3.1. Pasting profile
The gelatinization temperature of arrowroot starch was determined using RVA by estimating the pasting temperature which follows the gelatinization temperature. The pasting temperature was indicated by the changes in viscosity before, during, and after the gelatinization process 36. The viscoamylograph (Fig. 1) indicates that the onset gelatinization temperature of arrowroot starch was 75.10°C. This result was in line with the report of 27 and 24 which showed gelatinization temperature of arrowroot starch of 74.8°C and 72.3–85.4°C, respectively. However, some authors also reported higher or lower temperatures. 23 revealed onset, peak, and final gelatinization temperature of 63.9, 71.0, and 81.3°C, respectively. While, 37 showed results of gelatinization temperature of 81.23–82.36°C. Variety of starch, the condition, and growing location of plants of the same species may results in different properties of starch 38. PS production with enzyme hydrolysis was conducted below the gelatinization temperature to assure enzyme activity on starch granules while maintaining its integrity during enzymolysis, This way, enzymatic activity on starch granules was ensured while maintaining its integrity during enzymolysis, enlarging the surface area by forming pores in the starch granules 2. Thus, hydrolysis temperature was determined at 60°C.
3.2. Effects of α-amylase concentration and incubation time on water and oil adsorption capacity of porous arrowroot starch
Before the optimization of PS production by CCD, a single factorial design of enzyme concentration, and incubation time was performed. Important functional characteristics of PS such as WAC and OAC, which significantly affect its application were determined (Fig. 2). The PS attributes was compared with their respective controls of non-modified starch (NS) which was conducted with the absence of enzyme to rule out the effects of incubation time only.
WAC, Water absorption capacity; OAC, Oil absorption capacity; NS, non-modified arrowroot starch with sodium phosphate buffer solution. Reaction conditions for enzyme concentration variable: reaction temperature 60°C; reaction time 6 h; α-amylase enzyme concentration 50, 100, 150, 200, and 300 U/g. Reaction conditions for incubation time variable: reaction temperature, 60°C; reaction time, 3, 6, 8, 10, and 12 h; α-amylase enzyme concentration 100 U/g. Values reported are mean of triplicate determinations; error bars are standard deviations. Values with different superscripts within an analysis are significantly different (p < 0,05)
Arrowroot PS generally exhibits a higher WAC and OAC than its control. The highest WAC was obtained with an α-amylase concentration of 100 U/g, and incubation time of 3, and 6 h. The increment of PS WAC is approximately 20% compared to the control starch. This rise of WAC is higher than previous report of 16% 39. Additionally, a further increase in enzyme concentration or incubation time shows a reduction in WAC. These results concur with previous findings 11, which reports a similar trend of rising, and reduction in maize PS hydrolyzed with α-amylase.
As for oil absorption properties, the arrowroot PS improved significantly after enzyme treatment, with a maximum value reached under the condition of enzyme activity of 100 U/g, and incubation time of 6 h. PS also exhibits greater OAC than water capacities. This result is higher than those achieved in porous corn starch by α-amylase treatment of approximately 96% 14. These results conform to a study by 40 that revealed an improved OAC on popcorn PS by α-amylase hydrolysis after 3 h, followed by a reduction after a longer incubation time. In the previous study, 11 found that OAC increases along with enzyme concentration increment. However, above 400 U/g of α-amylase results in deduced absorption.
Enhanced adsorption properties of PS may be related to the enlargement of surface area due to pores formation by α-amylase action, allowing water to bind with the granules. Additionally, improving the accessibility of oil with a higher viscosity to enter the pore channel 11,40. Moreover, improved absorption capacities may be related to the hydrophilic/ hydrophobic nature of the starch. 41 suggested that the oil and water absorption of PS is considered as a non-selective physical adsorption, due to its ability to trap, and retains both oil, and water. Thus, excessive decomposition of starch due to an increase in enzyme concentration or incubation time possibly destroys pores and forms larger craters, hindering entrapment and retainment of both water and oil.
3.3. Optimization of porous starch characteristics using CCD
The CCD design was used to evaluate interactions between enzyme concentration (U/g) and incubation time (h) which affected PS characteristics and to obtain the optimized process condition of aforementioned modifier compound. WAC and OAC were chosen as the response factors for optimization to acquire the highest adsorption capacity for arrowroot PS. Based on the results, enzyme concentrations of 50, 100, and 150 U/g, and incubation times of 3, 6, and 9 h were then applied as -1, 0, and + 1 values of the independent variables of the CCD matrix. The experimental design, and its results were presented in Table 1.
