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Caffeic acid grafted chitosan as a novel dual-functional stabilizer for food-grade emulsions and additive antioxidant property
Food Hydrocolloids 95 (2019) 168–176
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Caffeic acid grafted chitosan as a novel dual-functional stabilizer for foodgrade emulsions and additive antioxidant property
T
Huri İlyasoğlua,1, Marcin Nadziejab, Zheng Guoa,∗ a b
Department of Engineering, Aarhus University, Gustav Wieds Vej 10, DK, 8000, Aarhus C, Denmark Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, DK, 8000, Aarhus C, Denmark
A R T I C LE I N FO
A B S T R A C T
Keywords: Caffeic acid Chitosan Dual-functional stabilizer Pickering emulsion Antioxidant
The aim of this study was to develop a novel dual-functional stabilizer for food-grade Pickering emulsions by grafting caffeic acid onto chitosan via a free radical mediated method. The grafting of caffeic acid onto chitosan was confirmed by UV–vis absorption spectra, FTIR spectra and 1H NMR spectra and DSC thermogram. The antioxidant properties of the caffeic acid grafted chitosan (CA-g-Ch) were measured by three in vitro assays. Caffeic acid-grafted chitosan displayed much better Fe2+ chelating property than both chitosan and caffeic acid, and retained comparable DPPH radical scavenging activity as caffeic acid. Moreover, the formation of the CA-gCh based emulsion was monitored by the turbidity, zeta potential, and particle size measurements. The CA-g-Ch polymers were found to be pH-responsive. The oil (linoleic acid) in water emulsions with the CA-g-Ch (as emulsifier/Pickering stabilizer) was prepared to examine its potential to stabilize Pickering emulsions, as well as its additive antioxidant properties. The results (zeta potential, mean droplet size, creaming index and TBARS values etc) demonstrated that the CA-g-Ch could act as a dual-functional stabilizer for Pickering emulsions. The emulsions made with the CA-g-Ch were found to be stable at a broad pH range of 3–6. Our findings proved that CA-g-Ch as novel dual-functional polymeric surfactant, may have great potential to find applications in food and pharmaceutical industries.
1. Introduction Pickering emulsions are stabilized by colloidal particles. Compared to conventional systems, Pickering emulsions allow for specific applications where increased stability and reduced toxicity are in demand (Liu, Wang, Zou, Wei, & Tong, 2012). Inorganic colloids, such as silica and clay, have been used for stabilizing Pickering emulsions (Rayner et al., 2014), however their use in food and pharmaceutical industries is limit due to a biocompatibility concern. Natural food grade polymers as Pickering emulsifiers are more acceptable for use in food and medical applications and therefore extensively studied as emulsion stabilizers. Polysaccharide-based particles including starch (Hong, Cheng, Gan, Lee, & Peh, 2018; Leal-Castenada et al., 2018), cellulose (Jia et al., 2015; Mikulcova, Bordes, Minarik, & Kasporkova, 2018), chitin (Barkhordari & Fathi, 2018; Tzoumaki, Moschakis, Kiosseoglou, & Biliaderis, 2011) and chitosan (Mwangi, Ho, Tey, & Chan, 2016; Wang & Heuzey, 2016) have been attempted to stabilize Pickering emulsions. They can be applied for the encapsulation of active food ingredients and enhancement of nutritional value or sensorial properties of foods.
Chitosan is an amino-polysaccharide obtained from partial deacetylation of chitin. Chitosan is biodegradable, biocompatible and nontoxic (Cheung, Ng, Wong, & Chan, 2015) and has been successfully used for stabilizing Pickering emulsions (Liu et al., 2012; Mwangi et al., 2016; Wei, Wang, Zou, Liu, & Tong, 2012). Chitosan has limited antioxidant capacity because there is no phenol-like functional group or conjugated structure in the molecule. Grafting of phenolic acids onto chitosan has been attempted to improve antioxidant activity of chitosan (Hu & Luo, 2016; Liu, Wen, Lu, Kan, & Jin, 2014; Schreiber, Bozell, Hayes, & Zivanovic, 2013; Woranuch & Yoksan, 2013; Xie, Hu, Wang, & Zeng, 2014). Although there are a lot of studies on the preparation of phenolic acids grafted chitosan, applications of these conjugates are limited. Applications of chitosan-based conjugates are primarily focused on the use of them as food coating or packaging materials (Hu & Luo, 2016). The application of phenolic acids grafted chitosan as a delivery system for food ingredients has not been intensively investigated. Caffeic acid, a type of phenolic acid, has excellent antioxidant properties. It has two phenol groups and a eCH=CHeCOOH group (Mathew, Abraham, & Zakaria, 2015). Grafting of caffeic acid
∗
Corresponding author. Department of Engineering, Aarhus University, Gustav Wieds Vej 10, Building, 3141, DK-8000, Aarhus C, Denmark. E-mail addresses: [email protected] (H. İlyasoğlu), [email protected] (M. Nadzieja), [email protected] (Z. Guo). 1 Department of Nutrition and Dietetics, Gümüşhane University, 29100 Gümüşhane, TURKEY. https://doi.org/10.1016/j.foodhyd.2019.04.043 Received 3 November 2018; Received in revised form 14 April 2019; Accepted 19 April 2019 Available online 20 April 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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2.4. FTIR analysis
onto chitosan could enhance its physicochemical and biological properties, thus functioning as a new ingredient. Specifically, it can be utilized as a chitosan-based stabilizer for food-grade Pickering emulsions, which can also be applied for the encapsulation of functional food ingredients, such as n-3 polyunsaturated fatty acids. Therefore, this study focused on design and synthesis of a novel dual-functional stabilizer from chitosan, possessing both antioxidant and emulsion-stabilizing properties for food-grade Pickering emulsions. The phenolic acid grafted chitosan has been synthesized using different methods including carbodiimide based chemical coupling method (Liu, Meng, Liu, Kan, & Jin, 2017; Woranuch & Yoksan, 2013) and free radical mediated grafting reaction (Lee, Woo, Ahn, & Je, 2014; Liu et al., 2015). The carbodiimide based coupling method requires use of toxic reagents. In contrast, the free radical method is a process that does not result in serious contamination from toxic reagents essentially used in the reaction of chemical coupling, which is safer and environmentally friendly and good for food application. In the present study, the caffeic acid grafted chitosan (CA-g-Ch) was synthesized by a free radical mediated method, and the synthesized compound was characterized using UV–vis, Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies and differential scanning calorimetry (DSC). The antioxidant activity of CA-gCh was evaluated by three in vitro assays. The antioxidant and emulsifying properties of CA-g-Ch were investigated by preparing emulsions with linoleic acid as oil phase. The emulsions were characterized by dynamic light scattering (DLS) and creaming stability studies; and the oxidative stability of the emulsions was determined by accelerated TBARS test. The effect of stabilizer concentration (0.05–1%) and pH (3–8) on the physical and oxidative stability of the emulsions were also evaluated.
FTIR spectra was recorded in transmittance mode in the 4000600 cm−1 region at resolution 4 cm−1 with 16 scans, using a spectrophotometer (QFA Flex, USA) equipped with deuterium triglycine sulfate detector. The attenuated total reflection mode was used. Spectra were analyzed using GRAMS/AI software. 2.5. NMR analysis Chitosan and CA-g-Ch were dissolved in D2O with 1% CD3COOD and caffeic acid was dissolved in CH3OD. 1H NMR spectra was recorded at room temperature using a Bruker Ascend 400 NMR system (Germany) at 400 MHz. 2.6. DSC analysis The thermal properties were analyzed using differential scanning calorimetry on a Pyris 6 DSC system (PerkinElmer). Approximately 5 mg of each sample was put into an aluminum pan and placed in the equipment under a purging atmosphere of nitrogen (20 mL min−1), with an empty pan as an inert reference. The heating and cooling profile were: (1) Isothermal heating at 25 °C for 3 min; (4) Heating to 450 °C at a rate of 10 °C min−1 (5) Cooling to 25 °C with rate of 10 °C min−1. The DSC scans was evaluated using Micro Cal Origin 8.6 software. 2.7. Determination of caffeic acid content The caffeic acid content of the CA-g-Ch was determined using FolinCiocalteu method. The CA-g-Ch was solved in water. A 100 μL of the sample solution was mixed with 0.50 mL of diluted Folin-Ciocalteu reagent, 0.4 mL of sodium carbonate (1 M) and 4 mL of distilled water. The absorbance of the mixture was measured at 760 nm after 1 h. The calibration curve was prepared with caffeic acid standard ranging from 0 to 100 mg/mL. The caffeic acid content was expressed as mg caffeic acid equivalents per g (CAE/g) of the CA-g-Ch.
2. Materials and methods 2.1. Chemicals Chitosan (Aldrich 448869, low molecular weight: 50–190 kDa, Degree of deacetylation: 75–85%), ascorbic acid, caffeic acid, DPPH (2,2-Diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), trolox, ferrozine, iron chloride, ferric chloride, sodium carbonate, sodium triacetate, Folin Ciocalteu reagent, acetic acid, hydrogen peroxide and solvents were obtained from Sigma-Aldrich (Søborg, Denmark).
