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Induced electric field intensification of acid hydrolysis of polysaccharides: Roles of thermal and non-thermal effects
Food Hydrocolloids 101 (2020) 105484
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Food Hydrocolloids journal homepage: http://www.elsevier.com/locate/foodhyd
Induced electric field intensification of acid hydrolysis of polysaccharides: Roles of thermal and non-thermal effects Dan-Dan Li a, b, Na Yang b, Yang Tao a, En-Bo Xu c, Zheng-Yu Jin b, Yong-Bin Han a, **, Xue-Ming Xu b, * a b c
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, Jiangsu Province, China College of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu Province, China College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, Zhejiang Province, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Electro-processing Degradation Mechanism Electrical properties Directional migration
The mechanism of induced electric field (IEF)-assisted acid hydrolysis was investigated by using guar gum, chitosan, and pectin as model polysaccharides. Roles of thermal and non-thermal effects were explored by temperature determination, HPSEC-MALLS-RI, SEM, FT-IR, XRD, and a rheometer. Results suggested that IEF affected the hydrolysis of polysaccharides by inducing thermal and non-thermal effects. Thermal effect contributed to the hydrolysis. As the excitation voltage increased, the thermal effect enhanced, with a temper ature increased by about 25 � C after IEF (75 V) treatment for 4 h. Non-thermal effect, induced by the rapid and directional migration of charged species, varied with the electrical properties of the polysaccharides. It enhanced the hydrolysis of guar gum and pectin but negatively impacted the chitosan due to their different migration directions under IEF. IEF-assisted hydrolysis damaged the structural compactness of polysaccharides and significantly decreased the viscosity of their solutions. This study clarified the hydrolysis mechanism of poly saccharides under IEF, providing a theoretical guide for the application of IEF in the chemical modification of biopolymers.
1. Introduction
of high voltage electric field to materials placed between two electrodes. The energy dissipated during PEF treatment not only degrades poly saccharides such as chitosan (Luo, Han, Zeng, Yu, & Kennedy, 2010) and starch (Han, Zeng, Zhang, & Yu, 2009), but also assists the acetylation of pectin (Ma & Wang, 2013) and starch (Hong, Chen, Zeng, & Han, 2016). Ohmic heating (OH) is an internal thermal energy generation technol ogy that electrical energy is dissipated into heat directly by passing electric current through sample (Knirsch, Alves dos Santos, Martins de Oliveira Soares Vicente, & Vessoni Penna, 2010). OH allows for faster and more uniform heating and induces an increase in the directed movement of charged molecules when compared with water-bath and microwave heating, thus accelerating organic syntheses such as a Diels-Alder cycloaddition, a nucleophilic substitution, an N-alkylation, and a Suzuki cross-coupling reaction (Cardoso et al., 2015; Pinto et al., 2013). Therefore, electric field based techniques may have potentials in enhancing the modification of polysaccharides. The combination of the electrical technique with acid hydrolysis is expected to have a
Natural polysaccharides are widely available, affordable, and sus tainable, thus having great potentials in food, medicine, and other in dustries. However, natural polysaccharides also have shortcomings, in particular low solubility, a quality which dramatically limits their po tential applications. Degradation is the simplest method for improving the properties and functions of polysaccharides, with degraded poly saccharides usually exhibiting a higher level of solubility than their natural counterparts. Degradation can be achieved by physical (Sar avana et al., 2018), chemical (Miao, Jiang, Zhang, Jin, & Mu, 2011), and biological (Ma et al., 2018) means, with acid hydrolysis being the simplest method. However, the compact and complex structure of native polysaccharides limits their hydrolysis efficiency. Electric field based techniques have gained increasing interests in food and biomaterial processing. Pulsed electric field (PEF) is a nonthermal technique that involves the application of short pulses (μs-ms)
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y.-B. Han), [email protected] (X.-M. Xu). https://doi.org/10.1016/j.foodhyd.2019.105484 Received 17 April 2019; Received in revised form 31 October 2019; Accepted 1 November 2019 Available online 4 November 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Typical chromatograms for native and IEF-assisted hydrolyzed guar gum (75 V, 400 Hz).
synergistic effect when applied to natural polysaccharides. However, the current applications of electro-techniques are based on the electropho resis of metal electrodes. The electrodes, when inserted into electrolyte solutions, especially acidic solutions, will result in severe sample contamination due to the occurrence of electrochemical reactions (Winkleman, Gates, McCarty, & Whitesides, 2005). The induced electric field (IEF) system is developed in the configu ration of a transformer by employing Faraday’s law of induction. Alternating excitation voltage in the primary coil causes a variable magnetic flux in the core of the transformer, thus inducing alternating electric voltage in the sample coil. Compared with PEF and OH, the generation of IEF is through coils and core rather than metal electrodes, thus avoiding electrode-corrosion-induced sample contamination. Pre vious studies confirmed that IEF significantly improved the hydrolysis of cellulose (Yang, Jin, Li, Jin, & Xu, 2017) and starch (Li et al., 2017). For example, IEF treatment (150 V at 50 � C for 24 h) increased the reducing sugar content in cellulose hydrolysates by 155.5% when compared with traditional heating (50 � C). Despite encouraging results, the mechanism of IEF-assisted hydrolysis remains unclear. The energy dissipated during PEF treatment can cause electro chemical reactions and the polarization of structural moieties, and thus PEF has potentials to modify the microstructure and functional prop erties of bio-macromolecules (Giteru, Oey, & Ali, 2018). Typically, a critical PEF treatment intensity is required to initiate structural, conformational and functional changes to bio-macromolecules. For example, Luo et al. (2010) demonstrated that the minimum intensity for production of pits on the surface of chitosan granules was 15 kV/cm. The intensity of IEF is much lower than the required critical intensity. Hence, the electrochemical reactions and the polarization of structural moieties of polysaccharides during IEF treatment may be ignored. Pinto et al. (2013) reported that OH induced electric current passing through sample and accelerated the directional migration of charged species, thus producing both thermal and non-thermal effects to affect chemical reactions. The energy dissipated to samples during OH processing is similar to that during IEF processing. Therefore, the induced alternating
voltage in the sample coil may induce thermal and non-thermal effects to alter the hydrolysis of polysaccharides. In this study, chitosan, pectin, and guar gum were selected as the model polysaccharides in order to investigate the underlying mechanism of IEF-assisted hydrolysis. The roles of the thermal and non-thermal effects were analyzed and the ef fects of IEF-assisted hydrolysis on the structural and physicochemical properties of polysaccharides were explored. This study clarified the mechanism of IEF-assisted hydrolysis, which would contribute to the development of other potential applications for IEF in chemical modification. 2. Materials and methods 2.1. Materials Chitosan (DD ¼ 85.83%, 200-mesh sieve) and guar gum (viscos ity ¼ 5200 � 150 mPa s, 200-mesh sieve) were of analytical grade and were purchased from Aladdin Co., Ltd. (Shanghai, CN). Pectin (DE ¼ 96.83%, pH ¼ 2.8 � 0.2 (1% in H2O), 200-mesh sieve) was of biochemical grade and was purchased from Sangon Biotech Co., Ltd. (Shanghai, CN). Hydrochloric acid, sodium hydroxide, and all other analytical chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Suchou, CN). 2.2. Acid hydrolysis of polysaccharides under IEF The schematic diagram of a 4-series IEF system has been reported in our previous study (Li et al., 2017). The core component of an IEF system is an o-core transformer, which consists of a primary copper coil, a magnetic core, and a glass spiral. The glass spiral is surrounded by a circulating water chamber, which controls the reaction temperature. During IEF treatment, liquid sample is pumped into the glass spiral, acting as the secondary coil of the transformer (sample coil). According to Ampere’s law, as an excitation voltage is imposed on primary coil, an alternating magnetic flux appears in magnetic core, and then an electric 2
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voltage is induced in sample coil. In this study, guar gum, pectin, or chitosan (3.2 g) was precisely weighed and then was slowly added to 0.15 mol/L or 1.5 mol/L HCl solutions (400 mL) with continuously stirring at 25 � C for 12 h. The obtained mixture was pumped into IEF system and then treated at the conditions: excitation voltage 15 V, 35 V, 55 V, and 75 V; frequency 400 Hz; flow rate 300 mL/min; reaction temperature not controlled or controlled at 50 � C; reaction time 0–24 h for guar gum and 0–60 h for chitosan and pectin (guar gum was sensitive to acid hydrolysis and exhibited extensive hydrolysis after treated by 0.15 mol/L HCl for 24 h, whereas chitosan and pectin were more stable in acid solutions and thus higher concentrated acid and longer reaction time were necessary). When the reaction temperature was not controlled, the changes in the temperature of polysaccharide-HCl mixtures were recorded by using a CEM DT-810 InfraRed Thermometer (Huashengchang Co., Ltd., Shenz hen, CN) to investigate the roles of thermal effect in IEF-assisted hy drolysis. Treatments at the same conditions but without excitation voltage (0 V) and frequency (0 Hz) were run as controls. In order to study the roles of non-thermal effect, a temperature (50 � C) higher than that caused by thermal effect of IEF was set by the circulating water bath, the temperature profiles of IEF treatment at 50 � C and continuousflow water-bath heating at 50 � C were recorded, and then the hydrolysis efficiency of polysaccharides during IEF treatment (75 V) and continuous-flow water-bath heating was compared by molecular weight determination. After treatments, the hydrolysis was terminated by adding 1 mol/L NaOH to adjust the pH of the reaction mixture to 7.0–8.0. When the reaction mixture was cooled to 25 � C, the resulting product was precipitated with anhydrous ethanol and the hydrolyzed polysaccharide was separated by filtration under vacuum. The solid was further washed with 80% ethanol (200 mL each time) until no Cl in effluent could be detected by 0.1 mol/L AgNO3. After filtration, the residue was dried at 45 � C for 24 h. The prepared samples were used for testing. 2.3. Molecular weight determination The molecular weight determination was performed by using a highperformance size-exclusion chromatography (HPSEC) system with two columns in series (Shodex OHpak SB-804 HQ and OHpak SB-802 HQ columns). The HPSEC system consists of an isocratic pump, a multiangle laser light-scattering (MALLS) detector, and a refractive index (RI) detector (Dawn DSP; Wyatt Technology Co., Ltd., Santa Barbara, USA). All columns were thermostated at 40 � C. Guar gum and pectin: the mobile phase was 0.1 mol/L NaNO3 and 0.02% NaN3 aqueous solution; polysaccharide was diluted to 0.2% wt. using mobile phase and filtered through 0.45 μm filter before injection. Chitosan: the mobile phase was 0.3 mol/L CH3COOH and 0.1 mol/L CH3COONa aqueous solution (pH ¼ 4.5); chitosan was diluted to 0.4% wt. using mobile phase and filtered through 0.45 μm filter before injection. Before analysis, the mobile phase was ultrasound for 30 min to degas and filtered through 0.45 μm filters. Samples (20 μL) were injected into HPSEC system and eluted at a flow rate of 0.6 mL/min for 70 min. Pullulan standards (P50 Mw ¼ 4.71 � 104 g/mol, P800 Mw ¼ 6.42 � 105 g/mol, Shodex standard P-82) were used to calibrate the columns. The electronic outputs of the MALLS and RI detectors were collected using ASTRA software (version 5.3.4, Wyatt Technology Co., Ltd., Santa Barbara, USA). 2.4. Output voltage determination Fig. 2. Effect of IEF treatment on the Mw of polysaccharides: (a) guar gum; (b) chitosan; and (c) pectin (Without temperature control).