Table 1
Experimental data of Central Composite Design for two-factor five-level response surface analyses to optimize arrowroot porous starch production using α-amylase
No.
|
Coded Value
|
Uncoded Value
|
WAC (%)
|
OAC (%)
|
Enzyme Concentration (x1)
|
Incubation Time (x2)
|
Enzyme Concentration (U/g)
|
Incubation Time (h)
|
1
|
-1
|
-1
|
50.00
|
3.00
|
75.20
|
90.72
|
2
|
-1
|
+ 1
|
50.00
|
9.00
|
75.04
|
78.92
|
3
|
+ 1
|
-1
|
150.00
|
3.00
|
78.84
|
86.86
|
4
|
+ 1
|
+ 1
|
150.00
|
9.00
|
58.86
|
85.34
|
5
|
-α
|
0
|
29.29
|
6.00
|
72.68
|
66.22
|
6
|
+ α
|
0
|
170.71
|
6.00
|
69.60
|
105.28
|
7
|
0
|
- α
|
100.00
|
1.76
|
82.96
|
87.72
|
8
|
0
|
+ α
|
100.00
|
10.24
|
61.48
|
77.56
|
9
|
0
|
0
|
100.00
|
6.00
|
100.30
|
105.24
|
10
|
0
|
0
|
100.00
|
6.00
|
97.38
|
93.62
|
11
|
0
|
0
|
100.00
|
6.00
|
95.60
|
105.22
|
12
|
0
|
0
|
100.00
|
6.00
|
95.54
|
98.26
|
13
|
0
|
0
|
100.00
|
6.00
|
93.50
|
102.06
|
The statistical analysis results of the ANOVA indicated a reasonable agreement with the experimental data based on the high values of R2 of WAC (97.99%) and OAC (74.93%). These justify that the experimental model was efficient for the present optimization study. A significant model terms were indicated by p-value \(\le\) 0.05. The pareto chart showed significant terms sorted from the highest to the least effect to the response. It was displayed in Fig. 3 that the enzyme concentration (p-value = 0.000) and incubation time (p-value = 0.000) had strong quadratic relations with WAC. The next significant effect was linear relation of incubation time (p-value = 0.000) with WAC, followed by the interaction of both variables (p-value = 0.007). As for OAC, variables that are considered significant effect by the highest are: the quadratic term of incubation time (p-value = 0.023), linear term of enzyme concentration (p-value = 0.038), and its quadratic term (p-value = 0.048).
To simplify the equation, the insignificant terms which were established by p-value > 0.05 may be eliminated due to minimal statistical and evident effect towards the design of experiments. By applying multiple regression analysis to the experimental data, second-order polynomial equations (Eqs. 6 and 7) were obtained for WAC and OAC to explain PS production.
$${Y}_{WAC\left(\%\right)}=-5.03+1.1628 {x}_{1}+17.26 {x}_{2}-0.005034 {x}_{1}^{2}-1.338 {x}_{2}^{2}-0.03303 {x}_{1} {x}_{2}$$
6
$${Y}_{OAC\left(\%\right)}=29.1+0.724 {x}_{1}-10.58 {x}_{2}-0.0029 {x}_{1}^{2}-0.978 {x}_{2}^{2}$$
7
Where YWAC and YOAC are the predicted yield of WAC and OAC, respectively, x1 and x2 are the coded values of enzyme concentration, and incubation time, respectively, x12 and x22 are the quadratic terms of the coded values, and x1x2 are the interaction of both variables. The response surface plot (Fig. 4) showed the relation between WAC and OAC response by enzyme concentration and incubation time. The plot displayed highest WAC value of > 90% may be achieved by approximately incubation time range of 3 to 7 h, and enzyme concentration range of 75 to 125 U/g. As for OAC, incubation time range of approximately 4–6 h and enzyme concentration of 100–150 U/g indicates highest OAC of > 100%.
From these results, the optimum response was calculated by Minitab statistical software. Optimized condition of 107.86 U/g enzyme concentration and 5.24 h of incubation time was predicted to obtain both maximum WAC, and OAC of 96.85% and 102.15%, respectively, with high composite desirability of 0.9182.
3.4. Characterization of optimized porous starch
The optimum condition PS was then characterized its water (WAC) and oil adsorption capacities (OAC), adsorption capacity of methylene blue (AR), amylose content, swelling power, solubility, morphological analysis through SEM, and crystalline structure through XRD and FTIR. Characterization of arrowroot PS are shown in Table 2.
