Structural and rheological properties of pectic polysaccharide extracted from Ulmus davidiana esterified by succinic acid

International Journal of Biological Macromolecules 120 (2018) 245–254

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Structural and rheological properties of pectic polysaccharide extracted from Ulmus davidiana esterified by succinic acid Yu-Ra Choi, Yun-Kyung Lee, Yoon Hyuk Chang ⁎ Department of Food and Nutrition, and Bionanocomposite Research Center, Kyung Hee University, Seoul 02447, Republic of Korea

a r t i c l e

i n f o

Article history: Received 29 May 2018 Received in revised form 13 August 2018 Accepted 19 August 2018 Available online 20 August 2018 Keywords: Ulmus davidiana Pectic polysaccharide Esterification Succinic acid Rheological properties

a b s t r a c t The present study was carried out to investigate the physicochemical and structural properties of pectic polysaccharide extracted from Ulmus davidiana (UDP) and to determine the physicochemical, structural, and rheological properties of esterified UDP with succinic acid (ES-UDP). The results indicated that UDP had high amounts of galacturonic acids and various neutral sugars, such as galactose, rhamnose, and glucose. UDP was identified as a low methoxyl pectin, consisting of 1,4-linked α-D-GalpA (the main backbone chain), supported by the results of Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction, and 1D Nuclear magnetic resonance (NMR) spectroscopy. In the FT-IR and XRD, no difference was detected between UPD and ES-UDPs. However, 1 H and 13C NMR spectra revealed that the new ester bonds were formed between a hydroxyl group of UDP and a carboxyl group of succinic acid during esterification. In the steady shear rheological analysis, the consistency index (K) of ES-UDP was significantly higher than that of UDP and increased significantly with increasing concentration of succinic acid. In the dynamic rheological analysis, the tan δ values of all ES-UDP solutions were significantly lower than those of the UDP solution. © 2018 Published by Elsevier B.V.

1. Introduction Chemical modification is the most commonly investigated modification method due to the non-destructive nature of several selected processes and the potential enhancement in the polymer functionality [1]. Modification can enable enhancement or introduction of key properties to the polysaccharides. The number of reactive sites for chemical modification increases with the number of hydroxyl groups in polymers [2]. There are various methods of chemical modification of polymers, but the most important methods are cross-linking, esterification, and etherification [3]. Such modifications of polysaccharides can enhance pasting, gelatinization, swelling, and solubility properties [4]. Esterification is one of the most common modification techniques that can be used to improve viscosity and to enhance the resistance to machinability, heating, shearing, and acid exposure. In general, polymers can form esters either through intra- or intermolecular linkage during esterification [5]. Polymer esters are esterified by various reactants such as acid anhydrides, octenyl succinic anhydride (OSA), dodecenyl succinic anhydride (DDSA) fatty acids, and polycarboxylic acid [6,7]. Esterification with OSA is a commonly applied method of polymer esterification [8]. Modification with polycarboxylic acids such as citric, adipic, and glutaric acid have also been used widely for

⁎ Corresponding author. E-mail address: [email protected] (Y.H. Chang).

https://doi.org/10.1016/j.ijbiomac.2018.08.094 0141-8130/© 2018 Published by Elsevier B.V.

polymers [6]. However, esterification using succinic acid has not been studied so far. Succinic acid has been used as a food additive, and as such is listed as a “Generally Recognized as Safe” food additive by the Food and Drug Administration (FDA). Succinic acid is a dicarboxylic acid and thus may promote esterification owing to the two carboxyl groups that can react with different polysaccharide chains. Thus, we hypothesized that succinic acid may play an important role for steady and dynamic shear rheological properties of polysaccharides owing to greater network development. The formation of adhesive and friction forces between succinic acid and polysaccharide surfaces were predicted, based on new intermolecular and/or intramolecular ester bridges between a hydroxyl group of polysaccharide and a carbxoylic group of the succinic acid after esterification. The physicochemical and rheological properties of polysaccharides esterified with succinic acid have not been reported so far. Ulmus davidiana (Ulmaceae) is naturally growing in North-East Asia such as Korea, Japan, and China. Especially, root barks of Ulmus davidiana have been used in traditional medicine to prevent inflammation, cancer, edema, and other ailments [9,10]. According to Lee et al. [11], water extract from Ulmus pumila L. root bark contains pectic polysaccharides which consist of various monosaccharides such as galacturonic acid, rhamnose, galactose, and glucose. Previous studies mostly focused on extraction, antioxidant, and anti-inflammatory activities of pectic polysaccharide extracted from Ulmus davidiana (UDP) [11]. However, no studies have been performed on the rheological properties of UDP. Moreover, the structural and physicochemical properties

