Enhancing hydrophobicity, strength and UV shielding capacity of starch film via novel co-cross-linking in neutral conditions

Starch films are developed as the biodegradable packaging materials to replace the petroleum-based materials in recent years. Thus, it is extremely beneficial to improve the hydrophobicity and mechanical strength of starch films, through a novel approach of co-cross-linking in neutral conditions, with glyoxal and AZC. In this work, systematic studies have been conducted to assess the performance of the co-cross-linked starch along with the control starch and starch cross-linked by glyoxal or AZC alone. Results showed that the co-cross-linked starch films exhibited significantly improved hydrophobicity and strength and the wet stress reached 1.53 MPa, compared to the control, glyoxal or AZC cross-linked starch films. More interestingly, the co-cross-linked film also demonstrated excellent UV shielding capacity and transmittance at visible wavelength range. The reaction mechanism was revealed based on the findings from UV, FT-IR and NMR spectra. This work established an innovative approach to improving the performance of starch film in neutral conditions for packaging applications.


Fabrication of cross-linked starch films
The 10% control starch solutions were prepared by dissolving 10 g soluble starch into 90 g water through heating at 908C for 2 h. The Gly-AZC starch solution was obtained through first heating at 608C for 1 h and then heating at 908C for another 1 h. The dosages of glyoxal and AZC were 10% (wt) and 5% (wt) to the dry starch, respectively. For comparison, the starch solution added by glyoxal or AZC alone was also prepared under the same condition, at a dosage of 15%. The as-prepared starch solution was poured into the hydrophobic Petri dishes and oven-dried at 408C for 2 h. Before characterization, the films were kept at 238C and a relative humidity (RH) of 50% for 2 days and then peeled off. The grammage of films was determined based on the amount of dry starch (g) over the unit area coated. The starch granules for the characterizations of FT-IR, NMR, TGA and XRD were obtained through grinding the starch samples using a solid grinder (FW100, Tianjin Taisite Corp, China) for 5 min after the as-prepared starch solutions were freeze-dried at 2808C and 15 Pa for 48 h.

Characterization
The water contact angle of starch film was measured by the sessile drop method using a contact angle analyser (Attention Theta, Biolin Scientific Corp, Sweden) at 258C and 50% RH with 4 ml of deionized water [30]. The reported values were measured at 15 s after the deposition of droplets and every sample was performed 10 times in different positions.
For the testing of mechanical properties, the starch films were cut into 5 mm Â 30 mm rectangular strips. Afterwards, their tensile stress and Young's modulus were measured at room temperature using a UTM6502 universal testing machine (Suns, Shenzhen, China) equipped with a 500 N load cell and pneumatic grips. The cross-head speed during tensile testing was 5 mm min 21 . The specific tensile stress and specific Young's modulus were calculated accordingly [31].
The water stability tests were conducted as follows. The starch films were immersed in deionized water and stirred for 5 min (600 rpm) at 258C and 608C, respectively. Then the photographs were taken to determine the water stability of starch films in water.
The swelling ratio of control and cross-linked starch film in water was also determined. Firstly, the cross-linked starch films were conditioned at 1058C for 4 h to remove the moisture and weighted as M 1 . Then the films were soaked in deionized water for 7 min and the swollen films were wiped gently using Kimwipes every minute and weighed as M 2 . The swelling ratio of starch films in water was calculated as follows: A UV-Vis spectrometer (TU-1900, Beijing Purkinje Corp., China) was used to characterize the light transmittance of the as-prepared starch films and record the adsorption of the reacted mixture solution of glyoxal and AZC from 200 to 800 nm, in accordance with the procedures in related literature [32][33][34].
The 650 FT-IR spectrometer (Tianjin GangDong Sci & Tech. D) was used on the control starch, glyoxal cross-linked starch, AZC cross-linked starch and Gly -AZC starch granules after grinding with KBr by co-adding over 16 scans, capturing spectra between 500 and 4000 cm 21 , at a resolution of 0.5 cm 21 . 13 C NMR of the starch granules dissolved in deuterium peroxide (D 2 O) were recorded on a Varian Unity 400 spectrometer operated at 400 MHz.
The thermal properties of the starch were examined using an SDT Q600 TGA System thermogravimetric analyser (TA, America). The sample weight was 1-10 mg and heated from 308C to 8008C under the nitrogen atmosphere at a heating rate of 108C/min and a nitrogen flow rate of 100 ml min 21 .
The Ultima X-ray diffractometer (Rigaku, Japan) was used to determine the crystallinity of starch samples over a 2u range from 58 to 508 with a scan step time of 30 s.
The surface of starch granules and the cross-sections of the starch films were observed using a Quanta 200 Environmental scanning electron microscope (FEI, America) after gold sputtering according to the reference [35]. The starch films were immersed in liquid nitrogen before fracturing and the starch granules were obtained using a solid grinder (FW100, Tianjin Taisite Corp, China) after freeze-drying. The surface topographical images of starch films were also examined using a Dimension Edge Atomic Force Microscope (Bruck, Germany) in tapping mode at 300 kHz with the starch film stuck on a silicon cantilever.