2.8. Determination of antioxidant activity Chitosan (2 mg), caffeic acid (2 mg), and CA-g-Ch (2 mg) were dispersed in ultrapure water (1 mL) with ultrasonication for 2 h. Antioxidant activity was measured using three vitro methods (DPPH, Fe+2 chelating, and FRAP assays). For the DPPH radical scavenging assay, a 50 μL of the sample solution was mixed with 950 μL of DPPH radical solution (100 μM). The absorbance was measured at 515 nm after 60 min at room temperature. The control sample was prepared with 50 μL of water instead of sample solution. The DPPH radical scavenging activity was calculated by following formula:
2.2. Preparation of caffeic acid grafted chitosan Chitosan (500 mg) was dissolved in acetic acid solution (2%, 50 mL) with stirring overnight. Hydrogen peroxide (1 M, 1 mL) and ascorbic acid (54 mg) were added to the chitosan solution, and then mixed at room temperature for 30 min. Caffeic acid (250 mg) was added to the reaction mixture and stirred at room temperature for 24 h. Washing with hot deionized water at 80 °C was repeated (up to 7 times) to remove most of unreacted caffeic acid before subjected to a dialysis process. The reaction mixture enclosed in a dialysis bag (14 kDa cut-off) was dialysed against deionized water. After 2 days’ dialysis, dialysate was freeze-dried.
Scavenging activity (%) = (AceAs)*100/Ac
(1)
Where Ac is the absorbance of control, and As is the absorbance of sample. For the Fe+2 chelating activity assay, a 300 μL of the sample solution was mixed with 120 μL iron chloride (3 mM) and ferrozine (3 mM). After 10 min, the absorbance was measured at 562 nm. The control sample was prepared with 300 μL of water with instead of sample solution. Fe+2 chelating activity was calculated by following formula:
2.3. UV–vis analysis UV–Vis spectra of chitosan, caffeic acid and CA-g-Ch were recorded by UV–Vis spectrophotometer (Varian, Carry 50 Bio) in the range of 200–500 nm. Chitosan and CA-g-Ch were dispersed in acetic acid solution (0.2%) and water, respectively; and stirred overnight until a complete dissolution. Caffeic acid was dissolved in ethanol for measurement. The UV–Vis spectra of the resulting solutions (25 μg/mL) were recorded.
Fe+2 chelating activity (%) = (AceAs)*100/Ac
(2)
Where Ac is the absorbance of control, and As is the absorbance of sample. For the FRAP assay; firstly, fresh FRAP reagent was prepared by mixing the following solutions (10:1:1): acetate buffer solution 169
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Fig. 1. DPPH radical scavenging activity (a), iron chelating power (b), and FRAP (c).
equivalent (TE).
(pH = 3.6), TPTZ solution in 40 mM HCl (10 mM) and FeCl3 (20 mM) solution, respectively. A 50 μL of the sample solution was mixed with 950 μL of FRAP reagent and the absorbance was measured at 595 nm after 20 min. The results were expressed as micromolar of Trolox 170
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Fig. 2. Transmittance at 600 nm of CA-g-Ch dispersion as a function of pH (a), zeta potential value of CA-g-Ch dispersion as a function of pH (b), and particle mean diameter of CA-g-Ch dispersion as a function of pH (c).
2.9. Characterization of CA-g-Ch
2.10. Emulsion preparation
CA-g-Ch (0.1%) was dispersed in water and stirred overnight. The pH of the dispersions (5.60) was adjusted to the value, ranging from 3 to 8, with 1 N NaOH or HCl. The transmittance at 600 nm was measured by UV–Vis spectrophotometer (Varian, Carry 50 Bio). Particle diameter and zeta potential were determined by a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C.
CA-g-Ch was dispersed in water and stirred overnight. Emulsions consisting of CA-g-Ch as stabilizer, linoleic acid (10%) as oil phase, antimicrobial agent (sodium azide) and water were prepared. To evaluate the effect of concentration on the physical stability of the emulsions, the emulsions with CA-g-Ch in the range of 0.05–1% were prepared. Firstly, the aqueous and lipid phase were stirred in a magnetic stirrer for 15 min at 1000 rpm. The resulting systems were
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Table 1 Mean droplet size, polydispersity index (PDI), zeta potential, creaming index (CI), and degree of oxidation of the emulsions with different stabilizer CA-g-Ch concentrations. Stabilizer concentration (%)
Mean
c 0.25 chitosan 0.05 0.10 0.25 0.50 1.00
456 583 540 400 343 378
a
± ± ± ± ± ±
a
droplet size (nm)
PDI
35 76 20 11 7 4
0.25 0.17 0.24 0.22 0.17 0.14
Zeta potential ± ± ± ± ± ±
0.04 0.01 0.03 0.05 0.04 0.01
a
(mV)
42.14 ± 1.3 40.0 ± 0.1 43.7 ± 1.2 41.1 ± 1.0 41.8 ± 0.3 36.2 ± 0.8
b
CI (%) 15.1 15.0 15.0 15.0 15.0 15.0
± ± ± ± ± ±
0.1 0.1 0.1 0.1 0.1 0.1
Degree of oxidation (%), TBARS test
70.1 ± 5.6 7.4 ± 0.9 10.1 ± 0.8 48.7 ± 7.1 51.9 ± 9.7 50.6 ± 5.8
a
Measurements were done on the same day of emulsion preparation. The control emulsion was prepared using Tween 20 (1%) as the emulsifier; and its degree of oxidation is defined as 100%. See the details for the measure conditions in 2. MATERIALS AND METHODS, 2.14. TBARS test. c The chitosan control with 0.25% chitosan as stabilizer and 1% Tween 20 as the emulsifier. b
room temperature with tap water. The absorbance of the colored complex was measured after centrifugation at 4000 rpm for 5 min. The degree of oxidation was calculated by following formula:
sonicated (Bandelin Sonopuls HD 2200) for 2 min at an amplitude of 70%, and a duty cycle of 0.5 s in an ice-bath. To evaluate the effect of pH on the physical stability of the emulsions, pH of the aqueous phase was adjusted to the value, ranging from 3 to 8, with 1 N NaOH or HCl.
Oxidation (%) = As*100/Ac
Where, As is the absorbance of the sample including stabilizer, Ac is the absorbance of the control emulsion prepared with Tween 20 (1%).
2.11. Emulsion properties (droplet size and zeta potential) The oil droplet size distribution and zeta potential were determined in a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C. The emulsions were diluted with deionized water (1:10). The size distribution of oil droplets was determined by DLS using non-invasive backscatter optics. Mean values of three measurements were calculated.
3. Results and discussion 3.1. UV spectra The UV spectra of chitosan (Ch), caffeic acid (CA), and caffeic acid grafted chitosan (CA-g-Ch) are presented in Fig. S1. UV spectra confirmed the grafting of caffeic acid onto chitosan. Chitosan exhibited no absorption peak at the studied wavelengths (200–500 nm) whereas caffeic acid gave two absorption bands at 298 nm and 325 nm, respectively. As the CA alone, the CA-g-Ch exhibited two absorption bands. The wavelengths of the UV–vis absorption peaks of the CA-g-Ch (297 nm and 324 nm) were close to those of the CA, implying the grafting of caffeic acid onto chitosan was successful.
2.12. Creaming stability Emulsions were transferred to clear vials (15 mL, ring 1.8 cm), and stored at room temperature for 1 week. The emulsions separated into two layers: a) an optically opaque “cream layer” at the top, b) turbid “serum layer” at the bottom. Creaming index (CI) was calculated in accordance with Equation (3). CI = 100* (Hc/HE)
(4)
(3)
3.2. FTIR spectra
Where, Hc is height of cream layer, and HE is height of emulsion. The FTIR spectra of Ch, CA, and CA-g-Ch are presented in Fig. S2. The FTIR spectra supported the grafting of caffeic acid onto chitosan. Chitosan showed characteristics bands around 3350 cm−1(OeH stretching), 2930-2830 cm−1 (CeH stretching), 1650 cm−1(amide I, C=O stretching), 1590 cm−1(NeH bending of primary amine), 1540 cm−1 (amide II, NeH bending), 1030 cm−1(CeOeC), and 890 cm−1(pyranose ring), respectively (Jiang et al., 2010; Kumirska et al., 2010). Caffeic acid exhibited characteristic peaks around 3400 cm−1 (OeH stretching), 2900-3000 cm−1 (CeH stretching), 1530 cm−1 (C=C stretching), 1350 cm−1 (OeH bending), and 1220 cm−1 (CeOH stretching) (Swislocka, 2013). Compared to the chitosan, the band at 1590 cm−1 was no longer visible in the spectra of the CA-g-Ch, indicating the amide bond formation between chitosan and caffeic acid. An increase in the intensity of the peaks around 12001600 cm−1 was observed, suggesting the grafting of caffeic acid onto chitosan as well.