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of esterified pectic polysaccharide extracted from Ulmus davidiana (ESUDP) are also unknown. Therefore, the present study aimed to produce ES-UDPs with different concentration of succinic acid usable for emulsifiers, gelling agents, and thickeners in food production. Thus, the objectives of the present study were to produce ES-UDPs using four different concentration of succinic acid (0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis UDP, respectively) and to determine structural properties (1H and 13C NMR, FT-IR, XRD, and SEM), and rheological properties (steady shear, frequency sweep, time sweep, and temperature sweep). 2. Materials and methods

dry solution and 3 mL of methanol was added to remove borohydrate. The procedure was repeated four times. Acetic anhydride (5 mL) was subsequently added to the mixture and incubated for an additional 60 min at 100 °C. The hydrolysate was then converted into the corresponding alditol acetates and analyzed with GC (G3440B, Agilent Technologies, Palo Alto, CA, USA) equipped with a HP-5 column (0.25 mm × 30 m × 0.25 μm, Agilent Technologies, Palo Alto, CA, USA) and a flame-ionization detector. Initial column temperature was held at 140 °C for 5 min, increased to 240 °C at 4 °C/min and maintained at 240 °C for 5 min. Nitrogen gas (N2) was used as carrier gas with flow rate of 1.0 mL/min. The neutral monosaccharides were estimated by the standards (rhamnose, arabinose, xylose, glucose, and galactose) purchased from Sigma Aldrich (St. Louis, MO, USA).

2.1. Materials 2.4. Preparation of esterified pectic polysaccharide with succinic acid The root of Ulmus davidiana was obtained from the local market of Kyungdong in Seoul, Korea. Succinic acid and acetic anhydride were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Monosaccharide standards (galactose, glucose, rhamnose, xylose and arabinose) and galacturonic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents were of analytical grade. 2.2. Extraction of pectic polysaccharide from root bark of Ulmus davidiana The extraction procedure of UDP was carried out following the methodology previously reported by Lee et al. [11] with slight modifications. Dried root bark of Ulmus davidiana (60 g) was crushed into small pieces of size (3 cm × 1 cm × 0.5 cm) and was mixed with distilled water (1:20, w/v). During extraction, temperature was maintained at 80 °C for 24 h using shaking water bath (BS-11, Jeio tech Co., Ltd., Daejeon, Korea). After cooling at room temperature, the suspension was centrifuged (Combi 408, Hanil BioMed Inc., Gwangju, Korea) at 3500 rpm for 10 min. To allow pectic polysaccharides precipitation, ethanol (95%) was added to the supernatant (3:1, v/v) and then kept at room temperature for 12 h. It was then separated by filtration using a Whatman No. 1 filter paper (GE Healthcare, Amersham, UK) and freeze dried to obtain pure pectic polysaccharide. 2.3. Characterization of UDP 2.3.1. Chemical composition The yield of UDP was measured as percentage of the dry weight of sample, which was calculated by using the formula: yield of UDP (% dry weight) = (weight of polysaccharide / weight of dried sample) × 100. The chemical composition of UDP including moisture content, ash content, protein content, and fat content were determined according to A.O.A.C. methods [12] by 925.09B, 923.03, 979.09, and 920.39C, respectively. The content of total sugar was determined by the phenol sulfuric acid method described by Dubois et al. [13]. 2.3.2. Monosaccharide composition The content of GalA was measured by the m-hydroxyphenyl colorimetric method described by Blumenkrantz et al. [14] with Dgalacturonic acid as the standard (Sigma-Aldrich Chemical Co., St. Louis, MO, USA). Degree of esterification was determined by titration method, as described by Wai et al. [15] with some modifications. The neutral monosaccharide composition of UDP was analyzed by GC method of Zhu et al. [16] with slightly modification. Briefly, approximately 5 mg of the sample was dissolved in an ampoule containing 3 mL of 4 M trifluoroacetic acid. The mixture was hydrolyzed at 121 °C for 6 h. After hydrolysis, trifluoroacetic acid was evaporated to dryness at 40 °C. Neutral sugar was reduced to alditol using 25 mg of sodium borohydride with 3 mL of distilled water at room temperature for 2 h. Acetic acid was added to solution to decompose excess sodium borohydride until bubble formation stopped. A stream of nitrogen gas was used to

