Tuning the Chemical and Thermo-Mechanical Properties of Cassava Starch Thin Films to Produce Biodegradable Packaging Materials


 Starch is a renewable resource and starch films play a vital role as an alternative for synthetic polymers in packaging applications. However, the films prepared from native starch fail to meet the process or product requirements due to high water absorption and inferior mechanical properties. In order to avoid these drawbacks, and to enhance the desired properties, starch can be modified using acid hydrolysis. In this study, the effects of acid hydrolysis time on the structural, thermal, and chemical properties of cassava starch and cassava starch thin films were investigated. Native cassava starch was hydrolyzed using 2.2 M hydrochloric acid with varying time intervals. With the increase of hydrolysis time, the relative crystallinity of cassava starch increased while the thermal decomposition temperature decreased in cassava starch. XRD and 13C-NMR spectrums results show that, cassava starch has been subjected to polymorphism changes from A (monoclinic cell) to B (hexagonal unit cell) due to the acid hydrolysis treatment. FTIR and TGA analysis showed that, the moisture absorbance of cassava starch decreased significantly as a result of the acid hydrolysis treatment. The acid hydrolyzed samples showed up to 31.79 % reduction of moisture content compared to the native starch samples. Also, the films prepared from acid hydrolyzed starch showed up to 25.12% reduction of water absorption compared to that prepared from native starch. Acid hydrolysis treatment significantly impacted the mechanical behavior of starch-based films. The tensile strength of cassava starch-based films improved continuously with acid hydrolysis time, reaching a maximum of 5.67 MPa. However, the ductility of the films was negatively impacted when the starch was subjected to acid hydrolysis. The films prepared using acid hydrolyzed starch showed a 12.78 % reduction of elongation compared to that prepared with native cassava starch, despite with minimum dependence of acid hydrolysis time.


Introduction
Starch is one of the most abundant polysaccharides found in plant storage organs such as seeds and swollen stems.
Granules of starch are semi crystalline particles which contain two homopolymers known as amylose and amylopectin.
Starch granule size, morphology, composition, and supramolecular structure are highly dependent on the plant source (Parker and Ring, 2001;Bertolini, 2010). Some of the main starch sources are corn, cassava, wheat, and potato. Components of cassava starch depend on the geographical locations, variety, and the age of the particular plants ( Morgan and Choct, 2016). Cassava starch typically contains 17 % of amylose and 83 % of amylopectin (Morgan and Choct, 2016). The physicochemical properties of starch depend on the ratio among amylose and amylopectin ( Corcuera et al., 2007 ;Sasaki et al., 2000;Duan et al., 2012). When compared with amylose, usually, amylopectin is a highly branched molecule and larger than amylose in size (Tang et al., 2006).
Starch plays a signi cant role as a raw material for the production of bioplastic, paper, textile, and in the food industries (Whistler et al., 2012). Hence, it is an excellent opportunity to add value to cassava which is grown in the tropical environment without any fertilizer (Wahyuningtiyas and Suryanto, 2017). Despite the fact that, native starch is used because of its thickening and gelling capacities, for a number of applications, properties of native starches fail to meet process or product requirements (Waterschoot et al., 2014). It is a well-known fact that, native cassava starch is not suitable for industrial applications due to its poor mechanical and thermal properties. Therefore, in order to avoid these natural limitations of native starch, starch modi cation is of high importance. Among widely used starch modi cation techniques, physical methods (moisture, heat, high pressure, and radiation), chemical methods (acid hydrolysis, cross-linking, esteri cation, etheri cation, etc.) and enzymatic methods play an important role (Chen et al., 2018;Shrestha and Halley, 2014 ;Bemiller, 2018). Acid hydrolysis is performed using either sulfuric acid or hydrochloric acid (Gerard et al., 2002). The acid modi cation changes the physical and chemical properties of starch in an effective way (Bemiller, 2018). Vermeylen et al., (2004) reported that acid hydrolysis has caused the changes of polymorphic transitions in the tapioca starch and these changes depend on the particular source of starch and the degree of acid hydrolysis. The ndings of Sakkara et al., (2019) indicate a reduction of hydrophilicity and moisture content of maize starch lms prepared under acidic pH. Zhang et al., (2019) have prepared the acid hydrolyzed pea starch and increase of tensile strength and reduction of water vapor permeability have been observed. Most of the previous studies on acid hydrolysis of starch were focused on the physiochemical changes with the acid hydrolysis temperature, stirring time, and acid concentration. However, none of these studies have investigated the impact of acid hydrolysis on both thermo-mechanical properties and water absorption of cassava starch based thin lms. Therefore, in this study, cassava starch was modi ed using hydrochloric acid (2.2 M) at different stirring times (30, 60 and 90 mins). The changes of structural, thermal and mechanical properties of the lms were investigated. The aim of this research was mainly focused on discovering appropriate acid hydrolysis conditions to prepare cassava starch-based lms with improved water resistance and thermo-mechanical properties.