2.13. Confocal laser scanning microscopy The droplet size distribution of the emulsions and the distribution of stabilizer in the emulsions were visualized by the confocal laser scanning microscopy (Zeiss LSM780). For imaging of the lipid phase, Nile red solution (10 μL, 1 mg/mL in acetone) was added to the emulsion (1 mL) and then the emulsion was gently stirred. Nile red molecules were excited at a wavelength of 514 nm, and fluorescence emission intensity was collected over 539–753 nm. For imaging of the stabilizer, Rhodamine 6G (10 μL, 1 mg/mL in water) was added to the emulsion (1 mL), and then the emulsion was gently stirred. Rhodamine 6G molecules were excited at a wavelength of 514 nm, and fluorescence emission intensity was collected over 517–696 nm. The images were analyzed using Zeiss LSM Image Browser software. 2.14. Thiobarbutiric acid reactive substances (TBARS) test
3.3. 1H NMR spectrum Secondary oxidation products were determined by TBARS test. Thiobarbutiric acid (TBA) solution including trichloroacetic acid (15 g), TBA (375 mg), HCl (1.76 mL, 12 N), and water (82.9 mL) was prepared. A 20 μL of the emulsions was diluted with water to 1 mL. To accelerate oxidation, 250 μL of FeSO4 (25 mM) was added to the emulsions, and then the mixture was stirred at 200 rpm for 15 min. A 2 mL of TBA solution was added to the mixture, and then incubated at boiling water bath for 15 min. The TBA formed colored complex were cooled to the
The NMR spectra of Ch, CA, and CA-g-Ch are presented in Fig. S3. Chitosan showed a single peak at 1.95 ppm, representing the methyl group of the N-acetyl glucosamine units. It also presented a single peak at 3.05 ppm (H-2), multiple peaks at 3.20–4.00 ppm (H-3 to H-6), a small single peak at 4.45 ppm (H-1) (Liu et al., 2014). The methine protons of caffeic acid gave signals at 6.24 ppm (H-h), 6.78 ppm (H-e), 6.91 ppm (H-f), 7.04 ppm (H-b), and 7.51 ppm (H-g), respectively 172
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Table 2 Mean droplet size, polydispersity index (PDI), zeta potential, creaming index (CI), and degree of oxidation of the emulsions with different pH. pH
Mean a droplet size (nm)
PDI
3 4 5 6
554 553 521 435
0.37 0.20 0.43 0.24
± ± ± ±
7 13 6 11
a
± ± ± ±
0.03 0.03 0.01 0.01
Zeta potential a (mV)
CI (%)
43.1 57.1 46.0 38.5
14.3 15.0 15.8 15.8
± ± ± ±
0.7 2.0 0.9 2.9
± ± ± ±
b
Degree of oxidation (%)
0.1 0.1 0.1 0.1
12.5 ± 3.8 10.5 ± 0.2 9.9 ± 0.9 8.1 ± 0.7
Emulsions: 0.1% CA-g-Ch, 10% oil load. a Measurements were done on the same day of sample preparation. b The control emulsion was prepared using Tween 20 (1%) as the stabilizer at pH 5.4; and its degree of oxidation is defined as 100%. See the details for the measure conditions in 2. MATERIALS AND METHODS, 2.14. TBARS test.
2014; Liu et al., 2014). 3.4. DSC thermogram The DSC thermogram of Ch, CA, and CA-g-Ch are presented in Fig. S4. Chitosan exhibited a broad endothermic peak (130–180 °C) and a deep exothermic peak (300 °C). The broad endothermic peak was previously attributed to the evaporation of water adsorbed and bound to the chitosan (Kittur, Prashanth, Sankar, & Tharanathan, 2002). The exothermic peak could be related to the thermal unfolding of chitosan chain stacking (Pereira, da Silva Agostini, Job, & Gonzalez., 2013). Compared to the chitosan (a deep exothermic peak at 300 °C), the CA-gCh had a shallow exothermic peak (around 280 °C), shifting to a lower temperature; indicating that the grafting reaction has loosen and broken the stack pack of chitosan chains by introducing caffeoyl groups. The DSC thermogram of the caffeic acid exhibited one exothermic peak (234 °C), representing melting point of caffeic acid. Our findings revealed that Ch and CA-g-Ch had different thermal behaviour, providing evidence for the grafting of caffeic acid onto chitosan and explanation for their different chemo-physical properties. 3.5. Caffeic acid content and antioxidant activity The grafting ratio of the CA-g-Ch was found to be as 99 ± 3 mg caffeic acid equivalent/g by the Folin Ciocalteu method. FTIR spectra suggested the conjugation of caffeic acid occurred at amino group of chitosan. The antioxidant activities of Ch, CA, and CA-g-Ch were measured using three in vitro assays (Fig. 1). Compared to the chitosan, the CA-g-Ch showed higher antioxidant activity. At the concentration less than 0.6 mg/mL, the CA had higher DPPH radical scavenging activity than the CA-g-Ch, whereas the CA-g-Ch exhibited similar DPPH radical scavenging activity at the concentration between 0.6 mg/mL and 1 mg/mL. At the concentration of 1 mg/mL, the DPPH radical scavenging activity of the CA-g-Ch was found to be more than 90%. The CA-g-Ch showed more FRAP than the chitosan, whereas it had lower FRAP than the CA. Our findings have revealed that caffeic acid grafting onto chitosan can enhance the antioxidant activity of chitosan and provide a new material for industrial applications. The modification of chitosan could improve its solubility and physicochemical properties, and thus providing excellent antioxidant properties as good as caffeic acid. Similar findings were reported for gallic acid grafted chitosan, which showed comparable DPPH radical scavenging activity at high concentrations as gallic acid and higher chelating ability than gallic acid (Xie, Hu, Wang, & Zheng, 2014).
Fig. 3. CSLM images of the emulsions including different stabilizer concentration (0.05–1%). Nile Red (red) and Rhodamine 6G (green). Scale bar 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
(Swislocka, 2013). Compared to the chitosan, the CA-g-Ch exhibited new peaks at 6.24–7.44 ppm, representing the methine protons of the caffeic acid, confirming the successful grafting of caffeic acid onto chitosan. Our results were consistent with the literature (Lee et al.,
3.6. Properties of CA-g-Ch The CA-g-Ch (1 mg/mL) was dissolved in the deionized water, and its pH value (5.6) was adjusted to 3, 4, 5, 6, 7, and 8 using 0.5 M NaOH 173
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emulsion upon precipitation (Mwangi et al., 2016). All emulsions showed similar creaming stability (CI:15%) at the end of storage, indicating that the concentration of CA-g-Ch had no significant influence on the creaming stability. These results may be attributed to a similar zeta potential value at all concentrations and polymeric structure of the CA-g-Ch. A higher zeta potential value provides resistance against droplet coalescence, and an emulsion system with a value higher than ± 30 mV is considered as stable (Mikulcova et al., 2018). The polymeric structure of the stabilizer may present steric hindrance, preventing droplet coalescence. Compared to the same concentration of CA-g-Ch, Ch based Pickering emulsion show similar Zeta potential and CI value but larger mean droplet size; indicating a better surface-active property of CA-g-Ch adsorbed at oil-water interface. CLSM images (Fig. 3.) revealed that the distribution of oil droplets was homogeneous in the emulsions. The oxidative stability of the emulsions was investigated by means of accelerated TBARS test. The emulsions with the CA-g-Ch exhibited better oxidative stability than the control emulsion prepared with both the Tween 20 (1%) and the chitosan control (Table 1). Due to the presence of eOH and eOe groups in chitosan for chelating function and positive surface-charge of the Ch-based emulsion, the chitosan control showed better antioxidation property (70%) than the Tween 20 control (100%). The degree of the oxidation varied from 7% to 52% (Table 1) compared to the control (100%). The emulsions with the CAg-Ch had positively charged droplets, which could electrostatically repel iron cations, and reduce the lipid oxidation rate (Kargar, Fayazmanesh, Alavi, Spyropoulous, & Norton, 2012). Moreover, the CA-g-Ch was found to possess good iron chelating ability, which might help retard lipid oxidation. The emulsion with a concentration of 0.05–0.1% had higher oxidative stability compared to the emulsion with a concentration of 0.25–1%. These findings may be related to prooxidant effect of the CA-g-Ch. Maurya and Devasagayam (2010) found that the caffeic acid showed antioxidant behaviour at low concentration and pro-oxidant behaviour at high concentration. The pro-oxidant behaviour of the caffeic acid was previously associated with iron reducing ability. From Table 1, the degree of oxidation increases as mean droplet size drops, which might indicate the increasing surface area/volume ratio increases the chance for the migration of Fe2+ from continuous water into oil phase. Polysaccharide-based particles including starch (Hong et al., 2018; Leal-Castenada et al., 2018), cellulose (Jia et al., 2015; Mikulcova et al., 2018), chitin (Barkhordari & Fathi, 2018; Tzoumaki et al., 2011) and chitosan (Mwangi et al., 2016; Wang, & Heuzey, 2016) were found to be effective in stabilizing microemulsions. Compared to these studies, the CA-g-Ch seemed to present some valuable properties, such as stabilizing oil-in-water nanoemulsion at low concentration or delay of lipid oxidation. It can be concluded that the grafting of caffeic acid onto chitosan may enhance its antioxidant and emulsifying properties, enabling a dual-functional stabilizer.