Esterified pectic polysaccharides with succinic acid (ES-UDP) were carried out using the method of Šubarić et al. [17] with some modifications. The mixture of succinic acid and acetic anhydride (1:4, w/w) was prepared by hot-dissolving. 50 g of UDP (dry basis) were introduced to a beaker and soaked with distilled water (1:20, w/v). The slurry was thoroughly mixed and adjusted to pH 9 using 1 M NaOH solution. Under continuous stirring and pH kept at 9, the mixture of succinic acid and acetic anhydride (1.5, 3.0, 4.5 and 6.0 mL per 50 g of dry basis UDP) was drop-wise. After the addition of the succinic acid and acetic anhydride, the pectic polysaccharide suspension was stirred for 1 h at room temperature and then added with 1 M NaOH solution to pH 5.4. The esterified pectic polysaccharides were rinsed with distilled water to remove reagent residues and washed by ethanol aqueous solutions with a concentration of 50% until it was neutral. The washed pectic polysaccharides were air-dried at 50°C for 24 h, ground into powder, and passed through a 150 mesh. The esterified UDP with four different concentrations of succinic acid (0.6, 1.2, 1.8, and 2.4 g per 100 g of dry basis UDP) were denoted by ES-UDP0.6, ES-UDP1.2, ES-UDP1.8, and ES-UDP2.4, respectively. 2.5. Structural properties 2.5.1. Scanning electron microscopy (SEM) SEM was carried out on a scanning electron microscope (S-4700, Hitachi CO., Tokyo, Japan). UDP and ES-UDPs were placed on double side carbon tape attached to a specimen holder and coated with platinum powder. The samples were examined at accelerating voltage of 10 kV and magnification of ×250. 2.5.2. Fourier transform infrared (FT-IR) spectroscopy The FT-IR spectra of UDP and its esterified derivatives were obtained using a Fourier-transform infrared spectrophotometer (Spectrum GX, Perkin Elmer, Massachusetts, USA). Each of the samples were mixed with potassium bromide (KBr), compressed into pellets and analyzed between 500 and 4000 cm−1. 2.5.3. X-ray diffraction (XRD) analysis The XRD patterns of UDP and the ES-UDPs were detected by a X-ray diffractometer (X'Pert PRO MPD, PANalytica, Netherlands) using Cu Kα radiation (λ = 1.5418 Å) in the range (2θ = 10°–80°). The samples were scanned from 10° to 50° diffraction angle (2θ) (step size 0.02° 2θ, time per step: 38.4 s). 2.5.4. 1D nuclear magnetic resonance (NMR) spectroscopy The dried UDP and ES-UDPs were dissolved in deuterium oxide (99.8 atom % D2O, Sigma Chemical Co., USA) at 85°C for 3 h and freeze-dried three times to replace the exchangeable protons with deuterons before finally dissolving in D2O for NMR analysis. The 1H and 13C NMR spectra of UDP and ES-UDPs were detected by a Bruker AMX 600 MHz spectrometer (Bruker BioSpin, Rheinstetten, Germany).

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2.6. Rheological properties 2.6.1. Preparation of pectic polysaccharide for rheological measurement UDP and ES-UDP dispersions (10%, w/w) were prepared by mixing each sample with distilled water. The UDP and ES-UDP dispersions were then stirred at a moderate speed for 30 min at room temperature, and then heated water both at 80°C for 1 h with constant mild agitation provided by a magnetic stirrer in order to avoid sedimentation and agglomeration. At the end of the heating period, the UDP and ES-UDP dispersions were cooled at room temperature and transferred to a rheometer plate to measure their rheological properties. 2.6.2. Steady shear rheological properties Steady shear rheological properties of the pectic polysaccharide samples were conducted with a controlled stress rheometer (MCR102, Anton paar, Graz, Austria), using a plate to plate system (diameter: 5 cm, gap: 0.5 mm). Samples were transferred to the rheometer plate at 20 °C. Steady shear data were obtained over the shear rate in the range of 0.1–1000 s−1. In order to describe the variation in the rheological properties of pectic polysaccharide solutions under steady shear, the data were fitted to the well-known power law (Eq. (1)) model, which is used extensively to describe the flow properties of non-Newtonian liquids in theoretical analysis as well as in practical engineering applications: σ ¼ Kγ_ n

