The effects of dual modification on functional, microstructural, and thermal properties of tapioca starch

Abstract The aim of this study was to investigate the effects of dual modification on the functional, microstructural, and thermal properties of tapioca starch. Tapioca starch was first hydrolyzed by 0.14 M HCl for 0, 6, 12, 18, and 24 hr and then hydroxypropylated by adding 0%, 10%, 20%, and 30% (v/w) propylene oxide. The degree of hydroxypropylation, solubility, water absorption, rheological, thermal, and microstructure characterization of dually modified tapioca starch was determined. Hydroxypropylation did not cause any considerable changes in the starch granular size and shape of tapioca starch. Acid hydrolysis disrupts the starch granules, and the starches with smaller sizes were produced. The degree of molar substitution (DS) of dual modified starches ranged from 0.118 to 0.270. The dual modified starches significantly had higher solubility than native starch (p < .05). Hydrolysis of starches with acid decreases swelling power while hydroxypropylation increases the swelling power. The results also showed lower gelatinization (To, Tp, Tc, and ΔH) and pasting parameters (the peak and final viscosity, peak time, and pasting temperature) for the dual modified starches than other treatments. In summary, this study showed that dually modified tapioca starch has potential application in dip molding and coating.

(WHC), they face limitations in industrial applications (Azaripour & Abbasi, 2020;Russ et al., 2016). Due to the high number of free hydroxyl groups on the structure of starch, these compounds can be easily modified by physical, chemical, biotechnological, or enzymatic methods as well as their combination (Ashogbon, 2020). These modifications are done to improve the texture, adhesion, gel clarity, film making, cooking properties, transparency of gel, freeze-thaw stability, reduction of retrogradation, the tendency to gel, and syneresis (Mehfooz et al., 2019;Zhang et al., 2021).
Chemical modification of starch structure includes cross-linking, acid hydrolysis, oxidation, acetylation, grafting, esterification, and dual modification (Haq et al., 2019), which are most used in industry due to easier control (Araghi et al., 2015;Li et al., 2018). Chemical modification of starches can be performed by a single method or a combination of different modification methods. The different properties of the chemically modified starches depend on their botanical source, the nature and number of substitutions, and reaction conditions (Chen et al., 2018).
One of the most practical chemical methods to modify the starches is acid hydrolysis, which improves the different properties of starch, such as improving the resistance to retrogradation, increases water solubility, modification of gelatinization temperatures, and increases hydroxyl groups. This method is often used in the dual modification of starch (Basilio-Cortés et al., 2019). This method is done by the controlled addition of an acid to an aqueous suspension of starch at a temperature in the normal temperature range slightly below the gelatinization temperature for a certain period (Hoover, 2000). In industry, acid hydrolysis is used to manufacture resistant starches and gelling agents (Aparicio-Saguilán et al., 2007).
Hydroxypropylation (HP), which is a chemical modification method, reduces the hydroxyl group's number in the structure of starch and weakens the hydrogen bonds between the granules. Increasing the degree of hydroxypropylation can increase the freeze-thaw stability, hydrophilicity, concentration, and clarity of paste (Fouladi & Mohammadi Nafchi, 2014;Park & Kim, 2020).
Today, dual modification of starch structure has attracted the most attention because studies have shown that often the use of single modification methods is not complete and effective to improve the application and processing properties of native starches (Fakharian et al., 2015;Li et al., 2018;Oladzadabbasabadi et al., 2017). Atichokudomchai and Varavinit (2003) investigated the effect of dual modification of tapioca starch by acid hydrolysis and cross-linking on pharmaceutical tablets and observed that the tablets prepared by dual modified starch were better than those prepared by acid-modified starch. The researchers also found that the use of dual modification (hydroxypropylation and cross-linking) improved the properties of taro starch better than using each of these modification methods alone (Hazarika & Sit, 2016).
Due to the confirmation of the effect of chemical modifications on improving the characteristics of starches, this study was conducted to investigate the effect of dual modification of tapioca starch by acid hydrolysis-hydroxypropylation on its physicochemical, rheological, and thermal properties.

