Preparation method of heat-resistant and digestion-resistant starch microspheres

Abstract

The invention belongs to the technical field of starch structure regulation and function modification, and provides a preparation method of heat-resistant and digestion-resistant starch microspheres. The method comprises the following steps: by taking high amylose as a raw material, carrying out acidolysis and debranching treatment to obtain an amylose mixed component, and further carrying out accurate grading on the amylose according to the molecular size through volume exclusion chromatography to obtain an amylose fraction with narrow polymerization degree distribution and uniform chain length. Then recrystallization induction is carried out on the fractions, autoclaving treatment is carried out under the condition that the water content is controlled, amylose molecules are subjected to ordered rearrangement, and the starch microspheres which are compact in structure and high in crystallinity are constructed. The prepared resistant starch microspheres have excellent thermal stability and enzymolysis resistance, and still keep higher resistant starch content after being boiled. The method realizes accurate regulation and control of the chain length of the amylose and synergistic improvement of heat resistance and digestion resistance, is suitable for the fields of functional food and nutrition and health, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of starch structure regulation and functional modification technology, and provides a method for preparing heat-resistant and digestion-resistant starch microspheres. Background Technology

[0002] Resistant starch (RS) is a class of starches and their degradation products that are not absorbed by the small intestine of healthy individuals but are fermented and utilized by intestinal microorganisms in the large intestine. It produces beneficial metabolites such as short-chain fatty acids, which have various physiological functions, including regulating blood sugar and improving gut health. Type III resistant starch (RS3) has attracted widespread attention due to its high thermal stability. However, a portion of Type III resistant starch is still digested after cooking (typically exceeding 40%) (CN115466759B), which reduces its functionality. Therefore, the development of RS3 with higher thermal stability and greater resistance to digestion holds great promise.

[0003] RS3 is formed through recrystallization of debranched starch; therefore, the degree of polymerization of amylose is the main factor affecting the formation and content of RS3. Existing technologies have improved the content of resistant starch by reducing the molecular weight distribution of starch through ethanol precipitation (CN115181767A), but the molecular weight distribution still largely overlaps and lacks precise control over the amylose chain length, making it difficult to achieve synergistic optimization of RS3's digestibility and heat resistance. Summary of the Invention

[0004] To address the shortcomings of existing RS3 microspheres, such as insufficient heat resistance and lack of precise structural control methods, this invention provides a method for preparing heat-resistant and digestible starch microspheres. By precisely controlling the degree of polymerization of amylose through gel chromatography, highly thermally stable digestible starch microspheres can be obtained.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows: The first aspect of the present invention provides a method for preparing heat-resistant and digestible starch microspheres, characterized by comprising the following steps: (1) Preparation of amylose mixture: After acid hydrolysis and debranching of high amylose, amylose mixture is obtained by precipitation; (2) Amylose fractionation: After dissolving the amylose mixture, fractionation is performed by size exclusion chromatography to obtain amylose fractions with different degrees of polymerization, and recrystallization is induced. (3) Pressure heat treatment: The recrystallized and induced amylose fractions are subjected to pressure heat treatment to obtain the heat-resistant and digestible starch microspheres.

[0006] Furthermore, the high amylose is high amylose pea starch; the acid hydrolysis treatment includes dispersing the high amylose in a hydrochloric acid solution, reacting at 40-60℃ for 10-30 hours, and separating after neutralization to obtain acid hydrolyzed starch.

[0007] Furthermore, the debranching treatment includes dispersing acid-hydrolyzed starch in a buffer solution, gelatinizing it by heating at high temperature, adding pullulanase and reacting at 40-60℃ for 10-30 hours, inactivating the enzyme, and then taking the supernatant.

[0008] Furthermore, the amylose mixture is obtained by adding ethanol to the supernatant for precipitation, allowing it to stand, separating and drying to obtain the mixture.