UV -vis spectra of the mixture solution of glyoxal and AZC
The adsorption of the as-prepared mixture solution of glyoxal and AZC was recorded with the UV-vis spectra, and the results are presented in figure 1. As depicted, the maximum adsorption wavelength of mixture solution shifts from 276 to 288 nm as the molar ratio of glyoxal to AZC varies from 1 : 1 to 6 : 1. According to our previous work (electronic supplementary material, figure S1), the glyoxal solution and AZC solution heated at 608C for 30 min appeared to be transparent and milky white, respectively; and such adsorption intensity was not observed around 288 nm in the UV-vis spectra. The 'white' colour in the AZC solution indicates the self-polymerization of AZC upon heating. Here we found that the solution mixture of glyoxal and AZC changes from white to light yellow with the increasing molar ratio in figure 1a. When the molar ratio rises to 2 : 1, the solution becomes clear and transparent without any precipitation. This can be attributed to the fact that the increased glyoxal dosage efficiently limits the self-polymerization of AZC through the Schiff-base reaction with AZC.
As the molar ratio increases, an obviously enhanced adsorption intensity appears at 282 nm due to the newly produced Schiff-base structure (O ¼ C-C ¼ N), indicating the successful reaction between the aldehyde and amino groups. As the glyoxal dosage further increases, the maximum adsorption wavelength shifts to 288 nm and the solution becomes dark yellow. It is confirmed that the Schiff-base reaction between glyoxal and AZC successfully occurs, and the nitrogen element in the AZC can be retained. Besides, the exhibited prominent UV adsorbing ability of glyoxal-AZC solution might be beneficial for improving the UV shielding capacity of the co-cross-linked starch films. Figure 1b illustrates the effect of reaction time on the UV-vis spectra of the mixture solution of glyoxal and AZC. As the reaction proceeds, the solution colour changes from light yellow to dark yellow; and enhanced absorbance intensity is observed, indicating an increased amount of Schiff-base structure. The turbidity phenomenon is not observed due to the polymerization of AZC, which indicates that the free 'Zr' in AZC have the chance to form the hydrogen linkage with the hydroxyl groups of starch. Thus, this result is extremely beneficial for increasing the cross-linking density of starch and improving its stress-transferring ability.

Cross-linking reactions via functional groups
To further confirm the co-cross-linking of starch induced by glyoxal and AZC, FT-IR spectra and 13 C NMR of the starch samples were collected, and the results are shown in figures 2 and 3. As can be seen from FT-IR spectra, the adsorption peak of -OH around 3450 cm 21 in the AZC sample becomes broader compared with the other samples, which can be attributed to the re-association of hydroxyls caused by the hydrogen linkage between Zr and hydroxyl groups [36]. The adsorption peak at 1640 cm 21 can be ascribed to the stretching vibration of hydrogen bonds [37]. As reported, the crosslinking reaction between aldehyde and hydroxyl groups can decrease the amount of hydrogen bonds and give rise to the amount of C-O linkages. Therefore, the intensity at 1640 cm 21 is decreased, whereas the intensity at 1020 cm 21 is enhanced in Glyoxal and Gly -AZC samples. The adsorption peak at 1740 cm 21 in the Gly -AZC sample is attributed to the stretching vibration of O ¼ C-C ¼ N, indicating the successful Schiff-base reaction between glyoxal and NH 3 . The chemical shifts and assignments of 13    rsos.royalsocietypublishing.org R. Soc. open sci. 5: 181206 confirms that NH 3 has been successfully covalently bonded to the glyoxal through the Schiff-base reaction. The signals of C-8 and C-7 at 95.4 ppm and 90.6 ppm are observed due to the acetalization between glyoxal and the hydroxyl groups of starch.