or 0.5 M HCl. As seen from Fig. 2a, the transmittance of the CA-g-Ch dispersions slightly decreased with increasing pH value in the range of 3–7. At these pH ranges, the transmittance of the solutions was higher than 90%, and the solutions were transparent. At pH 8, the transmittance of the solution sharply decreased (< 50%), and the solution became turbid. When the pH value of the CA-g-Ch solution could revert to the value below 7 using 0.5 M HCl, the solution became transparent again. These findings revealed that the CA-g-Ch was a pH-responsive polymer as the chitosan (Mwangi et al., 2016; Liu et al., 2012). The amino groups of the CA-g-Ch could be protonated at low pH (< 7), converting the polymer to a water-soluble cationic polyelectrolyte. The zeta potential values of the CA-g-Ch (Fig. 2b) supported this hypothesis. At high pH (> 7), the amino groups of the CA-g-Ch could be deprotonated, and the polymer could turn into insoluble form (Kumirska et al., 2010; Liu et al., 2012). The zeta potential values of the CA-g-Ch dispersions were found to decrease with increasing pH value (Fig. 2b). The CA-g-Ch dispersions could carry strong positive charge (> +30) at the pH values, ranging from 3 to 5, whereas it could carry weak positive charge (< +20) at the pH value higher than 6. These findings can be due to the presence of amino groups on the caffeic acid grafted chitosan's backbone. Chitosan has amino groups, that are protonated or deprotonated depending on the pH value of the medium (Kumirska et al., 2010). The mean diameter of the CA-g-Ch dispersions was less than 1 μm at the pH values between 3 and 7. However, it sharply increased with an increase in the pH value, ranging from 7 to 8, and reached to the highest value (3.5 μm). These findings may be attributed to the flocculation of the CA-g-Ch dispersions, depending on the pH value. At above pH 6, the CA-g-Ch dispersion had weak positive charges, implying lower electrostatic repulsion between the CA-g-Ch dispersions. Higher electrostatic repulsion enhances the physical stability of dispersion (Yin, Chu, Kobayashi, & Nakasihima, 2009). 3.7. Emulsions Homogenous and white emulsions were obtained by employing the high-energy input from ultrasonic probe to mix oil and aqueous phases and the emulsion properties including zeta potential, mean droplet size, polydispersity index (PDI), creaming index (CI), and oxidative stability were determined. Linoleic acid was selected as the oil phase to evaluate the ability of the CA-g-Ch to prevent lipid oxidation. 3.7.1. Effect of CA-g-Ch concentration on the stability of emulsions To investigate the effect of concentration on the physical and oxidative stability of emulsions, the emulsions with different concentration CA-g-Ch (0.05%, 0.1%, 0.25%, 0.50% and 1%) were prepared and characterized as shown in Table 1. The chitosan control with 0.25% chitosan as stabilizer and 1% Tween 20 as the emulsifier is also presented. All emulsions had strong positive charges, ranging from +36 to +44, implying possible high electrostatic repulsion between oil droplets. Higher electrostatic repulsion is known to enhance the physical stability of an emulsion by improving its anti-aggregation (Dickinson, 2009). The positive charges of the emulsions could be related to the presence of primary amino groups in Ch and CA-g-Ch. The mean droplet size of the emulsions varied from 343 nm to 583 nm. The mean droplet size of the emulsions decreased with increasing stabilizer concentration. With increasing stabilizer concentration in the aqueous phase, an increase in the number of adsorbed particles at the oil-water interface could result in a reduction in the droplet size. The adsorbed particles at the oil-water interface could cover much larger area per volume to reduce droplet size (Yan et al., 2017). The PDI values of the emulsions ranged from 0.17 to 0.25, indicating a narrow size distribution. The phase separation was observed during 1 week of storage at the room temperature to determine the creaming stability of the emulsions. A white cream layer at the top was observed at the first day. Formation of creaming is a characteristic property of Pickering
3.7.2. Effect of pH on the stability of emulsions The pKa value of glucosamine is 12.27, pKb is 1.72. pKa of chitosan showed a slightly decreasing from 6.51 to 6.39 (Wang et al., 2006), depending on the molecular weight. Caffeic acid pKa value is 4.62. As some amine groups in CA-g-Ch were modified by caffeic acid, so the pKa value is CA-g-Ch is higher than Ch but lower than glucosamine, as the density of eNH2 group in glucosamine is much higher than in CA-gCh. However, we have not measured the apparent pKa of the CA-g-Ch in the present study. The effect of pH value on the stability of the emulsions was evaluated as the CA-g-Ch dispersions were found to be soundly pH-responsive. The pH value of the freshly prepared emulsion (0.1% CA-g-Ch, and 10% oil load) was adjusted to 3, 4, 5, 6, 7, and 8, respectively. The emulsions were broken when the pH was adjusted to 7 or 8. Coalescence, flocculation or aggregation of oil droplets could occur when electrostatic repulsion between oil droplets is low (Mikulcova et al., 2018). An increase in the pH value above pKa of the 174
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References
amino groups could result in the droplet coalescence as surface charge density decreased. At the pH 7 or 8, the CA-g-Ch dispersions had a much lower zeta potential value (< +13). The emulsifying properties at low pH are presented in Table 2. All emulsions had strong positive charges (> +30), ranging from +39 to +57. The mean droplet size of the emulsions varied from 435 nm to 554 nm, indicating that the CA-g-Ch had ability to stabilize oil-in-water emulsion at nanometric scale. The mean droplet size slightly decreased with pH increasing from 3 to 6. The polydispersity index of the emulsion was in the range of 0.20–0.43, indicating narrow to moderate size distribution. The phase separation was observed during 1 week of storage at the room temperature in the determination of the creaming stability of the emulsions. The CI values of the emulsion ranged from 14.3% to 15.8%. The emulsion with pH 3 showed higher creaming stability followed by the emulsion with pH 4. Both emulsions (pH 5 and pH 6) showed similar CI values. Our findings indicated that the CA-g-Ch had ability to stabilize oil-in-water emulsion by being adsorbed to the oil-water interface at the pH values between 3 and 6. The oxidative stability of the emulsions was determined by the accelerated TBARS test to investigate the influence of pH on the oxidative stability of the emulsions. The control emulsion was prepared using Tween 20 (1%) as a stabilizer. The degree of oxidation was found to be ranged from 8% to 13% compared to the control (100%), indicating that the CA-g-Ch had potent ability to prevent oxidation in the emulsions between pH 3 and pH 6. The oxidative stability of the emulsions slightly increased with increasing pH value. A lower degree of oxidation observed in the emulsion stabilized with the CA-g-Ch could be due to two reasons. Firstly, CA-g-Ch showed both good radical scavenging activity and iron chelating ability as antioxidant. Secondly, it could act as surface-active emulsifier/stabilizer capable to be localized at oil-inwater interface. Wei and Gao (2016) reported that the chitosan chlorogenic acid conjugate inhibited the β-carotene degradation in the oil-in-water emulsion due to its excellent antioxidant properties. A similar finding was reported for the emulsion stabilized with the chitosan-EGCG (epigallocatechin-3-gallate) (Lei, Liu, Yuan, & Gao, 2014). It may be thus concluded that the CA-g-Ch could exhibit excellent antioxidant activity in the emulsion system as other chitosan-phenolic acid conjugates.
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Application of spectroscopic methods for structural analysis of chitin and chitosan. Marine Drugs, 8, 1567–1636. Leal-Castenada, E. J., Garcia-Tejeda, Y., Hernandez-Sanchez, H., Alamilla-Beltran, L., Tellez-Medina, D. I., Calderon-Dominguez, G., et al. (2018). Pickering emulsions stabilized with native and lauroylated amaranth starch. Food Hydrocolloids, 80, 177–185. Lee, D.-S., Woo, J.-Y., Ahn, C.-B., & Je, J.-Y. (2014). Chitosan hydroxycinnamic acid conjugates : Preparation, antioxidant and antimicrobial activity. Food Chemistry, 148, 97–104. Lei, F., Liu, F., Yuan, F., & Gao, Y. (2014). Impact of chitosan-ECGC conjugates on physical stability of β-carotene emulsion. Food Hydrocolloids, 39, 163–170. Liu, J., Meng, C.-G., Liu, S., Kan, J., & Jin, C.-H. (2017). Preparation and characterization of protocatechuic acid grafted chitosan films with antioxidant activity. Food Hydrocolloids, 63, 457–466. Liu, H., Wang, C., Zou, S., Wei, Z., & Tong, Z. (2012). Simple, reversible emulsion system switched by pH on the basis of chitosan without hydrophobic modification. Langmuir, 28, 11017–11024. Liu, J., Wen, X.-Y., Lu, J.-F., Kan, J., & Jin, C.-H. (2014). Free radical mediated grafting of chitosan with caffeic and ferulic acids: Structures and antioxidant activity. International Journal of Biological Macromolecules, 65, 97–106. Liu, J., Wu, H.-T., Lu, J.-F., Wen, X.-Y., Kan, J., & Jin, C.-H. (2015). Preparation and characterization of novel phenolic acid (hydroxybenzoic and hydroxycinnamic acid derivatives) grafted chitosan microspheres with enhanced adsorption properties for Fe (II). Chemical Engineering Journal, 262, 803–813. Mathew, S., Abraham, T. E., & Zakaria, Z. A. (2015). Reactivity of phenolic compounds towards free radicals under in vitro conditions. Journal of Food Science & Technology, 52, 5790–5798. Maurya, D. K., & Devasagayam, T. P. A. (2010). Antioxidant and prooxidant nature of hydroxycinnamic acid derivatives ferulic and caffeic acid. Food and Chemical Toxicology, 48, 3369–3373. Mikulcova, V., Bordes, R., Minarik, A., & Kasporkova, V. (2018). Pickering oil-in-water emulsions stabilized by carboxylated cellulose nanocrystals-Effect of the pH. Food Hydrocolloids, 80, 60–67. Mwangi, W. W., Ho, K.-W., Tey, B.-T., & Chan, E.-S. (2016). Effects of environmental factors on the physical stability of pickering-emulsions stabilized by chitosan particles. Food Hydrocolloids, 60, 543–550. Pereira, F. S., da Silva Agostini, D. B., Job, A. E., & Gonzalez, E. R. P. (2013). Thermal studies of chitin and chitosan derivatives. Journal of Thermal Analysis and Colorimetry, 114, 321–327. Rayner, M., Marku, D., Eriksson, M., Sjöö, M., Dejmek, P., & Wahlgren, M. (2014). Biomass-based particles for the formulation of Pickering type emulsions in food and topical applications. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 458, 48–62. Schreiber, S. B., Bozell, J. J., Hayes, D. G., & Zivanovic, S. (2013). Introduction of primary antioxidant activity to chitosan for application as a multifunctional food packaging material. Food Hydrocolloids, 33, 207–214. Swislocka, R. (2013). Spectroscopic (FTIR, FT-Raman, UV absorption, 1H and 13C NMR) and theoretical (in B3LYP/6-311++G** level) studies on alkali metal salts of caffeic acid. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 100, 21–30. Tzoumaki, M. V., Moschakis, T., Kiosseoglou, V., & Biliaderis, C. G. (2011). Oil-in-water emulsions stabilized by chitin nanocrystal particles. Food Hydrocolloids, 25, 1521–1529. Wang, Q. Z., Chen, X. G., Liu, N., Wang, S. X., Liu, C. S., Meng, X. H., et al. (2006). Protonation constants of chitosan with different molecular weight and degree of deacetylation. Carbohydrate Polymers, 65, 194–201. Wang, X.-Y., & Heuzey, M.-C. (2016). Chitosan-based conventional and Pickering emulsions with long-term stability. Langmuir, 32, 929–936. Wei, Z., & Gao, Y. (2016). Physicochemical properties of β-carotene emulsions stabilized
4. Conclusion A novel dual-functional stabilizer, caffeic acid grafted chitosan (CAg-Ch), was developed and applied for preparation of Pickering emulsions. The CA-g-Ch was synthesized by ascorbic acid-initiated grafting of caffeic acid onto chitosan. The CA-g-Ch product showed better Fe+2 chelating property than caffeic acid and comparable DPPH radical scavenging activity with caffeic acid. The CA-g-Ch polymeric surfactant demonstrated ability to stabilize oil-in-water nanoemulsion in the pH range of 3–6. The CA-g-Ch polymeric surfactant also had potential to retard lipid oxidation. The CA-g-Ch polymeric surfactant may open a new route for the preparation of food-grade Pickering emulsions. Acknowledgement The authors thank The Scientific and Technological Research Council of Turkey (TÜBİTAK), Turkey, for financial support of Huri İlyasoğlu (Grant No. TÜBİTAK 2219 International Post Doctoral Research Fellowship Programme). Sampson Anankanbil from Aarhus University is thanked for his kind help with the NMR analyses. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodhyd.2019.04.043. 175
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antioxidant activities and alters rheological properties of the copolymer. Journal of Agricultural and Food Chemistry, 62, 9128–9136. Yan, H., Chen, X., Song, H., Li, J., Feng, Y., Shi, Z., et al. (2017). Synthesis of bacterial cellulose and bacterial cellulose nanocrystals for their applications in the stabilization of olive oil Pickering emulsion. Food Hydrocolloids, 72, 127–135. Yin, L.-J., Chu, B.-S., Kobayashi, I., & Nakasihima, M. (2009). Performance of selected emulsifiers and their combinations in the preparation of β-carotene nanodispersions. Food Hydrocolloids, 23, 1617–1622.
