Method for preparing high-resistance starch through synergy of double enzymes

Abstract

The invention relates to a method for preparing high-resistance starch through synergy of double enzymes, and belongs to the technical field of enzyme engineering and food processing engineering. The method comprises the following steps: preparing starch emulsion from starch and water, heating and gelatinizing the starch emulsion, and performing constant-temperature water bath; adding starch branching enzyme for water bath enzymolysis, performing boiling water bath enzyme deactivation and cooling; adding cane sugar, stirring and dissolving, adding amylosucrase for water bath enzymolysis, and adding absolute ethyl alcohol to terminate the reaction; and separating out solids, washing and freeze-drying to obtain the high-resistance starch. Through the synergistic effect of the starch branching enzyme and the amylosucrase, the production efficiency of the resistant starch is remarkably improved, the structural characteristics of the product are optimized, B-type crystals with high-stability double-helix conformation are formed, the digestion resistance characteristic is remarkably enhanced, various cereal raw materials with low amylose content can be effectively treated, and the raw material adaptability is high.

Description

Technical Field

[0001] This invention relates to a method for the synergistic preparation of highly resistant starch using two enzymes, belonging to the fields of enzyme engineering and food processing engineering technology. Background Technology

[0002] With increasing public awareness of health and growing demand for chronic disease prevention and control, resistant starch (RS) is finding increasingly widespread applications in the food, medical, and nutritional fields due to its unique physiological functions and processing characteristics. In health foods, resistant starch can effectively lower the glycemic index (GI) of food, making it suitable for dietary interventions in people with diabetes and obesity. Clinical studies have shown that daily intake of 40 g of resistant starch can significantly reduce intrahepatic triglyceride levels in patients with non-alcoholic fatty liver disease and improve weight and lipid metabolism, thus it is widely used in meal replacement products and low-calorie snacks. In food processing, resistant starch has low water-holding capacity, high stability, and a neutral taste, making it an ideal functional additive. For example, adding it to fried foods can reduce fat absorption by 5.0%–13.2% while enhancing product crispness. In the field of medical nutrition, resistant starch is used in the dietary management of patients with metabolic diseases, as an enteral nutrition formula and postoperative supplement, providing dietary fiber while avoiding blood sugar fluctuations.

[0003] The production of resistant starch mainly employs physical, chemical, and enzymatic methods, aiming to improve the crystallinity of amylose by regulating its molecular structure. Mainstream technologies include the pressurization-enzyme coupling method, bio-enzymatic chain extension technology, and composite modification processes. The pressurization-enzyme coupling method gelatinizes starch emulsion under high temperature and pressure, followed by debranching with a compound enzyme and low-temperature recrystallization to form a highly resistant structure, achieving a yield of 25-40%, but it is energy-intensive and dependent on high-amylose raw materials. The bio-enzymatic chain extension technology uses starch sucrase to extend the amylopectin structure, followed by debranching and retrogradation to obtain resistant starch. This reduces dependence on high-amylose, lowers production costs by approximately 50%, and achieves a yield of over 45%, but enzyme activity limitations affect actual efficiency. The composite modification process combines oxidation and microwave-assisted treatment, increasing resistant starch content without chemical residues, but uneven energy distribution in the equipment may lead to yield fluctuations. Currently, there is no method that can simultaneously reduce raw material dependence and increase the yield of resistant starch. Summary of the Invention

[0004] [Technical Issues] Current technologies lack a method for simultaneously reducing raw material dependence, increasing resistant starch yield, and modifying highly resistant starch. There is a need for a highly resistant starch modification method with broad raw material adaptability, reducing the content of fast and slow-digestible starches, and increasing both the yield and content of resistant starch.

[0005] [Technical Solution] To address the problems existing in the prior art, this invention introduces starch sucrase into the starch branching enzyme system to form a dual-enzyme synergistic catalytic system, which significantly improves the efficiency of resistant starch production and provides a more efficient new method for the enzymatic preparation of highly resistant starch.

