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Synthesis and characterization of cassava starch with maleic acid derivatives by etherification reaction
Accepted Manuscript Title: Synthesis and characterization of cassava starch with maleic acid derivatives by etherification reaction Authors: Samuel H. Clasen, Carmen M.O. Muller, ¨ Alexandre L. Parize, Alfredo T.N. Pires PII: DOI: Reference:
S0144-8617(17)31161-X https://doi.org/10.1016/j.carbpol.2017.10.016 CARP 12865
To appear in: Received date: Revised date: Accepted date:
8-5-2017 2-10-2017 3-10-2017
Please cite this article as: Clasen, Samuel H., Muller, ¨ Carmen MO., Parize, Alexandre L., & Pires, Alfredo T.N., Synthesis and characterization of cassava starch with maleic acid derivatives by etherification reaction.Carbohydrate Polymers https://doi.org/10.1016/j.carbpol.2017.10.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and characterization of cassava starch with maleic acid derivatives by etherification reaction Samuel H. Clasena*, Carmen M. O. Müllerb, Alexandre L. Parizec, Alfredo T. N. Piresd a
Chemistry Department – Polymeric Materials Research Group, Federal University of Santa
Catarina Campus Florianópolis, P.O. Box 476, 88040-900 Florianópolis, SC, Brazil, e-mail address:[email protected], telephone number +5548984182654. b
Department of Food Science and Technology, Federal University of Santa Catarina Campus
Florianópolis, 88034-001 Florianópolis, SC, Brazil, e-mail address: [email protected]. c
Chemistry Department – Polymeric Materials Research Group, Federal University of Santa
Catarina Campus Florianópolis, P.O. Box 476, 88040-900 Florianópolis, SC, Brazil, e-mail address: [email protected]. d
Chemistry Department – Polymeric Materials Research Group, Federal University of Santa
Catarina Campus Florianópolis, P.O. Box 476, 88040-900 Florianópolis, SC, Brazil, e-mail address: [email protected].
*
Corresponding author: E-mail address: [email protected] (Samuel H. Clasen) Phone: + 55 48 3271-2313
Highlights
Etherification reaction of cassava starch.
Modification of the starch fobicity.
Determination of the degree of substitution from 1H-NMR analyzes.
Potential of modified starch for use in bioactive packaging. 1
Potential of modified starch for use as encapsulation medium.
Abstract Cassava starch was grafted with three different esters by the etherification reaction and its modification was characterized by 1H-NMR, FTIR, DSC, SEM, XDR, contact angle and SLS. The samples grafted with diethyl maleate, dipropyl maleate, and dibutyl maleate showed DS values of 2.3, 1.0 and 2.0, respectively, determined from 1H-NMR analysis and confirmed by FTIR analysis, with the appearance of bands at 1721, 1550 and 1126 cm-1. The FTIR, XRD, SEM and DSC results indicated a change in the intra and intermolecular hydrogen interactions in the grafted starch when compared to native starch. Based on the contact angles, it was observed that the macromolecular starch chain acquired hydrophobic characteristics through the substitution of the hydrogens with di maleate esters. The characteristics acquired by grafted starch allow it to be used for the encapsulation of bioactive molecules for the production of bioactive packages and the production of biodegradable packages. Keywords: Starch modification, etherification, biodegradable packaging, bioactive packaging 1 – Introduction Starch constitutes the carbohydrate reserve of many plants, and it is found in leaves (chloroplasts) and in the reproductive organ (amyloplasts). It is extracted commercially from grains such as corn and rice and from tubers and roots like potato and cassava. In the food industry, it is used in soups, sauces, baking products, confectionery products, and dairy products, among others. It can also be applied in pharmaceuticals, textiles, fuels, biodegradable packaging materials, and thin films of thermoplastics, among other items 2
(Kaur, Ariffin, Bhat, & Karim, 2012). Its application is due to its action as a thickener, emulsifier, gel-former and stabilizer, modifying the texture of the emulsions, and also as a pharmaceutical excipient (Wiacek & Dul, 2015). Starch is a biopolymer composed of glucose as repeat units. There are, however, two different structures, amylose and amylopectin (Han, 2005). An important aspect to note is that it is a renewable, low cost, widely availability material, which can replace the petrochemical polymers currently used in industry (Mensitieri et al., 2011; Müller, Pires, & Yamashita, 2012; Ye et al., 2017). As is the case in several biopolymers, starch exhibits the characteristic of hydrophilicity, which assists in biodegradability, but also impairs other properties such as water vapor permeability and interactions with hydrophobic substances (Soares, Yamashita, Müller, & Pires, 2013; Yu, Dean, & Li, 2006). Starch modification is a methodology used in several areas to change the micro and macroscopic characteristics. Sonication and ultrasound can be used to alter the gel-forming properties of starch (Ashokkumar, 2015). Starch modification can also be used for the encapsulation of natural substances, such as Melissa Officinalis (Mourtzinos, Papadakis, Igoumenidis, & Karathanos, 2011). Among the various starch modification objectives, enhancing the compatibility of starch with hydrophobic materials in polymeric blends intended for the production of packaging materials is a notable example (Xiong et al., 2014; Zuo et al., 2015). There are several starch modification techniques, including: i) chemical modification; ii) physical modification; iii) enzymatic modification; and iv) genetic modification (Kaur et al., 2012; Zhu, 2015). Within each class of modification exists a great diversity, for instance, possible chemical modifications include the reactions of esterification and etherification, of which the former are more frequently used than the latter. 3
The chemical modification of starch is of great interest when the surface acquires hydrophobic characteristics. When this characteristic is achieved, starch can be used in several situations, as emulsions, pharmaceutical excipients, hydrophobic polymer blends and others. In this context, the aim of this study was to prepare starch derivatives by the grafting reaction of diethyl maleate, dipropyl maleate and dibutyl maleate (derived from maleic acid) in the macromolecular chain of starch by etherification reactions (Figure 1), based on previous studies (Bien, Wiege, Warwel, & Addition, 2001; Wokadala, Emmambux, & Ray, 2014). This etherification reaction mechanism has been selected so that the two R groups present in the reactant are available to promote a surface interaction with another hydrophobic polymer material. After the etherification reaction, the degree of substitution and the physicochemical properties of the modified starch will be evaluated. The grafted starch must present a high degree of substitution, thus providing hydrophobic characteristics for the macromolecular chain for future applications in the area of biodegradable packaging and in the encapsulation of active principles for bioactive packaging and drug delivery.