A pair of platinum electrodes connected to an AC millivoltmeter (UT631; UniTrend Group Limited Co., Ltd., Hong Kong, CN) was inserted into the two ends of sample coil to determine the output voltage of IEF system. The determination was performed at excitation voltages of 15 V, 35 V, 55 V, and 75 V and a frequency of 400 Hz. 3
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Table 1 Electrical parameters of induced electric field system when different polysaccharides acted as the secondary coil. Excitation voltage (V)
Excitation current (A)
Power (W)
15 35 55 75
0 0.261 0.473 0.687
0 7 18 37
Output voltage (V) Guar gum
Chitosan
Pectin
Expected value
3.13 � 0.43Da 7.41 � 0.31Ca 11.88 � 0.62Ba 15.89 � 0.74Aa
3.17 � 0.36Da 7.53 � 0.61Ca 11.84 � 0.52Ba 17.04 � 1.32Aa
3.01 � 0.42Da 7.51 � 0.38Ca 11.81 � 0.45Ba 16.01 � 0.42Aa
3.75 8.75 13.75 18.75
Means in the same row with different superscript lowercase letter and same column with different superscript uppercase letter indicate significant difference (P < 0.05).
hydration. After that, sample was transferred to the rheometer plate and the excess sample was wiped off by using a straw. Steady shear tests were performed over the shear rate range of 0.01–100 s 1. Before each test, a time waiting process of 60 s was applied to equilibrate the stress and temperature of hydrolyzed polysaccharides (25 � C). 2.6. Statistical analysis All experiments were performed in triplicates. Data were expressed as mean � standard deviation. Fisher’s least significant difference (LSD) test was used to compare means at the 5% significance level (IBM SPSS Statistics 20, Somers, USA). 3. Results and discussion 3.1. IEF enhancement of acid hydrolysis of polysaccharides The molecular characteristics of guar gum, chitosan, and pectin during IEF-assisted hydrolysis were determined by HPSEC-MALLS-RI (Fig. 1, Fig. S1, and Fig. S2). The MALLS intensity was applied to calculate the molecular weight, and the RI gave a signal proportional to the concentration (Yi et al., 2016). During IEF-assisted hydrolysis, the molar mass of guar gum dramatically decreased, accompanied by a falling MALLS intensity and a shift of RI peak to larger elution volumes (Fig. 1). These results suggested that the guar gum chains were degraded to lower molecular weights. Chitosan and pectin showed similar varia tion trends (Fig. S1 and Fig. S2). The effects of IEF on the Mw of poly saccharides were illustrated in Fig. 2. Without IEF treatment, the Mw of polysaccharides remained unchangeable, whereas the Mw significantly decreased with IEF treatment, suggesting that IEF enhanced the hy drolysis of polysaccharides. As the excitation voltage was increased, the hydrolysis of polysaccharides was enhanced. After IEF treatment at 15 V and 75 V for 24 h, the Mw of guar gum decreased by 2.43 � 105 g/mol and 16.11 � 105 g/mol, respectively (Fig. 2a). Chitosan and pectin showed similar variation trends with guar gum, but they exhibited less extensive hydrolysis because they were more stable in acid solution (Fig. 2b and Fig. 2c). Luo et al. (2010) and Han, Zeng, Yu, Zhang, and Chen (2009) reported that PEF could cause the degradation of chitosan and starch. Zhou, Jin, Yang, Xie, and Xu (2017) suggested that the hy drolysis of corn starch enhanced with increasing excitation voltage of IEF treatment.
Fig. 3. Temperature profiles of polysaccharide-HCl mixtures during IEF treat ment at 15 V, 35 V, 55 V, and 75 V (Without temperature control). G represents guar gum, C represents chitosan, P represents pectin.
2.5. Characteristics of hydrolyzed polysaccharides 2.5.1. Fourier infrared spectroscopy (FT-IR) The FT-IR spectra of native and hydrolyzed chitosan, guar gum, and pectin were recorded from a KBr disk (500 mg) containing finely ground sample (25 mg) on a Bruker GER400 spectrometer (Bruker Co., Ltd., GER) at a wavenumber range of 4000-400 cm 1. 2.5.2. X-ray diffraction (XRD) XRD patterns were performed by a X-ray diffractometer of Bruker D8-Advance type (Bruker AXS Co., Ltd., Karlsruhe, GER) with Cu-Kα filtered radiation (wavelength 1.5405 Å) radiator operating at 30 kV and 10 mA. The scans ranged from 5� to 50� with a step width of 0.02� . 2.5.3. Scanning electron microscope (SEM) The granular structure of native and partially hydrolyzed poly saccharides was observed by a SU 1510 SEM (Hitachi High-Technologies Co., Ltd., JP). The applied accelerating voltage is 5.00 kV. Prior to observation, the polysaccharide powder was mounted on an aluminum specimen holder with double-sided adhesive tape and then coated with gold under vacuum evaporation.
3.2. Possible mechanism of IEF-assisted hydrolysis of polysaccharides 3.2.1. Electrical characteristics of polysaccharides When polysaccharide-HCl mixture that was electrically conductive was pumped into the IEF system, it acted as the secondary coil. An alternating voltage would be produced in the secondary coil if an exci tation voltage was imposed on the primary coil (Heathcote, 2007). Ac – NS/NP), the induced cording to Ampere’s circuital theorem (US/UP– voltage in the secondary coil (US) was linearly related to the excitation voltage (UP) and the ratio of primary coil turns (NP) to secondary coil turns (NS). The excitation electric signal of IEF was sinusoidal alter nating, with voltages of 15 V–75 V and a frequency of 400 Hz. The NS
2.5.4. Rheological measurements Steady shear rheological tests on a TA DHR-3 Rheometer (TA In struments Co., Ltd., USA) were applied to characterize the native and hydrolyzed polysaccharides. The tests were performed by using cone plate geometry (60 mm) at a gap of 116 μm. Guar gum and pectin were added to distilled water at a concentration of 1% wt. and 5% wt. with continuously stirring, respectively, while chitosan (4% wt.) was dis solved in 1% CH3COOH solution. Polysaccharide solution was then stirred at room temperature (25 � C) for 12 h to confirm complete 4
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Fig. 4. The electrical signal of IEF system and the possible migration ways of charged ions under IEF.
the expected value when using liquid sample, which exhibited signifi cant levels of impedance, as the secondary coil of IEF system. As shown in Table 1, the output voltage was lower than the expected value when polysaccharide-HCl mixture acted as the secondary coil of IEF system. As excitation voltage increased from 15 V to 75 V, the output voltage of guar gum increased from 3.133 V to 15.92 V. No significant difference in the output voltage was observed when using different polysaccharide-HCl mixtures as the secondary coil. The increased output voltage could induce higher electrical current passing through polysaccharide-HCl mixtures, thus enhancing thermal effect. In addi tion, the charged species exhibited higher migration rate under IEF as the output voltage increased. 3.2.2. Roles of thermal effect on IEF-assisted hydrolysis of polysaccharides Thermal effect commonly exists in electro-processing due to the conversion of electric energy into heat energy. The magnitude of the temperature increase of a product is crucial in determining the efficiency of electro-processing. The equation for calculating the thermal effect induced by IEF processing has not been proposed, but the increase in the temperature during IEF processing is considered to depend on the electrical current density. Due to the lack of technical means, the ionic current in liquid sample (secondary coil) cannot be determined. How ever, the electric current in primary coil, which is proportional to the induced current in sample coil, can be obtained from the power source. When excitation voltage increased from 15 V to 75 V, the electric current in primary coil increased from 0 to 0.687 A, suggesting that thermal effect existed in IEF-assisted hydrolysis (Table 1). Fig. 3 confirmed that IEF treatment caused thermal effect. The temperature of all
Fig. 5. Temperature profiles of polysaccharide-HCl mixtures during IEF treat ment at 50 � C and water-bath heating at 50 � C.
