Regular Papers
The Formation of Resistant Starch during Acid Hydrolysis of High-amylose Corn Starch
2013 Volume 60 Issue 2 Pages 123-130
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2013 Volume 60 Issue 2 Pages 123-130
Four types of corn starch (waxy corn starch (WC), normal corn starch (NC), high-amylose corn starch class 5 (HAC class 5) and high-amylose corn starch class 7 (HAC class 7)) were hydrolyzed with 1.5% hydrochloric acid and the resistant starch (RS) content was measured. The acid-hydrolyzed HAC class 5 and class 7 show significantly higher RS content. The change in RS content, X-ray crystallinity, molecular size distribution, thermal property and appearance of HAC class 7 after up to 100 h acid hydrolysis were analyzed. The RS content increased to 69.3 from 40.5% at 16 h and then decreased gradually, while crystallinity continued to increase during acid hydrolysis. The fractionation profiles indicated that with the decrease of the amylose fraction correlating well with the increase in RS content. Granules hydrolyzed for 24 h retained their natural shape, whereas after 100 h hydrolysis granules were damaged. The enzyme-digested residues of native and acid-hydrolyzed HAC class 7 were recovered. The crystalline regions of HAC class 7 acid-hydrolyzed for 24 h were only slightly digested by enzymatic treatment, whereas native and HAC class 7 acid-hydrolyzed for 100 h were extensively digested. Thermal analysis showed the transition peak in high temperature region increased with acid hydrolysis and this peak was also shown in enzyme-digested residues. Acid hydrolysis may influence RS content by two mechanisms: (1) moderate acid hydrolysis increases the crystalline regions and thus resistance to enzymatic digestion of the hydrolyzed granules and (2) excess acid hydrolysis damages granular structure and decreases resistance to enzymatic digestion.
Resistant starch (RS) is defined as the sum of starch and starch degradation products that are not absorbed in the small intestine of healthy individuals. This indigestibility is attributed to the structure of the starch and has been classified into four types. Type-1 RS is starch that is resistant to digestion because it is entrapped in plant tissue and thus is physically inaccessible to digestive enzymes. Type-2 RS is native raw starch granules containing B-type crystals, which are resistant to enzymatic digestion. Type-3 RS is retro-graded amylose, while Type-4 is chemically modified starch which interferes with enzymatic digestion.1) A wide range of health benefits of RS have been suggested such as prebiotic effects and the suppression of glycemic increases.
Various methods have been reported in the literature for analyzing RS content in various foods and starches.2) 3) The Prosky (AOAC 985.29) and related methods (such as AOAC 991.43) are analytical methods for dietary fiber4) 5) and have been used to analyze RS content. However, some of these methods cannot detect RS with precision because some of the RS can be gelatinized and solubilized due to boiling in water during the enzymatic digestion process.3) In contrast, in the AOAC 2002.02 analytical method6) for RS, enzymatic digestion is carried out at 37°C for 16 h and provides results that correlate well with in vivo analysis.
High-amylose corn starch (HAC) is obtained from corn containing the amylose extender (ae) genotype. It is the major source of RS and is classified as type-2 RS. HAC is different from normal or waxy corn starch due to its higher amylose content, crystal structure (B-type) and morphological properties (elongated granules).1) 7)
Acids such as hydrochloric and sulfuric hydrolyze glycoside linkages and modify starch properties such as viscosity and solubility, and thus have been widely used in the starch industry to improve food texture, as well as in the paper industry. These acid-modified starches are typically generated by mild acid hydrolysis by heating 36‒40% solids at 40‒60°C for one to several hours.8) To produce Naegeli’s amylodextrin and Lintnerized starch, starch is treated with 15% sulfuric acid at room temperature for 30 days and with 7.5% hydrochloric acid at room temperature for 7 days, respectively. This removes the amorphous and some of the crystalline regions, resulting in acid-resistant crystalline starch.9) Acid hydrolysis also alters the structure of native starch, namely the molecular mass, X-ray diffraction pattern and crystallinity, susceptibility to annealing, gelatinization transition temperature, and the content of double helices.9)
The enzymatic resistance of a starch granule depends on the granule’s structure, especially the ratio of amylose to amylopectin, the type of crystal and its crystallinity, its granular structure, the retrogradation of amylose, and the chain length of the amylopectin.1) Various treatments have been attempted to increase RS content by changing the starch structure such as hydrothermal treatment, annealing and retrogradation to increase crystallinity and packing density. In contrast, there have been few studies on the effect of acid hydrolysis for increasing the RS content of HAC.10) 11)
In this study the effect of mild acid hydrolysis on the RS content of HAC was extensively analyzed by the AOAC 2002.02 method, as was the structure of RS in HAC.
