Dodecenylsuccinic Anhydride Derivatives of Gum Karaya - American

Mar 23, 2015 - Institute for Nanomaterials, Advanced Technologies and Innovation, Technical University of Liberec, Studentská 1402/2, 461 17 ... Natu...
9 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/JAFC

Dodecenylsuccinic Anhydride Derivatives of Gum Karaya (Sterculia urens): Preparation, Characterization, and Their Antibacterial Properties Vinod Vellora Thekkae Padil,*,† Chandra Senan,§ and Miroslav Č erník† †

Downloaded via UNIV OF SUSSEX on July 24, 2018 at 08:15:59 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Institute for Nanomaterials, Advanced Technologies and Innovation, Technical University of Liberec, Studentská 1402/2, 461 17 Liberec 1, Czech Republic § Centre for Water-Soluble Polymers, Applied Science, Engineering and Computing, Glyndwr University, Wrexham LL11 2AW, Wales, United Kingdom ABSTRACT: Esterifications of the tree-based gum, gum karaya (GK), using dodecenylsuccinic anhydride (DDSA) were carried out in aqueous solutions. GK was deacetylated using alkali treatment to obtain deacetylated gum karaya (DGK). The DGK and its DDSA derivative were characterized using gel permeation chromatography/multiangle laser light scattering (GPC/MALLS), attenuated total reflectance−Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), proton nuclear magnetic resonance spectroscopy (1H NMR), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) analysis, and rheological studies. The degree of substitution was found to be 10.25% for DGK using 1H NMR spectroscopy. The critical aggregation concentration of DDSA-DGK was determined using dye solubilization and surface tension methods. The antibacterial activity of the DDSA-DGK derivative was then investigated against Gram-negative Escherichia coli and Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus. The DDSA-DGK derivative has the potential for use as a stabilizing agent in food and nonfood applications. It can also be developed as an antibacterial agent. KEYWORDS: tree polysaccharides, deacetylated gum karaya, DDSA (dodecenylsuccinic anhydride) derivative, thermal study, rheological property, antibacterial activity

■

INTRODUCTION Exudate gums are hydrocolloids with complex molecular structures that are hydrophilic in nature and are extracted from trees. They have been used in the food, pharmaceutical, adhesive, and textile industries both to stabilize emulsions and to enhance thickening for hundreds of years. The important tree-based polysaccharides available in the markets are gum arabic (GA), gum karaya (GK), gum tragacanth (GT), guar gum (GG), and kondagogu gum (KG). Extensive research has been carried out on various aspects of these tree gum polysaccharides, which include their availability, molecular weight distribution, chemical structures, and food and nonfood applications.1−4 The physicochemical, structural, and rheological properties, coupled with the occurrence and production of food and nonfood applications of GK (Sterculia urens), have been widely studied by various research groups.5−10 GK is a partially acetylated polysaccharide that has a branched structure and high molecular mass of ∼16 × 106 Da.6 It is categorized under “substituted rhamno-galacturonoglycans” (pectic) type tree gums.6 This gum contains approximately 60% neutral sugars (viz., rhamnose and galactose) and 40% acidic sugars (viz., glucuronic acid and galacturonic acids) as well as 8% acetyl groups.11,12 GK is a good emulsification agent, a consequence of its acid stability, high viscosity, and ability to form suspensions and bind water. The importance of polymeric surfactants based on natural hydrocolloids is increasing due to their nontoxicity, biodegradability, availability, and superior physicochemical properties. © 2015 American Chemical Society

Long-chain alkyl and alkenyl dicarboxylic acid anhydrides, namely, octenylsuccinic anhydrdide (OSA) and dodecenylsuccinic anhydride (DDSA), have recently been used for synthesizing derivatives of starch, inulin, and konjac glucomannan.13−16 Furthermore, these OSA and DDSA derivatives of natural polysaccharides have displayed superior abilities in promoting the stabilization, encapsulation, interfacial, thermal, emulsification, and rheological properties.17,18 Natural hydrocolloid gums obtained from trees such as GA, GG, KG, and GK have been used as food additives and pharmaceutical ingredients for centuries. However, little work has been performed on these gums in relation to the synthesis of their DDSA or OSA derivatives. Neither has evaluation of their potential in food and other industrial applications (as polymeric surfactants) or their antibacterial effectiveness been carried out. In very recent times, GA has been chemically modified using DDSA, and it was established that the derivative of this gum possessed enhanced emulsification properties compared to the native form of the gum.19 In the present investigation, the molecular weight distribution of DGK was studied using gel permeation chromatography/multiangle laser light scattering (GPC/MALLS). Additionally, we synthesized the DDSA derivative of DGK in aqueous media and subsequently evaluated its properties using Received: Revised: Accepted: Published: 3757

November 28, 2014 March 9, 2015 March 23, 2015 March 23, 2015 DOI: 10.1021/jf505783e J. Agric. Food Chem. 2015, 63, 3757−3765