[0009] Furthermore, the size exclusion chromatography fractionation includes dissolving the mixed components in DMSO, precipitating them, dissolving them in urea solution, filtering and loading the sample, using urea solution as the eluent, and collecting the eluent; the recrystallization induction includes adding ethanol to the eluent, allowing it to stand for recrystallization, and then separating and drying.

[0010] Furthermore, the pressure heat treatment includes preparing recrystallized amylose into a starch slurry with a moisture content of 20-40%, subjecting it to high pressure treatment for 0.5-2 hours, and drying it after cooling to obtain the starch microspheres; the high pressure treatment temperature is 100-140℃.

[0011] Furthermore, the average degree of polymerization (DP) of the amylose fraction is 10-70, preferably 25-35.

[0012] The second aspect of this invention is to provide heat-resistant and digestion-resistant starch microspheres, characterized in that they are prepared by the method described in the first aspect, wherein the starch microspheres are spherical particles with a type B crystalline structure, a relative crystallinity of 20-50%, a gelatinization temperature of 80-150°C, and a resistant starch content of 40-90%; preferably, the degree of polymerization (DP) of the amylose in the microspheres is 25, 35, or 47. Furthermore, the starch microspheres have an average degree of polymerization (DP) of 25, and after pressure heat treatment, the resistant starch content is ≥80%, and the gelatinization peak temperature is ≥120℃.

[0013] A third aspect of the present invention is to provide the application of the heat-resistant and digestive-resistant starch microspheres described in the second aspect in the preparation of food, pharmaceuticals or health products, characterized in that they are used to regulate blood sugar, improve intestinal health or as a heat-resistant functional ingredient.

[0014] The beneficial effects of this invention include: 1) Achieve precise control of amylose chain length: Amylose is fractionated by size exclusion chromatography, which significantly narrows the molecular weight distribution and enables precise control of the degree of polymerization of amylose, overcoming the problems of wide chain length distribution and uncontrollable structure in the existing technology.

[0015] 2) Significantly improves the heat resistance and digestibility of resistant starch: Through the synergistic effect of recrystallization and pressure heat treatment, starch microspheres with dense structure and high crystallinity are constructed, so that the obtained resistant starch still maintains a high resistant starch content under high temperature cooking conditions, which is significantly better than conventional RS3 products.

[0016] 3) The method is highly controllable and has good application adaptability: The process route of this invention is clear and the parameters are adjustable. It is applicable to different amylose raw materials, and the resulting products are suitable for use in functional foods, nutritional preparations and other fields, and have good industrial application potential. Attached Figure Description

[0017] Figure 1 Molecular weight distribution of recrystallized amylose at different molecular weights; Figure 2 XRD patterns of recrystallized amylose at different molecular weights; Figure 3 Microstructure diagrams of recrystallized amylose starch at different molecular weights; Figure 4 The molecular weight distribution (A) and XRD pattern (B) of starch obtained by gravity induction of DP35 RS3 prepared without size exclusion chromatography. Detailed Implementation

[0018] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0019] Example 1: Preparation of heat-resistant and digestible starch microspheres 1. Preparation of amylose mixture (1) Acid hydrolysis: Accurately weigh 50g of high amylose pea starch and disperse it in 0.8M hydrochloric acid solution to prepare a 30% (w / w starch on a dry basis) starch slurry. Then, react the slurry in a constant temperature shaker at 50℃ and 160rpm for 20 h. Adjust the pH to 7.0 with 4.0M sodium hydroxide to terminate the hydrolysis. 3000× g Centrifuge for 10 minutes, wash the precipitate three times each with distilled water and anhydrous ethanol, and dry it at 40°C overnight to obtain acid-hydrolyzed starch; (2) Debranching treatment: Acid-hydrolyzed starch was dispersed at 6.25% (w / w, starch on dry basis) in 0.01 M sodium acetate buffer (pH 4.0). The starch emulsion bath was heated at 120℃ for 1 h, followed by reaction in a 121℃ autoclave for 1 h. After cooling to 50℃, pullulanase was added at 0.13 mL / g based on the dry weight of starch. The mixture was reacted in a 50℃, 160 rpm air bath constant temperature shaker for 24 h, followed by autoclaving at 121℃ for 30 min to inactivate the enzyme. g Centrifuge for 10 minutes and collect the supernatant; (3) Obtaining the amylose mixture: Add anhydrous ethanol to the supernatant (volume ratio of supernatant to ethanol 1:2), let stand at 4°C for 12 hours, and centrifuge at 3000×g for 10 minutes; wash the precipitate three times each with distilled water and anhydrous ethanol, dry at 40°C overnight, grind through a 125 μm sieve to obtain the amylose mixture.