ð1Þ

where σ is the shear stress (Pa), γ_ is the shear rate (s−1), K is the consistency index (Pa·sn), and n is the flow behavior index (dimensionless). The apparent viscosity (ηa,100) at 100 s−1 was calculated using the magnitudes of K and n. 2.6.3. Dynamic shear rheological properties 2.6.3.1. Frequency sweep. Dynamic rheological properties of all samples were conducted with a rheometer (MCR-102, Anton paar, Graz, Australia), using a plate to plate system (diameter: 5 cm, gap: 0.5 mm). Frequency sweep tests were performed the range of 0.63–62.8 rad/s at 1% strain, which was in the linear viscoelastic region. Frequency sweep tests were also conducted at 20°C. A rheoplus data analysis software (32 V3.40.) was used to obtain the experimental data and for calculating the storage modulus (G′), loss modulus (G″), complex viscosity (η*), and tan δ (G″/G′). 2.6.3.2. Time sweep. For time sweep measurements in the aging process, UDP and ES-UDP samples were loaded onto the rheometer plate at 4°C. The exposed sample edge was covered with a thin layer of light paraffin oil to prevent evaporation during measurements. The G′ and G″ values were monitored during aging at 4 °C for 1 h at 6.28 rad/s and 1% strain (within the linear viscoelastic region). 2.6.3.3. Temperature sweep. The temperature sweep measurements were performed at a strain value of 0.01 (1%) (within the linear viscoelastic region), while the frequency was fixed at 6.28 rad/s. To investigate the effect of temperature on the rheological properties of UDP and ES-UDP samples, a program was set up. Samples were transferred to the rheometer plate at 4°C. The exposed sample edge was covered with a thin layer of light paraffin oil to prevent evaporation during measurements. Briefly, the programs included a cooling step with a linear temperature decrease from 90 to 4°C, using cooling rate of 2°C/min. 2.7. Statistical analysis All statistical analyses were performed using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). Analysis of variance (ANOVA) was performed using the general linear models (GLM) procedure to determine

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Table 1 Chemical composition and monosaccharide composition of pectic polysaccharide extracted from Ulmus davidiana (UDP). Chemical composition

UDP

Moisture (%) Ash (%) Protein (%) Fat (%) Total sugar (% w/w) Monosaccharide composition (mol %) Galactose Rhamnose Glucose Galacturonic acid

8.16 ± 0.31 8.53 ± 0.21 3.45 ± 0.15 0.35 ± 0.27 79.30 ± 0.59 26.15 ± 0.39 14.34 ± 0.07 8.77 ± 0.12 44.91 ± 1.95

significant differences among the samples. Means were compared by using Fisher's least significant difference (LSD) procedure. Significance was defined at the 5% level. 3. Results and discussion 3.1. Chemical composition and monosaccharide composition The yield and chemical composition of UDP are summarized in Table 1. The yield of UDP was 9.6% of the total mass. Moisture, ash, protein, and fat contents of UDP were 8.16, 8.53, 3.45, and 0.35%, respectively. The total sugar content (79.3%) of UDP showed that carbohydrates were the main constituent in UDP. Pectins can be grouped into high methoxyl pectin (HMP, DE N 50%) and low-methoxyl pectin (LMP, DE b 50%), based on the degree of esterification (DE) [18]. Therefore, in the present study, UDP was categorized as LMP because its DE value is below 50%. The monosaccharide composition of UDP is presented in Table 1. According to the galacturonic acid (GalA) contents (44.91 mol%) of UDP, it was obvious that UDP mainly consisted of homogalacturonan, which is known to be a linear backbone composed of the smooth region of pectin. Different monosaccharides (galactose, rhamnose, and glucose) were found in UDP in a mole % of 26.15:14.34:8.77, respectively. These findings were in concordance with the previous study, which showed that the major monosaccharide in polysaccharides extracted from bark of Ulmus pumila L. was galacturonic acid, followed by galactose, rhamnose, and glucose [11]. The high amounts of galacturonic acid and rhamnose could indicate the presence of both homogalacturonan and rhamnogalacturonan in UDP. The presence of galactose and glucose in UDP could indicate the existence of side chains, such as galactan and glucan. Furthermore, the considerable amounts of galactose suggest the existence of highly branched galactan in the side chain of UDP. 3.2. Structural properties 3.2.1. Scanning electron microscopy (SEM) The morphology of the UDP granules after esterification was examined by SEM (Fig. 1). There were no pronounced changes between UDP and ES-UDPs. The shape of all samples was spherical and square. The surface of all samples was rough. Hongbo et al. [19] reported that the particle morphology of modified sesbania gum did not differ from that of the native. Accordingly, the results of the present study suggest that the esterified with succinic acid did not affect the morphology of UDP. 3.2.2. Fourier transform infrared (FT-IR) spectroscopy FT-IR spectra can be an effective technique for examining the functional groups in the chemical structures of various materials [20]. To investigate the changes that occurs in UDP after esterification, the FT-IR spectra (500–4000 cm−1) of UDP and ES-UDPs are presented in Fig. 2. The FT-IR spectrum of UDP was not remarkably changed after esterified

