Inhibition Of Starch Digestion Research Articles

Beta-Glucan From Barley Attenuates Post-prandial Glycemic Response by Inhibiting the Activities of Glucose Transporters but Not Intestinal Brush Border Enzymes and Amylolysis of Starch

Beta (β)-glucan (BG) from cereal grains is associated with lowering post-prandial blood glucose but the precise mechanism is not well-elucidated. The main aim of this study was to understand the mechanism through which BG from barley affects post-prandial glycemic response. Waffles containing 0, 1, 2, and 3 g barley BG and the same amount of available carbohydrate (15 g) were fed to the TIM-1 dynamic gastrointestinal digestion system to study the effect of BG on starch hydrolysis. Intestinal acetone powder and Xenopus laevis oocytes were used to study BG's effect on mammalian intestinal α-glucosidase and glucose transporters. The presence of BG did not significantly affect the in vitro starch digestion profiles of waffles suggesting that BG does not affect α-amylase activity. Intestinal α-glucosidase and glucose transport activities were significantly (p < 0.0001) inhibited in the presence of barley BG. Interestingly, BG viscosity did not influence α-amylase, α-glucosidase, GLUT2, and SGLT1 activities. This study provides the first evidence for the mechanism by which BG from barley attenuates post-prandial glycemic response is via alteration of α-glucosidase, GLUT2, and SGLT1 activity, but not amylolysis of starch. The decrease in post-prandial blood glucose in the presence of BG is likely a consequence of the interaction between BG and membrane active proteins (brush border enzymes and glucose transporters) as opposed to the commonly held hypothesis that increased viscosity caused by BG inhibits starch digestion.

The Inhibitory Effects of Hericium erinaceus β-glucan on in vitro Starch Digestion.

β-glucan has attracted extensive attention due to its health promoting effects, such as lowering the blood sugar and lipids levels, and enhancing immunity. In this study, three different β-glucans (HEBG-1, HEBG-2, HEBG-3) were obtained from Hericium erinaceus by sodium hydroxide, β-1,3-glucanase and β-1,6-glucanase, respectively. The effects of the glucans on in vitro digestion of wheat starch were investigated by Englyst method. We found that addition of HEBGs significantly reduced the digestibility of starch, showing as decreased RDS and pGI, and increased SDS and RS content. In addition, the inhibitory effects positively correlated with the molecular weight of HEBG. The triple helix structure in HEBG plays important roles in inhibiting starch digestion. And β-1,3- glucan showed stronger inhibitor effects than those of β-1,6- glucan. This study unravels the mechanism of HEBG on inhibition of starch digestion and provides a theoretical understanding for the application of edible mushroom β-glucan to the development of low glycemic index starchy foods.

In Vitro and In Vivo Antihyperglycemic Activities of Medicinal Mushrooms (Agaricomycetes) from India.

Recent research focuses on exploring natural resources to improve the management of type 2 diabetes and to reduce the precarious health effects of synthetic drugs. This investigation aimed to appraise the antihyperglycemic potential of hydroalcoholic (70% ethanol) extracts of Inonotus pachyphloeus, Phellinus allardii, Ph. fastuosus, Ph. gilvus, Ph. sanfordii, and Ph. torulosus. Antihyperglycemic potential was screened using an in vitro inhibition of enzymatic starch digestion assay model. The amount of glucose liberation was determined using the 3,5-dinitrosalicylic acid method. Mushroom extracts showed a concentration-dependent inhibition of α-amyalse and α-glucosidase and a consequent decrease in glucose liberation. Extracts of Ph. fastuosus (half-maximal inhibitory concentration [IC50] = 27.33 ± 1.45 mg/mL) and Ph. sanfordii (IC50 = 30.33 ± 0.88 mg/mL) causing comparable inhibition of α-amyalse and α-glucosidase and decreased glucose liberation were evaluated in vivo through oral starch tolerance and oral glucose tolerance tests using Wistar albino rats. Acarbose (10 mg/kg body weight) was used as a positive control. The extracts of Ph. fastuosus and Ph. sanfordii (100, 200, and 400 mg/kg body weight) showed a dose-dependent decrease in blood glucose concentration, and this decrease was greater in starch-fed rats than in glucose-loaded rats. Ph. fastuosus and Ph. sanfordii extracts (200 and 400 mg/kg body weight) significantly reduced postprandial hyperglycemic peaks in rats challenged with excess starch and glucose. This decrease was statistically comparable to acarbose with Ph. fastuosus extract (400 mg/kg body weight). Thus, it may be concluded that the antihyperglycemic effect of Ph. fastuosus and Ph. sanfordii is mediated by inhibition of starch digestion (inhibition of α-amylase and α-glucosidase). Hence, Ph. fastuosus and Ph. sanfordii can be developed as natural antidiabetic drugs after detailed pharmacological studies.