Materials. Commercial grade waxy corn starch (WC), normal corn starch (NC), high-amylose corn starch class 7 (HAC class 7) and high-amylose corn starch class 5 (HAC class 5) were obtained from J-OILMILLS, Inc. (Tokyo, Japan). HAC class 7 contains 1.3% lipid. All other chemicals were of the highest grade commercially available.
Measurement of the amylose content. The amylose content of these starches was measured using a slight modification of the official Corn Refiners Association Method B-26. Starch sample (100 mg, dry weight basis) was dispersed in 1 mL of ethanol, then 10 mL of 1 N sodium hydroxide was added. The suspension was dissolved completely in a water-bath at 70°C for 1 h and then added water to 100 mL. An aliquot of this starch solution (0.1 mL) was mixed with 50 mL of water, 0.1 mL of 0.1 N hydrochloric acid and 2.5 mL of iodine solution (0.2% iodine, 2.0% potassium iodide), added water to 100 mL, stored in the dark for 2 h, then the absorbance was determined at 660 nm. WC, NC and HAC with known amylose content were used as standards.
Acid hydrolysis. Starch samples were hydrolyzed in hydrochloric acid solution at 50°C for 24 h. The concentration of starch and hydrochloric acid in the reaction mixture was 32 and 1.5% (w/w), respectively. After incubation, the suspension was adjusted to pH 5.0 with 3% (w/w) sodium hydroxide to stop the reaction. The suspension was washed several times with distilled water, dehydrated using a vacuum filter, then dried at 40°C overnight. The dried starch was ground and passed through a 250 μm screen. The yield of starch was calculated from the weight percentage of the obtained starch residue to the weight of the starting starch material.
Time course analysis of acid-hydrolyzed HAC class 7. The conditions used to hydrolyze HAC class 7 were as described above. Aliquots were removed at 2, 4, 6, 10, 16, 24, 30, 48, 72 and 100 h of acid hydrolysis and the pH was adjusted to 5.0. Each suspension was washed several times with distilled water, dehydrated using a vacuum filter, and dried at 40°C overnight. The dried starch was ground and passed through a 250 μm screen.
Measurement of resistant starch content. The RS content of the samples was determined using a Resistant Starch Assay Kit (Megazyme International Ireland Limited, Bray, Ireland), approved as the AOAC official method 2002.02 (AOAC 2002.02).6) Starch samples were incubated with pancreatic α-amylase and amyloglucosidase for 16 h at 37°C. The residue was recovered by centrifugation and washed with 50% ethanol. RS in the recovered residue was dissolved in 2 M potassium hydroxide, the solution was neutralized with acetate buffer, and the starch was digested to glucose with amyloglucosidase. The amount of glucose was measured with glucose oxidase/peroxidase reagent. The RS content (%, w/w) was the ratio of RS in the test sample on a dry weight basis. The yield of RS was calculated as the change in the amount of RS compared to the starting native HAC class 7 as follows:
Yield of RS =
{(RS content of residual starch after acid hydrolysis)/
(Yield of starch)}/(RS content of native starch)
X-ray diffraction and crystallinity. Starch samples were equilibrated in a chamber at 100% relative humidity for 3 days at 35°C. Measurement of the X-ray diffraction patterns of the samples was carried out using an X-ray diffractometer (RINT-1100, Rigaku Corp., Tokyo, Japan). Starch samples were mixed with 5% calcium fluoride as an internal standard to calculate crystallinity according to the method of Hamanishi et al.12) The scanning region of the 2-theta angle (2θ) was from 3 to 30° at a speed of 2°/min. The X-ray diffractogram was smoothed using a smoothing function followed by removing the baseline using the optional analysis program (Ver.1.203) shipped with the diffractometer. The peak area was the region surrounded by the smoothed diffractogram and baseline, and was analyzed by Image J software (National Institutes of Health, USA). The crystallinity of the starch was calculated from the relative peak area of the calcium fluoride.
