Optimisation of the Preparation of Sweet Potato Resistant Starch by Dry Heating with Pectin

Abstract

The technological conditions for preparing sweet potato resistant starch (RS) by utilising the synergy of dry heating with pectin were optimised. By applying a response surface method, the influences of concentration of pectin, dry-heating treatment time, and dry-heating treatment temperature on starch anti-digestibility of sweet potato starches were analysed. The results showed that the optimal technological conditions were as follows: the content of pectin, dry-heating time, and dry-heating temperature were 2.18%, 2.09 h, and 121.73 °C, respectively. The RS content in sweet potato starches prepared under these conditions was within 18.48% ± 0.73%. The result obtained through differenti0al scanning calorimetry (DSC) revealed that the heat enthalpy (ΔH) of sweet potato starch increased from 2.475 to 3.698 J/g after being subjected to dry heating with pectin. Moreover, the result of in vitro digestibility tests suggested that the glycemic index of sweet potato RS prepared through dry heating with pectin was 69.09.

Introduction

Resistant starch (RS) cannot be degraded, digested, and absorbed by amylase in the human digestive tract but can be alcoholised by microorganisms in the colon. RS show various effects, including regulating blood glucose, and preventing cardio-cerebrovascular disease, colon cancer, and rectal cancer. As a novel functional food ingredient with low calorific value (LC), RS has become a research focus among those interested in functional foodstuffs (Chung and Augustin, 2010; Zeng et al., 2015; Al-Mana et al., 2018; Gabriel et al., 2018).

Starches can be divided into three types – RDS, SDS, and RS – which are distinguished according to the difference required for their digestion. Hyperglycaemia likely occurs after RDS is digested and absorbed by the human body to increase in vivo blood glucose level. For some glucose-sensitive patients, such as those with diabetes mellitus and hypertension, the digestion and absorption of RDS likely triggers metabolic syndrome. SDS is digested continuously after an initially rapid phase of digestion, so it can maintain the characteristics of hyperglycaemia. RS is not absorbed in the small intestine but only reacted with microorganisms in the colon to generate short chain fatty acids through fermentation: this is beneficial to human health, therefore, SDS and RS can both improve the nutritional quality of carbohydrates.

Dry heating as a method for physically modifying starches is used to heat starches to render them anhydrous or relatively anhydrous (i.e., with a gravimetric moisture content of less than 1%) in a dry environment. Dry heating is a promising production method for modifying starches due to its advantages, i.e., having no residual chemical reagents, and being both safe and clean (Ke et al., 2019; Lee et al., 2017; Oh et al., 2018). In recent years, it has been found that blending starches and edible gum in dry-heat conditions can increase the viscosity of gelatinised starches, delay starch retrogradation, and improve food quality or the stability of food systems (Lim et al., 2002; Lim et al., 2003; Su et al., 2018). Pectin exhibits advantages such as its abundance, proven safety, absence of need for chemical residues, and non-polluting nature (Chaliha et al., 2018). Previous research has revealed that blended pectin and starches show favourably slow digestible properties after being subjected to dry heating (Bao et al., 2018; Oh et al., 2019; Chen et al., 2019).

At present, the research on preparing starches by exploiting the synergy of dry heating with pectin mainly focuses on improving the quality of application of starches. By contrast, the research on increasing anti-digestibility of starches prepared by the above synergy is sparse. In the test, by taking sweet potato starches as raw materials, based on a single-factor experiment, the technological conditions for preparing RS from sweet potatoes through the synergy of dry heating with pectin were optimised by utilising a response surface analysis. It is expected to provide a reference for industrial production of RS in sweet potatoes.

Materials and Methods

Reagents    Sweet potato starch was provided by the Saiwengfu Agriculture Development Limited Company, Shanghai. LM pectin (DE value is 42) was provided by the Tang Ruisi Food Materials Company (Wichita, USA). α-amylase and glucoamylase were kindly provided by Fuyuan Biological Science and Technology Limited Company, Zhengzhou, Henan. All the other chemicals used were of analytical grade.

Preparation of RS in sweet potatoes through synergy of dry heating with pectin    Pectin (2.0 g) and distilled water (84 mL) were put into a conical flask and stirred until the pectin was completely dissolved. Sweet potato starch (49 g) was then added to the uniformly mixed solution, and the mixture was stirred for 1 h at room temperature (25 °C). The solution was then oven-dried at 40 °C until its moisture content was less than 10%. This dried blend was then dry-heated for 2 h at 120 °C in an oven to obtain the dry-heat treated sample. The residue was then ground and filtered through a sieve with an aperture size of 0.154 mm (Chen et al., 2016). The starches treated by use of dry heating without addition of pectin were considered as the b-sps control group.

