showed new peaks between
275
δ 6.2 and 7.6 ppm, which was attributed to aromatic proton signals. Thus, it is evidenced that
276
the structure of the synthesized chitosan-caffeic acid conjugate is similar with those
277
synthesized using the grafting methods.
278
DSC thermogram
279
The DSC thermogram of the chitosan, caffeic acid, and C-CAC are shown in Figure 4. The
280
chitosan showed an endothermic peak between 193 and 206 °C, relating to the evaporation of
281
adsorbed and bound water. A similar endothermic event was observed in the C-CAC between
282
156 and 175 °C. The peak areas, and the start and end points of peaks differed, indicating that
283
the chitosan and chitosan-caffeic acid conjugate had distinctive water holding capacity. The
284
chitosan showed an exothermic peak at 304 °C, which can be associated with the
285
decomposition of chitosan (Woranuch and Yoksan, 2013). This exothermic peak shifted to
286
lower temperature (∼260 °C) in the DSC thermogram of the synthesized C-CAC, suggested
287
that the coupling of caffeic acid onto chitosan indeed resulted in the obstruction of chitosan
288
chain packing (Pasanphan and Chirachanchi, 2008). The obstruction of chitosan chain
289
packing could reduce the thermal stability of the C-CAC.
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decreased the thermal stability of chitosan conjugates, which was attributed to the
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The graft copolymerization
ACCEPTED MANUSCRIPT incorporation of functional groups, obstructing the chitosan chain packing (Liu et al., 2014).
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Caffeic acid had an endothermic peak at 228 °C, corresponding to the melting temperature.
293
The melting temperature peak of caffeic acid was not seen in the DSC thermogram of C-
294
CAC, suggesting that caffeic acid is chemically incorporated onto chitosan, not physically
295
adsorbed onto chitosan chains as free molecules.
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Antioxidant activity
297
The antioxidant activity of the C-CAC was evaluated using three in vitro assays (Table 1).
298
The DPPH and ABTS free radicals have been widely used for measuring the scavenging
299
activities of antioxidant compounds. The half inhibition concentration (IC50) values of DPPH
300
and ABTS radical scavenging activities were determined in the range of 0.01-1 mg ml-1 for
301
the caffeic acid and C-CAC, and in the range of 1-10 mg ml-1 for the chitosan, respectively.
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The FRAP value was determined as ascorbic acid equivalents (AAE). The results of DPPH,
303
ABTS and FRAP assays showed the incorporation of caffeic acid into the chitosan enhanced
304
the antioxidant activity. The C-CAC showed significant DPPH radical scavenging activity
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(IC50: 370 µg ml-1), ABTS radical scavenging activity (IC50: 172 µg ml-1) and FRAP (55 µM
306
AAE) compared to the chitosan that has IC50 value over 10 mg ml-1. These findings were
307
compatible with Aytekin et al. (2011), who found the DPPH IC50 value of the chitosan-caffeic
308
acid conjugate was <1 mg ml-1, and that of the chitosan was >10 mg ml-1. However, the IC50
309
values of the DPPH and ABTS radical scavenging activity were higher than the values
310
reported by Lee et al. (2014).
311
The amount of C-CAC needed was 4 times higher than caffeic acid to achieve the same
312
scavenging activity in terms of IC50 values, which can be ascribed to the dilution effect when
313
caffeic acid is chemically bound to chitosan; on the other hand, C-CAC retains the antioxidant
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property of caffeic acid as a new polymeric antioxidant.
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Emulsion properties
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emulsion was prepared, and its emulsion characteristics were compared with a chitosan based
318
emulsion. White and homogenous emulsions were obtained upon preparation. The emulsion
319
with chitosan was more viscous than that with C-CAC.
320
The mean droplet size, polydispersity index (PDI) and zeta-potential values of the emulsions
321
were determined (Table 2). The emulsions ranged in size from 1.8 to 3.1 µm. The emulsion
322
with C-CAC showed lower mean droplet size compared to the emulsion with chitosan,
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indicating that the modification of chitosan with caffeic acid reduced the droplet size. Similar
324
findings were reported for β-carotene emulsions stabilized with chitosan-chlorogenic acid
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conjugate (Wei and Gao, 2016) and chitosan-epigallocatechin-3-gallate (EGCG) conjugate
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(Lei et al., 2014). Wei and Gao (2016) stated that chitosan conjugates could be adsorbed at
327
the oil-in-water interface more easily than chitosan due to the improvement of amphipathy
328
after the grafting reaction. The amphipathy of the chitosan could be enhanced with the
329
incorporation of caffeic acid onto chitosan, which may make it better adsorbed at an oil-in-
330
water interface; reducing mean droplet size. Another possible reason for this reduction may
331
be due to the difference in the viscosity of the emulsions. Lower viscosity of the aqueous
332
phase may allow more absorption of the stabilizer at the oil-water interface during emulsion
333
preparation (Tzoumaki et al., 2011).
334
The polydispersity index (PDI) represents the size distribution of particles in a colloidal
335
system. It changes from 0 to 1 and a lower value generally reflects a more stable emulsion.
336
The emulsion with C-CAC showed a moderate size distribution, whereas the size distribution
337
of the emulsion with the chitosan was broad. It suggested that the introduction of caffeic acid
338
onto chitosan can reduce its size distribution in a colloidal system, and thus it can enhance the
339
stability of an emulsion. Zeta potential is an important factor affecting the stability of an
340
emulsion. A higher zeta potential value provides better electrostatic repulsion between the
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emulsions varied from 55.6 to 60.4. Chitosan is a cationic polysaccharide (Peniche et al.,
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2013), where amino groups give a positive zeta potential value. C-CAC showed a smaller
344
magnitude of positive charge than the chitosan, which may be explained by less available
345
protonated amino groups due to the amide bonds that resulted from conjugation of chitosan
346
with caffeic acid. As observed in a previous study, the β-carotene emulsion stabilized with
347
chitosan-EGCG conjugate was found to have a lower zeta potential value than that stabilized
348
with the native chitosan (Lei et al., 2014).
349
The CLSM images of the emulsions are shown in Figure 5. The distribution of oil droplets
350
seemed more homogeneous in the emulsion with C-CAC compared to that in the emulsion
351
with chitosan. The distribution of the stabilizer was also more homogeneous in the emulsion
352
with C-CAC. The green background in the images of Figure 5 a’ & b’ indicated the presence
353
of dissolved chitosan or C-CAC molecules in the continuous water phase. The size
354
distribution is shown in the supplementary file (S1).
355
The phase separation of the emulsions was observed during storage at 4 °C for 2 wk to
356
evaluate the creaming stability of the emulsions. A cream layer was observed on the top of the
357
emulsions at the 1st day for the C-CAC emulsion, and at the 2nd day for the chitosan emulsion,
358
and the creaming index value was found to be 12% for the C-CAC and 8% for the chitosan.
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The phase separation did not develop further during storage, indicating that the emulsions had
360
good creaming stability. The oxidative stability of the emulsions was evaluated using the
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TBARS test with accelerated conditions. The control emulsion was prepared using Tween 20
362
(1%), and the oxidation rate of the emulsions were compared with the control emulsion. The
363
emulsion with the C-CAC (oxidation rate: 31.3%) was found to be more oxidatively stable
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than that with the chitosan (oxidation rate: 57.8%). These results can support the improved
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antioxidant efficiency of the synthesized C-CAC in an oil-in-water emulsion. The finding may
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the C-CAC could locate at the oil-in-water interface and act as an antioxidant. The application
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of chitosan-grafted-vanillic acid as a wall material for microencapculation of sardine oil was
369
investigated by Vishnue et al. (2017), who reported that microencapsulated oil had the lowest
370
TBARS value due to excellent antioxidant properties of chitosan-grafted-vanillic acid.
371
Chitosan-chlorogenic acid conjugate was found to inhibit the degradation of β-carotene in
372
emulsions stored at 55 °C and under UV-light due to its strong free radical scavenging
373
activity (Wei and Gao, 2016).
