The effect of dual modification with annealing and Heat Moisture Treatment (HMT) on physicochemical properties of jack bean starch (Canavalia ensiformis)

In this study, the effect of the dual modification with annealing and Heat Moisture Treatment (HMT) on the physicochemical properties of jack bean starch was investigated. Native jack bean starch was modified by the annealing method with a moisture ratio of 1:2 and 1:3 at 50°C, 55°C, and 60°C for 24 hrs. Furthermore, the annealing method was combined with HMT at 30% moisture at 110°C for 16 hrs. The results showed that the dual modification process decreased the whiteness, amylose content, swelling power, solubility, setback viscosity, and breakdown viscosity of the jack bean starch. On the contrary, it led to an increase in the water adsorption, paste clarity, pasting temperature and stability ratio of the starch. The stability ratio of native jack bean starch, the sample treated by the single annealing, and the dual modification were 0.56, 0.62, and 0.86, respectively, indicating that the sample treated with dual modification was more stable at high temperature and shear stress. The dual modification did not affect the crystalline x ray diffraction patterns. The findings emphasized the potential of using a dual modification to alternate the characteristic of jack bean starch.


Introduction
Starch, a polysaccharide that consists mainly of amylose and amylopectin stored in the form of granules, is widely used in the food industry (Cabrera-Canales et al., 2020). It was reported that in 2018 the income from the starch industry was approximately 7.7 billion (Yao et al., 2019). Thus, the exploration of the novel potential source of native starch is still being carried out by scientists (Sudheesh et al., 2020). Jack bean (Canavalia ensiformis) is a potential source of starch and worth investigating due to its abundance and availability, particularly in Africa, Asia, the West Indies, Latin America, and India (Akande, 2016). Jack bean contains high carbohydrates (about 60%), making it a valuable source of starch for the industry. However, native starch is mostly unstable at high temperature or shear force, and also relatively easy to gelatinize and form a gel. These characteristics do not meet industrial needs (Yao et al., 2019). Hence, modification of starch is required mainly to fine-tune its characteristic in order to use it for a specific purpose in the food industry, including as a thickener, gelling agent, binder, stabilizer, emulsifier, or encapsulation material (Cabrera-Canales et al., 2020).
Hydrothermal modification using annealing and Heat Moisture Treatment (HMT) has been widely used to modify starch since these methods do not destroy the starch granule. Annealing is a physical modification in the presence of excess water at a temperature above the glass transition but below the gelatinization temperature, while HMT is a physical modification of the starch with a moisture content of 10-30% at a temperature above the gelatinization temperature (da Rosa Zavareze and Dias, 2011).
According to Kittipongpatana and Kittipongpatana (2011), annealing can reduce the swelling power and solubility of the starch and improved starch stability at high temperatures and shear force. In the other study by Chung et al., (2009), HMT improved paste properties of the starch, starch molecular interaction, and crystallinity of the starch.
Although many studies investigated the effect of annealing and HMT individually, there is still a lack of information about the effect of both combinations of annealing and HMT, particularly on jack bean starch. Thus, the objective of this study was to investigate the effect of the dual modification on the physicochemical

Starch extraction
Jack beans were obtained from the local market in Surakarta (Central Java, Indonesia). The extraction of jack bean starch in this study followed the method of Ríos-Ríos et al. (2016) with minor modifications. Briefly, 500 g of jack beans were soaked in sodium metabisulfite at a concentration of 0.45% for 24 hrs at a ratio of 1 : 3 (w/v). Afterwards, it was peeled and milled to reduce particle size and form jack bean paste. The jack bean paste was then squeezed and precipitated for 24 hrs to obtain wet starch. The wet starch was washed three times with distilled water and then dried in a cabinet drier at 40°C for 24 hrs. The dried starch was crushed and sieved to 100 mesh size.

Hydrothermal modification
Starches were subjected to one-step annealing with the method of Ríos-Ríos et al. (2016) with slight modification. Native jack bean starch (60 g) was mixed with distilled water 120 mL (ratio 1:2), and 180 mL (ratio 1:3) in a sealed glass container. The samples were then incubated at 50°C, 55°C, and 60°C for 24 hrs in a water bath. After the incubation, the samples were decanted and air-dried at 40°C for 24 hrs. After that, the starches were crushed and sieved to 100 mesh size. The jack bean starch was obtained by only the annealing method and is considered single-annealed jack bean starch.
The modification process was then continued by the HMT method. HMT was performed based on the method of Chatpapamon et al. (2019). Accurately-weighed of annealed jack bean starch (40 g) were put into a petri dish. The moisture content of the starch was increased to 30% by adding the appropriate amount of distilled water. The petri dish was then sealed and incubated at 4°C for 24 hrs. Afterwards, it was placed in the oven at 110°C for 16 hrs. Finally, the samples were air-dried at 40°C for 24 hrs, milled and sieved to get 100 mesh size dualmodified jack bean starch.