by chitosan-chlorogenic acid complexes. LWT Food Science and Technolog, 71, 295–301. Wei, Z., Wang, C., Zou, S., Liu, H., & Tong, Z. (2012). Chitosan nanoparticles as particular emulsifier for preparation of novel pH-responsive Pickering emulsions and PLGA microcapsules. Polymer, 53, 1229–1235. Woranuch, S., & Yoksan, R. (2013). Preparation, characterization and antioxidant property of water-soluble ferulic acid grafted chitosan. Carbohydrate Polymers, 96, 495–502. Xie, M., Hu, B., Wang, Y., & Zeng (2014). Grafting of gallic acid onto chitosan enhances
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Caffeic acid grafted chitosan as a novel dual-functional stabilizer for foodgrade emulsions and additive antioxidant property
T
Huri İlyasoğlua,1, Marcin Nadziejab, Zheng Guoa,∗ a b
Department of Engineering, Aarhus University, Gustav Wieds Vej 10, DK, 8000, Aarhus C, Denmark Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, DK, 8000, Aarhus C, Denmark
A R T I C LE I N FO
A B S T R A C T
Keywords: Caffeic acid Chitosan Dual-functional stabilizer Pickering emulsion Antioxidant
The aim of this study was to develop a novel dual-functional stabilizer for food-grade Pickering emulsions by grafting caffeic acid onto chitosan via a free radical mediated method. The grafting of caffeic acid onto chitosan was confirmed by UV–vis absorption spectra, FTIR spectra and 1H NMR spectra and DSC thermogram. The antioxidant properties of the caffeic acid grafted chitosan (CA-g-Ch) were measured by three in vitro assays. Caffeic acid-grafted chitosan displayed much better Fe2+ chelating property than both chitosan and caffeic acid, and retained comparable DPPH radical scavenging activity as caffeic acid. Moreover, the formation of the CA-gCh based emulsion was monitored by the turbidity, zeta potential, and particle size measurements. The CA-g-Ch polymers were found to be pH-responsive. The oil (linoleic acid) in water emulsions with the CA-g-Ch (as emulsifier/Pickering stabilizer) was prepared to examine its potential to stabilize Pickering emulsions, as well as its additive antioxidant properties. The results (zeta potential, mean droplet size, creaming index and TBARS values etc) demonstrated that the CA-g-Ch could act as a dual-functional stabilizer for Pickering emulsions. The emulsions made with the CA-g-Ch were found to be stable at a broad pH range of 3–6. Our findings proved that CA-g-Ch as novel dual-functional polymeric surfactant, may have great potential to find applications in food and pharmaceutical industries.
1. Introduction Pickering emulsions are stabilized by colloidal particles. Compared to conventional systems, Pickering emulsions allow for specific applications where increased stability and reduced toxicity are in demand (Liu, Wang, Zou, Wei, & Tong, 2012). Inorganic colloids, such as silica and clay, have been used for stabilizing Pickering emulsions (Rayner et al., 2014), however their use in food and pharmaceutical industries is limit due to a biocompatibility concern. Natural food grade polymers as Pickering emulsifiers are more acceptable for use in food and medical applications and therefore extensively studied as emulsion stabilizers. Polysaccharide-based particles including starch (Hong, Cheng, Gan, Lee, & Peh, 2018; Leal-Castenada et al., 2018), cellulose (Jia et al., 2015; Mikulcova, Bordes, Minarik, & Kasporkova, 2018), chitin (Barkhordari & Fathi, 2018; Tzoumaki, Moschakis, Kiosseoglou, & Biliaderis, 2011) and chitosan (Mwangi, Ho, Tey, & Chan, 2016; Wang & Heuzey, 2016) have been attempted to stabilize Pickering emulsions. They can be applied for the encapsulation of active food ingredients and enhancement of nutritional value or sensorial properties of foods.
Chitosan is an amino-polysaccharide obtained from partial deacetylation of chitin. Chitosan is biodegradable, biocompatible and nontoxic (Cheung, Ng, Wong, & Chan, 2015) and has been successfully used for stabilizing Pickering emulsions (Liu et al., 2012; Mwangi et al., 2016; Wei, Wang, Zou, Liu, & Tong, 2012). Chitosan has limited antioxidant capacity because there is no phenol-like functional group or conjugated structure in the molecule. Grafting of phenolic acids onto chitosan has been attempted to improve antioxidant activity of chitosan (Hu & Luo, 2016; Liu, Wen, Lu, Kan, & Jin, 2014; Schreiber, Bozell, Hayes, & Zivanovic, 2013; Woranuch & Yoksan, 2013; Xie, Hu, Wang, & Zeng, 2014). Although there are a lot of studies on the preparation of phenolic acids grafted chitosan, applications of these conjugates are limited. Applications of chitosan-based conjugates are primarily focused on the use of them as food coating or packaging materials (Hu & Luo, 2016). The application of phenolic acids grafted chitosan as a delivery system for food ingredients has not been intensively investigated. Caffeic acid, a type of phenolic acid, has excellent antioxidant properties. It has two phenol groups and a eCH=CHeCOOH group (Mathew, Abraham, & Zakaria, 2015). Grafting of caffeic acid
∗
Corresponding author. Department of Engineering, Aarhus University, Gustav Wieds Vej 10, Building, 3141, DK-8000, Aarhus C, Denmark. E-mail addresses: [email protected] (H. İlyasoğlu), [email protected] (M. Nadzieja), [email protected] (Z. Guo). 1 Department of Nutrition and Dietetics, Gümüşhane University, 29100 Gümüşhane, TURKEY. https://doi.org/10.1016/j.foodhyd.2019.04.043 Received 3 November 2018; Received in revised form 14 April 2019; Accepted 19 April 2019 Available online 20 April 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.
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2.4. FTIR analysis
onto chitosan could enhance its physicochemical and biological properties, thus functioning as a new ingredient. Specifically, it can be utilized as a chitosan-based stabilizer for food-grade Pickering emulsions, which can also be applied for the encapsulation of functional food ingredients, such as n-3 polyunsaturated fatty acids. Therefore, this study focused on design and synthesis of a novel dual-functional stabilizer from chitosan, possessing both antioxidant and emulsion-stabilizing properties for food-grade Pickering emulsions. The phenolic acid grafted chitosan has been synthesized using different methods including carbodiimide based chemical coupling method (Liu, Meng, Liu, Kan, & Jin, 2017; Woranuch & Yoksan, 2013) and free radical mediated grafting reaction (Lee, Woo, Ahn, & Je, 2014; Liu et al., 2015). The carbodiimide based coupling method requires use of toxic reagents. In contrast, the free radical method is a process that does not result in serious contamination from toxic reagents essentially used in the reaction of chemical coupling, which is safer and environmentally friendly and good for food application. In the present study, the caffeic acid grafted chitosan (CA-g-Ch) was synthesized by a free radical mediated method, and the synthesized compound was characterized using UV–vis, Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopies and differential scanning calorimetry (DSC). The antioxidant activity of CA-gCh was evaluated by three in vitro assays. The antioxidant and emulsifying properties of CA-g-Ch were investigated by preparing emulsions with linoleic acid as oil phase. The emulsions were characterized by dynamic light scattering (DLS) and creaming stability studies; and the oxidative stability of the emulsions was determined by accelerated TBARS test. The effect of stabilizer concentration (0.05–1%) and pH (3–8) on the physical and oxidative stability of the emulsions were also evaluated.