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374 CONCLUSION
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Water soluble chitosan-caffeic acid conjugate was synthesized using a chemical modification
377
of chitosan. The modification of chitosan enhanced its antioxidant activity and water
378
solubility. The synthesized C-CAC showed enhanced capacity to stabilize fish oil-in-water
379
emulsion, and reduce lipid oxidation rate in the emulsion, confirming that the C-CAC could
380
have a dual function as emulsifier and antioxidant. In conclusion, the synthesized chitosan
381
conjugate may be proposed as a polymeric emulsifier with additive antioxidant property that
382
may find applications in food related sectors (Ngo and Kim, 2010; Yang et al., 2016; Wu et
383
al., 2016; Vishnue et al. 2017; Ceylan et al. 2017a & 2017b).
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ACKNOWLEDGEMENT
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The authors thank The Scientific and Technological Research Council of Turkey (TÜBİTAK)
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for financial support of Huri İlyasoğlu (TÜBİTAK 2219 International Post Doctoral Research
388
Fellowship Programme). Sampson Anankanbil and Marcin Nadzieja from Aarhus University
389
are thanked for their kind help with the NMR and CSLM analyses, respectively.
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Conflict of Interest
392
The authors confirm that they have no conflicts of interest with respect to the work described
393
in this manuscript.
394 REFERENCES
396
Aytekin, A.O., Morimura, S., & Kida, K. (2011). Synthesis of chitosan-caffeic acid
397
derivatives and evaluation of their antioxidant activities. Journal of Bioscience and
398
Bioengineering, 111, 212-216.
399
Bozic, M., Gorgieva, S., & Kokol, V. (2012). Laccase mediated functionalization of chitosan
400
by caffeic and gallic acids for modulating antioxidant and antimicrobial properties.
401
Carbohydrate Polymers, 87, 2388-2398.
402
Ceylan, Z., Sengor, G.F.U., Sağdıç, O., Yilmaz, M.T. (2017a) A novel approach to extend
403
microbiological stability of sea bass (Dicentrarchus labrax) fillets coated with electrospun
404
chitosan nanofibers. L W T - Food Science and Technolog, 79, 367-375.
405
Ceylan, Z., Sengor, G.F.U., Yilmaz, M.T. (2017b) A novel approach to limit chemical
406
deterioration of gilthead sea bream (Sparus aurata) fillets: Coating with electrospun
407
nanofibers as characterized by molecular, thermal, and microstructural properties. Journal of
408
Food Science, 82, 1163-1170.
409
Choi, C.Y., Kim S.B., Pak, P.K., Yoo, D.II., & Chung, Y.S. (2007). Effect of N-acylation on
410
structure and properties of chitosan fibers. Carbohydrate Polymers, 68, 122-127.
411
Falkeborg, M., & Guo, Z. (2015). Dodecenyl succinylated alginate (DSA) as a novel dual-
412
function emulsifier for improved fish oil-in-water emulsions. Food Hydrocolloids, 46, 10-18.
413
Hu, Q., & Luo, Y. (2016). Polyphenol-chitosan conjugates: Synthesis, characterization, and
414
applications. Carbohydrate Polymers, 151, 624-639.
AC C
EP
TE D
M AN U
SC
RI PT
395
17
ACCEPTED MANUSCRIPT Lee, M-Y., Hong, K.J., Kajiuchi, T., & Yang, J-W. (2005). Synthesis of chitosan-based
416
polymeric surfactants and their adsorption properties for heavy metals and fatty acids.
417
International Journal of Biological Macromolecule, 36, 152-158.
418
Lee, D-S., Woo, J-Y., & Ahn, C-B., & Je, J-Y. (2014). Chitosan hydroxycinnamic acid
419
conjugates: Preparation, antioxidant and antimicrobial activity. Food Chemistry, 148, 97-104.
420
Lin, H-Y., & Chou, C-C. (2004). Antioxidant activities of water-soluble disaccharide chitosan
421
derivatives. Food Research International, 37, 883-889.
422
Lei, F., Liu, F., Yuan, F. & Gao, Y. (2014). Impact of chitosan-EGCC conjugates on
423
physicochemical stability of β-carotene emulsion. Food Hydrocolloids, 39, 163-170.
424
Liu, J., Wen, X-Y., Lu, J-F., Kan, J., & Jin, C-H. (2014). Free radical mediated grafting of
425
chitosan with caffeic and ferulic acids: Structures and antioxidant activity. International
426
Journal of Biological Macromolecules, 65, 97-106.
427
Liu, J. Wu, H-T., Lu, J-F., Wen, X-Y., Kan, J., & Jin, C-H. (2015). Preparation and
428
characterization of novel phenolic acid (hydroxybenzoic and hydroxycinnamic acid
429
derivatives) grafted chitosan microspheres with enhanced adsorption properties for Fe (II).
430
Chemical Engineering Journal, 262, 803-813.
431
Liu, J., Meng, C-G., Liu, S., Kan, J., & Jin, C-H. (2017). Preparation and characterization of
432
protocatechuic acid grafted chitosan films with antioxidant activity. Food Hydrocolloids, 63,
433
457-466.
434
Mathew, S., Abraham, T.E., & Zakaria, Z.A. (2015). Reactivity of phenolic compounds
435
towards free radicals under in vitro conditions. Journal of Food Science and Technology, 52,
436
5790-5798.
437
Manivasagan, P., Bharathiraja, S., Moorthy, M.S., Oh, Y-O, Seo, H. & Oh, J. (2017). Marine
438
biopolymer-based nanomaterials as a novel platform for theranostic applications. Polymer
439
Reviews, 57, 631-667
AC C
EP
TE D
M AN U
SC
RI PT
415
18
ACCEPTED MANUSCRIPT Ngo, D-H., & Kim, S-K. (2014). Antioxidant effects of chitin, chitosan and their derivatives.
441
Advances in Food and Nutrition Research, 73, 15-31.
442
Nunes, C., Maricato, E., Cunha, A., Nunes, A., da Silva, J.A.P., & Coimbra, M.A. (2013).
443
Chitosan-caffeic acid-genipin films presenting enhanced antioxidant activity and stability in
444
acidic media. Carbohydrate Polymers, 91, 246-253
445
Ogawa, S., Decker, E.A., & McClements, D.C. (2013). Production and characterization of
446
o/w emulsions containing cationic droplets stabilized by lecithin-chitosan membranes.
447
Journal of Agricultural and Food Chemistry, 51, 2806-2812
448
Pasanphan, W., & Chirachanchi, S. (2008). Conjugation of gallic acid onto chitosan: An
449
approach for green and water-based antioxidant. Carbohydrate Polymers, 72, 169-177.
450
Peniche, C., Argüelles-Monal, W., Peniche, H., & Acosta, N. (2003). Chitosan: An attractive
451
biopolymer for microencapsulation. Macromolecular Bioscience, 3, 511-520
452
Pillai, C.K.S., Paul, W., & Sharma, C.P. (2009). Chitin and chitosan polymers: Chemistry,
453
solubility and fiber formation. Progress in Polymer Science, 34, 641-678.
454
Rinaudo, M. (2006). Chitin and chitosan: Properties and applications. Progress in Polymer
455
Science,31, 603-632
456
Rodrigues M.R. (2005). Synthesis and investigation of chitosan derivatives formed by
457
reaction with acyl chlorides. Journal of Carbohydrate Chemistry, 24, 41-54.
458
Schreiber, S.B., Bozell, J.J., Hayes, D.G. & Zivanovic, S. (2013). Introduction of primary
459
antioxidant activity to chitosan for application as a multifunctional food packaging material.
460
Food Hydrocolloids, 33, 207-214
461
Shelma, R., & Sharma, C.P. (2010). Acyl modified chitosan derivatives for oral delivery of
462
insulin and curcumin. Journal of Material Science and Material Medicine, 21, 2133-2140.