Chemical analysis
The Standard Association of Official Analytical Chemistry method, AOAC (2012), was adopted for determining moisture, amylose, and amylopectin contents of native, single-annealed and dual-modified jack bean starch.

Colour analysis
The colour analysis method was carried out using a colour reader (CR-300, Kinoca Minolta, Japan). Based on the obtained L*, a*, and b* values, the whiteness index (WI) was calculated using Equation 1.

Swelling power and solubility
Swelling power and solubility analysis was conducted according to the protocol of Kittipongpatana and Kittipongpatana (2011). Briefly, 0.1 g of starches were placed on a test tube and then mixed with 10 mL of distilled water using a vortex for 1 min. The samples were then heated at 95°C for 10 mins in a water bath and then followed by air-cooling to reach room temperature. Afterwards, centrifugation at 3000 rpm was performed for 15 mins. Aliquots (5 mL) of the supernatant obtained from centrifugation were dried to a constant weight at 120°C. The swelling power of the samples was calculated using Equation 2 while the solubility was calculated using Equation 3.

Water adsorption
Water adsorption analysis was carried out following the method of Ríos-Ríos et al. (2016). Shortly, 0.1 g of starch was diluted with 10 mL distilled water and then incubated at room temperature for 30 mins. After that, the suspension was centrifuged at 2120 rpm for 15 mins and then weighed. The amount of water that was absorbed by starch is described as water adsorption.

Paste clarity
Paste clarity analysis was done referring to the method of Reddy and Seib (1999). Jack bean starches (0.5 g) were with 5 mL of distilled water in a test tube and followed by shaking at 1500 rpm for 30 secs using a vortex. The samples were placed in a water bath at 95°C for 30 mins. After cooling to an ambient temperature, the absorbance was measured at the wavelength of 650 nm using a spectrophotometer (Shimadzu model UV-3600, Shidmazu Corp, Kyoto, Japan).

X-ray diffraction
X-ray diffractogram of the native and modified starch was obtained from X-ray diffractometer D8 Advance, Bruker AXS, Billerica, MA, the USA at 40 kV at the scanning rate of 1 o /min, at the diffraction of 2Ɵ = 10 o -30 o (Wang et al., 2014).

Pasting properties
Pasting properties were determined using a rapid visco-analyzer (RVA) Super 3 (Newport Scientific, Warriewood, Australia) following the protocol of Wang et al. (2014). Jack bean starch (3 g) was added into RVA canisters and then added with 25 mL of distilled water. Peak Viscosity (PV), Breakdown Viscosity (BD), Trough Viscosity (TV), Pasting temperature, Final Viscosity (FV), and Setback Viscosity (SB) were obtained from the RVA data.

Statistical analysis
Data were analysed statistically using analysis of variance (ANOVA) using SPSS V.20 (IBM, USA). The DMRT test, with a significance threshold of ρ < 0.05, was used to compare the difference among mean values. Table 1 and Figure 1 show the colour properties of the native and modified starch. Native starch exhibited L*, a*, b*, and whiteness values of 90.48, 0.53, 17.17, and 80.36, respectively. After single annealing and dual modification were performed, the whiteness and lightness substantially decreased. As shown in Table 1, this phenomenon is because the values of a* and b* substantially increased, and therefore significantly higher (p < 0.05) than native starch. Modification using the single annealing and the dual modification decreased the whiteness of jack bean starch to a range of 66.8 and 71.7. These results are consistent with the results reported by Suriya et al. (2019) who work with elephant foot yam starch. It was reported that the dual modification of jack bean starch resulted in significantly lower whiteness values than single annealing. A similar phenomenon has also been reported for sago and potato starch (Hrynets et al., 2017).

Colour properties
According to Suriya et al. (2019), the decrease in the whiteness of modified starch may be due to the Maillard reaction during hydrothermal treatment. As jack bean starch contained amino acid, sugar reduction, and protein, the Maillard reaction was indeed possible (Kittipongpatana and Kittipongpatana, 2011). Furthermore, in the dual modification, Heat Moisture Treatment was also involved causing a nonenzymatic reaction between hydroxyl groups and amino acid, therefore resulting in the browning of the polymer. The heating process also induced a Maillard reaction, and thus an increase in heating temperature resulted in a decrease in the whiteness of the starch (Hrynets et al., 2017). As colour is generally an important attribute in food (Muhammad et al., 2020), the change of the colour of the jack bean starch during the modification process should be taken into account by the food industries.

