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gelatin-based materials functionalized by pomegranate peel extract
Carbohydrate Polymers 228 (2020) 115386
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Rheological and antioxidant properties of chitosan/gelatin-based materials functionalized by pomegranate peel extract
T
⁎
Mirella R.V. Bertoloa, , Virginia C.A. Martinsa, Marilia M. Hornb, Lívia B. Brenellic,d, Ana M.G. Plepisa a
São Carlos Institute of Chemistry (IQSC), University of São Paulo (USP), São Carlos, São Paulo, Brazil Macromolekulare Chemie und Makromolekulare Materialien, Universität Kassel, Kassel, Germany c Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, São Paulo, Brazil d Interdisciplinary Center of Energy Planning, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Gelatin Pomegranate peel extract Rheology Antioxidant Food coating
Biopolymer-based materials are potential candidates for food coatings application. In this study, pomegranate (Punica granatum L.) peel extract (PPE) at different concentrations was incorporated to chitosan/gelatin gels and the rheological, antioxidant and structural properties were evaluated. Due to its high phenolic content, PPE enhanced the antioxidant capacity of chitosan/gelatin mixtures. PPE addition extended linear viscoelastic range and enabled the samples to easily flow under the applied shear rate. Rheological properties indicated that both viscosity and activation energy of materials containing natural compounds are highly dependent on temperature. Scanning electron microscopy (SEM) images revealed the influence of PPE concentration in the scaffolds pores size. Findings of this study proved that PPE was capable to improve the functional characteristics of chitosan/ gelatin-based materials enhancing the desired properties for their potential application as food coatings.
1. Introduction Chitosan is a cationic polysaccharide which can be obtained by the deacetylation of chitin, a polymer found in the exoskeleton of crustaceans and insects (Nair, Saxena, & Kaur, 2018). Due to its biodegradability, biocompatibility, antimicrobial activity and non-toxicity, chitosan is considered a very promising and eco-friendly material for different purposes (Dutta, Tripathi, Mehrotra, & Dutta, 2009; Kanatt, Rao, Chawla, & Sharma, 2012; Nair et al., 2018). Moreover, chitosanbased materials (CB) can be designed in different forms, including gels, films and porous scaffolds. These materials can trap essential oils and bioactive compounds in their structure (Aloui et al., 2014), which enables their use in diverse fields, such as food coatings and drug-controlled release systems (Tang, Guan, Yao, & Zhu, 2014). Regarding that, many studies explored the use of chitosan-based systems associated with essential oils or plant extracts. The applicability of these systems is based on their potential biological and
antimicrobials activity, antifungals and antioxidants properties (Silalahi, Situmorang, Patilaya, & Silalahi, 2016; Sugita, Wijaya, Syahbirin, Dewi, & Sugita, 2017; Yuan, Lv, Tang, Zhang, & Sun, 2016; Yuan, Chen, & Li, 2016). Following this concept, the development of chitosan-edible films and coating materials containing essential oils and plant extracts represents an alternative to substitute non-renewable sources (Nair et al., 2018). Lozano-Navarro et al. (2018) described the preparation of chitosan/ starch films associated with natural antioxidants, such as anthocyanins from pomegranate and blueberry, resveratrol from grape and carvacrol from oregano, concluding that these materials exhibited promising positive properties for food packaging applications. In another study, Kanatt et al. (2012) prepared chitosan-polyvinyl alcohol films containing mint extract and pomegranate peel extract, which can serve for food packaging materials with desired antioxidants properties. Although there are researches which describe the use of CB as food coatings, strategies are currently being formulated to improve their
Abbreviations: 1HNMR, proton nuclear magnetic resonance spectroscopy; C2G1, chitosan/gelatin, 2:1 mixture; C2G1E100, chitosan/gelatin, 2:1 mixture, incorporated with 100 mg of PPE; C2G1E150, chitosan/gelatin, 2:1 mixture, incorporated with 150 mg of PPE; C2G1E200, chitosan/gelatin, 2:1 mixture, incorporated with 200 mg of PPE; C2G1E50, chitosan/gelatin, 2:1 mixture, incorporated with 50 mg of PPE; DPPH, 2,2-diphenyl-1-picrylhydrazyl; GAE, gallic acid equivalent; IC50, sample concentration necessary to inhibit 50% of DPPH radical; LVR, linear viscoelastic region; PPE, pomegranate peel extract; SEM, scanning electron microscopy; TPC, total phenolics content ⁎ Corresponding author at: Av. Trabalhador São Carlense, 400, São Carlos, SP, Zip code 13566-590, Brazil. E-mail address: [email protected] (M.R.V. Bertolo). https://doi.org/10.1016/j.carbpol.2019.115386 Received 3 July 2019; Received in revised form 25 September 2019; Accepted 25 September 2019 Available online 28 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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rinsing and drying at 60 °C overnight. The yield of the reaction was 27.8%, which was calculated from the initial mass of squid pens and agrees with a previous study (Kurita, 2006). Pomegranate peel extract (PPE) was extracted according to the procedure adapted from Yuan, Lv et al. (2016), Yuan, Chen et al. (2016). Initially, the peels were removed from the fruits, washed, lyophilized and crushed to obtain a thin powder. Then, the powder (1 g) was dissolved in a 60% (v/v) hydroethanolic solution (60% ethanol/ 40% water), in the solid:liquid ratio of 1:30 (w/v). The extraction procedure was carried out for 1 h at 50 °C, ensuring an extraction yield of 51.6% which was determined relative to the initial mass of peel powder. The solution was filtered and lyophilized until constant weight, which means that the extract was completely dried. The final product was a reddish extract.
functionality and reduce the limitation of chitosan low solubility at neutral pH, as well as its poor mechanical resistance and low elasticity. To improve CB functional properties, the combination with proteins or other polysaccharides has been described (Nowzari, Shábanpour, & Ojagh, 2013). Thus, gelatin is a polypeptide that can be considered a suitable option to associate with CB improving not only the mechanical but also, the functional properties. Gelatin is obtained by the partial hydrolysis of collagen and it is a valuable component to produce biodegradable materials for medical and food areas (Nair et al., 2018). Pomegranate (Punica granatum L.) is a highly nutritious fruit which contains many bioactive compounds even in its inedible parts, such as peel and seeds. Pomegranate peel is considered an agro-waste (Ismail, Sestili, & Akhtar, 2012; Malviya & Jha, 2014; Nair et al., 2018), and it is reported as a potential source of antioxidant compounds, besides exhibiting antifungal, antimicrobial and antibacterial activities (Malviya & Jha, 2014). The peels represent approximately 60% of the total weight of pomegranate fruit (Lansky & Newman, 2007), which provides higher antioxidant capacity and higher total phenolic content in comparison with the amount found in the pulp (Li et al., 2006). Compounds as tannins, alkaloids, flavonoids and organic acids are responsible for the health benefits previously reported for pomegranate peel, including treatment and prevention against cancer, cardiovascular diseases, diabetes, erectile disfunctions, as well as protection against ultraviolet radiation. Also, studies using pomegranate peel extract for the treatment of Alzheimer’s disease, arthritis and obesity were already reported (Cook & Samman, 2006; Lansky & Newman, 2007; Mohammad & Kashani, 2012; Qu, Pan, & Ma, 2010; Zhang, Fu, & Zhang, 2011). The interest of using chitosan-based systems associated with pomegranate peel (Kanatt et al., 2012; Nair et al., 2018; Yuan, Lv et al., 2016, Yuan, Chen et al., 2016) has also been growing. Nevertheless, there is no literature related to chitosan/gelatin/pomegranate peel association, although they are excellent candidates for the development of systems with high antioxidant capacity. Moreover, its rheological properties should be evaluated to predict their stability under certain conditions of temperature, viscosity, shear and stress rate (Razmkhah, Mohammad, & Razavi, 2017). It is assumed that the addition of phenolic components from pomegranate peel extract increases the antioxidant properties of the polymeric system, which improves its potential application as a biobased material for food coating technology. However, linkages of phenolic groups with both polymers are expected, which may change their main rheological characteristics, such as viscosity, elasticity and gel strength. Thus, this paper focuses on an unprecedented study about how the incorporation of pomegranate peel extract can affect the structural, rheological and antioxidant properties of a chitosan and gelatin polymeric system.
2.2. Solutions and samples preparation A 1% (w/w) chitosan gel was prepared by dissolution of the polysaccharide in a 1% acetic acid (HAc) aqueous solution under stirring at room temperature for 24 h. Gelatin solution with concentration of 1% (w/w) was obtained in deionized water under constant stirring at 60 °C for 30 min. After heating, the solution was placed at 4 °C for 3 h to enable the gelation process. Chitosan/gelatin control, labelled as C2G1, was prepared by mixing both solutions at 2:1 (w/w) proportion at 45 °C. PPE hydroethanolic solutions (50%, v/v) were prepared at concentrations of 50, 100, 150 and 200 mg mL−1. A series of chitosan/ gelatin and PPE solutions was prepared by adding 1 mL of each PPE solution to the chitosan/gelatin 2:1 mixture under constant stirring at 45 °C. Samples were labelled as C2G1E50, C2G1E100, C2G1E150 and C2G1E200, respectively. To prevent the dilution effect, 1 mL of a 50% (v/v) hydroethanolic solution without PPE was added to the control sample (C2G1). 2.3. Methods 2.3.1. Chitosan characterization Chitosan acetylation degree (6.7%) was determined by proton nuclear magnetic resonance spectroscopy (1H NMR) according to the method developed and validated by Lavertu et al. (2003). The molecular weight (327 kDa) was determined by capillary viscosimetry procedure (Rinaudo, 2006) in which flow time measurements were performed on an AVS-306 (SCHOTT) viscosimeter coupled to a universal TITRONIC automatic diluter (SCHOTT). 2.3.2. Fourier-transform infrared spectroscopy (FTIR) FTIR was employed to examine the chemical structure of the components. Analysis was performed in films by casting of diluted chitosan/ gelatin mixtures and drying at room temperature following storage in a desiccator with NaOH(s). PPE spectrum was obtained using KBr pellets. FTIR spectra were obtained using a Shimadzu IR Affinitu-1 at 4000 400 cm−1 interval with 4 cm−1 resolution.