Rice varieties with a high endosperm lipid content have reduced starch digestibility and increased γ-oryzanol bioaccessibility.

The amount and distribution of rice endosperm lipids can influence starch digestibility and nutritional properties of white rice. However, this aspect has been poorly investigated thus far. We investigated the digestion properties of five rice varieties and common rice having different lipid contents (8.1-24.2 g kg-1) showing that the lipid content is positively correlated with the resistant starch content and negatively correlated with digestion extent (C∞) and estimated glycemic index (eGI). After non-starch lipid (NSL) removal from selected high-lipid mutants (ALK3 and RS4), C∞ was significantly enhanced compared to native samples when digested by α-amylase, while this phenomenon was not observed in low-lipid rice (GZ93). When pancreatin was used, starch digestion was only delayed; triglycerides were gradually hydrolyzed by pancreatic lipase and the lipids-starch complex became no longer resistant to hydrolysis by α-amylase. These results indicated that rice endosperm lipids inhibited starch digestion, by transforming part of the starch into a slowly digestible starch fraction. High-lipid mutants also had a higher total amount of, and more bioaccessible, γ-oryzanol than low-lipid varieties. This study indicates that high-lipid white rice has great potential in designing functional rice-based foods, combining a relatively lower eGI and a high γ-oryzanol content.

New food compositions to increase the content of phenolic compounds in extrudates

Phenolic compounds are linked to a number of health benefits, including antioxidant, antibacterial, antiglycaemic, antiviral, anticarcinogenic, anti-inflammatory and vasodilatory properties. To improve a great loss of phenolics during extrusion, researchers have investigated incorporating functional ingredients into the extrusion input mixture. Other reasons for the addition of active ingredients are the re-use of by-products from food technology, decreasing the calorie content of extruded food, inhibition of starch digestion, and the colour change of the gluten-free products. The paper presents 28 examples of new designs for extrusion based on rice, corn, cassava, sorghum, and lentil flours and on other crops, together with the analyses of phenolics. The present results show the highest total phenolic content in sorghum among cereals, and lentil flour and orange peel powder among mixtures for extrusion to prepare extrudates. The highest content of total flavonols was found in the mixture containing corn and freeze-dried red and purple potatoes.

Natural edible materials made of protein-functionalized aerogel particles for postprandial hyperglycemia management

α-Amylase inhibitors (α-AIs) delay digestion of dietary starch by inhibiting α-amylase in the gut, thereby reducing the postprandial glycemia, which is beneficial to the patients with obesity and diabetes. The proteinaceous α-AIs from wheat can effectively control starch digestion and regulate postprandial hyperglycemia. However, their gastric intolerance remains a challenge, which limits its commercial production and industrial application. In this study, sodium alginate/chitosan aerogels loaded with wheat protein α-AIs were prepared and evaluated as potential transportation and protection matrices for important components in food or pharmaceutical applications. Specifically, the biodegradable aerogel cross-linked with sodium alginate-chitosan-calcium chloride, has a large surface area and open porous structure, which can adsorb staple wheat proteins as an integrated edible material to block around 88,660 U/g of α-amylase activity. The aerogel particles were able to protect the activity of wheat α-AIs in the stomach, leading to the slow passage of the wheat α-AIs through the small intestine to inhibit starch digestion more effectively. Animal experiments further showed that the postprandial blood glucose levels in rats were effectively controlled through delayed increase, after administration of wheat protein-functionalized aerogel particles loaded with wheat α-AIs, which are natural biological macromolecules. This is a novel, safe, and economical method for the prevention and pretreatment of diabetes.

Evaluation of the Antidiabetic and Insulin Releasing Effects of A. squamosa, Including Isolation and Characterization of Active Phytochemicals.