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Fig. 1. Scanning electron microphotograph of pectic polysaccharide extracted from Ulmus davidiana (UDP) and esterified pectic polysaccharide with succinic acid (ES-UDP). UDP, ESUDP0.6, ES-UDP1.2, ES-UDP1.8, and ES-UDP2.4 represented esterified pectic polysaccharide with four different concentrations of succinic acid (0, 0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis, respectively).

with succinic acid. Chen et al. [21] and Mudgil et al. [22] also reported that no distinct difference was obtained between the spectra of native pectin and modified pectin. All samples showed a vibrational stretching-OH peak at the region of 3400–3450 cm−1, a stretching vibration aliphatic-CH peak at the region of 2933 cm−1, and a bending vibrational CH3 peak at 1418 cm−1 [23]. Interestingly, two distinct peaks at 1611 and 1720 cm−1 were found in all samples. Monsoor [24] reported that bands between 1600 and 1800 cm−1 are typically associated with different types of pectin. Absorption bands at 1600–1650 and 1700–1750 cm−1 are kbown to indicate non-esterified and methyl esterified carboxyl groups, respectively [25]. Chen et al. [21] also reported that peaks at 1616–1634 and 1745–1750 cm−1 in native sugar beet pectin and modified sugar beet pectin were associated with non-esterified and esterified groups, respectively. Thus, it was indicated in the present study that two distinguished bands at 1611 and 1720 cm−1 in all samples can be associated with the non-esterified and methyl esterified carboxyl groups, respectively.

3.2.3. X-ray diffraction (XRD) analysis X-ray diffraction (XRD) was performed to determine where ester linkages occurred in UDP after esterification. The XRD patterns of UDP and ESUDPs are shown in Fig. 3. The XRD patterns revealed that UDP and ESUDPs were entirely amorphous and had unique diffraction peaks at 17° (2θ). The XRD patterns of all ES-UDPs were not considerably different from that of UDP. This result observed that succinic acid had no significant effect on the UDP during the process of esterification, as esterification occurred primarily in the amorphous regions [26]. Moreover, Cui et al. [27] reported that modification did not occur in the crystalline region of pectin nanofibers. Therefore, it was indicated in the present study that esterified with succinic acid occurred mainly in the amorphous region and did not change the crystalline region of UDP.

3.2.4. 1H NMR spectroscopy Signals of 1H NMR spectra were assigned as completely as possible, based on monosaccharide analysis, linkage analysis, and chemical shifts,

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Fig. 2. Fourier transform-infrared (FT-IR) spectra of pectic polysaccharide extracted from Ulmus davidiana (UDP) and esterified pectic polysaccharide with succinic acid (ES-UDP). UDP, ESUDP0.6, ES-UDP1.2, ES-UDP1.8, and ES-UDP2.4 represented esterified pectic polysaccharide with four different concentrations of succinic acid (0, 0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis, respectively).

as reported in literature [11]. The 1H NMR spectra of UDP and ES-UDPs are presented in Fig. 4. In the anomeric region, the signals of all samples at around 5.21, 5.06 and 4.96 ppm were assigned to H-1 of 2,4-linked αL-Rhap, methyl esterified and non-esterified 4-linked α-D-GalpA, respectively [28,29]. Two signals at 3.78 and 4.10 ppm were associated with H-2 and H-3 of both non-esterified and methyl esterified 1,4 linked α-D-GalpA [30,31]. The very weak signals at around 2.00 ppm (2.07 and 1.91 ppm) were attributed to acetyl groups binding at O-2 and O-3 of GalpA, respectively [32].

However, 1H NMR spectra of ES-UDPs produced additional peaks at 2.38 and 1.85 ppm. The new peak at 2.38 ppm could be associated with a methylene chain of succinic acid. Namazi et al. [33] found that a new peak at 2.10–2.25 ppm was associated with the methylene group beside the carbonyl group in hydrophobically modified potato and waxy maize starch using long-chain fatty acids. Furthermore, Dulong et al. [34] reported that carboxymethylpullulan cross-linked with a mixture of ferulic acid and adipic acid dihydrazyde produced a new signal at about 2.36 ppm by methylene chain of acid adipic dihydrazyde. The

Fig. 3. XRD patterns of pectic polysaccharide extracted from Ulmus davidiana (UDP) and esterified pectic polysaccharide with succinic acid (ES-UDP). UDP, ES-UDP0.6, ES-UDP1.2, ES-UDP1.8, and ES-UDP2.4 represented esterified pectic polysaccharide with four different concentrations of succinic acid (0, 0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis, respectively).