2. Experimetal 2.1. Materials All the solvents and the reagents were of analytical grade and used without further purification. Doryteuthis spp squid pens were obtained at Miami Comércio e Exportação de Pescados Ltda in Cananéa-SP and were used as a source of β-chitin. Gelatin (type A, swine, ∼300 bloom) was purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). Pomegranate fruits were acquired in the local commerce of São Carlos – SP- Brazil. β-chitin extracted from squid pens was used to produce chitosan by means of the deacetylation process, following the procedure adapted from Horn, Martins, Maria, and Plepis (2009)). Briefly, squid pens were immersed in a 0.3 mol L−1 NaOH solution at 80 °C for 1 h to remove proteins and chitin was isolated as the main product. Finally, the second step consisted of an alkaline deacetylation performed by an immersion of chitin in a 40% (w/w) NaOH solution at 80 °C for 3 h, followed by
2.3.3. Antioxidant assays 2.3.3.1. PPE total phenolics content (TPC). The TPC of pomegranate peel extract was determined using the Folin-Ciocalteu method, as described by Singleton, Orthofer, and Lamuela-Raventós (1999), with some modifications. In this procedure, gallic acid was used as an internal standard. The Folin-Ciocalteu method is used to determine the total phenolic content of plant extracts and essential oils. Folin's Reagent is mixture of tungstate and molybdates, which in alkaline medium are reduced due to the presence of a phenolic compound. The final products are phenolate anions, a bluish chromogen with a maximum UV absorbance at 765 nm. A 96-well microplate was filled in with 25 μL of PPE hydroethanolic solutions at different concentrations, 150 μL of ultrapure water and 2
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2.3.4.3. Steady shear measurements. The steady shear flow properties were measured at a shear rate of 0.4 to 1000 s−1 at different temperatures (15, 20, 25, 30, 35 and 40 °C). Cross model (Eq. (4)) was used to fit the experimental flow curves (Razmkhah et al., 2017):
25 μL of Folin-Ciocalteu reagent. The microplate was stirred for 20 s and stand at room temperature for 5 min. Then, 100 μL of 7% (w/w) sodium carbonate solution was added to each well to ensure the reaction, and the microplate was stored protected from light. After 2 h, the absorbance at 765 nm was measured on a SpectraMax M3 (Molecular Devices) UV–vis spectrophotometer with SoftMax Pro 6.1 software. The results were expressed as equivalent mg of gallic acid (mg GAE) per mg of extract. Following the same procedure, the TPC determination in C2G1E200 mixture was performed to evaluate if phenolic content changes after incorporation in chitosan/gelatin mixture.
η − η∞ 1 = η0 − η∞ (1 + (kγ )n)
In Eq. (4), η0 is the zero-shear viscosity (Pa s), η∞ is the viscosity limit at infinite shear (Pa s), γ is the shear rate (s−1) and k and n are constants, k being the consistency index (s) and n the rate index (dimensionless). The effect of temperature on apparent viscosity of the mixtures was determined according to an Arrhenius-type model (Eq. (5)) (Steffe, 1996), using the values of η0 determined by the fitting of experimental flow curves:
2.3.3.2. DPPH antioxidant assay. The free radical scavenging antioxidant capacity of PPE was evaluated by the DPPH (2,2diphenyl-1-picrylhydrazyl) radical assay, according to the method described by Pal et al. (2017) with some modifications. First, a 25 mmol L−1 radical solution, violet colored, was prepared in ethanol. Then, 80 μL of the radical solution was mixed with 190 μL of ethanol and 10 μL of PPE and C2G1E200 sample at different concentrations. The samples absorbance was measured at 517 nm, and the decay was monitored for 30 min. A SpectraMax M3 (Molecular Devices) UV–vis spectrophotometer with SoftMax Pro 6.1 software was employed in the measurement. The DPPH inhibition percent of each sample was calculated using Eq. (1):
%inhibition = ((Abs blank − Abs final )/(Abs blank )) x 100
ln η0 = ln A – (Ea/ R) x (1/ T )
2.3.5. Scanning electron microscopy (SEM) SEM measurement was carried out to analyze the internal structure of chitosan/gelatin mixture and to verify the changes in the morphology after PPE addition at different concentrations. The samples prepared as described before were placed in Teflon® plates, frozen in liquid nitrogen and freeze-dried in an Edwards Freeze Dryer Modulyo equipment (Edwards High Vacuum International), to obtain the porous scaffolds. Samples were covered with a 6 mm thickness gold layer on a Coating System BAL-TEC MED 020 metallizer (BAL-TEC, Liechtenstein). The pressure in the chamber was 0.02 mbar, the current of 60 mÅ and the deposition rate of 0.6 nm s−1. The equipment was a ZEISS LEO 440 (Cambridge, England) with an OXFORD detector (model 7060), electron beam of 20 kV. The software UTHSCSA Image Tool version 3.0 was used to measure the scaffolds porous size. At least 15 different points were used to calculate the average porous size in micrography with magnification of 500×.
(1)
2.3.4. Rheological measurements The rheological measurements were conducted in an AR-1000 N (TA Instruments) controlled stress rheometer, using a cone/plate geometry of stainless-steel of 20 mm in diameter, angle of 2° and a fixed gap of 69 μm. A Peltier system was used for temperature control. All the rheological measurements were performed in triplicate.
2.4. Statistical analysis The Shapiro-Wilk test was used to verify data distribution, while differences in the TPC values were analyzed using the Student t-test. Rheological results were examined using analysis of variance (ANOVA), followed by Tukey’s test. Significance level was set at 5% in all cases.
2.3.4.1. Strain sweep measurements. Strain sweep tests were performed in the amplitude range of 0.05–500 Pa at constant frequency (1.0 Hz) and temperature (25 °C) to determine the linear viscoelastic region (LVR) in which the elastic (G’) and viscous (G’’) moduli are obtained as a function of the strain. Rheology Advantage Data Analysis, version V5.7.0 (TA Instruments Ltd.) was employed to analyze the results of strain sweep tests, as G’LVR (elastic modulus at critical strain), γL (the critical strain, that is the highest strain value to which the sample is subjected before leaving LVR), tanδ (the ratio of viscous and elastic modulus, that indicates if the samples behave as liquids or solids). Moreover, the difference between viscous and elastic moduli in a fixed value of strain (G’’- G’) was determined as well (Naji-tabasi, Mohammad, & Razavi, 2017; Razmkhah et al., 2017).
3. Results and discussion 3.1. Fourier-transform infrared spectroscopy (FTIR) The FTIR spectra of chitosan, gelatin and PPE are shown in Fig. 1. The wide band at 3385 cm−1 in the chitosan spectrum (Fig. 1a) corresponds to OeH and NeH stretching. The characteristics bands at 1665 and 1580 cm-1 can be assigned to the amide-I and amide-II, respectively, while the band at 1384 cm−1 corresponds to eCH2 bending. The bands at 1160, 1082 and 1027 cm-1 are attributed to the symmetrical and non-symmetrical stretching of CeOeC linkages (Pereda, Ponce, Marcovich, Ruseckaite, & Martucci, 2011; Qiao, Ma, Zhang, & Yao, 2017; Wang et al., 2003). In the gelatin spectrum (Fig. 1b), the characteristics absorption bands at 3320, 1661, 1549 and 1244 cm−1 correspond, respectively, to amide A (OeH and NeH vibrations), amide I (C]O axial deformation), amide II (CeN and NeH stretching) and amide III (CeN and NeH stretching) (Liu et al., 2012, Jridi et al., 2014; Qiao et al., 2017; Staroszczyk, Sztuka, Wolska, & Wojtasz-paja, 2014; Sokolan et al., 2015). Fig. 1c shows the spectrum obtained for PPE. The broad band at
2.3.4.2. Frequency sweep measurements. Frequency sweep tests were performed at a frequency range of 0.1 to 100 rad s−1 at 5% of strain in the LVR region and constant temperature of 25 °C. The power law Eqs. (2) and (3) describes the frequency dependence of G’ and G’’: n′
G′′ = K ′′ (ω)
n′′
(5)
Where Ea is the activation energy (kJ mol−1), R is the universal gas constant (8.314 kJ mol−1 K−1) and T is the absolute temperature (K). Ea is determined from the slope of a ln η0 vs 1/T curve.
Where Abs blank is the absorbance at λ =517 nm, 30 min after the time without the addition of antioxidant and Abs final is the absorbance at λ =517 nm, 30 min after the time in the presence of antioxidant. Using the mathematic adjust (y = a + b.cx) in the curves of DPPH inhibition versus sample concentration, the exponent x was determined as the IC50 DPPH (the concentration of antioxidant which reduces the free radical DPPH• about 50%).
G′ = K ′ (ω)
(4)
(2) (3)
In Eqs. (2) and (3), k’ and k’’ are constants. The slopes (n’ and n’’) provide some viscoelastic characteristics of food materials (Özkan, Xin, & Chen, 2002). 3
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Fig. 1. FTIR spectra of: (a) chitosan, (b) gelatin and (c) PPE.
3387 cm−1 is attributed to the OeH stretching of phenolic compounds of the extract. At 2935 cm-1 the band is referred to CeH stretching of methyl and methoxy groups, as well as methyl and methylene groups from carboxylic acids. The band at 1728 cm−1 is typical of C]O stretching in carbonyl compounds, while its nearby band at 1616 cm−1 can be attributed to C]C linkages in alkenes and aromatics (Nisha, Tamileaswari, & Jesurani, 2015).
PPE, in agreement with previous studies that used three different types of pomegranate (276–413 mg GAE/g extract) (Derakhshan et al., 2018). In another paper, Malviya and Jha (2014)) described a TPC value dependence according to the extraction procedure, ranging from 297 to 435 mg tannin acid equivalent/g extract. Even the literature provides distinct values which are related with different extraction methods and pomegranate type, in most cases the high TPC values found for pomegranate are a reflect of its high antioxidant capacity (Derakhshan et al., 2018). The TPC value for C2G1E200 was 188 mg GAE/g extract, which is 2.5-fold lower than observed for pure PPE. This reduction may indicate the existence of hydrogen bonds between the phenolic hydroxyls of the several compounds present in the extract (anthocyanins, ellagic acids, tannins, etc.) with the amine, amide and hydroxyl groups of chitosan, as well as the carbonyl, carboxyl and amides groups of gelatin. Due to the formation of these bonds, the amount of free phenolic hydroxyls available to react with Folin-Ciocalteu reagent decreased, which is the responsible for the reduction in the TPC value. Nevertheless, the amount of 188 mg GAE/g extract indicates that there are phenolic hydroxyls available to enhance the expected antioxidant activity of chitosan/gelatin mixture.