463
Tzoumaki, M.V., Moschakis, T., Kiosseoglou, V., & Biliaderis, C.S. (2011). Oil-in-water
464
emulsions stabilized by chitin nanocrystal particles. Food Hydrocolloids, 25, 1521-1529.
AC C
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M AN U
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ACCEPTED MANUSCRIPT Wei, Z. & Gao, Y. (2016). Physicochemical properties of β-carotene emulsions stabilized by
466
chitosan-chlorogenic acid complexes. LWT - Food Science and Technolog, 71, 295-301.
467
Vishnu, K.V., Chatterjee, N.S., Ajeeshkumar, K.K., Lekshmi, R.G.K., Tejpal, C.S., Mathew,
468
S., & Ravinshankar, C.N. (2017). Microencapsulation of sardine oil: Application of vanillic
469
acid grafted chitosan as a bio-functional wall material. Carbohydrate Polymers, 174, 540-548.
470
Woranuch, S., & Yoksan, R. (2013). Preparation, characterization and antioxidant property of
471
water-soluble ferulic acid grafted chitosan. Carbohydrate Polymers, 96, 495-502.
472
Wu, C., Fu, S., Xiang, Y., Yuan, C., Hu, Y., Chen, S. (2016). Effect of chitosan gallate
473
coating on the quality maintenance of refrigerated (4 °C) pomfret fruit (Pampus argentus).
474
Food and Bioprocess Technology, 9, 1885-1893.
475
Xie, M., Hu, B., Wang, Y., & Zeng (2014). Grafting of gallic acid onto chitosan enhances
476
antioxidant activities and alters rheological properties of the copolymer. Journal of
477
Agricultural and Food Chemistry, 62, 9128-9136.
478
Yang, C., Han, B., Zheng, Y., Liu, L., Li, C., Sheng, S. (2016). The quality changes of
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postharvest mulberry fruit treated by chitosan-g-caffeic acid during cold storage. Journal of
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Food Science, 81, C881-C888.
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Table 1. Antioxidant activity of chitosan, caffeic and chitosan-caffeic conjugate
Sample
Chitosan > 10000±0 > 10000±0 Caffeic acid 77±3 35.2±0.1 Chitosan-caffeic acid 370±20 172±0.1 55±7c conjugate a : The concentration of chitosan, 10,000 µg ml-1; b : The concentration of caffeic acid, 10 µg ml-1; c: The concentration of chitosan-caffeic acid conjugate, 100 µg ml-1.
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FRAP (µM ascorbic acid equivalent) 8.8±1.3a 127±3b
ABTS (IC50:µg ml-1)
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DPPH (IC50:µg ml-1)
503 504 505 506 21
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Table 2. Emulsion properties of chitosan and chitosan-caffeic acid conjugate Stabilizer
Mean droplet size (µm) 3.1±0.5 1.8±0.3
Chitosan Chitosan-caffeic acid conjugate
1.00±0.002 0.41±0.01
Zeta potential (mV) 60±2 56±2
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PDI
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Scheme 1. The reaction scheme for the synthesis of chitosan-caffeic acid conjugate
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Fig. 1. UV-Vis spectra of chitosan (a), caffeic acid (b), and chitosan-caffeic acid conjugate (c)
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Fig. 2. FTIR spectra of chitosan (a), caffeic acid (b), and chitosan-caffeic acid conjugate (c)
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Fig. 3. NMR spectra of chitosan (a), caffeic acid (b), and chitosan-caffeic acid conjugate (c)
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Fig. 4. DSC thermogram of chitosan (a), caffeic acid (b), and chitosan-caffeic acid conjugate
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(c)
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Fig. 5. CSLM images of chitosan-based (a & a’) and chitosan-caffeic acid conjugate-based (b
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& b’) emulsions: Nile red (stained red, a & b) and Rhodamine 6G (stained green, a’ & b’).
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Scale bars are 50 µm.
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Figure 1. 283
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Absorbance
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Figure 2.
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Wavenumber (cm-1)
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Figure 3.
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Figure 4.
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Temperature (°C)
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Figure 5
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Chitosan based
Chitosan-caffeic acid conjugate based
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S2212-4292(18)30936-2
DOI:
https://doi.org/10.1016/j.fbio.2019.04.007
Reference:
FBIO 406
To appear in:
Food Bioscience
Received Date: 24 September 2018 Revised Date:
14 April 2019
Accepted Date: 14 April 2019
Please cite this article as: İlyasoğlu H. & Guo Z., Water soluble chitosan-caffeic acid conjugates as a dual functional polymeric surfactant, Food Bioscience (2019), doi: https://doi.org/10.1016/ j.fbio.2019.04.007. 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.
ACCEPTED MANUSCRIPT 1 2 3 4 5 6
Water soluble chitosan-caffeic acid conjugates as a dual functional polymeric surfactant Running title: Chitosan-caffeic acid conjugates as a polymeric surfactant Huri İlyasoğlu 1,2, Zheng Guo 1, a*
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1
Department of Engineering, Aarhus University, DK-8000 Aarhus C, DENMARK
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Department of Nutrition and Dietetics, Gümüşhane University, 29100 Gümüşhane,
TURKEY
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a*
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Zheng Guo
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Department of Engineering, Aarhus University, Gustav Wieds Vej 10,
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DK-8000 Aarhus C, DENMARK
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E-mail: [email protected]
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Tel.: +45 8715 5528; fax: +45 8715 0201.
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Corresponding Author:
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ACCEPTED MANUSCRIPT ABSTRACT
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A water soluble chitosan-caffeic acid conjugate (C-CAC) was prepared by chemical
24
modification of chitosan using activated caffeoyl with dimethyl aminopyridine as a catalyst.
25
The C-CAC was characterized using UV-visible spectrometry, Fourier transform infrared,
26
nuclear magnetic resonance spectroscopies and differential scanning calorimetry. The
27
caffeoyl content in the C-CAC was 65 mg/g. To study the introduction of a new function into
28
chitosan, the antioxidant activities of the synthesized C-CAC was determined using three in
29
vitro
30
ethylbenzothiazoline-6-sulfonic acid)), and ferric reducing antioxidant power. The half
31
inhibition concentration values for DPPH and ABTS radicals were 370 and 172 µg/ml,
32
respectively; showing its good radical scavenging activity. The emulsifying properties of the
33
C-CAC were investigated by preparing 10% fish oil-in-water emulsions. C-CAC not only
34
showed a good ability to stabilize oil-in-water emulsions, but also was more effective in
35
retarding lipid oxidation compared to chitosan; implying that the synthesized C-CAC was
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functioning in an oil-in-water emulsion system as both emulsifier and antioxidant. Chitosan-
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phenolic conjugates, as a biopolymer-based surfactant, may represent a promising ingredient
38
for enabling stable oxidation-sensitive oil-in-water emulsions.
39
Keywords: Caffeic acid, chitosan, chitosan-caffeic acid conjugate, fish oil
(2,2-diphenyl-1-picrylhydrazyl),
ABTS
(2,2′-azino-bis(3-
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DPPH
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assays:
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ACCEPTED MANUSCRIPT INTRODUCTION
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Marine biopolymers have interesting intrinsic properties (Manivasagan et al., 2017), such as
43
easily available, flexible, biodegradable, biocompatible, and non-toxic. Chitin is one of the
44
most abundant biopolymers, comprised of N-acetylglucosamine monomers. Chitosan, the
45
most important derivative of chitin, is obtained by deacetylation of chitin using alkali
46
treatment (concentrated sodium hydroxide) or enzyme treatment (chitin deacetylase)
47
(Rinaudo,
48
biodegradability, and non-toxicity, which makes it a good material for a wide range of
49
industrial applications including biomedical, cosmetics and food (Ngo and Kim, 2010; Yang
50
et al., 2016; Wu et al., 2016). Ceylan et al. (2017a, b) reported some innovative applications
51
of chitosan with electrospun chitosan nanofibers to coat sea bass fillets to delay
52
microbiological spoilage and to coat sea bream fillets to limit their chemical deterioration.
