Preparation method of non-debranching enzyme sensitive starch-based alpha-glucan

Abstract

The invention discloses a preparation method of non-debranching enzyme sensitive starch-based alpha-glucan, and belongs to the technical field of food industry. The invention aims to realize efficient conversion of various different starch-based anti-digestion alpha-glucan without debranching enzyme sensitive structures so as to promote product industrialization. The method comprises a starch liquefaction process, a debranching process and a transglycosylation process, solves the problems of high viscosity and complex components of a high-concentration starch system, and is convenient for efficiently modifying starch by glycosyltransferase to synthesize starch-based anti-digestion alpha-glucan with different structures and properties.

Description

Technical Field

[0001] This invention relates to a method for preparing non-debranching enzyme-sensitive starch-based α-glucan, belonging to the field of food industry technology. Background Technology

[0002] Alpha-glucan is a homopolysaccharide composed of glucose units linked by α-glycosidic bonds, widely found in nature. Starch-based anti-digestive α-glucan is a class of α-glucans with anti-digestive properties synthesized from starch using 4,3 / 6-α-glycosyltransferases through a specific process. Due to their unique physiological functions and nutritional value, this type of polysaccharide has received widespread attention in food science and nutrition in recent years. Its anti-digestive properties allow it to resist enzymatic degradation in the human digestive tract, thereby delaying the release and absorption of glucose. Simultaneously, it exerts a prebiotic effect, promoting the proliferation of beneficial gut bacteria and potentially bringing various health benefits such as lowering blood sugar and improving lipid metabolism. Due to its structural diversity, α-glucan exhibits rich physicochemical properties and possesses excellent dietary fiber and prebiotic potential, making it a highly promising functional polysaccharide.

[0003] However, current research on starch-based resistant α-glucans is mainly concentrated in the laboratory stage, mostly conducted in low-concentration starch systems. This differs significantly from the high-concentration conditions in actual industrial production, making it difficult to directly apply existing research results to large-scale production. Therefore, there is an urgent need for an efficient starch conversion process to transform starch into deep-processed products with higher nutritional value and application efficacy, thereby achieving high-value and diversified applications of starch. In starch conversion, linear dextrin obtained by introducing debranching enzymes is more likely to serve as a substrate for 4,3 / 6-α-glycosyltransferases.

[0004] α-Glucan contains (α1→4) glycosidic bonds and (α1→6) glycosidic bonds. When the proportion of (α1→6) glycosidic bonds is higher than 80%, it has extremely strong digestive resistance (ensuring the effectiveness of prebiotics), excellent solubility and solution stability, making it very suitable for fields that require clear, low-viscosity, and high-efficiency prebiotic formulations.

[0005] Considering whether the structural bonds of the starch-based resistant α-glucan prepared in the reaction are degraded by debranching enzymes, and the impact of linear dextrin generated during liquefaction and debranching on the composition and yield of the 4,3 / 6-α-glycosyltransferase product, different process routes and reaction conditions need to be designed for products with specific structures to obtain high-yield target resistant α-glucan. This invention lays a solid foundation for the large-scale industrial production of starch-based resistant α-glucan. Summary of the Invention

[0006] To overcome the aforementioned shortcomings, this invention provides a high substrate concentration conversion method for starch-based resistant α-glucans. The aim is to achieve efficient conversion of various starch-based resistant α-glucans without debranching enzyme-sensitive structures, thereby promoting the industrialization of the products. This method includes starch liquefaction, debranching, and glycosyltransferase processes, addressing the problems of high viscosity and complex composition in high-concentration starch systems. It facilitates efficient modification of starch by glycosyltransferases to synthesize starch-based resistant α-glucans with different structures and properties.

[0007] This invention provides a method for preparing non-debranching enzyme-sensitive starch-based resistant α-glucan using a high-concentration substrate. The method involves first liquefying starch with a liquefying enzyme, followed by enzyme inactivation. Based on the target product's insensitivity to debranching enzymes, debranching enzymes and glycosyltransferases are selected to simultaneously perform debranching and glycosyltransferase reactions on starch. After the reaction is completed, the enzymes are inactivated, the mixture is centrifuged, and the supernatant is then subjected to alcohol precipitation, dialyzed, and dried to obtain the target product, starch-based resistant α-glucan.