3.2. Antioxidant assays 3.2.1. PPE total phenolics content (TPC) The phenolic content of a vegetable extract is a good indicator of its antioxidant potential, since the phenolic hydroxyls can act as reducing agents of free radicals, as hydrogens donors and even as singlet oxygen quenchers (Chang et al., 2011; Malviya & Jha, 2014). Quantification was performed using Folin-Ciocalteu reagent in which phenols form the blue colored phosphomolybdic-phosphotungstic-phenol complex in alkaline media. Table 1 shows the TPC values for pure PPE and PPE into C2G1E200 mixture, which was chosen for comparison due to its highest concentration of PPE. Statistical analysis shows a significant difference The TPC value of 492 mg GAE/g extract was determined for pure Table 1 Antioxidant capacity for pure PPE and C2G1E200 mixture. Sample PPE C2G1E200
TPC (mg GAE/g extract) a
492 ± 82 188 ± 13b
3.2.2. DPPH antioxidant assay A widely used method to evaluate the antioxidant activity of natural compounds is the scavenging of the stable DPPH radical. The phenolics found in the peel extract react with DPPH and convert it to 2,2-diphenyl-1-picrylhydrazyne which shows a characteristic absorbance peak at 517 nm. Fig. 2 shows the DPPH inhibition curves (in percentage) for the radical scavenging activity of pure PPE and C2G1E200 sample. In both cases, a strong correlation between their antioxidant
IC50 4.07(μg mL−1) 20.59 (mg mL−1).
For TPC values, the different superscript letters (a–b) shows significant difference between the samples (P < 0.05). 4
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Fig. 2. % DPPH inhibition versus sample concentration curves for: (a) PPE and (b) C2G1E200 mixture. The polynomial adjustment of the data allowed the estimated IC50 values of the samples.
activity and concentration was observed (R2 = 0.99). The IC50 value (Table 1), which is the concentration where 50% scavenging of the free radical is obtained, was found to be 4.07 μg mL−1 and 20.59 mg mL−1 for PPE and C2G1E200, respectively. Pal et al. (2017) established the IC50 value of 16.78 μg mL−1 for pomegranate peel, while Okonogi, Duangrat, and Anuchpreeda (2007) reported a value around 3 μg mL−1. These differences between the values reported in literature can be also explained by the type of pomegranate used and the extraction method adopted. Phenolic compounds are effective hydrogens donors which is the reason of its good antioxidant properties (Malviya & Jha, 2014). This explains the low IC50 value found for PPE, which also presented a high content of phenolic compounds. Regarding to C2G1E100 mixture, the IC50 value corroborates with the TPC value of 188 mg GAE/g extract, showing that even with the possible bonds formed between the phenolics hydroxyls from PPE and the groups from chitosan and gelatin, the remaining hydroxyls were able to improve the antioxidant capacity of the chitosan/gelatin system.
region, as G’’ > G’. The G’LVR values (the elastic modulus at the limit of LVR) showed a significant decrease from mixture C2G1 to C2G1E50, continuing to diminish according to the increase in PPE concentration (C2G1E200 significantly different from the two first samples). This result indicates a decrease in the elastic behavior of the mixtures with PPE addition to chitosan/gelatin system. Critical strain γL, depends on the molecular architecture of polymer molecules and can be related with the deformability of the samples (Anvari, Tabarsa, Cao, & You, 2016). According to Table 2, it is evident a linear dependence of γL regarding with PPE concentration (Fig. S2), reflecting in an extension of LVR. Loss tangent (tanδ = G’’/G’) is a fundamental parameter in oscillation analysis since it indicates the characteristic of materials. Solutions with tanδ > 1, which means G’’ > G’, are classified as viscous, while samples with tanδ < 1 (G’ > G’’) are elastic. If tanδ > 0.1, the sample is not a true gel, ranging from a behavior of high concentrated solution and a real gel (Mandala, Savvas, & Kostaropoulos, 2004; Razmkhah et al., 2017). For the studied samples, tanδ values (Table 2) showed a significant (P > 0.05) increase ranging from 1.25 (C2G1) to 2.16 (C2G1E50), which is another evidence of the enhancement of the viscous behavior with PPE addition and also another prove that the samples cannot be considered true gels (tanδ > 0.1). The G’’ - G’ values at a fixed strain of 5% (Table 2) show that both the addition and the increase of PPE leads to an increase in the viscous behavior of the samples. Moreover, this rise is more than the double compared with the control sample (C2G1) (Naji-tabasi et al., 2017). Furthermore, the G’’ - G’ values agree with the observed tan δ behavior.
3.3. Rheological measurements Rheological measurements are useful to understand the relationship between structure and function of the polymeric mixtures (Kurt, 2019). As the biopolymer-based materials prepared in this study are potential candidates for food coatings purposes, spreadability, thickness and the uniformity of the covering surface are properties which must be evaluated. For example, changes in viscous and elastic properties implies modification in structural strength and stability. On the other hand, an increase in the viscosity means the necessity to improve the shear rate to force the flow of the material. All these properties can be determined by rheology.
3.3.2. Frequency sweep measurements The behavior of G’ and G’’ moduli against the frequency range (Fig. 4) allows the classification of the samples, which can be dilute solutions, concentrated solutions or gels (Steffe, 1996). For gels, G’ > G’’ in all the studied frequency range; if G’’ > G’ and the moduli get closer to each other at higher frequency values, the solution is diluted. Otherwise, if G’’ > G’ and the moduli cross inside the frequency range, the solution is concentrated (Naji-tabasi et al., 2017). Frequency sweep dependency of G’ and G’’ moduli (Fig. 4) showed that all the mixtures are concentrated polymers solutions as already suggested by tanδ values (> 0.1) in strain sweep measurements, in the section 3.3.1. Table 3 shows the parameters obtained from frequency sweep measurements, in which G’ crossover is the value when G’= G’’ and ω crossover is the frequency at which this occurs. Comparing C2G1 and C2G1E50 mixtures, it was observed a shift in ωcrossover from 27.30 to 31.81 rad s−1, as well as an increase in G’crossover values from 21.18 to 26.88 Pa. However, considering the statistical analysis (Table 3), both parameters are not significantly different but show a tendency to increase
3.3.1. Strain sweep measurements Strain sweep measurements were performed to determine the LVR of the samples (mixtures showed in Fig. S1), i.e. the region in which G’ and G’’ moduli are constant, independent of the applied strain. The amplitude sweep measurements provide information about material structural strength such as sample stability, since stable solutions may remain in the LVR region over greater strain than unstable ones (Steffe, 1996). The end limit of LVR region is characterized by the non-linearity of G’ and G’’ moduli under deformation (Fadavi, Mohammadifar, Zargarran, Mortazavian, & Komeili, 2014). Fig. 3 shows the representative curve of the mean values of data points for respective G´ and G´´ moduli as a function of strain. The parameters obtained from strain sweep measurements are summarized in Table 2. All the samples showed a liquid-like behavior in the LVR 5
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Fig. 3. (a) strain sweep dependence of G’ and G’’ moduli at f =1 Hz; (b) highlighting the LVR.
The Power Law model parameters for the mixtures are reported in Table 3. As described in the literature the n’ value is an indicator of the sample nature (Balaghi, Amin, & Zargaraan, 2011; Razavi, Cui, & Ding, 2016). When n’ = 0, the sample is considered a covalent gel, whereas for n’ > 0, the gel is described as physical gel. On the other hand, if n’ values are low (near to zero), it means G’ does not change with the applied frequency. In this study, the n’ values for the mixtures were closer to 1 which is related with a viscous gel behavior. Furthermore, for all the mixtures n’ was higher than n’’, indicating that G’ increased faster than G’’ in the studied frequency range (Fig. 4). When PPE is added to the mixtures, both k’ and k’’ values decreased according to the PPE concentration suggesting that the gel network became weaker due to the formation of new interactions between the polymers and the phenolic groups from the extract. Frequency sweep assay findings evidenced that the samples are not real gels but are concentrated and viscous polymers solutions.
Table 2 Critical strain (γL), G’ modulus at the LVR limit (G’LVR), loss tangent value (tanδ) and the difference between G’’ and G’ moduli at 5% strain. Parameters determined by strain sweep measurements at f = 1 Hz and T = 25 °C. Sample
γL (%)
C2G1 C2G1E50 C2G1E100 C2G1E150 C2G1E200
24.99 38.67 49.40 60.94 78.36
G' ± ± ± ± ±
1.46e 0.32d 0.97c 1.17b 0.91a
LVR
6.02 3.18 2.67 2.27 2.09
(Pa)
± ± ± ± ±
tanδ
1.01a 0.11b 0.18b,c 0.10b,c 0.07c
1.25 2.16 2.25 2.59 2.48
G’’- G’ ± ± ± ± ±
0.16d 0.04c 0.10b,c 0.12a 0.09a,b
1.48 3.66 3.95 3.90 3.41
*
± ± ± ± ±
(Pa) 0.86b 0.57a 0.11a 0.36a 0.23a
In the same column, values with the same superscript letter (a–e) were no significantly different (P > 0.05). * Values obtained at 5% strain.
3.3.3. Steady shear measurements Fig. 5 shows the viscosity curves as a function of the applied shear rate to the samples at 25 °C for all the mixtures (Fig. 5a) and for C2G1E100 sample at temperatures ranging from 15 to 40 °C (Fig. 5b). As expected for polymers solutions, a shear thinning behavior was observed in both cases, which means the decrease of viscosity values according to the increase of shear rate. In fact, an increase in shear rate leads to higher ordering of the polymer chains, which tend to orientate toward the applied stress. The more oriented the chains, the lower the viscosity. The pseudoplastic (or shear thinning) behavior of the samples was successfully described by the Cross model (Eq. (4)) that has provided a good adjustment of the experimental data. The parameters obtained by the Cross Model are summarized in Table 4. The zero-shear viscosity parameter is directly related with the number of linkages between the polymers molecules (Razavi et al., 2016; Razmkhah et al., 2017) where higher η0 values imply in a higher number of interactions. The decrease of η0 values from 2.18 (C2G1) to 1.56 Pa s (C2G1E200) confirms that PPE addition weakened the
Fig. 4. Frequency sweep dependency of G’ and G’’ moduli at 5% of strain and T = 25 °C for chitosan/gelatin/PPE mixtures.
according to PPE addition and PPE concentration increase. Frequency sweep curves were adjusted using the Power Law Eqs. (2) and (3) which is the model that best described the experimental data.