53
However, chitosan is only soluble in dilute acids below pH 6.0, which limits its usage in
54
industrial applications (Pillai et al., 2009). Therefore, the chemical structure of chitosan has
55
been modified to improve its solubility to expand the range of utilization. Chitosan has amino
56
functional groups that provide the possibility of enabling many chemical reactions such as
57
acetylation, alkylation, grafting, etc. N-acetylation of chitosan using carboxylic acid
58
anhydrides (Choi et al., 2007; Lee et al., 2005), or carboxylic acid chlorides (Rodrigues, 2005;
59
Shelma and Sharma, 2010), and N-alkylation of chitosan with various disaccharides (Lin and
60
Chou, 2004) has been done.
61
Chitosan has antioxidant activity (Ngo and Kim, 2010). However, its poor solubility and poor
62
H-bond donating ability limits its antioxidant efficiency (Xie et al., 2014). To overcome these
63
drawbacks, phenolic acids have been introduced into the chitosan structure using several
64
methods, including carbodiimide based chemical coupling (Liu et al., 2017; Pasanphan and
65
Chirachanchi, 2008; Woranuch and Yoksan, 2013), free radical mediated grafting (Lee at al.,
Chitosan
has
important
properties
such
as
biocompatibility,
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2006).
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ACCEPTED MANUSCRIPT 2014; Liu et al., 2014; Liu et al., 2015), and enzyme catalyzed grafting (Bozic et al., 2015).
67
The introduction of phenolic acids onto the chitosan structure provides better water-solubility
68
and stronger antioxidant activity. A covalent bond is formed between the phenolic acid and
69
the amino group of chitosan, leading to the breaking of some intermolecular and
70
intramolecular hydrogen bonds. The crystallinity is reduced, and the solubility properties are
71
improved. Moreover, the phenolic hydroxyl groups in phenolic acids may enhance the
72
antioxidant properties of the chitosan (Huo and Luo, 2016).
73
The synthesis of polyphenol-chitosan conjugates added phenolic groups onto chitosan
74
improved both physicochemical and biological properties, which may help open up new
75
applications in the food industry. These polyphenol-conjugates can be used as food coating
76
agents (Yang et al., 2016; Wu et al., 2016), food packaging materials (Nunes et al., 2013;
77
Schreiber et al., 2013), and encapsulation agents of functional dietary ingredients (Vishnu et
78
al., 2017). Emulsion systems can be used for the encapsulation of functional food ingredients.
79
An emulsion is a thermodynamically unstable system. To prepare kinetically stable
80
emulsions, emulsifiers must be added before homogenization. There is no single emulsifier
81
that can be used in all food emulsion systems (Ogawa et al., 2003). Therefore, the
82
development of each food emulsion system depends on the selection of appropriate
83
emulsifiers. Oil-in-water emulsions as delivery systems can enable incorporation of
84
polyunsaturated fatty acids into foods. Polyunsaturated fatty acids are prone to oxidation.
85
Lipid oxidation in emulsions is related to interfacial area, which enables contacts between the
86
oil and pro-oxidation compounds in the aqueous phase. Antioxidants at the interfacial area
87
can retard lipid oxidation. Emulsifiers with antioxidant properties would help protect against
88
lipid oxidation (Falkeborg and Guo, 2015). Caffeic acid (3,4-hydroxycinnamic acid), a
89
naturally phenolic compound, shows good radical scavenging activity owing to its structure
90
including multiple phenolic hydroxyl groups and a -CH=CH-COOH group (Mathew et al.,
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ACCEPTED MANUSCRIPT 2015). The introduction of caffeic acid onto the chitosan structure can provide a new matrix
92
with antioxidant properties. Chitosan-caffeic acid conjugate (C-CAC) may act as both
93
emulsifier and antioxidant in food emulsion systems. However, their applications in food
94
emulsion systems have not been fully investigated. Therefore, this study focused on designing
95
a dual functional emulsifier from the chitosan conjugate. The preparation of caffeic acid
96
grafted chitosan was reported (Aytekin et al, 2011; Nunes et al., 2013). However, the grafting
97
methods are complicated. For the synthesis of chitosan-caffeic acid conjugate, a simpler and
98
faster grafting method was developed. The water-soluble C-CAC was synthesized from
99
chitosan with caffeic acid chloride using dimethyl aminopyridine (DMAP) as the catalyst. The
100
C-CAC was characterized using spectroscopic techniques (UV-Vis, FTIR and NMR) and
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DSC. The antioxidant activity of the C-CAC was determined using three in vitro assays:
102
DPPH, ABTS and FRAP (ferric reducing antioxidant power). The emulsifying properties
103
(droplet size, zeta potential, and creaming stability) of the C-CAC were evaluated in 10% fish
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oil-in-water emulsion. The oxidative stability of the emulsion was also investigated using the
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thiobarbutiric acid reactive substances (TBARS) test.
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MATERIALS AND METHODS
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Materials
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Chitosan (Aldrich 448869, low molecular weight: 50-190 kDa, degree of deacetylation: 75-
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85%), caffeic acid, DMAP, thionyl chloride, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS
111
(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), TPTZ (2,4,6-
112
Tris(2-pyridyl)-s-triazine), sodium triacetate, sodium azide and solvents were obtained from
113
Sigma-Aldrich (Søborg, Denmark). Fish oil (refined and bleached sardine oil) was provided
114
in dry ice by Marine Bioproducts A/S (Bergen, Norway), and it was immediately stored at -40
115
°C and used within 6 months. Its fatty acid composition was 35.2% saturated fatty acids,
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117
fatty acids. The free fatty acids were <0.05% according to the manufacturer.
118
Preparation of caffeic acid chloride
119
Caffeic acid was converted to caffeic acid chloride using thionyl chloride. Caffeic acid (20
120
mmol) was placed in a two-necked round bottom flask. The flask was fitted with a condenser,
121
and then placed in an oil bath at 70 °C. Thionyl chloride (120 mmol) was gradually added
122
using a funnel over 10 min. The reaction was maintained at 90 °C with agitation at 400 rpm
123
for 2 hr, the excess thionyl chloride was distilled off.
124
Preparation of chitosan-caffeic acid conjugate
125
Chitosan (0.4 g) was dissolved in 1% acetic acid solution (50 ml) with overnight stirring at
126
room temperature (20 °C). Caffeic acid chloride (0.8 g) and DMAP (1.8 g) were then added to
127
the chitosan solution. The reaction was done at room temperature for 6 hr. The reaction
128
mixture was precipitated with acetone, and centrifugated at 12,500 g (Sigma 2-16 Centrifuge,
129
Buch & Holm A/S, Herlev, Denmark) for 10 min. The precipitate was washed with methanol
130
a few times to remove excess caffeic acid chloride. The product C-CAC was dried for 24 hr.
131
Solubility test
132
Approximately 5 mg of C-CAC was mixed with 1 ml of different solvents. They were stirred
133
using an ultrasonic bath at full power (M1800 Ultrasonic Cleaner, Branson, Mentor, OH,
134
USA) for 2 hr. Water, acetic acid (1%) methanol, 100% ethanol, acetone, and chloroform
135
were used as solvents. Ultraviolet-visible (UV-Vis) spectra analysis of chitosan, caffeic acid
136
and chitosan-caffeic conjugate were determined (Cary 50 Bio, Varian, Walnut Creek, CA,
137
USA) in the range of 200-500 nm.
138
Fourier transform infrared (FTIR) analysis
139
FTIR spectra were obtained in absorbance mode in the 4000-600 cm-1 region at a resolution 4
140
cm-1 with 16 scans, using a spectrophotometer (Q-Interline, Tollese, Denmark) equipped with
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directly. Spectra were analyzed using GRAMS/AI software (purchased in 2001 from Alfasoft
143
Ltd., Luton, Bedfordshire, UK).
144
Nuclear magnetic resonance (NMR) analysis
145
Chitosan and chitosan-caffeic acid conjugate were dissolved in D2O with 1% CD3COOD
146
(Sigma-Aldrich, St. Louis, MO, USA) and caffeic acid was solved in CH3OD. 1H NMR
147
spectra were obtained using a Bruker Ascend 400 NMR equipment (Bruker Daltonics Inc.,
148
Billerica, MA, USA) at 400 MHz with tetramethylsilane as an internal standard.