Measurement of digestibility of starches    During the test, 200 mg of starch samples were weighed and put into a test tube, in which 15 mL of 0.2 mol/L sodium acetate buffer (pH 4.8) was added. The mixture was gelatinised for 10 min in a boiling bath and then cooled at room temperature. Afterwards, 10 mL of porcine pancreas α-amylase (290 U/mL) and glucoamylase (15 U/mL) were added into the cooled mixture and then vibrated (at 150 rpm) in a thermostatic water-bath at 40 °C. After separately being subjected to hydrolysis for 20 min and 120 min, 0.5 mL of hydrolysate was taken out into which 4 mL of anhydrous ethanol was added for enzyme deactivation. Subsequently, the mixture was centrifuged and the glucose content in the supernate was measured by using the DNS method. The mass fractions of rapidly digestible starches (RDS), slowly digestible starches (SDS), and RS in samples can be calculated by using the following formulae:   

  

  

where: G₂₀ and G₁₂₀ denote the amounts (mg) of glucose generated by hydrolysing starch solution with the amylase for 20 and 120 min, respectively; FG denotes the content (mg) of free glucose in starches before enzymatic hydrolysis; TS denotes the total starch content (mg) in samples.

Optimisation of technology for preparing RS through synergy of dry heating with pectin    Based on a single factor experiment, response surface optimisation was undertaken by taking the content of RS (RS%) as the response value. By taking three factors (pectin concentration (%), dry-heating time (h), and dry-heating temperature (°C)) as reference factors, a central composite design was used. Moreover, by taking X₁, X₂, and X₃ as representatives, the experimental design based on the response surface method was undertaken by utilising the Box-Behnken method in Design-Expert (Version 8.0.6) software (Qi et al., 2018). The details are summarised in Table 1.

Table 1. Variables and their levels for central composite design.

Variable	Symbol	Coded-variable level
		−1	0	1
Pectin concentration (%)	X1	1	2	3
Dry-heating time (h)	X2	1	2	3
Dry-heating temperature (°C)	X3	110	120	130

Measurement of thermodynamic properties of starch samples    During the test, 0.5 g of starch samples to be measured were weighed, to which 1 mL of distilled water was added to prepare uniform starch suspension with a concentration of 30%. Afterwards, 8 to 12 mg of starch suspension samples were placed in a stainless crucible and thermodynamic properties of starch were determined with a differential scanning calorimeter (DSC, Q200, TA Instruments, USA). The cuvette was placed on the sample holder of the instrument after pressing the samples therein and sealing them (an empty stainless crucible was used as a control). Finally, the measurement was conducted according to the experimentally designed process. The test parameters were as follows: a heating rate of 10 °C/min, a temperature-scanning range of 20 to 100 °C, and a rate of injection of nitrogen of 50 mL/min. The characteristic parameters for calculating heat included T_o, T_p, T_c, and ΔH.

Measurement of rate of digestion of starches    During the test, 0.2 g samples of sweet potato starches were weighed and put in a test tube, to which 15 mL of sodium acetate buffer (at a pH of 5.2) was added. After uniformly stirring the mixture, 10 mL of porcine pancreas α-amylase (290 U/mL) and glucoamylase (15 U/mL) were added. Afterwards, the test tube was synchronously heated and vibrated (at 150 rpm) in a thermostatic water-bath at 37 °C. The reaction process was ended after hydrolysis for 0, 30, 60, 90, 120, 150, and 180 minutes, respectively. Next, the mixture was stood and allowed to cool to room temperature. Subsequently, 1 mL of reagent was removed to measure the reducing sugar content by using 3,5-dinitrosalicylic acid colorimetry (Englyst et al., 1992). Moreover, the rate of hydrolysis of starches was calculated according to the following formula:   

where, G_t denotes the amount (mg) of glucose generated after hydrolysis with amylase for time t.

Statistical analysis    Statistical analysis was carried out using SAS 8.0. All measurements were repeated three times. Statistical analysis was carried out using Turkey method. Differences were considered significant at p < 0.01. Data were plotted using Origin 8.0 software (Origin Lab, Northampton, USA).