Chemical properties
As presented in Table 1, the moisture content of the native jack bean starch in this study was 10.56%. The result was lower than the moisture content of the native jack bean starch in the study of Lawal and Adebowale (2005), which was about 12.56%. The different moisture content of the starch might be affected by different geographical and starch sources and also the starch production method. Modification could reduce the moisture content of the jack bean starch. The application of dual modification resulted in significantly lower moisture content than a single annealing method. The dual modification resulted in moisture content in the range of 3.48-3.86%, while the single modification resulted in moisture content in the range of 9.57-9.83%. The high temperature during the modification process could have a significant impact on the reduction of moisture content during modification (Obi et al., 2016).
In this study, the single and dual modification had just a limited effect on the amylose content and thus the amylopectin content of jack bean starch, which remained at around 25%. Notwithstanding the report of Chung et al. (2009), the modification could reduce the amylose content of corn starch by about 0.2-0.9% depending on the modification method. A decrease in the helical complex may cause a decrease in amylose content as a result of limited amylose that can form long helical from  Table 2 presents the swelling and solubility values of the native and modified jack bean starches. The result showed that the native starch had a swelling power value of 8.57 g/g. The single annealing and the dual modification caused a decrease in swelling power values. The dual modification resulted in a more pronounced swelling power reduction to around 3.67-4.33 g/g compared to the single modification (5.68-6.34 g/g). The modification is known to lower the swelling power of starch due to the interaction of amylose-amylose and amylose-amylopectin as well as crystalline perfection, which might decrease the swelling ability (da Rosa Zavareze and Dias, 2011). This result is in accordance with a study by Chung et al. (2009) reporting the reduction of swelling power of corn starch after modification using the combination of annealing and HMT.

Swelling power and solubility
Besides the swelling power, the modification resulted in a lower solubility. The solubility of native starch was recorded at 7.27%, while the single annealed and the dual modified starch had a solubility of about 5.1 -5.7% and 4-4.3%, respectively. This finding is in agreement with the previous reports on annealed corn starch, annealed rice starch, and the dual-modified corn starch (Chung et al., 2009;Dias et al., 2010;Ariyantoro et al., 2018). Annealing caused a reorientation of the starch granules, and an increase in the interaction of amylose and amylopectin made starch granules more rigid, and thus decreased the solubility of the jack bean starch. The solubility of the starch is influenced by amylose content, amylose-lipid complex, amylose-amylose interaction, and amylose-amylopectin interaction. In the dual modification, HMT additionally led to a reinforcement interaction between amylose and the crystalline area, increasing the crystalline complex. HMT also caused an expansion of the amorphous region. The amorphous region caused by starch remains undissolved, and thus this situation can decrease the solubility of the starch Rafiq et al., 2016).

Paste clarity
The clarity of the jack bean starch paste increased after the single annealing, and the dual modification treatment from 62.9% to around 74-77% (Table 2). This finding is in agreement with the previous studies focusing on the modified corn starch and the modified barley starch Ariyantoro et al., 2018). Paste clarity of the starch is influenced by the water absorption capability (WAC). A high WAC value resulted in a high transmittance of the starch (Gani et al., 2010). As also shown in this study, an increase in WAC was also directly proportional to an increase the paste clarity. The hydrothermal modification increased the paste clarity, which is strongly correlated with the solubility of the starch. The decrease in solubility caused an increase in paste clarity because of the limited granules leached out and also due to the interaction between amylose-amylose and amylose-amylopectin (Gani et al., 2010). Swelling power also influenced the clarity of the jack bean paste. A decrease in swelling power led to a lower retrogradation, and subsequently, a higher transmittance of the starches (Mendes et al., 2018).