Table 3 Frequency dependence of elastic (G’) and viscous (G’’) moduli of chitosan/gelatin mixtures with and without PPE (γ = 5%, T = 25 °C). Sample
ω
C2G1 C2G1E50 C2G1E100 C2G1E150 C2G1E200
27.30 31.81 32.57 34.77 31.60
crossover
± ± ± ± ±
(rad s−1)
G’
3.69ª 3.56a 3.80a 2.80a 1.87a
21.18 26.88 20.30 22.21 17.11
crossover
± ± ± ± ±
(Pa)
1.67a,b 1.97ª 2.44b 3.36a,b 1.05b
k’ 1.339 1.737 1.087 0.918 1.047
n' ± ± ± ± ±
0.138b 0.201a 0.099c 0.117c 0.165c
0.824 0.774 0.811 0.885 0.767
± ± ± ± ±
0.029a,b 0.047a,b 0.029a,b 0.057a 0.047b
R2 (G’)
k’’
0.995 0.986 0.984 0.989 0.967
3.221 3.868 2.530 2.545 2.012
In the same column, values with the same superscript letter (a–d) were no significantly different (P > 0.05). 6
R2 (G’’)
n’’ ± ± ± ± ±
0.061a,b 0.215a 0.171b,c 0.519b,c 0.245c
0.555 0.543 0.585 0.601 0.609
± ± ± ± ±
0.013c,d 0.007d 0.003ª,b,c 0.016ª,b 0.012a
0.993 0.990 0.994 0.994 0.995
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Fig. 5. (a) curves of viscosity versus shear rate for all the mixtures at 25 °C; (b) curves of viscosity versus shear rate for C2G1E100 mixture for temperatures ranging from 15 °C to 40 °C.
3.4. Scanning electron microscopy (SEM)
linkages between chitosan and gelatin polymers. Although the overall effect was a decrease in η0 values with increasing PPE concentration, it was observed a slight increase for C2G1E100 sample when compared to C2G1E50; even so, they were statistically similar within the considered confidence interval (P > 0.05). In fact, this effect is stronger for samples with high PPE concentration (C2G1E150 and C2G1E200) which showed a significant decrease in η0 values in comparison with control sample (C2G1), confirmed by statistical analysis. The evaluation of the rheological properties as function of temperature is necessary to understand the behavior of the chitosan/gelatin/PPE mixtures. As their potential application as food coatings, the temperature is the principal factor that may affect the quality and the deterioration of coated fruits. Fig. 5b shows the viscosity curves versus shear rate of C2G1E100 mixture at different temperatures. The viscosity dependence of the temperature was used to calculate the activation energy of the C2G1E100 mixture applying the Arrhenius equation (Eq. (5)). Basically, the activation energy (Ea) is defined as the energy required for the molecule of a fluid to move freely and its value depends on factors such as polymer nature and concentration (Abbastabar, Azizi, Adnani, & Abbasi, 2014; Anvari et al., 2016; Razmkhah et al., 2017). The curves of ln η0 versus 1/T for the mixtures with and without PPE (Fig. S3) were used for Ea calculation (Table 4). The Arrhenius model showed a satisfactory fitting of the temperature-viscosity relation as can be noticed by R2 values reported in Table 4. Comparison of the Ea values as a function of extract concentration (Fig. S4) showed a decrease in this parameter after PPE addition, from 39.27 kJ mol−1 in C2G1 to 15.89 kJ mol−1 in C2G1E200. Therefore, the mixtures with higher amount of PPE were less sensitive to temperature changes and were easier to flow. In all rheological measurements a slightly decrease in C2G1E200 properties was observed which can be related with a limit of PPE that can be added in this polymeric system. However, antioxidant properties were preserved that enable the use of the developed material for the desired application.
The surface properties of freeze-dried scaffold (Fig. S5) were observed using SEM. The SEM images of all mixtures are shown in Fig. 6A–E at 500× of magnification. The surface morphology characteristics exhibits differences in pore size, with a clear increase related to the PPE addition ranging from 0 to 100 mg (C2G1E100 scaffold) as observed in the sequence images of Fig. 6A–C. The addition of 150 mg (Fig. 6D) and 200 mg (Fig. 6E) of pomegranate peel extract affects differently the pore size and a decrease in this parameter was observed. The measured values of the mean pore diameters are summarized in Table S1. The reason for that behavior could be a saturation of the linkages of chitosan/gelatin polymeric system with phenolic components for high PPE concentrations. At high magnification of 15,000x (Fig. 6F) it is evident the presence of PPE adhered to the chitosan/gelatin network as exemplified by the C2G1E200 scaffold surface. 4. Conclusions This study focused on the development and the characterization of chitosan/gelatin-based materials incorporated with pomegranate peel extract, aiming the reuse of this agroindustry residue in potential valueadded applications. To evaluate if PPE addition actually contributed with significant changes in the structural and functional characteristics of the material, rheological and antioxidant properties of the obtained mixtures were explored. PPE incorporation in the chitosan/gelatin polymeric systems enhanced their antioxidant capacity, as it was already expected due to PPE high phenolic content. Regarding to rheology, PPE addition increased the materials viscous behavior and their stability against the strain applied, which is a promising result for eventual applications of them as fruits edible coatings: low viscous behavior and low strength materials might not adhere well on fruit´s surface. These characteristics would suggest the use of large amount of material and more expensive coating techniques, instead of dip the fruit
Table 4 η0, k and n parameters obtained by Cross-model (at 25 °C) and Ea and R2values obtained by Arrhenius equation from steady shear measurements. Sample
η0 (Pa.s)
C2G1 C2G1E50 C2G1E100 C2G1E150 C2G1E200
2.18 1.74 1.91 1.58 1.56
± ± ± ± ±
k (s) 0.37a 0.11a,b 0.12a,b 0.10b 0.10b
0.080 0.052 0.058 0.036 0.069
n ± ± ± ± ±
0.032a 0.003a 0.009a 0.004a 0.032a
0.705 0.732 0.745 0.784 0.689
Ea ± ± ± ± ±
0.043a 0.038a 0.034a 0.057a 0.069a
In the same column, values with the same superscript letter (a–b) were no significantly different (P > 0.05). * Ea and R2 are related with Arrhenius equation. 7
*
(kJ mol−1)
39.27 25.60 24.11 17.83 15.89
R2
*
0.977 0.924 0.994 0.958 0.981
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Fig. 6. SEM photomicrographs of the surface of: (A) C2G1, (B) C2G1E50, (C) C2G1E100, (D) C2G1E150, (E) C2G1E200, magnification of 500×, and (F) C2G1E200, magnification of 15,000×.
Author’s contributions
into the mixture. The frequency sweep measurements indicated that PPE interacts with chitosan and gelatin chains resulting in a network with very weak gel strength, which helped us to better understand the interactions of the phenolic compounds once exposed to the polymer matrix. For this reason, mixtures with high amount of PPE were easier to flow and showed lower activation energy values, in which the addition of 200 mg of PPE corresponds to an activation energy of 15.89 kJ mol −1. Furthermore, it was evident that flow properties were affected by PPE concentration and temperature. Overall, it was demonstrated a step forward in exploring the potential use of renewable and highly antioxidant materials like PPE to improve the structural, functional and antioxidant characteristics of a well-known polymer blend, applied in a variety of fields. It is believed that this study opens a range of new studies to be conducted in the future, according to the intended application of the developed materials.
VCAM and MRVB conceived the study. VCAM and MRVB designed the experiments. MRVB and VCAM contributed to the experimental work. LBB and MRVB contributed to the antioxidant assays. MRVB, VCAM, MMH, LBB and AMGP contributed to the results interpretation. AMGP supervised the final version. All authors read and approved the final manuscript.
Funding This work was supported by the National Council for Scientific and Technological Development (CNPq) contract number 140406/2019-0 for MRVB and by the São Paulo Research Foundation (FAPESP) contract number 2017/15477-8 for LBB.