[0008] The present invention also provides a method for preparing starch-based resistant α-glucan, the method comprising the following steps: (1) Add liquefying enzyme to starch suspension to carry out liquefaction reaction; the mass fraction (w / w) of starch in starch suspension is 1~30%.

[0009] (2) Add debranching enzyme and transglycosylation enzyme to the starch obtained in step (1) and react to obtain the reaction product.

[0010] The present invention also provides a method for preparing a starch-based resistant α-glucan containing a non-debranching enzyme-sensitive structure, the method comprising the following steps: (1) Add 2 U / g to the starch suspension 干淀粉 ~4 U / g 干淀粉 The β-cyclodextrin glucosyltransferase was subjected to a liquefaction reaction under the following conditions: the temperature was raised to 100°C over a period of 50 to 70 minutes and then held for 30 to 50 minutes; the starch mass fraction (w / w) in the starch suspension was 1 to 30%; after the liquefaction reaction, the temperature was lowered to 60°C. (2) Pullulanase and 4,6-α-glucosyltransferase are added simultaneously to the reaction system obtained in step (1) for enzymatic hydrolysis. The amount of pullulanase added is 15 U / g. 干淀粉 ~25 U / g 干淀粉 The amount of 4,6-α-glucosyltransferase added was 30 U / g. 干淀粉 ~35 U / g 干淀粉The enzymatic hydrolysis conditions are: 40~45℃, 20~30 h, enzyme inactivation after hydrolysis to obtain the reactant; the reactant is centrifuged and the supernatant is collected, dialyzed and dried to obtain starch-based α-glucan.

[0011] In one embodiment of the present invention, the starch mass fraction (w / w) in the starch suspension is 1%~2%, 2%~3%, 3%~4%, 4%~5%, 5%~6%, 6%~7%, 7%~8%, 8%~9%, 9%~10%, 10%~11%, 11%~12%, 12%~13%, 13%~14%, 14%~15%, 15%~16%, 16%~17%, 17%~18%, 18%~19%, 19%~20%, 21%~22%, 22%~23%, 23%~24%, 24%~25%, 25%~26%, 26%~27%, 27%~28%, 28%~29%, or 29%~30%.

[0012] In one embodiment of the present invention, in step (1), the β-cyclodextrin glucosyltransferase is derived from... Alkalophilic Bacillus SP. 1011, NCBI number: AAA22308.1.

[0013] In one embodiment of the present invention, the amount of β-cyclodextrin glucosyltransferase added is 2.1 U / g. 干淀粉 2.2 U / g 干淀粉 2.3 U / g 干淀粉 2.4 U / g 干淀粉 2.5 U / g 干淀粉 2.6 U / g 干淀粉 2.7 U / g 干淀粉 2.8 U / g 干淀粉 2.9 U / g 干淀粉 3.0 U / g 干淀粉 3.1 U / g 干淀粉 3.2 U / g 干淀粉 3.3 U / g 干淀粉 3.4 U / g 干淀粉 3.5 U / g 干淀粉 3.6 U / g 干淀粉 3.7 U / g 干淀粉 3.8 U / g 干淀粉 3.9 U / g 干淀粉 Or 4.0 U / g 干淀粉 .

[0014] In one embodiment of the present invention, the reaction conditions in step (1) are: heating to 50°C, raising the temperature of the solution from 50°C to 100°C within 1 hour, keeping it at that temperature for 30 minutes, and then cooling it to 60°C after liquefaction.

[0015] In one embodiment of the present invention, in step (2), the pullulanase is derived from: Bacillus thermophilic US10, NCBI number: AJ315595.1; the 4,6-α-glucosyltransferase described is derived from... Lactobacillus reuteri 121 GtfB, NCBI No.: AAU08014.2; In one embodiment of the present invention, in step (2), the amount of pullulanase added is 15 U / g. 干淀粉 16U / g 干淀粉 17 U / g 干淀粉 18 U / g 干淀粉 19 U / g 干淀粉 20 U / g 干淀粉 21 U / g 干淀粉 22 U / g 干淀粉 23 U / g 干淀粉 24 U / g 干淀粉 Or 25 U / g 干淀粉 .