[0020] 2. Amylose grading (1) Sample preparation: The amylose mixture was suspended in DMSO at 0.1 g / mL and boiled in water for 20 minutes to dissolve it; anhydrous ethanol was added (the volume ratio of solution to ethanol was 1:10), and the mixture was centrifuged at 3000×g for 10 minutes. The precipitate was dissolved in 60 mL of 8 M urea solution and filtered through a 0.45 μm filter membrane. (2) Size exclusion chromatography fractionation: Load the filtrate onto a size exclusion chromatography column, use 1 M urea solution as the eluent, flow rate 2 mL / min, and automatically collect the eluent in 20 mL / tube; (3) Recrystallization induction: Add anhydrous ethanol to the eluent (eluent to ethanol volume ratio 1:10), let stand at 4℃ for 24 hours to recrystallize, centrifuge at 3000×g for 10 minutes, wash the precipitate 3 times with anhydrous ethanol, dry at 40℃ for 12 hours, and grind for later use.

[0021] 3. Pressure Heat Treatment (PHT) The recrystallized amylose product was placed in a glass container and prepared into a starch slurry with a moisture content of 30% (starch on a dry basis). It was then allowed to equilibrate overnight at room temperature. After high-pressure treatment at 121°C for 1 hour, cooled to room temperature for 2 hours, and dried at 40°C overnight, the product was then ground to obtain heat-resistant and digestible starch microspheres.

[0022] Example 2 Performance testing of heat-resistant and digestible starch microspheres 1. The molecular structure of recrystallized amylose was characterized by size exclusion chromatography (SEC). The molecular weight of total starch was determined using a tandem GRAM 30 and GRAM 3000 SEC analytical column. Approximately 2 mg of RS3 sample was dissolved in 1 mL of DMSO solution containing 0.5% w / w lithium bromide. The mobile phase was DMSO containing 0.5% w / w lithium bromide, and the flow rate was set at 0.3 mL / min. Peak molecular weights in the range of 180–1.22 × 10⁻⁶ were used. 6 The pullulan standard of Da was used for calibration.

[0023] Separation using a size exclusion chromatography (SEC) column separated the DP16-mixed sample into seven fractions (DP10, DP13, DP18, DP25, DP35, DP47, and DP70). With increasing elution time, the amylose chain length gradually shortened, consistent with the size exclusion principle: larger molecules have shorter retention times in the pores of the packing material and are therefore eluted earlier. The molecular weight distribution of each fraction exhibited a single and narrower symmetrical peak, indicating higher homogeneity of the fractions compared to the mixed components. This narrowing of the distribution range demonstrates effective separation based on molecular size. Figure 1 ).

[0024] 2. The crystalline structure of recrystallized amylose was characterized by X-ray diffraction (XRD). The relative crystallinity (RC) of the sample was calculated by scanning the sample from 1° to 40° (2θ) at a rate of 2° / min under Cu-Kα radiation (λ = 0.15406 nm) at 40 kV and 40 mA.