Table 2
Characterization of arrowroot porous starch on water adsorption capacity (WAC), oil adsorption capacity (OAC), methylene blue adsorption capacity (AR), amylose content, swelling power and solubility.
Sample
|
WAC (%)
|
OAC (%)
|
AR (%)
|
Amylose (%)
|
Solubility (%)
|
Swelling Power (g/g)
|
NS
|
76.65 ± 7.40b
|
69.62 ± 1.98a
|
23.18 ± 1.60b
|
40.76 ± 1.84a
|
7.04 ± 1.85b
|
7.00 ± 0.41a
|
Arrowroot PS
|
96.67 ± 3.24a
|
103.06 ± 2.01a
|
25.01 ± 1.84a
|
36.56 ± 0.85b
|
88.92 ± 1.83a
|
0.04 ± 0.01b
|
Values are in Means ± SD (n = 3). Values followed by different letters within a column are significantly different (p-value < 0.05).
3.4.1. Adsorption capacity
Table 2 present the WAC, OAC, and AR of arrowroot PS and its respective control sample (NS). After enzymatic treatment, the WAC, OAC and AR were enhanced. This result of WAC and OAC are in agreement with reports by 15 which shows increasing WAC in wheat, rice, potato, and cassava PS after enzymatic treatment. Arrowroot PS displayed a higher WAC than maize PS with AA of ~ 80% and similar value to maize PS with glucoamylase of ~ 95% 11. High WAC characteristic of arrowroot PS may support PS application to adsorb or carry water soluble component. An increase in OAC also reported in maize PS after α-amylase treatment 11,41. The high OAC indicates ability of PS to encapsulate oil by simple plating to prevent it from oxidation, in addition to probiotic encapsulation 42. This study found that arrowroot PS showed a higher OAC than other commercial tuber roots PS of previous reports. 43 showed OAC of porous sweet potato starch with AA and glucoamylase modification of ~ 43.84%, while 15 studied OAC of potato and cassava PS of ~ 48% and ~ 67%, respectively.
The increasing AR is in accordance with reports by 34. They suggested that the adsorption capacity of maize starch PS using α-amylase showed better value than that of native maize. The greater adsorption capacity might be related to pores size that formed on the starch granule as a consequence of α-amylase hydrolysis, which allows trapping of methylene blue, considering its rectangular geometry, and small dimension (length x width x thickness 1.6 x 0.84 x 0.47 nm) 34. Additionally, formed pores may increase specific surface area and enhance adsorption sites of dyes, allowing higher AR with mechanism of electrostatic interaction between cationic MB and negative charged surface of pores 44. Nevertheless, the increment of arrowroot PS AR was smaller than in study by 34 of 92.5% of rice PS. Methylene blue was used as a drug or dye model to analyze decolorization performance of PS. This result suggest that arrowroot PS may not be suitable for dyes adsorption, and more appropriate to be used as adsorbent or carrier for hydrophilic and hydrophobic compounds.
3.4.2. Amylose contents
The amylose content result is shown in Table 2. Native arrowroot starch contained 40.76 ± 1.84% apparent amylose, thus classified as high amylose starch 45. This value concurs with previous finding by 19 and 46 of 40.86% and 42.01%, respectively. The action of α-amylase showed evident decrease of amylose. Reduction in amylose content by α-amylase treatment in various starch sources has been reported in several findings by 15,47. These results suggest that the endo-action of α-amylase preferentially attacks the amylose chain, consequently release maltose and glucose 10.
3.4.3. Solubility and swelling power
The solubility and swelling power of arrowroot starch are shown in Table 2. Improving solubility serves as one of the main aims of PS production. A higher index of solubility indicates a better pore structure of PS and easier application of PS 4,11. Enzymatic treatment of α-amylase on arrowroot starch shows significantly improved solubility compared to the non-modified starch Nonetheless, this result reported higher solubility than previous reports by 17) of 82.92% in porous edible canna starch with AA hydrolysis, and 11 of 9.94% in porous maize starch modified with α-amylase. Enhanced solubility may be related to hole-formation after enzymatic treatment of endo-enzyme acting α-amylase, allowing starch granules to expand freely, and release more soluble compounds such as oligosaccharides and dextrin. Moreover, AA action to shorten amylopectin side chain may promote solubilization 4,47.