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Consent for publication
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The authors have consented for publication. Ethics approval and consent to participate Not applicable. Declaration of Competing Interest The authors declare they have no competing interests. Acknowledgments The authors would like to thank the Center of Analytical Chemical Analysis of IQSC/USP, for all the infrastructure available for FTIR, SEM and 1H NMR analysis, as well as the Brazilian Biorenewables National Laboratory (LNBR/CNPEM), where the antioxidants assays were developed. We also thank Prof. Dr. Sérgio Paulo Campana Filho (IQSC/ USP) for the access of the viscometer SCHOTT equipment and his postdoctoral student Dr. Danilo M. dos Santos (IQSC/USP) for his help during the measurements. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2019.115386. References Abbastabar, B., Azizi, M. H., Adnani, A., & Abbasi, S. (2014). Determining and modeling rheological characteristics of quince seed gum. Food Hydrocolloids, 70, 1–6. Aloui, H., Khwaldia, K., Licciardello, F., Mazzaglia, A., Muratore, G., Hamdi, M., et al. (2014). Efficacy of the combined application of chitosan and Locust Bean Gum with different citrus essential oils to control postharvest spoilage caused by Aspergillus flavus in dates (2020) International Journal of Food Microbiology, 170, 21–28. Anvari, M., Tabarsa, M., Cao, R., & You, S. (2016). Compositional characterization and rheological properties of an anionic gum from Alyssum homolocarpum seeds. Food Hydrocolloids, 52, 766–773. Balaghi, S., Amin, M., & Zargaraan, A. (2011). Compositional analysis and rheological characterization of gum tragacanth exudates from six species of Iranian Astragalus. Food Hydrocolloids, 25(7), 1775–1784. Chang, L., Juang, L., Wang, B., Wang, M., Tai, H., Hung, W., et al. (2011). Antioxidant and antityrosinase activity of mulberry (Morus alba L.) twigs and root bark. Food and Chemical Toxicology, 49(4), 785–790. Cook, N. C., & Samman, S. (2006). Flavonoids Chemistry, metabolism, cardioprotective effects, and dietary sources. The Journal of Nutritional Biochemistry, 7, 66–76 1996. Derakhshan, Z., Ferrante, M., Tadi, M., Ansari, F., Heydari, A., Sadat, M., et al. (2018). Antioxidant activity and total phenolic content of ethanolic extract of pomegranate peels, juice and seeds. Food and Chemical Toxicology, 114(2), 108–111. Dutta, P. K., Tripathi, S., Mehrotra, G. K., & Dutta, J. (2009). Perspectives for chitosan based antimicrobial films in food applications. Food Chemistry, 114(4), 1173–1182. Fadavi, G., Mohammadifar, M. A., Zargarran, A., Mortazavian, A. M., & Komeili, R. (2014). Composition and physicochemical properties of Zedo gum exudates from Amygdalus scoparia. Carbohydrate Polymers, 101, 1074–1080. Horn, M. M., Martins, V. C. A., Maria, A., & Plepis, D. G. (2009). Interaction of anionic collagen with chitosan: Effect on thermal and morphological characteristics. Carbohydrate Polymers, 77(2), 239–243. Ismail, T., Sestili, P., & Akhtar, S. (2012). Pomegranate peel and fruit extracts: A review of potential anti-inflammatory and anti-infective effects. Journal of Ethnopharmacology, 143(2), 397–405. Jridi, M., Hajji, S., Ben, H., Lassoued, I., Mbarek, A., Kammoun, M., et al. (2014). International Journal of Biological Macromolecules Physical, structural, antioxidant and antimicrobial properties of gelatin – chitosan composite edible films. International Journal of Biological Macromolecules, 67, 373–379. Kanatt, S. R., Rao, M. S., Chawla, S. P., & Sharma, A. (2012). Active chitosan e polyvinyl alcohol films with natural extracts. Food Hydrocolloids, 29(2), 290–297. Kurita, K. (2006). Mini-review chitin and chitosan: Functional biopolymers from marine crustaceans. Marine Biotechnology, 8, 203–226. Kurt, A. (2019). Rheology of film-forming solutions and physical properties of differently deacetylated salep glucomannan film. Food and Health, 5(3), 175–184. Lavertu, M., Xia, Z., Serreqi, A. N., Berrada, M., Rodrigues, A., Wang, D., et al. (2003). A validated 1H NMR method for the determination of the degree of deacetylation of chitosan. Journal of Pharmaceutical and Biomedical Analysis, 32(6), 1149–1158. Lansky, E. P., & Newman, R. A. (2007). Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. Journal of
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Rheological and antioxidant properties of chitosan/gelatin-based materials functionalized by pomegranate peel extract
T
⁎
Mirella R.V. Bertoloa, , Virginia C.A. Martinsa, Marilia M. Hornb, Lívia B. Brenellic,d, Ana M.G. Plepisa a
São Carlos Institute of Chemistry (IQSC), University of São Paulo (USP), São Carlos, São Paulo, Brazil Macromolekulare Chemie und Makromolekulare Materialien, Universität Kassel, Kassel, Germany c Brazilian Biorenewables National Laboratory (LNBR), Brazilian Center for Research in Energy and Materials (CNPEM), Campinas, São Paulo, Brazil d Interdisciplinary Center of Energy Planning, State University of Campinas (UNICAMP), Campinas, São Paulo, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Gelatin Pomegranate peel extract Rheology Antioxidant Food coating
Biopolymer-based materials are potential candidates for food coatings application. In this study, pomegranate (Punica granatum L.) peel extract (PPE) at different concentrations was incorporated to chitosan/gelatin gels and the rheological, antioxidant and structural properties were evaluated. Due to its high phenolic content, PPE enhanced the antioxidant capacity of chitosan/gelatin mixtures. PPE addition extended linear viscoelastic range and enabled the samples to easily flow under the applied shear rate. Rheological properties indicated that both viscosity and activation energy of materials containing natural compounds are highly dependent on temperature. Scanning electron microscopy (SEM) images revealed the influence of PPE concentration in the scaffolds pores size. Findings of this study proved that PPE was capable to improve the functional characteristics of chitosan/ gelatin-based materials enhancing the desired properties for their potential application as food coatings.
1. Introduction Chitosan is a cationic polysaccharide which can be obtained by the deacetylation of chitin, a polymer found in the exoskeleton of crustaceans and insects (Nair, Saxena, & Kaur, 2018). Due to its biodegradability, biocompatibility, antimicrobial activity and non-toxicity, chitosan is considered a very promising and eco-friendly material for different purposes (Dutta, Tripathi, Mehrotra, & Dutta, 2009; Kanatt, Rao, Chawla, & Sharma, 2012; Nair et al., 2018). Moreover, chitosanbased materials (CB) can be designed in different forms, including gels, films and porous scaffolds. These materials can trap essential oils and bioactive compounds in their structure (Aloui et al., 2014), which enables their use in diverse fields, such as food coatings and drug-controlled release systems (Tang, Guan, Yao, & Zhu, 2014). Regarding that, many studies explored the use of chitosan-based systems associated with essential oils or plant extracts. The applicability of these systems is based on their potential biological and
antimicrobials activity, antifungals and antioxidants properties (Silalahi, Situmorang, Patilaya, & Silalahi, 2016; Sugita, Wijaya, Syahbirin, Dewi, & Sugita, 2017; Yuan, Lv, Tang, Zhang, & Sun, 2016; Yuan, Chen, & Li, 2016). Following this concept, the development of chitosan-edible films and coating materials containing essential oils and plant extracts represents an alternative to substitute non-renewable sources (Nair et al., 2018). Lozano-Navarro et al. (2018) described the preparation of chitosan/ starch films associated with natural antioxidants, such as anthocyanins from pomegranate and blueberry, resveratrol from grape and carvacrol from oregano, concluding that these materials exhibited promising positive properties for food packaging applications. In another study, Kanatt et al. (2012) prepared chitosan-polyvinyl alcohol films containing mint extract and pomegranate peel extract, which can serve for food packaging materials with desired antioxidants properties. Although there are researches which describe the use of CB as food coatings, strategies are currently being formulated to improve their
Abbreviations: 1HNMR, proton nuclear magnetic resonance spectroscopy; C2G1, chitosan/gelatin, 2:1 mixture; C2G1E100, chitosan/gelatin, 2:1 mixture, incorporated with 100 mg of PPE; C2G1E150, chitosan/gelatin, 2:1 mixture, incorporated with 150 mg of PPE; C2G1E200, chitosan/gelatin, 2:1 mixture, incorporated with 200 mg of PPE; C2G1E50, chitosan/gelatin, 2:1 mixture, incorporated with 50 mg of PPE; DPPH, 2,2-diphenyl-1-picrylhydrazyl; GAE, gallic acid equivalent; IC50, sample concentration necessary to inhibit 50% of DPPH radical; LVR, linear viscoelastic region; PPE, pomegranate peel extract; SEM, scanning electron microscopy; TPC, total phenolics content ⁎ Corresponding author at: Av. Trabalhador São Carlense, 400, São Carlos, SP, Zip code 13566-590, Brazil. E-mail address: [email protected] (M.R.V. Bertolo). https://doi.org/10.1016/j.carbpol.2019.115386 Received 3 July 2019; Received in revised form 25 September 2019; Accepted 25 September 2019 Available online 28 September 2019 0144-8617/ © 2019 Elsevier Ltd. All rights reserved.
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rinsing and drying at 60 °C overnight. The yield of the reaction was 27.8%, which was calculated from the initial mass of squid pens and agrees with a previous study (Kurita, 2006). Pomegranate peel extract (PPE) was extracted according to the procedure adapted from Yuan, Lv et al. (2016), Yuan, Chen et al. (2016). Initially, the peels were removed from the fruits, washed, lyophilized and crushed to obtain a thin powder. Then, the powder (1 g) was dissolved in a 60% (v/v) hydroethanolic solution (60% ethanol/ 40% water), in the solid:liquid ratio of 1:30 (w/v). The extraction procedure was carried out for 1 h at 50 °C, ensuring an extraction yield of 51.6% which was determined relative to the initial mass of peel powder. The solution was filtered and lyophilized until constant weight, which means that the extract was completely dried. The final product was a reddish extract.
functionality and reduce the limitation of chitosan low solubility at neutral pH, as well as its poor mechanical resistance and low elasticity. To improve CB functional properties, the combination with proteins or other polysaccharides has been described (Nowzari, Shábanpour, & Ojagh, 2013). Thus, gelatin is a polypeptide that can be considered a suitable option to associate with CB improving not only the mechanical but also, the functional properties. Gelatin is obtained by the partial hydrolysis of collagen and it is a valuable component to produce biodegradable materials for medical and food areas (Nair et al., 2018). Pomegranate (Punica granatum L.) is a highly nutritious fruit which contains many bioactive compounds even in its inedible parts, such as peel and seeds. Pomegranate peel is considered an agro-waste (Ismail, Sestili, & Akhtar, 2012; Malviya & Jha, 2014; Nair et al., 2018), and it is reported as a potential source of antioxidant compounds, besides exhibiting antifungal, antimicrobial and antibacterial activities (Malviya & Jha, 2014). The peels represent approximately 60% of the total weight of pomegranate fruit (Lansky & Newman, 2007), which provides higher antioxidant capacity and higher total phenolic content in comparison with the amount found in the pulp (Li et al., 2006). Compounds as tannins, alkaloids, flavonoids and organic acids are responsible for the health benefits previously reported for pomegranate peel, including treatment and prevention against cancer, cardiovascular diseases, diabetes, erectile disfunctions, as well as protection against ultraviolet radiation. Also, studies using pomegranate peel extract for the treatment of Alzheimer’s disease, arthritis and obesity were already reported (Cook & Samman, 2006; Lansky & Newman, 2007; Mohammad & Kashani, 2012; Qu, Pan, & Ma, 2010; Zhang, Fu, & Zhang, 2011). The interest of using chitosan-based systems associated with pomegranate peel (Kanatt et al., 2012; Nair et al., 2018; Yuan, Lv et al., 2016, Yuan, Chen et al., 2016) has also been growing. Nevertheless, there is no literature related to chitosan/gelatin/pomegranate peel association, although they are excellent candidates for the development of systems with high antioxidant capacity. Moreover, its rheological properties should be evaluated to predict their stability under certain conditions of temperature, viscosity, shear and stress rate (Razmkhah, Mohammad, & Razavi, 2017). It is assumed that the addition of phenolic components from pomegranate peel extract increases the antioxidant properties of the polymeric system, which improves its potential application as a biobased material for food coating technology. However, linkages of phenolic groups with both polymers are expected, which may change their main rheological characteristics, such as viscosity, elasticity and gel strength. Thus, this paper focuses on an unprecedented study about how the incorporation of pomegranate peel extract can affect the structural, rheological and antioxidant properties of a chitosan and gelatin polymeric system.