Figure 1: Starch etherification reaction scheme.
2 – Materials and methods 2.1 – Materials
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Cassava starch (Manihot esculento), with 22.5 ± 2.5 % of amylose and 14.4 ± 0.6 % of moisture, was supplied by Indemil (Diadema-SP, Brazil). Maleic acid (analytical grade), acetic acid (analytical grade) and sodium hydroxide (analytical grade) were supplied by Vetec (Duque de Caxias-RJ, Brazil). Ethanol, 1-propanol, 1-butanol (anhydrous), Amberlist 15 hydrogen and dialysis tubing (D9402) were supplied by Sigma-Aldrich (Brazil). Hydrogen peroxide (35 %) and sulfuric acid (analytical grade) were supplied by Lafan (Várzea Paulista-SP, Brazil). All of the chemical reagents were used without further purification. 2.2 – Synthesis of precursors for starch chemical modification Initially, three different precursors, diethyl maleate, dipropyl maleate and dibutyl maleate, were synthetized for the starch chemical modification. The esterification reaction was performed according to the method described by (Yadav & Thathagar, 2002) with some modifications. The reaction medium, maleic acid, alcohol (ethanol, propanol or butanol) and catalyst (Amberlyst 15 hydrogen) remained under stirring and reflux for 4 h. The product obtained was then purified and characterized by infrared spectroscopy (FTIR) and nuclear magnetic resonance of hydrogen (1H-NMR). 2.3 – Starch modification The starch grafted reaction scheme is shown in Figure 1, where R is the alkyl groups. The etherification procedure was carried out according to Wokadala et al., 2014 with modifications. For this, 10 g of granular starch was dispersed in 70 mL of water, 9 mL of NaOH (3 mol L-1) was added and the solution was maintained at 110 °C. The precursor in peracetic acid solution was added to the gelatinized starch, and maintained under reflux for 100 min. Next, the starch was precipitated with ethanol and the dispersion centrifuged under a gravitational force of 1900 x g for 25 min. The extracted starch was maintained in a
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desiccator for 20 days, washed with diethyl ether and dialyzed to remove the unreacted components. The products were characterized by proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), determination of the contact angle, differential scanning calorimetry (DSC), X-ray diffraction (XDR) and static light scattering (SLS). 2.4– Characterization 2.4.1 – 1H-NMR and FT-IR analyses The precursors and modified starch were analyzed by infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (1H-NMR). The FTIR spectra of the samples were obtained on a PRESTIGE 21 spectrophotometer (Shimadzu, Japan), equipped with a multiple reflectance accessory, with a resolution of 4 cm-1, and performing 20 scans. The FTIR measurements were carried out in KBr or as a thin film on an Si support. The nuclear magnetic resonance was recorded on a Varian AS400 spectrometer. The samples were dissolved in CDCl3 (precursors) or DMSO-d6 (starch samples) for the 1H-NMR analysis. 2.4.2 – Scanning electron microscopy (SEM) The granular native starch and grafted starch were deposited on a carbon tape and placed in a desiccator containing silica for 24 h. The specimens were coated with gold to avoid charging by the electron beam and analyzed by scanning electron microscopy (JSM6701F, JEOL). 2.4.3 – Contact angle The influence of water and diiodomethane on the surface hydrophilicity was observed by contact angle measurements using a Ramé-Hart 250-F1 (USA). The sessile drop experiment consists of placing water or diiodomethane on the grafted starch surface. 6
The formed drop spreads until it attains an equilibrium state, and a finite contact angle formed by the static drop with the surface can be determined by computer software (DropImage). Measurements were taken on the left and right sides of the 3L droplet profile for each liquid probe. 2.4.4 – Differential scanning calorimetry (DSC) DSC curves were obtained on a Shimadzu 50 (DSC-50 - Shimadzu, Japan) by heating from –50 °C to 250 °C at 5 °C min-1 in a nitrogen atmosphere (50 mL min-1). Only one heating cycle was used in all of the thermo analysis experiments. 2.4.5 – X-Ray Diffraction The granular native starch and grafted starch were deposited on the sample holder. The analysis was performed with a Philips X’Pert diffractometer (Netherlands), using copper radiation K (k = 1.5418 Å), voltage of 40 kV and operation current of 30 mA. All assays were performed with 2 = 2° and 2 = 50°, pitch of 0.05 °/s. The crystallinity index (CI) was determined by Equation 1, where Ac is the crystalline area and Aa is the amorphous halo area. 𝐶𝐼 =
𝐴𝑐 𝐴𝑐 +𝐴𝑎
× 100
Equation 1
2.4.6 – Static light scattering (SLS) The molar mass of native starch and grafted starch was determined by static light scattering (SLS) measurements. An ALV/CGS-3 (Germany) light scattering spectrometer was used, with an ALV/LSE-7004 multiple- digital correlator equipped with a 22 mW red helium-neon linearly polarized laser ( = 632.8 nm). The solvent was DMSO, with dn/dc equal to 0.066 mL g-1, and the starch solution concentrations were 1.0 x10-4, 3.0 x10-4, 6.5 x10-4, 1.0 x10-3, 3.0 x10-3 g mL-1. All samples were systematically assayed at different 7
scattering angles from 30° to 150° with 3° of increment. Data were collected digitally, using ALV Correlator Control software. The molar mass was determined using the ALV/Fit & Plot software.
3 – Results and Discussion 3.1 –Proton nuclear magnetic resonance (1H-NMR) and infrared spectroscopy (FTIR) Figures S1 and S2 show the 1H-NMR and FTIR of the synthetized diethyl maleate, dipropyl maleate and dibutyl maleate, respectively. These compounds showed ester groups, sp2 and sp3 carbons, with similar absorption bands to unsaturated esters (C-O-C), in the range of 1300 to 1220 cm-1, sp2 carbon (C = C) at 1644 cm-1 and sp3 carbons -CH2- and CH3 at 2965 and 2879 cm-1, respectively. All FTIR spectra of the analyzed precursors showed an absence of OH absorption band at 3500 cm-1, suggesting that the esterification reaction occurred in the hydroxyls group present in maleic acid, as shown in Figure S2. In the 1H-NMR spectrum of diethyl maleate, Figure S2, there were three hydrogen peaks, as expected due to symmetry, in the unsaturated carbon. A triplet with a chemical shift at 1.29 ppm was characteristic of sp3 carbon (-CH3), and a quartet with a chemical shift at 4.22 ppm was characteristic of a sp3 carbon (-CH2-) attached to a carbonyl group. A singlet with a chemical shift at 6.22 ppm was characteristic of the sp2 carbon (-CH = CH-), linked to a carbonyl group. In the spectrum of dipropyl maleate, in addition to the peaks discussed above, there was a further sextuplet signal with a chemical shift at 1.68 ppm, characteristic of sp3 carbon (-CH2-), as expected. As the spectrum of dibutyl maleate showed, we can observe one more sextuplet signal with a chemical shift at 1.40 ppm characteristic of the sp3 carbon (-CH2-). The precursors were obtained as expected.