[0016] In one embodiment of the present invention, the starch substrate includes potato starch, tapioca starch, corn starch, pea starch, etc.

[0017] In one embodiment of the present invention, the enzyme inactivation method includes high-temperature enzyme inactivation, acid-base enzyme inactivation, and ethanol enzyme inactivation.

[0018] In one embodiment of the present invention, the molecular weight cutoff of the dialysis bag is 1000 Da or higher.

[0019] In one embodiment of the present invention, the drying method includes freeze drying, atmospheric pressure drying, spray drying, drum drying, or microwave drying.

[0020] In one embodiment of the present invention, in step (2), the enzymatic hydrolysis conditions are: reaction in a water bath at 40°C for 24 h.

[0021] In one embodiment of the present invention, in step (2), the amount of 4,6-α-glucosyltransferase added is 30 U / g. 干淀粉 31 U / g 干淀粉 32 U / g 干淀粉 33 U / g 干淀粉 34 U / g 干淀粉35 U / g 干淀粉 .

[0022] This invention provides a starch-based resistant α-glucan prepared by the above method.

[0023] The present invention also provides the application of the above-mentioned starch-based resistant α-glucan in the food, pharmaceutical and biological fields.

[0024] Beneficial effects (1) The α-glucan obtained by the method of the present invention has an α-glucan yield of more than 85%.

[0025] (2) The method of this invention uses starch as a substrate, which has the advantages of wide availability and low cost, opening up a new pathway for the high-value conversion of starch and realizing the high-concentration synthesis of starch-based resistant α-glucan. The entire process uses enzymatic conversion technology, which has the characteristics of high efficiency, high specificity, cleanliness and safety, and can be applied in food production. The product conversion rate is high, the product composition is uniform, the purity is high, the by-products are few, and the product structure is clear and well-defined.

[0026] (3) The conversion process provided by the present invention enables the efficient synthesis of a variety of α-glucans with different structures that do not contain debranching enzyme sensitivity, providing technical guidance for industrialization. These starch-based anti-digestive α-glucans can be added to food and medicine as potential dietary fiber or prebiotics. Attached Figure Description

[0027] Figure 1 : Conversion process flow diagram.

[0028] Figure 2 : Glycosidic bond composition analysis of P1 ( 1 H NMR).

[0029] Figure 3 Enzymatic fingerprint of P1. S, standard (G1-G8); P, product P1; 1-6, P1 is hydrolyzed by α-amylase (1), β-amylase (2), endoglucosidase (3), exoglucosidase (4), pullulanase (5) and isopulanase (6), respectively. Detailed Implementation

[0030] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0031] The following embodiments involve methods for detecting bond types: Weigh 10 mg of the lyophilized reaction product, add 1.5 mL of heavy water (D2O), heat in a boiling water bath for 60 min to fully exchange the heavy water, and then lyophilize. Repeat the above operation twice. Then, redissolve the sample in 500 μL of D2O containing tris(trimethylsilyl) phosphate (TMSP). Measure the relative contents of (α1→4), (α1→3), and (α1→6) glycosidic bonds in the sample using an Avance NEO 600MHz fully digital nuclear magnetic resonance spectroscopy (1H NMR). Based on the peak areas at chemical shifts of 5.41 ppm, 5.37 ppm, and 4.97 ppm (corresponding to the anomeric proton signals of (α1→4), (α1→3), and (α1→6) glycosidic bonds, respectively), the relative contents of (α1→4), (α1→3), and (α1→6) glycosidic bonds in the sample were preliminarily calculated.

[0032] Example 1: Preparation of starch-based resistant α-glucan Includes the following steps: (1) Preparation of substrate: A certain amount of potato starch was weighed and dispersed in sodium acetate buffer (25 mM, pH 5.0, containing 1 mM CaCl2) to prepare a 30% (w / w) starch solution.