Crystallographic structures of control and cross-linked starch granules
The X-ray diffraction patterns for both control and cross-linked starch granules are shown in figure 4. The starches exhibit two strong characteristic peaks at around 2u value of 17.068 and 22.148 and a weak peak at near 2u value of 19.78. Compared with control starch, the AZC sample shows an obviously decreased intensity of reflection at 2u ¼ 19.78, indicating the loss of ordered structure in starch. It is indicated that the AZC cross-linking with the starch molecules brings a significant reduction to the crystallinity of starch. This might be attributed to the decreased amount of the inter-and intra-molecular hydrogen bonds in the starch. By contrast, decreased crystallinity is not observed in the Gly-AZC sample which is further confirmation that the addition of glyoxal can efficiently limit the self-polymerization of AZC.

Thermal properties of starch granules
Thermal properties of the control and cross-linked starches are characterized by thermogravimetric analysis (TGA). As shown in figure 5a, the control and cross-linked starch samples present two distinct stages. The first weight loss occurring between 1008C and 1908C is attributed to the loss of free and combined water in the starch granules. The cross-linking gives an obvious increase in the hydrophobicity of starch granules, and thus a lower mass loss of water is seen in the cross-linked starch samples.  In the second process, the initial decomposition temperature of the starch samples after cross-linking begins at 1908C; by contrast, the control starch is still stable even at around 3008C. This phenomenon might be related to the loose structure of starch due to cross-linking, which is accorded with the decreased crystallinity ( figure 4). However, the decomposed mass of the cross-linked starch is much lower than that of control starch, which is also reflected in the DTG curves (figure 5b). Compared with the control starch, the intensity of cross-linked starch (Glyoxal, AZC and Gly -AZC) at the maximum degradation temperature is much lower. Besides, the maximum weight loss (MWS) peak in the AZC and Gly -AZC samples appeared at 2828C and 2918C, respectively, in comparison to that (3118C) of control starch. It seems that the aggregation of AZC created by its self-polymerization might destroy the ordered structure of the starch chain, thus resulting in the decreased maximum decomposition temperature (MDT); while the addition of glyoxal can limit the self-polymerization of AZC to some extent, thus giving a higher MDT than the AZC cross-linked starch. The thermal decomposition peak appearing in the Glyoxal sample at 2188C might be ascribed to the degradation of the volatile structure in the glyoxal cross-linked starch.
The cross-linking gives rise to the starch mass remaining after 6008C, as compared to the control starch (figure 5a). As seen, the residual contents in the AZC, Glyoxal and Gly -AZC samples at 6008C reach as high as 39, 23 and 20%, respectively, which is much higher than that of the control sample (6%). The results are accorded with the decreased decomposition weight loss at the corresponding MDT (figure 5b), indicating the improved thermal stability after cross-linking. Figure 6 shows the stability of the starch film in water at 258C and 608C under constant stirring. It is observed that an obvious crack appeared on the surface of the Control and Glyoxal sample after 10 min at 258C (see Stirring I), while the AZC and Gly -AZC sample remains stable and keeps its intact original shape under the same conditions. It is indicated that the AZC cross-linking can effectively improve the water stability of the starch film. Because of the formation of the compact network structure after the cross-linking, the fractured regions previously accessible to water are reduced in the starch film completely or partly cross-linked by AZC. After stirring for 10 min at 608C (see Stirring II), the Control sample disintegrates partly and the aqueous solution becomes turbid; while the Glyoxal sample still dissolves. This can be ascribed to the unreacted glyoxal in neutral conditions or the produced loose structure, thus water molecules move towards the inner parts of starch film readily and proceed with the disintegration of films, especially at a high temperature. The AZC and Gly -AZC films are curled, but still keep their original shape without any disintegration. It can be concluded that the water stability of the starch films cross-linked by glyoxal and AZC is significantly improved.