and NP values were 23 and 92, respectively. Therefore, the expected induced voltage was 3.75 V, 8.75 V, 13.75 V, and 18.75 V (Table 1). The induced voltage could be determined by a Voltmeter, which formed a closed loop with the secondary coil. For coils that were made of ideal conductor materials, the determined value was expected to be same as theoretical value. However, the output voltage was typically lower than 5
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polysaccharide-HCl mixtures rapidly increased in the first 4 h and then remained constant during IEF treatment. According to Joule’s law, the density of electric current is closely related to the intensity of electric field and the electrical conductivity of sample. As the excitation voltage increased, the temperature signifi cantly increased (Fig. 3). After IEF treatment at 15 V and 75 V for 4 h, the temperature of polysaccharides increased by about 5 � C and about 25 � C, respectively. During PEF-assisted acetylation of potato starch, the tem perature of the reaction mixture increased from 30 � C to 39 � C when the strength of electric field increased from 2 kV/cm to 5 kV/cm (Hong et al., 2016). Han et al. (2012) also reported that the increase in tem perature of tapioca starch suspension was positively related to the in crease in PEF strength. Different polysaccharides exhibited different charge content, and thus the thermal effect of differently charged polysaccharides during IEF treatment slightly differed from each other. Compared with the excitation voltage, the electrical conductivity of sample might play a secondary role in the thermal effect. After being subjected to IEF treatment at 75 V for 4 h, the temperature of chitosan, pectin, and guar gum was 45.1 � C, 44.2 � C, and 43 � C, respectively. This result was in accordance with the output voltage results which slightly decreased in the sequence: chitosan > pectin > guar gum (Table 1). Kanjanapongkul (2017) used OH to cook rice and observed that the magnitude of the temperature increase varied with the electrical conductivity. Heat plays a vital role in the modification of polysaccharides. High temperatures increase the mobility of molecular chains and helical structures, which leads to the structural alteration of the amorphous and crystalline regions in polysaccharide granules (BeMiller & Huber, 2015), thus allowing chemical reagents and enzymes attacking polysaccharide chains more rapidly. Heat is also known to influence molecular motion, where, at high temperatures, faster rate of movement tends to increase the interparticle collisions among reaction substances, thus enhancing chemical reactions. Moreover, the hydrolysis of polysaccharides is an endothermic process. Therefore, the increase in temperature is expected to enhance the hydrolysis of polysaccharides. Compared with the con trols, the Mw of guar gum, chitosan and pectin all decreased more rapidly with increasing excitation voltage of IEF treatment (Fig. 2). The variation trends of Mw were in accordance with those of temperature, suggesting that IEF might improve the hydrolysis process by inducing thermal effect. Pinto et al. reported that OH allowed for faster and more uniform heating compared with oil bath and microwave, thus short ening the reaction time and increasing the yields of organic syntheses (Pinto et al., 2013). 3.2.3. Roles of non-thermal effect on IEF-assisted hydrolysis of polysaccharides Pinto et al. (2013) suggested that alternating electric fields induced the rapid and reciprocating movement of charged species, altered the collision opportunities among reaction substances, changed mass transfer, and thus affected the overall yield and reaction time of organic syntheses. Hong et al. (2016) reported that the high electric potential difference and changed electric field direction induced by PEF acceler ated the reciprocating motion of reaction ions, leading to a more effective collision rate and a higher degree of acetylation. Souza et al. (2010) suggested that moderate electric field enhanced the mechanical and thermal properties of chitosan films/coatings by inducing a more ordered structure and a clearly higher crystallinity, owing to the direc tional migration of chitosan under the electric field. During IEF treat ment, alternating excitation voltage imposed on the primary coil induced an alternating bipolar current in the sample coil (Fig. 4). Hence, the induced electric signal showed a periodic variation, with continu ously increased electric voltage during 0–1/4 T (T represents the time necessary for a periodically varying induced electric voltage), gradually decreased electric voltage during 1/4–1/2 T, increased electric voltage but in the opposite direction during 1/2–3/4 T, and finally decreased voltage in 3/4-1 T (Fig. 4). Without IEF (t ¼ 0, 1/2 T, and 1 T),
Fig. 6. Changes in the Mw of polysaccharides subjected to IEF treatment (75 V) at 50 � C and water-bath heating at 50 � C: (a) guar gum; (b) chitosan; and (c) pectin.
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Fig. 7. SEM images of native and hydrolyzed polysaccharides: (a) guar gum; (b) chitosan; (c) pectin; (1) native polysaccharide; (2) IEF-assisted hydrolyzed poly saccharide (15 V); (3) IEF-assisted hydrolyzed polysaccharide (35 V); (4) IEF-assisted hydrolyzed polysaccharide (55 V); (5) IEF-assisted hydrolyzed polysaccharide (75 V); (6) hydrolyzed polysaccharide treated by continuous-flow water-bath heating at 50 � C.
polysaccharides, hydrogen ions, and other molecules exhibited Brow nian movement and thus were symmetrically distributed. With IEF, charged species exhibited alternating motions due to a varying electric field direction. Positively charged molecules (hydrogen ion and chito san) migrated in the same direction as the electric field, negatively charged molecules migrated in the opposite direction of the field, and IEF had a negligible effect on the migration of neutrally charged sub strates such as guar gum. The collision opportunities between hydrogen ion and polysaccharide might vary with the charge types of poly saccharide, thus causing different hydrolysis behaviors under IEF. The Mw results shown in Fig. 6 confirmed that non-thermal effect varied with the polysaccharide types. When investigating the role of non-thermal effects on IEF-assisted hydrolysis, the reaction temperature was controlled at 50 � C, a
temperature higher than that induced by IEF treatment (75 V). As shown in Fig. 5, the temperature profiles of polysaccharide-HCl mixtures dur ing IEF treatment at 50 � C and continuous-flow water-bath heating at 50 � C were similar. However, the changes in Mw were slightly different, suggesting that a non-thermal effect might exist during IEF-assisted hydrolysis. IEF accelerated the migration of hydrogen ions but showed no significant effect on the migration of guar gum, and hence hydrogen ions were expected to attack the guar gum chains more rapidly. Compared with continuous-flow water-bath heating at 50 � C, IEF at 50 � C slightly decreased the Mw of guar gum (Fig. 6a). However, the decrease was not statistically significant, which might be attributed to the relative low electric field intensity of current IEF system. Fig. 6b suggested that the non-thermal effect seemed to weaken the hydrolysis of chitosan, which might be attributed to the same migration direction of 7
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chitosan and hydrogen ion under the electric field. However, the final Mw were at the same level as that treated by continuous-flow water-bath heating. Wu, Zivanovic, Hayes, and Weiss (2008) suggested that the hydrolysis rate of chitosan varied with the degree of acetylation of chitosan during sonication treatment, but the final Mw of different chitosan tended to be similar. Typically, the Mw of chitosan reduced rapidly at initial stage but then slowly decreased with further hydrolysis due to the semi-crystal structure of chitosan. After hydrolysis for 36 h, the amorphous part in chitosan granule was hydrolyzed and the remaining tight crystal part started to be damaged. As a result, the hy drolysis rate decreased and no significant difference between IEF treatment at 50 � C and continuous-flow water-bath heating at 50 � C was observed. Pectin is a negatively charged polysaccharide in distilled water, but it tends to be neutrally charged when dispersed in acid so lutions. Hydrogen ion exhibits much higher migration speed under IEF than pectin because pectin has much lower charge density. Therefore, hydrogen ions may attack pectin more rapidly under IEF. Fig. 6c showed that pectin exhibited a lower hydrolysis rate during initial 12-h period but the Mw of IEF-treated pectin decreased more after hydrolysis for 60 h when compared with continuous-flow water-bath heating. The different phenomenon between guar gum and pectin might be due to their different dissolved state in acid solution. 3.3. Changes in structural and physicochemical properties of polysaccharides induced by IEF-assisted hydrolysis 3.3.1. SEM Zhu (2018) reviewed the recent papers about the applications of electric field based techniques in starch modification and concluded that PEF, OH, and IEF treatments all damaged the granular structure of starch. SEM micrographs of native and hydrolyzed polysaccharides at magnifications of 200� are depicted in Fig. 7. Fig. 7a shows that native guar gum possessed a tough surface morphology in the form of nodules and flakes (Fig. 7a1). After being treated by IEF-assisted hydrolysis, the particle size distribution of guar gum was broadened, because some native granules were broken down into smaller pieces and some of these pieces congregated (Fig. 7a2-a5). This effect was enhanced with increasing excitation voltage of IEF treatment. A similar phenomenon was observed for pectin (Fig. 7c1-c5). In comparison with native chi tosan, the IEF-assisted hydrolyzed samples aggregated to form large particles and pits and pores emerged on the surface of these large par ticles (Fig. 7b2-b5). The changes in the granular structure of poly saccharides during continuous-flow water-bath heating (Fig. 7a6, Fig. 7b6, and Fig. 7c6) were similar to those during IEF-assisted hy drolysis, suggesting that IEF might replace traditional heating to be an effective method for polysaccharide degradation. The damaged granular structure allowed enzymes and chemical reagents entering granular interior easily and therefore might contribute to the modification of polysaccharides. 3.3.2. FT-IR The effect of IEF-assisted hydrolysis on the chemical structure of guar gum, chitosan, and pectin was investigated by FT-IR (Fig. 8). No major structural differences in the spectra of native and IEF-assisted hydro lyzed guar gum could be seen (Fig. 8a). Acid hydrolysis performed with continuous-flow water-bath heating also showed no significant effect on the chemical structure of guar gum, which was in accordance with the results reported by Mudgil, Barak, and Khatkar (2012). After IEF-assisted hydrolysis, no new peaks appeared in the FT-IR spectra of chitosan (Fig. 8b) and pectin (Fig. 8c). However, the degree of acety lation (DA) of chitosan and the degree of esterification (DE) of pectin were changed by the hydrolysis. Previous study suggested that A1655/A1070 was suitable for evaluating the DA of chitosan ranging from 0 to 100% as it displayed various advantages including no broadening, no shoulder issues, and it also remained unchanged regardless of water content (Kasaai, 2008). The ratio increased from 0.73 (native chitosan)
Fig. 8. FT-IR spectra of polysaccharides subjected to acid hydrolysis with IEF treatment and continuous-flow water-bath heating at 50 � C: (a) guar gum, (b) chitosan, and (c) pectin.
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Table 2 Data for FT-IR spectra and XRD patterns of polysaccharides subjected to acid hydrolysis with IEF treatment and continuous-flow water-bath heating at 50 � C. native FT-IR XRD
A1655/A1070 A1749/(A1749þA1630)) Peak 1 Peak 2
15 V IEF c
0.73 � 0.03 0.47 � 0.04b 11.07 � 0.21a 20.00 � 0.04a
35 V IEF bc
55 V IEF a
0.89 � 0.07 0.59 � 0.06ab 10.19 � 0.04b 20.09 � 0.12a
1.09 � 0.05 0.61 � 0.06ab 10.19 � 0.12b 20.09 � 0.12a
75 V IEF a
1.11 � 0.11 0.61 � 0.12ab 10.19 � 0.12b 20.09 � 0.12a
water bath a
1.11 � 0.04 0.69 � 0.05a 10.19 � 0.13b 20.09 � 0.09a
1.01 � 0.02ab 0.61 � 0.04ab 10.19 � 0.09b 20.09 � 0.04a
Means in the same row with different superscript lowercase letter indicate significant difference (P < 0.05).