[0033] (2) Liquefaction reaction: Add 2 U of β-cyclodextrin glucosyltransferase (β-CGTase) per gram of dry starch. (Source: Alkalophilic) Bacillus Add β-cyclodextrin glucosyltransferase to the starch solution according to the dosage of sp. 1011 (NCBI number: AAA22308.1), heat in a water bath with stirring (speed: 200 rpm), slowly raise the temperature of the solution from 50℃ to 100℃ (within 1 h), keep it at this temperature for 30 minutes, and then cool it down to 60℃ after liquefaction is completed.

[0034] (3) Debranching and transformation reactions occur simultaneously: Add 20 U pullulanase (source: [missing information]) per gram of dry starch (based on the potato starch weighed in step (1)). Bacillus thermoleovorans The amount of pullulanase and 30 U of pullulanase added to the liquefied starch solution obtained in step (1) were determined by adding US10 (NCBI number: AJ315595.1) and NCBI number: AJ315595.1. Lactobacillus reuteri121 GtfB type 4,6-α-glucosyltransferase (Lr121GtfB, NCBI No.: AAU08014.2) was reacted in a 40℃ water bath for 24 h. After the reaction was completed, it was boiled in a water bath for 15 min.

[0035] (4) Centrifuge the reactants (8000 × g, 4℃, 20 min), collect the supernatant, dialyze with deionized water for 48 h to remove oligosaccharides (molecular weight cutoff 1000 Da), freeze dry, collect, weigh, grind, and obtain product P1.

[0036] Example 2: Preparation of starch-based resistant α-glucan 1. Experimental Method: (1) Specifically the same as in Example 1, except that the reaction time in the water bath in step (3) is adjusted to 12 h, 18 h and 36 h respectively, to obtain products P2, P3 and P4.

[0037] (2) Specifically the same as in Example 1, except that the amount of pullulanase added in step (3) is adjusted to 5 U, 10 U, 30 U and 50 U respectively, to obtain products P5, P6, P7 and P8.

[0038] (3) Specifically the same as in Example 1, except that step (3) is adjusted. Lactobacillus reuteri The amounts of 121 GtfB type 4,6-α-glucosyltransferase added were 10 U, 20 U, 40 U, ​​and 50 U, respectively, yielding products P9, P10, P11, and P12.

[0039] (4) Specifically the same as in Example 1, except that the liquefaction process in step (2) is adjusted to: liquefaction holding time is 1 h, 2 h, and the products obtained are P13 and P14.

[0040] (5) Specifically the same as in Example 1, except that the amount of β-cyclodextrin glucosyltransferase added in step (2) is adjusted to 1 U, 5 U, and 10 U, and the products obtained are P15, P16, and P17.

[0041] 2. Experimental Results: The bond type ratio and α-glucan yield of the products of Example 1 and α-glucan-1~17 were tested, and the results are shown in Table 1 below. The α-glucan yield was calculated as follows: the mass of the dried product was recorded as m1, the dry starch mass was recorded as m2, and the yield was m1 / m2*100%.

[0042] Table 1: Results

[0043] The results show: (1) As can be seen from the results of P1~P4, with the increase of conversion time, the proportion of digestible (α1→6) glycosidic bonds increases, and the yield of the target product also increases; when more than 24 h, the enzyme activity is lost, and the proportion of (α1→6) glycosidic bonds and the yield of the target product remain basically unchanged. (2) The results from P1, P5~P8 show that pullulanase at low addition levels (5U, 10U) cannot completely debranch starch, resulting in a higher proportion of (α1→4) glycosidic bonds. Pullulanase at high addition levels (20U, 30U, 50U) debranchs too quickly, and the linear substrate cannot be absorbed in time. Lactobacillus reuteri 121 GtfB type 4,6-α-glucosyltransferase is used to regenerate, resulting in a higher proportion of (α1→4) glycosidic bonds and a lower yield of the target product; (3) From the results of P1, P9~P12, it can be seen that low addition amounts (10U, 20U) Lactobacillus reuteri 121GtfB type 4,6-α-glucosyltransferase cannot utilize debranched linear substrates in a timely manner, resulting in a high proportion of (α1→4) glycosidic bonds in the product and a low yield of the target product. High addition amounts (30U, 40U, 50U) are necessary. Lactobacillus reuteri The increase in the (α1→6) glycosidic bond ratio and the yield of the target product prepared by 121 GtfB type 4,6-α-glucosyltransferase was not significant. For economic reasons, an excessively high amount of enzyme was not selected.