Molecular size distribution of starch. The molecular size distribution of starch was analyzed by gel-permeation chromatography (GPC) using an HPLC system and refractive-index (RI) detector (HPLC-RI). The HPLC-RI system (Tosoh Corp., Tokyo, Japan) consisted of an isocratic pump (DP-8020), a degasser (SD-8022), an auto sampler (AS-8022), a column oven (CTO-10A), an RI detector (RI-8021), two analytical columns (TSK-gel α-M (7.8×300 mm)× 2) and a guard column (TSK-GUARD COLUMN α (6.0 × 40 mm)). The eluent was 90% (w/v) DMSO in 5 mM sodium nitrate. The starch sample (5.0 mg) was dispersed in the eluent (5.0 mL) in a boiling-water bath for 2 min, cooled at room temperature, filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane. More than 95% of the total sugar in all samples passed through the filter. Then the filtrates were injected into the HPLC system. The molecular weight distribution was estimated from a standard curve obtained using glucose, maltose and pullulan standards (P-5, P-10, P-20, P-50, P-100, P-200, P-400 and P-1300, Showa Denko K.K., Tokyo, Japan). The temperature of the columns was maintained at 40°C and the flow rate was 0.5 mL/min. The peak molecular weight (Mp) was used as the molecular weight of the compound.
Fractionation of acid-hydrolyzed HAC class 7. GPC fractionation was carried out on the acid-hydrolyzed HAC class 7 after enzymatic debranching according to the method of Funami et al.13) Fractions from the GPC profile were divided according to the following absorption maxima (λmax) of the glucan-iodine complexes in each tube: Fr. I, λmax ≥ 620 nm; intermediate Fr., 620 nm > λmax ≥ 600 nm; Fr. II, 600 nm > λmax ≥ 540 nm; Fr. III, 540 nm > λmax.
Enzymatic digestion of HAC class 7. Native and acid-hydrolyzed HAC class 7 samples were enzymatically digested and the residues were analyzed. The enzyme solution was same composition with measurement of RS content: 1.0% pancreatin (E-PANAA, Megazyme International Ireland Limited), 300 U of amyloglucosidase (E-AMGDF, Megazyme International Ireland Limited) and 10 mM maleate buffer (pH 6.0) with 0.3% calcium chloride and 0.2% sodium azide. The samples were centrifuged for 10 min at 1,800 × G. The starch samples (11 g, dry base) were suspended in enzyme solution (440 mL) and incubated at 37°C for 16 h with moderate stirring, then 440 mL of 99.5% ethanol was added to the suspension. The residue was collected using suction filtration and washed with 880 mL of 50% ethanol, air-dried at 40°C, ground, and passed through a 250 μm screen.
Thermal property. Thermal property of starches was analyzed using a differential scanning calorimeter (DSC) (Thermal analyzing system WS002, MAC Science Ltd., Fukui, Japan) equipped with a cooling unit (CU9440, MAC Science Ltd.). Fifteen milligrams (dry weight basis) of starch sample was weighed in silver pan and then distilled water was added to make 25% (w/w) starch suspension. The pan was hermetically sealed and stored overnight at 4°C. Sample was heated at a rate of 10°C/min from 5 to 150°C. Sample was then quench-cooled and immediately rescanned at the same conditions. An empty pan served as the reference. Onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and transition enthalpy (ΔH) were measured. DSC analyses were performed in triplicate.