FTIR spectra was recorded in transmittance mode in the 4000600 cm−1 region at resolution 4 cm−1 with 16 scans, using a spectrophotometer (QFA Flex, USA) equipped with deuterium triglycine sulfate detector. The attenuated total reflection mode was used. Spectra were analyzed using GRAMS/AI software. 2.5. NMR analysis Chitosan and CA-g-Ch were dissolved in D2O with 1% CD3COOD and caffeic acid was dissolved in CH3OD. 1H NMR spectra was recorded at room temperature using a Bruker Ascend 400 NMR system (Germany) at 400 MHz. 2.6. DSC analysis The thermal properties were analyzed using differential scanning calorimetry on a Pyris 6 DSC system (PerkinElmer). Approximately 5 mg of each sample was put into an aluminum pan and placed in the equipment under a purging atmosphere of nitrogen (20 mL min−1), with an empty pan as an inert reference. The heating and cooling profile were: (1) Isothermal heating at 25 °C for 3 min; (4) Heating to 450 °C at a rate of 10 °C min−1 (5) Cooling to 25 °C with rate of 10 °C min−1. The DSC scans was evaluated using Micro Cal Origin 8.6 software. 2.7. Determination of caffeic acid content The caffeic acid content of the CA-g-Ch was determined using FolinCiocalteu method. The CA-g-Ch was solved in water. A 100 μL of the sample solution was mixed with 0.50 mL of diluted Folin-Ciocalteu reagent, 0.4 mL of sodium carbonate (1 M) and 4 mL of distilled water. The absorbance of the mixture was measured at 760 nm after 1 h. The calibration curve was prepared with caffeic acid standard ranging from 0 to 100 mg/mL. The caffeic acid content was expressed as mg caffeic acid equivalents per g (CAE/g) of the CA-g-Ch.
2. Materials and methods 2.1. Chemicals Chitosan (Aldrich 448869, low molecular weight: 50–190 kDa, Degree of deacetylation: 75–85%), ascorbic acid, caffeic acid, DPPH (2,2-Diphenyl-1-picrylhydrazyl), TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), trolox, ferrozine, iron chloride, ferric chloride, sodium carbonate, sodium triacetate, Folin Ciocalteu reagent, acetic acid, hydrogen peroxide and solvents were obtained from Sigma-Aldrich (Søborg, Denmark).
2.8. Determination of antioxidant activity Chitosan (2 mg), caffeic acid (2 mg), and CA-g-Ch (2 mg) were dispersed in ultrapure water (1 mL) with ultrasonication for 2 h. Antioxidant activity was measured using three vitro methods (DPPH, Fe+2 chelating, and FRAP assays). For the DPPH radical scavenging assay, a 50 μL of the sample solution was mixed with 950 μL of DPPH radical solution (100 μM). The absorbance was measured at 515 nm after 60 min at room temperature. The control sample was prepared with 50 μL of water instead of sample solution. The DPPH radical scavenging activity was calculated by following formula:
2.2. Preparation of caffeic acid grafted chitosan Chitosan (500 mg) was dissolved in acetic acid solution (2%, 50 mL) with stirring overnight. Hydrogen peroxide (1 M, 1 mL) and ascorbic acid (54 mg) were added to the chitosan solution, and then mixed at room temperature for 30 min. Caffeic acid (250 mg) was added to the reaction mixture and stirred at room temperature for 24 h. Washing with hot deionized water at 80 °C was repeated (up to 7 times) to remove most of unreacted caffeic acid before subjected to a dialysis process. The reaction mixture enclosed in a dialysis bag (14 kDa cut-off) was dialysed against deionized water. After 2 days’ dialysis, dialysate was freeze-dried.
Scavenging activity (%) = (AceAs)*100/Ac
(1)
Where Ac is the absorbance of control, and As is the absorbance of sample. For the Fe+2 chelating activity assay, a 300 μL of the sample solution was mixed with 120 μL iron chloride (3 mM) and ferrozine (3 mM). After 10 min, the absorbance was measured at 562 nm. The control sample was prepared with 300 μL of water with instead of sample solution. Fe+2 chelating activity was calculated by following formula:
2.3. UV–vis analysis UV–Vis spectra of chitosan, caffeic acid and CA-g-Ch were recorded by UV–Vis spectrophotometer (Varian, Carry 50 Bio) in the range of 200–500 nm. Chitosan and CA-g-Ch were dispersed in acetic acid solution (0.2%) and water, respectively; and stirred overnight until a complete dissolution. Caffeic acid was dissolved in ethanol for measurement. The UV–Vis spectra of the resulting solutions (25 μg/mL) were recorded.
Fe+2 chelating activity (%) = (AceAs)*100/Ac
(2)
Where Ac is the absorbance of control, and As is the absorbance of sample. For the FRAP assay; firstly, fresh FRAP reagent was prepared by mixing the following solutions (10:1:1): acetate buffer solution 169
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Fig. 1. DPPH radical scavenging activity (a), iron chelating power (b), and FRAP (c).
equivalent (TE).
(pH = 3.6), TPTZ solution in 40 mM HCl (10 mM) and FeCl3 (20 mM) solution, respectively. A 50 μL of the sample solution was mixed with 950 μL of FRAP reagent and the absorbance was measured at 595 nm after 20 min. The results were expressed as micromolar of Trolox 170
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Fig. 2. Transmittance at 600 nm of CA-g-Ch dispersion as a function of pH (a), zeta potential value of CA-g-Ch dispersion as a function of pH (b), and particle mean diameter of CA-g-Ch dispersion as a function of pH (c).
2.9. Characterization of CA-g-Ch
2.10. Emulsion preparation
CA-g-Ch (0.1%) was dispersed in water and stirred overnight. The pH of the dispersions (5.60) was adjusted to the value, ranging from 3 to 8, with 1 N NaOH or HCl. The transmittance at 600 nm was measured by UV–Vis spectrophotometer (Varian, Carry 50 Bio). Particle diameter and zeta potential were determined by a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C.
CA-g-Ch was dispersed in water and stirred overnight. Emulsions consisting of CA-g-Ch as stabilizer, linoleic acid (10%) as oil phase, antimicrobial agent (sodium azide) and water were prepared. To evaluate the effect of concentration on the physical stability of the emulsions, the emulsions with CA-g-Ch in the range of 0.05–1% were prepared. Firstly, the aqueous and lipid phase were stirred in a magnetic stirrer for 15 min at 1000 rpm. The resulting systems were
171
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Table 1 Mean droplet size, polydispersity index (PDI), zeta potential, creaming index (CI), and degree of oxidation of the emulsions with different stabilizer CA-g-Ch concentrations. Stabilizer concentration (%)
Mean
c 0.25 chitosan 0.05 0.10 0.25 0.50 1.00
456 583 540 400 343 378
a
± ± ± ± ± ±
a
droplet size (nm)
PDI
35 76 20 11 7 4
0.25 0.17 0.24 0.22 0.17 0.14
Zeta potential ± ± ± ± ± ±
0.04 0.01 0.03 0.05 0.04 0.01
a
(mV)
42.14 ± 1.3 40.0 ± 0.1 43.7 ± 1.2 41.1 ± 1.0 41.8 ± 0.3 36.2 ± 0.8
b
CI (%) 15.1 15.0 15.0 15.0 15.0 15.0
± ± ± ± ± ±
0.1 0.1 0.1 0.1 0.1 0.1
Degree of oxidation (%), TBARS test
70.1 ± 5.6 7.4 ± 0.9 10.1 ± 0.8 48.7 ± 7.1 51.9 ± 9.7 50.6 ± 5.8
a
Measurements were done on the same day of emulsion preparation. The control emulsion was prepared using Tween 20 (1%) as the emulsifier; and its degree of oxidation is defined as 100%. See the details for the measure conditions in 2. MATERIALS AND METHODS, 2.14. TBARS test. c The chitosan control with 0.25% chitosan as stabilizer and 1% Tween 20 as the emulsifier. b
room temperature with tap water. The absorbance of the colored complex was measured after centrifugation at 4000 rpm for 5 min. The degree of oxidation was calculated by following formula:
sonicated (Bandelin Sonopuls HD 2200) for 2 min at an amplitude of 70%, and a duty cycle of 0.5 s in an ice-bath. To evaluate the effect of pH on the physical stability of the emulsions, pH of the aqueous phase was adjusted to the value, ranging from 3 to 8, with 1 N NaOH or HCl.
Oxidation (%) = As*100/Ac
Where, As is the absorbance of the sample including stabilizer, Ac is the absorbance of the control emulsion prepared with Tween 20 (1%).
2.11. Emulsion properties (droplet size and zeta potential) The oil droplet size distribution and zeta potential were determined in a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C. The emulsions were diluted with deionized water (1:10). The size distribution of oil droplets was determined by DLS using non-invasive backscatter optics. Mean values of three measurements were calculated.
3. Results and discussion 3.1. UV spectra The UV spectra of chitosan (Ch), caffeic acid (CA), and caffeic acid grafted chitosan (CA-g-Ch) are presented in Fig. S1. UV spectra confirmed the grafting of caffeic acid onto chitosan. Chitosan exhibited no absorption peak at the studied wavelengths (200–500 nm) whereas caffeic acid gave two absorption bands at 298 nm and 325 nm, respectively. As the CA alone, the CA-g-Ch exhibited two absorption bands. The wavelengths of the UV–vis absorption peaks of the CA-g-Ch (297 nm and 324 nm) were close to those of the CA, implying the grafting of caffeic acid onto chitosan was successful.