[0044] (4) As can be seen from the results of P1, P13~P14, for liquefaction, extending the liquefaction holding time produces more small sugars, which is not conducive to the debranching of pullulanase, resulting in a higher proportion of (α1→4) glycosidic bonds in the product. (5) The results of P1, P15~P17 show that the addition of low amount (1U) of β-cyclodextrin glucosyltransferase leads to incomplete liquefaction of high-concentration starch, resulting in clumps and insufficient subsequent conversion, low (α1→6) glycosidic bond ratio and low yield of target product. Excessive small sugars generated from high addition levels (10U) cannot be utilized, which also has an adverse effect on the formation of (α1→6) glycosidic bonds and the yield of the target product.

[0045] Example 3: Preparation of starch-based resistant α-glucan 1. Effects of enzymes from different sources (1) Specifically the same as in Example 1, except that the β-cyclodextrin glucosyltransferase (liquefying enzyme) in step (2) is adjusted to: Thermoproteus uzoniensis 4-α-glucosyltransferase of the source (NCBI ID: WP_013679179.1), Niallia circulans β-cyclodextrin glucosyltransferase of this source (NCBI ID: CAA48401.1) Geobacillus stearothermophilus β-cyclodextrin glucosyltransferase (NCBI code: CAA41772.1) from s and α-amylase from Aspergillus oryzae (HS code: A9857) were used to prepare products P18, P19, P20, and P21, respectively. The results are shown in Table 2. The results show that the yield of α-glucan is relatively low when using glucosyltransferase or α-amylase from other sources.

[0046] (2) Specifically the same as in Example 1, except that pullulanase in step (3) is adjusted to Anoxybacillus Pullulanase derived from sp. LM18-11 (NCBI ID: AEW23439.1), Acidopullulytic Bacillus The source of pullulanase (NCBI number: CAC60156.1) Klebsiella pneumoniae Pullulanase (NCBI ID: STR29919.1) was used to prepare products P22, P23, and P24, respectively. The results are shown in Table 2. The results show that the yield of α-glucan is relatively low when pullulanase from other sources is used.

[0047] (3) Specifically the same as in Example 1, except that the glycosyltransferases in step (3) are adjusted to be respectively Lactobacillus fermentum NCC 2970 GtfB type 4,3-α-glucosyltransferase (Lf2970 GtfB, NCBI No.: AOR73699.1) Fructilactobacillus sanfranciscensis TMW11304 GtfB type 4,6-α-glucosyltransferase (NCBI No.: WP103434638.1); products P25 and P26 were prepared respectively; the results are shown in Table 2.

[0048] (4) Specifically the same as in Example 1, except that the β-cyclodextrin glucosyltransferase liquefying enzyme in step (2) is adjusted to: Thermoproteus uzoniensis The source of 4-α-glucosyltransferase (NCBI ID: WP_013679179.1) and the pullulanase in step (3) were adjusted. Anoxybacillus Pullulanase from sp. LM18-11 (NCBI ID: AEW23439.1) was used to prepare product P27; the results are shown in Table 2.

[0049] (5) Specifically the same as in Example 1, except that the β-cyclodextrin glucosyltransferase liquefying enzyme in step (2) is adjusted to: Thermoproteus uzoniensis The source of 4-α-glucosyltransferase (NCBI ID: WP_013679179.1) and the glycosyltransferase in step (3) are adjusted as follows: Lactobacillus fermentumThe NCC 2970 GtfB type 4,3-α-glucosyltransferase (Lf2970 GtfB, NCBI No.: AOR73699.1) was used to prepare product P28; the results are shown in Table 2.

[0050] (6) Specifically the same as in Example 1, except that pullulanase in step (3) is adjusted to Anoxybacillus Pullulanase from sp. LM18-11 (NCBI ID: AEW23439.1), adjust the glycosyltransferase in step (3) to Lactobacillus fermentum The NCC 2970 GtfB type 4,3-α-glucosyltransferase (Lf2970 GtfB, NCBI No.: AOR73699.1) was used to prepare product P29; the results are shown in Table 2.