Article

Journal of Agricultural and Food Chemistry

Synthesis of DDSA Derivative of DGK. DGK (15 g, lyophilized dry powder) was suspended in deionized water (100 mL) with gentle agitation and placed on a roller overnight (12 h). The pH of the solution was adjusted to 8.5 by adding 5% NaOH. A weighed quantity of DDSA (5 g) dissolved in absolute ethanol (30 mL) was slowly added to the gum solution. All reactions were performed in 500 mL, three-necked round-bottom flasks placed in a water bath to maintain a temperature of 25 °C. A propeller mixer (Heidolph type: STI) with a PTFE centrifugal stirrer shaft (6 mm diameter, 400 mm length) was used for continuous mixing of the solutions. The pH of the reaction mixtures was adjusted using 5% NaOH and a peristaltic pump. The reaction was continued for 7−8 h until the pH became stable at 8.5. The resultant product was neutralized with 5% HCl to pH 6.0 before being freeze-dried, yielding a white powder. The final product was purified by Soxhlet extraction for 6 h using cyclohexane as solvent. Finally, the DDSA-DGK was dried in an oven overnight at 70 °C. SEM Analysis. The composition and morphology of the native GK, DGK, and DDSA-DGK were studied using a scanning electron microscope with a beam current of 12−40 nA and an acceleration voltage of 0.02−30 kV. The complete detection system is composed of an In-lens energy and angle selective backscatter detector (EsB), fourquadrant solid-state backscattered detector (AsB), and conventional secondary electron detector (Everhardt-Thornley) (Carl Zeiss, Ultra/ Plus, Munich, Germany). Specimens were permanently mounted onto stubs using slow-drying Araldite or silver dag. The stubs with the specimens were then sputter-coated with a thin layer of gold to make the specimens conductive under high-vacuum conditions. The processed specimens were then subjected to SEM analysis. ATR-FTIR Spectrometry. ATR-FTIR (Nicolet IZ10, Thermo Scientific, USA) was used to characterize the functional groups of GK, DGK, and DDSA-DGK. The spectrometer was equipped with a multireflection, variable angle, and horizontal ATR accessory. Determination of the Degree of Substitution (DS). The DS of DDSA-DGK was determined by titration and 1H NMR methods. The titration procedure adopted for the determination of the DS for DDSA-DGK was as reported for gum arabic and starch.13,19 In brief, 1 g of DDSA-DGK was accurately weighed into a 50 mL beaker, after which 20 mL of deionized water was added. The resulting solution was magnetically stirred until complete dissolution was achieved. Subsequently, 0.05 M NaOH was added slowly until the solution pH reached 9.0. The resulting solution was then titrated against 0.05 M HCl. A control reaction was also performed with unmodified DGK. All of the experiments were carried out in triplicate, and average values were noted. The DS was determined using the equation

a variety of techniques. The morphologies of the native forms of gums were compared with their DDSA derivatives using SEM analysis. DGK and its DDSA derivatives were characterized using attenuated total reflectance−Fourier transform infrared spectroscopy (ATR-FTIR), 1H NMR, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) analysis, and rheological studies. The critical aggregation concentration (CAC) was determined by means of dye solubilization and surface tension. We tested the antibacterial properties of DDSA-DGK using Gram-positive and -negative bacteria and believe this to be the first instance of such experimentation.

■

MATERIALS AND METHODS

Materials. The GK sample was procured from Girijan Co-operative Corp. (GCC), Hyderabad, India. (2-Dodecen-1-yl)succinic anhydride (DDSA), sodium nitrate, sodium iodide, hydrochloric acid, cyclohexane, and absolute ethanol were obtained from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Sudan IV (dye content 80%) was obtained from Eastman Kodak Co. (Rochester, NY, USA). Deionized water was used for all analyses. Preparation of DGK. The GK was powdered in a high-speed mechanical blender and later sieved using a bin (mesh size = 250 μm), so as to obtain fine and uniform samples. One gram portions of GK powder were accurately weighed and dispensed into clean glass beakers containing 1 L of deionized water. The gum solutions were placed on magnetic stirrers at room temperature and gently stirred overnight, after which they were allowed to stand at room temperature for 12 h to separate out any undissolved matter. The resulting gum solutions were subsequently centrifuged to obtain clear solutions. Three volumes of each of the gum solutions was deacetylated by mixing with 1 volume of 1 M NaOH. After incubation for 6 h at room temperature (with gentle agitation on a magnetic stirrer), 1 volume of 1 M HCl was added to neutralize the solution to a final pH of 7.0. The resulting DGK solution was dialyzed (dialysis tubing DTV 12000.09.000; Mw range = 12−14 kDa, Medicell International Ltd., London, UK) extensively against deionized water to remove residual salts. The centrifuged DGK solution was dialyzed against deionized water. After dialysis, the gum solution was centrifuged to remove any residual precipitates, and the clear solutions so obtained were freezedried and stored until further use. Deacetylation was monitored by FTIR analysis.4 Determination of Molecular Mass Distributions of DGK. The molecular mass distributions of DGK were determined using the technique of gel permeation chromatography (GPC) linked to multiangle laser light scattering (MALLS). Sodium nitrate (0.1 M) containing 0.005% sodium azide (biocide) was utilized as the eluent, and the solution was filtered using a GSWP 0.45 μm nylon filter (Millipore) and degassed by means of a vacuum degasser (CS 615; Cambridge Scientific Instruments, UK) before use. The samples of DGK (0.2 wt %) were prepared in 0.1 M NaNO3 solution and left overnight on a roller to achieve complete dissolution. The GPC system consisted of a Suprema 3000 column with these specifications: dimensions, 300 mm × 8 mm; bead size, 10 μm; and pore size, 100 Å. The column was protected by a guard column (Polymer Standards Service GmbH, Mainz, Germany). The flow rate was set to 0.5 mL/ min using a Waters Corp. HPLC pump in conjunction with a Rheodyne 7125 model injection system (loop volume = 200 μL). A Dawn DSP Laser Photometer and OPTILAB DSP Interferometric Refractometer (Wyatt Technology Corp., Santa Barbara, CA, USA) were used as detectors. The DGK samples were filtered through 0.45 μm nylon syringe filters before being injected into the HPLC column. All measurements were performed in triplicate. Molecular mass distributions and rms radius moments of the DGK were determined using the designated Astra software for Windows (4.90.08, QELSS 2. XX). Data were fitted using a first-order polynomial and the Zimm model. The refractive index increment (dn/dc) value for DGK was determined to be 0.140 mL/g.20