149
Differential scanning calorimetry (DSC) analysis
150
The thermal properties were measured on a Pyris 6 DSC system (Perkin-Elmer Inc., San Jose,
151
CA, USA). Approximately 5 mg of each sample was put into an aluminum pan and placed in
152
the equipment under a purging atmosphere of nitrogen (20 ml min−1), with an empty pan as an
153
inert reference. The heating and cooling profile were: (1) Isothermal heating at 25 °C for 3
154
min; (2) Heating to 450 °C at 10 °C min−1 (3) Cooling to 25 °C at 10 °C min−1. The DSC
155
scans were evaluated using Micro Cal Origin 8.6 software (purchased in 2000 from OriginLab
156
Corp., Northampton, MA, USA).
157
Determination of caffeic acid content in chitosan-caffeic acid conjugate.
158
The caffeic acid content of the C-CAC was determined using a Folin-Ciocalteu method. The
159
C-CAC was dissolved in water. A 100 µl sample was mixed with 0.50 ml of diluted Folin-
160
Ciocalteu reagent, 0.4 ml of 1 M sodium carbonate and 4 ml of distilled water. The
161
absorbance of the mixture was measured at 760 nm after 1 hr. The calibration curve was
162
prepared with caffeic acid from 0 to 100 mg ml-1. The caffeic acid content was expressed as
163
mg caffeic acid equivalents (CAE)/g of C-CAC.
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Antioxidant activity
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assays).
167
For the DPPH radical scavenging assay, a 50 µl sample was mixed with 950 µl of DPPH
168
radical solution (100 µM). The absorbance was measured at 515 nm after 60 min at room
169
temperature. The DPPH radical scavenging activity was calculated as:
170
Scavenging activity (%) = (Ac-As)*100/Ac
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(1)
Where Ac is the absorbance of control, and As is the absorbance of sample.
172
For the ABTS radical scavenging assay; an ABTS stock solution was prepared by reacting 7
173
mM ABTS (ABTS•+) radical cation solution with 2.45 mM potassium persulfate solution. The
174
stock solution was left in the dark at room temperature for 16 hr. The stock solution was
175
diluted with 100% ethanol to obtain an absorbance of 0.70 (± 0.02) AU at 734 nm. A 50 µl
176
sample was mixed with 950 µl of ABTS•+ solution and the absorbance was measured at 734
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nm after 10 min. The ABTS radical scavenging activity was calculated as:
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Where Ac is the absorbance of control, and As is the absorbance of sample.
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For the FRAP assay, fresh FRAP reagent was prepared by mixing the following solutions
181
(10:1:1): acetate buffer solution (pH = 3.6), 10 mM TPTZ solution in 40 mM HCl and 20 mM
182
FeCl3 solution, respectively. A 50 µl of the sample solution was mixed with 950 µl of FRAP
183
reagent and the absorbance was measured at 595 nm after 20 min. The results were expressed
184
as µm ascorbic acid equivalents (AAE) using an ascorbic acid (AA) calibration curve.
185
Emulsion preparation
186
The C-CAC and chitosan (used as stabilizer) were dispersed in the buffer (100 mM acetic
187
acid, pH = 3), and stirred overnight. Emulsions consisting of 1% stabilizer, 0.02%
188
antimicrobial agent sodium azide, 88.9% buffer, and 10% fish oil were prepared. Fish oil
189
(10%) was added to the aqueous phase during stirring at 6000 rpm with a homogenizer
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191
min at 10,000 rpm.
192
Emulsion properties (droplet size and zeta potential)
193
The oil droplet size distribution and zeta potential were determined in a Zetasizer Nano ZS
194
(Malvern Instruments, Malvern, UK) at 25 °C. The emulsions were diluted with buffer (1:10).
195
The size distribution of oil droplets was determined using DLS using non-invasive backscatter
196
optics. Mean values of three measurements were calculated using Archimedes software
197
(purchased in 2001 from Particle Metrology System, Malvern, UK).
198 199
Confocal laser scanning microscopy (CLSM)
200
The droplet size distribution of the emulsions and the distribution of stabilizer in the
201
emulsions were visualized using a confocal laser scanning microscopy (Zeiss LSM 510, Jena,
202
Germany). Nile red solution (10 µl, 1 mg ml-1 in acetone) was added to the emulsion (1 ml) to
203
observe the lipid phase, and then the emulsion was gently stirred. Nile red molecules were
204
excited at a wavelength of 514 nm, and fluorescence emission intensity was collected over
205
539-753 nm. Rhodamine 6G (10 µl, 1 mg ml-1 in water) was added to the emulsion (1 ml) to
206
observe the lipid phase, and then the emulsion was gently stirred. Rhodamine 6G molecules
207
were excited at a wavelength of 514 nm, and fluorescence emission intensity was collected
208
over 517-696 nm. The images were analysed using Zeiss LSM Image Browser software.
209
Creaming stability
210
Emulsions were transferred to clear vials (15 ml, r: 1.8 cm), and stored at 4 °C for two wk.
211
The emulsions separated into two layers: a) an optically opaque “cream layer” at the top and
212
b) turbid “serum layer” at the bottom. Creaming index (CI) was calculated using Equation 3.
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CI = 100* (Hc/HE)
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Where, Hc is the height of cream layer, and HE is the height of emulsion using a meter stick
215
with 1 mm markings. 9
ACCEPTED MANUSCRIPT TBARS assay
217
TBA (thiobarbutiric acid) solution including 15 g trichloroacetic acid, 375 mg TBA, 1.76 ml
218
of 12 N HCl, and 82.9 ml of deionized water (generated from Milli-Q® CLX 7000 water
219
purification systems, Merck KGaA, Darmstadt, Germany) was prepared. A 20 µl sample of
220
the emulsions was diluted with water to 1 ml. To accelerate oxidation, 250 µl of 25 mM
221
FeSO4 was added to the emulsions, and then the mixture was stirred at 200 rpm for 15 min.
222
TBA solution (2 ml) was added to the mixture, and then incubated at boiling water bath for 15
223
min. The TBA colored complexes were cooled to room temperature with tap water. The
224
absorbance of the colored complex was measured after centrifugation at 12,500 g for 5 min.
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The degree of oxidation was calculated as:
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Oxidation (%) = As*100/Ac
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Where As is the absorbance of the sample including stabilizer, Ac is the absorbance of the
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control emulsion prepared with Tween 20 (Mol. Wt. ∼1228; Sigma-Aldrich, St. Louis).
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RESULTS AND DISCUSSION
231
Preparation of chitosan-caffeic acid conjugate
232
The chitosan-caffeic acid derivative (C-CAC) was synthesized using DMAP as the catalyst
233
(Scheme 1). The caffeic acid content of the C-CAC was determined as 65±2 mg CAE/g by the
234
Folin-Ciocalteu method. This value was close to the value (73.4 mg CAE/g) reported by Liu
235
et al. (2014), who used a free radical mediated grafting method. It was also in the range (13-
236
150 mg CAE/g) of the chitosan-caffeic acid conjugate synthesized using a grafting method
237
with EDC as the coupling reagent (Aytekin et al., 2011); therefore, it can be concluded that
238
the proposed method can give similar yields as more complex methods, and provide sufficient
239
synthesis of chitosan-caffeic acid conjugate including similar caffeic acid content obtained
240
using the grafting methods.
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The C-CAC was soluble in water and 1% acetic acid solution. The C-CAC was soluble in
242
water at a concentration of up to 10 mg ml-1. The reduction of the chitosan crystallinity and
243
hydrophilicity of caffeic acid hydroxyl groups is the driving force for improved water
244
solubility of C-CAC.
245
solvents such as methanol, ethanol, acetone and chloroform.