Water stability and water resistance of cross-linked starch films
Starch is an inherently hydrophilic polymer that can easily absorb moisture due to the presence of hydroxyl groups; but this feature weakens its mechanical properties. The swelling ratio of cross-linked starch films with different cross-linkers in water is shown in figure 7a. Swelling does occur after the diffusion of water within the starch polymer network. The cross-linking can lead to the formation of a rsos.royalsocietypublishing.org R. Soc. open sci. 5: 181206 compact and rigid network, which limits the absorption and diffusion of water molecules [38]. From figure 7a, it can be seen that the swelling ratios of the Glyoxal, AZC and Gly -AZC samples are decreased obviously compared with the Control sample, particularly in the first 2 min. Specifically, Glyoxal sample keeps intact shape until 5 min, while the AZC and Gly -AZC samples do not disintegrate even after 7 min. Meanwhile, the swelling ratio of Gly -AZC decreases obviously, compared with the other samples, indicating the formation of a highly cross-linked structure in the network induced by the novel co-cross-linking system consisting of glyoxal and AZC. The above phenomenon can be clearly illustrated from the optical images of these four samples impregnated in water for 2 h at 258C. As seen in figure 7b, the Control sample almost dissolves in water and presents a transparent state. The Glyoxal sample swells and dissolves partly, without sufficient wet-strength to keep its shape intact. By contrast, the AZC and Gly-AZC film is insoluble in water, suggesting the efficient strengthening of starch polymer networks caused by the crosslinking. The AZC presents a compact and intact shape without any sign of swelling and disintegration. Its wet tensile strength reaches to 0.66 MPa and represents the satisfied cross-linking performance. This is more prominent in the Gly-AZC sample: the film keeps its original shape without any sign of swelling and the wet tensile stress reaches to 1.53 MPa, indicating that the cocross-linking induces efficient linkages with hydroxyl groups of starch, and helps form a dense network that can limit the penetration of water molecules into the inner parts of starch films.
The surface hydrophobic properties of the cross-linked starch films are illustrated through the contact angle (CA) measurements. As presented in figure 7c,d, the initial water contact angle of control starch film (Control sample) is only 42.048 and decreases to 34.068 within the recording time (180 s), exhibiting a highly hydrophilic nature. The glyoxal cross-linked starch film almost doesn't improve the hydrophobicity compared with the control starch, and the initial water contact angle is only 50.918 and decreases to 41.278 at 11 s, then becomes stable until 180 s. Therefore, the cross-linking through rsos.royalsocietypublishing.org R. Soc. open sci. 5: 181206 glyoxal alone does not contribute to the improved surface hydrophobicity of starch. In contrast, the AZC starch film presents excellent hydrophobic properties with the initial CA of 99.458. Besides, the water contact angle remains high and still over 908 (92.378) at 180 s. The starch film co-cross-linked by glyoxal and AZC also exhibits improved surface hydrophobicity, compared with the Control sample.