DS =

MDDSA MV × 100% (W − MDDSA )MV

(1)

where V is the volume of the titer, M is the molarity of the HCl solution, MDDSA is the molecular weight of the DDSA (266 g/mol), and W is the weight of the DDSA-DGK sample. 1 H NMR Spectroscopy. The samples (DGK and DDSA-DGK, 5 mg each) were dissolved in D2O or DMSO-d6 and placed in NMR tubes. The 1H NMR spectra were measured using a 500 MHz spectrometer (Bruker DRX-500, Rheinstetten, Germany) at 70 °C. The DS was determined by comparing the intensity (I) of the peak at δ 0.89 due to the −CH3 group from the terminal end of the alkenyl chains with that of anomeric protons of the (1→4)-α-D-GalA-(1→2)α-L-Rhap units of DGK from the 1H NMR spectra by using the following equation: DS =

[1/3 × I(DDSA,methyl group)] [I(1 → 4)‐α‐D‐GalA − (1 → 2)‐α‐L‐Rhap,anomeric protons plus reducing end)]

× 100%

(2) The present study was compared to the determination of DS using H NMR of DDSA and OSA derivatives of various polysaccharides such as inulin,15 konjac glucomannan,16 hyaluronic acid,21,22 and starch.13,23,24 DSC. DSC experiments on DGK and DDSA-DGK were carried out using a Pyris Diamond S6 DSC (PerkinElmer, Boston, MA, USA). 1

3758

DOI: 10.1021/jf505783e J. Agric. Food Chem. 2015, 63, 3757−3765

Article

Journal of Agricultural and Food Chemistry The sample was heated from 30 to 400 °C with temperature increments of 10 °C in a nitrogen atmosphere, at a flow rate of 20 mL/ min. Each sample (5 mg) was crimped in an aluminum DSC pan. TG Analysis. The thermal stability and composition of the DGK and its DDSA derivative were determined using a Mettler Toledo apparatus (TGA/SDTA851, Columbus, OH, USA). The experimental atmosphere was nitrogen at a flow rate of 20 mL/min. The sample was heated from 30 to 800 °C. Critical Aggregation Concentration (CAC). The CAC was determined using dye solubilization and surface tension techniques. Dye Solubilization. CAC was determined by the dye solubilization technique using Sudan IV. A total of 10 mg of dye was added to 10 mL of the DGK and DDSA-DGK, at various concentrations in deionized water. The samples were mixed overnight at room temperature and filtered through Mille-GP 0.22 μm filters (Millipore Ireland Ltd.) into disposable, UV-grade, 10 mm path length cuvettes (CXA-110-0053, Fisher Scientific Ltd., Loughborough, UK). The absorbances of the solutions were measured at 510 nm using a Lambda 25 UV−vis spectrometer (PerkinElmer). All of the measurements were carried out in triplicate. The CAC was determined from the increase in absorbance. A blank solution of DGK was used as the control. Surface Tension Measurements. The Du Noüy ring method was used to determine the surface tension of solutions of DGK and the DDSA-DGK derivative at different concentrations using a Kruss K8 surface tensiometer (Krüss GmbH, Hamburg, Germany). Measurements were carried out in triplicate, and the values were reported as means ± SD. The CAC was determined from the inflection in the plot of the surface tension versus concentration. Rheological Study. Rheological measurements of both aqueous DGK and DDSA-DGK were performed using a rheometer (Physica MCR 51, Anton Paar GmbH, Graz, Austria). The solutions were characterized for their steady shear viscosity function, η(r), using a unidirectional steady-shear flow, with shear rate ranging from 1 to 700 s−1 at 25 °C. Antibacterial Activity Tests. Bacterial Strains and Culture Media. The bacterial strains of Gram-negative Escherichia coli (CCM 3954) and Pseudomonas aeruginosa (CCM 3955) and Gram-positive Staphylococcus aureus (CCM 3953) used in this study were obtained from the Czech Collection of Microorganisms, Masaryk University Brno, Czech Republic. Bacterial suspensions were always prepared fresh by growing a single colony overnight at 37 °C in a nutrient broth. All agar plates were freshly prepared before antibacterial testing. A sterilized cotton swab was dipped into the culture suspension, and the cells were spread homogeneously over the agar plates. These plates were immediately used for antibacterial activity tests. Determining the Zone of Inhibition (ZOI). We determined the antibacterial activity of the DDSA derivative of DGK. The DDSADGK solutions (1, 2, 3, 4, and 10% each of DDSA-DGK) were pipetted onto sterilized membrane filters and placed on inoculated agar plates. In addition, 6 mm diameter circles of filter paper were also placed directly onto the inoculated agar plates. Similarly, samples of DGK solution (10%) without DDSA were used as controls. The samples and inoculated agar plates were then incubated for 24 h at 37 °C. The ZOI was determined as being the total diameter (mm) of DDSA-DGK filter paper sample plus the halo-zone where bacterial growth was inhibited. All measurements were performed in triplicate for the DDSA-DGK solutions, and average values were reported. Statistical Analysis. All of the tests were performed in triplicate. Analysis of variance (ANOVA) was performed, and all experimental values were reported as means ± SD (n = 3). A Duncan test was conducted to examine significant differences among experimental mean values (P < 0.05).

