Green preparation method of resistant starch RS3 with hypoglycemic function

Abstract

The invention discloses a green method for preparing resistant starch RS3 with a blood sugar reducing function, and belongs to the technical field of food. The starch is used as a raw material, a new compound enzyme combination of alpha-amylase, pullulanase and saccharifying enzyme is adopted, the adding sequence of the enzymes is optimized, a green enzyme method process is utilized, the production process flow is simplified, the glycemic index value of the obtained starch is smaller than 55, and the starch can be used as low-glycemic-index food. According to the method, the content of RS3 prepared from starch which is wide in production range and low in cost through an enzyme method process is increased, and the method has great application potential in production.

Description

Technical Field

[0001] The invention relates to a method for green preparation of resistant starch RS3 with blood sugar lowering function, belonging to the technical field of food. Background Art

[0002] Resistant starch usually refers to starch that is not digested in the small intestine. It has special health benefits and functional properties, such as lowering blood sugar, preventing obesity, and improving intestinal flora. Among them, type III resistant starch (RS3) is formed by recrystallization of straight chain starch and branched chain starch during cooling and regeneration after starch gelatinization. It is also called retrograde starch. It has the ability to resist enzymatic hydrolysis and thermal stability. It is widely used in the food industry. It can reduce insulin response, relieve type II diabetes, regulate the body's energy intake, and control body weight. Therefore, it has broad prospects in human health diet. The book "Carbohydrates in Human Nutrition" jointly published by the World Health Organization and other institutions in 1998 pointed out: "The discovery and research progress of resistant starch is an important achievement in the study of the relationship between carbohydrates and health in recent years", and highly praised its importance to human health. Therefore, type III resistant starch (RS3) has received widespread attention from researchers.

[0003] The enzymes used in the enzymatic method are usually derived from natural organisms, are biodegradable and environmentally friendly, and will not introduce harmful chemicals, ensuring the naturalness and safety of the product. At the same time, the enzymatic reaction is highly specific and selective, and can accurately act on specific parts of starch molecules to improve the yield and quality of resistant starch. In the process of preparing RS3 by enzymatic method, the reaction conditions are easy to control, the process is simple and highly repeatable, which helps to achieve large-scale production and industrial application, meets the requirements of modern food safety and environmental protection, and also meets the market demand for natural and healthy products. Pullulanase is a debranching enzyme that specifically hydrolyzes α-1,6 glycosidic bonds in starch. Debranching can significantly increase the proportion of amylose in starch, promote more recrystallization formation in the retrogradation stage, and this process is crucial to increase the yield of RS. Therefore, pullulanase has become one of the most commonly used enzymes in the enzymatic preparation of RS3. α-Amylase (α-Amylases) is an endonuclease that randomly hydrolyzes the α-1,4 glycosidic bonds inside starch to generate more short chains, which promotes recrystallization during the retrogradation process. Glucoamylase is an exoenzyme that can hydrolyze α-1,3, α-1,4 and α-1,6 glycosidic bonds from the non-reducing ends of starch molecules. It is widely used in the production of glucose syrup and the saccharification step in the production of bioethanol. Studies have shown that the compound enzyme treatment method can increase the yield of resistant starch. Yang Yuexi, Zhou Ying and others prepared RS3 by combining α-amylase and pullulanase, which effectively improved the enzymatic resistance and RS content of RS3 compared with single enzymes. However, current research mainly focuses on the preparation of RS3 by combining pullulanase with α-amylase, and the combined use of other types of enzymes still has great application potential.

[0004] CN103819568B discloses a method for preparing resistant starch RS3, and resistant starch RS3 is obtained through seven operation steps. Through slurry adjustment, autoclave, debranching, centrifugation, crystallization, sugar washing and fluidized bed boiling drying, heavy phase resistant starch (RS content of 30.9%, product yield of 47.6%) and light phase resistant starch (RS content of 61.7%, product yield of 33.9%) are finally obtained, but the operation method is too complicated and is not conducive to large-scale industrial promotion.