Optical and mechanical properties analysis
The transmittance and optical properties of the control and cross-linked starch films are exhibited in figure 8. As shown, the Gly -AZC starch film presents excellent transmittance and the 'logo' image can be seen clearly. The cross-linking reaction gives an obvious visual colour difference in the Gly -AZC sample (yellow) compared with the other starch films (colourless and transparent). The yellow colour in the Gly -AZC sample is attributed to the presence of the Schiff-base structure. More importantly, the Gly-AZC sample still exhibits excellent transmittance at visible wavelength range. Figure 8a depicts the light transmittance through the films as a function of wavelength. As seen, the transmittance at 600 nm is around 80% for these starch films except for the AZC sample. This might be attributed to the increased starch granule size due to the self-polymerization of AZC. For the Gly-AZC sample, the transmittance at 300 nm shows an obvious decrease, compared with other starch films. This is accorded with the result of UV-vis spectra in figure 1. The produced Schiff-base structure can adsorb the light at 300 nm and thus give a decrease to the transmittance correspondingly. It is noteworthy that the excellent UV shielding capacity between the 300 nm-330 nm ranges might be very beneficial for broadening the application fields of starch films in the packaging industry. The capacity for both excellent transmittance at a visible wavelength range and UV shielding capacity is extremely important for food packaging materials.
Tensile stress (s) and the specific tensile stress (s/r) of starch films are presented in figure 8b, where r is the density. The (s/r) of Glyoxal film is 7.37 kN m kg 21  rsos.royalsocietypublishing.org R. Soc. open sci. 5: 181206 Control sample. In AZC, it only increases by 108.22%. The difference might be attributed to the decreased amount of linking bonds in starch network cross-linked by AZC compared with the Glyoxal sample, as well as the relatively weaker hydrogen linkage in the AZC sample against the covalent bonding in the Glyoxal sample. For the Gly-AZC sample, the specific tensile stress (6.53 KN m kg 21 ) increases by 123.63%. Clearly, the strengthening effect of glyoxal is better than that of AZC at the same dosage. In fact, glyoxal has a lower molecular weight and a higher number of reactive groups that can form the covalent bonds with the hydroxyl groups of starch in comparison with AZC. Moreover, the weight ratio of glyoxal to AZC is also closely related to the strength performance of starch film cross-linked by the current novel co-cross-linking system, which has also been studied in subsequent works. The relatively high modulus (E) of cross-linked starch films is shown in figure 8c. Glyoxal and AZC have been reported to improve the elastic modulus of cellulose-based materials. As expected, the addition of glyoxal and AZC gives higher tensile modulus than the control starch film. For better comparison, the specific tensile modulus (E/r) takes the density (r) variation into account. The E/r of Gly-AZC increases by 137.92% compared with the Control film. The increase ratio in the dual-crosslinking system is not as high as the Glyoxal sample but is higher than the AZC sample. The SEM and AFM images of starch are shown in figure 9. As shown, the cross-linked starch after freezedrying exhibits obvious different morphology compared with the Control sample. The starch granules after cross-linking present obvious connection, compared with the control starch, clearly visible in the SEM images (in figure 9a-d). As shown, the control starch granules are individual or separated with diameters of around 150 mm -300 mm (figure 9b and electronic supplementary material, figure S2a). However, the Glyoxal, AZC and Gly -AZC starch present a flaky shape, which is dramatically different to the un-cross-linked control starch granules (electronic supplementary material, figure S3a and S3b and figure 9b). Electronic supplementary material, figure S2 shows the cross-section images of starch films and there appear 'hills and valleys' in the fracture of Control and AZC film samples; while the cross-sections of the Glyoxal and Gly -AZC samples exhibit relatively smooth surfaces, indicating the increased rigidity after the co-cross-linking (electronic supplementary material, figure S2d and S2f ). This change in the cross-section morphology after co-cross-linking can also be observed in figure 9c,d. From the AFM images, it is clear that the surface of Gly-AZC starch film is more homogeneous and the height is only 14 nm, while the roughness of the control films is relatively higher. As seen in electronic supplementary material, figures S3c and S3d, the addition of glyoxal in Glyoxal film give a much more homogeneous surface than that of AZC film. This indicates that the addition of glyoxal efficiently limits the self-polymerization of AZC when co-cross-linked with starch and contributes to the formation of a uniform morphology in starch films.

Conclusion
In this assessing work, a starch film with enhanced hydrophobicity, strength and excellent transmittance at the visible light range and UV shielding ability was obtained via a novel co-cross-linking system. Based on the experimental findings, the following conclusions were achieved: 1. The addition of glyoxal and AZC significantly improved the hydrophobicity and strength of the co-cross-linked starch. The E/r of Gly -AZC increased by 137.92%, compared with the Control. The wet tensile stress reached to 1.53 MPa and the initial CA was 71.058. Moreover, the excellent transmittance at visible wavelength range and UV shielding capacity were observed. 2. The UV, FT-IR spectra and 13 C NMR confirmed the presence of Schiff-base reaction in the co-crosslinking system. XRD and TG analysis showed that the addition of glyoxal avoided the weakened crystallinity structure and the decreased MDT of co-cross-linked starch to some extent, via limiting the self-polymerization of AZC. SEM and AFM images indicated that a rigid and uniform starch film was formed after the co-cross-linking.

Ethics. The study was approved by the Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest
Resources, Nanjing, China and experiments related to the animals were not involved in this work.
Data accessibility. The datasets supporting this article have been uploaded as part of the electronic supplementary material.