■

Table 1. The data were obtained using GPC/MALLS and fitted with first-order polynomials using the Zimm method. The Table 1. Estimated Molar Mass Distributions and Average Mean Square Radius Moments of DGK Using GPC/MALLS and Data Fitting by First-Order Polynomials: Zimm Methoda Mn (g/mol)b

Mw (g/mol)c

Mz (g/mol)d

Rn (nm)e

Rw (nm)f

Rz (nm)g

1.537 × 106

1.827 × 106

2.062 × 106

87.0

91.8

95.3

Eluent, 0.1 M NaNO3; flow rate, 0.5 mL/min.; dn/dc, DGK, 0.140 mL/g. bMn, number-average molecular mass. cMw, weight-average molecular mass. dMz, z-average molecular mass. eRn, number−average mean square radius. fRw, weight-average mean square radius. gRz, zaverage mean square radius. a

elution profiles of DGK juxtaposed against their corresponding molecular weight distributions (derived using GPC in conjunction with light scattering and refractive index detectors) are also shown in Figure 1. For DGK, the weight-average molecular weight was ascertained to be 1.827 × 106. The weight-average molecular mass of DGK was found to be higher compared to other tree gums reported in the literature.20,25 Preparation and Characterization of DDSA Derivative of DGK. Esterification of DGK. The scheme for esterification of DGK with DDSA is presented in Figure 2. The major parameters that affected the formation of DDSA derivatives of the DGK were the concentration ratio of DDSA and DGK, pH, concentration of NaOH solution, reaction time, and temperature.13−15 In the present study, the DDSA-DGK ratio was maintained at 3:1; the pH of the reaction mixture was kept between 8 and 9 using 3% NaOH; and the temperature was held between 25 and 30 °C for 8 h. Confirmation of Esterification by ATR-FTIR. ATR-FTIR spectra of GK, DGK, and the DDSA-DGK are presented in Figure 3. Spectra of GK and DGK displayed peaks at 3300 cm−1 (relating to the OH group) and also peaks in the fingerprint region (corresponding to −C−O−C− stretching vibrations of various sugar moieties present in the gums) at 1159, 1082, and 1014 cm−1, respectively. The band at 2923 cm−1 represents the characteristic vibration due to C−H stretching. The DDSA-DGK spectrum indicates that the peaks at 1726 cm−1 represent the −COOR group. The latter group is absent in the spectra of DGK and is the result of ester bond formation between the −COOH groups of DDSA and the −OH groups of the DGK in the derivatization process (Figure 3c). Compared with DGK spectra, the new absorption bands at 1726 and 1572 cm−1 appear after DDSA esterification of DGK corresponding to the CO stretching vibration of an ester group and the asymmetric stretching vibration of a carbonyl group, respectively (Figure 3c). Furthermore, the predominant band at 2923 cm−1 representing aliphatic C−H stretching vibrations suggested the formation of DDSA-DGK (Figure 3c). The prominent peak observed in the DDSA derivative of DGK was a reflection of the high degree of substitution. Similar types of results were reported in the literature when corn starch,13 inulin,15 and gum arabic19 were derivatized with DDSA to form DDSA derivatives of these polysaccharides. SEM Analysis of GK, DGK, and DDSA-DGK. SEM images relating to the surface morphology of GK, DGK, and the DDSA-DGK derivative are shown in Figure 4. Irregular particles were observed on the surface of GK (Figure 4A),

RESULTS AND DISCUSSION

Molecular Mass Distribution of DGK. The estimated molar mass distributions (weight-average, Mw; number-average, Mn and z-average, Mz) and average mean square radius moments (Rn, Rw, and Rz, all in nm) of DGK are presented in 3759

DOI: 10.1021/jf505783e J. Agric. Food Chem. 2015, 63, 3757−3765

Article

Journal of Agricultural and Food Chemistry

Figure 1. Refractive index and weight-average molecular mass GPC elution profiles of DGK.

Figure 2. Schematic representation of reaction between DDSA and DGK and formation of the DDSA-DGK derivative.

and flake-like structures were displayed for DGK due to deacetylation of GK (Figure 4B). Significant changes, namely, highly porous structures and cavities, were observed after DDSA derivatization of DGK (Figure 4C). SEM analysis showed that the DDSA treatment caused some changes in the structure of the DGK. The SEM images of DDSA-DGK (Figure 4C) exhibited slightly rough surfaces with greater porosity, and many holes were observed, probably due to the DDSA treatment occurring on the surface of the DGK. This indicated that the esterification reaction had taken place. Similar types of morphological analysis have been reported for octenylsuccinic anhydride modified early “Indica” rice starch.26 Determination of the DS Using Titration and 1H NMR Spectroscopy Methods. The DS of DDSA-DGK was determined to be 10.29% by means of titration using eq 1.

1

H NMR spectra of DGK and DDSA-DGK are presented in Figure 5. The DS can be calculated from the intensity of DDSA groups’ signals as a ratio of the DGK anomeric protons, which are used as an internal reference. The proton signals at δ 3.2− 3.7 were very broad and complex. They were attributed to protons of various sugar moieties present in the DGK.4,27 Compared to the DGK, the DDSA-DGK derivative had several additional signals (Figure 5). It was seen that the peak at δ 0.89 in the 1H NMR spectrum corresponded to the methyl protons of the DDSA group.13,15 The peaks from δ 1.28 to 2.62 were due to the methylene and methenyl groups of the DDSA.13,15 There was a multiplex at δ 5.54 and a shoulder with multiple peaks around at δ 5.4. These peaks were ascribed to protons of the double bond in the DDSA group. Signals from δ 4.5 to 5.2 observed in DGK were assigned to the anomeric proton.27 3760

DOI: 10.1021/jf505783e J. Agric. Food Chem. 2015, 63, 3757−3765

Article

Journal of Agricultural and Food Chemistry

Figure 3. ATR-FTIR spectra of (a) GK, (b) DGK, and (c) DDSADGK.