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Figure 2 shows the 1H-NMR spectrum of the native cassava starch. The peak at 2.5 ppm was related to the proton of DMSO-D6 used as solvent, and an intense peak at 3.3 ppm was characteristic of the water present in solvent medium. The peaks referring to hydrogen numbered 2 to 6 (Figure 2) were partially masked by the peak due to water present in the solvent medium. Hydrogen 1 (linked to the 1-4 alpha glycosidic carbon bond) and 1' (linked to the alpha 1-6 glycosidic carbon bond) were similar and exhibited a chemical shift at around 5 ppm, related to a neighborhood surrounded by oxygen atoms with high electronic density. The hydroxylic hydrogens (7, 8 and 9) showed different chemical displacements, although they were connected at the oxygen, due to their adjacent group. Due to its adjacent group with lower electronic density, hydrogen 7 showed a chemical shift at 4.5 ppm, and hydrogens 8 and 9 had a similar adjacent group and higher electron density due to the oxygen atoms of the glycosidic bonds, displacing the peaks to 5.5 ppm (Tizzotti, Sweedman, Tang, Schaefer, & Gilbert, 2011; Xiong et al., 2014; A. Zhang et al., 2013).
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Figure 2: 1H-NMR spectrum of native cassava starch. The numbers in the peaks correspond to the hydrogen numbers in the above structure. Figure 4 shows the 1H-NMR spectra of native starch, starch-diethyl maleate, starchdipropyl maleate and starch-dibutyl maleate, in which we can observe displaced peaks related to the hydroxyl groups of the starch, and appearance of peaks between 0.5 and 1.5 ppm, probably related to the presence of hydrogens of sp3 carbon bonded to sp3 carbons. The decrease in the peak intensities, related to the hydroxyl groups of the macromolecular chain of the starch with the appearance of sp3 hydrogens, indicates the efficiency of the etherification reaction. The FTIR spectra of grafted starch showed two absorption bands at 1721 cm-1 1550 cm-1 related to the C = O stretching and CH stretching adjacent to carbonyl and oxygen, respectively. Another absorption band at 1126 cm-1, characteristic of the C-O-C stretching of the ester group, was present in the spectra of the precursors and was not observed in the infrared spectrum of the native starch. These absorption bands at 1721 and 1126 cm-1 were characteristic of groups present in the precursor molecules, indicating the effective grafting reaction.
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Figure 3: 1H-NMR spectrum of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate The 1H-NMR data were further used to determine the substitution degree of the precursors in the macromolecule starch, using the peak in 3.6 ppm as a reference and evaluating the intensity of hydroxyl group (de Graaf, Lammers, & Et Al., 1995; Wokadala et al., 2014). The degrees of substitution of starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate were 2.3, 1.0, and 2.0, respectively. The substitution reaction was directed at the hydroxyls attached to the glycosidic ring, as verified by the 1H-NMR spectra (Figure 4, chemical shift 5.5 ppm). This reaction preference was due to the fact that the glycosidic ring stabilized the electronic density of oxygen, facilitating the attack of oxygen in the epoxy ring of the reaction medium.
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Figure 4: FTIR spectrum of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate. It can also be observed in the FTIR spectra (Figure 4) that the OH stretching band (~ 3400 cm-1) was broad and highly symmetrical in the native starch spectrum. However, the spectra of the grafted starches showed an asymmetric band, displacing its absorption band at higher wavenumbers. This displacement at higher wavenumber was related to the higher energy required for OH stretching, indicating that the hydroxyl groups still present in the graft starch were not compromised by hydrogen bonds compared to the native starch (Liang & Ludescher, 2015; C. Zhang et al., 2017). These data complemented the characterization of the etherification reaction and indicated a decrease in the hydrophilicity of the starch after the modification reaction. 3.2 – Scanning electron microscopy (SEM)
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Figure 5 shows the SEM micrographs of the native starch and grafted starches. The native starch presented a semicrystalline granular form with an oval shape and a size range between 5 and 15 μm (Zhu, 2015).
Figure 5: Scanning electron macroscopy images of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate. After the modification process, the starch granules presented changes in their shape and surface morphology. This process was not related to the graft reaction but to the rupture of the residual crystal structure of the starch process followed by recrystallization, and the starch did not maintain its granular form. The surface morphology of the granules presented a greater roughness and an irregular structure after the modification, when compared with the native starch. This change in starch roughness may be related to a decrease in intra and intermolecular interactions between the hydroxyl groups present in the macromolecule repeating unit, influencing the degree of crystallinity in the samples (Chauhan et al., 2015).
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3.3 – XRD characterization The crystallinity index for the cassava starch (36 %) was in agreement with the value obtained by authors using the same experimental method, as shown in Figure 6 (Garrido, Schnitzler, Zortéa, de Souza Rocha, & Demiate, 2012). The diffractogram of native starch showed strong reflections at 2θ, equal to 15.2º , 17.2º, 18,2º, and 23.0º , and weaker unsolved peaks can be observed at 11.4º, 19.9º, 26,2º and 31.5º. The reflections presented in the XRD pattern were in agreement with other authors, suggesting that the starch crystalline structure was of A-type related to a monoclinic lattice (Lin, Li, Long, Su, & Huang, 2014). The disappearance of the crystal peaks (15° and 18°) and the decrease in the peak at 22° indicate that the crystallinity of the native starch was affected during the process, which implies that the hydrogen bond of the starch was disrupted. After the etherification reaction, the samples showed a decrease in the crystallinity degree, which corroborates the DSC results. The crystallization form of the grafted starch changed because it did not present an orientation in the retrogradation to the reestablishment of the hydrogen bonds. The modified starch samples showed a new diffraction peak at 6° and a shift in the peak at 23° to 21°, which implies that new crystalline regions were formed in the modified starch ethers.