Starch morphology. Scanning electron microscopy (SEM) images of starch samples were taken using a scanning electron microscope (JSL-6510LV, JEOL Ltd., Tokyo, Japan). The surface of the starches was sputter-coated with gold using an ion sputter (JFC-1100, JEOL Ltd.) and viewed at accelerating voltage of 10 kV.
Acid hydrolysis of corn starches with different amylose content. Table 1 shows the RS content and yield of native and acid-hydrolyzed starches with different amylose content after 24 h acid hydrolysis. Before acid hydrolysis, HAC class 5 and class 7 had high RS content whereas WC and NC were almost completely digested. Evans et al.14) reported that the RS content of WC, common corn starch, HAC class 5 and HAC class 7, determined using the method of Englyst,15) were 5.1, 24.4, 66.0 and 69.5%, respectively. Our results measured using the AOAC 2002.02 method provided lower values. The discrepancy appears to be due to differences in the analytical method used to quantify RS and in the digestion time: the AOAC 2002.02 method requires longer digestion (16 h) than the Englyst method (2 h).
Effect of acid hydrolysis for 24 h on RS content and yields of corn starches containing different amounts of amylose.
RS content was obviously higher in the acid hydrolytes, except for WC. Acid-hydrolyzed HAC class 5 and HAC class 7 showed significantly higher RS content than acid-hydrolyzed WC and NC. HAC class 5 and class 7 hydrolytes provided B-type diffraction patterns whereas WC and NC hydrolytes provided A-type patterns. In general, starches that produce a B-type X-ray diffraction pattern are more resistant to acid hydrolysis than A-type starches due to their high density of amorphous lamellae and the stability of the crystals.16) 17) 18) It has been reported, however, that RS content does not increase following acid hydrolysis. For example, lintnerized starch is easily digested by α-amylase: its granule structure decomposes under severe conditions (2.2 M HCl, about 15 days), allowing α-amylase to penetrate the internal structure more readly.18) 19) Using milder acid hydrolysis conditions, Brumovsky et al. reported that hydrolysis with 0.1% HCl at 25°C for up to 78 h had little effect on the RS content of HAC class 7, as analyzed using the Prosky and Englyst methods.10) We propose two reasons why their results are different from ours. First, their reaction temperature (25°C) was lower than ours (50°C), so HAC class 7 was insufficiently hydrolyzed to increase the RS content. Second, the two studies used different methods to quantify enzyme digestibility: we used the AOAC 2002.02 method rather than the Englyst or Prosky method (AOAC 985.29 method). The AOAC 2002.02 and AOAC 985.29 methods employ different digestion conditions2) 3) thereby providing different degrees of digestibility of acid-hydrolyzed HAC.
The yields of acid-hydrolyzed starches were higher if the amylose content was higher. The yield of RS is the ratio of the RS content in acid-hydrolyzed starch to the RS content in the starting starch. An RS yield of over 100% indicates that acid hydrolysis increased the amount of RS above the amount of RS in the original starch. The RS content in the acid-hydrolyte of NC, HAC class 5 and HAC class 7 was higher than the RS content in the original starches, showing that RS is found not only in the crystalline region remaining following removal of the amorphous regions, but is also generated during acid hydrolysis.
Time course analysis of acid-hydrolyzed HAC class 7. HAC class 7 was hydrolyzed with 1.5% hydrochloric acid at 50°C for up to 100 h in order to investigate the structural changes caused by acid hydrolysis. HAC class 7 and all the acid hydrolytes provided B-type X-ray diffraction patterns. There was no difference in diffraction patters, although the relative intensity of each peak increased depending on the degree of acid hydrolysis (Figs. 3 A, C, E). Table 2 summarizes the RS content, yield of starch, yield of RS, peak molecular weight (Mp), and X-ray crystallinity of native and acid-hydrolyzed HAC class 7. RS content changed in three steps: where RS content increased (～16 h), where RS content was maximum (16‒30 h), and where RS content slowly decreased (30 h～). During the first step, the RS content and crystallinity of acid-hydrolyzed HAC class 7 increased remarkably from 40.5 to 69.3% and 15.9 to 23.3%, respectively, whereas the yield of starch slightly decreased (100 to 96.8%). Since acid hydrolysis occurs preferentially in the amorphous rather than the crystalline regions, this result indicates that the amorphous regions may be degraded and new crystalline regions resistant to enzymatic hydrolysis may be formed. In the second and third step (16‒100 h), RS content was first stable, then decreased, even though the crystallinity continued to increase (23.3 to 30.2%). The yield of starch decreased further (96.8 to 80.6%), perhaps because the starch chains were shorter and thus more soluble and more difficult to recrystallize.