Sample
The ratio of native jack bean and water

Water absorption
Water absorption is defined to be the amount of water needed for starch to swell. It can also be defined as the percentage of water that could be absorbed by the starch (Takagi et al., 2017). Modification with single annealing and combined annealing-HMT caused an increase in water absorption ( Table 2). The water absorption of the starch obtained by the dual modification (171.40 -187.56%) was higher than that of single annealing (120.41 -155.38%) and native starch (112.19%). As afore-mentioned, annealing increased the crystalline complex on starch granules.
Interestingly, the increase in water ratio and temperature of the annealing process linearly increased the water absorption. This phenomenon occurred in both modified starch prepared by single annealing and dual modification. According to Adebowale et al. (2005), the increase in water absorption is correlated with the increase of binding sites and interaction between the amorphous region and the crystalline region Furthermore, in the dual modification, HMT caused an expansion of the amorphous region, broke the hydrogen bonds making the water easily penetrate, and thus increased the water absorption of the starch granules (Rafiq et al., 2016). It has indicated that the hydrophilic tendency increased with hydrothermal modification annealing and HMT . Figure 2 illustrates the XRD patterns of the native and modified jack bean starches. The native and modified starches showed a characteristic C-type diffraction pattern, with a rate peak 2θ at 15.39° and 23.29° and a double peak at 2θ = 17.42° dan 18.23°. In general, the crystallinity of starch contained 10-20 glucose. The XRD pattern shows a double helix on crystalline polymorphs as A-type and B-type. The C-type starch is a combination of A-type and B-type. The crystallinity of the starch is influenced by size granule, double helix orientation, and double helix interaction (Wang and Copeland, 2015). The result obtained in this study indicated that annealing and HMT did not alter the diffraction pattern of jack bean starch. The C-type XRD of jack bean has also been previously reported in other studies . As the XRD pattern of native and jack bean starches remained unaffected after treatment, this result confirms the study of Suriya et al. (2019), stating that HMT did not change the crystalline structures of starch. A similar result was also found on the dual modified -HMT corn starch studied by Chung et al. (2009). According to Singh et al. (2009), the XRD pattern is influenced by amyloseamylopectin chains, lipid content, amylose-lipid complex, and size granules. Table 3 presents the pasting properties of the native and modified jack bean starches. The native jack bean starch had a pasting point at 82.45°C. The temperature was slightly increased to about 83-87°C after the single annealing and dual modification were applied. The dual modification resulted in a higher temperature point than the single annealing modification and native starch. The Breakdown Viscosity (BV) value of native starch was observed at 749 cP, while the BV values of the starch were modified with annealing at native starch and a water ratio of 1:3 combined with HMT treatment was at the lowest point at 490 cP. This point was obtained by the annealing temperature at a temperature of 50 and 55°C

Pasting properties
. This result indicated that starch was more stable at high temperatures and shear force. The comparable results were also obtained in modified cassava, and pinhao starch (Klein et al., 2013). This phenomenon could be possible because the dual modification could strengthen the interaction between amylose and amylopectin. This is also strongly correlated with the   Marta and Tensiska (2017).
Trough Viscosity (TV) and Final Viscosity (FV) parameters tended to increase after the starch modification. However, a decrease in the TV and FV value was still shown, particularly in the starch modified by dual technique with native starch and water ratio of 1:3 at 50°C. The TV value was recorded at 630 cP which was lower than the TV value of the native starch (749 cP). A previous study by Cahyana et al. (2019) also reposted a decrease in the TV value of modified banana flour. Lower in TV value indicated that the starch is more stable on shear force because it strengthens the interaction between amylose-amylopectin causing a decrease in swelling power value (Wang et al., 2014;. The stability ratio (SR) of native jack bean starch was 0.56. The highest SR value was 0.86, which was obtained by the annealing technique with native starch and water ratio of 1:3 at 50°C combined with HMT. The SR value, calculated by TV/PV, indicates the stability of starch to heat and shear stress. Starch with an SR value lower than 0.65 is unstable under high temperature and shear stress (Shafie et al., 2016). Hence, the modified starch obtained in this study has high stability on high temperature and shear stress. The SR values were indeed affected by the TV and PV values of the starch. The modification decreased both the TV value from 1702 cP to 1120 cP and the PV value from 630 cP to 953 cP.
Furthermore, it was shown that single and dual modification also decreased the Final Viscosity (FV) and Setback Viscosity (SB) values of jack bean starch. For instance, the SB value of native jack bean starch was 2658 cP). The dual modification resulted in a more pronounced reduction of SB value than the single annealing modification, which was at the level of 420 cP -2091 cP and 525 cP-2789 cP, respectively. Similar results were also obtained on the modification of corn starch (Chung et al., 2009). According to , starch with lower SB values has a lower retrogradation ability. Also, a decrease in SB value indicated the existence of intact granules that is resistant to fragmentation, thus improving the stability of the granules (Chung et al., 2009). The combination of annealing and HMT decreased the SB values more effectively than the single annealing modification. SB value is dependent on various factors like swelling power capability, size granules, and amylose content (Klein et al., 2013).

Conclusion
In conclusion, the effect of dual modification of annealing and Heat Moisture Treatment was more pronounced than the single annealing method regardless of the ratio of native jack bean and water as well as the modification temperature. The dual modification can be employed to increase the gelatinization temperature, through Viscosity and peak viscosity of jack bean starch. The dual modification caused a decrease in Breakdown, Setback Viscosity, and Final Viscosity without changing its crystalline type pattern. The modified jack bean starch prepared by dual modification with starch and water ratio of 1:3 treated at 55°C had a better pasting property. The starch was identified to have high stability on high temperature and shear stress as indicated by SR values of 0.86. The dual modification can be considered an effective method to improve the stability of jack bean starch, and thus the modified starch can be potentially used in the food industry.