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Figure 6: X-ray diffractograms of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate.
3.4 –Differential scanning calorimetry (DSC) The melting enthalpy, related to the energy required for the rupture of the residual crystal structure of the starch, underwent a significant reduction after the grafting of the precursors (Table 1). This reduction did not have a significant influence on the changes in the grafted group, but instead showed a tendency toward a reduction with the carbonic chain of the grafted group. The change in the melting enthalpy occurred because of the rupture of the intra and intermolecular hydrogen bonds of the starch. The melting enthalpy is related to a crystallinity of the starch, and the lower degree of crystallinity the lower the melting enthalpy will be. This corroborates the proposal that a substitution reaction decreases the interactions between the hydroxyl group of the repeat unit of starch, thus decreasing the residual crystallinity of the native starch and consequently the melting enthalpy. 15
The melting peak temperature differed for each grafted starch sample. The starchdiethyl maleate showed no significant decrease in relation to native starch, but the starchdipropyl maleate and starch-dibutyl maleate samples showed a reduction. This behavior is attributed to the degree of crystallinity of the grafted starch. Table 1: Melting temperature, melting enthalpy, molar mass and crystallization index of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate. Sample Native starch Starch-diethyl maleate Starch-dipropyl maleate Starch-dibutyl maleate
Tm (°C) Hm (J.g-1) Mw (g.mol-1) 108 31 1.0 x 108 108 16 1.2 x 106 94 21 1.0 x 106 95 20 4.2 x 106
CI (%) 36 30 28 29
3.5 – Contact angle Table 2 shows the data for the contact angles of the modified starch samples obtained with polar solvent (water) and an apolar solvent (diiodomethane). The contact angles for the apolar solvent presented lower values compared to the contact angles for the water. These data indicate that the etherified starch had a more hydrophobic character. The higher affinity of the apolar solvent (diiodomethane) in the sample compared to the polar solvent (water) was shown by a smaller angle of contact between the drop of the liquid and the sample surface. The contact angle values corroborated the data reported above, indicating effective starch modification and showing that the grafting of apolar groups in the starch backbone chain led to grafted starch samples with an apolar character. Table 2: Contact angles for starch-diethyl maleate, starch-dipropyl maleate and starchdibutyl maleate. Sample
Water (°)
Diiodomethane (°)
Starch-diethyl maleate Starch-dipropyl maleate Starch-dibutyl maleate
31.8 ± 0.1 30.0 ± 0.1 32.1 ± 0.2
23.8 ± 0.2 29.3 ± 0.2 25.1 ± 0.1
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3.6 – Static light scattering (SLS) The molar mass of the native starch obtained from the SLS analysis presented values in the order of 1.0 x 108 g mol-1 (Table 1). These values are close to those obtained in the literature for cassava starch and other sources obtained by chromatography analysis (GPC) (Israkarn, Na Nakornpanom, & Hongsprabhas, 2014; Kowittaya & Lumdubwong, 2014; Miao et al., 2015). The analyses were performed using DMSO as a solvent to decrease the aggregation forming effect, since samples analyzed with DMSO show a better dispersion of the starch macromolecules compared with starch solution in water (Kowittaya & Lumdubwong, 2014). The etherified starch samples showed a 100-fold reduction in the molar mass, regardless of the grafted group. This indicates that the substitution reaction mechanism did not interfere in the reduction of the molar mass of the starch. The reduction of the molar mass of the starch is related to the depolymerization reaction caused by the basic treatment used in the synthesis (Misman, Azura, & Hamid, 2015). 4 – Conclusions The etherification reactions of starch from maleic acid derivatives showed a high degree of substitution, with a mean of two substitutions at each repeat unit. Of the three reactive hydroxyls groups present in the starch repeat unit, it can be seen from the 1H-NMR analysis that the hydroxyls attached directly to the glycosidic ring were preferentially substituted because the glycosidic ring stabilized the reaction intermediately, thus allowing the maleic acid derivatives to attack. Grafting of the maleic acid derivatives was affected by intra- and intermolecular interactions, thus decreasing the melting enthalpy of the modified starch. The modification
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also changed the crystal structure and decreased the degree of crystallinity (%) compared to native starch. The surfaces of the grafted starch samples had an irregular shape with greater roughness and a greater hydrophobicity resulting from the grafting. The molar mass of the starch was reduced by the selected synthesis method, where the starch undergoes basic hydrolysis in the presence of sodium hydroxide. This hydrolysis reduced the molar mass of the starch approximately one-hundred-fold. The modification of the starch surface added a hydrophobic character and thus amplified its potential uses and applications. Modified starch can be used as a compatibilizer in polymer blends with hydrophobic polymers for the production of sheets and films for biodegradable and bioactive packaging, in the food industry and in the encapsulation of natural substances or hydrophobic drugs, among other applications. Acknowledgements The authors are grateful for the financial support provided by CAPES and CNPq.