Annona squamosa is generally referred to as a ‘custard apple’. Antidiabetic actions of hot water extract of Annona squamosa (HWAS) leaves together with isolation of active insulinotropic compounds were studied. Insulin release, membrane potential and intracellular Ca2+ were determined using BRIN-BD11 cells and isolated mouse islets. 3T3L1 adipocytes and in vitro models were used to determine cellular glucose uptake, insulin action, starch digestion, glucose diffusion, DPP-IV activity and glycation. Glucose intolerant high-fat fed rats were used for in vivo studies. Active compounds were isolated and characterized by HPLC, LCMS and NMR. HWAS stimulated insulin release from clonal β-cells and mouse islets. Using fluorescent indicator dyes and modulators of insulin secretion, effects could be attributed to depolarization of β-cells and influx of Ca2+. Secretion was stimulated by isobutylmethylxanthine (IBMX), tolbutamide or 30 mM KCl, indicating additional non-KATP dependent pathways. Extract stimulated cellular glucose uptake and insulin action and inhibited starch digestion, protein glycation, DPP-IV enzyme activity and glucose diffusion. Oral HWAS improved glucose tolerance and plasma insulin in high-fat fed obese rats. Treatment for 9 days with HWAS (250 mg/5 mL/kg), partially normalised energy intake, body weight, pancreatic insulin content, and both islet size and beta cell mass. This was associated with improved oral glucose tolerance, increased plasma insulin and inhibition of plasma DPP-IV activity. Isolated insulinotropic compounds, including rutin (C27H30O16), recapitulated the positive actions of HWAS on beta cells and in vivo glucose tolerance and plasma insulin responses. Annona squamosa is attractive as a dietary adjunct in treatment of T2DM and as a source of potential antidiabetic agents including rutin.

Inhibition of starch digestion by flavonoids: Role of flavonoid-amylase binding kinetics

In this study, kinetics of binding between α-amylase and green tea flavonoids were investigated by fluorescence quenching (FQ). Their effect on α-amylase inhibition was evaluated. Whereas epicatechin (EC) and epigallocatechin (EGC) exhibited slow binding kinetics (in the order of minutes), epicatechin gallate (ECG) and epigallocatechin gallate (ECGC) exhibited very rapid binding (in the order of seconds) with Human Salivary α-amylase (HSA) and Porcine Pancreatic α-amylase (PPA). EGCG reached maximum inhibition of HSA and PPA with short incubation time whereas maximum inhibition of HSA and PPA by EC was reached only after 45 to 60 min of incubation. Similar results with ECG and EGC, but not in line with FQ kinetics, highlighted possible interferences of starch-flavonoid interaction in the binding and inhibition process. These results suggest that incubation times of enzymes and flavonoids shall be evaluated prior to enzyme inhibition testing in order to ensure consistent and reliable results.

Inhibition of starch digestion: The role of hydrophobic domain of both α-amylase and substrates

The physicochemical mechanism of starch digestion is very complicated since it may be affected by the non-valence interactions of the amylase inhibitor with the substrate or the enzyme. The role of hydrophobic interaction in the process of starch digestion is not clear. In this study, pluronics (PLs) with different hydrophobicity were used as model amphiphilic compounds to study their inhibition on starch digestion using multi-spectroscopic methods. The results showed that the hydrophobic nature of PLs changed starch structure, but it had a greater effect on the structure of α-amylase by exposing more tryptophan residues and increasing α-helix and β-sheet contents. Further investigation by using different chain-length fatty acids confirmed the results. The finding in this study is informative to design and fabricate α-amylase inhibitors for controlling starch digestion at the molecular level.