Materials
Conc. HCl and NaOH were purchased from Sigma Aldrich Co. (St. Louis, MO). All the reagents used were of analytical reagent grade and used without further puri cation. Amylose and Amylopectin were purchased from Sigma Aldrich (Merck Group, Germany).

Extraction of the cassava starch
Cassava starch was extracted in the laboratory according to the wet method described by Benesi et al., (2004). The extracted -dried cassava starch was passed through a 100-mesh sieve before using for further experiments. Moisture, protein, fat, ash, and the crude ber content of the cassava starch were determined using Association of O cial Analytical Chemists (AOAC) 920. 36, 984.13, 948.22, 923.03 (2000), and AOAC 962.09(2005) standards, respectively. All the analyses were carried out in triplicate. The percentage data depicted in Table 1 are the means of triplicates.

Acid hydrolysis
In order to obtain acid hydrolyzed cassava starch, the method described by Chung et al., (2003)  X-ray diffraction (XRD) The X-ray diffraction patterns were obtained for native cassava starch and acid hydrolyzed cassava starch, using a diffractometer (Rigaku Ultima IV) with the Scintillation counter detector at 40 kV and 30 mA with K-α lter. The XRD spectra were recorded over an angular range (2θ) of 2 to 45 o and the scan rate was maintained at 2 o /min. The relative crystallinity (RC) of scattering spectra was calculated as the ratio of the sharp peaks (crystalline peaks) to the total peaks (Both crystalline and Amorphous line) shown in Eq. 1 according to the method described by the (Utrilla-coello et al., 2014).
Where Ac is the Value of the area under the curve corresponding to the crystalline portion and At is the total area of the diffractograms Solid-state Carbon-13 nuclear magnetic resonance ( 13 C-NMR) The 13 C CP-MAS solid-state NMR spectra were obtained by using a Bruker Avance III 400 NMR spectrometer. The samples were spun at a rate of 5 kHz in a 4 mm zirconia rotor. The 13 C CP-MAS NMR spectra were recorded using a Total Suppression of Spinning Sidebands (TOSS), pulse sequence. The chemical shifts were externally referenced to 176.03 ppm for the carbonyl carbon of glycine.

Starch Granules Morphology
Native cassava starch and modi ed starch granules morphology were studied using Scanning Electron Microscope (SEM) (ZEISS, Germany). The morphologies were evaluated at 2500× magni cation and samples were coated with gold, and then examined at an acceleration potential of 10 kV. Thermogravimetric Analysis (TGA) Thermal degradation and moisture content of all the samples were evaluated using the TGA 5500 (TA instruments, USA) under the nitrogen atmosphere. 100 µl platinum crucibles were used with a heating rate of 10 o C /min while maintaining the temperature range from 25 o C to 650 o C (Stawski, 2008). Thermal degradation temperature and the moisture content were calculated using the TRIOS software. Fourier Transforms Infrared spectroscopy (FTIR) Characteristic peaks for the cassava starch and modi ed cassava starch were obtained using FTIR Spectrum two (PerkinElmer, USA) equipped with ATR re ectance cell. Spectra were observed in the ranging 500-4000 cm -1 . For each sample, 4 scans were taken at a resolution of 4 cm -1 . FTIR spectra were analyzed using the Origin software.

Preparation of Cassava starch based thin lms
Thin lms were prepared by using the method adapted by Belibi et al., (2014). Initially, native and acid hydrolyzed cassava starch (5 % (w/w) total lm solution) was dispersed separately in distilled water with the corresponding content of glycerol (30 wt.% on the dry starch basis) at room temperature and stirred for 10 minutes on a magnetic stirrer. After that, mixtures were stirred at 80 o C for 45 minutes. Then the lms were obtained by casting the hot suspension into petri dishes. These dishes were left at room temperature for 6 hours to allow bubbles to dissipate. Then, the samples were dried in an oven with air circulation at 65°C for ve hours. The dishes were kept in a desiccator and nally, the dry lms were removed from the dishes.

Mechanical Test
Mechanical property measurements (Tensile strength, and Percentage Elongation at break) were obtained according to After 2 hours, all the specimens were removed from water, dried with a cloth, and immediately weighed (M 1 ). The water absorption data of the lms were obtained by soaking them in water for 2 hours. After that, those samples were soaked in water for another 24 hours and weighted (M 2 ). The water absorption capacities of the thin lms were calculated as follows.
Results And Discussion Starch is a semi-crystalline natural polymeric material where it has both amorphous and crystalline regions. The amorphous regions of starch granules mainly consist of amylose and some branches of amylopectin, while the crystalline areas are made out of amylopectin (Bertolini, 2010). The relative crystallinity of starch depends on the environmental conditions of the cultivation which determines the ratio between amylose and amylopectin (Stawski,2008). Lemos et al., (2018) andBeninca et al., (2013) have reported that the relative crystallinity of cassava starch as 17.5 % and 22.65%, respectively. However, the relative crystallinity of Sri Lankan cassava starch has not been reported yet. In this study, the relative crystallinity of Sri Lankan cassava starch determined by XRD was 23.87%.  -coello et al., 2014). Note that these four peaks are visible for both native and acid hydrolyzed cassava starch samples. As indicated in Fig. 1 and Table 2, cassava starch granules subjected to acid hydrolysis increases the relative crystallinity along with the hydrolysis time. Similar results were reported by Zhang et al., (2019) where the relative crystallinity increased in pea starch with the hydrolyzed time. The crystalline lamella is more resistance to chemical reactions than the amorphous region and this phenomenon re ects the crystallinity data shown in Table 2. According to XRD data, the highest crystallinity (33.3 %) was observed for the acid hydrolyzed cassava starch sample hydrolyzed for 90 mins.
Usually, acid hydrolysis causes the change of crystalline polymorphs of both A and B type starch (Wang et al., 2015). These acid hydrolyzed cassava starch samples show B-crystallites compared to that of their native cassava starch peaks according to the data depicted in Table 2. Therefore, it is conclusive that, acid hydrolysis could improve not only total crystallinity of cassava starch, but also the crystallinity level of B-crystallites. This improvement in crystallinity is demonstrated by the increased peak intensity at ∼31 ˚ (2θ) as depicted in Fig. 1. Moreover, Garcia et al., (1996) and Vermeylen et al., (2004) have reported the change of polymorphs of cassava starch from A to B, and it has been well matched with the present work-study. Two hypotheses have been proposed in order to describe this change of polymorphism in this study. The rst one is, some A-type crystallites could be metastable, and the removal of a part of Atype crystallites may cause the reorganization of remaining chains into more stable crystalline B-type (Fig. 2). The second one is based on the assumption that, there might be some B-type polymorphs in native cassava starch that are too small to be detected by XRD (Wang et al., 2015). Carbon-13 Nuclear Magnetic Resonance ( 13 C-NMR) Molecular order in starch granules can be determined by using both X-ray diffraction and 13 C solid-state NMR (Gidley, 2014).
However, by using XRD, it is not able to detect either the double helices or whether they are packed in the short-range distance or not. 13 C solid-state NMR is mostly used to analyze the molecular structure of starch granules at the single chain (amorphous) and double helix (ordered) (Gidley and Bociek, 1985). 13 C solid-state NMR of C-1 resonance gives information about the crystalline and the non-crystalline nature of the starch chains. Although the information about the structure type of the starch granules is given by the multiplicity (Triplet or doublet) of the C-1 resonance, the 13 C-NMR resonance for C-1 (90-110 ppm) is related to the crystalline structure of starch granules. A-type starch shows a triplet at C-1 resonance (The two-fold axis generates the double helix and the C-1 peak in Atype starch spectra is a triplet). B-type starch shows a doublet at C-1 resonance. Although for C-type starch, C-1 resonances mainly depend on the relative proportion of A-and B-type polymorphs (Atichokudomchai et al., 2004), Fig. 3 shows that the native cassava starch consists only of A-type starch (hexagonal arrangement of the double helices) (Gidley, 2014). Several signi cant changes have occurred in the molecular structure of starch granules during acid hydrolysis. It can be monitored by using 13 C-NMR spectra (Fig. 3). In the C-1 region of the spectrum, (90-110 ppm) it shows the doublet which indicates the changing of crystalline structure into B type with the variation of acid hydrolysis time. The signal at 81-82 ppm (C-4) indicates the amorphous regions of cassava starch. Changes in the 13 C -NMR spectra also occur in the C-2, C-3, C-4 and C-5 regions (68-78 ppm), where the signals have become increasingly sharper. The same observation was also reported by Atichokudomchai et al., (2001) for tapioca starch.