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Wiacek, A. E., & Dul, K. (2015). Effect of surface modification on starch/phospholipid wettability. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 480, 351–359. http://doi.org/10.1016/j.colsurfa.2015.01.085 Wokadala, O. C., Emmambux, N. M., & Ray, S. S. (2014). Inducing PLA/starch compatibility through butyl-etherification of waxy and high amylose starch. Carbohydrate Polymers, 112, 216–224. http://doi.org/10.1016/j.carbpol.2014.05.095 Xiong, Z., Ma, S., Fan, L., Tang, Z., Zhang, R., Na, H., & Zhu, J. (2014). Surface hydrophobic modification of starch with bio-based epoxy resins to fabricate highperformance polylactide composite materials. Composites Science and Technology, 94, 16–22. http://doi.org/10.1016/j.compscitech.2014.01.007 Yadav, G. D., & Thathagar, M. B. (2002). Esterification of maleic acid with ethanol over cation-exchange resin catalysts. Reactive and Functional Polymers, 52(2), 99–100. http://doi.org/10.1016/S1381-5148(02)00086-X Ye, F., Miao, M., Lu, K., Jiang, B., Li, X., & Cui, S. W. (2017). Structure and physicochemical properties for modified starch-based nanoparticle from different maize varieties. Food Hydrocolloids. http://doi.org/10.1016/j.foodhyd.2016.12.041 Yu, L., Dean, K., & Li, L. (2006). Polymer blends and composites from renewable resources. Progress in Polymer Science, 31(6), 576–602. http://doi.org/10.1016/j.progpolymsci.2006.03.002 Zhang, A., Zhang, Z., Shi, F., Ding, J., Xiao, C., Zhuang, X., … Chen, X. (2013). Disulfide crosslinked PEGylated starch micelles as efficient intracellular drug delivery platforms. Soft Matter, 9, 2224–2233. http://doi.org/10.1039/c2sm27189c Zhang, C., Li, F., Li, J., Wang, L., Xie, Q., Xu, J., & Chen, S. (2017). A new biodegradable composite with open cell by combining modified starch and plant fibers. Materials & Design, 120, 222–229. http://doi.org/10.1016/j.matdes.2017.02.027 Zhu, F. (2015). Composition, structure, physicochemical properties, and modifications of cassava starch. Carbohydrate Polymers, 122, 456–480. http://doi.org/10.1016/j.carbpol.2014.10.063 Zuo, Y. F., Gu, J., Qiao, Z., Tan, H., Cao, J., & Zhang, Y. (2015). Effects of dry method esterification of starch on the degradation characteristics of starch/polylactic acid composites. International Journal of Biological Macromolecules, 72, 391–402. http://doi.org/10.1016/j.ijbiomac.2014.08.038
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S0144-8617(17)31161-X https://doi.org/10.1016/j.carbpol.2017.10.016 CARP 12865
To appear in: Received date: Revised date: Accepted date:
8-5-2017 2-10-2017 3-10-2017
Please cite this article as: Clasen, Samuel H., Muller, ¨ Carmen MO., Parize, Alexandre L., & Pires, Alfredo T.N., Synthesis and characterization of cassava starch with maleic acid derivatives by etherification reaction.Carbohydrate Polymers https://doi.org/10.1016/j.carbpol.2017.10.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis and characterization of cassava starch with maleic acid derivatives by etherification reaction Samuel H. Clasena*, Carmen M. O. Müllerb, Alexandre L. Parizec, Alfredo T. N. Piresd a
Chemistry Department – Polymeric Materials Research Group, Federal University of Santa
Catarina Campus Florianópolis, P.O. Box 476, 88040-900 Florianópolis, SC, Brazil, e-mail address:[email protected], telephone number +5548984182654. b
Department of Food Science and Technology, Federal University of Santa Catarina Campus
Florianópolis, 88034-001 Florianópolis, SC, Brazil, e-mail address: [email protected]. c
Chemistry Department – Polymeric Materials Research Group, Federal University of Santa
Catarina Campus Florianópolis, P.O. Box 476, 88040-900 Florianópolis, SC, Brazil, e-mail address: [email protected]. d
Chemistry Department – Polymeric Materials Research Group, Federal University of Santa
Catarina Campus Florianópolis, P.O. Box 476, 88040-900 Florianópolis, SC, Brazil, e-mail address: [email protected].
*
Corresponding author: E-mail address: [email protected] (Samuel H. Clasen) Phone: + 55 48 3271-2313
Highlights
Etherification reaction of cassava starch.
Modification of the starch fobicity.
Determination of the degree of substitution from 1H-NMR analyzes.
Potential of modified starch for use in bioactive packaging. 1
Potential of modified starch for use as encapsulation medium.
Abstract Cassava starch was grafted with three different esters by the etherification reaction and its modification was characterized by 1H-NMR, FTIR, DSC, SEM, XDR, contact angle and SLS. The samples grafted with diethyl maleate, dipropyl maleate, and dibutyl maleate showed DS values of 2.3, 1.0 and 2.0, respectively, determined from 1H-NMR analysis and confirmed by FTIR analysis, with the appearance of bands at 1721, 1550 and 1126 cm-1. The FTIR, XRD, SEM and DSC results indicated a change in the intra and intermolecular hydrogen interactions in the grafted starch when compared to native starch. Based on the contact angles, it was observed that the macromolecular starch chain acquired hydrophobic characteristics through the substitution of the hydrogens with di maleate esters. The characteristics acquired by grafted starch allow it to be used for the encapsulation of bioactive molecules for the production of bioactive packages and the production of biodegradable packages. Keywords: Starch modification, etherification, biodegradable packaging, bioactive packaging 1 – Introduction Starch constitutes the carbohydrate reserve of many plants, and it is found in leaves (chloroplasts) and in the reproductive organ (amyloplasts). It is extracted commercially from grains such as corn and rice and from tubers and roots like potato and cassava. In the food industry, it is used in soups, sauces, baking products, confectionery products, and dairy products, among others. It can also be applied in pharmaceuticals, textiles, fuels, biodegradable packaging materials, and thin films of thermoplastics, among other items 2
(Kaur, Ariffin, Bhat, & Karim, 2012). Its application is due to its action as a thickener, emulsifier, gel-former and stabilizer, modifying the texture of the emulsions, and also as a pharmaceutical excipient (Wiacek & Dul, 2015). Starch is a biopolymer composed of glucose as repeat units. There are, however, two different structures, amylose and amylopectin (Han, 2005). An important aspect to note is that it is a renewable, low cost, widely availability material, which can replace the petrochemical polymers currently used in industry (Mensitieri et al., 2011; Müller, Pires, & Yamashita, 2012; Ye et al., 2017). As is the case in several biopolymers, starch exhibits the characteristic of hydrophilicity, which assists in biodegradability, but also impairs other properties such as water vapor permeability and interactions with hydrophobic substances (Soares, Yamashita, Müller, & Pires, 2013; Yu, Dean, & Li, 2006). Starch modification is a methodology used in several areas to change the micro and macroscopic characteristics. Sonication and ultrasound can be used to alter the gel-forming properties of starch (Ashokkumar, 2015). Starch modification can also be used for the encapsulation of natural substances, such as Melissa Officinalis (Mourtzinos, Papadakis, Igoumenidis, & Karathanos, 2011). Among the various starch modification objectives, enhancing the compatibility of starch with hydrophobic materials in polymeric blends intended for the production of packaging materials is a notable example (Xiong et al., 2014; Zuo et al., 2015). There are several starch modification techniques, including: i) chemical modification; ii) physical modification; iii) enzymatic modification; and iv) genetic modification (Kaur et al., 2012; Zhu, 2015). Within each class of modification exists a great diversity, for instance, possible chemical modifications include the reactions of esterification and etherification, of which the former are more frequently used than the latter. 3
The chemical modification of starch is of great interest when the surface acquires hydrophobic characteristics. When this characteristic is achieved, starch can be used in several situations, as emulsions, pharmaceutical excipients, hydrophobic polymer blends and others. In this context, the aim of this study was to prepare starch derivatives by the grafting reaction of diethyl maleate, dipropyl maleate and dibutyl maleate (derived from maleic acid) in the macromolecular chain of starch by etherification reactions (Figure 1), based on previous studies (Bien, Wiege, Warwel, & Addition, 2001; Wokadala, Emmambux, & Ray, 2014). This etherification reaction mechanism has been selected so that the two R groups present in the reactant are available to promote a surface interaction with another hydrophobic polymer material. After the etherification reaction, the degree of substitution and the physicochemical properties of the modified starch will be evaluated. The grafted starch must present a high degree of substitution, thus providing hydrophobic characteristics for the macromolecular chain for future applications in the area of biodegradable packaging and in the encapsulation of active principles for bioactive packaging and drug delivery.