[0025] The DP16 (mixed component) sample exhibited a typical A-type crystalline structure, with major diffraction peaks at 2θ values ​​of 15.3°, 17.3°, 18.2°, and 23.2°, and a relative crystallinity (RC) as high as 48.0%. After fractionation, under the same recrystallization conditions, RS3 samples with different degrees of polymerization prepared from narrow-distribution amylose showed significant crystal form transformations. DP70 RS3 showed new characteristic diffraction peaks at 13.1° and 20.1°, corresponding to V... 6h The crystalline structure is attributed to the fact that the long, straight starch chains hinder their alignment and double-helix recombination, thereby promoting the formation of single-helix inclusion complexes with ethanol molecules.

[0026] The results are as follows Figure 2As shown, with decreasing average degree of polymerization, the RS3 samples with medium chain lengths (DP47, DP35, and DP25) exhibit a type B crystalline structure, showing a strong diffraction peak at 17.2° 2θ and weak diffraction peaks at 15.0°, 22.4°, and 24.3°. This result is consistent with previous studies: high solids content and shorter chain lengths induce type A crystal formation, while the opposite conditions promote type B crystal formation. However, the RC values ​​of the type B RS3 samples (29.1%-31.7%) are significantly lower than those of the type A samples (48.0%), which may be because the double helix arrangement in the type B crystalline structure is relatively loose, leading to a corresponding increase in the inter-helix water molecule content.

[0027] When the chain length was further shortened, the RS3 samples of DP18 and DP13 exhibited a mixed crystal structure of type B and type V, showing type B crystallization at 17.2° and type V crystallization at 13.8° and 21.0°, respectively. 6a V-shaped crystals. This may be because some amylose chains are too short to form a stable double helix, and instead adopt a single helix conformation, thus forming a V-shaped crystal structure. Notably, the DP10 RS3 sample only exhibits a V-shaped crystal structure. 6h Crystalline structure. The lower RC value of the DP10 sample may be due to its small crystallites, which prevent the generation of obvious XRD diffraction peaks. These results indicate that the degree of polymerization of amylose chains during recrystallization has a significant impact on the polymorphic structure of RS3.

[0028] After PHT treatment, the crystal types of DP16 and DP25 did not change, but the RC was significantly increased.

[0029] 3. The microstructure of recrystallized amylose was characterized using scanning electron microscopy (SEM). The sample was evenly coated onto the adsorption strip and then sputtered with gold. The sample was observed under an accelerating voltage of 3.0 kV. The surface morphology of the sample was observed by randomly selecting a region and magnifying it by 2000 times.

[0030] The results are as follows Figure 3As shown, RS3 prepared from DP16 mixed with amylose exhibits a dense and irregular aggregate structure. This is attributed to its wide chain length distribution, where excessively short and excessively long amylose molecules interfere with the self-assembly process, leading to heterogeneous nucleation and crystal growth. Compared to DP16 RS3, DP70 RS3 exhibits loosely arranged, non-uniformly sized irregular aggregates. This can be attributed to the entanglement and steric hindrance of long amylose chains, which restrict molecular mobility and disrupt the ordered self-assembly process for forming dense, regular particles. In contrast, RS3 samples from DP47, DP35, and DP25 exhibit well-defined spherical particles. For samples with shorter chain lengths (DP18 and DP13), their RS3 aggregates exhibit an irregular and loosely arranged structure. This may be attributed to the coexistence of V-type and B-type crystals, where the single-helix V-type structure interferes with the arrangement and stacking of the B-type double helix, thereby disrupting the overall molecular orientation. Regarding DP10, its RS3 product was observed to consist of aggregated, irregular particles, likely due to the lack of anisotropic molecular arrangement in its locally ordered V-shaped crystal structure. This suggests that differences in chain length distribution lead to the formation of aggregates with distinct morphologies, possibly related to different self-assembly pathways. PHT treatment had no significant effect on the morphology of DP16 and DP25 particles.