RS content, yield of starch, yield of RS, Mp and crystallinity of native and acid-hydrolyzed HAC class 7.
The Mp of the starches rapidly decreased by about 90% in the first step. However, after RS content peaked, the Mp decreased slowly in the second and third steps (Table 2). The Mp, indicating maximum RS content, spanned 9.0 × 103 to 1.2 × 104 and corresponded to DP 56‒74. Nakazawa et al. reported that Naegeli dextrin generated during 10 days hydrolysis of HAC class 7 showed a main peak at DP 75 and a small peak at DP 15. This main peak may multiple branched chains, because DP 13‒15 was the main peak of debranched Naegeli dextrin generated from HAC class 7.20)
There have been very few publications regarding the effect of acid hydrolysis on RS content, although there have been reports that the combination of acid hydrolysis and gelatinization, annealing, recrystallization and retrogradation are effective for increasing RS21) 22) 23) presumably due to acid hydrolysis facilitating structural changes in HAC into a more ordered structure. Our results show that the RS content of HAC class 7 only increases significantly upon hydrolysis with acid, perhaps due to the reaction conditions used or to annealing during the 50°C reaction.
Fractionation of acid-hydrolyzed HAC class 7 using gel permeation chromatography. HAC class 7, both native and acid-hydrolyzed for up to 30 h, were debranched with isoamylase and fractionated using GPC. Figure 1 shows the elution profiles of native and acid-hydrolyzed HAC class 7 debranched by isoamylase; the percentages of the fractions are shown in Table 3. The percentage of amylose, the component in Fr. I, decreased and disappeared at 16 h. Fr. II and III, which consist of B2 or longer chains of amylopectin and the A or B1 chains of amylopectin, respectively, became the dominant fractions. This finding agreed with the report of Inouch et al.16) showing that aeo2 (high-amylose) corn starch was more slowly solubilized by acid than o2 (normal) and wx (waxy) corn starches, and that Fr. I was rapidly digested by acid. The content of Fr. I inversely correlated with the RS content (Fig. 2). These data imply that amylose in HAC class 7 is susceptible to acid hydrolysis and forms double helixes with the component of Fr. II and III comprised of degraded amylose and amylopectin. These double helices may form crystals, which are resistant to enzymatic hydrolysis.
Elution profiles of native and acid-hydrolyzed HAC debranched using isoamylase.
, Native HAC class 7; 
, HAC class 7 acid-hydrolyzed for 2 h; 
, HAC class 7 acid-hydrolyzed for 10 h; 
, HAC class 7 acid-hydrolyzed for 30 h.
Percentages of fractions of debranched native and acid-hydrolyzed HAC class 7 as determined by GPC.
Correlation between RS content and the Fr. I content of acid-hydrolyzed HAC class 7.
The RS content shown in Table 2 and the Fr. I content shown in Table 3 were plotted. The coefficient of liner correlation (r2) was 0.95.
Enzymatic digestion of acid-hydrolyzed HAC class 7. Native HAC class 7, and HAC class 7 hydrolyzed in acid for 24 and 100 h, were digested with pancreatic α-amylase and amyloglucosidase for 16 h at 37°C. The residues after enzymatic digestion were collected to obtain more information about the resistance of native and acid-hydrolyzed HAC class 7 to enzymatic digestion. The enzymatic digestion conditions were same as those used in the resistant starch assay method (AOAC 2002.02), and the collected residues corresponded to 100% RS. The yields of residue after enzymatic digestion of native, 24 and 100 h acid-hydrolyzed HAC class 7 were 48.1, 71.7 and 65.8%, respectively (Table 4). These yields are slightly higher than the RS content (40.5% for native HAC class 7, 68.7% for 24 h acid-hydrolyzed, 62.5% for 100 h acid-hydrolyzed HAC class 7, Table 2). This may be due to differences in the scale of the AOAC 2002.02 reaction for the various samples, or due to the presence of minor components such as lipid, protein and ash in the residues.
The yield and crystallinity of enzyme-digested residues.
＊1Yield of starch by enzymatic digestion. ＊2Product-based crystallinity after enzymatic digestion is calculated from the relative peak area of the X-ray diffractogram of the enzyme digested residues. ＊3Material-based crystallinity after enzymatic digestion is the ratio of weight of the crystalline region of the enzyme-digested residue to weight of the starch material before enzymatic digestion. This is calculated from: (Product-based crystallinity after enzymatic digestion (%)) × (Yield of starch (%))/100.
X-ray diffraction analysis showed no difference in the diffraction patterns between the pre-digested residue and enzyme-digested residue (Fig. 3). The product-based crystallinity after enzymatic digestion is defined as the ratio of weight of the crystalline regions in the enzyme-digested residues to weight of the total enzyme-digested residue. The material-based crystallinity after enzymatic digestion is the ratio of weight of the crystalline region of the enzyme-digested residue to weight of the starch material before enzymatic digestion (Table 4). In native HAC class 7, the product-based crystallinity of the enzyme-digested residue increased (15.9 to 21.2%) due to enzymatic digestion, but the material-based crystallinity decreased (15.9 to 10.2%). These results show that the crystalline region of native HAC class 7 decreased due to the low yield of starch in enzymatic digestion (48.1%). In contrast, the crystalline region of HAC class 7 hydrolyzed in acid for 24 h decreased only slightly (21.8 to 20.6%). These results show that the crystalline regions of HAC class 7 hydrolyzed in acid for 24 h are more resistant to enzyme attack than the crystalline regions of unhydrolyzed HAC class 7. Interestingly, the crystalline region of HAC class 7 hydrolyzed in acid for 100 h decreased (30.2 to 20.3%) following enzymatic digestion, suggesting that the enzyme resistance of the crystalline region of 100 h acid-hydrolyzed HAC class 7 is weaker than that of 24 h acid-hydrolyzed HAC class 7, despite its higher crystallinity.
X-ray diffraction patterns of native and acid-hydrolyzed HAC class 7 and their enzyme-digested residues.
(A) Native HAC class 7, (B) enzyme-digested residue of native HAC class 7, (C) HAC class 7 acid-hydrolyzed for 24 h, (D) enzyme-digested residue of HAC class 7 acid-hydrolyzed for 24 h, (E) HAC class 7 acid-hydrolyzed for 100 h, (F) enzyme-digested residue of HAC class 7 acid-hydrolyzed for 100 h.
Figure 4 shows the change in molecular weight distribution following enzymatic digestion. The molecular weight distribution of the residue following enzymatic digestion of native HAC class 7 provided two peaks: a main peak at Mp 6.2 × 103 (DP 38) and a smaller peak at Mp 1.7 × 105 (DP 1049), which is the same molecular peak obtained with native HAC class 7. Jiang et al. reported similar results following GPC analysis of the RS residue of HAC class 7, which is the fraction resistant to α-amylase hydrolysis. Their analysis showed two major groups of components: larger components (average DP 840‒951) consisting of partially hydrolyzed amylose, partially hydrolyzed amylopectin and an intermediate fragment of amylopectin, and smaller components (average DP 59‒74) consisting of liner short chains.7)
Molecular size distributions of native and acid-hydrolyzed HAC class 7 and its enzyme-digested residues.
(A) Native HAC class 7, (B) HAC class 7 acid-hydrolyzed for 24 h, (C) HAC class 7 acid-hydrolyzed for 100 h. , Before enzymatic digestion; , after enzymatic digestion.
The molecular weight distribution of HAC class 7 hydrolyzed in acid for 24 and 100 h provided residues after enzymatic digestion that grouped into a single peak. The Mp of HAC class 7 acid hydrolyzed for 24 and 100 h were 9.0 × 103 and 5.1 × 103, while the molecular weights of the residues following enzymatic digestion were 6.8 × 103 and 4.6 × 103, respectively. The molecular weights of these peaks were similar to that of the main peak of the enzyme-digested residue of native HAC class 7. The higher molecular weight fraction (1.7 × 105) of enzyme-digested residue of native HAC class 7 was not apparent in the elution profile of acid-hydrolyzed HAC class 7 (Fig. 4). Pancreatic α-amylase acts both on amorphous and crystalline regions, whereas acid hydrolyzes preferentially attacks amorphous regions. Our results show that acid would preferentially hydrolyze the amorphous lamella which connects amylopectin clusters; consequently, the larger molecular weight peaks disappear rapidly compared to when HAC class 7 is subjected to enzymatic hydrolysis.
Thermal property of acid-hydrolyzed HAC class 7 and its enzyme-digested residues. The DSC thermograms of native and acid-hydrolyzed HAC class 7 are shown in Fig. 5 (a) and thermal properties are shown in Table 5. The endothermic transition of native HAC class 7 ranged from 66.7 to 126.6°C, and peaked at 99.9°C. This transition enthalpy may be amylose-lipid complex as reported by Shi et al.24) HAC class 7 acid-hydrolyzed for 24 h showed bimodal peaks. The peak ranged below 110°C was shown in native HAC class 7, but the peak ranged from 110 to 137.5°C was not shown in native HAC class 7. The endothermic transition of HAC class 7 acid-hydrolyzed for 100 h had broader temperature range from 65.8 to 147.0°C and major peak was shown at 125.7°C. The broad thermograms of HAC class 7 acid-hydrolyzed for 100 h were similar to that of Naegeli dextrin and lintnerlized starch of HAC class 7 previously reported by Jiang et al.25) and Shi et al.24) Jiang et al. suggested that the relatively high temperature endothermic transitions were attributed to long double helices of amylose. Shi et al. suggested that the broad endothermic enthalpy of lintnerized HAC could be due to disordering of double helices of short chains from amylopectin, double helices of long chains from free amylose, and V-helix residues of amylose-lipid complex. In our result, the endothermic transition above 110°C of acid-hydrolyzed HAC class 7 might be due to long double helices of amylose.
DSC thermograms of native and acid-hydrolyzed HAC class 7 and its enzyme-digested residues.
(a) Before enzymatic digestion, (b) After enzymatic digestion, (A) Native HAC class 7, (B) HAC class 7 acid-hydrolyzed for 24 h, (C) HAC class 7 acid-hydrolyzed for 100 h. The broken line in thermogram used as baseline.
Thermal properties of native and acid-hydrolyzed HAC class 7 and its enzyme-digested residues.
*1Peak temperature <110°C. *2Peak temperature ≥110°C. *3 Not detected.
Figure 6 (a) shows the thermograms of immediate rescanning of native and acid-hydrolyzed HAC class 7 and thermal properties are shown in Table 5. The transition peak area increased with acid hydrolysis and was larger than corresponding area at first scanning. The peak area increased in relatively higher temperature at HAC class 7 acid-hydrolyzed for 24 h, whereas the transition peak was shifted to lower temperature range at HAC class 7 acid-hydrolyzed for 100 h. The increase of enthalpy appears to arise from increase of interaction and refolding between amylose, amylopectin and inner lipid during quench cooling. The transition enthalpy above 110°C may contain newly formed complex within relatively long chain helices of amylose, while the peak area below 110°C may be affected by double helices with short chain length amylose in addition to amylose-lipid complex. Chung et al. reported the enthalpy of amylose-lipid complex increased with the acid hydrolysis of HAC.22) The acid hydrolysis might make amylose chain easy to move and produce new complex with lipid.