246
UV-Vis spectra
247
UV-Vis spectra of the chitosan, caffeic acid and chitosan-caffeic acid conjugate are shown in
248
Figure 1. Chitosan had no absorption peak in the range of 200-500 nm. Caffeic acid had two
249
absorption peaks at 298 and 326 nm. Chitosan-caffeic acid conjugate showed two absorption
250
peaks at 283 and 322 nm, respectively. The UV-Vis absorption peaks of the C-CAC shifted to
251
shorter wavelengths, indicating a blue shift. Molecular structure changes could lead to the
252
blue shift due to the incorporation of caffeic acid onto chitosan with amide bonds.
253
FTIR spectra
254
The FTIR spectra of the chitosan, caffeic acid, and chitosan-caffeic acid conjugate are shown
255
in Figure 2. The chitosan-caffeic acid conjugate showed characteristic amide bands (amide I:
256
1640 cm-1, amide II: 1550 cm-1 and amide III: 1300 cm-1), as for chitosan (Liu et al., 2015).
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The main difference between chitosan and C-CAC was observed at the amide bands. The
258
peak height of the amide bands was higher in the C-CAC than that in the chitosan, indicating
259
that higher amide stretching could occur due to amide bond formation between chitosan and
260
caffeic acid. Moreover, the amide I and II bands shifted to lower wavenumber with the
261
incorporation of caffeic acid moieties into chitosan, supporting the formation of NH-CO
262
groups.
263
The reduction in the absorption at 1305 and 1415 cm-1 was observed with the incorporation of
264
caffeic acid onto chitosan, which was previously attributed to the formation of a covalent
265
linkage between caffeic acid and chitosan (Liu et al., 2014)
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The synthesized conjugates were not soluble in common organic
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ACCEPTED MANUSCRIPT NMR spectrum
267
The 1H NMR spectrum of the chitosan, caffeic acid and C-CAC are shown in Figure 3. In the
268
chitosan, a peak representing protons of N-acetylglucosamine was observed at δ 1.9 ppm, and
269
chitinous backbone gave a single peak at δ 3.1 ppm, multiple peaks between δ 3.3 and δ 4.1
270
ppm, and a small peak at δ 4.5 ppm. In addition to these peaks, the C-CAC showed new
271
peaks between δ 6.3 and δ 7.6 ppm, corresponding to methine protons of caffeic acid,
272
confirming the coupling of caffeic acid moieties into the chitosan. These findings were
273
consistent with the literature (Aytekin et al., 2011; Liu et al., 2014). In addition to the
274
characteristic peaks of chitosan, the chitosan-grafted-caffeic acid showed new peaks between
275
δ 6.2 and 7.6 ppm, which was attributed to aromatic proton signals. Thus, it is evidenced that
276
the structure of the synthesized chitosan-caffeic acid conjugate is similar with those
277
synthesized using the grafting methods.
278
DSC thermogram
279
The DSC thermogram of the chitosan, caffeic acid, and C-CAC are shown in Figure 4. The
280
chitosan showed an endothermic peak between 193 and 206 °C, relating to the evaporation of
281
adsorbed and bound water. A similar endothermic event was observed in the C-CAC between
282
156 and 175 °C. The peak areas, and the start and end points of peaks differed, indicating that
283
the chitosan and chitosan-caffeic acid conjugate had distinctive water holding capacity. The
284
chitosan showed an exothermic peak at 304 °C, which can be associated with the
285
decomposition of chitosan (Woranuch and Yoksan, 2013). This exothermic peak shifted to
286
lower temperature (∼260 °C) in the DSC thermogram of the synthesized C-CAC, suggested
287
that the coupling of caffeic acid onto chitosan indeed resulted in the obstruction of chitosan
288
chain packing (Pasanphan and Chirachanchi, 2008). The obstruction of chitosan chain
289
packing could reduce the thermal stability of the C-CAC.
290
decreased the thermal stability of chitosan conjugates, which was attributed to the
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The graft copolymerization
ACCEPTED MANUSCRIPT incorporation of functional groups, obstructing the chitosan chain packing (Liu et al., 2014).
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Caffeic acid had an endothermic peak at 228 °C, corresponding to the melting temperature.
293
The melting temperature peak of caffeic acid was not seen in the DSC thermogram of C-
294
CAC, suggesting that caffeic acid is chemically incorporated onto chitosan, not physically
295
adsorbed onto chitosan chains as free molecules.
296
Antioxidant activity
297
The antioxidant activity of the C-CAC was evaluated using three in vitro assays (Table 1).
298
The DPPH and ABTS free radicals have been widely used for measuring the scavenging
299
activities of antioxidant compounds. The half inhibition concentration (IC50) values of DPPH
300
and ABTS radical scavenging activities were determined in the range of 0.01-1 mg ml-1 for
301
the caffeic acid and C-CAC, and in the range of 1-10 mg ml-1 for the chitosan, respectively.
302
The FRAP value was determined as ascorbic acid equivalents (AAE). The results of DPPH,
303
ABTS and FRAP assays showed the incorporation of caffeic acid into the chitosan enhanced
304
the antioxidant activity. The C-CAC showed significant DPPH radical scavenging activity
305
(IC50: 370 µg ml-1), ABTS radical scavenging activity (IC50: 172 µg ml-1) and FRAP (55 µM
306
AAE) compared to the chitosan that has IC50 value over 10 mg ml-1. These findings were
307
compatible with Aytekin et al. (2011), who found the DPPH IC50 value of the chitosan-caffeic
308
acid conjugate was <1 mg ml-1, and that of the chitosan was >10 mg ml-1. However, the IC50
309
values of the DPPH and ABTS radical scavenging activity were higher than the values
310
reported by Lee et al. (2014).
311
The amount of C-CAC needed was 4 times higher than caffeic acid to achieve the same
312
scavenging activity in terms of IC50 values, which can be ascribed to the dilution effect when
313
caffeic acid is chemically bound to chitosan; on the other hand, C-CAC retains the antioxidant
314
property of caffeic acid as a new polymeric antioxidant.
315
Emulsion properties
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317
emulsion was prepared, and its emulsion characteristics were compared with a chitosan based
318
emulsion. White and homogenous emulsions were obtained upon preparation. The emulsion
319
with chitosan was more viscous than that with C-CAC.
320
The mean droplet size, polydispersity index (PDI) and zeta-potential values of the emulsions
321
were determined (Table 2). The emulsions ranged in size from 1.8 to 3.1 µm. The emulsion
322
with C-CAC showed lower mean droplet size compared to the emulsion with chitosan,
323
indicating that the modification of chitosan with caffeic acid reduced the droplet size. Similar
324
findings were reported for β-carotene emulsions stabilized with chitosan-chlorogenic acid
325
conjugate (Wei and Gao, 2016) and chitosan-epigallocatechin-3-gallate (EGCG) conjugate
326
(Lei et al., 2014). Wei and Gao (2016) stated that chitosan conjugates could be adsorbed at
327
the oil-in-water interface more easily than chitosan due to the improvement of amphipathy
328
after the grafting reaction. The amphipathy of the chitosan could be enhanced with the
329
incorporation of caffeic acid onto chitosan, which may make it better adsorbed at an oil-in-
330
water interface; reducing mean droplet size. Another possible reason for this reduction may
331
be due to the difference in the viscosity of the emulsions. Lower viscosity of the aqueous
332
phase may allow more absorption of the stabilizer at the oil-water interface during emulsion
333
preparation (Tzoumaki et al., 2011).
334
The polydispersity index (PDI) represents the size distribution of particles in a colloidal
335
system. It changes from 0 to 1 and a lower value generally reflects a more stable emulsion.