[0031] 4. The thermal properties of recrystallized amylose were characterized using differential scanning calorimetry (DSC). The sample (10 mg) was mixed with distilled water at a ratio of 1:3 (w / v), sealed, and equilibrated at room temperature for 2 hours. The sample was then heated from 20 °C to 160 °C at a rate of 10 °C / min, using an empty sample pan as a reference. It was then rapidly cooled to 20 °C at a rate of 20 °C / min, and then heated again from 20 °C to 160 °C at a rate of 10 °C / min for a second scan. The initial gelatinization temperature To, peak gelatinization temperature Tp, and final gelatinization temperature Tc of the sample were obtained using differential scanning calorimetry.

[0032] The results are shown in Table 1. The mixed component DP16 RS3 exhibits a wide gelatinization temperature range, from 93.9 to 139.9°C, with an enthalpy change of 13.5 J / g. In contrast, the RS3 sample prepared from the homogeneous chain length component shows a lower gelatinization temperature, attributed to differences in crystalline structure. Type A crystals are thermodynamically more stable than type B or type V crystals. The melting temperature of RS3 gradually increases with decreasing chain length, reaching a maximum at DP25, and then decreases further with decreasing chain length. This trend indicates that amylose chains with an average DP of 25 are most favorable for forming thermally stable crystalline structures.

[0033] Compared to the unmodified sample, the PHT-modified RS3 sample exhibited a significantly increased gelatinization temperature. This improvement can be attributed to the rearrangement of the double helix structure induced by PHT treatment, resulting in a more compact arrangement of crystalline and amorphous regions within the particles, thus conferring higher thermal stability. Compared to PHT-modified DP16 RS3, DP25 RS3 modification not only showed a higher gelatinization temperature but also exhibited a narrower melting temperature range. This difference stems from the more uniform chain length distribution of DP25, which is conducive to the formation of a highly ordered and structurally uniform crystal arrangement.

[0034] Table 1 Thermal properties of recrystallized amylose (Note: Different letters in each column indicate significant differences between the data groups.) p <0.05) 5. The Englyst method was used to determine the resistant starch content of recrystallized amylose. 600 mg of starch was dispersed in 15 mL of distilled water and boiled in a water bath for 10 minutes. Then, 5 mL of acetate buffer was added and the mixture was equilibrated at 37°C for 15 minutes. Enzymatic hydrolysis was then performed using a mixture of α-amylase (30 U / mg) and amyloglucosidase (260 U / ml). The glucose content released at 20 and 120 minutes of hydrolysis was quantified using the GOPOD kit (Megazyme D-glucose Assay Kit), and the contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) were calculated accordingly. All experiments were performed in triplicate.

[0035] Due to sample yield limitations, the in vitro digestibility of DP16, DP35, DP25, DP18, and DP13RS3 samples was determined using the Englyst method. The results are shown in Table 2. Cooked DP16 RS3 exhibited 52.9% RDS, 4.2% SDS, and 40.4% RS. The relatively high enzyme resistance of DP16 RS3 may be related to its higher gelatinization temperature (Table 1), which limits the expansion of starch granules during cooking. Compared to DP16 RS3, cooked DP35 and DP25 showed significantly lower digestibility, with RS contents of 50.3% and 52.9%, respectively. Their highly uniform molecular orientation facilitated the formation of ordered double helices, which assembled into crystals with greater structural stability than DP16 RS3, thus significantly reducing the sensitivity to digestive enzymes. In contrast, cooked DP18 and DP13 RS3 had lower resistant starch contents and higher rapidly digestible starch contents. The lower enzyme resistance of DP18 and DP13 RS3 can be attributed to their irregular molecular orientation, which leads to the formation of heterogeneous and thermally unstable crystalline structures. Therefore, these samples exhibit higher gelatinization after cooking, loss of structural integrity, and an inability to rapidly recombine into enzyme-resistant crystals during cooling. Among all components, the DP25 RS3 sample had the highest resistant starch content (52.9%) and the highest gelatinization temperature, indicating superior structural stability.