to 1.01 (continuous-flow water-bath heating), 0.89 (15 V), 1.09 (35 V), 1.11 (55 V), and 1.11 (75 V), respectively (Table 2). Chatjigakis et al. (1998) reported that there was a linear relationship between the DE value and the ratio of the area underneath the peak 1749 cm 1 over the sum of the areas underneath the two peaks, at 1749 cm 1 and 1630 cm 1. The ratio (A1749/(A1749þA1630)) was calculated and the value was 0.47, 0.61, 0.59, 0.61, 0.61, and 0.69 for native pectin, water-bath-treated pectin, 15 V-treated pectin, 35 V-treated pectin, 55 V-treated pectin, and 75 V-treated pectin, respectively (Table 2). Although more extensive hydrolysis was obtained with continuous-flow water-bath heating at 50 � C, IEF treatment (35–75 V) increased the DA values and DE values. IEF allowed hydrogen ions to attack poly saccharide chains more directionally, which might cause the more se lective hydrolysis of polysaccharides. Hernoux-Villi� ere et al. (2013) reported that there was glucose selectivity with a rate of saccharification of up to 46% when a simultaneous high-frequency electromagnetic field/ultrasound-assisted procedure was applied to catalyze the con version of starch-based industrial waste into sugars.
shear rates, supporting that the low molecular weight polysaccharide fragments were formed. However, for pectin samples, a pseudo-plastic behavior was observed over the tested shear rate range. This phenom enon contributed to a rearrangement in the structure of the associated complexes, which were disrupted as a result of shearing. Gamonpilas, Krongsin, Methacanon, and Goh (2015) suggested that pectin disper sions exhibited Newtonian behavior at low concentrations (<0.2%) and pseudo-plastic behavior at high concentrations (>1%). In addition, the shear thinning behavior of pectin enhanced with hydrolysis, especially €m, 2005) at high shear rates. Previous work (Kjøniksen, Hiorth, & Nystro proposed that a variation in the branching of pectin resulted in a different shear rate dependence of viscosity. FT-IR spectra illustrated that the DE value of pectin was changed by hydrolysis and thus might cause the changes in the shear thinning behavior (Fig. 8c). The viscosity of all polysaccharides significantly decreased with acid hydrolysis (Fig. 9a, Fig. 9b, and Fig. 9c). For example, the viscosity of guar gum decreased by 1 order of magnitude after IEF treatment at 75 V for 24 h. IEF accelerated the migration rate of charged hydrogen ions, enhanced the friction between hydrogen protons and polysaccharides, and improved the cleavage of backbone as well as side chains, thus effectively decreasing the viscosity of polysaccharide solutions. The presence of lower molecular weight polysaccharide fragments was confirmed by HPSEC analysis (Figs. 2, Fig. 3, and Fig. 6). Compared with IEF treatment (without temperature control), continuous-flow waterbath heating at 50 � C caused more extensive hydrolysis, thus causing lower steady shear viscosity. The hydrolyzed polysaccharides with low Mw and low viscosity might be used as water-soluble dietary fibers or as water-soluble antibacterial agents, which had a wide range of uses in both clinical nutrition and medicine (Slavin & Greenberg, 2003).
3.3.3. XRD X-ray diffraction was performed in order to explore the changes in crystal form and crystallinity of guar gum, chitosan, and pectin during acid hydrolysis (Fig. 9). IEF-assisted hydrolysis showed no significant effect on the crystal structure of guar gum (Fig. 9a) and pectin (Fig. 9c). Native and partially hydrolyzed guar gum samples displayed low overall crystallinity, with reflections at 17.2� and 20.1� . Previous studies also suggested that acid hydrolysis had no significant effect on the crystal structure of guar gum (Mudgil et al., 2012). Native and hydrolyzed pectin were semi-crystalline, with two principal diffraction peaks at 2θ of about 13� and of about 21� . However, the crystal structure of chitosan tended to transfer from “Form II” to “Form I00 with IEF-assisted hydro lysis. In the XRD spectra of native chitosan, a strong reflection at 20.00� and a weak reflection at 11.07� were observed (Fig. 9b), which indicated that native chitosan displayed a pattern equivalent to the “Form II” crystal structure. After hydrolysis, the reflection at around 11.07� shif ted to a smaller angle (10.19� ). However, the reflection at around 20.00� shifted to a slightly larger angle (20.09� ). These shifts demonstrated that the crystal structure of hydrolyzed chitosan tended to conform to the “Form I00 crystal structure. The “Form II” crystal structure usually dis played a more constrained chain conformation, whereas “Form I00 had a more extended chain structure (Qun, Ajun, & Yong, 2007), meaning that IEF-assisted hydrolysis lessened the structural compactness of chitosan. The damaged crystal structure allowed chemical reagents and enzymes attacking polysaccharides easier, thus contributing to their chemical and biological modification. Similar changes in the crystal structure of polysaccharides were observed for polysaccharides treated by continuous-flow water-bath heating.
4. Conclusions IEF affected the hydrolysis of polysaccharides by inducing thermal and non-thermal effects. During IEF treatment, the temperature of polysaccharide-acid solutions increased, thus contributing to the hy drolysis of polysaccharides. As the excitation voltage was increased, the thermal effect was enhanced. The electrical properties of poly saccharides showed no significant effects on the thermal effect. The nonthermal effect, caused by the rapid migration of charged species, also affected the hydrolysis of polysaccharides. Different polysaccharides exhibited different electrical properties, migrated at different direction under IEF, and thus led to different non-thermal effect. Compared with continuous-flow water-bath heating, IEF enhanced the hydrolysis of guar gum and pectin but showed a negative effect on chitosan. After IEFassisted hydrolysis, the granular surface of the samples was destroyed, the chemical structure of the samples was slightly altered, the crystal structure of pectin and guar gum remained unchanged whereas the structural compactness of chitosan was partially lost, and the viscosity of all polysaccharides significantly decreased. Notably, the DE of pectin and DA of chitosan treated by IEF were slightly higher than those treated by continuous-flow water-bath heating, suggesting that IEF might allow for a more selective hydrolysis of polysaccharides. This study explored the mechanism of IEF-assisted acid hydrolysis of polysaccharides, which might provide a theoretical guide for the application of IEF in the chemical modification of biopolymers.
3.3.4. Viscosity Fig. 10 shows typical viscosity vs. shear rate plots of native and hy drolyzed guar gum, chitosan, and pectin solutions upon exposure to continuous-flow water-bath heating at 50 � C and IEF-assisted hydrolysis at excitation voltages of 15 V–75 V. A Newtonian region at low shear rates and a shear thinning region at higher shear rates were observed for both native guar gum and chitosan, whereas the Newtonian behavior of hydrolyzed guar gum and chitosan was extended to the higher values of 9
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Fig. 10. Steady shear viscosity of polysaccharides subjected to acid hydrolysis with IEF treatment and continuous-flow water-bath heating at 50 � C: (a) guar gum, (b) chitosan, and (c) pectin.
Declaration of competing interest The authors declare no conflict of interest.
Fig. 9. XRD patterns of polysaccharides subjected to acid hydrolysis with IEF treatment and continuous-flow water-bath heating at 50 � C: (a) guar gum, (b) chitosan, and (c) pectin.
Acknowledgement This work was financially supported by the Natural Science 10
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Foundation of Jiangsu Province (No. BK20190523 and No. BK20170182), the National Natural Science Foundation of China (No. 31701522) and a Special Fund for Talents’ Introduction Program of Jiangsu Superiority Disciplines (No. 080/80900605).