[0051] (7) Adjust the β-cyclodextrin glucosyltransferase liquefying enzyme in step (2) to: Thermoproteus from Usonia The source of 4-α-glucosyltransferase (NCBI ID: WP_013679179.1) and the pullulanase in step (3) were adjusted. Anoxybacillus Pullulanase from sp. LM18-11 (NCBI ID: AEW23439.1), adjust the glycosyltransferase in step (3) to Lactobacillus fermentum The NCC 2970 GtfB type 4,3-α-glucosyltransferase (Lf2970 GtfB, NCBI No.: AOR73699.1) was used to prepare product P30; the results are shown in Table 2.

[0052] 2. The experimental results are shown in Table 2.

[0053] Table 2: Experimental Results

[0054] Example 4: Preparation of starch-based digestible α-glucan under different substrate concentrations Specifically, the process is the same as in Example 1, except that the starch solution concentration in step (1) is adjusted to 10% (w / w) and 20% (w / w) to prepare product-10% and product-20%; The bond type ratio of the product and the yield of α-glucan were determined according to the method in Example 2.

[0055] The results are shown in Table 3: Table 3: Results

[0056] Example 5: Identification of starch-based resistant α-glucan (1) Glycosidic bond composition Weigh 10 mg of the lyophilized reaction product P1, add 1.5 mL of heavy water (D2O), heat in a boiling water bath for 60 min to fully exchange the heavy water, and then lyophilize. Repeat the above operation twice. Then, redissolve the sample in 500 μL of D2O containing tris(trimethylsilyl) phosphate (TMSP). Measure the relative contents of (α1→4) and (α1→6) glycosidic bonds in the sample using an Avance NEO 600 MHz fully digital nuclear magnetic resonance spectroscopy (Proton nuclear magnetic resonance spectroscopy, 1H NMR). Based on the peak areas at chemical shifts of 5.37 ppm and 4.97 ppm (corresponding to the anomeric proton signals of (α1→4) and (α1→6) glycosidic bonds, respectively), the relative contents of (α1→4) and (α1→6) glycosidic bonds in the sample were preliminarily calculated.

[0057] The nuclear magnetic resonance spectrum of the sample is as follows Figure 2 The proportion of (α1→6) glycosidic bonds is 86.3%, and the proportion of (α1→4) glycosidic bonds is 13.7%.

[0058] (2) Enzyme fingerprint The P1 product was dissolved in NaAc-HAc buffer (25 mM, pH 5.0, containing 1 mM CaCl2) to prepare a product stock solution (6 mg / mL). Excess of [unspecified ingredient] was added to each solution. Aspergillus oryzae α-Amylase, sweet potato β-amylase 、 Chaetomium sp. Endoglucosidase, Streptococcus mutans Exo-dextrosease, Bacillus acidophilic pullulanase and Aspergillus niger Isoprolanase was digested at 37 °C for 48 hours.

[0059] Take 1-2 μL of the enzymatically digested sample and spot it onto a TLC silica gel plate. Develop the sample in a developing solvent system (ethyl acetate / isopropanol / water = 3:1:1, v / v / v) until the TLC silica gel plate is completely wetted. Remove the TLC plate, dry the solvent, and stain with a methanol reagent containing 0.3% (w / v) naphthylethylenediamine hydrochloride and 5% (w / v) sulfuric acid. Heat at 160 °C for color development.

[0060] Enzyme fingerprint analysis is Figure 3 .

[0061] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing starch-based α-glucan containing a non-debranching enzyme-sensitive structure, characterized in that, The method includes the following steps: (1) Add 2 U / g to the starch suspension 干淀粉 ~4 U / g 干淀粉 The β-cyclodextrin glucosyltransferase was subjected to a liquefaction reaction under the following conditions: the temperature was raised to 100°C over a period of 50 to 70 minutes and then held for 30 to 50 minutes; the starch mass fraction (w / w) in the starch suspension was 1 to 30%; after the liquefaction reaction, the temperature was lowered to 60°C. (2) Pullulanase and 4,6-α-glucosyltransferase are added simultaneously to the reaction system obtained in step (1) for enzymatic hydrolysis. The amount of pullulanase added is 15 U / g. 干淀粉 ~25 U / g 干淀粉 The amount of 4,6-α-glucosyltransferase added was 30 U / g. 干淀粉 ~35 U / g 干淀粉 The enzymatic hydrolysis conditions are: 40~45℃, 20~30 h, enzyme inactivation after hydrolysis to obtain the reactant; the reactant is centrifuged and the supernatant is collected, dialyzed and dried to obtain starch-based α-glucan.