2.2. Solutions and samples preparation A 1% (w/w) chitosan gel was prepared by dissolution of the polysaccharide in a 1% acetic acid (HAc) aqueous solution under stirring at room temperature for 24 h. Gelatin solution with concentration of 1% (w/w) was obtained in deionized water under constant stirring at 60 °C for 30 min. After heating, the solution was placed at 4 °C for 3 h to enable the gelation process. Chitosan/gelatin control, labelled as C2G1, was prepared by mixing both solutions at 2:1 (w/w) proportion at 45 °C. PPE hydroethanolic solutions (50%, v/v) were prepared at concentrations of 50, 100, 150 and 200 mg mL−1. A series of chitosan/ gelatin and PPE solutions was prepared by adding 1 mL of each PPE solution to the chitosan/gelatin 2:1 mixture under constant stirring at 45 °C. Samples were labelled as C2G1E50, C2G1E100, C2G1E150 and C2G1E200, respectively. To prevent the dilution effect, 1 mL of a 50% (v/v) hydroethanolic solution without PPE was added to the control sample (C2G1). 2.3. Methods 2.3.1. Chitosan characterization Chitosan acetylation degree (6.7%) was determined by proton nuclear magnetic resonance spectroscopy (1H NMR) according to the method developed and validated by Lavertu et al. (2003). The molecular weight (327 kDa) was determined by capillary viscosimetry procedure (Rinaudo, 2006) in which flow time measurements were performed on an AVS-306 (SCHOTT) viscosimeter coupled to a universal TITRONIC automatic diluter (SCHOTT). 2.3.2. Fourier-transform infrared spectroscopy (FTIR) FTIR was employed to examine the chemical structure of the components. Analysis was performed in films by casting of diluted chitosan/ gelatin mixtures and drying at room temperature following storage in a desiccator with NaOH(s). PPE spectrum was obtained using KBr pellets. FTIR spectra were obtained using a Shimadzu IR Affinitu-1 at 4000 400 cm−1 interval with 4 cm−1 resolution.
2. Experimetal 2.1. Materials All the solvents and the reagents were of analytical grade and used without further purification. Doryteuthis spp squid pens were obtained at Miami Comércio e Exportação de Pescados Ltda in Cananéa-SP and were used as a source of β-chitin. Gelatin (type A, swine, ∼300 bloom) was purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). Pomegranate fruits were acquired in the local commerce of São Carlos – SP- Brazil. β-chitin extracted from squid pens was used to produce chitosan by means of the deacetylation process, following the procedure adapted from Horn, Martins, Maria, and Plepis (2009)). Briefly, squid pens were immersed in a 0.3 mol L−1 NaOH solution at 80 °C for 1 h to remove proteins and chitin was isolated as the main product. Finally, the second step consisted of an alkaline deacetylation performed by an immersion of chitin in a 40% (w/w) NaOH solution at 80 °C for 3 h, followed by
2.3.3. Antioxidant assays 2.3.3.1. PPE total phenolics content (TPC). The TPC of pomegranate peel extract was determined using the Folin-Ciocalteu method, as described by Singleton, Orthofer, and Lamuela-Raventós (1999), with some modifications. In this procedure, gallic acid was used as an internal standard. The Folin-Ciocalteu method is used to determine the total phenolic content of plant extracts and essential oils. Folin's Reagent is mixture of tungstate and molybdates, which in alkaline medium are reduced due to the presence of a phenolic compound. The final products are phenolate anions, a bluish chromogen with a maximum UV absorbance at 765 nm. A 96-well microplate was filled in with 25 μL of PPE hydroethanolic solutions at different concentrations, 150 μL of ultrapure water and 2
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2.3.4.3. Steady shear measurements. The steady shear flow properties were measured at a shear rate of 0.4 to 1000 s−1 at different temperatures (15, 20, 25, 30, 35 and 40 °C). Cross model (Eq. (4)) was used to fit the experimental flow curves (Razmkhah et al., 2017):
25 μL of Folin-Ciocalteu reagent. The microplate was stirred for 20 s and stand at room temperature for 5 min. Then, 100 μL of 7% (w/w) sodium carbonate solution was added to each well to ensure the reaction, and the microplate was stored protected from light. After 2 h, the absorbance at 765 nm was measured on a SpectraMax M3 (Molecular Devices) UV–vis spectrophotometer with SoftMax Pro 6.1 software. The results were expressed as equivalent mg of gallic acid (mg GAE) per mg of extract. Following the same procedure, the TPC determination in C2G1E200 mixture was performed to evaluate if phenolic content changes after incorporation in chitosan/gelatin mixture.
η − η∞ 1 = η0 − η∞ (1 + (kγ )n)
In Eq. (4), η0 is the zero-shear viscosity (Pa s), η∞ is the viscosity limit at infinite shear (Pa s), γ is the shear rate (s−1) and k and n are constants, k being the consistency index (s) and n the rate index (dimensionless). The effect of temperature on apparent viscosity of the mixtures was determined according to an Arrhenius-type model (Eq. (5)) (Steffe, 1996), using the values of η0 determined by the fitting of experimental flow curves:
2.3.3.2. DPPH antioxidant assay. The free radical scavenging antioxidant capacity of PPE was evaluated by the DPPH (2,2diphenyl-1-picrylhydrazyl) radical assay, according to the method described by Pal et al. (2017) with some modifications. First, a 25 mmol L−1 radical solution, violet colored, was prepared in ethanol. Then, 80 μL of the radical solution was mixed with 190 μL of ethanol and 10 μL of PPE and C2G1E200 sample at different concentrations. The samples absorbance was measured at 517 nm, and the decay was monitored for 30 min. A SpectraMax M3 (Molecular Devices) UV–vis spectrophotometer with SoftMax Pro 6.1 software was employed in the measurement. The DPPH inhibition percent of each sample was calculated using Eq. (1):
%inhibition = ((Abs blank − Abs final )/(Abs blank )) x 100
ln η0 = ln A – (Ea/ R) x (1/ T )
2.3.5. Scanning electron microscopy (SEM) SEM measurement was carried out to analyze the internal structure of chitosan/gelatin mixture and to verify the changes in the morphology after PPE addition at different concentrations. The samples prepared as described before were placed in Teflon® plates, frozen in liquid nitrogen and freeze-dried in an Edwards Freeze Dryer Modulyo equipment (Edwards High Vacuum International), to obtain the porous scaffolds. Samples were covered with a 6 mm thickness gold layer on a Coating System BAL-TEC MED 020 metallizer (BAL-TEC, Liechtenstein). The pressure in the chamber was 0.02 mbar, the current of 60 mÅ and the deposition rate of 0.6 nm s−1. The equipment was a ZEISS LEO 440 (Cambridge, England) with an OXFORD detector (model 7060), electron beam of 20 kV. The software UTHSCSA Image Tool version 3.0 was used to measure the scaffolds porous size. At least 15 different points were used to calculate the average porous size in micrography with magnification of 500×.
(1)
2.3.4. Rheological measurements The rheological measurements were conducted in an AR-1000 N (TA Instruments) controlled stress rheometer, using a cone/plate geometry of stainless-steel of 20 mm in diameter, angle of 2° and a fixed gap of 69 μm. A Peltier system was used for temperature control. All the rheological measurements were performed in triplicate.
2.4. Statistical analysis The Shapiro-Wilk test was used to verify data distribution, while differences in the TPC values were analyzed using the Student t-test. Rheological results were examined using analysis of variance (ANOVA), followed by Tukey’s test. Significance level was set at 5% in all cases.
2.3.4.1. Strain sweep measurements. Strain sweep tests were performed in the amplitude range of 0.05–500 Pa at constant frequency (1.0 Hz) and temperature (25 °C) to determine the linear viscoelastic region (LVR) in which the elastic (G’) and viscous (G’’) moduli are obtained as a function of the strain. Rheology Advantage Data Analysis, version V5.7.0 (TA Instruments Ltd.) was employed to analyze the results of strain sweep tests, as G’LVR (elastic modulus at critical strain), γL (the critical strain, that is the highest strain value to which the sample is subjected before leaving LVR), tanδ (the ratio of viscous and elastic modulus, that indicates if the samples behave as liquids or solids). Moreover, the difference between viscous and elastic moduli in a fixed value of strain (G’’- G’) was determined as well (Naji-tabasi, Mohammad, & Razavi, 2017; Razmkhah et al., 2017).
3. Results and discussion 3.1. Fourier-transform infrared spectroscopy (FTIR) The FTIR spectra of chitosan, gelatin and PPE are shown in Fig. 1. The wide band at 3385 cm−1 in the chitosan spectrum (Fig. 1a) corresponds to OeH and NeH stretching. The characteristics bands at 1665 and 1580 cm-1 can be assigned to the amide-I and amide-II, respectively, while the band at 1384 cm−1 corresponds to eCH2 bending. The bands at 1160, 1082 and 1027 cm-1 are attributed to the symmetrical and non-symmetrical stretching of CeOeC linkages (Pereda, Ponce, Marcovich, Ruseckaite, & Martucci, 2011; Qiao, Ma, Zhang, & Yao, 2017; Wang et al., 2003). In the gelatin spectrum (Fig. 1b), the characteristics absorption bands at 3320, 1661, 1549 and 1244 cm−1 correspond, respectively, to amide A (OeH and NeH vibrations), amide I (C]O axial deformation), amide II (CeN and NeH stretching) and amide III (CeN and NeH stretching) (Liu et al., 2012, Jridi et al., 2014; Qiao et al., 2017; Staroszczyk, Sztuka, Wolska, & Wojtasz-paja, 2014; Sokolan et al., 2015). Fig. 1c shows the spectrum obtained for PPE. The broad band at
2.3.4.2. Frequency sweep measurements. Frequency sweep tests were performed at a frequency range of 0.1 to 100 rad s−1 at 5% of strain in the LVR region and constant temperature of 25 °C. The power law Eqs. (2) and (3) describes the frequency dependence of G’ and G’’: n′
G′′ = K ′′ (ω)
n′′
(5)
Where Ea is the activation energy (kJ mol−1), R is the universal gas constant (8.314 kJ mol−1 K−1) and T is the absolute temperature (K). Ea is determined from the slope of a ln η0 vs 1/T curve.
