Physical Properties of Films Based on Gelatin and Dialdehyde Starch with Different Oxidation Degrees

In this study, dialdehyde starch (DAS) was prepared by oxidizing corn starch using potassium periodate as the oxidant. The aldehyde contents in DAS were determined using titration with hydroxylamine and were found proportional to the molar ratio of KIO4:C6H10O5. Fourier transform infrared spectroscopy was used to confirm the breakage of the bond between C2 and C3 in anhydroglucose units and the formation of -CHO group in DAS. X-ray diffractometry showed a decrease in crystallinity of starch after periodate oxidation. Thermogravimetric analysis showed reduced thermal stability of DAS in comparison with the native starch. The prepared DAS was blended with gelatin to form films by casting method. Increasing aldehyde contents in DAS from 0 to 12.2% resulted in decreased moisture contents, solubility in water, and the swelling capacity of gelatin-DAS films. The value of tensile strength and elongation at break of the gelatin-DAS films rose when the aldehyde content was low (0-3.5%), but declined when the aldehyde content was high (3.5-12.2%). Slight periodate oxidation is, therefore, an effective way to enhance the mechanical characteristics of blend films from DAS and other biopolymers.


INTRODUCTION
Nowadays, the use of petrochemical-based plastics causes nagative impacts on ecosystem because it take many years for them to decompose. To solve this problem, the development and applications of biodegradable polymeric materials, especially those from renewable resources, are needed. Among natural resources, starch is the most commonly used polymers because it is inexpensive, non-toxic, renewable, and biodegradable 2 . However, practical applications of starch as a plastic material were limited due to its disadvantages, such as poor mechanical properties and hydrophilic character 3 .
Dialdehyde starch (DAS) is an industrial starch derivative, in which the C 2 -C 3 bonds of the anhydroglucose units are cleaved and the CH-OH groups at these atoms are converted to CH=O aldehyde groups 4 . When blended with other biopolymers to form films, aldehyde groups in DAS can crosslink with other functional groups (-OH and -NH 2 ) and hence enhance the physicomechanical properties of the blend films. Moreno et al., reported that the improvement of hydrophobic and mechanical characteristics of the gelatin-DAS films is related to the formation of crosslinking processes in polymeric matrices while increasing the hydrophobicity of the film 5 .
DAS is commonly prepared by periodate oxidizing starch with NaIO 4 as the most widely used reagent 4 . Despite lower price compared to NaIO 4 , KIO 4 is rarely used as the oxidizing agent to prepare DAS, possibly due to its low solubility. In this study, we used KIO 4 to prepare DAS from corn starch and evaluated the influence of the oxidation degrees of starch on physico-mechanical properties of gelatin-DAS blend films.

Preparation of dialdehyde starch (DAS)
The corn starch of 3.0 g and various quantities of KIO 4 were suspended in distilled water to obtain various molar ratios of KIO 4 :C 6 H 10 O 5 = 0.1, 0.2, and 0.3 ( Table 1). The pH of solution was adjusted to 3 by adding 0.1 M HCl. Then the mixtures were vigorously stirred at 35°C for 4 horus. After that, they were filtered and rinsed ten times (10 × 10 mL) with distilled water. The products were dehydrated in a convection oven at 508°C for 48 h to obtain DAS with different aldehyde contents. stirred at 50°C. During the reaction, the pH gradually decreased due to the release of HCl from NH 2 OH.HCl. Therefore, the 0.1 M NaOH was continuously added from a buret to remain pH = 5. After 2 h, pH of the reaction mixture stopped decreasing, indicating the end of the reaction. The total volume of 0.1 M NaOH used VDAS was recorded. A blank determination was also conducted for 0.2 g of the native corn starch instead of DAS and the volume of 0.1 NaOH used was also recorded (Vblank). The content of aldehyde in DAS was evaluated using Eq (1) 1 .