Knirsch, M. C., Alves dos Santos, C., Martins de Oliveira Soares Vicente, A. A., & Vessoni Penna, T. C. (2010). Ohmic heating-a review. Trends in Food Science & Technology, 21 (9), 436–441. Li, D., Yang, N., Jin, Y., Guo, L., Zhou, Y., Xie, Z., et al. (2017). Continuous-flow electroassisted acid hydrolysis of granular potato starch via inductive methodology. Food Chemistry, 229, 57–65. Luo, W. B., Han, Z., Zeng, X. A., Yu, S. J., & Kennedy, J. F. (2010). Study on the degradation of chitosan by pulsed electric fields treatment. Innovative Food Science & Emerging Technologies, 11(4), 587–591. Ma, S., & Wang, Z. H. (2013). Pulsed electric field-assisted modification of pectin from sugar beet pulp. Carbohydrate Polymers, 92(2), 1700–1704. Ma, F., Wang, D., Zhang, Y., Li, M., Qing, W., Tikkanen-Kaukanen, C., et al. (2018). Characterisation of the mucilage polysaccharides from Dioscorea opposita Thunb. with enzymatic hydrolysis. Food Chemistry, 245, 13–21. Miao, M., Jiang, B., Zhang, T., Jin, Z., & Mu, W. (2011). Impact of mild acid hydrolysis on structure and digestion properties of waxy maize starch. Food Chemistry, 126(2), 506–513. Mudgil, D., Barak, S., & Khatkar, B. (2012). X-ray diffraction, IR spectroscopy and thermal characterization of partially hydrolyzed guar gum. International Journal of Biological Macromolecules, 50(4), 1035–1039. Pinto, J., Silva, V. L. M., Silva, A. M. G., Silva, A. M. S., Costa, J. C. S., Santos, L. M. N. B. F., et al. (2013). Ohmic heating as a new efficient process for organic synthesis in water. Green Chemistry, 15(4), 970–975. Qun, G., Ajun, W., & Yong, Z. (2007). Effect of reacetylation and degradation on the chemical and crystal structures of chitosan. Journal of Applied Polymer Science, 104 (4), 2720–2728. Saravana, P. S., Cho, Y. N., Patil, M. P., Cho, Y. J., Kim, G. D., Park, Y. B., et al. (2018). Hydrothermal degradation of seaweed polysaccharide: Characterization and biological activities. Food Chemistry, 268, 179–187. Slavin, J. L., & Greenberg, N. A. (2003). Partially hydrolyzed guar gum: Clinical nutrition uses. Nutrition, 19(6), 549–552. Souza, B. W. S., Cerqueira, M. A., Martins, J. T., Casariego, A., Teixeira, J. A., & Vicente, A. A. (2010). Influence of electric fields on the structure of chitosan edible coatings. Food Hydrocolloids, 24(4), 330–335. Winkleman, A., Gates, B. D., McCarty, L. S., & Whitesides, G. M. (2005). Directed selfassembly of spherical particles on patterned electrodes by an applied electric field. Advanced Materials, 17(12), 1507–1511. Wu, T., Zivanovic, S., Hayes, D. G., & Weiss, J. (2008). Efficient reduction of chitosan molecular weight by high-intensity ultrasound: Underlying mechanism and effect of process parameters. Journal of Agricultural and Food Chemistry, 56(13), 5112–5119. Yang, N., Jin, Y., Li, D., Jin, Z., & Xu, X. (2017). A reconfigurable fluidic reactor for intensification of hydrolysis at mild conditions. Chemical Engineering Journal, 313, 599–609. Yi, J., Njoroge, D. M., Sila, D. N., Kinyanjui, P. K., Christiaens, S., Bi, J., et al. (2016). Detailed analysis of seed coat and cotyledon reveals molecular understanding of the hard-to-cook defect of common beans (Phaseolus vulgaris L.). Food Chemistry, 210, 481–490. Zhou, Y., Jin, Y., Yang, N., Xie, Z., & Xu, X. (2017). Electrofluid enhanced hydrolysis of maize starch and its impacts on physical properties. RSC Advances, 7(31), 19145–19152. Zhu, F. (2018). Modifications of starch by electric field based techniques. Trends in Food Science & Technology, 75, 158–169.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodhyd.2019.105484. References BeMiller, J. N., & Huber, K. C. (2015). Physical modification of food starch functionalities. Annual Review of Food Science and Technology, 6, 19–69. Cardoso, M. F.d. C., Gomes, A. T. P. C., Silva, V. L. M., Silva, A. M. S., Neves, M. G. P. M. S., da Silva, F.d. C., et al. (2015). Ohmic heating assisted synthesis of coumarinyl porphyrin derivatives. RSC Advances, 5(81), 66192–66199. Chatjigakis, A. K., Pappas, C., Proxenia, N., Kalantzi, O., Rodis, P., & Polissiou, M. (1998). FT-IR spectroscopic determination of the degree of esterification of cell wall pectins from stored peaches and correlation to textural changes. Carbohydrate Polymers, 37(4), 395–408. Gamonpilas, C., Krongsin, J., Methacanon, P., & Goh, S. M. (2015). Gelation of pomelo (Citrus maxima) pectin as induced by divalent ions or acidification. Journal of Food Engineering, 152(Supplement C), 17–23. Giteru, S. G., Oey, I., & Ali, M. A. (2018). Feasibility of using pulsed electric fields to modify biomacromolecules: A review. Trends in Food Science & Technology, 72, 91–113. Han, Z., Zeng, X. A., Fu, N., Yu, S. J., Chen, X. D., & Kennedy, J. F. (2012). Effects of pulsed electric field treatments on some properties of tapioca starch. Carbohydrate Polymers, 89(4), 1012–1017. Han, Z., Zeng, X. A., Yu, S. J., Zhang, B. S., & Chen, X. D. (2009). Effects of pulsed electric fields (PEF) treatment on physicochemical properties of potato starch. Innovative Food Science & Emerging Technologies, 10(4), 481–485. Heathcote, M. J. (2007). 1-Transformer theory. J & P transformer book (Thirteenth Editon). Oxford: Newnes. Hernoux-Villi� ere, A., Lassi, U., Hu, T., Paquet, A., Rinaldi, L., Cravotto, G., et al. (2013). Simultaneous microwave/ultrasound-assisted hydrolysis of starch-based industrial waste into reducing sugars. ACS Sustainable Chemistry & Engineering, 1(8), 995–1002. Hong, J., Chen, R., Zeng, X. A., & Han, Z. (2016). Effect of pulsed electric fields assisted acetylation on morphological, structural and functional characteristics of potato starch. Food Chemistry, 192, 15–24. Kanjanapongkul, K. (2017). Rice cooking using ohmic heating: Determination of electrical conductivity, water diffusion and cooking energy. Journal of Food Engineering, 192, 1–10. Kasaai, M. R. (2008). A review of several reported procedures to determine the degree of N-acetylation for chitin and chitosan using infrared spectroscopy. Carbohydrate Polymers, 71(4), 497–508. Kjøniksen, A. L., Hiorth, M., & Nystr€ om, B. (2005). Association under shear flow in aqueous solutions of pectin. European Polymer Journal, 41(4), 761–770.
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Food Hydrocolloids journal homepage: http://www.elsevier.com/locate/foodhyd
Induced electric field intensification of acid hydrolysis of polysaccharides: Roles of thermal and non-thermal effects Dan-Dan Li a, b, Na Yang b, Yang Tao a, En-Bo Xu c, Zheng-Yu Jin b, Yong-Bin Han a, **, Xue-Ming Xu b, * a b c
College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, Jiangsu Province, China College of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu Province, China College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, Zhejiang Province, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Electro-processing Degradation Mechanism Electrical properties Directional migration
The mechanism of induced electric field (IEF)-assisted acid hydrolysis was investigated by using guar gum, chitosan, and pectin as model polysaccharides. Roles of thermal and non-thermal effects were explored by temperature determination, HPSEC-MALLS-RI, SEM, FT-IR, XRD, and a rheometer. Results suggested that IEF affected the hydrolysis of polysaccharides by inducing thermal and non-thermal effects. Thermal effect contributed to the hydrolysis. As the excitation voltage increased, the thermal effect enhanced, with a temper ature increased by about 25 � C after IEF (75 V) treatment for 4 h. Non-thermal effect, induced by the rapid and directional migration of charged species, varied with the electrical properties of the polysaccharides. It enhanced the hydrolysis of guar gum and pectin but negatively impacted the chitosan due to their different migration directions under IEF. IEF-assisted hydrolysis damaged the structural compactness of polysaccharides and significantly decreased the viscosity of their solutions. This study clarified the hydrolysis mechanism of poly saccharides under IEF, providing a theoretical guide for the application of IEF in the chemical modification of biopolymers.
1. Introduction
of high voltage electric field to materials placed between two electrodes. The energy dissipated during PEF treatment not only degrades poly saccharides such as chitosan (Luo, Han, Zeng, Yu, & Kennedy, 2010) and starch (Han, Zeng, Zhang, & Yu, 2009), but also assists the acetylation of pectin (Ma & Wang, 2013) and starch (Hong, Chen, Zeng, & Han, 2016). Ohmic heating (OH) is an internal thermal energy generation technol ogy that electrical energy is dissipated into heat directly by passing electric current through sample (Knirsch, Alves dos Santos, Martins de Oliveira Soares Vicente, & Vessoni Penna, 2010). OH allows for faster and more uniform heating and induces an increase in the directed movement of charged molecules when compared with water-bath and microwave heating, thus accelerating organic syntheses such as a Diels-Alder cycloaddition, a nucleophilic substitution, an N-alkylation, and a Suzuki cross-coupling reaction (Cardoso et al., 2015; Pinto et al., 2013). Therefore, electric field based techniques may have potentials in enhancing the modification of polysaccharides. The combination of the electrical technique with acid hydrolysis is expected to have a
Natural polysaccharides are widely available, affordable, and sus tainable, thus having great potentials in food, medicine, and other in dustries. However, natural polysaccharides also have shortcomings, in particular low solubility, a quality which dramatically limits their po tential applications. Degradation is the simplest method for improving the properties and functions of polysaccharides, with degraded poly saccharides usually exhibiting a higher level of solubility than their natural counterparts. Degradation can be achieved by physical (Sar avana et al., 2018), chemical (Miao, Jiang, Zhang, Jin, & Mu, 2011), and biological (Ma et al., 2018) means, with acid hydrolysis being the simplest method. However, the compact and complex structure of native polysaccharides limits their hydrolysis efficiency. Electric field based techniques have gained increasing interests in food and biomaterial processing. Pulsed electric field (PEF) is a nonthermal technique that involves the application of short pulses (μs-ms)
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y.-B. Han), [email protected] (X.-M. Xu). https://doi.org/10.1016/j.foodhyd.2019.105484 Received 17 April 2019; Received in revised form 31 October 2019; Accepted 1 November 2019 Available online 4 November 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Typical chromatograms for native and IEF-assisted hydrolyzed guar gum (75 V, 400 Hz).