In regards to swelling power, arrowroot PS exhibits lower swelling properties relative to control samples. The reduced swelling power indicated the stronger binding forces within the granules 48. This result is in agreement with reports by 47 and 11 on rice and maize porous starch with AA modification. This phenomenon might be attributed to: (1) endo-action of AA randomly attacks amorphous region of the starch and reducing amylose content which plays role in granule swelling, (2) weakened overall structure of the starch granules due to enzymatic hydrolysis that formed a porous structure 47, (3) hydrolysis results generate more hydrophobic surfaces at the interior wall of the pores, thus preventing water binding, (4) AA action produces dextrin that is naturally exhibits less swelling property during pasting process 49, and (5) more ordered double-helical segments of amylopectin side chain may be formed by internal molecular rearrangement, consequently preventing water to enter and swell the granules 11.
3.4.4. Morphology of porous starch
The SEM images of NS starch granules (Fig. 5) revealed elliptical and polygonal shapes with large granules sizes, and some small granules were in spherical shapes. Generally, the granules reveal a smooth surface, with few small indentations and truncated shapes are occasionally seen on the granules. These irregular shapes of truncates and dents might be due to natural mechanical defects as also reported in arrowroot starch 27 and tapioca starch granules 26. These structures may serve as weak points in the granules structure owing to its softer edges, thus enabling enzyme penetration into the starch granule interior and promoting susceptibility 28. This truncated and spherical shape of native arrowroot starch granules also reported by 27 in arrowroot starch and by 26 in cassava starch. The size of NS starch granules measured by SEM is shown in Table 3. The size of the granules are similar to that reported by 23 of 8.6–42.0 µm in arrowroot starch.
SEM micrograph displayed that α-amylase successfully produce porous arrowroot starch. Porous arrowroot starch showed surface erosion of starch granules into large and shallow craters, followed by the formation of deep canals into the interior of granules in some pores (Fig. 6). This phenomenon showed hydrolysis pattern of exo-corrosion due to random endo-attacks of AA to α-(1,4) glycosidic linkages, aside those nearby branching points of α-(1,6), thereby resulting in large cavities in PS granules 11. The rather smooth surface may hinder enzyme adsorption to the granules, thus the hydrolysis process occurs from the outside to the central of the granules, creating a large cavity that extends to the center of the granules 1. 39 reported similar pattern in cassava PS through exo-hydrolysis. Additionally, 50 explained that AA tends to erode the surface or penetrate granule through the weak points of the surface, hence the rough surface and large craters.
Enzymatic treatment of α-amylase in arrowroot PS also revealed that native starch hydrolysis was not homogenous. This phenomenon was also mentioned by 35 and 51 in regular rice PS and potato starch PS, respectively. Differences in starch granules morphology, starch granular organization, mode and the concentration of enzyme may contribute to this non-homogenous hydrolysis 51. AA hydrolysis of potato starch was more focused at periphery end and vertical axis, hence creating a large crate-like pores, and in some granules resulting in fissures and surface-pitting, while leaving other granules intact 51.
Table 3 displayed that the pore size increased. However, no significant change was found for granule size. This result may suggest that arrowroot PS with AA produce macropores (> 50 nm) 11.
Table 3
Morphological and crystalline characteristics of NS and porous arrowroot starch
Sample
|
Granule size range (µm)
|
Granule size mean (µm)
|
Pore size range (µm)
|
Pore size mean (µm)
|
Relative Crystallinity (%)
|
R1047/1022
(cm− 1/ cm− 1)
|
NS
|
10.50–35.42
|
23.04 ± 9.24
|
0.41–2.23
|
1.15 ± 0.59
|
29.20
|
1.1679 ± 0.0011
|
Arrowroot PS
|
17.09–34.12
|
25.78 ± 7.11
|
1.02–3.68
|
2.07 ± 1.10
|
28.00
|
1.2017 ± 0.0005
|
3.4.5. Crystalline structure
The crystalline structures of non-modified and PS were determined by XRD and FTIR analysis. The X-ray diffractograms of non-modified arrowroot starch and PS are presented in Fig. 6A. It was found that NS exhibits CA type. This CA type comprise of both A- and B- type crystallinities with a lower B/A polymorph ratio, hence displaying a closer type to A 52. The NS diffractogram shows characteristics of A-type crystalline of strong peaks at 15° and 23° 2θ, a shoulder peak at 17° and 18° 2θ, and weaker peaks at 10°, 11.5°, 20°, 26.5°, and 30.5°. Whilst the B-type crystalline characteristics was evidenced by the peak at 5° 2θ 40,53. This C-type crystalline arrowroot starch is in accord with 21. Although some sources mentioned varied crystallinity types of arrowroot starch, including A-type 18,22,53 and B-type 19,46. These various crystallinity types might be due to environmental differences in the growing plants. Tuber starch grown in an arid area predominantly exhibit an A-type crystalline structure attributable to the release of water out of the crystalline structure and the rearrangement of amylopectin into a more ordered arrangement. Meanwhile, those grown in wet areas tend to show a B-type crystalline structure 37.