Results and Discussion

The influence of synergy of dry heating with pectin on digestibility of sweet potato starches

The influence of the pectin concentration on digestibility of sweet potato starches    According to the above experimental methods, sweet potato starches prepared through synergy of dry heating with pectin were prepared. The influence of the pectin concentration on the in vitro digestibility of sweet potato starches is shown in Table 2.

Table 2. Influence of the pectin concentration on digestibility of sweet potato starches.

Pectin concentration (%)	RDS (%)	SDS (%)	RS (%)
0	69.58±0.8a	21.87±1.0c	8.15±1.1c
1	59.31±1.1c	27.18±0.9ab	13.31±1.0ab
2	55.34±1.0c	28.82±1.1a	15.44±0.8a
3	60.88±1.5b	26.57±0.6b	12.35±0.9b
4	62.18±1.7b	25.83±0.4b	11.79±1.2b
5	63.28±0.7b	25.17±0.8b	11.35±1.0b

It can be seen from Table 2 that the RDS content in starch samples treated by dry-heating was higher than that subjected to dry-heating with pectin. This indicated that the starch samples processed by dry-heating with addition of different masses of pectin can decrease the rate of starch digestion This was probably because the contact probability between amylase and starches was reduced by dry-heating treatment of starches blended with pectin, thus causing that a part of the amylase to be obstructed. As a result, the RS and SDS contents in starches prepared through dry-heating with pectin both increased. On condition that the pectin concentration was 2%, the anti-digestion rate of starches was greater than that (7.29%) under b-sps. When the added amount of pectin was excessively high, the anti-digestion rate of starches gradually decreased.

The influence of pH of blends on digestibility of sweet potato starches    The influence of pH of blends on the in vitro digestibility of sweet potato starches is summarised in Table 3.

Table 3. Influence of pH of blends on digestibility of sweet potato starches.

pH	RDS (%)	SDS (%)	RS (%)
b-sps	69.58±0.8a	21.87±1.0b	8.15±1.1c
5	63.35±0.9b	23.38±1.3b	12.87±1.2b
7	55.34±1.0c	28.82±1.1a	15.44±1.8a
9	63.12±1.2b	23.05±1.1b	13.43±1.0ab
11	67.67±0.6a	21.78±0.8b	10.15±0.7bc

As shown in Table 3, b-sps starches contained a high RDS content, implying that the samples showed a rapid digestion rate within 20 min in the in vitro digestion test. The amounts of RDS in starch samples treated by synergy of dry-heating with pectin were all less than those under b-sps. At a pH of 7, the SDS content was the lowest. Compared with b-sps, the contents of SDS and RS being subjected to synergy of dry-heating with pectin both increased. When the pH was 7, the SDS and RS contents in starch samples were the highest. This indicated that the starch samples in this condition can most effectively hinder amylase from entering the interior of the starch granules, thus decreasing the contact area between amylase and starches, showing the strongest resistance to enzymatic hydrolysis; however, when constantly increasing the pH, the RDS content in starch samples gradually increased while the SDS and RS contents gradually decreased.

The influence of dry-heating time on digestibility of sweet potato starches    The influence of dry-heating time on in vitro digestibility of sweet potato starches is summarised in Table 4.

Table 4. Influence of dry-heating time on digestibility of sweet potato starches.

Dry-heating time (h)	RDS (%)	SDS (%)	RS (%)
b-sps	69.58±0.8a	21.87±1.0c	8.15±1.1b
1	61.93±1.0cd	27.81±0.9ab	9.86±0.7b
2	55.34±1.0e	28.82±1.1a	15.44±0.8a
3	60.06±1.1d	24.96±0.8bc	14.58±0.6a
4	65.65±0.6b	23.66±1.0c	10.29±1.1b
5	64.36±0.7bc	24.95±1.1bc	10.29±0.9b

It can be seen from Table 4 that RDS content in the blend of pectin and starches decreased at first and then increased with increasing dry-heating time while SDS and RS contents exhibited a contrary trend. As dry-heating continued, the digestion rate of RDS, that is, the percentage of RDS digested within 20 min during in vitro digestion simulation was the lowest at a dry-heating time of 2 h while the SDS content (digested within 20 to 120 min) was higher than those in other starch samples. The highest RS content was found in starch samples by dry-heating the blend of pectin and sweet potato starches for 2 h, that is, that content of the part of the starch samples which failed to be digested by the human body after rapid and slow digestion was the highest. This implied that there was an optimal resistance to enzymatic hydrolysis when the dry-heating time was 2 h.