| Dual modification of tapioca starch by acid hydrolysis-hydroxypropylation
In order to dual modification of tapioca starch, acid hydrolysis (AH) was performed first, followed by hydroxypropylation (HP). For acid hydrolysis of tapioca starch, about 40% of starch solution was prepared by adding 400 g of starch to a solution of 0.14 N hydrochloric acid at 50°C, and the final weight was 1,000 g. The resulting suspension was placed at 50°C for different periods of time (6, 12, 18, and 24 hr) to prepare starch with different molecular weights. The resulting suspension was stirred in a rotary incubator (Jeio Tech SI-. 600R, South Korean) at 200 rpm to prevent the formation of sediment. After certain periods of time (6, 12, 18, and 24 hr), the starch solutions were neutralized with sodium hydroxide (1% NaOH) until their pH reached 5.5. The samples were washed with distilled water (twice their volume), and then, they were filtered through filter paper (Whatman No. 4). The starch samples were placed in an oven (Memmert, Germany) at 40°C overnight to dry completely (Abdorreza et al., 2012).
To prepare hydroxypropylated tapioca starch, 20% w/v sodium sulfate was added to the 20% w/v hydrolyzed starch solution and stirred. Using 5% sodium hydroxide, the pH of the solution was adjusted above 10.5. Propylene oxide was added as an etherifying agent in different proportions (10%, 20%, and 30% (based on the dry weight of starch)). The samples were then capped and stirred at 20-25°C for 30 min. The resulting solution was then stirred in a rotary incubator at 200 rpm to prevent the formation of sediment. After that, using 10% hydrochloric acid, the pH of the suspension was reduced to 5.5. Samples were immediately washed with distilled water until their sulfate content was negative. By the time the moisture content reached about 10%, the samples were dried in an oven at 40°C. After grinding the starch samples, they were passed through a sieve with a size of 250 µm (Aminian et al., 2013).

| Investigation of the granular structure of starches
Scanning electron microscopy (SEM) (Leica Cambridge, England) was used to determining the appearance of starch granules. First, a very small amount of the sample (about 1 g) was glued to a circular metal base made of aluminum, and the surface of the sample was covered with a layer of gold by passing the sputter coater device through a stream of argon gas. The samples were then examined under a microscope with an electric potential of 15 kV (Majzoobi et al., 2012).

| Determination of molar substitution (MS)
In order to estimation of molar substitution, 100 mg of the modified sample was poured into a 100-ml volumetric flask, and 25 ml of 0.5 M sulfuric acid was added. The resulting mixture was placed in a water bath until the color of the solution was light. The solution was immediately cooled and then reached 100 ml with distilled water.
1 mg of the solution was pipetted into a 25-ml graduated test tube.
The test tube was placed in hot water and then transferred to an ice bath. 0.6 ml of ninhydrin solution (3% of ninhydrin in 5% Na 2 S 2 O 5 ) was added to the tube and stirred well. It was then placed in a water bath at 25°C for 1 hr. Finally, with concentrated sulphuric acid, it reached a volume of 25 ml. The resulting solution was transferred to a spectrophotometer (Shimadzu, Japan) cell, and its absorbance was read at 590 nm. MS of starch was calculated as follow: where w is the equivalent hydroxypropyl in 100 g of starch, and M is the molecular weight of C 3 H 6 O (Fouladi & Mohammadi Nafchi, 2014).

| Measurement of solubility and water uptake of starches
In order to measure the solubility of starch in water, 1 g of sample was weighed and poured into a 50 ml plastic centrifuge tube. Then, 30 ml of distilled water was added to the tubes, and tubes were kept in a Bain-marie at 95°C for 30 min with stirring. The sample temperature was brought to room temperature by cold water for 5 min and centrifuged (Hettich Mikro R220, Germany) at 700 g for 15 min. The supernatant clear liquid was carefully transferred to a container of specified weight and dried at 120°C for 4 hr. After drying, the samples were weighed, and the water solubility was calculated using the following equation (Abdorreza et al., 2012): To determine the water uptake of starches, the centrifuge tubes used in the solubility test were weighed along with the sediments deposited in them after the supernatant was removed, and the amount of water uptake was calculated using the following equation

| Investigation of starch pasting properties
Changes in the viscosity of aqueous solutions of starch during heating were investigated by a rapid viscosity analyzer (RVA) (Newport Scientific, Warriewood, Australia). 4 g of starch sample was added to 25 g of distilled water. The desired speed was 960 rpm in the beginning and 160 rpm at the resting stage of the sample. In this experiment, the starch was first kept at 50°C for 1 min and then rapidly increased to 95°C (at a speed of 14°C per minute) and remained at this temperature for about 3.3 min. It was then cooled to 50°C for 4 min and finally kept at this temperature for 2 min. The pasting properties studied in this study included pasting temperature (°C), time to reach peak viscosity (min), peak viscosity (cP), and final viscosity (cP) (Karim et al., 2008).