2.12. Creaming stability Emulsions were transferred to clear vials (15 mL, ring 1.8 cm), and stored at room temperature for 1 week. The emulsions separated into two layers: a) an optically opaque “cream layer” at the top, b) turbid “serum layer” at the bottom. Creaming index (CI) was calculated in accordance with Equation (3). CI = 100* (Hc/HE)
(4)
(3)
3.2. FTIR spectra
Where, Hc is height of cream layer, and HE is height of emulsion. The FTIR spectra of Ch, CA, and CA-g-Ch are presented in Fig. S2. The FTIR spectra supported the grafting of caffeic acid onto chitosan. Chitosan showed characteristics bands around 3350 cm−1(OeH stretching), 2930-2830 cm−1 (CeH stretching), 1650 cm−1(amide I, C=O stretching), 1590 cm−1(NeH bending of primary amine), 1540 cm−1 (amide II, NeH bending), 1030 cm−1(CeOeC), and 890 cm−1(pyranose ring), respectively (Jiang et al., 2010; Kumirska et al., 2010). Caffeic acid exhibited characteristic peaks around 3400 cm−1 (OeH stretching), 2900-3000 cm−1 (CeH stretching), 1530 cm−1 (C=C stretching), 1350 cm−1 (OeH bending), and 1220 cm−1 (CeOH stretching) (Swislocka, 2013). Compared to the chitosan, the band at 1590 cm−1 was no longer visible in the spectra of the CA-g-Ch, indicating the amide bond formation between chitosan and caffeic acid. An increase in the intensity of the peaks around 12001600 cm−1 was observed, suggesting the grafting of caffeic acid onto chitosan as well.
2.13. Confocal laser scanning microscopy The droplet size distribution of the emulsions and the distribution of stabilizer in the emulsions were visualized by the confocal laser scanning microscopy (Zeiss LSM780). For imaging of the lipid phase, Nile red solution (10 μL, 1 mg/mL in acetone) was added to the emulsion (1 mL) and then the emulsion was gently stirred. Nile red molecules were excited at a wavelength of 514 nm, and fluorescence emission intensity was collected over 539–753 nm. For imaging of the stabilizer, Rhodamine 6G (10 μL, 1 mg/mL in water) was added to the emulsion (1 mL), and then the emulsion was gently stirred. Rhodamine 6G molecules were excited at a wavelength of 514 nm, and fluorescence emission intensity was collected over 517–696 nm. The images were analyzed using Zeiss LSM Image Browser software. 2.14. Thiobarbutiric acid reactive substances (TBARS) test
3.3. 1H NMR spectrum Secondary oxidation products were determined by TBARS test. Thiobarbutiric acid (TBA) solution including trichloroacetic acid (15 g), TBA (375 mg), HCl (1.76 mL, 12 N), and water (82.9 mL) was prepared. A 20 μL of the emulsions was diluted with water to 1 mL. To accelerate oxidation, 250 μL of FeSO4 (25 mM) was added to the emulsions, and then the mixture was stirred at 200 rpm for 15 min. A 2 mL of TBA solution was added to the mixture, and then incubated at boiling water bath for 15 min. The TBA formed colored complex were cooled to the
The NMR spectra of Ch, CA, and CA-g-Ch are presented in Fig. S3. Chitosan showed a single peak at 1.95 ppm, representing the methyl group of the N-acetyl glucosamine units. It also presented a single peak at 3.05 ppm (H-2), multiple peaks at 3.20–4.00 ppm (H-3 to H-6), a small single peak at 4.45 ppm (H-1) (Liu et al., 2014). The methine protons of caffeic acid gave signals at 6.24 ppm (H-h), 6.78 ppm (H-e), 6.91 ppm (H-f), 7.04 ppm (H-b), and 7.51 ppm (H-g), respectively 172
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Table 2 Mean droplet size, polydispersity index (PDI), zeta potential, creaming index (CI), and degree of oxidation of the emulsions with different pH. pH
Mean a droplet size (nm)
PDI
3 4 5 6
554 553 521 435
0.37 0.20 0.43 0.24
± ± ± ±
7 13 6 11
a
± ± ± ±
0.03 0.03 0.01 0.01
Zeta potential a (mV)
CI (%)
43.1 57.1 46.0 38.5
14.3 15.0 15.8 15.8
± ± ± ±
0.7 2.0 0.9 2.9
± ± ± ±
b
Degree of oxidation (%)
0.1 0.1 0.1 0.1
12.5 ± 3.8 10.5 ± 0.2 9.9 ± 0.9 8.1 ± 0.7
Emulsions: 0.1% CA-g-Ch, 10% oil load. a Measurements were done on the same day of sample preparation. b The control emulsion was prepared using Tween 20 (1%) as the stabilizer at pH 5.4; and its degree of oxidation is defined as 100%. See the details for the measure conditions in 2. MATERIALS AND METHODS, 2.14. TBARS test.
2014; Liu et al., 2014). 3.4. DSC thermogram The DSC thermogram of Ch, CA, and CA-g-Ch are presented in Fig. S4. Chitosan exhibited a broad endothermic peak (130–180 °C) and a deep exothermic peak (300 °C). The broad endothermic peak was previously attributed to the evaporation of water adsorbed and bound to the chitosan (Kittur, Prashanth, Sankar, & Tharanathan, 2002). The exothermic peak could be related to the thermal unfolding of chitosan chain stacking (Pereira, da Silva Agostini, Job, & Gonzalez., 2013). Compared to the chitosan (a deep exothermic peak at 300 °C), the CA-gCh had a shallow exothermic peak (around 280 °C), shifting to a lower temperature; indicating that the grafting reaction has loosen and broken the stack pack of chitosan chains by introducing caffeoyl groups. The DSC thermogram of the caffeic acid exhibited one exothermic peak (234 °C), representing melting point of caffeic acid. Our findings revealed that Ch and CA-g-Ch had different thermal behaviour, providing evidence for the grafting of caffeic acid onto chitosan and explanation for their different chemo-physical properties. 3.5. Caffeic acid content and antioxidant activity The grafting ratio of the CA-g-Ch was found to be as 99 ± 3 mg caffeic acid equivalent/g by the Folin Ciocalteu method. FTIR spectra suggested the conjugation of caffeic acid occurred at amino group of chitosan. The antioxidant activities of Ch, CA, and CA-g-Ch were measured using three in vitro assays (Fig. 1). Compared to the chitosan, the CA-g-Ch showed higher antioxidant activity. At the concentration less than 0.6 mg/mL, the CA had higher DPPH radical scavenging activity than the CA-g-Ch, whereas the CA-g-Ch exhibited similar DPPH radical scavenging activity at the concentration between 0.6 mg/mL and 1 mg/mL. At the concentration of 1 mg/mL, the DPPH radical scavenging activity of the CA-g-Ch was found to be more than 90%. The CA-g-Ch showed more FRAP than the chitosan, whereas it had lower FRAP than the CA. Our findings have revealed that caffeic acid grafting onto chitosan can enhance the antioxidant activity of chitosan and provide a new material for industrial applications. The modification of chitosan could improve its solubility and physicochemical properties, and thus providing excellent antioxidant properties as good as caffeic acid. Similar findings were reported for gallic acid grafted chitosan, which showed comparable DPPH radical scavenging activity at high concentrations as gallic acid and higher chelating ability than gallic acid (Xie, Hu, Wang, & Zheng, 2014).
Fig. 3. CSLM images of the emulsions including different stabilizer concentration (0.05–1%). Nile Red (red) and Rhodamine 6G (green). Scale bar 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
(Swislocka, 2013). Compared to the chitosan, the CA-g-Ch exhibited new peaks at 6.24–7.44 ppm, representing the methine protons of the caffeic acid, confirming the successful grafting of caffeic acid onto chitosan. Our results were consistent with the literature (Lee et al.,
3.6. Properties of CA-g-Ch The CA-g-Ch (1 mg/mL) was dissolved in the deionized water, and its pH value (5.6) was adjusted to 3, 4, 5, 6, 7, and 8 using 0.5 M NaOH 173
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emulsion upon precipitation (Mwangi et al., 2016). All emulsions showed similar creaming stability (CI:15%) at the end of storage, indicating that the concentration of CA-g-Ch had no significant influence on the creaming stability. These results may be attributed to a similar zeta potential value at all concentrations and polymeric structure of the CA-g-Ch. A higher zeta potential value provides resistance against droplet coalescence, and an emulsion system with a value higher than ± 30 mV is considered as stable (Mikulcova et al., 2018). The polymeric structure of the stabilizer may present steric hindrance, preventing droplet coalescence. Compared to the same concentration of CA-g-Ch, Ch based Pickering emulsion show similar Zeta potential and CI value but larger mean droplet size; indicating a better surface-active property of CA-g-Ch adsorbed at oil-water interface. CLSM images (Fig. 3.) revealed that the distribution of oil droplets was homogeneous in the emulsions. The oxidative stability of the emulsions was investigated by means of accelerated TBARS test. The emulsions with the CA-g-Ch exhibited better oxidative stability than the control emulsion prepared with both the Tween 20 (1%) and the chitosan control (Table 1). Due to the presence of eOH and eOe groups in chitosan for chelating function and positive surface-charge of the Ch-based emulsion, the chitosan control showed better antioxidation property (70%) than the Tween 20 control (100%). The degree of the oxidation varied from 7% to 52% (Table 1) compared to the control (100%). The emulsions with the CAg-Ch had positively charged droplets, which could electrostatically repel iron cations, and reduce the lipid oxidation rate (Kargar, Fayazmanesh, Alavi, Spyropoulous, & Norton, 2012). Moreover, the CA-g-Ch was found to possess good iron chelating ability, which might help retard lipid oxidation. The emulsion with a concentration of 0.05–0.1% had higher oxidative stability compared to the emulsion with a concentration of 0.25–1%. These findings may be related to prooxidant effect of the CA-g-Ch. Maurya and Devasagayam (2010) found that the caffeic acid showed antioxidant behaviour at low concentration and pro-oxidant behaviour at high concentration. The pro-oxidant behaviour of the caffeic acid was previously associated with iron reducing ability. From Table 1, the degree of oxidation increases as mean droplet size drops, which might indicate the increasing surface area/volume ratio increases the chance for the migration of Fe2+ from continuous water into oil phase. Polysaccharide-based particles including starch (Hong et al., 2018; Leal-Castenada et al., 2018), cellulose (Jia et al., 2015; Mikulcova et al., 2018), chitin (Barkhordari & Fathi, 2018; Tzoumaki et al., 2011) and chitosan (Mwangi et al., 2016; Wang, & Heuzey, 2016) were found to be effective in stabilizing microemulsions. Compared to these studies, the CA-g-Ch seemed to present some valuable properties, such as stabilizing oil-in-water nanoemulsion at low concentration or delay of lipid oxidation. It can be concluded that the grafting of caffeic acid onto chitosan may enhance its antioxidant and emulsifying properties, enabling a dual-functional stabilizer.