336
The emulsion with C-CAC showed a moderate size distribution, whereas the size distribution
337
of the emulsion with the chitosan was broad. It suggested that the introduction of caffeic acid
338
onto chitosan can reduce its size distribution in a colloidal system, and thus it can enhance the
339
stability of an emulsion. Zeta potential is an important factor affecting the stability of an
340
emulsion. A higher zeta potential value provides better electrostatic repulsion between the
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ACCEPTED MANUSCRIPT dispersed particles, increasing the stability of an emulsion. The zeta potential values of the
342
emulsions varied from 55.6 to 60.4. Chitosan is a cationic polysaccharide (Peniche et al.,
343
2013), where amino groups give a positive zeta potential value. C-CAC showed a smaller
344
magnitude of positive charge than the chitosan, which may be explained by less available
345
protonated amino groups due to the amide bonds that resulted from conjugation of chitosan
346
with caffeic acid. As observed in a previous study, the β-carotene emulsion stabilized with
347
chitosan-EGCG conjugate was found to have a lower zeta potential value than that stabilized
348
with the native chitosan (Lei et al., 2014).
349
The CLSM images of the emulsions are shown in Figure 5. The distribution of oil droplets
350
seemed more homogeneous in the emulsion with C-CAC compared to that in the emulsion
351
with chitosan. The distribution of the stabilizer was also more homogeneous in the emulsion
352
with C-CAC. The green background in the images of Figure 5 a’ & b’ indicated the presence
353
of dissolved chitosan or C-CAC molecules in the continuous water phase. The size
354
distribution is shown in the supplementary file (S1).
355
The phase separation of the emulsions was observed during storage at 4 °C for 2 wk to
356
evaluate the creaming stability of the emulsions. A cream layer was observed on the top of the
357
emulsions at the 1st day for the C-CAC emulsion, and at the 2nd day for the chitosan emulsion,
358
and the creaming index value was found to be 12% for the C-CAC and 8% for the chitosan.
359
The phase separation did not develop further during storage, indicating that the emulsions had
360
good creaming stability. The oxidative stability of the emulsions was evaluated using the
361
TBARS test with accelerated conditions. The control emulsion was prepared using Tween 20
362
(1%), and the oxidation rate of the emulsions were compared with the control emulsion. The
363
emulsion with the C-CAC (oxidation rate: 31.3%) was found to be more oxidatively stable
364
than that with the chitosan (oxidation rate: 57.8%). These results can support the improved
365
antioxidant efficiency of the synthesized C-CAC in an oil-in-water emulsion. The finding may
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ACCEPTED MANUSCRIPT be explained two ways. First, the C-CAC showed good radical scavenging activity. Second,
367
the C-CAC could locate at the oil-in-water interface and act as an antioxidant. The application
368
of chitosan-grafted-vanillic acid as a wall material for microencapculation of sardine oil was
369
investigated by Vishnue et al. (2017), who reported that microencapsulated oil had the lowest
370
TBARS value due to excellent antioxidant properties of chitosan-grafted-vanillic acid.
371
Chitosan-chlorogenic acid conjugate was found to inhibit the degradation of β-carotene in
372
emulsions stored at 55 °C and under UV-light due to its strong free radical scavenging
373
activity (Wei and Gao, 2016).
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374 CONCLUSION
376
Water soluble chitosan-caffeic acid conjugate was synthesized using a chemical modification
377
of chitosan. The modification of chitosan enhanced its antioxidant activity and water
378
solubility. The synthesized C-CAC showed enhanced capacity to stabilize fish oil-in-water
379
emulsion, and reduce lipid oxidation rate in the emulsion, confirming that the C-CAC could
380
have a dual function as emulsifier and antioxidant. In conclusion, the synthesized chitosan
381
conjugate may be proposed as a polymeric emulsifier with additive antioxidant property that
382
may find applications in food related sectors (Ngo and Kim, 2010; Yang et al., 2016; Wu et
383
al., 2016; Vishnue et al. 2017; Ceylan et al. 2017a & 2017b).
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ACKNOWLEDGEMENT
386
The authors thank The Scientific and Technological Research Council of Turkey (TÜBİTAK)
387
for financial support of Huri İlyasoğlu (TÜBİTAK 2219 International Post Doctoral Research
388
Fellowship Programme). Sampson Anankanbil and Marcin Nadzieja from Aarhus University
389
are thanked for their kind help with the NMR and CSLM analyses, respectively.
390 16
ACCEPTED MANUSCRIPT 391
Conflict of Interest
392
The authors confirm that they have no conflicts of interest with respect to the work described
393
in this manuscript.
394 REFERENCES
396
Aytekin, A.O., Morimura, S., & Kida, K. (2011). Synthesis of chitosan-caffeic acid
397
derivatives and evaluation of their antioxidant activities. Journal of Bioscience and
398
Bioengineering, 111, 212-216.
399
Bozic, M., Gorgieva, S., & Kokol, V. (2012). Laccase mediated functionalization of chitosan
400
by caffeic and gallic acids for modulating antioxidant and antimicrobial properties.
401
Carbohydrate Polymers, 87, 2388-2398.
402
Ceylan, Z., Sengor, G.F.U., Sağdıç, O., Yilmaz, M.T. (2017a) A novel approach to extend
403
microbiological stability of sea bass (Dicentrarchus labrax) fillets coated with electrospun
404
chitosan nanofibers. L W T - Food Science and Technolog, 79, 367-375.
405
Ceylan, Z., Sengor, G.F.U., Yilmaz, M.T. (2017b) A novel approach to limit chemical
406
deterioration of gilthead sea bream (Sparus aurata) fillets: Coating with electrospun
407
nanofibers as characterized by molecular, thermal, and microstructural properties. Journal of
408
Food Science, 82, 1163-1170.
409
Choi, C.Y., Kim S.B., Pak, P.K., Yoo, D.II., & Chung, Y.S. (2007). Effect of N-acylation on
410
structure and properties of chitosan fibers. Carbohydrate Polymers, 68, 122-127.
411
Falkeborg, M., & Guo, Z. (2015). Dodecenyl succinylated alginate (DSA) as a novel dual-
412
function emulsifier for improved fish oil-in-water emulsions. Food Hydrocolloids, 46, 10-18.
413
Hu, Q., & Luo, Y. (2016). Polyphenol-chitosan conjugates: Synthesis, characterization, and
414
applications. Carbohydrate Polymers, 151, 624-639.
AC C
EP
TE D
M AN U
SC
RI PT
395
17
ACCEPTED MANUSCRIPT Lee, M-Y., Hong, K.J., Kajiuchi, T., & Yang, J-W. (2005). Synthesis of chitosan-based
416
polymeric surfactants and their adsorption properties for heavy metals and fatty acids.
417
International Journal of Biological Macromolecule, 36, 152-158.
418
Lee, D-S., Woo, J-Y., & Ahn, C-B., & Je, J-Y. (2014). Chitosan hydroxycinnamic acid
419
conjugates: Preparation, antioxidant and antimicrobial activity. Food Chemistry, 148, 97-104.
420
Lin, H-Y., & Chou, C-C. (2004). Antioxidant activities of water-soluble disaccharide chitosan
421
derivatives. Food Research International, 37, 883-889.
422
Lei, F., Liu, F., Yuan, F. & Gao, Y. (2014). Impact of chitosan-EGCC conjugates on
423
physicochemical stability of β-carotene emulsion. Food Hydrocolloids, 39, 163-170.
424
Liu, J., Wen, X-Y., Lu, J-F., Kan, J., & Jin, C-H. (2014). Free radical mediated grafting of
425
chitosan with caffeic and ferulic acids: Structures and antioxidant activity. International
426
Journal of Biological Macromolecules, 65, 97-106.
427
Liu, J. Wu, H-T., Lu, J-F., Wen, X-Y., Kan, J., & Jin, C-H. (2015). Preparation and
428
characterization of novel phenolic acid (hydroxybenzoic and hydroxycinnamic acid
429
derivatives) grafted chitosan microspheres with enhanced adsorption properties for Fe (II).
430
Chemical Engineering Journal, 262, 803-813.
431
Liu, J., Meng, C-G., Liu, S., Kan, J., & Jin, C-H. (2017). Preparation and characterization of
432
protocatechuic acid grafted chitosan films with antioxidant activity. Food Hydrocolloids, 63,
433
457-466.