Figure 4. SEM micrographs of the surfaces of (A) GK, (B) DGK, and (C) DDSA-DGK, indicating the morphological changes during esterification.

After DDSA modification, the additional signals appeared in the δ 0.89−3.00 region of the DDSA-DGK spectrum. 1H NMR spectroscopy was utilized for the determination of the DS of the OSA/DDSA anhydrides to many polysaccharides such as inulin,15 konjac glucomannan,16 hyaluronic acid,21,22 and starch,13,23,24 and the present study is in agreement with these reported methods in the literature. It was not possible to determine which hydroxyl moieties the DDSA had reacted with or how evenly distributed the modification had been along the polymer chain. According to earlier studies on starches, both primary and secondary hydroxyl groups of the glucose repeating unit had been observed to react with the OSA/DDSA. This was also the case for other starches, hyaluronic acid, and most other poly-

saccharides, as reported in the literature.21−24 In the DGK molecule, there are three hydroxyl groups available for chemical modification. They are located at the C-2, C-3, and C-6 positions. Hydroxyl groups at C-2 and C-6 are more active than those at C-3 because of their steric attributes. The extent of alkenyl chain incorporation (DS) was calculated from the ratio of the peak areas at δ 0.89 to the corresponding area from δ 3 to 5.5, due to the contribution of DGK, which we established had a DS of 10.25%. In the present investigation, the values of the DS for DDSA-DGK obtained by titration and 1H NMR methods were 10.29 and 10.25%, respectively. Therefore, the figures obtained from the DS calculation by using both methods complement each other very well. These results are 3761

DOI: 10.1021/jf505783e J. Agric. Food Chem. 2015, 63, 3757−3765

Article

Journal of Agricultural and Food Chemistry

release of alkyl chains from the DDSA. The second major degradation step shown by the DDSA derivative of DGK occurred at a peak decomposition temperature of 275 °C, this representing the bulk of the weight loss. This substantial weight reduction was due to breakages of main-chain linkages of DGK at this temperature. A similar behavior was reported for TGA of carboxymethyl starch/quaternary ammonium salt complexes, where two-step weight losses caused by the release of alkyl moieties associated with surfactant and the starch matrix were observed.28 Thus, TGA indicates clearly that pristine DGK is more stable than its corresponding DDSA derivative. However, the DDSA derivatized samples exhibited lower weight loss until approximately 200 °C, owing possibly to the reduced water absorption of the modified samples. DGK in contrast showed a higher weight loss until 200 °C. However, the overall rate of decomposition of DDSA-DGK was faster compared to DGK and gave a low char yield of 20%. The comparative yield for pristine DGK was twice this value. DSC. Figure 7 shows DSC thermal curves for both DGK and its derivative, DDSA-DGK. The DSC profiles reflect the

Figure 5. 1H NMR spectra recorded for DGK and DDSA-DGK.

in agreement with those obtained for DDSA derivatives of starch,13 inulin,15 and gum arabic19 previously reported in the literature. TGA. The TGA and derivative thermogravimetric analysis (DTG) plots of DGK and the DDSA-DGK derivative are presented in Figure 6. TGA was performed in an inert nitrogen atmosphere. From the TGA curves, it is evident that the DDSA-DGK derivative decomposed in two major weight-loss steps as compared to DGK, which decomposed in a single step. The first stage of decomposition occurred in DDSA-DGK at 219 °C. This is attributed to breakage of ester bonds between DDSA and the gum at the aforementioned temperature and the

Figure 7. DSC thermograms of DGK and DDSA-DGK.

occurrence of exothermic and endothermic changes with increasing temperature. Thermograms for the DDSA-DGK indicate a broad endothermic transition over the range of 60− 140 °C with a melting peak at around 93 °C. This can be attributed to the increased thermoplastic nature of DDSA modified DGK brought about by plasticizing alkenyl groups, probably through internal plasticization. The results suggest that the DDSA-DGK derivative has better thermoplasticity compared to pristine DGK. The decreased enthalpy value of the first transition (Figure 7) also suggests greater interaction between DGK and DDSA. DGK does show a very mild transition in this range, if viewed individually. The improved thermoplastic nature effected by alkyl group modified surfactants and the adoption of simple methodologies have been reported earlier.28 Both samples exhibited exothermic peaks at higher temperatures, at 276 °C for DGK and at 319 °C for DDSA-DGK. These results suggest that modification by DDSA had improved the thermal stability. The enthalpy changes associated with the first and second transitions were 63 and −116 J/g for DDSA-DGK. In comparison, the DGK gave corresponding figures of 415 and −203 J/g. Furthermore, the results obtained indicate that hydrophobic groups of the DDSA reagent had weakened hydrogen bond formation in the DDSADGK derivative. This in turn enhanced structural flexibility, altered the gelatinization temperature, and brought about a

Figure 6. TGA and DGT curves as a function of temperature for DGK and DDSA-DGK. 3762

DOI: 10.1021/jf505783e J. Agric. Food Chem. 2015, 63, 3757−3765

Article

Journal of Agricultural and Food Chemistry

Rheological Properties. The flow behavior of DGK and DDSA-DGK at different concentrations (1, 2, and 3% w/w) was monitored, and the apparent viscosities were plotted as a function of shear rate. It was observed that for both DGK and DDSA-DGK in deionized water, the apparent viscosity increased with rising concentrations of both formulations (Figure 10). The apparent viscosity of the DDSA-DGK

reduction in enthalpy, as had also been reported for OSA derivatives of starch.29 CAC. Dye Solubilization. Absorbance versus concentration plots for DGK and DDSA-DGK along with Sudan IV dye are presented in Figure 8. The absorbance of DGK does not