or 0.5 M HCl. As seen from Fig. 2a, the transmittance of the CA-g-Ch dispersions slightly decreased with increasing pH value in the range of 3–7. At these pH ranges, the transmittance of the solutions was higher than 90%, and the solutions were transparent. At pH 8, the transmittance of the solution sharply decreased (< 50%), and the solution became turbid. When the pH value of the CA-g-Ch solution could revert to the value below 7 using 0.5 M HCl, the solution became transparent again. These findings revealed that the CA-g-Ch was a pH-responsive polymer as the chitosan (Mwangi et al., 2016; Liu et al., 2012). The amino groups of the CA-g-Ch could be protonated at low pH (< 7), converting the polymer to a water-soluble cationic polyelectrolyte. The zeta potential values of the CA-g-Ch (Fig. 2b) supported this hypothesis. At high pH (> 7), the amino groups of the CA-g-Ch could be deprotonated, and the polymer could turn into insoluble form (Kumirska et al., 2010; Liu et al., 2012). The zeta potential values of the CA-g-Ch dispersions were found to decrease with increasing pH value (Fig. 2b). The CA-g-Ch dispersions could carry strong positive charge (> +30) at the pH values, ranging from 3 to 5, whereas it could carry weak positive charge (< +20) at the pH value higher than 6. These findings can be due to the presence of amino groups on the caffeic acid grafted chitosan's backbone. Chitosan has amino groups, that are protonated or deprotonated depending on the pH value of the medium (Kumirska et al., 2010). The mean diameter of the CA-g-Ch dispersions was less than 1 μm at the pH values between 3 and 7. However, it sharply increased with an increase in the pH value, ranging from 7 to 8, and reached to the highest value (3.5 μm). These findings may be attributed to the flocculation of the CA-g-Ch dispersions, depending on the pH value. At above pH 6, the CA-g-Ch dispersion had weak positive charges, implying lower electrostatic repulsion between the CA-g-Ch dispersions. Higher electrostatic repulsion enhances the physical stability of dispersion (Yin, Chu, Kobayashi, & Nakasihima, 2009). 3.7. Emulsions Homogenous and white emulsions were obtained by employing the high-energy input from ultrasonic probe to mix oil and aqueous phases and the emulsion properties including zeta potential, mean droplet size, polydispersity index (PDI), creaming index (CI), and oxidative stability were determined. Linoleic acid was selected as the oil phase to evaluate the ability of the CA-g-Ch to prevent lipid oxidation. 3.7.1. Effect of CA-g-Ch concentration on the stability of emulsions To investigate the effect of concentration on the physical and oxidative stability of emulsions, the emulsions with different concentration CA-g-Ch (0.05%, 0.1%, 0.25%, 0.50% and 1%) were prepared and characterized as shown in Table 1. The chitosan control with 0.25% chitosan as stabilizer and 1% Tween 20 as the emulsifier is also presented. All emulsions had strong positive charges, ranging from +36 to +44, implying possible high electrostatic repulsion between oil droplets. Higher electrostatic repulsion is known to enhance the physical stability of an emulsion by improving its anti-aggregation (Dickinson, 2009). The positive charges of the emulsions could be related to the presence of primary amino groups in Ch and CA-g-Ch. The mean droplet size of the emulsions varied from 343 nm to 583 nm. The mean droplet size of the emulsions decreased with increasing stabilizer concentration. With increasing stabilizer concentration in the aqueous phase, an increase in the number of adsorbed particles at the oil-water interface could result in a reduction in the droplet size. The adsorbed particles at the oil-water interface could cover much larger area per volume to reduce droplet size (Yan et al., 2017). The PDI values of the emulsions ranged from 0.17 to 0.25, indicating a narrow size distribution. The phase separation was observed during 1 week of storage at the room temperature to determine the creaming stability of the emulsions. A white cream layer at the top was observed at the first day. Formation of creaming is a characteristic property of Pickering
3.7.2. Effect of pH on the stability of emulsions The pKa value of glucosamine is 12.27, pKb is 1.72. pKa of chitosan showed a slightly decreasing from 6.51 to 6.39 (Wang et al., 2006), depending on the molecular weight. Caffeic acid pKa value is 4.62. As some amine groups in CA-g-Ch were modified by caffeic acid, so the pKa value is CA-g-Ch is higher than Ch but lower than glucosamine, as the density of eNH2 group in glucosamine is much higher than in CA-gCh. However, we have not measured the apparent pKa of the CA-g-Ch in the present study. The effect of pH value on the stability of the emulsions was evaluated as the CA-g-Ch dispersions were found to be soundly pH-responsive. The pH value of the freshly prepared emulsion (0.1% CA-g-Ch, and 10% oil load) was adjusted to 3, 4, 5, 6, 7, and 8, respectively. The emulsions were broken when the pH was adjusted to 7 or 8. Coalescence, flocculation or aggregation of oil droplets could occur when electrostatic repulsion between oil droplets is low (Mikulcova et al., 2018). An increase in the pH value above pKa of the 174
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References
amino groups could result in the droplet coalescence as surface charge density decreased. At the pH 7 or 8, the CA-g-Ch dispersions had a much lower zeta potential value (< +13). The emulsifying properties at low pH are presented in Table 2. All emulsions had strong positive charges (> +30), ranging from +39 to +57. The mean droplet size of the emulsions varied from 435 nm to 554 nm, indicating that the CA-g-Ch had ability to stabilize oil-in-water emulsion at nanometric scale. The mean droplet size slightly decreased with pH increasing from 3 to 6. The polydispersity index of the emulsion was in the range of 0.20–0.43, indicating narrow to moderate size distribution. The phase separation was observed during 1 week of storage at the room temperature in the determination of the creaming stability of the emulsions. The CI values of the emulsion ranged from 14.3% to 15.8%. The emulsion with pH 3 showed higher creaming stability followed by the emulsion with pH 4. Both emulsions (pH 5 and pH 6) showed similar CI values. Our findings indicated that the CA-g-Ch had ability to stabilize oil-in-water emulsion by being adsorbed to the oil-water interface at the pH values between 3 and 6. The oxidative stability of the emulsions was determined by the accelerated TBARS test to investigate the influence of pH on the oxidative stability of the emulsions. The control emulsion was prepared using Tween 20 (1%) as a stabilizer. The degree of oxidation was found to be ranged from 8% to 13% compared to the control (100%), indicating that the CA-g-Ch had potent ability to prevent oxidation in the emulsions between pH 3 and pH 6. The oxidative stability of the emulsions slightly increased with increasing pH value. A lower degree of oxidation observed in the emulsion stabilized with the CA-g-Ch could be due to two reasons. Firstly, CA-g-Ch showed both good radical scavenging activity and iron chelating ability as antioxidant. Secondly, it could act as surface-active emulsifier/stabilizer capable to be localized at oil-inwater interface. Wei and Gao (2016) reported that the chitosan chlorogenic acid conjugate inhibited the β-carotene degradation in the oil-in-water emulsion due to its excellent antioxidant properties. A similar finding was reported for the emulsion stabilized with the chitosan-EGCG (epigallocatechin-3-gallate) (Lei, Liu, Yuan, & Gao, 2014). It may be thus concluded that the CA-g-Ch could exhibit excellent antioxidant activity in the emulsion system as other chitosan-phenolic acid conjugates.
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4. Conclusion A novel dual-functional stabilizer, caffeic acid grafted chitosan (CAg-Ch), was developed and applied for preparation of Pickering emulsions. The CA-g-Ch was synthesized by ascorbic acid-initiated grafting of caffeic acid onto chitosan. The CA-g-Ch product showed better Fe+2 chelating property than caffeic acid and comparable DPPH radical scavenging activity with caffeic acid. The CA-g-Ch polymeric surfactant demonstrated ability to stabilize oil-in-water nanoemulsion in the pH range of 3–6. The CA-g-Ch polymeric surfactant also had potential to retard lipid oxidation. The CA-g-Ch polymeric surfactant may open a new route for the preparation of food-grade Pickering emulsions. Acknowledgement The authors thank The Scientific and Technological Research Council of Turkey (TÜBİTAK), Turkey, for financial support of Huri İlyasoğlu (Grant No. TÜBİTAK 2219 International Post Doctoral Research Fellowship Programme). Sampson Anankanbil from Aarhus University is thanked for his kind help with the NMR analyses. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodhyd.2019.04.043. 175
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