Where Abs blank is the absorbance at λ =517 nm, 30 min after the time without the addition of antioxidant and Abs final is the absorbance at λ =517 nm, 30 min after the time in the presence of antioxidant. Using the mathematic adjust (y = a + b.cx) in the curves of DPPH inhibition versus sample concentration, the exponent x was determined as the IC50 DPPH (the concentration of antioxidant which reduces the free radical DPPH• about 50%).
G′ = K ′ (ω)
(4)
(2) (3)
In Eqs. (2) and (3), k’ and k’’ are constants. The slopes (n’ and n’’) provide some viscoelastic characteristics of food materials (Özkan, Xin, & Chen, 2002). 3
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Fig. 1. FTIR spectra of: (a) chitosan, (b) gelatin and (c) PPE.
3387 cm−1 is attributed to the OeH stretching of phenolic compounds of the extract. At 2935 cm-1 the band is referred to CeH stretching of methyl and methoxy groups, as well as methyl and methylene groups from carboxylic acids. The band at 1728 cm−1 is typical of C]O stretching in carbonyl compounds, while its nearby band at 1616 cm−1 can be attributed to C]C linkages in alkenes and aromatics (Nisha, Tamileaswari, & Jesurani, 2015).
PPE, in agreement with previous studies that used three different types of pomegranate (276–413 mg GAE/g extract) (Derakhshan et al., 2018). In another paper, Malviya and Jha (2014)) described a TPC value dependence according to the extraction procedure, ranging from 297 to 435 mg tannin acid equivalent/g extract. Even the literature provides distinct values which are related with different extraction methods and pomegranate type, in most cases the high TPC values found for pomegranate are a reflect of its high antioxidant capacity (Derakhshan et al., 2018). The TPC value for C2G1E200 was 188 mg GAE/g extract, which is 2.5-fold lower than observed for pure PPE. This reduction may indicate the existence of hydrogen bonds between the phenolic hydroxyls of the several compounds present in the extract (anthocyanins, ellagic acids, tannins, etc.) with the amine, amide and hydroxyl groups of chitosan, as well as the carbonyl, carboxyl and amides groups of gelatin. Due to the formation of these bonds, the amount of free phenolic hydroxyls available to react with Folin-Ciocalteu reagent decreased, which is the responsible for the reduction in the TPC value. Nevertheless, the amount of 188 mg GAE/g extract indicates that there are phenolic hydroxyls available to enhance the expected antioxidant activity of chitosan/gelatin mixture.
3.2. Antioxidant assays 3.2.1. PPE total phenolics content (TPC) The phenolic content of a vegetable extract is a good indicator of its antioxidant potential, since the phenolic hydroxyls can act as reducing agents of free radicals, as hydrogens donors and even as singlet oxygen quenchers (Chang et al., 2011; Malviya & Jha, 2014). Quantification was performed using Folin-Ciocalteu reagent in which phenols form the blue colored phosphomolybdic-phosphotungstic-phenol complex in alkaline media. Table 1 shows the TPC values for pure PPE and PPE into C2G1E200 mixture, which was chosen for comparison due to its highest concentration of PPE. Statistical analysis shows a significant difference The TPC value of 492 mg GAE/g extract was determined for pure Table 1 Antioxidant capacity for pure PPE and C2G1E200 mixture. Sample PPE C2G1E200
TPC (mg GAE/g extract) a
492 ± 82 188 ± 13b
3.2.2. DPPH antioxidant assay A widely used method to evaluate the antioxidant activity of natural compounds is the scavenging of the stable DPPH radical. The phenolics found in the peel extract react with DPPH and convert it to 2,2-diphenyl-1-picrylhydrazyne which shows a characteristic absorbance peak at 517 nm. Fig. 2 shows the DPPH inhibition curves (in percentage) for the radical scavenging activity of pure PPE and C2G1E200 sample. In both cases, a strong correlation between their antioxidant
IC50 4.07(μg mL−1) 20.59 (mg mL−1).
For TPC values, the different superscript letters (a–b) shows significant difference between the samples (P < 0.05). 4
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Fig. 2. % DPPH inhibition versus sample concentration curves for: (a) PPE and (b) C2G1E200 mixture. The polynomial adjustment of the data allowed the estimated IC50 values of the samples.
activity and concentration was observed (R2 = 0.99). The IC50 value (Table 1), which is the concentration where 50% scavenging of the free radical is obtained, was found to be 4.07 μg mL−1 and 20.59 mg mL−1 for PPE and C2G1E200, respectively. Pal et al. (2017) established the IC50 value of 16.78 μg mL−1 for pomegranate peel, while Okonogi, Duangrat, and Anuchpreeda (2007) reported a value around 3 μg mL−1. These differences between the values reported in literature can be also explained by the type of pomegranate used and the extraction method adopted. Phenolic compounds are effective hydrogens donors which is the reason of its good antioxidant properties (Malviya & Jha, 2014). This explains the low IC50 value found for PPE, which also presented a high content of phenolic compounds. Regarding to C2G1E100 mixture, the IC50 value corroborates with the TPC value of 188 mg GAE/g extract, showing that even with the possible bonds formed between the phenolics hydroxyls from PPE and the groups from chitosan and gelatin, the remaining hydroxyls were able to improve the antioxidant capacity of the chitosan/gelatin system.
region, as G’’ > G’. The G’LVR values (the elastic modulus at the limit of LVR) showed a significant decrease from mixture C2G1 to C2G1E50, continuing to diminish according to the increase in PPE concentration (C2G1E200 significantly different from the two first samples). This result indicates a decrease in the elastic behavior of the mixtures with PPE addition to chitosan/gelatin system. Critical strain γL, depends on the molecular architecture of polymer molecules and can be related with the deformability of the samples (Anvari, Tabarsa, Cao, & You, 2016). According to Table 2, it is evident a linear dependence of γL regarding with PPE concentration (Fig. S2), reflecting in an extension of LVR. Loss tangent (tanδ = G’’/G’) is a fundamental parameter in oscillation analysis since it indicates the characteristic of materials. Solutions with tanδ > 1, which means G’’ > G’, are classified as viscous, while samples with tanδ < 1 (G’ > G’’) are elastic. If tanδ > 0.1, the sample is not a true gel, ranging from a behavior of high concentrated solution and a real gel (Mandala, Savvas, & Kostaropoulos, 2004; Razmkhah et al., 2017). For the studied samples, tanδ values (Table 2) showed a significant (P > 0.05) increase ranging from 1.25 (C2G1) to 2.16 (C2G1E50), which is another evidence of the enhancement of the viscous behavior with PPE addition and also another prove that the samples cannot be considered true gels (tanδ > 0.1). The G’’ - G’ values at a fixed strain of 5% (Table 2) show that both the addition and the increase of PPE leads to an increase in the viscous behavior of the samples. Moreover, this rise is more than the double compared with the control sample (C2G1) (Naji-tabasi et al., 2017). Furthermore, the G’’ - G’ values agree with the observed tan δ behavior.
3.3. Rheological measurements Rheological measurements are useful to understand the relationship between structure and function of the polymeric mixtures (Kurt, 2019). As the biopolymer-based materials prepared in this study are potential candidates for food coatings purposes, spreadability, thickness and the uniformity of the covering surface are properties which must be evaluated. For example, changes in viscous and elastic properties implies modification in structural strength and stability. On the other hand, an increase in the viscosity means the necessity to improve the shear rate to force the flow of the material. All these properties can be determined by rheology.
3.3.2. Frequency sweep measurements The behavior of G’ and G’’ moduli against the frequency range (Fig. 4) allows the classification of the samples, which can be dilute solutions, concentrated solutions or gels (Steffe, 1996). For gels, G’ > G’’ in all the studied frequency range; if G’’ > G’ and the moduli get closer to each other at higher frequency values, the solution is diluted. Otherwise, if G’’ > G’ and the moduli cross inside the frequency range, the solution is concentrated (Naji-tabasi et al., 2017). Frequency sweep dependency of G’ and G’’ moduli (Fig. 4) showed that all the mixtures are concentrated polymers solutions as already suggested by tanδ values (> 0.1) in strain sweep measurements, in the section 3.3.1. Table 3 shows the parameters obtained from frequency sweep measurements, in which G’ crossover is the value when G’= G’’ and ω crossover is the frequency at which this occurs. Comparing C2G1 and C2G1E50 mixtures, it was observed a shift in ωcrossover from 27.30 to 31.81 rad s−1, as well as an increase in G’crossover values from 21.18 to 26.88 Pa. However, considering the statistical analysis (Table 3), both parameters are not significantly different but show a tendency to increase
3.3.1. Strain sweep measurements Strain sweep measurements were performed to determine the LVR of the samples (mixtures showed in Fig. S1), i.e. the region in which G’ and G’’ moduli are constant, independent of the applied strain. The amplitude sweep measurements provide information about material structural strength such as sample stability, since stable solutions may remain in the LVR region over greater strain than unstable ones (Steffe, 1996). The end limit of LVR region is characterized by the non-linearity of G’ and G’’ moduli under deformation (Fadavi, Mohammadifar, Zargarran, Mortazavian, & Komeili, 2014). Fig. 3 shows the representative curve of the mean values of data points for respective G´ and G´´ moduli as a function of strain. The parameters obtained from strain sweep measurements are summarized in Table 2. All the samples showed a liquid-like behavior in the LVR 5
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Fig. 3. (a) strain sweep dependence of G’ and G’’ moduli at f =1 Hz; (b) highlighting the LVR.
The Power Law model parameters for the mixtures are reported in Table 3. As described in the literature the n’ value is an indicator of the sample nature (Balaghi, Amin, & Zargaraan, 2011; Razavi, Cui, & Ding, 2016). When n’ = 0, the sample is considered a covalent gel, whereas for n’ > 0, the gel is described as physical gel. On the other hand, if n’ values are low (near to zero), it means G’ does not change with the applied frequency. In this study, the n’ values for the mixtures were closer to 1 which is related with a viscous gel behavior. Furthermore, for all the mixtures n’ was higher than n’’, indicating that G’ increased faster than G’’ in the studied frequency range (Fig. 4). When PPE is added to the mixtures, both k’ and k’’ values decreased according to the PPE concentration suggesting that the gel network became weaker due to the formation of new interactions between the polymers and the phenolic groups from the extract. Frequency sweep assay findings evidenced that the samples are not real gels but are concentrated and viscous polymers solutions.