[0006] The core mechanism of the synergistic effect of the two enzymes lies in the fact that starch branching enzyme lays a crucial structural foundation for the effective modification of starch sucrase, while starch sucrase further constructs starch products with significantly slow digestibility based on this foundation. The specific process is as follows: Starch branching enzyme first acts on starch, hydrolyzing α-1,4 glycosidic bonds and recombining them with α-1,6 glycosidic bonds, thereby introducing new branching points into the starch molecule. This treatment significantly increases the branching density and generates a large number of new non-reducing ends. Subsequently, starch sucrase utilizes these newly generated ends as action sites, using sucrose as a glycosyl donor, to efficiently extend the outer chains of amylopectin. The sequential treatment by the two enzymes is not a simple additive process: starch branching enzyme provides abundant reaction targets for starch sucrase by increasing branching density, significantly improving the modification efficiency of starch sucrase; while starch sucrase further enhances the complexity and spatial size of the starch molecule structure by extending the chain length. The synergistic effect of the two ultimately forms a highly branched starch structure with longer outer chains. This complex and massive branched structure physically hinders the binding of starch-digesting enzymes (such as α-amylase) to the internal action sites of starch through steric hindrance, thus significantly slowing down the digestion rate. As a result, the content of rapidly digestible starch (RDS) decreases significantly, while the proportion of slowly digestible starch (SDS) and resistant starch (RS) increases significantly, making the final product a high-quality low-GI feedstock.

[0007] The first objective of this invention is to provide a method for the synergistic preparation of highly resistant starch using two enzymes, comprising the following steps: S1. Prepare a starch emulsion by mixing starch and water, heat the starch emulsion until gelatinized, and then place it in a constant temperature water bath. S2. Add starch branching enzyme and hydrolyze in a water bath, then inactivate the enzyme in a boiling water bath and cool. S3. Add sucrose and stir to dissolve, add starch sucrase and hydrolyze in water bath, add anhydrous ethanol to terminate the reaction; S4. The solid is separated, washed, and freeze-dried to obtain highly resistant starch.

[0008] In one embodiment of the present invention, in step S1, the starch concentration of the starch emulsion is 3-15 wt%; preferably 3-6 wt%.

[0009] In one embodiment of the present invention, in step S1, the gelatinization conditions are 96~99℃ and gelatinization time is 25~35 min.

[0010] In one embodiment of the present invention, in step S1, the temperature of the constant temperature water bath is 40~55℃, and the time of the constant temperature water bath is 15~25 min.

[0011] In one embodiment of the present invention, in step S2, the amount of starch branching enzyme added is 15~35 U / g dry starch.

[0012] In one embodiment of the present invention, in step S2, the enzymatic hydrolysis time is 3-5 h; and the water bath temperature is 50-55℃.

[0013] In one embodiment of the present invention, in step S2, the temperature is cooled to a temperature that does not significantly reduce the enzyme activity of starch sucrase; the temperature is then cooled to 20~45°C.

[0014] In one embodiment of the present invention, in step S3, the mass of sucrose added is 2.5 to 3.5 times the mass of starch.

[0015] In one embodiment of the present invention, in step S3, the amount of starch sucrase added is 40~60 U / g dry starch.

[0016] In one embodiment of the present invention, in step S3, the temperature of the water bath is 38~42℃; and the enzymatic hydrolysis time is 3~5h.

[0017] In one embodiment of the present invention, in step S3, the volume of anhydrous ethanol is 2 to 4 times the volume of the reaction liquid.

[0018] In one embodiment of the present invention, in step S3, the product is washed with deionized water and separated by centrifugation, repeated 2 to 5 times. This washes away fructose to avoid affecting the determination of the resistant starch content in the product.

[0019] A second objective of this invention is to provide a highly resistant starch prepared by the above method.

[0020] A third objective of this invention is to provide applications of the aforementioned highly resistant starch in the food and pharmaceutical fields.

[0021] Beneficial effects: This invention significantly improves the production efficiency of resistant starch through the "branching-extension" cascade reaction mechanism of a dual-enzyme system, enabling the content of resistant starch in the final product to be increased to 60% or more.

[0022] This invention optimizes the structural characteristics of the product through the synergistic action of starch branching enzyme and starch sucrase, forming B-type crystals with a highly stable double-helix conformation. This significantly enhances the anti-digestion properties and provides a high-quality raw material for the development of low-GI foods. The synergistic modification by the two enzymes precisely regulates the branching chain length and debranching degree of starch molecules, producing linear segments with concentrated chain length distribution and regular molecular arrangement. These segments are more likely to self-assemble through hydrogen bonds during retrogradation, forming tightly packed and ordered double-helix microcrystals, thereby greatly enhancing their resistance to enzymatic hydrolysis and giving the product high and stable anti-digestion properties. In terms of particle structure, this chemical homogeneity promotes the formation of particles with concentrated size distribution, higher crystallinity, and denser morphology. This significantly improves the thermal stability of the produced high-resistant starch (higher gelatinization temperature) and maintains good physical stability during processing and digestion.