As for α-amylase modified porous arrowroot starch, XRD spectra display diffraction peaks at 15°, 23°, doublets at 17° and 18°, and the absence of a peak at 5° which indicate an A-type of crystallinity 40. This showed an alteration of CA- to A-type of crystalline. A small alteration in the average chain length of amylopectin may induce a significant impact in crystal type and crystallinity 2. The weight-average chain-lengths of the amylopectin ranges of short (23–29), intermediate (26–29) and long (30–44) chain-length fraction that respectively represents A, C, and B-type crystallinity 54. AA action on arrowroot starch may reduce the amylopectin chain length from intermediate (C-type) to short (A-type) fraction, thereby the altering crystallinity type from CA to A. Similar phenomenon of AA in decreasing amylopectin long chain and increasing short chain-length fraction was also reported by 18.
Along with the changes of crystallinity type, a slight reduction in peaks sharpness were observed. This result suggested a decrease in relative crystallinity compared to the non-modified arrowroot starch. NS sample displayed relative crystallinity with similar value to study by 53 of arrowroot starch crystallinity of 29.10%. The reduction of crystallinity degree might be due to easier access for α-amylase as endo-enzymes to hydrolyze α-1,4-glycosidic linkages in amylose and amylopectin which both participate in the crystalline structure of starch granules 10. Meanwhile, 55 hypothesize that endo-action of AA allows easier access to the internal structure of starch granule and readily hydrolyze both amorphous and imperfect crystalline region. Thus, resulting in a decrease in crystalline proportion
The FTIR spectra of non-modified arrowroot starch and PS are shown in Fig. 6B. Both reveal characteristics of starch, no difference in the position of characteristic absorption peaks, and no new bands generated. Although, there were changes in the absorption intensities due to pores formation and a decrease in the granular starch density. Similar behavior was reported by 34 in rice PS. These findings indicate that enzymatic action does not alter the type of chemical groups found in native starch molecules or generate new chemical group 34,41,56.
The broad band at 3620 to 3070 cm− 1 is assigned to O-H group absorption. Intensification of this peak in PS indicated higher water absorption, which also shown by increasing WAC of arrowroot PS. Peaks at 2928.38 and 2892.70 cm− 1 signify absorption of C-H group and responsible for lipophilic nature of the material 22,41. In PS, this peak shows intensification and higher OAC than NS. These findings confirm that enzymatic hydrolysis may generate more hydrophilic and hydrophobic groups 40,41. The FTIR spectra also contain other characteristic peaks: 1641.13 cm− 1 (C-O bending absorption associated with O-H group or tightly water bound in the starch), 1450.21 cm− 1 (CH2 symmetric deformation), 1414.53 cm− 1 (C-H symmetrical scissoring of CH2OH moiety), 1337.00 cm− 1 (carboxyl group bending), 1148.40 cm− 1 (C-C, C-O stretching), 1076.09 cm− 1 (C-O functional groups), 992.20 cm− 1 (C-O stretching), whilst 926.63, 860.10 and 761.74 cm− 1 are assigned to C-O-C ring vibration of carbohydrate 30,57. These peaks showed characteristics of starch and was in agreement with reports of FTIR spectra of arrowroot by 58.
The IR absorbances at 1047 cm− 1 and 1022 cm− 1 are characteristic of the crystalline and amorphous structure in starch, respectively. The ratio (R) of 1047/1022 cm− 1 is ascribed as an index of the crystalline to amorphous proportion in the starches and represents as the hydrogen bonds strength and the double-helical structures stability 34,41. Arrowroot PS shows a higher R1047/1022 (Table 3), which conveys better strength and stability due to a higher short-range order and more ordered domain 11,34. The R1047/1022 of non-modified and arrowroot PS are similar to those reported by 41 of 1.17 for native corn starch and 1.22 for corn PS. An increase in R1047/1022 value after enzymatic treatment indicates that enzymatic hydrolysis preferentially attacks the amorphous region of starch granules rather than crystalline regions, in addition that enzymatic susceptibility is higher in the amorphous region 41.