DAS characterization
FTIR spectra of DAS were measured with using PerkinElmer MIR/NIR Frontier. The resolution was at 2 cm -1 . The scanning region was 400-4000 cm -1 .
X-ray diffraction (XRD) patterns of DAS were measured by diffractometer (Brucker D2) at ambient temperature. The scanning region was 10-90° of 2θ. The equipment was operated with CuKα wavelength of 1.54056 angstroms.
Thermal decomposition of DAS was investigated by thermogravimetric analyser TA-200 (TA Instruments). The mass of the sample was continuously recorded while being heated in temperature range of 30 -350°C. The heating rate was 10°C/minute. These experiments were carried out under a nitrogen flow (100 mL/min) to help protect the samples from oxidative degradation.

Preparation of gelatin-DAS films
Gelatin (Ge) of 1.5 g was first completely dissolved in 5 mL distilled water by stirring at 80°C. Then 3 g of DAS, 2 mL of glycerol and the prepared gelatin solution were added to 25 mL of distilled water. The blended Ge-DAS with glycerol were mixed using magnetic stirrer at 100 °C during 30 minute. After that the mixtures were left to cool down during 10 min and then tranfered into Petri dishes (15 g/dish). The Petri dishes with the mixtures were dehydrated at room temperature for 72 hours. The obtained films were peeled off and placed in a desiccator with a saturated NaBr solution to equilibriate the moisture in the films. A film with the same composition but using native corn starch (NS) instead of DAS was used as the control. Determination of aldehyde content A 0.25 M hydroxylamine solution was prepared from NH 2 OH.HCl. The pH value of solution was adjusted to 5 by using sodium hydroxide 0.1 M. Dried DAS of 0.2 g was then added to 25 mL of the prepared hydroxylamine solution and magnetically

Characterization of Ge-DAS films Mechanical testing
The tensile tester (Testometric, UK) was used to investigated the mechanical characteristics of Ge-NS and Ge-DAS films. The instrument was operated with a crosshead speed of 0.8 mm/s (ASTM Standard method D882-12).

Moisture content, solubility and swelling capacity in water
Film samples of 2 × 2-cm 2 were weighed (m o ) and then dehydrated in an oven at temperature of 55 o C for 24 hours. The weight of the prepared film samples were tested (m 1 ). Then these samples were immersed in distilled water during 2 h at ambient temperature. After that, the films samples were removed and the surface water was cleaned using a filter paper. After weighing, the films (m 2 ) were kept dry at temperature of 55 o C during 24 horus. Then these films samples were weighed (m 3 ).
The moisture content (MC) of the Ge-NS and Ge-DAS films was determined using Eq (2): The solubility (S) of the Ge-NS and Ge-DAS films was determined using Eq (3): Swelling capacity (SW) of the Ge-NS and Ge-DAS films was determined using Eq 4: Table 2 shows that the aldehyde content of DAS was proportional to the molar ratio of KIO 4 :anhydroglucose C 6 H 10 O 5 units in corn starch. The aldehyde content of DAS increased as the molar ratio of KIO 4 /glucose increased. In comparison with the study of Yu et al., with using NaIO 4 as the oxidant, it was shown that the oxidation degree of starch in our study was lower, which might be due to the lower solubility of KIO 4 comparing with NaIO 4 6 .

FTIR
FTIR was applied to investigate the changes in structure when native starch was oxidized to DAS (Figure 1).