[0005] CN115595346B discloses a method for preparing pea type III resistant starch, which is also applicable to the preparation of resistant starch RS3 in wheat, corn, and sweet potato. Resistant starch RS3 is obtained through five steps, and the yield of resistant starch RS3 is finally obtained in corn starch by acid hydrolysis, gelatinization, debranching, aging, annealing or autoclaving, which is 3.7 times that of the original corn starch resistant starch RS3. However, the temperature of the entire reaction system is as high as 121°C and is accompanied by a long hydration and drying process, which has high energy consumption and is not environmentally friendly, and is not conducive to green production.

[0006] CN 115466759 A discloses a method for preparing RS3 resistant starch by an "extrusion-debranching" method. Although this method has a simple process and a high output, it requires special equipment and may require a high initial investment cost using a twin-screw extruder, especially if a specific design or configuration is required to adapt to the method and the operation of the twin-screw extruder may be accompanied by a problem of high energy consumption, especially if the process needs to maintain high temperature and high speed.

[0007] Therefore, it is necessary to develop a method for preparing resistant starch RS3 with low raw material cost, simple operation, and green environmental protection. Summary of the invention

[0008] In order to solve at least one of the above problems, the present invention has developed a method for preparing resistant starch RS3 with low raw material cost, simple operation and green environmental protection. The present invention utilizes green enzymatic process, by adopting a new combination of complex enzymes of alpha-amylase, pullulanase and saccharifying enzyme, and optimizing the order of enzyme addition, by pre-gelatinization, enzymolysis and purification to increase the content of RS3 in starch. The present invention uses starch with a wide range of sources, easy to obtain and low cost in production as the preparation object, and adopts methods such as Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) to study and compare native starch (NS) and pullulanase, alpha-amylase-pullulanase, saccharifying enzyme in different order. The structural characteristics and physicochemical properties of RS3 under the three enzymes combined treatment, compare the RS content, thermodynamic properties, swelling power and solubility of different treatment groups. The present invention provides a new enzymatic preparation idea for the preparation of RS3, and provides a theoretical basis for the application of resistant starch in the food industry.

[0009] The purpose of the present invention is to provide a green and environmentally friendly method for preparing resistant starch RS3 with blood sugar lowering function.

[0010] The present invention also provides a method for preparing resistant starch with high RS content, high crystallinity, low swelling power, high solubility and low eGI value, the method comprising the following steps:

[0011] (1) adding fungal α-amylase to the gelatinized starch milk, performing enzymolysis at 45 to 55° C. for 15 to 20 minutes, and then cooling to obtain an enzymolysis solution 1;

[0012] (2) adding pullulanase to the enzymolysis solution 1 obtained in step (1), performing enzymolysis at 45-50° C. for 12 hours, and cooling to obtain enzymolysis solution 2; adding saccharifying enzyme to the enzymolysis solution 2, performing enzymolysis at 55-60° C. for 25-30 minutes, and cooling to obtain a composite enzymolysis solution;

[0013] or adding saccharifying enzyme to the enzymolysis solution 1 obtained in step (1), performing enzymolysis at 55-60° C. for 25-30 min, and then cooling to obtain enzymolysis solution 2; adding pullulanase to the enzymolysis solution 2, performing enzymolysis at 45-50° C. for 12 hours, and then cooling to obtain a composite enzymolysis solution;

[0014] (3) After adjusting the pH of the composite enzymatic hydrolyzate prepared in step (2), adding a thermostable α-amylase for enzymatic hydrolysis, and then precipitating with alcohol and drying to obtain resistant starch.

[0015] In one embodiment of the present invention, in step (1), the gelatinized starch milk is prepared by mixing starch and water to obtain a starch milk with a solid-liquid ratio of 1 to 1.5:4 to 4.5, adjusting the pH to 5.5 to 6.5, and gelatinizing at 80 to 85° C. and 200 to 300 rpm for 20 to 25 minutes to obtain the gelatinized starch milk.

[0016] In one embodiment of the present invention, in step (1), the starch includes but is not limited to: corn starch, tapioca starch, wheat starch, rice starch or potato starch.