Starch Granules Morphology
The average diameter of the native cassava starch granules obtained from SEM ranged from 12 to 20 µm and regular ovalor sphere-like shapes with smooth surfaces (Fig. 4). AH 0.5, AH 1.0 and AH 1.5 (see Fig. 4, b, c and d) and b) samples exhibited surface erosion on the surfaces of the starch granules. Note that the smaller sizes cassava granules showed the fragmentation and deformation due to the acid hydrolysis. According to the Fig. 4with the increased acid hydrolysis time, the surface erosion and fragmentation increased. As previously mentioned, it is caused by the attack of acid to the amorphous regions. It is suggested that, the amorphous regions on the periphery of the granule were preferentially attacked by acid during the acid hydrolysis treatment process.
Thermogravimetric Analysis (TGA) The thermogravimetric (TG) and corresponding differential thermogravimetric (DTG) pro les of native and acid hydrolyzed cassava starch samples are shown in Figure 5.  Table 3) decreased with increase of acid hydrolysis time due to the reduction of thermal stability of cassava starch during acid hydrolysis. Thermal stability mainly depends on the crystallinity and the amylopectin content of the starch (Miao et al., 2011). Liu et al., (2009) has also reported a decrease in the decomposition temperature with the decrease of amylose content. According to our results, we also believe that, the reduction of amylose content by the acid hydrolysis could be the main reason for the decrease of both decomposition temperature and weight loss.

Fourier Transforms Infrared spectroscopy (FTIR)
FTIR spectrums of acid hydrolyzed starch were compared with the native cassava starch as shown in Fig. 6. According to

Biodegradability test
The biodegradation rate with the hydrolsysis time is shown in Fig. 8. In soil, water diffuses into the cassava starch chain causing swelling and enhancing biodegradation due to increases in microbial growths. Then microorganisms attack and consume the cassava starch leading to a fracture of the lm structure. Also, the burial environment parameters (temperature, pH, moisture, nutrition) have a great impact on the biodegradation of lms (Seligra et al., 2016). According to the data in The reason is due to the reduction of the amorphous region as explained previously.

Water absorption
Starch is sensitive to water and this behavior limits their industrial applications. However, water absorption of starch materials is of high importance to promote their biodegradability. The bio-based materials must be hydrophobic for industrial applications but they must absorb water after using, to promote degradability since; most microorganisms are e cient in high moisture environments (Nguyen et al., 2016). There was a signi cant positive correlation between acid hydrolysis time and water absorption. The water absorption of lms is demonstrated in Fig. 9. Native cassava thin lms have high water uptake rates at 2 hrs. and 24 hrs. immersed time periods. The most remarkable result to emerge from the data is that, the acid hydrolysis has created a positive impact on the reduction of water absorption. The reason is due to the reduction of the amylose amount in starch. It leads to a lot of industrial applications such as biodegradable lm preparation for packaging materials. Comparison with other studies

Conclusions
This study has investigated the effect of acid hydrolysis treatment on the structural, thermal, and mechanical properties of  Page 17/18 TGA/DTG curves for the native (CS) and acid hydrolyzed cassava starch (AH 0.5, AH 1.0 and AH 1.5 are the 30, 60 and 90 mins stirred cassava starch at 45 oC, respectively.

Supplementary Files
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