Figure 8. Concentration dependence of the UV−vis absorbances of DGK and DDSA-DGK in the presence of Sudan IV dye.

change over the concentration range studied, confirming that there is no interaction between DGK and the dye. For the DDSA-DGK sample, the absorbance values increase significantly above a certain concentration, indicating the formation of aggregates (Figure 8). The CAC was determined to be 0.1% for DDSA-DGK, the results obtained being in agreement with CAC values reported for DDSA derivatives of inulin15 and gum arabic.19 Surface Tension Measurements. The surface tensions of DDSA-DGK solutions are plotted as a function of concentration in Figure 9. The surface tension of the DGK derivative

Figure 10. Plots of viscosity as a function of shear rate for DGK and DDSA-DGK of different concentrations (1, 2, and 3%).

derivative was significantly higher than that of DGK alone at the same concentration. This indicated that DDSA derivatization had indeed occurred and had resulted in a higher molecular weight. The higher viscosity values reported for OSA derivatives of starch30,31 are in agreement with the results of our present study. It was also noted that the viscosities of both DGK and DDSA-DGK decreased sharply with increasing shear rate for all concentrations, eventually reaching a constant value at higher shear rates. The behavior was ascribed to the formation of aggregates through hydrogen bonding and polymer entanglement, producing a highly ordered arrangement of stiff molecules. This ultimately resulted in high viscosity at low shear rates, as reported in the literature for gum kondagogu.32 Antibacterial Properties. We tested the antibacterial activity of the DDSA-DGK against Gram-negative E. coli and P. aeruginosa and Gram-positive S. aureus bacteria. The results show that the control sample without DDSA does not display any antibacterial activity. Figure 11 and Table 2 summarize the results ZOI were determined. The ZOI for Gram-negative E. coli and P. aeruginosa and Gram-positive S. aureus were 8.2 ± 0.8, 8.0 ± 0.8, and 8.2 ± 0.7 mm, respectively, for 1 wt % concentration of DDSA-DGK. The ZOI for DDSA-DGK against E. coli, P. aeruginosa, and S. aureus were seen to increase when the concentration of DDSADGK rose from 1 to 10 wt % in all three cases. Furthermore, the antibacterial ZOI for DDSA-DGK against Gram-negative E. coli and P. aeruginosa and Gram-positive S. aureus were observed to be almost equal for all three tested microorganisms of different bacterial strains (Table 2). These results were in good agreement with earlier reported investigations about antibacterial properties of gum tragacanth/ PVA nanofibers, Ag/montmorillonite/chitosan bionanocomposites, and PVA/carboxymethyl-chitosan/Ag-nanowires.33−35 The present study suggests that antibacterial properties of DDSA-DGK are enhanced with increasing concentration. The improved antimicrobial action of DDSA-DGK may be related

Figure 9. Surface tension of DDSA-DGK as a function of concentration.

was found to decrease with increasing concentration, but there was a distinct inflection in the curve, corresponding to the CAC. This was attributable to the formation of micelle-type aggregates brought about by hydrophobic association of the molecules through the alkyl chains, occurring at a concentration of 0.1%. This value is in good agreement with the result obtained from the dye solubilization experiment (Figure 8). The surface tension was found to be ∼30−35 mN/m at the CAC. The experimental values obtained concur with the CAC values reported in the literature for DDSA derivatives of inulin15 and gum arabic.19 3763

DOI: 10.1021/jf505783e J. Agric. Food Chem. 2015, 63, 3757−3765

Article

Journal of Agricultural and Food Chemistry

agents,2 the development of DDSA derivatized gums has potentially two major benefits, namely, as stabilizers and antibacterial agents in food and nonfood applications.

■

AUTHOR INFORMATION

Corresponding Author

*(Vinod V.T.P.) E-mail: [email protected] or [email protected]. Phone: +420 607 393 502. Fax: +420 485 353 445. Funding

The research reported in this paper was financially supported by the Ministry of Education, Youth and Sports within the framework of the targeted support of the “National Programme for Sustainability I”; the OPR & DI project (LO 1201), and the Centre for Nanomaterials, Advanced Technologies and Innovation CZ.1.05/2.1.00/01.0005. Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED GK, gum karaya; DGK, deacetylated gum karaya; DDSA, (2dodecen-1-yl)succinic anhydride; OSA, octenylsuccinic anhydride; DS, degree of substitution; GPC/MALLS, gel permeation chromatography/multiangle laser light scattering; ATRFTIR, attenuated total reflectance−Fourier transform infrared spectroscopy; SEM, scanning electron microscopy; 1H NMR, proton nuclear magnetic resonance spectroscopy; TGA, thermogravimetric analysis; DSC, differential scanning calorimetry; CAC, critical aggregation concentration; ZOI, zones of inhibition

■

Figure 11. Bacterial activity with zones of inhibition for various concentrations of DDSA-DGK (1, 2, 3, 4, and 10%): (A) E. coli; (B) P. aeruginosa; (C) S. aureus.