[0017] In one embodiment of the present invention, the addition amount of the fungal α-amylase is 2-4 U / g starch.

[0018] In one embodiment of the present invention, in step (2), after the pH of the enzymatic hydrolyzate 1 obtained in step (1) is adjusted to 5.8-6.2, pullulanase or saccharifying enzyme is added;

[0019] The added amount of the pullulanase is 25-35 U / g starch; the added amount of the saccharifying enzyme is 200-300 U / g starch.

[0020] In one embodiment of the present invention, in step (3), after adjusting the pH of the composite enzymatic hydrolysate to 5.8-6.2, a thermostable α-amylase is added, and after enzymatic hydrolysis at 85-95° C. for 1.5-2 min, ethanol is added, stirred, and then centrifuged; the obtained precipitate is dried to obtain resistant starch.

[0021] In one embodiment of the present invention, the addition amount of the thermostable α-amylase is 2 to 4 U / g starch; the drying includes but is not limited to normal pressure drying, vacuum freeze drying, and microwave drying.

[0022] In one embodiment of the present invention, the fungal α-amylase includes but is not limited to the fungal α-amylase with the catalog number G8291; the saccharifying enzyme includes but is not limited to the saccharifying enzyme with the catalog number S10018-100mL; the pullulanase includes but is not limited to the pullulanase with the catalog number P875969-100g.

[0023] In one embodiment of the present invention, the method comprises the following steps:

[0024] (1) Pregelatinization: Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4. The pH was adjusted to 6, and the mixture was stirred at 80° C. for 20 min to obtain a gelatinized starch milk.

[0025] (2) Enzymolysis: Add fungal α-amylase (article number: G8291) to the gelatinized starch milk obtained in step (1) in an amount of 4 U / g and an enzyme activity of 100,000 U / g, perform enzymolysis at 50°C for 15 min, treat at 95°C for 20 min, and then cool to obtain enzymolysis solution 1. After adjusting the pH of the obtained enzymolysis solution 1 to 5, add pullulanase (article number: P875969-100g) to the enzymolysis solution in an amount of 30 U / g and an enzyme activity of 1,000 U / g, perform enzymolysis at 46°C for 12 h, treat at 95°C for 20 min, and then cool to obtain enzymolysis solution 2. Add saccharifying enzyme (Cat. No.: S10018-100mL) to the obtained enzymatic solution 2, the addition amount is 200U / mL, the enzyme activity is 100000U / mL, after enzymatic hydrolysis at 58°C for 30min, treat at 95°C for 20min, cool to room temperature, and obtain a composite enzymatic solution. The obtained composite enzymatic solution is stored at 4°C for 24h.

[0026] (3) Purification: After standing at 4°C for 24 h, adjust the pH to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, add thermostable α-amylase at a dosage of 1400 U / g and an enzyme activity of 40000 U / g, and hydrolyze at 90°C for 1.5 h. Add 95% ethanol at a ratio of 1:4 (v:v) and stir for 4 h; centrifuge at 8000 r / min for 5 min at room temperature, discard the supernatant, retain the precipitate, wash with 95% ethanol, dry and sieve to a mesh size of 100.

[0027] In one embodiment of the present invention, the starch milk in step (1) is prepared by mixing commercially available common starch with deionized water at a material-liquid ratio of 1:4.

[0028] In one embodiment of the present invention, the fungal α-amylase in step (2) has an enzyme activity of 100,000 U / g and is purchased from Beijing Solebow Technology Co., Ltd.; the pullulanase has an enzyme activity of 1,000 U / g and is purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; and the saccharifying enzyme is purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0029] In one embodiment of the present invention, the thermostable α-amylase in step (3) has an enzyme activity of 40,000 U / g and is purchased from Beijing Solebow Technology Co., Ltd.

[0030] The present invention also provides resistant starch prepared by the method.

[0031] The present invention also provides the use of the above method or the above resistant starch in preparing low eGI products, or in food and pharmaceutical products.

[0032] Beneficial Effects

[0033] The present invention develops a method for preparing resistant starch RS3 which has low raw material cost, simple operation and is green and environmentally friendly.