Inhibition of Starch Digestion by Dallic Acid and Alkyl Gallates

Abstract Objectives Retardation of starch digestion is an effective way of optimizing glycemic response. Certain bioactive food components inhibit starch digestion by binding with starch digestive enzymes or starch molecules in the digestive tract. Inclusion complexation between starch and guest compounds is a specific non-covalent binding mode and may contribute to a lower digestibility of starch. The aim of this study was to examine the inhibitory effects of alkyl gallates on in vitro starch digestion and the mechanism of inhibition. Methods Raw and cooked high amylose maize starch (HAMS) and potato starch (PS) were tested for in vitro enzymatic digestion. Gallic acid (GA) and alkyl gallates, including butyl (BG), octyl (OG), dodecyl (DG), hexadecyl (HG), and octadecyl (SG) gallates, were either added during digestion or processed to form inclusion complexes with starch prior to digestion. Starch digestibility profiles, represented by rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) contents, were determined. Results GA and alkyl gallates significantly increased (P &amp;lt; 0.05) RS content in raw starches. Cooked starches had significantly lower RS contents than raw starches. GA and BG increased RS content in cooked HAMS, while all compounds except for SG increased RS content in cooked PS. DG and HG caused a significant increase in SDS contents in cooked HAMS but not in raw starches and cooked PS. Significant increases in SDS and RS contents were seen in HAMS inclusion complexes with DG and HG, compared with uncomplexed starch. Conclusions The bioactive food components tested could all inhibit starch digestion either by inhibiting the enzymes or by forming starch inclusion complex. As alkyl chain length increased, their inhibition on enzymatic activity decreased, but their ability to bind starch increased. While cooking makes starch more digestible, adding certain bioactive food components could compensate for RS loss, suggesting a practical way of modulating glycemic response. Funding Sources USDA National Institute for Food and Agriculture.

Inhibition of starch digestion by gallic acid and alkyl gallates

As phenolic compounds, alkyl gallates may inhibit the activity of digestive enzymes for starch. Furthermore, their alkyl chains may facilitate starch inclusion complexation and results in an increased resistant starch (RS) content or slowly digestible starch (SDS) content, which may further retard starch digestion. The significance of such inhibition is that the rate of starch hydrolysis into glucose is reduced, thereby preventing hyperglycemia and related metabolic diseases. This study examined the inhibitory effects on in vitro enzymatic digestion of starch by gallic acid and five alkyl gallates of varying alkyl chain lengths, i.e., butyl gallate, octyl gallate, dodecyl gallate, hexadecyl gallate, and octadecyl gallate. Raw and cooked potato starch (PS) and high-amylose maize starch (HAMS) were tested. For both types of raw starch, gallic acid and all the alkyl gallates significantly (p < 0.05) increased RS content except for octadecyl gallate. In the case of cooked starch, the RS contents were markedly decreased (p < 0.05) as compared to those in raw starch, because gelatinization caused an overall greater susceptibility to enzymatic digestion. The reduction in RS content was less in cooked HAMS than that in cooked PS, due to a higher gelatinization temperature range of HAMS. The aforementioned effect of gallates on RS content was also found for cooked PS, while cooked HAMS experienced increased RS only with gallic acid and butyl gallate. Overall, for the digestion of both starches, regardless of raw or cooked, gallic acid and alkyl gallates with shorter chains demonstrated the strongest inhibitory effects. Our findings indicate that shorter alkyl gallates are effective in inhibiting enzymatic digestion of starch and therefore may have potential in modulating glycemic response.

A Review of Rice Starch Digestibility: Effect of Composition and Heat‐Moisture Processing

Rice grain as the staple food for more than half the global population provides 20% of global human calorie requirements. Starch digestion rate is an important consideration for rice consumers who are concerned about health, particularly those with diabetes. Understanding the effect of rice grain composition on starch digestion may assist the growing and processing of rice for preferred eating quality and starch digestibility. Amylose content is regarded as the key determinant of rice starch digestibility, however, starch digestibility differs between rice varieties with similar amylose content indicating the possible role of non‐starch components in determining rice starch digestibility. The current understanding of the role of starch and non‐starch compounds in determining rice starch digestion has been reviewed here. Starch digestion rate is dependent upon starch granule size, starch crystallinity, amylose content and amylopectin chain length and non‐starch components such as fibre, protein, lipid and polyphenols. Non‐starch components may complex with starch and inhibit starch digestion by affecting starch properties or by producing a protective layer around starch or by having antagonistic effects on digestive enzymes. Hydrothermal processing of rice can alter the effects of starch and non‐starch components on starch digestion.