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1-Rhap (1H), 2-GalpAMe (1H), 3-GalpA (1H), 4-GalA (2H), 5-GalA (3H), 6-COCH3, 7-Rhap-CH3 Fig. 4. 1H NMR spectra of pectic polysaccharide extracted from Ulmus davidiana (UDP) and esterified pectic polysaccharide with succinic acid (ES-UDP). UDP, ES-UDP0.6, ES-UDP1.2, ESUDP1.8, and ES-UDP2.4 represented esterified pectic polysaccharide with four different concentrations of succinic acid (0, 0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis, respectively).

signals of ES-UDPs at around 1.86 ppm could be related to the new ester linkages between hydroxyl groups of UDP and carboxyl groups of succinic acid. Kwak et al. [35] also reported that β-cyclodextrin crosslinked with adipic acid found a new signal at 1.85 ppm which indicated new ester linkages between hydroxyl groups of β-cyclodextrin and carboxyl groups of adipic acid. Thus, our results confirm the successful esterification of UDP with succinic acid, based on the present study, the presence of new peaks (methylene group and ester linkages) in ESUDPs using 1H NMR spectra. 3.2.5. 13C NMR spectroscopy Detailed structural properties of all samples were determined using 13 C NMR spectroscopy experiments [11]. As shown by 13C NMR spectra of all samples (Fig. 5), the signals at 99.50 and 98.33 ppm corresponded to anomeric C-1 of methyl esterified and non-esterified 4-linked α-DGalpA, respectively [36,37]. The rhamnose C-1 signal at 96.68 ppm was assigned to 2,4-linked α-L-Rhap [38]. The signals at 175.19 and 174.33 ppm indicated the C-6 of the carboxyl groups of 4-linked α-DGalpA, and the existence of two carboxyl signals confirmed the presence of nonesterified and methyl esterified 1,4-linked α-D-GalpA [28,31]. For the bands of 75.66 and 69.32 ppm in this study, these were assigned to C-2 and C-4 of β-D-Galp, respectively. The differences between UDP and ES-UDP samples were the presence of signals at around 33.89 and 23.12 ppm in ES-UDP, which were absent in the spectrum for UDP. Kwak et al. [35] observed new peaks (33.89 and 23.12 ppm) of ester linkages after crosslinking of βcyclodextrin with adipic acid, which were not present in native βcyclodextrin, and they concluded that the two peaks were associated with ester linkages between carboxyl groups of adipic acid and hydroxyl groups of β-cyclodextrin. Therefore, in the present study, two new peaks at 33.89 and 23.12 ppm in ES-UDP could suggest the formation

of ester linkages between hydroxyl groups of UDP and carboxyl groups of succinic acid. 3.3. Rheological properties 3.3.1. Steady shear rheological properties The experimental results of shear stress and shear rate data fitted the power law models with high determination coefficients (R2 = 0.99) as shown in Table 2. The apparent viscosity (ηa,100) and consistency index (K) of the ES-UDP solution were higher than those of the UDP solution, and increased significantly with increasing concentrations of succinic acid (Table 2). Similar trends of the ηa,100 and K values were also observed in other modified polysaccharides, such as modified tapioca starch with STMP/STPP [39], modified xanthan gum using poly(maleic anhydride/1-octadecene) [40], esterified rice starch with octenyl succinic anhydride [31], and cross-linked potato starch with adipic acid [41]. Based on the findings obtained from the steady shear rheological properties, it was indicated that the esterified UDP samples can be shear thinning fluids and their steady shear rheological properties can be apparently improved by increasing the concentration of succinic acid (0.6–2.4 g succinic acid per 100 g of dry basis UDP). 3.3.2. Dynamic shear rheological properties 3.3.2.1. Frequency sweep. Generally, G′ (elastic modulus) is a measure of the energy stored in a material or recoverable per cycle of deformation, and G″ (viscous modulus) is a measure of energy lost through viscous dissipation per cycle of deformation. The complex viscosity (η*) is a measure of the overall resistance to flow. Thus, G′ indicates the solidlike properties and elastic contribution, and G″ indicates liquid-like properties for a viscoelastic material [42]. Wongsagonsup et al. [39]

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251

Ester linkage

ES-UDP2.4

ES-UDP1.8

ES-UDP1.2

ES-UDP0.6 6

3 UDP

5

2 4

1

10

7 9

8

1-COOH (6C), 2-GalAMe (1C), 3-GalA (1C), 4-Rhap (1C), 5-Galp (2C), 6-GalA (3C), 7-Galp (4C), 8-COCH3, 9-COCCH3, 10-Rhap-CH3 Fig. 5. 13C NMR spectra of pectic polysaccharide extracted from Ulmus davidiana (UDP) and esterified pectic polysaccharide with succinic acid (ES-UDP). UDP, ES-UDP0.6, ES-UDP1.2, ESUDP1.8, and ES-UDP2.4 represented esterified pectic polysaccharide with four different concentrations of succinic acid (0, 0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis, respectively).