synergistic effect when applied to natural polysaccharides. However, the current applications of electro-techniques are based on the electropho resis of metal electrodes. The electrodes, when inserted into electrolyte solutions, especially acidic solutions, will result in severe sample contamination due to the occurrence of electrochemical reactions (Winkleman, Gates, McCarty, & Whitesides, 2005). The induced electric field (IEF) system is developed in the configu ration of a transformer by employing Faraday’s law of induction. Alternating excitation voltage in the primary coil causes a variable magnetic flux in the core of the transformer, thus inducing alternating electric voltage in the sample coil. Compared with PEF and OH, the generation of IEF is through coils and core rather than metal electrodes, thus avoiding electrode-corrosion-induced sample contamination. Pre vious studies confirmed that IEF significantly improved the hydrolysis of cellulose (Yang, Jin, Li, Jin, & Xu, 2017) and starch (Li et al., 2017). For example, IEF treatment (150 V at 50 � C for 24 h) increased the reducing sugar content in cellulose hydrolysates by 155.5% when compared with traditional heating (50 � C). Despite encouraging results, the mechanism of IEF-assisted hydrolysis remains unclear. The energy dissipated during PEF treatment can cause electro chemical reactions and the polarization of structural moieties, and thus PEF has potentials to modify the microstructure and functional prop erties of bio-macromolecules (Giteru, Oey, & Ali, 2018). Typically, a critical PEF treatment intensity is required to initiate structural, conformational and functional changes to bio-macromolecules. For example, Luo et al. (2010) demonstrated that the minimum intensity for production of pits on the surface of chitosan granules was 15 kV/cm. The intensity of IEF is much lower than the required critical intensity. Hence, the electrochemical reactions and the polarization of structural moieties of polysaccharides during IEF treatment may be ignored. Pinto et al. (2013) reported that OH induced electric current passing through sample and accelerated the directional migration of charged species, thus producing both thermal and non-thermal effects to affect chemical reactions. The energy dissipated to samples during OH processing is similar to that during IEF processing. Therefore, the induced alternating
voltage in the sample coil may induce thermal and non-thermal effects to alter the hydrolysis of polysaccharides. In this study, chitosan, pectin, and guar gum were selected as the model polysaccharides in order to investigate the underlying mechanism of IEF-assisted hydrolysis. The roles of the thermal and non-thermal effects were analyzed and the ef fects of IEF-assisted hydrolysis on the structural and physicochemical properties of polysaccharides were explored. This study clarified the mechanism of IEF-assisted hydrolysis, which would contribute to the development of other potential applications for IEF in chemical modification. 2. Materials and methods 2.1. Materials Chitosan (DD ¼ 85.83%, 200-mesh sieve) and guar gum (viscos ity ¼ 5200 � 150 mPa s, 200-mesh sieve) were of analytical grade and were purchased from Aladdin Co., Ltd. (Shanghai, CN). Pectin (DE ¼ 96.83%, pH ¼ 2.8 � 0.2 (1% in H2O), 200-mesh sieve) was of biochemical grade and was purchased from Sangon Biotech Co., Ltd. (Shanghai, CN). Hydrochloric acid, sodium hydroxide, and all other analytical chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. (Suchou, CN). 2.2. Acid hydrolysis of polysaccharides under IEF The schematic diagram of a 4-series IEF system has been reported in our previous study (Li et al., 2017). The core component of an IEF system is an o-core transformer, which consists of a primary copper coil, a magnetic core, and a glass spiral. The glass spiral is surrounded by a circulating water chamber, which controls the reaction temperature. During IEF treatment, liquid sample is pumped into the glass spiral, acting as the secondary coil of the transformer (sample coil). According to Ampere’s law, as an excitation voltage is imposed on primary coil, an alternating magnetic flux appears in magnetic core, and then an electric 2
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voltage is induced in sample coil. In this study, guar gum, pectin, or chitosan (3.2 g) was precisely weighed and then was slowly added to 0.15 mol/L or 1.5 mol/L HCl solutions (400 mL) with continuously stirring at 25 � C for 12 h. The obtained mixture was pumped into IEF system and then treated at the conditions: excitation voltage 15 V, 35 V, 55 V, and 75 V; frequency 400 Hz; flow rate 300 mL/min; reaction temperature not controlled or controlled at 50 � C; reaction time 0–24 h for guar gum and 0–60 h for chitosan and pectin (guar gum was sensitive to acid hydrolysis and exhibited extensive hydrolysis after treated by 0.15 mol/L HCl for 24 h, whereas chitosan and pectin were more stable in acid solutions and thus higher concentrated acid and longer reaction time were necessary). When the reaction temperature was not controlled, the changes in the temperature of polysaccharide-HCl mixtures were recorded by using a CEM DT-810 InfraRed Thermometer (Huashengchang Co., Ltd., Shenz hen, CN) to investigate the roles of thermal effect in IEF-assisted hy drolysis. Treatments at the same conditions but without excitation voltage (0 V) and frequency (0 Hz) were run as controls. In order to study the roles of non-thermal effect, a temperature (50 � C) higher than that caused by thermal effect of IEF was set by the circulating water bath, the temperature profiles of IEF treatment at 50 � C and continuousflow water-bath heating at 50 � C were recorded, and then the hydrolysis efficiency of polysaccharides during IEF treatment (75 V) and continuous-flow water-bath heating was compared by molecular weight determination. After treatments, the hydrolysis was terminated by adding 1 mol/L NaOH to adjust the pH of the reaction mixture to 7.0–8.0. When the reaction mixture was cooled to 25 � C, the resulting product was precipitated with anhydrous ethanol and the hydrolyzed polysaccharide was separated by filtration under vacuum. The solid was further washed with 80% ethanol (200 mL each time) until no Cl in effluent could be detected by 0.1 mol/L AgNO3. After filtration, the residue was dried at 45 � C for 24 h. The prepared samples were used for testing. 2.3. Molecular weight determination The molecular weight determination was performed by using a highperformance size-exclusion chromatography (HPSEC) system with two columns in series (Shodex OHpak SB-804 HQ and OHpak SB-802 HQ columns). The HPSEC system consists of an isocratic pump, a multiangle laser light-scattering (MALLS) detector, and a refractive index (RI) detector (Dawn DSP; Wyatt Technology Co., Ltd., Santa Barbara, USA). All columns were thermostated at 40 � C. Guar gum and pectin: the mobile phase was 0.1 mol/L NaNO3 and 0.02% NaN3 aqueous solution; polysaccharide was diluted to 0.2% wt. using mobile phase and filtered through 0.45 μm filter before injection. Chitosan: the mobile phase was 0.3 mol/L CH3COOH and 0.1 mol/L CH3COONa aqueous solution (pH ¼ 4.5); chitosan was diluted to 0.4% wt. using mobile phase and filtered through 0.45 μm filter before injection. Before analysis, the mobile phase was ultrasound for 30 min to degas and filtered through 0.45 μm filters. Samples (20 μL) were injected into HPSEC system and eluted at a flow rate of 0.6 mL/min for 70 min. Pullulan standards (P50 Mw ¼ 4.71 � 104 g/mol, P800 Mw ¼ 6.42 � 105 g/mol, Shodex standard P-82) were used to calibrate the columns. The electronic outputs of the MALLS and RI detectors were collected using ASTRA software (version 5.3.4, Wyatt Technology Co., Ltd., Santa Barbara, USA). 2.4. Output voltage determination Fig. 2. Effect of IEF treatment on the Mw of polysaccharides: (a) guar gum; (b) chitosan; and (c) pectin (Without temperature control).
A pair of platinum electrodes connected to an AC millivoltmeter (UT631; UniTrend Group Limited Co., Ltd., Hong Kong, CN) was inserted into the two ends of sample coil to determine the output voltage of IEF system. The determination was performed at excitation voltages of 15 V, 35 V, 55 V, and 75 V and a frequency of 400 Hz. 3
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Table 1 Electrical parameters of induced electric field system when different polysaccharides acted as the secondary coil. Excitation voltage (V)
Excitation current (A)
Power (W)
15 35 55 75
0 0.261 0.473 0.687
0 7 18 37
Output voltage (V) Guar gum
Chitosan
Pectin
Expected value
3.13 � 0.43Da 7.41 � 0.31Ca 11.88 � 0.62Ba 15.89 � 0.74Aa
3.17 � 0.36Da 7.53 � 0.61Ca 11.84 � 0.52Ba 17.04 � 1.32Aa
3.01 � 0.42Da 7.51 � 0.38Ca 11.81 � 0.45Ba 16.01 � 0.42Aa
3.75 8.75 13.75 18.75
Means in the same row with different superscript lowercase letter and same column with different superscript uppercase letter indicate significant difference (P < 0.05).
hydration. After that, sample was transferred to the rheometer plate and the excess sample was wiped off by using a straw. Steady shear tests were performed over the shear rate range of 0.01–100 s 1. Before each test, a time waiting process of 60 s was applied to equilibrate the stress and temperature of hydrolyzed polysaccharides (25 � C). 2.6. Statistical analysis All experiments were performed in triplicates. Data were expressed as mean � standard deviation. Fisher’s least significant difference (LSD) test was used to compare means at the 5% significance level (IBM SPSS Statistics 20, Somers, USA). 3. Results and discussion 3.1. IEF enhancement of acid hydrolysis of polysaccharides The molecular characteristics of guar gum, chitosan, and pectin during IEF-assisted hydrolysis were determined by HPSEC-MALLS-RI (Fig. 1, Fig. S1, and Fig. S2). The MALLS intensity was applied to calculate the molecular weight, and the RI gave a signal proportional to the concentration (Yi et al., 2016). During IEF-assisted hydrolysis, the molar mass of guar gum dramatically decreased, accompanied by a falling MALLS intensity and a shift of RI peak to larger elution volumes (Fig. 1). These results suggested that the guar gum chains were degraded to lower molecular weights. Chitosan and pectin showed similar varia tion trends (Fig. S1 and Fig. S2). The effects of IEF on the Mw of poly saccharides were illustrated in Fig. 2. Without IEF treatment, the Mw of polysaccharides remained unchangeable, whereas the Mw significantly decreased with IEF treatment, suggesting that IEF enhanced the hy drolysis of polysaccharides. As the excitation voltage was increased, the hydrolysis of polysaccharides was enhanced. After IEF treatment at 15 V and 75 V for 24 h, the Mw of guar gum decreased by 2.43 � 105 g/mol and 16.11 � 105 g/mol, respectively (Fig. 2a). Chitosan and pectin showed similar variation trends with guar gum, but they exhibited less extensive hydrolysis because they were more stable in acid solution (Fig. 2b and Fig. 2c). Luo et al. (2010) and Han, Zeng, Yu, Zhang, and Chen (2009) reported that PEF could cause the degradation of chitosan and starch. Zhou, Jin, Yang, Xie, and Xu (2017) suggested that the hy drolysis of corn starch enhanced with increasing excitation voltage of IEF treatment.
Fig. 3. Temperature profiles of polysaccharide-HCl mixtures during IEF treat ment at 15 V, 35 V, 55 V, and 75 V (Without temperature control). G represents guar gum, C represents chitosan, P represents pectin.
2.5. Characteristics of hydrolyzed polysaccharides 2.5.1. Fourier infrared spectroscopy (FT-IR) The FT-IR spectra of native and hydrolyzed chitosan, guar gum, and pectin were recorded from a KBr disk (500 mg) containing finely ground sample (25 mg) on a Bruker GER400 spectrometer (Bruker Co., Ltd., GER) at a wavenumber range of 4000-400 cm 1. 2.5.2. X-ray diffraction (XRD) XRD patterns were performed by a X-ray diffractometer of Bruker D8-Advance type (Bruker AXS Co., Ltd., Karlsruhe, GER) with Cu-Kα filtered radiation (wavelength 1.5405 Å) radiator operating at 30 kV and 10 mA. The scans ranged from 5� to 50� with a step width of 0.02� . 2.5.3. Scanning electron microscope (SEM) The granular structure of native and partially hydrolyzed poly saccharides was observed by a SU 1510 SEM (Hitachi High-Technologies Co., Ltd., JP). The applied accelerating voltage is 5.00 kV. Prior to observation, the polysaccharide powder was mounted on an aluminum specimen holder with double-sided adhesive tape and then coated with gold under vacuum evaporation.