434
Mathew, S., Abraham, T.E., & Zakaria, Z.A. (2015). Reactivity of phenolic compounds
435
towards free radicals under in vitro conditions. Journal of Food Science and Technology, 52,
436
5790-5798.
437
Manivasagan, P., Bharathiraja, S., Moorthy, M.S., Oh, Y-O, Seo, H. & Oh, J. (2017). Marine
438
biopolymer-based nanomaterials as a novel platform for theranostic applications. Polymer
439
Reviews, 57, 631-667
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EP
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M AN U
SC
RI PT
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ACCEPTED MANUSCRIPT Ngo, D-H., & Kim, S-K. (2014). Antioxidant effects of chitin, chitosan and their derivatives.
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Advances in Food and Nutrition Research, 73, 15-31.
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Nunes, C., Maricato, E., Cunha, A., Nunes, A., da Silva, J.A.P., & Coimbra, M.A. (2013).
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Chitosan-caffeic acid-genipin films presenting enhanced antioxidant activity and stability in
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acidic media. Carbohydrate Polymers, 91, 246-253
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Ogawa, S., Decker, E.A., & McClements, D.C. (2013). Production and characterization of
446
o/w emulsions containing cationic droplets stabilized by lecithin-chitosan membranes.
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Journal of Agricultural and Food Chemistry, 51, 2806-2812
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Pasanphan, W., & Chirachanchi, S. (2008). Conjugation of gallic acid onto chitosan: An
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approach for green and water-based antioxidant. Carbohydrate Polymers, 72, 169-177.
450
Peniche, C., Argüelles-Monal, W., Peniche, H., & Acosta, N. (2003). Chitosan: An attractive
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biopolymer for microencapsulation. Macromolecular Bioscience, 3, 511-520
452
Pillai, C.K.S., Paul, W., & Sharma, C.P. (2009). Chitin and chitosan polymers: Chemistry,
453
solubility and fiber formation. Progress in Polymer Science, 34, 641-678.
454
Rinaudo, M. (2006). Chitin and chitosan: Properties and applications. Progress in Polymer
455
Science,31, 603-632
456
Rodrigues M.R. (2005). Synthesis and investigation of chitosan derivatives formed by
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reaction with acyl chlorides. Journal of Carbohydrate Chemistry, 24, 41-54.
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Schreiber, S.B., Bozell, J.J., Hayes, D.G. & Zivanovic, S. (2013). Introduction of primary
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antioxidant activity to chitosan for application as a multifunctional food packaging material.
460
Food Hydrocolloids, 33, 207-214
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Shelma, R., & Sharma, C.P. (2010). Acyl modified chitosan derivatives for oral delivery of
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insulin and curcumin. Journal of Material Science and Material Medicine, 21, 2133-2140.
463
Tzoumaki, M.V., Moschakis, T., Kiosseoglou, V., & Biliaderis, C.S. (2011). Oil-in-water
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emulsions stabilized by chitin nanocrystal particles. Food Hydrocolloids, 25, 1521-1529.
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ACCEPTED MANUSCRIPT Wei, Z. & Gao, Y. (2016). Physicochemical properties of β-carotene emulsions stabilized by
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chitosan-chlorogenic acid complexes. LWT - Food Science and Technolog, 71, 295-301.
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Vishnu, K.V., Chatterjee, N.S., Ajeeshkumar, K.K., Lekshmi, R.G.K., Tejpal, C.S., Mathew,
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S., & Ravinshankar, C.N. (2017). Microencapsulation of sardine oil: Application of vanillic
469
acid grafted chitosan as a bio-functional wall material. Carbohydrate Polymers, 174, 540-548.
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Woranuch, S., & Yoksan, R. (2013). Preparation, characterization and antioxidant property of
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water-soluble ferulic acid grafted chitosan. Carbohydrate Polymers, 96, 495-502.
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Wu, C., Fu, S., Xiang, Y., Yuan, C., Hu, Y., Chen, S. (2016). Effect of chitosan gallate
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coating on the quality maintenance of refrigerated (4 °C) pomfret fruit (Pampus argentus).
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Food and Bioprocess Technology, 9, 1885-1893.
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Xie, M., Hu, B., Wang, Y., & Zeng (2014). Grafting of gallic acid onto chitosan enhances
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antioxidant activities and alters rheological properties of the copolymer. Journal of
477
Agricultural and Food Chemistry, 62, 9128-9136.
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Yang, C., Han, B., Zheng, Y., Liu, L., Li, C., Sheng, S. (2016). The quality changes of
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postharvest mulberry fruit treated by chitosan-g-caffeic acid during cold storage. Journal of
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Food Science, 81, C881-C888.
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Table 1. Antioxidant activity of chitosan, caffeic and chitosan-caffeic conjugate
Sample
Chitosan > 10000±0 > 10000±0 Caffeic acid 77±3 35.2±0.1 Chitosan-caffeic acid 370±20 172±0.1 55±7c conjugate a : The concentration of chitosan, 10,000 µg ml-1; b : The concentration of caffeic acid, 10 µg ml-1; c: The concentration of chitosan-caffeic acid conjugate, 100 µg ml-1.
SC
489 490
M AN U
491 492 493 494
499 500 501 502
EP
498
AC C
497
TE D
495 496
FRAP (µM ascorbic acid equivalent) 8.8±1.3a 127±3b
ABTS (IC50:µg ml-1)
RI PT
485 486 487 488
DPPH (IC50:µg ml-1)
503 504 505 506 21
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Table 2. Emulsion properties of chitosan and chitosan-caffeic acid conjugate Stabilizer
Mean droplet size (µm) 3.1±0.5 1.8±0.3
Chitosan Chitosan-caffeic acid conjugate
1.00±0.002 0.41±0.01
Zeta potential (mV) 60±2 56±2
RI PT
510 511
PDI
512
AC C
EP
TE D
M AN U
SC
513
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ACCEPTED MANUSCRIPT Figure Captions
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Scheme 1. The reaction scheme for the synthesis of chitosan-caffeic acid conjugate
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Fig. 1. UV-Vis spectra of chitosan (a), caffeic acid (b), and chitosan-caffeic acid conjugate (c)
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Fig. 2. FTIR spectra of chitosan (a), caffeic acid (b), and chitosan-caffeic acid conjugate (c)
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Fig. 3. NMR spectra of chitosan (a), caffeic acid (b), and chitosan-caffeic acid conjugate (c)
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Fig. 4. DSC thermogram of chitosan (a), caffeic acid (b), and chitosan-caffeic acid conjugate
520
(c)
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Fig. 5. CSLM images of chitosan-based (a & a’) and chitosan-caffeic acid conjugate-based (b
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& b’) emulsions: Nile red (stained red, a & b) and Rhodamine 6G (stained green, a’ & b’).
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Scale bars are 50 µm.
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ACCEPTED MANUSCRIPT Scheme 1.
RI PT
525
M AN U
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526
527
EP
530
AC C
529
TE D
528
24
ACCEPTED MANUSCRIPT 531
Figure 1. 283
322
326
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Absorbance
RI PT
298
532 533 534
AC C
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TE D
Wavelenght (nm)
25
ACCEPTED MANUSCRIPT 535
Figure 2.
1550
M AN U
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1300
RI PT
1640
1650
1560
540
EP
539
Wavenumber (cm-1)
AC C
536 537 538
TE D
1415 1305
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ACCEPTED MANUSCRIPT 541
Figure 3.
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a
542
543
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b
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ACCEPTED MANUSCRIPT
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c
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544 545 546
Chemical shift (ppm)
547
AC C
EP
TE D
548
28
ACCEPTED MANUSCRIPT
RI PT
Figure 4.
M AN U TE D
554
EP
553
Temperature (°C)
AC C
550 551 552
SC
Heat flow(Endo up)
549
29
ACCEPTED MANUSCRIPT 555
Figure 5
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Chitosan based
Chitosan-caffeic acid conjugate based
b
a’
b’
RI PT
a
562
EP
561
AC C
558 559 560
TE D
M AN U
SC
557
30