Figure 1: Starch etherification reaction scheme.
2 – Materials and methods 2.1 – Materials
4
Cassava starch (Manihot esculento), with 22.5 ± 2.5 % of amylose and 14.4 ± 0.6 % of moisture, was supplied by Indemil (Diadema-SP, Brazil). Maleic acid (analytical grade), acetic acid (analytical grade) and sodium hydroxide (analytical grade) were supplied by Vetec (Duque de Caxias-RJ, Brazil). Ethanol, 1-propanol, 1-butanol (anhydrous), Amberlist 15 hydrogen and dialysis tubing (D9402) were supplied by Sigma-Aldrich (Brazil). Hydrogen peroxide (35 %) and sulfuric acid (analytical grade) were supplied by Lafan (Várzea Paulista-SP, Brazil). All of the chemical reagents were used without further purification. 2.2 – Synthesis of precursors for starch chemical modification Initially, three different precursors, diethyl maleate, dipropyl maleate and dibutyl maleate, were synthetized for the starch chemical modification. The esterification reaction was performed according to the method described by (Yadav & Thathagar, 2002) with some modifications. The reaction medium, maleic acid, alcohol (ethanol, propanol or butanol) and catalyst (Amberlyst 15 hydrogen) remained under stirring and reflux for 4 h. The product obtained was then purified and characterized by infrared spectroscopy (FTIR) and nuclear magnetic resonance of hydrogen (1H-NMR). 2.3 – Starch modification The starch grafted reaction scheme is shown in Figure 1, where R is the alkyl groups. The etherification procedure was carried out according to Wokadala et al., 2014 with modifications. For this, 10 g of granular starch was dispersed in 70 mL of water, 9 mL of NaOH (3 mol L-1) was added and the solution was maintained at 110 °C. The precursor in peracetic acid solution was added to the gelatinized starch, and maintained under reflux for 100 min. Next, the starch was precipitated with ethanol and the dispersion centrifuged under a gravitational force of 1900 x g for 25 min. The extracted starch was maintained in a
5
desiccator for 20 days, washed with diethyl ether and dialyzed to remove the unreacted components. The products were characterized by proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), determination of the contact angle, differential scanning calorimetry (DSC), X-ray diffraction (XDR) and static light scattering (SLS). 2.4– Characterization 2.4.1 – 1H-NMR and FT-IR analyses The precursors and modified starch were analyzed by infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (1H-NMR). The FTIR spectra of the samples were obtained on a PRESTIGE 21 spectrophotometer (Shimadzu, Japan), equipped with a multiple reflectance accessory, with a resolution of 4 cm-1, and performing 20 scans. The FTIR measurements were carried out in KBr or as a thin film on an Si support. The nuclear magnetic resonance was recorded on a Varian AS400 spectrometer. The samples were dissolved in CDCl3 (precursors) or DMSO-d6 (starch samples) for the 1H-NMR analysis. 2.4.2 – Scanning electron microscopy (SEM) The granular native starch and grafted starch were deposited on a carbon tape and placed in a desiccator containing silica for 24 h. The specimens were coated with gold to avoid charging by the electron beam and analyzed by scanning electron microscopy (JSM6701F, JEOL). 2.4.3 – Contact angle The influence of water and diiodomethane on the surface hydrophilicity was observed by contact angle measurements using a Ramé-Hart 250-F1 (USA). The sessile drop experiment consists of placing water or diiodomethane on the grafted starch surface. 6
The formed drop spreads until it attains an equilibrium state, and a finite contact angle formed by the static drop with the surface can be determined by computer software (DropImage). Measurements were taken on the left and right sides of the 3L droplet profile for each liquid probe. 2.4.4 – Differential scanning calorimetry (DSC) DSC curves were obtained on a Shimadzu 50 (DSC-50 - Shimadzu, Japan) by heating from –50 °C to 250 °C at 5 °C min-1 in a nitrogen atmosphere (50 mL min-1). Only one heating cycle was used in all of the thermo analysis experiments. 2.4.5 – X-Ray Diffraction The granular native starch and grafted starch were deposited on the sample holder. The analysis was performed with a Philips X’Pert diffractometer (Netherlands), using copper radiation K (k = 1.5418 Å), voltage of 40 kV and operation current of 30 mA. All assays were performed with 2 = 2° and 2 = 50°, pitch of 0.05 °/s. The crystallinity index (CI) was determined by Equation 1, where Ac is the crystalline area and Aa is the amorphous halo area. 𝐶𝐼 =
𝐴𝑐 𝐴𝑐 +𝐴𝑎
× 100
Equation 1
2.4.6 – Static light scattering (SLS) The molar mass of native starch and grafted starch was determined by static light scattering (SLS) measurements. An ALV/CGS-3 (Germany) light scattering spectrometer was used, with an ALV/LSE-7004 multiple- digital correlator equipped with a 22 mW red helium-neon linearly polarized laser ( = 632.8 nm). The solvent was DMSO, with dn/dc equal to 0.066 mL g-1, and the starch solution concentrations were 1.0 x10-4, 3.0 x10-4, 6.5 x10-4, 1.0 x10-3, 3.0 x10-3 g mL-1. All samples were systematically assayed at different 7
scattering angles from 30° to 150° with 3° of increment. Data were collected digitally, using ALV Correlator Control software. The molar mass was determined using the ALV/Fit & Plot software.