[0034] (1) The present invention adopts a new combination of complex enzymes of α-amylase, pullulanase and saccharifying enzyme, optimizes the order of enzyme addition, utilizes green enzymatic technology, and simplifies the production process.

[0035] (2) The present invention increases the content of RS3 prepared by enzymatic process using starch which is widely available and low in cost. The eGI of RS3-1 and RS3-2 groups to which α-amylase is preferentially added is significantly reduced, and the glycemic index value of the obtained starch is less than 55. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 FT-IR patterns for NS, RS3-1, RS3-2, RS3-3, RS3-4, RS3-5, RS3-6, RS3-7, RS3-8, RS3-9, RS3-10, RS3-11 and RS3-12.

[0037] Figure 2 These are the XRD patterns of NS, RS3-1, RS3-2, RS3-3, RS3-4, RS3-5, RS3-6, RS3-7, RS3-8, RS3-9, RS3-10, RS3-11 and RS3-12. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions in the art. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art.

[0039] The experimental materials involved in the following examples are as follows:

[0040] Commercially available starch, Shanghai Kuanchu Food Co., Ltd.; fungal α-amylase (item number: G8291, enzyme activity: 100000U / g) & thermostable α-amylase (item number: A8751, enzyme activity: 40000U / g), Beijing Solebaugh Technology Co., Ltd.; saccharifying enzyme (item number: S10018-100mL, enzyme activity: 100000U / mL), pancreatic α-amylase (item number: S31302-50ku, enzyme activity: 12U / mg), bile extract (item number: S31352-100g )&Pancreatin (Cat. No.: P7340-25g, enzyme activity: 4000U / g), Shanghai Yuanye Biotechnology Co., Ltd.; Pullulanase (Cat. No.: P875969-100g, enzyme activity: 1000U / g), Shanghai MacLean Biochemical Technology Co., Ltd.; Resistant starch detection kit, Megazyme, Ireland; Salivary α-amylase (Cat. No.: A0521-500UN, enzyme activity: 500U / g), Sigma-Aldrich Biochemical Technology Co., Ltd.; All other chemical reagents were of analytical grade.

[0041] The RS involved in the following embodiments is the part of starch that has the ability to resist enzymolysis;

[0042] The NS control group involved in the following examples is a normal starch (NS) raw material.

[0043] The instruments and equipment involved in the following embodiments are as follows:

[0044] SY-2230 constant temperature water bath shaker, Crystal Technology and Industries, USA; TM4000+ scanning electron microscope, CR21N centrifuge, Hitachi, Japan; Nicolet iS5 Fourier infrared spectrometer, Thermo Fisher Scientific, USA; multifunctional X-ray diffractometer, Bruker, Germany; MQX200 fluorescence microplate reader, Bettersize Instruments, USA, etc.

[0045] The detection methods involved in the following embodiments are as follows:

[0046] Determination of RS content in resistant starch:

[0047] Using the Irish Megazyme resistant starch kit, accurately weigh 100 mg of sample and put it into a culture tube, add 4 mL of pancreatic α-amylase (enzyme activity: 10 mg / mL) and saccharifying enzyme (AMG) (enzyme activity: 300 U / mL) to mix and prepare solution 1, incubate at 37 ° C with shaking (100 rpm) for 16 hours, and use 99% ethanol to stop the reaction; 1500g / min, 10min, centrifuge at room temperature, discard the supernatant, retain the precipitate and wash it several times with 50% ethanol, washing method: shaking. Add 2 mol / L KOH solution, and add 0.1 mL of AMG (enzyme activity: 3300 U / mL), and completely hydrolyze the precipitate in a 50 ° C water bath for 20 minutes, add 3 mL of GOPOD reagent to measure the glucose content of the precipitate, and further convert it into RS content.

[0048]

[0049] Where ΔE is the absorbance of the control reagent blank, F is the absorbance-to-μg conversion factor divided by the determined GOPOD absorbance of 100 μg of D-glucose, and W is the dry weight of the sample.

[0050] Infrared spectrum:

[0051] Use an infrared spectrometer at a wave number of 500 to 4000 cm -1 , resolution 4cm -1 The samples were analyzed under different conditions.