2. The method according to claim 1, characterized in that, The starch suspension contains starch with a mass fraction of 1%~2%, 2%~3%, 3%~4%, 4%~5%, 5%~6%, 6%~7%, 7%~8%, 8%~9%, 9%~10%, 10%~11%, 11%~12%, 12%~13%, 13%~14%, 14%~15%, 15%~16%, 16%~17%, 17%~18%, 18%~19%, 19%~20%, 21%~22%, 22%~23%, 23%~24%, 24%~25%, 25%~26%, 26%~27%, 27%~28%, 28%~29%, or 29%~30%.

3. The method according to claim 1 or 2, characterized in that, In step (1), the β-cyclodextrin glucosyltransferase is derived from... Alkalophilic Bacillus sp. 1011; Preferably, the addition amount of the β-cyclodextrin glucanotransferase is 2.1 U / g 干淀粉 , 2.2 U / g 干淀粉 , 2.3 U / g 干淀粉 , 2.4 U / g 干淀粉 , 2.5 U / g 干淀粉 , 2.6 U / g 干淀粉 , 2.7 U / g 干淀粉 , 2.8 U / g 干淀粉 , 2.9 U / g 干淀粉 , 3.0 U / g 干淀粉 , 3.1 U / g 干淀粉 , 3.2 U / g 干淀粉 , 3.3 U / g 干淀粉 , 3.4 U / g 干淀粉 , 3.5 U / g 干淀粉 , 3.6 U / g 干淀粉 , 3.7 U / g 干淀粉 , 3.8 U / g 干淀粉 , 3.9 U / g 干淀粉 or 4.0 U / g 干淀粉 .

4. The method according to any one of claims 1 to 3, characterized in that, In step (1), the reaction conditions are: heating to 50°C, raising the temperature of the solution from 50°C to 100°C within 1 hour, keeping it at that temperature for 30 minutes, and then cooling it to 60°C after liquefaction.

5. The method according to any one of claims 1 to 4, characterized in that, In step (2), the pullulanase is derived from: Bacillus thermoleovorans US10; the 4,6-α-glucosyltransferase is derived from... Lactobacillus reuteri 121 GtfB.

6. The method according to any one of claims 1 to 5, characterized in that, In step (2), the amount of pullulanase added is 15 U / g. 干淀粉 16 U / g 干淀粉 17 U / g 干淀粉 18 U / g 干淀粉 19 U / g 干淀粉 20 U / g 干淀粉 21 U / g 干淀粉 22 U / g 干淀粉 23 U / g 干淀粉 24 U / g 干淀粉 Or 25 U / g 干淀粉 .

7. The method according to any one of claims 1 to 6, characterized in that, The enzyme inactivation methods include high-temperature enzyme inactivation, acid-base enzyme inactivation, and ethanol enzyme inactivation. Preferably, the dialysis molecular weight cutoff is 1000 Da or higher; Preferably, the drying method includes freeze drying, atmospheric pressure drying, spray drying, drum drying, or microwave drying.

8. The method according to any one of claims 1 to 7, characterized in that, In step (2), the enzymatic hydrolysis conditions are: reaction in a 40°C water bath for 24 h.

9. The method according to any one of claims 1 to 8, characterized in that, In step (2), the amount of 4,6-α-glucosyltransferase added is 30 U / g. 干淀粉 31 U / g 干淀粉 32 U / g 干淀粉 33 U / g 干淀粉 34 U / g 干淀粉 35 U / g 干淀粉 .

10. The application of the starch-based α-glucan prepared by any one of the methods of claims 1 to 9 in the preparation of food, biological products, and pharmaceuticals.

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