[0036] After PHT treatment, the RDS content of DP16 and DP25 decreased significantly, while the RS content increased significantly. The significant enhancement of the anti-enzymatic hydrolysis performance of PHT-modified RS3 is mainly attributed to the following mechanisms: (1) At the molecular level, PHT treatment promotes partial structural reorganization of the amylose chain, forming a more ordered and thermally stable crystalline region, thereby reducing the sensitivity to enzymatic hydrolysis; (2) At the particle level, the treated starch particles exhibit a smoother and denser outer layer structure, effectively inhibiting the swelling of starch particles and reducing solubility, thereby further reducing its digestibility. Compared with the PHT-modified DP16 mixed sample, the resistant starch content of the DP25 modified sample was significantly higher (84.6% vs 61.2%), which mainly depends on two key factors: First, the longer and more uniformly oriented amylose chains in DP25 RS3 have better molecular mobility under PHT conditions, which promotes rapid recrystallization and the formation of a highly ordered and perfect crystal structure. This conclusion is confirmed by its higher gelatinization temperature and narrower melting temperature range. Secondly, the PHT-modified DP25 forms a more regular and dense starch granule structure with a relatively smaller specific surface area, which effectively reduces enzyme accessibility.

[0037] Table 2. In vitro starch digestibility of recrystallized amylose (Note: Different letters in each column indicate significant differences between the data groups.) p <0.05) Example 3: Differences between DP35 RS3 and DP25-PHT DP35 RS3 was prepared according to the method described in the inventor's previously published literature (Influence of fractions with different molecular weight distributions from high-amylose starches on their digestibility after recrystallization), summarized as follows: 1. Preparation of DP35 RS3 (1) Acid hydrolysis: Accurately weigh 50g of high amylose pea starch and disperse it in 0.8M hydrochloric acid solution to prepare a 30% (w / w starch on a dry basis) starch slurry. Then, react the slurry in a constant temperature shaker at 50℃ and 160rpm for 20 h. Adjust the pH to 7.0 with 4.0M sodium hydroxide to terminate the hydrolysis. 3000× g Centrifuge for 10 minutes, wash the precipitate three times each with distilled water and anhydrous ethanol, and dry it at 40°C overnight to obtain acid-hydrolyzed starch; (2) Debranching treatment: Acid-hydrolyzed starch was dispersed at 6.25% (w / w, starch on dry basis) in 0.01 M sodium acetate buffer (pH 4.0). The starch emulsion bath was heated at 120℃ for 1 h, followed by reaction in a 121℃ autoclave for 1 h. After cooling to 50℃, pullulanase was added at 0.13 mL / g based on the dry weight of starch. The mixture was reacted in a 50℃, 160 rpm air bath constant temperature shaker for 24 h, followed by autoclaving at 121℃ for 30 min to inactivate the enzyme. g Centrifuge for 10 minutes and collect the precipitate. (3) Recrystallization treatment: recrystallize by standing at 4℃ for 24 hours. Wash three times with anhydrous ethanol, dry at 40℃ for 12 hours, and grind for later use.

[0038] Molecular weight distribution and XRD patterns are as follows Figure 4 As shown in Figures AB, the DP35 starch chain length distribution is wider than that of the amylose fraction obtained by gel chromatography. The DP35 RS3 sample exhibits a type B crystalline structure, with diffraction peaks at 2θ of 5.3°, 15.0°, 17.0°, and 23.0°, and an RC of 43.0%. Scanning electron microscopy results showed that DP35 mainly consists of expanded starch granules / residues and amylose precipitated from the expanded granules during gelatinization. These substances form blocky aggregates during recrystallization due to hydrogen bonding interactions and physical entanglement.

[0039] Table 3 shows that DP35 exhibits a lower ΔH compared to DP25-PHT. This may be due to the presence of unbranched amylopectin in DP35, which interferes with the orderly stacking of amylose molecules and prevents the formation of a dense, complete crystalline structure. Therefore, the energy required to melt these crystalline regions is lower.