[0023] The dual-enzyme system of this invention has strong adaptability to raw materials. It is not only suitable for corn starch, but can also effectively process various grain raw materials with low amylose content, thus broadening the raw material sources for resistant starch production and having significant industrialization advantages. Attached Figure Description

[0024] Figure 1 The diagram shows the chain length distribution of corn starch in Examples 1 and 2, Comparative Examples 1 and 2, and untreated corn starch.

[0025] Figure 2 This is a comparison diagram of the chain length distribution of Example 1, 2, Comparative Examples 1, 2 and untreated corn starch.

[0026] Figure 3 Comparison of chain length distributions showing the effects of different substrate concentrations on the synergistic preparation of highly resistant starch using a dual-enzyme method.

[0027] Figure 4 Photograph of the high-resistant starch prepared in Example 1. Detailed Implementation

[0028] Test method: 1. Determination of RDS, SDS, and RS The in vitro digestion method used is based on the Englyst in vitro simulated digestion method. Based on different starch digestion rates, starch can be classified into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). RDS refers to the portion that can be rapidly digested in the small intestine within 20 minutes; SDS refers to the portion that can be completely digested and absorbed in the small intestine within 20-120 minutes, but at a slower rate; RS refers to the portion that cannot be digested and absorbed by the human small intestine within 120 minutes, but can be utilized by microbial fermentation in the large intestine. The formula for calculating resistant starch content is as follows:

[0029]

[0030]

[0031]

[0032] Where: G: glucose content in the solution; A: absorbance of the sample; A0: absorbance of the blank; A st Absorbance of the standard product; A b : Absorbance values ​​of 50 μL and 1.5 mL GOPOD translation; m: Sample mass approximately 200 mg; G0: Free glucose content in starch; G 20 : Glucose content produced after amylase hydrolysis for 20 min; G 120 The glucose content produced after amylase hydrolysis for 120 min.

[0033] 2. Chain length distribution determination First, accurately weigh 50 mg of starch sample and dissolve it in 5 mL of dimethyl sulfoxide (DMSO), shaking thoroughly until completely dissolved. Add 1 mL of this solution to 5 mL of anhydrous ethanol, stir well, and centrifuge at 4000 × g for 10 min. Collect the precipitate and redissolve it in 2 mL of 50 mM sodium acetate buffer solution (pH 3.5). Gelatinize the mixture in a boiling water bath for 10 min, then transfer it to a 50°C constant temperature water bath shaker for 15 min to equilibrate. Finally, add 100 μL of isoamylase (10000 U / mL) and perform a debranching reaction under the same conditions for 12 h. The chain length distribution of the sample was then measured by high-performance anion exchange chromatography.

[0034] Example 1 A method for preparing highly resistant starch using a dual-enzyme synergistic approach includes the following steps: S1. Prepare a starch emulsion with a starch concentration of 5 wt% by adding water to commercially available corn starch. Heat the starch emulsion at 98℃ for 30 min to gelatinize it, and then heat it in a constant temperature water bath at 50℃ for 20 min. S2. Add GT-GBE with 25 U / g dry starch, enzymatically hydrolyze in a 50℃ water bath for 4 h, inactivate the enzyme in a boiling water bath for 30 min, and cool to room temperature; the starch branching enzyme GT-GBE is derived from Geobacillus stearothermophilus. S3. Add sucrose at three times the weight of starch, stir until fully dissolved, place in a water bath at 40°C, add 50 U / g dry starch AS, and enzymatically hydrolyze in a water bath for 4 h. Add anhydrous ethanol at three times the volume of the reaction liquid to terminate the reaction. The starch sucrase AS is derived from Neisseria polysaccharide. S4. Separate the solid, wash with deionized water and centrifuge to remove the liquid. Repeat the washing-centrifugation process 3 times, place in a -80℃ freezer and then freeze-dry for 48 h.

[0035] Photograph of the high-resistant starch prepared in Example 1 is shown below. Figure 4 As shown.

[0036] Example 2 The method for preparing highly resistant starch by dual enzyme synergy differs from Example 1 only in that the starch concentration of the starch emulsion is 10 wt%.

[0037] Comparative Example 1 The method for preparing resistant starch using AS enzyme differs from Example 2 only in that step S2 is omitted.