| Investigation of thermal properties of starches
Thermal properties of starch samples, including onset temperature (To), peak temperature (Tp), ending temperature (Te), and gelatinization enthalpy (ΔH), were determined using a differential scanning calorimeter (DSC) (DSC-Q100, TA Instruments, New Castle, DE, USA). For this purpose, the suspension of starch in distilled water in a ratio of 3:1 (w/w) was weighed in a high-pressure resistance steel container for the device and kept at room temperature for one day to reach equilibrium. Then, the container containing the sample was placed inside the DSC device, and while heating the sample from 10 to 115°C, the sample was scanned. An empty container of the device was used as a control. The thermal properties of starch samples were calculated and reported from the DSC curves (Majzoobi & Beparva, 2013).

| Statistical analysis
Analytical data from tests were analyzed by using one-way analysis of variance (ANOVA) and Duncan multiply range test to identify the significant difference between samples at p < .05 using SPSS software version 22.0.  F I G U R E 2 Hydroxypropyl substitution of native and acid hydrolyzed tapioca starches. Bars represents of substitution mean ± SD (n = 3) of hydroxypropyl group. Different letters show significant difference at 5% level of probability. The numbers after "HP" represent the propylene oxide ratio (%), and the numbers after "AH" represent the time (hours) of acid hydrolysis

| Molar substitution of hydroxypropyl groups (MS)
In this study, the reaction efficiency of hydroxypropylation and acid hydrolysis was investigated by the degree of molar substitution of hydroxyl groups inside the starch granules. MS involves measuring the average number of hydroxyl groups per unit of anhydroglucose produced by substitution groups (Karim et al., 2008). The

| Water solubility of modified tapioca starch
The starch solubility values of native and modified tapioca starch samples are given in Table 1

| Water uptake of modified tapioca starch
The amount or strength of water absorption indicates the hydration capacity and the rate of reaction between the starch chains in the amorphous regions and the crystalline regions. Table 1 shows the water uptake percentage values of native and modified tapioca starch samples. As the results demonstrated, the hydroxypropylation process led to a significant increase in water uptake by starch granules and increasing the oxidizing agent from 10% to 30% also significantly increased water uptake percentage (p < .05). However, acid hydrolysis of starch samples showed an opposite effect on water uptake, so that increasing the time of hydrolysis significantly reduced the amount of water uptake of starch samples (p < .05). The lowest water uptake percentage TA B L E 1 Water solubility and water absorption of tapioca starches (native and modified) The values are mean ± SD (n = 3). Different letters show significant difference at 5% level of probability between values in same columns. The numbers after "HP" represent the propylene oxide ratio (%), and the numbers after "AH" represent the time (hours) of acid hydrolysis.

Reduction of water uptake of starch granules by acid hydrolysis
can be attributed to partial degradation of granule structure by acid treatment. Because due to the destruction of starch granules, their water holding capacity is reduced, and as a result, the water uptake is reduced. Increasing the water uptake of hydroxypropylated starches is due to the fact that due to the entry of hydroxypropyl groups into the starch chains, intramolecular and intermolecular hydrogen bonds are destroyed, and the water uptake of granules increases. Higher water uptake in hydroxypropylated starches is also related to the hydrophilic nature of hydroxypropyl groups (Pal et al., 2002). Sriburi et al. (1999) found that the presence of ascorbic acid on starch caused a series of changes, including a decrease in the swelling strength of granules, followed by a decrease in its water uptake.
However, Okunlola and Akingbala (2013) demonstrated that acid hydrolysis of Chinese sweet potato starch increased the water uptake and solubility of starch granules. Mehboob et al. (2021) also found an increase in the water holding capacity of sorghum starch due to the introduction of hydroxypropyl groups into the starch structure. Increased water uptake percentage of different starches due to the hydroxypropylation process has been reported by different researchers (Karim et al., 2008;Park & Kim, 2020;Tehkhunmag et al., 2008).

| Effects of modification on pasting properties of tapioca starch
The pasting properties of native and modified tapioca starch samples are compared in was reduced by dual modifying the structure of the starch (p < .05).
The effect of hydroxypropylation process on reducing the peak viscosity was greater than the acid hydrolysis so that in hydroxypropylated treatments alone, the peak viscosity was significantly lower than acid hydrolyzed treatments (p < .05). The lowest peak and final viscosities were obtained in HP30AH24 modified starch (2,004.8 and 1,912.5 cP, respectively). Dual modified starches showed lower final viscosity than hydrolyzed and hydroxypropylated starches.  = 3). Different letters show significant differences at 5% level of probability between values in the same columns. The numbers after "HP" represent the propylene oxide ratio (%), and the numbers after "AH" represent the time (hours) of acid hydrolysis.