and Yoneya et al. [43] reported that cross-linked tapioca starches prepared with up to 1.0% STMP/STPP concentration and cross-linked potato starches prepared with up to 0.05% POCl3 concentration showed the considerable increase in dynamic values compared with native ones. Thus, it was suggested in the present study that esterification with succinic acid can enhance the viscoelastic properties of UDP. The tan δ values of all samples ranged from 1.25 to 1.56 (Table 3), demonstrating that all samples were more viscous than elastic. The tan δ values of all ES-UDP solutions were significantly lower than those of the UDP solution, and significantly decreased with increasing concentrations of succinic acid. These findings indicated that the viscous properties of the ES-UDP solutions were less pronounced than those of the UDP solution. 3.3.2.2. Time sweep. Changes in G′ and G″ of UDP and ES-UDPs solutions during aging for 1 h at 4 °C are shown in Fig. 6. During aging, G′ and G″ values of ES-UDPs were substantially higher than those of UDP (apart from ES-UDP0.6). For all samples, G′ and G″ values did not remarkably changed during aging for 1 h. This phenomenon seems to confirm that

Table 2 Apparent viscosity (ηa,100), consistency index (K) and flow behavior index (n) of pectic polysaccharide extracted from Ulmus davidiana (UDP) and esterified pectic polysaccharide with succinic acid (ES-UDP) at 20°C1. Sample

UDP ES-UDP0.62 ES-UDP1.2 ES-UDP1.8 ES-UDP2.4 1

Apparent viscosity

Consistency index n

Flow behavior index

ηa,100 (Pa·s)

K (Pa·s )

n (−)

3.53 ± 0.04e 3.66 ± 0.07d 3.98 ± 0.04c 4.71 ± 0.07b 5.30 ± 0.01a

56.75 ± 2.05d 58.68 ± 2.28d 65.03 ± 2.51c 89.82 ± 1.46b 110.86 ± 0.41a

0.40 ± 0.00a 0.40 ± 0.00a 0.39 ± 0.01a 0.36 ± 0.00b 0.34 ± 0.00c

R2

0.99 0.99 0.99 0.99 0.99

Values with different letters within the same column differ significantly (p b 0.05). UDP, ES-UDP0.6, ES-UDP1.2, ES-UDP1.8, and ES-UDP2.4 represented esterified pectic polysaccharide with four different concentrations of succinic acid (0, 0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis, respectively). 2

solutions of ES-UDPs as well as UDP solution were stable during storage at refrigeration temperature (4 °C). For UDP and ES-UDP0.6 solution, G″ values were higher than G′ values during all the aging time, indicating that viscous properties predominated over the elastic component G′. However, ES-UDP1.2, ES-UDP1.8, and ES-UDP2.4 solutions had elastic properties rather than viscous properties because G′ values were higher than G″ values during all the aging time, representing a progressive reinforcement of the polymer networks [44]. 3.3.2.3. Temperature sweep. The temperature dependence of G′ and G″ during cooling from 90 to 4°C for UDP and ES-UDP solutions is shown in Fig. 7. During the initial cooling phase, G′ and G″ values increased as temperature decreased, reaching a maximum value at 4°C. The increase of G′ can be related to the decrease in fluidity with decreasing temperature. Alternatively, it may also be caused by energy dissipation owing to molecular movements and increased intermolecular interactions [45]. Li et al. [46] reported that the G′ and G″ values of modified potato starch increased with decreasing temperature because of the strengthened molecular mobility. The temperature at which the curves of G′ and G″ crossover of, also called sol-gel transition temperature, is defined as the gelling point [47]. Table 3 Storage modulus (G′), loss modulus (G″), complex viscosity (η*), and tan δ at 6.28 rad/s of pectic polysaccharide extracted from Ulmus davidiana (UDP) and esterified pectic polysaccharide with succinic acid (ES-UDP) at 20 °C1. Sample

G′ (Pa)

G″ (Pa)

η* (Pa·s)