The influence of dry-heating temperature on digestibility of sweet potato starches    The influence of dry-heating temperature on the in vitro digestibility of sweet potato starches is summarised in Table 5.

Table 5. Influence of dry-heating temperature on digestibility of sweet potato starches.

Dry-heating temperature (°C)	RDS (%)	SDS (%)	RS (%)
b-sps	69.58±0.8a	21.87±1.0b	8.15±1.1c
100	66.47±1.0b	23.13±1.2b	10.00±0.5bc
110	63.82±1.1b	24.63±0.8b	11.15±0.9bc
120	55.34±1.0c	28.82±1.1a	15.44±0.8a
130	63.68±0.9b	23.34±0.8b	12.58±1.0b
140	65.53±1.0b	23.64±0.9b	10.43±1.1bc

As shown in Table 5, the dry-heating temperature affected the in vitro digestibility of sweet potato starch samples: with increasing dry-heating temperature, the anti-digestion rates of sweet potato starch samples prepared by dry-heating with pectin all increased at first and then decreased. The highest RS content was found at a dry-heating temperature of 120 °C, indicating that starch molecules were bonded so closely with pectin molecules that it inhibited the access of amylase, thus generating more RS. As the dry-heating temperature was constantly increased, the RS content in starch samples gradually decreased, which implied that an overly-high temperature was unfavourable for generating RS.

Optimisation of the technology for preparing RS from sweet potatoes by synergy of dry-heating with pectin

Experimental design based on response surface method and results    Response surface optimisation was designed according to the scheme of central composite design based on Box-Behnken. Each test was conducted three times and the mean values calculated. The experimental design and result, as well as an analysis of variance, may be seen in Tables 6 and 7.

Table 6. Design and results of central composite test.

	Variable level			Response
	X1 (%)	X2 (h)	X3 (°C)	RS (%)
1	3	2	110	16.57
2	2	2	120	18.74
3	3	3	120	16.75
4	1	2	110	15.37
5	2	3	110	16.83
6	1	3	120	15.45
7	1	2	130	15.48
8	3	2	130	17.28
9	1	1	120	15.58
10	2	2	120	19.10
11	2	1	130	16.48
12	3	1	120	15.83
13	2	2	120	18.48
14	2	1	110	15.72
15	2	3	130	17.83

Table 7. Analysis of variance results of quadratic regression model and factor.

Source	Sum of squares	df	Mean square	F value	Prob > F
Model	28.37	9	3.15	22.18	0.0002
Residual	0.99	7	0.14
Lack of Fit	0.72	3	0.24	3.53	0.1273
Pure error	0.27	4	6.8×10−2
Cor total	29.36	16
	R2=0.9628

According to the result of variance analysis obtained through use of a quadratic regression model (Table 7), the regression coefficient R² was 0.9628 and the lack of fit showed P = 0.1273 which was greater than 0.05, indicating that the error of the regression model was low.

Determination of technological parameters for preparing RS from sweet potatoes through synergy of dry-heating with pectin    The comprehensive regression analysis and simulation was conducted based on Design Expert software. On this basis, it can be obtained that the optimal technological parameters for preparing RS from sweet potatoes through synergy of dry-heating with pectin were shown as follows: a pectin concentration of 2.18%, a dry-heating time of 2.09 h, and a dry-heating temperature of 121.73 °C. For ease of operation, the aforementioned parameters were modified to: 2.2%, 2.1 hm and 122 °C, respectively.

To verify the rationality of the prediction result, the test was repeated three times at optimal preparation conditions as obtained through comprehensive analysis by means of response surface optimisation. The mean RS content was 18.48% ± 0.73%, which was consistent with the predicted value (18.868%). This indicated that the equation was favourably fitted with actual conditions, which validated the correctness of the established model.

Thermodynamic properties    The influence of synergy of dry-heating with pectin on thermodynamic properties of sweet potato starch pastes is shown in Figure 1 and Table 8.

Fig. 1.

Influence of synergy of dry-heating with pectin on thermodynamic properties of sweet potato starch pastes.

Table 8. Influence of synergy of dry-heating with pectin on thermodynamic properties of sweet potato starch pastes.