3 – Results and Discussion 3.1 –Proton nuclear magnetic resonance (1H-NMR) and infrared spectroscopy (FTIR) Figures S1 and S2 show the 1H-NMR and FTIR of the synthetized diethyl maleate, dipropyl maleate and dibutyl maleate, respectively. These compounds showed ester groups, sp2 and sp3 carbons, with similar absorption bands to unsaturated esters (C-O-C), in the range of 1300 to 1220 cm-1, sp2 carbon (C = C) at 1644 cm-1 and sp3 carbons -CH2- and CH3 at 2965 and 2879 cm-1, respectively. All FTIR spectra of the analyzed precursors showed an absence of OH absorption band at 3500 cm-1, suggesting that the esterification reaction occurred in the hydroxyls group present in maleic acid, as shown in Figure S2. In the 1H-NMR spectrum of diethyl maleate, Figure S2, there were three hydrogen peaks, as expected due to symmetry, in the unsaturated carbon. A triplet with a chemical shift at 1.29 ppm was characteristic of sp3 carbon (-CH3), and a quartet with a chemical shift at 4.22 ppm was characteristic of a sp3 carbon (-CH2-) attached to a carbonyl group. A singlet with a chemical shift at 6.22 ppm was characteristic of the sp2 carbon (-CH = CH-), linked to a carbonyl group. In the spectrum of dipropyl maleate, in addition to the peaks discussed above, there was a further sextuplet signal with a chemical shift at 1.68 ppm, characteristic of sp3 carbon (-CH2-), as expected. As the spectrum of dibutyl maleate showed, we can observe one more sextuplet signal with a chemical shift at 1.40 ppm characteristic of the sp3 carbon (-CH2-). The precursors were obtained as expected.
8
Figure 2 shows the 1H-NMR spectrum of the native cassava starch. The peak at 2.5 ppm was related to the proton of DMSO-D6 used as solvent, and an intense peak at 3.3 ppm was characteristic of the water present in solvent medium. The peaks referring to hydrogen numbered 2 to 6 (Figure 2) were partially masked by the peak due to water present in the solvent medium. Hydrogen 1 (linked to the 1-4 alpha glycosidic carbon bond) and 1' (linked to the alpha 1-6 glycosidic carbon bond) were similar and exhibited a chemical shift at around 5 ppm, related to a neighborhood surrounded by oxygen atoms with high electronic density. The hydroxylic hydrogens (7, 8 and 9) showed different chemical displacements, although they were connected at the oxygen, due to their adjacent group. Due to its adjacent group with lower electronic density, hydrogen 7 showed a chemical shift at 4.5 ppm, and hydrogens 8 and 9 had a similar adjacent group and higher electron density due to the oxygen atoms of the glycosidic bonds, displacing the peaks to 5.5 ppm (Tizzotti, Sweedman, Tang, Schaefer, & Gilbert, 2011; Xiong et al., 2014; A. Zhang et al., 2013).
9
Figure 2: 1H-NMR spectrum of native cassava starch. The numbers in the peaks correspond to the hydrogen numbers in the above structure. Figure 4 shows the 1H-NMR spectra of native starch, starch-diethyl maleate, starchdipropyl maleate and starch-dibutyl maleate, in which we can observe displaced peaks related to the hydroxyl groups of the starch, and appearance of peaks between 0.5 and 1.5 ppm, probably related to the presence of hydrogens of sp3 carbon bonded to sp3 carbons. The decrease in the peak intensities, related to the hydroxyl groups of the macromolecular chain of the starch with the appearance of sp3 hydrogens, indicates the efficiency of the etherification reaction. The FTIR spectra of grafted starch showed two absorption bands at 1721 cm-1 1550 cm-1 related to the C = O stretching and CH stretching adjacent to carbonyl and oxygen, respectively. Another absorption band at 1126 cm-1, characteristic of the C-O-C stretching of the ester group, was present in the spectra of the precursors and was not observed in the infrared spectrum of the native starch. These absorption bands at 1721 and 1126 cm-1 were characteristic of groups present in the precursor molecules, indicating the effective grafting reaction.
10
Figure 3: 1H-NMR spectrum of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate The 1H-NMR data were further used to determine the substitution degree of the precursors in the macromolecule starch, using the peak in 3.6 ppm as a reference and evaluating the intensity of hydroxyl group (de Graaf, Lammers, & Et Al., 1995; Wokadala et al., 2014). The degrees of substitution of starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate were 2.3, 1.0, and 2.0, respectively. The substitution reaction was directed at the hydroxyls attached to the glycosidic ring, as verified by the 1H-NMR spectra (Figure 4, chemical shift 5.5 ppm). This reaction preference was due to the fact that the glycosidic ring stabilized the electronic density of oxygen, facilitating the attack of oxygen in the epoxy ring of the reaction medium.
11
Figure 4: FTIR spectrum of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate. It can also be observed in the FTIR spectra (Figure 4) that the OH stretching band (~ 3400 cm-1) was broad and highly symmetrical in the native starch spectrum. However, the spectra of the grafted starches showed an asymmetric band, displacing its absorption band at higher wavenumbers. This displacement at higher wavenumber was related to the higher energy required for OH stretching, indicating that the hydroxyl groups still present in the graft starch were not compromised by hydrogen bonds compared to the native starch (Liang & Ludescher, 2015; C. Zhang et al., 2017). These data complemented the characterization of the etherification reaction and indicated a decrease in the hydrophilicity of the starch after the modification reaction. 3.2 – Scanning electron microscopy (SEM)
12
Figure 5 shows the SEM micrographs of the native starch and grafted starches. The native starch presented a semicrystalline granular form with an oval shape and a size range between 5 and 15 μm (Zhu, 2015).
Figure 5: Scanning electron macroscopy images of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate. After the modification process, the starch granules presented changes in their shape and surface morphology. This process was not related to the graft reaction but to the rupture of the residual crystal structure of the starch process followed by recrystallization, and the starch did not maintain its granular form. The surface morphology of the granules presented a greater roughness and an irregular structure after the modification, when compared with the native starch. This change in starch roughness may be related to a decrease in intra and intermolecular interactions between the hydroxyl groups present in the macromolecule repeating unit, influencing the degree of crystallinity in the samples (Chauhan et al., 2015).