Hericium Erinaceus β-glucan modulates in vitro wheat starch digestibility

The mechanism of Hericium Erinaceus β-glucan inhibiting starch digestion has been seldom studied. In this study, the mechanism of inhibited starch digestion was explored in the context of interaction between β-glucans of different molecular weights (Mw) and starch. The rates of starch digestion and glucose release decreased with increased Hericium erinaceus β-glucan (HEBG) Mw. HEBG reduced leached amylose but increased peak, minimum and final viscosity. In laser confocal scanning microscopy experiments, HEBG was observed to coat the starch. The mechanisms of HEBG modulating glucose release and starch digestion might be due to increase system viscosity and coat starch. We address the specific mechanisms by which starch granules interact with β-glucan molecules and thus contribute to further applications to develop starchy foods with low predicted Glycemic Index (pGI).

In vitro potential of flavonoids from tartary buckwheat on antioxidants activity and starch digestibility

SummaryThe effects of various flavonoids of Tartary buckwheat (rutin‐enhanced flavonoid extract [REFE and quercetin‐enhanced flavonoid extract [QEFE]) and individual flavonoids (rutin, quercetin and kaempferol) on the antioxidant activity, inhibition of α‐glucosidases and α‐amylase and starch digestibility were evaluated. Quercetin possessed the highest antioxidant activity and inhibition of α‐glucosidases and α‐amylase activity followed by kaempferol and rutin. REFE and QEFE have similar antioxidant and inhibition of α‐amylase activities, but QEFE has much higher α‐glucosidases inhibition than REFE. Tartary buckwheat flour has the lowest content of rapidly digestible starch and predicted glycaemic index (pGI) compared to maize flour, wheat flour and rice flour. Addition of rutin and quercetin to wheat flour showed a weak or no effect on digestion inhibition, but they inhibited starch digestion under solid complex conditions. Our results may help explain the benefits of supplementing the diet with food rich in flavonoids.

The Mechanisms of Alpha-Amylase Inhibition by Flavan-3-Ols and the Possible Impacts of Drinking Green Tea on Starch Digestion.

Many studies have shown that flavan-3-ols inhibit mammalian alpha-amylases but the published IC50 and Ki values vary up to a thousand times. We therefore tested the effects of 6 pure flavan-3-ols-abundant in green tea-on the activity of pure porcine pancreatic alpha-amylase (PPA) under steady-state kinetic conditions. We used both amylose and maltopentaose as substrates, along with spectrophotometry and chromatography as analytical tools, respectively. A Docking approach was also used to probe the interaction between PPA and each flavan-3-ol. The results showed that the 6 flavan-3-ols inhibit amylose hydrolysis with Ki comprised between 7 and 34μM, according to a mixed inhibition profile for gallocatechin gallate, and a competitive inhibition profile for the 5 other flavanols. Only the galloyl-containing flavan-3-ols inhibited the maltopentaose hydrolysis with a Ki of about 30μM according to a noncompetitive profile. We conclude that dietary flavan-3-ols could inhibit starch digestion nonnegligibly. The results of the docking trials were concordant with the kinetic data and have noticeably revealed that the cis-flavan-3-ols epigallocatechin gallate and epicatechin gallate bind similarly to PPA, involving π-stacking with Trp59.

(-)-Epigallocatechin-3-gallate (EGCG) inhibits starch digestion and improves glucose homeostasis through direct or indirect activation of PXR/CAR-mediated phase II metabolism in diabetic mice.

As a major component of green tea, (-)-epigallocatechin-3-gallate (EGCG) has attracted interest from scientists owing to its potential to combat a variety of human diseases including abnormal glucose metabolism in obesity and diabetes. This study aims to (1) evaluate the molecular mechanism of EGCG in starch digestion before EGCG absorption; (2) investigate the link between PXR/CAR-mediated phase II metabolism and glucose homeostasis after EGCG is transported to small intestine and liver. EGCG suppressed starch hydrolysis both in vitro and in vivo. Molecular simulation results demonstrated that EGCG could bind to the active site of α-amylase and α-glucosidase, acting as an inhibitor. In addition, the anti-diabetic action of EGCG was investigated in high fat diet and STZ-induced type 2 diabetes. EGCG improved glucose homeostasis and inhibited the process of gluconeogenesis (PEPCK and G-6-Pase) and lipogenesis (SREBP-1C, FAS and ACC1) in the liver. Meanwhile, EGCG treatment activated PXR/CAR, accompanied by upgrading PXR/CAR-mediated phase II drug metabolism enzyme expression in small intestine and liver, involving SULT1A1, UGT1A1 and SULT2B1b. Dietary polyphenol EGCG could serve as a promising PXR/CAR activator and therapeutic intervention in diabetes.