3.2. Possible mechanism of IEF-assisted hydrolysis of polysaccharides 3.2.1. Electrical characteristics of polysaccharides When polysaccharide-HCl mixture that was electrically conductive was pumped into the IEF system, it acted as the secondary coil. An alternating voltage would be produced in the secondary coil if an exci tation voltage was imposed on the primary coil (Heathcote, 2007). Ac – NS/NP), the induced cording to Ampere’s circuital theorem (US/UP– voltage in the secondary coil (US) was linearly related to the excitation voltage (UP) and the ratio of primary coil turns (NP) to secondary coil turns (NS). The excitation electric signal of IEF was sinusoidal alter nating, with voltages of 15 V–75 V and a frequency of 400 Hz. The NS
2.5.4. Rheological measurements Steady shear rheological tests on a TA DHR-3 Rheometer (TA In struments Co., Ltd., USA) were applied to characterize the native and hydrolyzed polysaccharides. The tests were performed by using cone plate geometry (60 mm) at a gap of 116 μm. Guar gum and pectin were added to distilled water at a concentration of 1% wt. and 5% wt. with continuously stirring, respectively, while chitosan (4% wt.) was dis solved in 1% CH3COOH solution. Polysaccharide solution was then stirred at room temperature (25 � C) for 12 h to confirm complete 4
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Fig. 4. The electrical signal of IEF system and the possible migration ways of charged ions under IEF.
the expected value when using liquid sample, which exhibited signifi cant levels of impedance, as the secondary coil of IEF system. As shown in Table 1, the output voltage was lower than the expected value when polysaccharide-HCl mixture acted as the secondary coil of IEF system. As excitation voltage increased from 15 V to 75 V, the output voltage of guar gum increased from 3.133 V to 15.92 V. No significant difference in the output voltage was observed when using different polysaccharide-HCl mixtures as the secondary coil. The increased output voltage could induce higher electrical current passing through polysaccharide-HCl mixtures, thus enhancing thermal effect. In addi tion, the charged species exhibited higher migration rate under IEF as the output voltage increased. 3.2.2. Roles of thermal effect on IEF-assisted hydrolysis of polysaccharides Thermal effect commonly exists in electro-processing due to the conversion of electric energy into heat energy. The magnitude of the temperature increase of a product is crucial in determining the efficiency of electro-processing. The equation for calculating the thermal effect induced by IEF processing has not been proposed, but the increase in the temperature during IEF processing is considered to depend on the electrical current density. Due to the lack of technical means, the ionic current in liquid sample (secondary coil) cannot be determined. How ever, the electric current in primary coil, which is proportional to the induced current in sample coil, can be obtained from the power source. When excitation voltage increased from 15 V to 75 V, the electric current in primary coil increased from 0 to 0.687 A, suggesting that thermal effect existed in IEF-assisted hydrolysis (Table 1). Fig. 3 confirmed that IEF treatment caused thermal effect. The temperature of all
Fig. 5. Temperature profiles of polysaccharide-HCl mixtures during IEF treat ment at 50 � C and water-bath heating at 50 � C.
and NP values were 23 and 92, respectively. Therefore, the expected induced voltage was 3.75 V, 8.75 V, 13.75 V, and 18.75 V (Table 1). The induced voltage could be determined by a Voltmeter, which formed a closed loop with the secondary coil. For coils that were made of ideal conductor materials, the determined value was expected to be same as theoretical value. However, the output voltage was typically lower than 5
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polysaccharide-HCl mixtures rapidly increased in the first 4 h and then remained constant during IEF treatment. According to Joule’s law, the density of electric current is closely related to the intensity of electric field and the electrical conductivity of sample. As the excitation voltage increased, the temperature signifi cantly increased (Fig. 3). After IEF treatment at 15 V and 75 V for 4 h, the temperature of polysaccharides increased by about 5 � C and about 25 � C, respectively. During PEF-assisted acetylation of potato starch, the tem perature of the reaction mixture increased from 30 � C to 39 � C when the strength of electric field increased from 2 kV/cm to 5 kV/cm (Hong et al., 2016). Han et al. (2012) also reported that the increase in tem perature of tapioca starch suspension was positively related to the in crease in PEF strength. Different polysaccharides exhibited different charge content, and thus the thermal effect of differently charged polysaccharides during IEF treatment slightly differed from each other. Compared with the excitation voltage, the electrical conductivity of sample might play a secondary role in the thermal effect. After being subjected to IEF treatment at 75 V for 4 h, the temperature of chitosan, pectin, and guar gum was 45.1 � C, 44.2 � C, and 43 � C, respectively. This result was in accordance with the output voltage results which slightly decreased in the sequence: chitosan > pectin > guar gum (Table 1). Kanjanapongkul (2017) used OH to cook rice and observed that the magnitude of the temperature increase varied with the electrical conductivity. Heat plays a vital role in the modification of polysaccharides. High temperatures increase the mobility of molecular chains and helical structures, which leads to the structural alteration of the amorphous and crystalline regions in polysaccharide granules (BeMiller & Huber, 2015), thus allowing chemical reagents and enzymes attacking polysaccharide chains more rapidly. Heat is also known to influence molecular motion, where, at high temperatures, faster rate of movement tends to increase the interparticle collisions among reaction substances, thus enhancing chemical reactions. Moreover, the hydrolysis of polysaccharides is an endothermic process. Therefore, the increase in temperature is expected to enhance the hydrolysis of polysaccharides. Compared with the con trols, the Mw of guar gum, chitosan and pectin all decreased more rapidly with increasing excitation voltage of IEF treatment (Fig. 2). The variation trends of Mw were in accordance with those of temperature, suggesting that IEF might improve the hydrolysis process by inducing thermal effect. Pinto et al. reported that OH allowed for faster and more uniform heating compared with oil bath and microwave, thus short ening the reaction time and increasing the yields of organic syntheses (Pinto et al., 2013). 3.2.3. Roles of non-thermal effect on IEF-assisted hydrolysis of polysaccharides Pinto et al. (2013) suggested that alternating electric fields induced the rapid and reciprocating movement of charged species, altered the collision opportunities among reaction substances, changed mass transfer, and thus affected the overall yield and reaction time of organic syntheses. Hong et al. (2016) reported that the high electric potential difference and changed electric field direction induced by PEF acceler ated the reciprocating motion of reaction ions, leading to a more effective collision rate and a higher degree of acetylation. Souza et al. (2010) suggested that moderate electric field enhanced the mechanical and thermal properties of chitosan films/coatings by inducing a more ordered structure and a clearly higher crystallinity, owing to the direc tional migration of chitosan under the electric field. During IEF treat ment, alternating excitation voltage imposed on the primary coil induced an alternating bipolar current in the sample coil (Fig. 4). Hence, the induced electric signal showed a periodic variation, with continu ously increased electric voltage during 0–1/4 T (T represents the time necessary for a periodically varying induced electric voltage), gradually decreased electric voltage during 1/4–1/2 T, increased electric voltage but in the opposite direction during 1/2–3/4 T, and finally decreased voltage in 3/4-1 T (Fig. 4). Without IEF (t ¼ 0, 1/2 T, and 1 T),
Fig. 6. Changes in the Mw of polysaccharides subjected to IEF treatment (75 V) at 50 � C and water-bath heating at 50 � C: (a) guar gum; (b) chitosan; and (c) pectin.
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Fig. 7. SEM images of native and hydrolyzed polysaccharides: (a) guar gum; (b) chitosan; (c) pectin; (1) native polysaccharide; (2) IEF-assisted hydrolyzed poly saccharide (15 V); (3) IEF-assisted hydrolyzed polysaccharide (35 V); (4) IEF-assisted hydrolyzed polysaccharide (55 V); (5) IEF-assisted hydrolyzed polysaccharide (75 V); (6) hydrolyzed polysaccharide treated by continuous-flow water-bath heating at 50 � C.
polysaccharides, hydrogen ions, and other molecules exhibited Brow nian movement and thus were symmetrically distributed. With IEF, charged species exhibited alternating motions due to a varying electric field direction. Positively charged molecules (hydrogen ion and chito san) migrated in the same direction as the electric field, negatively charged molecules migrated in the opposite direction of the field, and IEF had a negligible effect on the migration of neutrally charged sub strates such as guar gum. The collision opportunities between hydrogen ion and polysaccharide might vary with the charge types of poly saccharide, thus causing different hydrolysis behaviors under IEF. The Mw results shown in Fig. 6 confirmed that non-thermal effect varied with the polysaccharide types. When investigating the role of non-thermal effects on IEF-assisted hydrolysis, the reaction temperature was controlled at 50 � C, a
temperature higher than that induced by IEF treatment (75 V). As shown in Fig. 5, the temperature profiles of polysaccharide-HCl mixtures dur ing IEF treatment at 50 � C and continuous-flow water-bath heating at 50 � C were similar. However, the changes in Mw were slightly different, suggesting that a non-thermal effect might exist during IEF-assisted hydrolysis. IEF accelerated the migration of hydrogen ions but showed no significant effect on the migration of guar gum, and hence hydrogen ions were expected to attack the guar gum chains more rapidly. Compared with continuous-flow water-bath heating at 50 � C, IEF at 50 � C slightly decreased the Mw of guar gum (Fig. 6a). However, the decrease was not statistically significant, which might be attributed to the relative low electric field intensity of current IEF system. Fig. 6b suggested that the non-thermal effect seemed to weaken the hydrolysis of chitosan, which might be attributed to the same migration direction of 7
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chitosan and hydrogen ion under the electric field. However, the final Mw were at the same level as that treated by continuous-flow water-bath heating. Wu, Zivanovic, Hayes, and Weiss (2008) suggested that the hydrolysis rate of chitosan varied with the degree of acetylation of chitosan during sonication treatment, but the final Mw of different chitosan tended to be similar. Typically, the Mw of chitosan reduced rapidly at initial stage but then slowly decreased with further hydrolysis due to the semi-crystal structure of chitosan. After hydrolysis for 36 h, the amorphous part in chitosan granule was hydrolyzed and the remaining tight crystal part started to be damaged. As a result, the hy drolysis rate decreased and no significant difference between IEF treatment at 50 � C and continuous-flow water-bath heating at 50 � C was observed. Pectin is a negatively charged polysaccharide in distilled water, but it tends to be neutrally charged when dispersed in acid so lutions. Hydrogen ion exhibits much higher migration speed under IEF than pectin because pectin has much lower charge density. Therefore, hydrogen ions may attack pectin more rapidly under IEF. Fig. 6c showed that pectin exhibited a lower hydrolysis rate during initial 12-h period but the Mw of IEF-treated pectin decreased more after hydrolysis for 60 h when compared with continuous-flow water-bath heating. The different phenomenon between guar gum and pectin might be due to their different dissolved state in acid solution. 3.3. Changes in structural and physicochemical properties of polysaccharides induced by IEF-assisted hydrolysis 3.3.1. SEM Zhu (2018) reviewed the recent papers about the applications of electric field based techniques in starch modification and concluded that PEF, OH, and IEF treatments all damaged the granular structure of starch. SEM micrographs of native and hydrolyzed polysaccharides at magnifications of 200� are depicted in Fig. 7. Fig. 7a shows that native guar gum possessed a tough surface morphology in the form of nodules and flakes (Fig. 7a1). After being treated by IEF-assisted hydrolysis, the particle size distribution of guar gum was broadened, because some native granules were broken down into smaller pieces and some of these pieces congregated (Fig. 7a2-a5). This effect was enhanced with increasing excitation voltage of IEF treatment. A similar phenomenon was observed for pectin (Fig. 7c1-c5). In comparison with native chi tosan, the IEF-assisted hydrolyzed samples aggregated to form large particles and pits and pores emerged on the surface of these large par ticles (Fig. 7b2-b5). The changes in the granular structure of poly saccharides during continuous-flow water-bath heating (Fig. 7a6, Fig. 7b6, and Fig. 7c6) were similar to those during IEF-assisted hy drolysis, suggesting that IEF might replace traditional heating to be an effective method for polysaccharide degradation. The damaged granular structure allowed enzymes and chemical reagents entering granular interior easily and therefore might contribute to the modification of polysaccharides. 3.3.2. FT-IR The effect of IEF-assisted hydrolysis on the chemical structure of guar gum, chitosan, and pectin was investigated by FT-IR (Fig. 8). No major structural differences in the spectra of native and IEF-assisted hydro lyzed guar gum could be seen (Fig. 8a). Acid hydrolysis performed with continuous-flow water-bath heating also showed no significant effect on the chemical structure of guar gum, which was in accordance with the results reported by Mudgil, Barak, and Khatkar (2012). After IEF-assisted hydrolysis, no new peaks appeared in the FT-IR spectra of chitosan (Fig. 8b) and pectin (Fig. 8c). However, the degree of acety lation (DA) of chitosan and the degree of esterification (DE) of pectin were changed by the hydrolysis. Previous study suggested that A1655/A1070 was suitable for evaluating the DA of chitosan ranging from 0 to 100% as it displayed various advantages including no broadening, no shoulder issues, and it also remained unchanged regardless of water content (Kasaai, 2008). The ratio increased from 0.73 (native chitosan)
Fig. 8. FT-IR spectra of polysaccharides subjected to acid hydrolysis with IEF treatment and continuous-flow water-bath heating at 50 � C: (a) guar gum, (b) chitosan, and (c) pectin.