[0052] X-ray diffraction:

[0053] Weigh 5 mg of the sample and place it at room temperature (25°C) and relative humidity 100% for 24 hours to characterize the crystal structure of the sample. Scan the sample at a diffraction angle of 3° to 40° at a voltage of 40 kV and a current of 44 mA at a scanning rate of 4° / min.

[0054] Swelling Power and Solubility:

[0055] Weigh 0.5g sample accurately, prepare 4% (w / v) starch suspension, stir at 95℃ for 30min, cool, and centrifuge at 5000r / min for 25min at room temperature. Take the supernatant and evaporate it at 105℃, then weigh it to calculate swelling power and solubility. The formula is as follows:

[0056]

[0057] Where P is the weight of the sediment, W is the dry weight of the sample, and A is the dry weight of the supernatant.

[0058] Predicted Glycemic Index (eGI):

[0059] Add 0.5g sample and 20mL sodium acetate buffer (0.2mol / L, pH 5.5) to a 50mL centrifuge tube and mix well. Then add 6mL of pancreatic α-amylase (enzyme activity 250U / mL) and saccharifying enzyme (enzyme activity 260U / mL) mixed enzyme solution, shake in a constant temperature water bath at 37°C, take 2mL of digestion solution at 0, 20, 40, 60, 90, 120, and 180min, respectively, and inactivate the enzyme in a boiling water bath for 10min. After cooling, centrifuge and dilute the supernatant to an appropriate concentration. Determine the glucose content in the digestion solution and calculate the starch hydrolysis rate. The starch hydrolysis glucose content at different times is recorded as G0, G20, G60, G90, G120, and G180. The in vitro digestion and hydrolysis curve of starch follows the first-order reaction equation. The starch hydrolysis index is obtained by integrating the area under the starch hydrolysis curve of the sample and the reference food (white bread). The estimated glycemic index is obtained based on the starch hydrolysis index. The specific calculation formula is as follows:

[0060]

[0061] eGI=39.71+0.549×HI (5)

[0062] Where:

[0063] HI – starch hydrolysis index;

[0064] AUC1——area under the starch hydrolysis curve of the sample;

[0065] AUC0 – area under the curve for hydrolysis of white bread starch;

[0066] eGI – glycemic index.

[0067] Example 1: Preparation of product (RS3-1) by adding fungal α-amylase, pullulanase and saccharifying enzyme successively

[0068] The specific steps are as follows:

[0069] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 6. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0070] (2) Adding fungal α-amylase (article number: G8291, 100000 U / g) to the gelatinized starch milk obtained in step (1) at an amount of 4 U / g 淀粉 , after enzymatic hydrolysis at 50°C for 15 min, treatment at 95°C for 20 min and cooling, enzymatic hydrolyzate 1 was obtained.

[0071] (3) After adjusting the obtained enzymatic hydrolyzate 1 to pH 5, pullulanase (catalog number: P875969-100g, 1000U / g) was added at an amount of 30U / g 淀粉 , enzymolyze at 46℃ for 12h, treat at 95℃ for 20min and then cool to obtain enzymolyzate 2.

[0072] (4) Add saccharifying enzyme (Cat. No.: S10018-100 mL, 100,000 U / mL) to the obtained enzymatic solution 2, with the addition amount being 200 U / g 淀粉 , after enzymatic hydrolysis at 58°C for 30 min, it was treated at 95°C for 20 min and cooled to room temperature to obtain a composite enzymatic hydrolysate.

[0073] (5) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0074] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0075] The product RS3-1 containing resistant starch was obtained.

[0076] Example 2: Preparation of product (RS3-2) by adding fungal α-amylase, saccharifying enzyme and pullulanase successively

[0077] The specific steps are as follows:

[0078] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 6. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0079] (2) Adding fungal α-amylase (article number: G8291, 100000 U / g) to the gelatinized starch milk obtained in step (1) at an amount of 4 U / g 淀粉 , after enzymatic hydrolysis at 50°C for 15 min, treatment at 95°C for 20 min and cooling, enzymatic hydrolyzate 1 was obtained.