REFERENCES

(1) Phillips, G. O.; Williams, P. A. Tree exudates gums: natural and versatile food additives and ingredients. Food Ingred. Anal. Int. 2001, 23, 26−28. (2) Verbeken, D.; Dierchx, S.; Dewettinck, K. Exudate gums: occurrence, production and applications. Appl. Microbiol. Biotechnol. 2003, 63, 10−21. (3) Pollard, M. A.; Kelley, R.; Fischer, P. A.; Windhab, E. J.; Eder, B.; Amado, R. Investigation of molecular weight distribution of LBG galactomannan for flours prepared from individual seeds, mixtures, and commercial samples. Food Hydrocolloids 2008, 22, 1596−1606. (4) Vinod, V. T. P.; Sashidhar, R. B.; Suresh, K. I.; Rama Rao, B.; Vijaya Saradhi, U. V. R.; Prabhakar Rao, T. Morphological, physiochemical and structural characterization of gum kondagogu (Cochlospermum gossypium): a tree gum from India. Food Hydrocolloids 2008, 22, 899−915. (5) Anderson, D. M. W.; McNab, C. G. A.; Anderson, C. G.; Braown, P. M.; Pringuer, M. A. Gum exudates from the genus Sterculia (gum karaya). Int. Tree Crops J. 1982, 2, 147−154. (6) Le Cerf, D.; Irinei, F.; Muller, G. Solution properties of gum exudates from Sterculia urens (karaya gum). Carbohydr. Polym. 1990, 13, 375−386.

to the balance of hydrophilicity/hydrophobicity and the increased surface roughness of DGK after derivatization with DDSA, the latter helping to increase contact between the surfaces of the material and the bacteria. The current work is in agreement with the antibacterial action of poly(propylene)modified Cu and Ag nanocomposites and polyethylene/ triclosan/chlorohexidene composites36,37 However, the antibacterial properties of DDSA are less well-known compared to those of Ag nanoparticles.38 The biocidal attributes and toxicity of Ag nanoparicles have also been studied in detail recently.39,40 With regard to nontoxicity, the present undertaking emphasizes that natural gum derivatized DDSA materials can be developed as the leading antibacterial agent, which could replace the toxic Ag and Cu of CuO-based polymeric materials in their role as antimicrobial agents. Given that gums are widely used in food and pharmaceutical applications as stabilizing and emulsifying

Table 2. Zone of Inhibition of Different Bacterial Strains Observed for DDSA-DGK zone of inhibitiona (mm) at DDSA-DGK concentration of

a

bacterial strain

1 wt %

2 wt %

3 wt %

4 wt %

10 wt %

E. coli P. aeruginosa S. aureus

8.1 ± 0.8 8.0 ± 0.8 8.2 ± 0.7

9.2 ± 0.6 10.5 ± 0.8 10.6 ± 0.8

10.2 ± 0.5 11.8 ± 0.7 12.0 ± 0.8

11.1 ± 0.8 12.8 ± 0.7 12.9 ± 0.6

14.2 ± 0.8 15.0 ± 0.6 15.0 ± 0.8

Values are means ± SD, n = 3. 3764

DOI: 10.1021/jf505783e J. Agric. Food Chem. 2015, 63, 3757−3765

Article

Journal of Agricultural and Food Chemistry (7) Whistler, R. L. Exudate gums. In Industrial Gums: Polysaccharides and Their Derivatives; Whistler, R. L., Bemiller, J. N., Eds.; Academic Press: San Diego, CA, USA, 1993; pp 318−337. (8) Weiping, W. Tragacanth and karaya. In Handbook of Gydrocolloids; Philips, G. O., Williams, P. A., Eds.; Woodhead: Cambridge, UK, 2000; pp 155−168. (9) Brito, A. C. F.; Silva, D. A.; de Paula, R. C. M.; Feitosa, J. P. A. Sterculia striata exudate polysaccharide: characterisation, rheological properties and comparison with Sterculia urens (karaya) polysaccharide. Polym. Int. 2004, 53, 1025−1032. (10) Brito, A. C. F.; Sierakowski, M. R.; Reicher, F.; Feitosa, J. P. A.; de Paula, R. C. M. Dynamic rheological study of Sterculia striata and karaya polysaccharides in aqueous solution. Food Hydrocolloids 2005, 19, 861−867. (11) Stephen, A. M.; Churms, S. C. Polysaccharides and mucilages. In Food Polysaccharides and Their Applications; Stephen, A. M., Ed.; Dekker: New York, 1995; pp 377−440. (12) Meer, W. In Handbook of Water-Soluble Polysaccharides and Resins; Davidson, R., Ed.; McGraw-Hill: New York, 1980; pp 3−4. (13) Chi, H.; Xu, K.; Xue, D.; Song, C.; Zhang, W.; Wang, P. Synthesis of dodecenyl succinic anhydride (DDSA) corn starch. Food Res. Int. 2007, 40, 232−238. (14) Zhou, J.; Ren, L.; Tong, J.; Xie, L.; Liu, Z. Surface esterification of corn starch films: Reaction with dodecenyl succinic anhydride. Carbohydr. Polym. 2009, 78, 888−893. (15) Kokubun, S.; Ratcliffe, I.; Williams, P. A. Synthesis, characterization and self-assembly of biosurfactants based on hydrophobically modified inulins. Biomacromolecules 2013, 14, 2830−2836. (16) Meng, F.; Zheng, L.; Wang, Y.; Liang, Y.; Zhong, G. Preparation and properties of konjac glucomannan octenyl succinate modified by microwave method. Food Hydrocolloids 2014, 38, 205−210. (17) Sweedman, M. C.; Tizzotti, M. J.; Schäfer, C.; Gilbert, R. G. Structure and physicochemical properties of octenyl succinic anhydride modified starches: a review. Carbohydr. Polym. 2013, 92, 905−920. (18) Segura-Campos, M.; Chel-Guerrero, L.; Betancur-Ancona, D. Synthesis and partial characterization of octenyl succinic starch from Phaseolus lunatus. Food Hydrocolloids 2008, 22, 1467−1474. (19) Wang, H.; Williams, P. A.; Senan, C. Synthesis, characterization and emulsification properties of dodecenyl succinic anhydride derivatives of gum Arabic. Food Hydrocolloids 2014, 37, 143−148. (20) Vinod, V. T. P.; Sashidhar, R. B. Solution and conformational properties of gum kondagogu (Cochlospermum gossypium) − a natural product with immense potential as a food additive. Food Chem. 2009, 116, 686−692. (21) Tommeraas, K.; Mellergaard, M.; Malle, B. M.; Skagerlind, P. New amphiphilic hyaluronan derivatives based on modification with alkenyl and aryl succinic anhydrides. Carbohydr. Polym. 2011, 85, 173− 179. (22) Eenschooten, C.; Guillaumie, F.; Kontogeorgis, M.; Stenby, E. H.; Abdellaoui, K. S. Preparation and structural characterisation of novel and versatile amphiphilic octenyl succinic anhydride-modified hyaluronic acid derivatives. Carbohydr. Polym. 2010, 79, 597−605. (23) Bai, Y.; Shi, Y.-C. Structure and preparation of octenyl succinic esters of granular starch, microporous starch and soluble maltodextrin. Carbohydr. Polym. 2011, 83, 520−527. (24) Tizzotti, M. J.; Sweedman, M. C.; Tang, D.; Schaefer, C.; Gilbert, R. G. New 1H NMR procedure for the characterization of native and modified food-grade starches. J. Agric. Food Chem. 2011, 59, 6913−6919. (25) Mahendran, T.; Williams, P. A.; Phillips, G. O.; Al-Assaf, S.; Baldwin, T. C. New insights into the structural characteristics of the arabinogalactan-protein (AGP) fraction of gum Arabic. J. Agric. Food Chem. 2008, 56, 9269−9276. (26) Song, X.; He, G.; Ruan, H.; Chen, Q. Preparation and properties of octenyl succinic anhydride modified early indica rice starch. Starch/ Staerke 2006, 58, 109−117. (27) Cheng, H. N.; Neiss, T. G. Solution NMR spectroscopy of food polysaccharides. Polym. Rev. 2012, 52, 81−114.

(28) Balsamo, V.; Lopez-Carrasquero, F.; Laredo, E.; Conto, K.; Contreras, J.; Feijoo, J. L. Preparation and thermal stability of carboxymethyl starch/quaternary ammonium salts complexes. Carbohydr. Polym. 2011, 83, 1680−1689. (29) Bao, J. S.; Xing, J.; Philips, D. L.; Corke, H. Physical properties of octenyl succinic anhydride modified rice, wheat, and potatoes starches. J. Agric. Food Chem. 2003, 51, 2283−2287. (30) Bhosale, R.; Singhal, R. Effect of octenylsuccinylation on physicochemical and functional properties of waxy maize and amaranth starches. Carbohydr. Polym. 2007, 68, 447−456. (31) Song, X.-Y.; Zhu, Z.; Li, Z.; Zhu, Z. Characteristic and application of octenyl succinic anhydride modified waxy corn starch in sausage. Starch /Staerke 2010, 62, 629−636. (32) Vinod, V. T. P.; Sashidhar, R. B.; Sarma, V. U. M.; Vijaya Saradhi, U. V. R. Compositional analysis and rheological properties of gum kondagogu (Cochlospermum gossypium): a tree gum from India. J. Agric. Food Chem. 2008, 56, 2199−2207. (33) Ranjbar-Mohammadi, M.; Bahrami, S. H.; Joghataei, M. T. Fabrication of novel nanofiber scaffolds from gum tragacanth/poly (vinyl alcohol) for wound dressing application: In vitro evaluation and antibacterial properties. Mater. Sci. Eng., C 2013, 33, 4935−4943. (34) Zhao, Y.; Zhou, Y.; Wu, X.; Wang, L.; Xu, L.; Wei, S. A facile method for electrospinning of Ag nanoparticles/poly (vinyl alcohol)/ carboxymethyl-chitosan nanofibers. Appl. Surf. Sci. 2012, 258, 8867− 8873. (35) Shameli, K.; Ahmad, M. B.; Zargar, M.; Yunus, W. M. Z.; Ibrahim, N. A.; Moghaddam, M. G. Synthesis and characterization of silver/montmorillonite/chitosan bionanocomposites by chemical reduction method and their antibacterial activity. Int. J. Nanomed. 2011, 6, 271−284. (36) Espana-Sanchez, B. L.; Avila-Orta, C. A.; Padilla-Vaca, F.; NeiraVelazquez, M. G.; Gonzalez-Moroues, P.; Rodriguez-Gonzalex, J. A.; Hernandez-Hernandez, E.; Rangel-Serrano, A.; Barriga-, E. D.; Yate, C. L.; Ziolo, R. F. Enhanced antibacterial activity of melt processed poly(propylene) Ag and Cu nanocomposites by argon plasma treatment. Plasma Processes Polym. 2014, 11, 353−365. (37) Popelka, A.; Novak, I.; Lehocky, M.; Chodak, I.; Sedliacik, J.; Gajtanska, M.; Sedliacikova, M.; Vesel, A.; Junkar, I.; Kleinova, A.; Spirkova, M.; Bilek, F. Anti-bacterial treatment of polyethylene by cold plasma for medical purposes. Molecules 2012, 17, 762−785. (38) Marambio-jones, C.; Hoek, E. M. V. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J. Nanopart. Res. 2010, 12, 1531−1551. (39) Liu, J.; Hurt, R. H. Ion release kinetics and particle persistence in aqueous nano-silver colloids. Environ. Sci. Technol. 2010, 44, 2169− 2175. (40) Kittler, S.; Greulich, C.; Diendorf, J.; Koller, M.; Epple, M. Toxicity of silver nanoparticles increases during storage because of slow dissolution under release of silver ions. Chem. Mater. 2010, 22, 4548−4554.

3765

DOI: 10.1021/jf505783e J. Agric. Food Chem. 2015, 63, 3757−3765