Fig. 1. FTIR spectra of native and dialdehyde starch
For NS, the main characteristic groups were the secondary -OH groups at the position of C 2 and C 3 , the primary -OH groups at the position of C 6 , and the D-pyranose ring structure. These structural characteristics in the infrared spectrogram are shown as follow. The broad peak at 3310 cm −1 correlates to the -OH stretching. The peak absorption observed at 2930 cm −1 is atributed to C-H stretching vibrations. The bound water in the starch was performed by the peak near 1640 cm -1 4,6-8 . The peaks in range of 1000-1200 cm -1 are attributed to the C-O bond of stretching and vibration 4 . The oxidation of starch by potassium periodate led to the linkage break at the position of C 2 and C 3 of the anhydroglucose units. The hydroxyl groups at C 2 -C 3 was taken place by aldehyde groups. The peak of the C-O bond stretching in C-O-H group in the anhydrogluscose ring of starch was observed near 1150 cm -1 . With the rising in contents of aldehyde in starch, the double peak of C-OH groups turned to a board band and gradually weakened. For DAS12.2 besides those peaks, there was an apperance of the peak around 1720 cm -1 , represented the C=O stretching and vibration 4 . This peak was not found in the spectra of DAS3.5 and DAS7.4 due to the low content of aldehyde groups in these samples.

XRD analysis
The method of X-ray diffractometry was applied to analyse the crystal structure of natural and dialdehyde starch (Figure 2). chain and formation of small crystallites exhibiting amorphous phase in DAS 4,6,9,10 .

Thermogravimetric analysis (TGA) and differential thermogravimetry (DTG)
TGA and DTG curves for NS and DAS12.2 are shown in Fig. 3. For DAS there were two stages of weight loss. The first stage around 30-120°C was related to the loss of water. The second one represented the decomposition of DAS with the maximum decomposition in the range of 249.6-270.8°C. For the native starch there were three stages of weight loss. The first stage around 30-120°C was related to the loss of water. The double peaks were attributed to the stages of starch decomposition. It was shown that the maximum decomposition was at a range of 269.0-292.3°C. With the existence of -CHO groups, the maximum peak shifted to region of lower temperature. It indicated the lower thermal stability of DAS in comparison with NS. This phenomenon might be related to the decline in evarage molecular weight of DAS 12.2 comparing to NS 11 . The hydroxyl groups at the position of C 2 and C 3 of anhydroglucose units are oxidized with the formation of aldehyde groups, hence destroying the hydrogen bonds between starch molecules (Scheme 1) 4 . Moreover, the oxidation using periodate was conducted at pH of 3, which might partially hydrolys the glycosidic bonds between glucose units in starch. These phenomena led to the cleavage in starch

Hydrophilicity of Ge-DAS films
The blend films containing DAS with higher aldehyde contents exhibited lower hydrophilicity, expressed in lower moisture content, solubility, and swelling capacity ( Table 3). The first reason is the lower hydrophilicity of carbonyl groups in DAS compared to hydroxyl groups in native starch 11 . The second reason is the crosslinks between the aldehyde groups in DAS with amino groups in gelatin (to form imine) and with hydroxyl groups in unoxidized fragments in DAS (to form acetal and cetal) 12 . These crosslinks tightened the film structure, hence lowering the the amount of moisture in the samples, hindering dissolution and suppressing the water diffusion into the film maxtric.  and -OH groups in starch 13 . However, for Ge-DAS films, the tensile properties are associated to the crosslinking reaction between -CHO and -NH 2 groups in a polymeric matrix. The change in crosslinking reaction in Ge-DAS matrix might not corespond to the change of the level of starch oxidation. It was related to the heterogeneous property due to containing of both hydroxyl and carbonyl in the polymeric chain. This phenomenon might produce hindrances to the formation new crosslinking reaction in the polymeric matrix5. Another possible reason for the decreases in TS and YM at high degree of oxidation is the depolymerization of starch 14 .

CONCLUSION
DAS was successfully prepared in a slightly acidic solution using KIO 4 as the periodate oxidant. The oxidation introduced aldehyde groups into the starch backbone, destroyed the crystallinity of starch, and decreased the thermal stability of starch. DAS was blended with gelatin to produce biodegradable films with lower hydrophilicity due to the crosslinking aldehyde groups in DAS with functional groups of gelatin. The value of elongation at break and tensile strength of Ge-DAS films increased at low and decreased at high degrees of starch oxidation. Therefore, controlling the degree of periodate oxidation is a way of altering the physicochemical properties of film from DAS and other polymers.