[0040] Table 3 Thermal properties of recrystallized high amylose pea starch Table 4 shows that the RS content of DP 35 RS3 is 74.5%, lower than that of DP25-PHT. This difference is directly related to the molecular structural characteristics of the two. DP35 contains a certain proportion of incompletely debranched amylopectin. This incomplete debranching molecular structure has a dual impact on the recrystallization process of starch molecules: on the one hand, the branched structure hinders the orderly arrangement of amylose molecules, slowing down the recrystallization rate; on the other hand, the presence of branches interferes with the integrity of the crystallization network formation, reducing the final degree of recrystallization. Simultaneously, incompletely gelatinized particles combine with amylose to form aggregates of uneven size, which creates more surface voids and edge sites, thereby increasing the contact area between starch and digestive enzymes, improving the efficiency of enzymatic reactions, and reducing the RS content.

[0041] Table 4. In vitro starch digestibility of recrystallized high amylose pea starch The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A method for preparing heat-resistant and digestible starch microspheres, characterized in that, Includes the following steps: (1) Preparation of amylose mixture: After acid hydrolysis and debranching of high amylose, amylose mixture is obtained by precipitation; (2) Amylose fractionation: After dissolving the amylose mixture, fractionation is performed by size exclusion chromatography to obtain amylose fractions with different degrees of polymerization, and recrystallization is induced. (3) Pressure heat treatment: The recrystallized and induced amylose fractions are subjected to pressure heat treatment to obtain the heat-resistant and digestible starch microspheres.

2. The method according to claim 1, characterized in that, The high amylose is high amylose pea starch; the acid hydrolysis treatment includes dispersing the high amylose in hydrochloric acid solution, reacting at 40-60℃ for 10-30 hours, and separating after neutralization to obtain acid hydrolyzed starch.

3. The method according to claim 1 or 2, characterized in that, The debranching process includes dispersing acid-hydrolyzed starch in a buffer solution, gelatinizing it by heating at high temperature, adding pullulanase and reacting at 40-60℃ for 10-30 hours, and then taking the supernatant after enzyme inactivation.

4. The method according to claim 1, characterized in that, The amylose mixture was obtained by adding ethanol to the supernatant to precipitate the amylose, allowing it to stand, separating and drying the mixture.

5. The method according to claim 1, characterized in that, The size exclusion chromatography fractionation includes dissolving the mixed components in DMSO, precipitating them, dissolving them in urea solution, filtering and loading the sample, using urea solution as the eluent, and collecting the eluent; the recrystallization induction includes adding ethanol to the eluent, allowing it to stand for recrystallization, and then separating and drying.

6. The method according to claim 1, characterized in that, The pressure heat treatment includes preparing recrystallized amylose into a starch slurry with a moisture content of 20-40%, subjecting it to high pressure treatment for 0.5-2 hours, and drying it after cooling to obtain the starch microspheres; the high pressure treatment temperature is 100-140℃.

7. The method according to claim 1, characterized in that, The average degree of polymerization (DP) of the amylose fraction is 10-70.

8. A heat-resistant and digestible starch microsphere, characterized in that, The starch microspheres prepared by the method of any one of claims 1-7 are spherical particles with a type B crystalline structure, a relative crystallinity of 20-50%, a gelatinization temperature of 80-150℃, and a resistant starch content of 40-90%.

9. The heat-resistant and digestible starch microspheres according to claim 8, characterized in that, The starch microspheres have an average degree of polymerization (DP) of 25, and after pressure heat treatment, the resistant starch content is ≥80%, and the gelatinization peak temperature is ≥120℃.

10. The application of the heat-resistant and digestion-resistant starch microspheres according to claim 8 or 9 in the preparation of food, pharmaceuticals, or health products, characterized in that, Used to regulate blood sugar, improve gut health, or as a heat-resistant functional ingredient.

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