[0080] (3) After the obtained enzymatic hydrolysate 1 was adjusted to pH 5, saccharifying enzyme (catalog number: S10018-100mL, 100000U / mL) was added to the enzymatic hydrolysate at an amount of 200U / g 淀粉, after enzymatic hydrolysis at 58°C for 30 min, it was treated at 95°C for 20 min and then cooled to obtain enzymatic hydrolyzate 2.

[0081] (4) Add pullulanase (catalog number: P875969-100g, 1000U / g) to the obtained enzymatic solution 2, and the addition amount is 30U / g 淀粉 After enzymatic hydrolysis at 46°C for 12 hours, the mixture was treated at 95°C for 20 minutes and cooled to room temperature to obtain a composite enzymatic hydrolysate.

[0082] (5) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0083] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0084] The product RS3-2 containing resistant starch was obtained.

[0085] Example 3: Preparation of product (RS3-3) by adding pullulanase, fungal α-amylase and saccharifying enzyme successively

[0086] The specific steps are as follows:

[0087] (1) Starch and deionized water were mixed as above to prepare starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 5. The mixture was pre-gelatinized at 80° C. and 200 rpm for 20 min to obtain gelatinized starch milk.

[0088] (2) Add pullulanase (product number: P875969-100g, 1000U / g) to the gelatinized starch milk obtained in step (1) at an amount of 30U / g 淀粉 After enzymatic hydrolysis at 46°C for 12 h, the mixture was treated at 95°C for 20 min and then cooled to obtain enzymatic hydrolyzate 1.

[0089] (3) After the obtained enzymatic hydrolysate 1 was adjusted to pH 6, fungal α-amylase (catalog number: G8291, 100000 U / g) was added to the enzymatic hydrolysate at an amount of 4 U / g 淀粉 , after enzymatic hydrolysis at 50°C for 15 min, treated at 95°C for 20 min and then cooled to obtain enzymatic hydrolyzate 2.

[0090] (4) After the obtained enzymatic hydrolysate 2 was adjusted to pH 5, saccharifying enzyme (catalog number: S10018-100 mL, 100000 U / mL) was added to the enzymatic hydrolysate at an amount of 200 U / g. 淀粉 , after enzymatic hydrolysis at 58°C for 30 min, it was treated at 95°C for 20 min and cooled to room temperature to obtain a composite enzymatic hydrolysate.

[0091] (5) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0092] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0093] The product RS3-3 containing resistant starch was obtained.

[0094] Example 4: Preparation of product (RS3-4) by adding pullulanase, saccharifying enzyme and fungal α-amylase successively

[0095] The specific steps are as follows:

[0096] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 5. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0097] (2) Add pullulanase (product number: P875969-100g, 1000U / g) to the gelatinized starch milk obtained in step (1) at an amount of 30U / g 淀粉 After enzymatic hydrolysis at 46°C for 12 h, the mixture was treated at 95°C for 20 min and then cooled to obtain enzymatic hydrolyzate 1.

[0098] (3) Add saccharifying enzyme (catalog number: S10018-100mL, 100000U / mL) to the obtained enzymatic solution 1, and the addition amount is 200U / g 淀粉 , after enzymatic hydrolysis at 58°C for 30 min, it was treated at 95°C for 20 min and then cooled to obtain enzymatic hydrolyzate 2.

[0099] (4) After the obtained enzymatic hydrolysate 2 was adjusted to pH 6, fungal α-amylase (Cat. No.: G8291, 100000 U / g) was added at an amount of 4 U / g 淀粉, after enzymatic hydrolysis at 50°C for 15 min, it was treated at 95°C for 20 min and cooled to room temperature to obtain a composite enzymatic hydrolysate.

[0100] (5) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0101] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0102] The product RS3-4 containing resistant starch was obtained.

[0103] Example 5: Preparation of product (RS3-5) by adding saccharifying enzyme, pullulanase and fungal α-amylase successively

[0104] The specific steps are as follows:

[0105] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 6. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0106] (2) