Effect of spray-drying and extrusion on physicochemical characteristics of sweet potato starch.

This study aimed to understand the physicochemical characteristics of sweet potato starch following spray-drying and extrusion processes for desirable applications. Spray-dried starch showed formation of agglomerates and decreased in average granular size from 16.5 µm of the native starch granules to 14.1 µm. Spray-drying reduced the percentage crystallinity from 25.3 to 22.6% and showed a slight decrease in the molecular weight of amylopectin from 3.1 to 2.6 × 108 g mol-1. In addition, changes in the pasting and gelatinization properties, higher final viscosity (454.4 RVU), and less enthalpy change (8.73 J g-1) were reported after spray-drying. Thus, spray-drying resulted in partially gelatinized starch, which can be selected for making more viscous products. Extruded sweet potato starch displayed an amorphous structure, showed total loss of crystallinity, and significant reduction in molecular weight of amylopectin to 0.41 × 108 g mol-1, reflecting complete gelatinization of starch granules during extrusion. Extruded starch showed significant changes in pasting properties, including a display cold viscosity (9.4 RVU). Therefore, extruded starch was suitable for products that require quick solubility and a low final viscosity. Thus, the spray-drying and extrusion processes produce sweet potato starches with particular characteristics that can be used for different and potential applications in industries.

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm.

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.

Macronutrients in Corn and Human Nutrition.

As the leading cereal crop in the world, corn (Zea mays L.) plays a significant role in human foods. The focus of this review is on the structures, properties, bioavailability, and health benefits of macronutrients in corn, including its starch, nonstarch polysaccharides, protein, and lipids. With a large number of naturally existent mutants, corn and corn-based food ingredients with varying macronutrient compositions and different chemical structures and physical properties are commercially available. The corn varieties that have attracted the most interest in human nutrition include high-amylose, high-protein, and high-oil corn. Chemical compositions and molecular structures of starch, nonstarch polysaccharides, protein, and lipids from different corn varieties are comprehensively reviewed here and related to their physical properties, bioavailability, and physiological effects on human health. Particular emphasis is placed upon the impacts of high-amylose corn flour/starch on postprandial glycemic/insulinemic responses, insulin sensitivity, satiety/appetite, lipid metabolism/obesity, colon health, and mineral absorption. The information presented in this article will be useful for the utilization of corn and the development of corn-based food ingredients to prepare nutritious food products to improve human health.

Preparation of gluten-free rice spaghetti with soy protein isolate using twin-screw extrusion.

The objective of this study was to investigate the effect of soy protein isolate on functional properties and consumer acceptance of gluten-free rice spaghetti (GFRS) made from rice flour. Dry-milled high-amylose (Chai Nat 1) rice flour was premixed with dry-milled waxy (RD 6) rice flour at a ratio of 90:10 (w/w) with the soy protein isolate (SPI) concentration varying between 0, 2.5, 5.0, 7.5, 10.0 %, db. The GFRS formulation was processed using a co-rotating twin-screw extruder up to 95 °C with a screw speed of 220 rpm, 32 % moisture content, and then dried at 40 °C. The GFRS samples were analyzed by differential scanning calorimetry (DSC), X-ray diffraction, scanning electron microscopy (SEM) and texture parameters. Increasing SPI decreased the starch retrogradation of GFRS, whereas the enthalpy change of the amylose-lipid complex increased and crystallinity decreased. SEM revealed that the surface of GFRS containing SPI was much more porous than that of GFRS without SPI. The cooked GFRS containing 5.0 % SPI showed the best eating quality with increased firmness and tensile strength, and decrease stickiness. The GFRS samples were evaluated on the bases of cooking qualities and sensory evaluation. The results showed that the GFRS containing 5.0 % SPI decrease the cooking time from 17.6 to 13.7 min and cooking loss from 25.4 to 17.0 %. Overall acceptability of cooked GFRS containing 5.0 % SPI was the highest among all GFRS samples.

Novel characteristics of a carbohydrate-binding module 20 from hyperthermophilic bacterium.

In this study, a gene fragment coding carbohydrate-binding module 20 (CBM20) in the amylopullulanase (APU) gene was cloned from the hyperthermophilic bacteria Thermoanaerobacter pseudoethanolicus 39E and expressed in Escherichia coli. The protein, hereafter Tp39E, possesses very low sequence similarity with the CBM20s previously reported and has no starch binding site 2. Tp39E did not demonstrate thermal denaturation at 50 °C; however, thermal unfolding of the protein was observed at 59.5 °C. A binding assay with Tp39E was conducted using various soluble and insoluble substrates, and starch was the best binding polysaccharide. Intriguingly, Tp39E bound, to a lesser extent, to soluble and insoluble xylan as well. The dissociation constant (K d) and the maximum specific binding (B max) of Tp39E to corn starch granules were 0.537 µM and 5.79 µM/g, respectively, at pH 5.5 and 20 °C. 99APU1357 with a Tp39E domain exhibited 2.2-fold greater activity than a CBM20-truncation mutant when starch granules were the substrate. Tp39E was an independently thermostable CBM and had a considerable effect on APU activity in the hydrolysis of insoluble substrates.

Physicochemical and morphological properties of starch from fresh waxy corn kernels.

The characteristics on physicochemical and morphological properties of starches were investigated in fresh waxy corn kernels. Starches were isolated from eight waxy corn genotypes at the immature kernel stage growing in Thailand. The starch content showed variation with genotypes and ranged from 77.76 to 90.97 %. Granule size distribution showed a two population of starch granules with peak values ranged from 0.8 to 1.1 µm (small) and 9.0 to 12.2 µm (large). Genotypes were also significantly different for average chain length (CL), unit chain length distribution and pasting properties. The small granule (<5 µm) was negatively correlated with CL and degree of polymerization (DP) 25-36 of amylopectin (-0.82 and -0.67, respectively, P < 0.01). And a strong relationship between trough and final viscosity was consistent with the contribution of pasting properties.

High-resolution time-of-flight mass spectrometry fingerprinting of metabolites from cecum and distal colon contents of rats fed resistant starch.

Time-of-flight mass spectrometry along with statistical analysis was utilized to study metabolic profiles among rats fed resistant starch (RS) diets. Fischer 344 rats were fed four starch diets consisting of 55 % (w/w, dbs) starch. A control starch diet consisting of corn starch was compared against three RS diets. The RS diets were high-amylose corn starch (HA7), HA7 chemically modified with octenyl succinic anhydride, and stearic-acid-complexed HA7 starch. A subgroup received antibiotic treatment to determine if perturbations in the gut microbiome were long lasting. A second subgroup was treated with azoxymethane (AOM), a carcinogen. At the end of the 8-week study, cecal and distal colon content samples were collected from the sacrificed rats. Metabolites were extracted from cecal and distal colon samples into acetonitrile. The extracts were then analyzed on an accurate-mass time-of-flight mass spectrometer to obtain their metabolic profile. The data were analyzed using partial least-squares discriminant analysis (PLS-DA). The PLS-DA analysis utilized a training set and verification set to classify samples within diet and treatment groups. PLS-DA could reliably differentiate the diet treatments for both cecal and distal colon samples. The PLS-DA analyses of the antibiotic and no antibiotic-treated subgroups were well classified for cecal samples and modestly separated for distal colon samples. PLS-DA analysis had limited success separating distal colon samples for rats given AOM from those not treated; the cecal samples from AOM had very poor classification. Mass spectrometry profiling coupled with PLS-DA can readily classify metabolite differences among rats given RS diets.

Ectopic expression of bacterial amylopullulanase enhances bioethanol production from maize grain.

KEY MESSAGE: Heterologous expression of amylopullulanase in maize seeds leads to partial starch degradation into fermentable sugars, which enhances direct bioethanol production from maize grain. Utilization of maize in bioethanol industry in the United States reached ±13.3 billion gallons in 2012, most of which was derived from maize grain. Starch hydrolysis for bioethanol industry requires the addition of thermostable alpha amylase and amyloglucosidase (AMG) enzymes to break down the α-1,4 and α-1,6 glucosidic bonds of starch that limits the cost effectiveness of the process on an industrial scale due to its high cost. Transgenic plants expressing a thermostable starch-degrading enzyme can overcome this problem by omitting the addition of exogenous enzymes during the starch hydrolysis process. In this study, we generated transgenic maize plants expressing an amylopullulanase (APU) enzyme from the bacterium Thermoanaerobacter thermohydrosulfuricus. A truncated version of the dual functional APU (TrAPU) that possesses both alpha amylase and pullulanase activities was produced in maize endosperm tissue using a seed-specific promoter of 27-kD gamma zein. A number of analyses were performed at 85 °C, a temperature typically used for starch processing. Firstly, enzymatic assay and thin layer chromatography analysis showed direct starch hydrolysis into glucose. In addition, scanning electron microscopy illustrated porous and broken granules, suggesting starch autohydrolysis. Finally, bioethanol assay demonstrated that a 40.2 ± 2.63 % (14.7 ± 0.90 g ethanol per 100 g seed) maize starch to ethanol conversion was achieved from the TrAPU seeds. Conversion efficiency was improved to reach 90.5 % (33.1 ± 0.66 g ethanol per 100 g seed) when commercial amyloglucosidase was added after direct hydrolysis of TrAPU maize seeds. Our results provide evidence that enzymes for starch hydrolysis can be produced in maize seeds to enhance bioethanol production.

Contributions of Dexter French (1918-1981) to cycloamylose/cyclodextrin and starch science.

Professor Dexter French (1918-1981) was an American chemist and biochemist at Iowa State College (University in 1959). He devoted his career to advance knowledge of polysaccharides and oligosaccharides, in particular starch, cyclodextrins, and enzymes. Cyclodextrins are oligosaccharides obtained from starch and are typically cage molecules with a hydrophobic cavity that can encapsulate other compounds nowadays the basis for many industrial applications. Since the 1960s, he has been recognized as an outstanding authority in the field of starches and cyclodextrins and has inspired researchers in laboratories around the world. This review, on the fortieth anniversary of his death, commemorates his remarkable contribution to starch and cyclodextrin chemistry. Firstly, we give an overview of his personal life and career. Secondly, we highlight some of the results on starch and cyclodextrins from Professor French and his group. A third part discusses his impact on the modern chemistry of cyclodextrins and starch.

Storage temperature and time affect the enzyme resistance starch and glycemic response of cooked noodles.

White-salted noodles are prepared, stored and consumed in various ways. However, relationships among cooking and storage conditions on nutritional functionality are not fully understood. The manuscript elucidates the mechanism of formation of resistant starch (RS) leading to slower digestion rate of variously cooked (boiled, steamed, stir-fried, fried and microwave heated) noodles followed by storage under different conditions (-18, 4 and 25 °C for 4, 24 and 48 h). RS content of noodles stored at 25 °C was higher than noodles stored at 4 °C, which was consistent with increases in the degree of crystallinity during storage. We showed that the residual moisture content primarily facilitated the mobility of starch chains and contributed towards the increase in RS associated with the decrease of enzyme susceptivity of noodles after storage. Evidence that supramolecular organization (helical structure and crystallinity) had a more pronounced effect than the macroscopic structure such as compactness or bulk density was also provided.

RS Content and eGI Value of Cooked Noodles (I): Effect of Cooking Methods.

Noodles are widely consumed in China, which can be cooked in different ways. The effects of different cooking methods (boiling, steaming, microwave heating, stir-frying and frying) on the resistance starch (RS) content and digestive properties (digestion rate, digestibility and estimated glycemic index (eGI) value) of noodles were investigated. The RS content was greatly affected by the cooking time, and it was varied when the noodles were optimally cooked using different cooking methods. The RS contents of the microwaved and stir-fried noodles were relatively high (0.59%-0.99%), but it was lower (0.43%-0.44%) in the boiled and steamed noodles. Microwaved noodles showed the slowest digestion rate and the lowest eGI. Due to the limited water within fried noodles, none RS was found in the fried noodles, whereas stir-fried noodles showed RS5 formation from the XRD and DSC results. Compared with boiled and steamed noodles, the microwaved noodles showed a more compact morphology without porous holes on the surface, whereas fried noodles showed irregular morphology. The results indicated that the digestive properties of noodles made with the same ingredients can be greatly altered by using different cooking methods, and the digestive properties of different cooked noodles are worthy of confirmation using in vivo analysis.

Effect of pH on complex formation between debranched waxy rice starch and fatty acids.

Complex formations between debranched waxy rice starch (DBS) and fatty acids (FA) of different hydrocarbon chain lengths (8:0, 10:0, 12:0, 14:0, 16:0, and 18:0) were studied in an aqueous solution by measuring the blue colour stained with iodine. The objective of this study was to understand the effects of the solubility and hydrophobicity of guest molecules (FA) on the complex formation with DBS. Lauric acid (12:0) displayed the greatest complex forming ability with DBS by showing the least blue colour developed with iodine. The effect of pH (3-7) on the DBS/FA complex formation was evaluated by measuring the iodine-scanning spectra of the mixture. Short-chain FA (8:0) displayed less complex formation at pH>or=5, above the pK(a) of fatty acid (approximately 4.8), which suggested that the charge formation of the short-chain FA caused a lower partitioning of the FA into the hydrophobic cavity of the DBS single helix. On the contrary, FA of 10:0-18:0 displayed an increased complex formation at pH>5, which could be attributed to increased solubility of these longer-chain FA at a dissociated and ionized form. The hydrocarbon chain length of the FA had an important impact on the extent of the complex formation. A FA that had a shorter hydrocarbon chain was more soluble in an aqueous solution and more readily formed a complex with DBS. At pH 6 and 7 (above the pK(a)), 10:0 formed less inclusion complexes with DBS than did 12:0. Iodine-scanning spectra showed that the absorbances of all iodine-stained DBS/FA solutions at higher wavelength were substantially lower than that of the iodine-stained DBS alone, suggesting that FA preferentially formed inclusion complexes with DBS of longer chains.

Oil and tocopherol content and composition of pumpkin seed oil in 12 cultivars.

Twelve pumpkin cultivars (Cucurbita maxima D.), cultivated in Iowa, were studied for their seed oil content, fatty acid composition, and tocopherol content. Oil content ranged from 10.9 to 30.9%. Total unsaturated fatty acid content ranged from 73.1 to 80.5%. The predominant fatty acids present were linoleic, oleic, palmitic, and stearic. Significant differences were observed among the cultivars for stearic, oleic, linoleic, and gadoleic acid content of oil. Low linolenic acid levels were observed (<1%). The tocopherol content of the oils ranged from 27.1 to 75.1 microg/g of oil for alpha-tocopherol, from 74.9 to 492.8 microg/g for gamma-tocopherol, and from 35.3 to 1109.7 microg/g for delta-tocopherol. The study showed potential for pumpkin seed oil from all 12 cultivars to have high oxidative stability that would be suitable for food and industrial applications, as well as high unsaturation and tocopherol content that could potentially improve the nutrition of human diets.

Effects of growth condition on the structure of glycogen produced in cyanobacterium Synechocystis sp. PCC6803.

Growth and glycogen production were characterized for Synechocystis sp. strain PCC6803 grown under continuous fluorescent light in four variations of BG-11 medium: either with (G+) or without (G-) 5mM glucose, and with a normal (N+, 1.5 g sodium nitrate/L) or a reduced (N-, 0.084 g sodium nitrate/L) nitrogen concentration. Glucose-supplemented BG-11 with a normal nitrogen concentration (N+G+) produced the highest growth rate and the greatest cell density. Although the maximum cell mass production was observed in the N+G+ medium, the highest glycogen yield (19.0mg/g wet cell mass) was achieved under the glucose-supplemented, nitrogen-limiting condition (N-G+). The addition of glucose enhanced cell growth, while nitrogen limitation apparently directed carbon flux into glycogen accumulation rather than cell growth. Transmission electron microscopic analysis showed that, under nitrogen-limiting conditions (N-G+), glycogen particles accumulated in large amounts and filled the cytosol of the cells. Analysis by high-performance size-exclusion chromatography further revealed that the glycogen produced in N-G+ medium had the longest average branch chain-length (DP10.4) among the conditions tested. When the yield and structure of glycogen were examined in different growth phases, the greatest yield (36.6 mg/g wet cell mass) and the longest branch chain-length (DP10.7) were observed 2 days after the fully grown cells in the N+G+ medium were transferred to the growth restricting (N-G+) medium.

Effects of adding corn oil and soy protein to corn starch on the physicochemical and digestive properties of the starch.

This study aimed to understand effects of adding corn oil (CO) and soy protein (SP) to corn starch on the physicochemical properties and digestive rates of annealed starch complex and mechanisms of interactions between corn starch (CS), CO and SP. Binary and ternary blends were prepared using CS mixed with CO (10%, dsb) and/or SP (10%, dsb) and incubated in a water bath at 50°C for 14h. Results showed that more agglomerates of the granules were in the ternary blends. With the addition of CO and/or SP, the CS displayed a decreased pasting temperature, an increased peak viscosity and a decreased enthalpy change of amylose-lipid complex dissociation. The CO can reinforce but SP hinder the annealing phenomenon. Results also showed that CO decreased retrogradation of CS, whereas SP increased it. The digestibility studies showed that the addition of CO and SP decreased the content of rapidly digestible starch and increased the sum of slowly digestible starch and resistant starch contents. SP displayed more impact on the digestibility of the ternary blends than CO. The physical barrier of CO, and amylose-lipid complex and protein-starch matrix can provide resistance to starch digestion.

Pysicochemical properties of Tibetan hull-less barley starch.

Objectives of this study were to (1) determine the starch physicochemical properties of two commercial Tibetan hull-less barley varieties, Beiqing (BQ) and Kangqing (KQ); and (2) understand the relationship between unique properties of the starches, their structures, and impacts of growing conditions. The BQ barleys were grown at a location with lower temperature and less rainfall compared with the KQ barleys. The BQ starches showed significantly lower onset-gelatinization temperature (54.1-54.9 °C), larger gelatinization-temperature range (9.4-10.6 °C), and higher peak-viscosities (138.9-153.9RVU) than the KQ starches (55.1-56.1 °C, 7.4-8.8 °C, and 63.4-64.7RVU, respectively). After a treatment with 2% sodium-dodecyl-sulphate solution, the KQ starches showed substantially greater increases in peak viscosities than the BQ starches. Annealing of starch and enhanced amylose-lipid complex formation, resulting from higher growing temperature during the development of the KQ starches, likely contributed to the differences in thermal and pasting properties between the BQ and KQ starches.

Dosage effects of Waxy gene on the structures and properties of corn starch.

The objective of this study was to understand dosage effects of the Waxy gene on the structures of amylose and amylopectin and on the properties of corn starch. Reciprocal crossing of isogenic normal and waxy corn lines was conducted to develop hybrids with different dosages (0, 1, 2, 3) of Waxy gene in the endosperm. The amylose content of starch and proportions of branch chains of DP 17-30 and extra-long branch chains (DP>100) of amylopectin were positively correlated with the Waxy-gene dosage. Proportions of short (DP<17) and long branch-chains (DP 30-80), however, were negatively correlated with the Waxy-gene dosage. The gelatinization conclusion-temperature and temperature-range of the starch were negatively correlated with the Waxy-gene dosage, indicating that amylose facilitated dissociation of the surrounding crystalline regions. These results helped us understand the function of granule-bound starch synthase I in the biosynthesis of amylose and amylopectin and impacts of Waxy-gene dosages on the properties of corn starch.

Characterization and development mechanism of Apios americana tuber starch.

Apios americana is a wild legume-bearing plant with edible tubers. Domestication of Apios is in progress because of the superior nutritional value and health benefits of the tuber. Objectives of this study were to: (1) characterize physicochemical properties of the Apios tuber starch; and (2) understand differences in starch structures and properties between the mother (seed) and child (progeny) tubers and the mechanism of starch development. Granules of the Apios tuber starch displayed ellipsoidal, rod, or kidney shape with diameter ranges of 1-30µm. The mother tuber starches displayed greater percentage crystallinity, larger gelatinization enthalpy-changes, longer branch-chain lengths of amylopectin, and lower pasting viscosity than their counterpart child tuber starches. The mother tuber starch of Apios 2127 displayed distinct two peaks of gelatinization, which were attributed to starch granules located at different regions of the tuber having different structures and properties. The mother tuber displayed more active starch biosynthesis in the periphery than in the center of the tuber.

Resistant Starch Alters the Microbiota-Gut Brain Axis: Implications for Dietary Modulation of Behavior.

The increasing recognition that the gut microbiota plays a central role in behavior and cognition suggests that the manipulation of microbial taxa through diet may provide a means by which behavior may be altered in a reproducible and consistent manner in order to achieve a beneficial outcome for the host. Resistant starch continues to receive attention as a dietary intervention that can benefit the host through mechanisms that include altering the intestinal microbiota. Given the interest in dietary approaches to improve health, the aim of this study was to investigate whether the use of dietary resistant starch in mice to alter the gut microbiota also results in a change in behavior. Forty-eight 6 week-old male Swiss-Webster mice were randomly assigned to 3 treatment groups (n = 16 per group) and fed either a normal corn starch diet (NCS) or diets rich in resistant starches HA7 diet (HA7) or octenyl-succinate HA7 diet (OS-HA7) for 6 week and monitored for weight, behavior and fecal microbiota composition. Animals fed an HA7 diet displayed comparable weight gain over the feeding period to that recorded for NCS-fed animals while OS-HA7 displayed a lower weight gain as compared to either NCS or HA7 animals (ANOVA p = 0.0001; NCS:HA7 p = 0.244; HA7:OS-HA7 p<0.0001; NCS:OS-HA7 p<0.0001). Analysis of fecal microbiota using 16s rRNA gene taxonomic profiling revealed that each diet corresponded with a unique gut microbiota. The distribution of taxonomic classes was dynamic over the 6 week feeding period for each of the diets. At the end of the feeding periods, the distribution of taxa included statistically significant increases in members of the phylum Proteobacteria in OS-HA7 fed mice, while the Verrucomicrobia increased in HA7 fed mice over that of mice fed OS-HA7. At the class level, members of the class Bacilli decreased in the OS-HA7 fed group, and Actinobacteria, which includes the genus Bifidobacteria, was enriched in the HA7 fed group compared to the control diet. Behavioral analysis revealed that animals demonstrated profound anxiety-like behavior as observed by performance on the elevated-plus maze with time spent by the mice in the open arm (ANOVA p = 0.000; NCS:HA7 p = 0.004; NCS:OS-HA7 p = 1.000; HA7:OS-HA7 p = 0.0001) as well as entries in the open arm (ANOVA p = 0.039; NCS:HA7 p = 0.041; HA7:OS-HA7 p = 0.221; NCS:OS-HA7 p = 1.000). Open-field behavior, a measure of general locomotion and exploration, revealed statistically significant differences between groups in locomotion as a measure of transitions across quadrant boundaries. Additionally, the open-field assay revealed decreased exploration as well as decreased rearing in HA7 and OS-HA7 fed mice demonstrating a consistent pattern of increased anxiety-like behavior among these groups. Critically, behavior was not correlated with weight. These results indicate that diets based on resistant starch can be utilized to produce quantifiable changes in the gut microbiota and should be useful to "dial-in" a specific microbiome that is unique to a particular starch composition. However, undesirable effects can also be associated with resistant starch, including lack of weight gain and increased anxiety-like behaviors. These observations warrant careful consideration when developing diets rich in resistant starch in humans and animal models.

Effects of alpha-amylase reaction mechanisms on analysis of resistant-starch contents.

This study aimed to understand differences in the resistant starch (RS) contents of native and modified starches obtained using two standard methods of RS content analysis: AOAC Method 991.43 and 2002.02. The largest differences were observed in native potato starch, cross-linked wheat distarch phosphate, and high-amylose corn starch stearic-acid complex (RS5) between using AOAC Method 991.43 with Bacillus licheniformis α-amylase (BL) and AOAC Method 2002.02 with porcine pancreatic α-amylase (PPA). To determine possible reasons for these differences, we hydrolyzed raw-starch granules with BL and PPA with equal activity at pH 6.9 and 37°C for up to 84 h and observed the starch granules displayed distinct morphological differences after the hydrolysis. Starches hydrolyzed by BL showed erosion on the surface of the granules; those hydrolyzed by PPA showed pitting on granule surfaces. These results suggested that enzyme reaction mechanisms, including the sizes of the binding sites and the reaction patterns of the two enzymes, contributed to the differences in the RS contents obtained using different methods of RS analysis.