TA B L E 2 Pasting properties of tapioca starches (natural and modified)
Peak viscosity is defined as the equilibrium point between swelling and solubility (increases viscosity) and the fracture and placement of polymer chains (reduces viscosity). Due to hydroxypropylation of starch, water absorption by granules is facilitated because the hydroxypropyl subgroup is a hydrophilic group and causes more water and starch connections. Increased water uptake and hydrophilic strength after hydroxypropylation increase the amylose release rate mainly in amorphous starch regions, and as a result, the solubility increases (Kaur et al., 2004;Lawal, 2009 The values are mean ± SE (n = 3). Different letters show significant differences at 5% level of probability between values in the same columns. To, onset temperature; Tp, peak temperature; Tc, completion temperature; ΔH, enthalpy. The numbers after "HP" represent the propylene oxide ratio (%), and the numbers after "AH" represent the time (hours) of acid hydrolysis.
TA B L E 3 DSC properties of tapioca starches (natural and modified) inside the starch chains. As a result, viscosity increases due to gel formation, known as the final viscosity (Perera & Hoover, 1999). The results of this study showed that the use of acid hydrolysis reduced the peak and final viscosities and the time to reach the peak viscosity. Smaller molecules often produce less viscosity, so the viscosity reduction was not unexpected due to the reduction in the size of the starch molecules due to acid hydrolysis. According to the findings of Thys et al. (2013), acid hydrolysis reduces the molecular weight of starch and increases free aldehyde groups, thus reducing viscosity and increasing starch solubility. Saberi et al. (2013) similarly showed that hydroxypropylation of wheat and barley starches reduced the peak viscosity, storage time, and pasting temperature of starches. Majzoobi and Beparva (2013) observed that acidic hydrolysis of wheat starch with acetic acid significantly reduced peak and final viscosities and time to reach peak viscosity, which was consistent with the results of the present study. Biduski et al. (2017) also observed that starches modified by acid hydrolysis and oxidation had a lower pasting temperature than natural sorghum starch, indicating starch depolymerization due to acid modification and oxidation.

| Effects of modification on thermal properties of tapioca starch
The thermal properties of native and modified tapioca starch samples are presented in Table 3. As can be seen in the The results of starch enthalpy also demonstrated that the hydroxypropylation led to a significant reduction in enthalpy, and with increasing the ratio of propylene oxide from 10% to 30%, the enthalpy also decreased significantly (p < .05). In general, acid hydrolysis had no significant effect on the enthalpy of tapioca starches.
Enthalpy values of different samples of tapioca starch ranged from 8.75 to 14.40 J/g. The enthalpy of gelatinization is a measure of the quality and quantity of crystals and the loss of molecular order within the granules (Cooke & Gidley, 1992). Low enthalpy actually indicates the structural order and less stability of the crystals. The entry of large groups of hydroxypropyl into the polymeric structure of starch destroys the double helix in the amorphous regions, thereby increasing the structural flexibility and decreasing the melting point (Perera et al., 1997). The hydroxypropylation also destroys intramolecular and intermolecular hydrogen bonds, reducing crystallinity in starch granules, increasing water access to starch granules, and reducing enthalpy (Liu et al., 1999;Woggum et al., 2015). Chen et al. (2021) reported a reduction in gelatinization temperature and enthalpy in dually modified maize starches modified by acid hydrolysis and hydroxypropylation. Lee and Yoo (2011) reported that hydroxypropylated sweet potato starches had lower gelatinization temperatures and enthalpy than native starch. Wang and Shi (2020) found that due to hydroxypropylation of waxy wheat, normal wheat, and waxy maize starches, their gelatinization temperatures and enthalpy significantly decreased. Gunaratne and Corke (2007) also reported that hydroxypropylation reduced the gelatinization temperatures and enthalpy of starch. They attributed this decrease to the fact that excessive amounts of hydrated granules require less force and energy for gelatinization.

| CON CLUS ION
Due to the low water solubility and tendency to retrograde, the use of native starches in industrial applications is often difficult. By introducing hydrophilic hydroxypropyl groups into the structure of tapioca starch, the water solubility and water uptake capacity of the resulting modified starch were significantly improved. Modification of tapioca starch in this study also improved the order of molecular structure and reduced the stability of granules. Therefore, it reduced the force and energy required to gelatinize the starch. According to the results obtained in this study, dually modified tapioca starch can be used in dip molding and coating industries.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author, upon reasonable request.