Tan δ

UDP ES-UDP0.62 ES-UDP1.2 ES-UDP1.8 ES-UDP2.4

26.43 ± 0.68e 29.85 ± 0.57d 34.82 ± 0.12c 49.14 ± 0.39b 52.26 ± 0.91a

41.12 ± 1.02e 44.03 ± 1.23d 46.75 ± 0.13c 62.57 ± 0.39b 65.34 ± 1.20a

7.76 ± 0.20e 8.44 ± 0.11d 9.25 ± 0.03c 12.63 ± 0.09b 13.28 ± 0.24a

1.56 ± 0.00a 1.48 ± 0.07b 1.34 ± 0.00c 1.27 ± 0.00d 1.25 ± 0.00e

1

Values with different letters within the same column differ significantly (p b 0.05). UDP, ES-UDP0.6, ES-UDP1.2, ES-UDP1.8, and ES-UDP2.4 represented esterified pectic polysaccharide with four different concentrations of succinic acid (0, 0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis, respectively). 2

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Fig. 6. Changes in storage (G′) and loss (G″) modulus of pectic polysaccharide extracted from Ulmus davidiana (UDP) and esterified pectic polysaccharide with succinic acid (ES-UDP) at 4°C during 1 h. UDP, ES-UDP0.6, ES-UDP1.2, ES-UDP1.8, and ES-UDP2.4 represented esterified pectic polysaccharide with four different concentrations of succinic acid (0, 0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis, respectively).

In the present study, the ES-UDP0.6 solution showed no cross-over point for G′ and G″, and G″ was greater than G′, indicating that the polysaccharide dispersion was in a more viscous-like state. Whereas, there was a cross-over point for ES-UDP solutions with concentration of 1.2, 1.8 and 2.4 g of succinic acid and the temperature was about 6, 8, and 10°C, respectively. Based on the results of structural, steady shear rheological and dynamic shear rheological properties, two mechanisms could account for the enhancement in rheological characteristics of esterified UDP samples. Increased values of steady shear rheological and dynamic shear rheological properties for ES-UDP samples might be explained by the introduction of new intermolecular and/or intramolecular esterified bridges between hydroxyl groups of UDP and carboxylic acid of succinic acid [48]. Seo et al. [49] reported that the steady shear (ηa,100 and K values) rheological and dynamic shear rheological (G′, G″, and η* values) properties increased with the increasing concentrations of modifying agents owing to greater network development, which is known to be facilitated by strong interactions between closed-packed molecules in

polysaccharide solutions. As shown previously in 1D NMR spectra (Figs. 4 and 5), the presence of new peaks in ES-UDP can explain the formation of ester linkages between hydroxyl groups of UDP and carboxyl groups of succinic acid during esterification of UDP with succinic acid. The esterification reagents can provide new bonds at random locations of UDP, which can then stabilize and strengthen the structure of ESUDP, thus leading to not only improved rheological properties, but also less sensitive processing conditions by shear and temperature than native one [5]. Acquarone and Rao [50] also reported that modification was intentionally directed to random locations in the polymer granule, which can stabilize and strengthen the granule, leading to significantly increased rheological properties. Moreover, it was suggested in the present study during the shearing, ES-UDP had greater resistant to deformation than UDP. In general, it is well-known that viscosity is the integral of all frictional resistances to flow and deformation. Thus, ES-UDP with a greater frictional resistance had higher steady and dynamic shear rheological properties. Therefore, it was suggested that the rheological properties were improved with increasing

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Fig. 7. Changes in storage (G′) and loss (G″) modulus during initial cooling from 90 to 4°C at a rate of 2 °C/min for pectic polysaccharide extracted from Ulmus davidiana (UDP) and esterified pectic polysaccharide with succinic acid (ES-UDP). UDP, ES-UDP0.6, ES-UDP1.2, ES-UDP1.8, and ES-UDP2.4 represented esterified pectic polysaccharide with four different concentrations of succinic acid (0, 0.6, 1.2, 1.8, and 2.4 g succinic acid per 100 g of dry basis, respectively).

concentrations of succinic acid owing to alterations of the structural properties of UDP by intermolecular and/or intramolecular esterified bridges. Moreover, ES-UDP can contribute to the functionally of food products and applied as a potential and alternative of thickener in food industry. 4. Conclusion The present study showed that esterified with succinic acid influenced the structural and rheological properties of UDP. In the structural properties, FT-IR spectra and XRD showed no differences between UDP and ES-UDPs. However, 1H and 13C NMR spectra revealed that the new ester bond between a hydroxyl group of UDP and a carbxoylic group of succinic acid can be formed during esterification. ES-UDP samples had higher shear-thinning behavior and their steady and dynamic shear rheological properties were improved by an increase in the concentration of succinic acid. It was suggested that the esterification with succinic acid can improve rheological properties of UDP, and the concentration of succinic acid can affect the structural properties of UDP due to intermolecular and/or intramolecular esterified bridges. Thus,

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