[0038] Comparative Example 2 The method for preparing resistant starch using GT-GBE enzyme differs from Example 2 only in that step S3 is omitted.

[0039] Comparative Example 3 Untreated corn starch, i.e., commercially available corn starch that has not undergone any processing.

[0040] Comparative Example 4 Debranched corn starch is produced by debranching commercially available corn starch using isoamylase.

[0041] The contents of rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) in the starches of Examples 1, 2, and Comparative Examples 1-3 were tested, and the results are shown in Table 1. Compared with untreated corn starch (Comparative Example 3), the RS content of the starches prepared from Examples 1, 2, and 1 were significantly increased. Comparative Examples 1 and 2 show that the synergistic effect of the two enzymes can increase the resistant starch content to 55% while significantly reducing the rapidly digestible starch content, thus providing dietary fiber without causing blood sugar fluctuations. However, Comparative Examples 1 and 2 only increased RS or SDS, while RDS and SDS remained at high levels, which could easily cause blood sugar fluctuations.

[0042] Table 1

[0043] Figure 1 The diagram shows the chain length distribution of starch in Examples 1 and 2, and Comparative Examples 1-4. Figure 2 This is a comparison chart of the chain length distribution of corn starch in Examples 1 and 2, and Comparative Examples 1, 2, and 4. The chain length distribution chart of unbranched corn starch is a mixture of complete, highly branched amylopectin molecules and linear amylose molecules; its chain length distribution is not concentrated and is used for experimental control. Figure 1 , Figure 2It can be seen that debranched corn starch and GT-GBE enzyme-treated corn starch have a high proportion of short chains and a low proportion of long chains. Dual-enzyme modification of corn starch significantly elongates the branched chains, reducing the proportion of short chains. Dual-enzyme modification promotes the association of long chains in starch molecules to form stable double helix structures, which then stack through hydrogen bonds, ultimately forming type B crystals. The binding sites of α-amylase in the human body cannot accommodate large, strong double helix fragments, thus the resulting starch exhibits resistance to enzymatic hydrolysis.

[0044] Figure 3 This is a comparison of chain length distributions showing the effect of different substrate concentrations on the two-enzyme synergistic method for preparing highly resistant starch. It can be seen that the lower the substrate concentration, the greater the proportion of long chains obtained by the two-enzyme synergistic method.

[0045] Example 3 The only difference from Example 1 is that the starch branching enzyme and starch sucrase used are commercially available.

[0046] The RDS, SDS, and RS contents of the high-resistant starch prepared in Example 3 are close to those in Examples 1 and 2.

[0047] Example 4 The only difference from Example 1 is that corn starch is replaced with potato starch.

[0048] The potato starch prepared in Example 4 had low RDS and SDS content and high RS content.

[0049] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for the synergistic production of high resistant starch by two enzymes, characterized in that, The method comprises the following steps: S1, preparing a starch emulsion by mixing starch and water, and heating the starch emulsion in a constant-temperature water bath; S2, adding a starch branching enzyme for water bath enzymolysis, boiling in a water bath to inactivate the enzyme, and cooling; S3, adding sucrose for stirring and dissolving, adding a starch sucrose enzyme for water bath enzymolysis, and adding anhydrous ethanol to terminate the reaction; S4, separating the solid, and washing and freeze-drying to obtain high-resistant starch.

2. The method of claim 1, wherein, In step S1, the starch concentration of the starch emulsion is 3-15 wt%.

3. The method of claim 1, wherein, In step S1, the temperature of the constant-temperature water bath is 40-55℃, and the time of the constant-temperature water bath is 15-25 min.

4. The method of claim 1, wherein, In step S2, the addition amount of the starch branching enzyme is 15-35 U / g of dry starch.

5. The method of claim 1, wherein, In step S2, the time of enzymolysis is 3-5 h, and the temperature of the water bath is 50-55℃.

6. The method of claim 1, wherein, In step S3, the addition amount of sucrose is 2.5-3.5 times the mass of the starch, and the addition amount of the starch sucrose enzyme is 40-60 U / g of dry starch.

7. The method of claim 1, wherein, In step S3, the temperature of the water bath is 38-42℃, and the time of enzymolysis is 3-5 h.

8. The method of claim 1, wherein, In step S3, the volume of the anhydrous ethanol is 2-4 times the volume of the reaction solution.

9. The high-resistant starch prepared by the method of any one of claims 1-8.

10. The high-resistant starch of claim 9 for use in the field of food and medicine.

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