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3.3 – XRD characterization The crystallinity index for the cassava starch (36 %) was in agreement with the value obtained by authors using the same experimental method, as shown in Figure 6 (Garrido, Schnitzler, Zortéa, de Souza Rocha, & Demiate, 2012). The diffractogram of native starch showed strong reflections at 2θ, equal to 15.2º , 17.2º, 18,2º, and 23.0º , and weaker unsolved peaks can be observed at 11.4º, 19.9º, 26,2º and 31.5º. The reflections presented in the XRD pattern were in agreement with other authors, suggesting that the starch crystalline structure was of A-type related to a monoclinic lattice (Lin, Li, Long, Su, & Huang, 2014). The disappearance of the crystal peaks (15° and 18°) and the decrease in the peak at 22° indicate that the crystallinity of the native starch was affected during the process, which implies that the hydrogen bond of the starch was disrupted. After the etherification reaction, the samples showed a decrease in the crystallinity degree, which corroborates the DSC results. The crystallization form of the grafted starch changed because it did not present an orientation in the retrogradation to the reestablishment of the hydrogen bonds. The modified starch samples showed a new diffraction peak at 6° and a shift in the peak at 23° to 21°, which implies that new crystalline regions were formed in the modified starch ethers.
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Figure 6: X-ray diffractograms of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate.
3.4 –Differential scanning calorimetry (DSC) The melting enthalpy, related to the energy required for the rupture of the residual crystal structure of the starch, underwent a significant reduction after the grafting of the precursors (Table 1). This reduction did not have a significant influence on the changes in the grafted group, but instead showed a tendency toward a reduction with the carbonic chain of the grafted group. The change in the melting enthalpy occurred because of the rupture of the intra and intermolecular hydrogen bonds of the starch. The melting enthalpy is related to a crystallinity of the starch, and the lower degree of crystallinity the lower the melting enthalpy will be. This corroborates the proposal that a substitution reaction decreases the interactions between the hydroxyl group of the repeat unit of starch, thus decreasing the residual crystallinity of the native starch and consequently the melting enthalpy. 15
The melting peak temperature differed for each grafted starch sample. The starchdiethyl maleate showed no significant decrease in relation to native starch, but the starchdipropyl maleate and starch-dibutyl maleate samples showed a reduction. This behavior is attributed to the degree of crystallinity of the grafted starch. Table 1: Melting temperature, melting enthalpy, molar mass and crystallization index of native starch, starch-diethyl maleate, starch-dipropyl maleate and starch-dibutyl maleate. Sample Native starch Starch-diethyl maleate Starch-dipropyl maleate Starch-dibutyl maleate
Tm (°C) Hm (J.g-1) Mw (g.mol-1) 108 31 1.0 x 108 108 16 1.2 x 106 94 21 1.0 x 106 95 20 4.2 x 106
CI (%) 36 30 28 29
3.5 – Contact angle Table 2 shows the data for the contact angles of the modified starch samples obtained with polar solvent (water) and an apolar solvent (diiodomethane). The contact angles for the apolar solvent presented lower values compared to the contact angles for the water. These data indicate that the etherified starch had a more hydrophobic character. The higher affinity of the apolar solvent (diiodomethane) in the sample compared to the polar solvent (water) was shown by a smaller angle of contact between the drop of the liquid and the sample surface. The contact angle values corroborated the data reported above, indicating effective starch modification and showing that the grafting of apolar groups in the starch backbone chain led to grafted starch samples with an apolar character. Table 2: Contact angles for starch-diethyl maleate, starch-dipropyl maleate and starchdibutyl maleate. Sample
Water (°)
Diiodomethane (°)
Starch-diethyl maleate Starch-dipropyl maleate Starch-dibutyl maleate
31.8 ± 0.1 30.0 ± 0.1 32.1 ± 0.2
23.8 ± 0.2 29.3 ± 0.2 25.1 ± 0.1
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3.6 – Static light scattering (SLS) The molar mass of the native starch obtained from the SLS analysis presented values in the order of 1.0 x 108 g mol-1 (Table 1). These values are close to those obtained in the literature for cassava starch and other sources obtained by chromatography analysis (GPC) (Israkarn, Na Nakornpanom, & Hongsprabhas, 2014; Kowittaya & Lumdubwong, 2014; Miao et al., 2015). The analyses were performed using DMSO as a solvent to decrease the aggregation forming effect, since samples analyzed with DMSO show a better dispersion of the starch macromolecules compared with starch solution in water (Kowittaya & Lumdubwong, 2014). The etherified starch samples showed a 100-fold reduction in the molar mass, regardless of the grafted group. This indicates that the substitution reaction mechanism did not interfere in the reduction of the molar mass of the starch. The reduction of the molar mass of the starch is related to the depolymerization reaction caused by the basic treatment used in the synthesis (Misman, Azura, & Hamid, 2015). 4 – Conclusions The etherification reactions of starch from maleic acid derivatives showed a high degree of substitution, with a mean of two substitutions at each repeat unit. Of the three reactive hydroxyls groups present in the starch repeat unit, it can be seen from the 1H-NMR analysis that the hydroxyls attached directly to the glycosidic ring were preferentially substituted because the glycosidic ring stabilized the reaction intermediately, thus allowing the maleic acid derivatives to attack. Grafting of the maleic acid derivatives was affected by intra- and intermolecular interactions, thus decreasing the melting enthalpy of the modified starch. The modification
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also changed the crystal structure and decreased the degree of crystallinity (%) compared to native starch. The surfaces of the grafted starch samples had an irregular shape with greater roughness and a greater hydrophobicity resulting from the grafting. The molar mass of the starch was reduced by the selected synthesis method, where the starch undergoes basic hydrolysis in the presence of sodium hydroxide. This hydrolysis reduced the molar mass of the starch approximately one-hundred-fold. The modification of the starch surface added a hydrophobic character and thus amplified its potential uses and applications. Modified starch can be used as a compatibilizer in polymer blends with hydrophobic polymers for the production of sheets and films for biodegradable and bioactive packaging, in the food industry and in the encapsulation of natural substances or hydrophobic drugs, among other applications. Acknowledgements The authors are grateful for the financial support provided by CAPES and CNPq.
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