Inhibition of Pancreatin-Induced Digestion of Cooked Rice Starch by Adzuki (Vigana angularis) Bean Flavonoids and the Possibility of a Decrease in the Inhibitory Effects in the Stomach.

Flavonoids of adzuki bean bind to starch when the beans are cooked with rice. The purpose of this study is to show that adzuki flavonoids can suppress pancreatin-induced digestion of cooked rice starch. The diethyl ether extract of water boiled with adzuki bean inhibited starch digestion, and quercetin and a cyanidin-catechin conjugate (vignacyanidin) but not taxifolin in the extract contributed to the inhibition. The order of their inhibitory effects (taxifolin < quercetin < vignacyanidin) suggested that the effects increased with an increase in their hydrophobicity. The diethyl ether extract also inhibited the starch digestion of cooked rice preincubated in artificial gastric juice, and the level of inhibition was decreased by nitrite. The decrease was due to nitrite-induced consumption of quercetin and vignacyanidin. Taking these results into account, we discuss mechanisms of quercetin- and vignacyanidin-dependent inhibition of starch digestion and the possibility of the decrease in their inhibitory effects by nitrite in the stomach.

Characteristics of defensin1 gene and designing structure to create resistant transgenic corn lines to weevils

Plant defensins are multifunctional proteins, inhibiting the growth of fungal, anti-bacterial, altering membrane channels, inhibiting activity of trypsin and α-amylase. Plant defensin consists of 18 groups in which the group 1 includes defensins to inhibit either α-amylase enzyme or trypsin. Defensins bind to the active site of α-amylase in the weevil gut, thus inhibit starch digestion in weevils. In this report, we present the results of cloning and determining the ZmDEF1 gene sequence isolated from mRNA and DNA of Sonla province local maize and LVN99 hybrid maize cultivar. The coding region of ZmDEF1 gene isolated from some maize samples had the size of 243 nucleotides, encoding 80 amino acids. Gen ZmDEF1 isolated from DNA had the size 345bp consists of two exons and one in tron (102 bp). The nucleotide sequences of ZmDEF1 gene (DNA) of the samples have 6 positions nucleotide difference, on exon 1 has two points difference (position 43, 53), on intron has a difference (position 150), on exon 2 has 3 nucleotide site difference (203, 263 and 297 position). Deduced amino acid sequences of defensin of the Sonla local maize sample has 8 cysteines to make 4 disulfide bridges, while LVN99 hybrid maize has 7 cysteines, which can formed only 3 disulfide bridges. Transformation vector pBetaPhaso-ZmDEF1 has been designed successfully, in which ZmDEF1 is controlled by seed specific Phasoline promoter. The correct insertion and expression of ZmDEF1 was examinated in transgenic tobacco plants throught PCR and RT-PCR, respectively. These results provide an firm evident for using the designed transformation vector to produce transgenic maỉze lines with an improved resistant ability to weevils.

Emblica officinalis Stimulates the Secretion and Action of Insulin and Inhibits Starch Digestion and Protein Glycation In vitro

Emblica officinalis Stimulates the Secretion and Action of Insulin and Inhibits Starch Digestion and Protein Glycation In vitro

In vitro analyses of resistant starch in retrograded waxy and normal corn starches

Gelatinized waxy and normal corn starches (40% starch) were subjected to temperature cycling between 4 and 30°C (1 day at each temperature) or isothermal storage (4°C) to induce retrogradation. The in vitro analysis methods that are currently used for the measurement of resistant starch (RS), i.e. Englyst, AACC 32-40 and Goni methods, were compared with homogenized retrograded starch gels and freeze-dried powders of the gels. RS contents obtained by the three analysis methods were in the following order: Goni>Englyst>AACC. Although different RS values were obtained among the analysis methods, similar trends in regards to the starch type and storage conditions could be observed. Little or no RS was found in freeze-dried powders of the retrograded starch gels and storage conditions had no effect, indicating that the physical state for RS analysis is important. More RS was found in normal corn starch gels than in waxy corn starch gels under identical storage conditions and in the gels stored under temperature cycling than those under isothermal storage (4°C), indicating that the presence of amylose inhibits starch digestion and the level of crystalline structure of re-crystallized amylopectin also affects the RS formation during retrogradation.