Table 2 Critical strain (γL), G’ modulus at the LVR limit (G’LVR), loss tangent value (tanδ) and the difference between G’’ and G’ moduli at 5% strain. Parameters determined by strain sweep measurements at f = 1 Hz and T = 25 °C. Sample
γL (%)
C2G1 C2G1E50 C2G1E100 C2G1E150 C2G1E200
24.99 38.67 49.40 60.94 78.36
G' ± ± ± ± ±
1.46e 0.32d 0.97c 1.17b 0.91a
LVR
6.02 3.18 2.67 2.27 2.09
(Pa)
± ± ± ± ±
tanδ
1.01a 0.11b 0.18b,c 0.10b,c 0.07c
1.25 2.16 2.25 2.59 2.48
G’’- G’ ± ± ± ± ±
0.16d 0.04c 0.10b,c 0.12a 0.09a,b
1.48 3.66 3.95 3.90 3.41
*
± ± ± ± ±
(Pa) 0.86b 0.57a 0.11a 0.36a 0.23a
In the same column, values with the same superscript letter (a–e) were no significantly different (P > 0.05). * Values obtained at 5% strain.
3.3.3. Steady shear measurements Fig. 5 shows the viscosity curves as a function of the applied shear rate to the samples at 25 °C for all the mixtures (Fig. 5a) and for C2G1E100 sample at temperatures ranging from 15 to 40 °C (Fig. 5b). As expected for polymers solutions, a shear thinning behavior was observed in both cases, which means the decrease of viscosity values according to the increase of shear rate. In fact, an increase in shear rate leads to higher ordering of the polymer chains, which tend to orientate toward the applied stress. The more oriented the chains, the lower the viscosity. The pseudoplastic (or shear thinning) behavior of the samples was successfully described by the Cross model (Eq. (4)) that has provided a good adjustment of the experimental data. The parameters obtained by the Cross Model are summarized in Table 4. The zero-shear viscosity parameter is directly related with the number of linkages between the polymers molecules (Razavi et al., 2016; Razmkhah et al., 2017) where higher η0 values imply in a higher number of interactions. The decrease of η0 values from 2.18 (C2G1) to 1.56 Pa s (C2G1E200) confirms that PPE addition weakened the
Fig. 4. Frequency sweep dependency of G’ and G’’ moduli at 5% of strain and T = 25 °C for chitosan/gelatin/PPE mixtures.
according to PPE addition and PPE concentration increase. Frequency sweep curves were adjusted using the Power Law Eqs. (2) and (3) which is the model that best described the experimental data.
Table 3 Frequency dependence of elastic (G’) and viscous (G’’) moduli of chitosan/gelatin mixtures with and without PPE (γ = 5%, T = 25 °C). Sample
ω
C2G1 C2G1E50 C2G1E100 C2G1E150 C2G1E200
27.30 31.81 32.57 34.77 31.60
crossover
± ± ± ± ±
(rad s−1)
G’
3.69ª 3.56a 3.80a 2.80a 1.87a
21.18 26.88 20.30 22.21 17.11
crossover
± ± ± ± ±
(Pa)
1.67a,b 1.97ª 2.44b 3.36a,b 1.05b
k’ 1.339 1.737 1.087 0.918 1.047
n' ± ± ± ± ±
0.138b 0.201a 0.099c 0.117c 0.165c
0.824 0.774 0.811 0.885 0.767
± ± ± ± ±
0.029a,b 0.047a,b 0.029a,b 0.057a 0.047b
R2 (G’)
k’’
0.995 0.986 0.984 0.989 0.967
3.221 3.868 2.530 2.545 2.012
In the same column, values with the same superscript letter (a–d) were no significantly different (P > 0.05). 6
R2 (G’’)
n’’ ± ± ± ± ±
0.061a,b 0.215a 0.171b,c 0.519b,c 0.245c
0.555 0.543 0.585 0.601 0.609
± ± ± ± ±
0.013c,d 0.007d 0.003ª,b,c 0.016ª,b 0.012a
0.993 0.990 0.994 0.994 0.995
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Fig. 5. (a) curves of viscosity versus shear rate for all the mixtures at 25 °C; (b) curves of viscosity versus shear rate for C2G1E100 mixture for temperatures ranging from 15 °C to 40 °C.
3.4. Scanning electron microscopy (SEM)
linkages between chitosan and gelatin polymers. Although the overall effect was a decrease in η0 values with increasing PPE concentration, it was observed a slight increase for C2G1E100 sample when compared to C2G1E50; even so, they were statistically similar within the considered confidence interval (P > 0.05). In fact, this effect is stronger for samples with high PPE concentration (C2G1E150 and C2G1E200) which showed a significant decrease in η0 values in comparison with control sample (C2G1), confirmed by statistical analysis. The evaluation of the rheological properties as function of temperature is necessary to understand the behavior of the chitosan/gelatin/PPE mixtures. As their potential application as food coatings, the temperature is the principal factor that may affect the quality and the deterioration of coated fruits. Fig. 5b shows the viscosity curves versus shear rate of C2G1E100 mixture at different temperatures. The viscosity dependence of the temperature was used to calculate the activation energy of the C2G1E100 mixture applying the Arrhenius equation (Eq. (5)). Basically, the activation energy (Ea) is defined as the energy required for the molecule of a fluid to move freely and its value depends on factors such as polymer nature and concentration (Abbastabar, Azizi, Adnani, & Abbasi, 2014; Anvari et al., 2016; Razmkhah et al., 2017). The curves of ln η0 versus 1/T for the mixtures with and without PPE (Fig. S3) were used for Ea calculation (Table 4). The Arrhenius model showed a satisfactory fitting of the temperature-viscosity relation as can be noticed by R2 values reported in Table 4. Comparison of the Ea values as a function of extract concentration (Fig. S4) showed a decrease in this parameter after PPE addition, from 39.27 kJ mol−1 in C2G1 to 15.89 kJ mol−1 in C2G1E200. Therefore, the mixtures with higher amount of PPE were less sensitive to temperature changes and were easier to flow. In all rheological measurements a slightly decrease in C2G1E200 properties was observed which can be related with a limit of PPE that can be added in this polymeric system. However, antioxidant properties were preserved that enable the use of the developed material for the desired application.
The surface properties of freeze-dried scaffold (Fig. S5) were observed using SEM. The SEM images of all mixtures are shown in Fig. 6A–E at 500× of magnification. The surface morphology characteristics exhibits differences in pore size, with a clear increase related to the PPE addition ranging from 0 to 100 mg (C2G1E100 scaffold) as observed in the sequence images of Fig. 6A–C. The addition of 150 mg (Fig. 6D) and 200 mg (Fig. 6E) of pomegranate peel extract affects differently the pore size and a decrease in this parameter was observed. The measured values of the mean pore diameters are summarized in Table S1. The reason for that behavior could be a saturation of the linkages of chitosan/gelatin polymeric system with phenolic components for high PPE concentrations. At high magnification of 15,000x (Fig. 6F) it is evident the presence of PPE adhered to the chitosan/gelatin network as exemplified by the C2G1E200 scaffold surface. 4. Conclusions This study focused on the development and the characterization of chitosan/gelatin-based materials incorporated with pomegranate peel extract, aiming the reuse of this agroindustry residue in potential valueadded applications. To evaluate if PPE addition actually contributed with significant changes in the structural and functional characteristics of the material, rheological and antioxidant properties of the obtained mixtures were explored. PPE incorporation in the chitosan/gelatin polymeric systems enhanced their antioxidant capacity, as it was already expected due to PPE high phenolic content. Regarding to rheology, PPE addition increased the materials viscous behavior and their stability against the strain applied, which is a promising result for eventual applications of them as fruits edible coatings: low viscous behavior and low strength materials might not adhere well on fruit´s surface. These characteristics would suggest the use of large amount of material and more expensive coating techniques, instead of dip the fruit
Table 4 η0, k and n parameters obtained by Cross-model (at 25 °C) and Ea and R2values obtained by Arrhenius equation from steady shear measurements. Sample
η0 (Pa.s)
C2G1 C2G1E50 C2G1E100 C2G1E150 C2G1E200
2.18 1.74 1.91 1.58 1.56
± ± ± ± ±
k (s) 0.37a 0.11a,b 0.12a,b 0.10b 0.10b
0.080 0.052 0.058 0.036 0.069
n ± ± ± ± ±
0.032a 0.003a 0.009a 0.004a 0.032a
0.705 0.732 0.745 0.784 0.689
Ea ± ± ± ± ±
0.043a 0.038a 0.034a 0.057a 0.069a
In the same column, values with the same superscript letter (a–b) were no significantly different (P > 0.05). * Ea and R2 are related with Arrhenius equation. 7
*
(kJ mol−1)
39.27 25.60 24.11 17.83 15.89
R2
*
0.977 0.924 0.994 0.958 0.981
Carbohydrate Polymers 228 (2020) 115386
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Fig. 6. SEM photomicrographs of the surface of: (A) C2G1, (B) C2G1E50, (C) C2G1E100, (D) C2G1E150, (E) C2G1E200, magnification of 500×, and (F) C2G1E200, magnification of 15,000×.
Author’s contributions
into the mixture. The frequency sweep measurements indicated that PPE interacts with chitosan and gelatin chains resulting in a network with very weak gel strength, which helped us to better understand the interactions of the phenolic compounds once exposed to the polymer matrix. For this reason, mixtures with high amount of PPE were easier to flow and showed lower activation energy values, in which the addition of 200 mg of PPE corresponds to an activation energy of 15.89 kJ mol −1. Furthermore, it was evident that flow properties were affected by PPE concentration and temperature. Overall, it was demonstrated a step forward in exploring the potential use of renewable and highly antioxidant materials like PPE to improve the structural, functional and antioxidant characteristics of a well-known polymer blend, applied in a variety of fields. It is believed that this study opens a range of new studies to be conducted in the future, according to the intended application of the developed materials.
VCAM and MRVB conceived the study. VCAM and MRVB designed the experiments. MRVB and VCAM contributed to the experimental work. LBB and MRVB contributed to the antioxidant assays. MRVB, VCAM, MMH, LBB and AMGP contributed to the results interpretation. AMGP supervised the final version. All authors read and approved the final manuscript.
Funding This work was supported by the National Council for Scientific and Technological Development (CNPq) contract number 140406/2019-0 for MRVB and by the São Paulo Research Foundation (FAPESP) contract number 2017/15477-8 for LBB.
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