DSC thermograms of immediate rescanning of native and acid-hydrolyzed HAC class 7 and its enzyme-digested residues.
(a) Before enzymatic digestion, (b) after enzymatic digestion, (A) native HAC class 7, (B) HAC class 7 acid-hydrolyzed for 24 h, (C) HAC class 7 acid-hydrolyzed for 100 h. The broken line in thermogram used as baseline.
Figure 5 (b) shows DSC thermograms of enzyme-digested residue of native and acid-hydrolyzed HAC. The transition enthalpies, especially areas of above 110°C, were larger than before enzymatic digestion. Figure 6 (b) shows immediate rescanning of enzyme-digested residues. The transition enthalpies were larger than before enzymatic digestion. Moreover, the shapes of thermograms of acid-hydrolyzed HAC were similar to that before enzymatic digestion. These results imply the area above 110°C, which is resistant to acid hydrolysis, is also resistant to enzymatic digestion.
Morphology of acid-hydrolyzed HAC class 7 and its enzyme-digested residue. SEM images of native HAC class 7 and HAC class 7 acid hydrolyzed for 24 and 100 h are shown in Figs. 7 (A)‒(C). Most granules of HAC class 7 acid hydrolyzed for 24 h (Fig. 7 (B)) retained the original shape of native HAC class 7 granules (Fig. 7 (A)), but a small number of damaged granules were present. In contrast, acid-hydrolysis of HAC class 7 for 100 h (Fig. 7 (C)) resulted in intensively damaged granules. Prolonged acid hydrolysis might corrode the granules, causing them to be damaged. The damaged granules have an increased surface area, and a part of interior of the granule was exposed. The outer region of HAC class 7 is more resistant to enzymatic digestion than the interior,14) 26) so the RS content of the damaged granules (100 h) is lower than that of normal granules (24 h).
SEM images of native HAC class 7, HAC class 7 acid-hydrolyzed for 24 and 100 h and their enzyme-digested residues.
The magnification of all samples was × 3,000. White bars, 5 μm. (A) Native HAC class 7, (B) HAC class 7 acid-hydrolyzed for 24 h, (C) HAC class 7 acid-hydrolyzed for 100 h, (D) enzyme-digested residue of native HAC class 7, (E) enzyme-digested residue of HAC class 7 acid-hydrolyzed for 24 h, (F) enzyme-digested residue of HAC class 7 acid-hydrolyzed for 100 h.
SEM images of the enzyme-digested granules of native HAC class 7 and HAC class 7 hydrolyzed in acid for 24 or 100 h are shown in Figs. 7 (D)‒(F). Native HAC class 7 granules after enzymatic digestion show many cavities or fissures on their surfaces. Granules of HAC class 7 acid hydrolyzed for 24 h followed by enzymatic digestion were similar to that of native HAC class 7 after enzymatic digestion, although more rounder granules remained than native HAC class 7 after enzymatic digestion, indicating that acid hydrolysis strengthened the outer regions of the granules which are naturally highly ordered and thus highly resistant to enzymatic digestion.14) 26) 27) Moderate acid hydrolysis digests amorphous amylose, which recrystallizes and forms new double helical crystals with amylose or amylopectin. The generated crystals provide a B-type diffraction pattern. These highly crystallized, acid-hydrolyzed HAC class 7 structures may have a more stable outer shell than native HAC class 7, and thus higher resistance to enzymatic degradation.
Most granules of HAC class 7 hydrolyzed in acid for 100 h, followed by enzymatic digestion, were more extensively damaged (Fig. 7 (F)). These cavities were deeper than that of HAC class 7 hydrolyzed in acid for 100 h (Fig. 7 (C)), indicating that excessive acid hydrolysis corroded the granules, resulting in an increase in surface area and exposure of internal regions which have lower enzymatic resistance. The destruction of the granules may reduce the resistance of both the whole granule and the crystalline regions towards enzymatic attack.