Sample	ΔH(J/g)	To (°C)	Tp (°C)	Tc (°C)	Tc-To (°C)
sps	2.475±0.02	61.12±0.03	71.25±0.00	80.76±0.13	19.64
b-sps	2.959±0.05	61.47±0.04	70.27±0.00	79.86±0.22	18.39
pec-sps	3.698±0.03	63.08±0.06	72.52±0.00	84.04±0.27	20.96

Note: sps, sweet potato starch; b-sps, control group; pec-sps, sweet potato resistant starch by dry heating with pectin

T_o and T_c represent the melting points of the weakest and strongest microcrystals in the amorphous region of starch granules, respectively. The difference between T_c and T_o reflects the diversity of crystals found in the starch granules. The greater the difference, the more diverse the crystals (Hyunjung et al., 2009). According to Figure 1 and Table 8, compared with native starches, T_o, T_c and ΔH of starches treated through dry-heating all increased; however, the gelatinisation temperature range (T_c − T_o) decreased compared to that of native sweet potato starches. The reason was that the dry-heating temperature was greater than the gelatinisation temperature, which caused the redistribution of the weakest microcrystal beams in the amorphous region of these starches, moreover, a high temperature can cause the double-helix structure of starches to unwind (Juansang et al., 2012). The initial, peak, and final gelatinisation temperatures, as well as the gelatinisation enthalpy, of starches processed through dry-heating with pectin all increased. This indicated that dry-heating expanded the crystal region of sweet potato starch granules to increase the degree of polymerisation of the starches and binding force in starch granules. Starch gelatinisation required absorption of more additional heat and therefore the gelatinisation temperature increased and the gelatinisation temperature range was broadened.

Determination of in vitro digestion rate    The in vitro digestion rate of sweet potato starch by dry-heating with pectin is shown in Figure 2.

Fig. 2.

In vitro digestion rate of sweet potato starches.

It can be seen from Figure 2 that the rate of hydrolysis of white bread was generally taken as a standard against which to compare the rates of digestion of starches among various samples. The digestion rate of native sweet potato starches at 30 min was slightly higher than that of white bread. After 30 min, their rates of digestion were similar, which indicated that, like white bread, native sweet potato starches were also easily hydrolysed by amylase and the glucose thus generated was easily absorbed in the human body to trigger hyperglycaemia. Compared with native starches, the starch samples treated by dry-heating showed a relatively slow rate of hydrolysis and the rates of hydrolysis in different stages were all lower than that of native sweet potato starches. After 90 min, the rate of hydrolysis tended to be stable, which was mainly because dry-heating damaged the internal spatial structure of such starches, resulting in breaking of molecular chains therein, therefore, moisture was able to penetrate, leading to swelling-induced fracture of the starch granules and the precipitation of amylose. Thereafter, the cooled amylose was re-crystallised while its crystals exhibited a strong resistance to digestive enzymes. Starches were not readily digested by enzymes and therefore there was a high RS content. Under the synergistic effect between pectin and dry-heating, the pectin molecules interacted with the amylose precipitated during dry-heating to form a more complex system, with a strong resistance to enzymatic hydrolysis, therefore, the rate of hydrolysis of the starches was low.

Table 9 lists the extent of hydrolysis and predicted glycemic index of gelatinised starches. The experimental result showed that the glycemic index of native sweet potato starches was higher than those of starch samples separately treated by dry-heating and synergistic dry-heating with pectin, which was 5.09 and 30.49 higher than those under b-sps and pec-sps. According to the rule of classification of glycemic index, native starches and starch samples treated through dry-heating all belonged to foodstuffs with a high glycemic index, moreover, the starch samples treated by synergy of dry-heating with pectin were subordinated to foodstuffs with a moderate blood glucose content, which can be moderately added into foodstuffs depending upon their end-consumer.

Table 9. The hydrolysis percentage of sweet potato starch sample (H₉₀) and predicted glycemic.

Sample	H90	GI
sps	75.18±1.2a	99.58
b-sps	56.39±1.5b	84.49
pec-sps	37.21±0.9c	69.09

Conclusions

By utilising a response surface analysis technique, the technological conditions for preparing RS from sweet potatoes through synergy of dry-heating with pectin were optimised. The test result obtained through DSC showed that the initial, peak, and final gelatinisation temperatures, as well as the gelatinisation enthalpy, all increased for sweet potato starches treated through the aforementioned synergy. The test result concerning in vitro digestion rate revealed that the rate of hydrolysis of sweet potato starches treated by synergy of dry-heating with pectin decreased and the predicted glycemic index was less than 70. Thus, synergy of dry-heating with pectin can improve the RS content in sweet potato starches.

Acknowledgements    This work was financially supported by the National Natural Science Foundation (Nos.31771941).

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