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Table 2 Data for FT-IR spectra and XRD patterns of polysaccharides subjected to acid hydrolysis with IEF treatment and continuous-flow water-bath heating at 50 � C. native FT-IR XRD
A1655/A1070 A1749/(A1749þA1630)) Peak 1 Peak 2
15 V IEF c
0.73 � 0.03 0.47 � 0.04b 11.07 � 0.21a 20.00 � 0.04a
35 V IEF bc
55 V IEF a
0.89 � 0.07 0.59 � 0.06ab 10.19 � 0.04b 20.09 � 0.12a
1.09 � 0.05 0.61 � 0.06ab 10.19 � 0.12b 20.09 � 0.12a
75 V IEF a
1.11 � 0.11 0.61 � 0.12ab 10.19 � 0.12b 20.09 � 0.12a
water bath a
1.11 � 0.04 0.69 � 0.05a 10.19 � 0.13b 20.09 � 0.09a
1.01 � 0.02ab 0.61 � 0.04ab 10.19 � 0.09b 20.09 � 0.04a
Means in the same row with different superscript lowercase letter indicate significant difference (P < 0.05).
to 1.01 (continuous-flow water-bath heating), 0.89 (15 V), 1.09 (35 V), 1.11 (55 V), and 1.11 (75 V), respectively (Table 2). Chatjigakis et al. (1998) reported that there was a linear relationship between the DE value and the ratio of the area underneath the peak 1749 cm 1 over the sum of the areas underneath the two peaks, at 1749 cm 1 and 1630 cm 1. The ratio (A1749/(A1749þA1630)) was calculated and the value was 0.47, 0.61, 0.59, 0.61, 0.61, and 0.69 for native pectin, water-bath-treated pectin, 15 V-treated pectin, 35 V-treated pectin, 55 V-treated pectin, and 75 V-treated pectin, respectively (Table 2). Although more extensive hydrolysis was obtained with continuous-flow water-bath heating at 50 � C, IEF treatment (35–75 V) increased the DA values and DE values. IEF allowed hydrogen ions to attack poly saccharide chains more directionally, which might cause the more se lective hydrolysis of polysaccharides. Hernoux-Villi� ere et al. (2013) reported that there was glucose selectivity with a rate of saccharification of up to 46% when a simultaneous high-frequency electromagnetic field/ultrasound-assisted procedure was applied to catalyze the con version of starch-based industrial waste into sugars.
shear rates, supporting that the low molecular weight polysaccharide fragments were formed. However, for pectin samples, a pseudo-plastic behavior was observed over the tested shear rate range. This phenom enon contributed to a rearrangement in the structure of the associated complexes, which were disrupted as a result of shearing. Gamonpilas, Krongsin, Methacanon, and Goh (2015) suggested that pectin disper sions exhibited Newtonian behavior at low concentrations (<0.2%) and pseudo-plastic behavior at high concentrations (>1%). In addition, the shear thinning behavior of pectin enhanced with hydrolysis, especially €m, 2005) at high shear rates. Previous work (Kjøniksen, Hiorth, & Nystro proposed that a variation in the branching of pectin resulted in a different shear rate dependence of viscosity. FT-IR spectra illustrated that the DE value of pectin was changed by hydrolysis and thus might cause the changes in the shear thinning behavior (Fig. 8c). The viscosity of all polysaccharides significantly decreased with acid hydrolysis (Fig. 9a, Fig. 9b, and Fig. 9c). For example, the viscosity of guar gum decreased by 1 order of magnitude after IEF treatment at 75 V for 24 h. IEF accelerated the migration rate of charged hydrogen ions, enhanced the friction between hydrogen protons and polysaccharides, and improved the cleavage of backbone as well as side chains, thus effectively decreasing the viscosity of polysaccharide solutions. The presence of lower molecular weight polysaccharide fragments was confirmed by HPSEC analysis (Figs. 2, Fig. 3, and Fig. 6). Compared with IEF treatment (without temperature control), continuous-flow waterbath heating at 50 � C caused more extensive hydrolysis, thus causing lower steady shear viscosity. The hydrolyzed polysaccharides with low Mw and low viscosity might be used as water-soluble dietary fibers or as water-soluble antibacterial agents, which had a wide range of uses in both clinical nutrition and medicine (Slavin & Greenberg, 2003).
3.3.3. XRD X-ray diffraction was performed in order to explore the changes in crystal form and crystallinity of guar gum, chitosan, and pectin during acid hydrolysis (Fig. 9). IEF-assisted hydrolysis showed no significant effect on the crystal structure of guar gum (Fig. 9a) and pectin (Fig. 9c). Native and partially hydrolyzed guar gum samples displayed low overall crystallinity, with reflections at 17.2� and 20.1� . Previous studies also suggested that acid hydrolysis had no significant effect on the crystal structure of guar gum (Mudgil et al., 2012). Native and hydrolyzed pectin were semi-crystalline, with two principal diffraction peaks at 2θ of about 13� and of about 21� . However, the crystal structure of chitosan tended to transfer from “Form II” to “Form I00 with IEF-assisted hydro lysis. In the XRD spectra of native chitosan, a strong reflection at 20.00� and a weak reflection at 11.07� were observed (Fig. 9b), which indicated that native chitosan displayed a pattern equivalent to the “Form II” crystal structure. After hydrolysis, the reflection at around 11.07� shif ted to a smaller angle (10.19� ). However, the reflection at around 20.00� shifted to a slightly larger angle (20.09� ). These shifts demonstrated that the crystal structure of hydrolyzed chitosan tended to conform to the “Form I00 crystal structure. The “Form II” crystal structure usually dis played a more constrained chain conformation, whereas “Form I00 had a more extended chain structure (Qun, Ajun, & Yong, 2007), meaning that IEF-assisted hydrolysis lessened the structural compactness of chitosan. The damaged crystal structure allowed chemical reagents and enzymes attacking polysaccharides easier, thus contributing to their chemical and biological modification. Similar changes in the crystal structure of polysaccharides were observed for polysaccharides treated by continuous-flow water-bath heating.
4. Conclusions IEF affected the hydrolysis of polysaccharides by inducing thermal and non-thermal effects. During IEF treatment, the temperature of polysaccharide-acid solutions increased, thus contributing to the hy drolysis of polysaccharides. As the excitation voltage was increased, the thermal effect was enhanced. The electrical properties of poly saccharides showed no significant effects on the thermal effect. The nonthermal effect, caused by the rapid migration of charged species, also affected the hydrolysis of polysaccharides. Different polysaccharides exhibited different electrical properties, migrated at different direction under IEF, and thus led to different non-thermal effect. Compared with continuous-flow water-bath heating, IEF enhanced the hydrolysis of guar gum and pectin but showed a negative effect on chitosan. After IEFassisted hydrolysis, the granular surface of the samples was destroyed, the chemical structure of the samples was slightly altered, the crystal structure of pectin and guar gum remained unchanged whereas the structural compactness of chitosan was partially lost, and the viscosity of all polysaccharides significantly decreased. Notably, the DE of pectin and DA of chitosan treated by IEF were slightly higher than those treated by continuous-flow water-bath heating, suggesting that IEF might allow for a more selective hydrolysis of polysaccharides. This study explored the mechanism of IEF-assisted acid hydrolysis of polysaccharides, which might provide a theoretical guide for the application of IEF in the chemical modification of biopolymers.
3.3.4. Viscosity Fig. 10 shows typical viscosity vs. shear rate plots of native and hy drolyzed guar gum, chitosan, and pectin solutions upon exposure to continuous-flow water-bath heating at 50 � C and IEF-assisted hydrolysis at excitation voltages of 15 V–75 V. A Newtonian region at low shear rates and a shear thinning region at higher shear rates were observed for both native guar gum and chitosan, whereas the Newtonian behavior of hydrolyzed guar gum and chitosan was extended to the higher values of 9
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Fig. 10. Steady shear viscosity of polysaccharides subjected to acid hydrolysis with IEF treatment and continuous-flow water-bath heating at 50 � C: (a) guar gum, (b) chitosan, and (c) pectin.
Declaration of competing interest The authors declare no conflict of interest.
Fig. 9. XRD patterns of polysaccharides subjected to acid hydrolysis with IEF treatment and continuous-flow water-bath heating at 50 � C: (a) guar gum, (b) chitosan, and (c) pectin.
Acknowledgement This work was financially supported by the Natural Science 10
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Foundation of Jiangsu Province (No. BK20190523 and No. BK20170182), the National Natural Science Foundation of China (No. 31701522) and a Special Fund for Talents’ Introduction Program of Jiangsu Superiority Disciplines (No. 080/80900605).
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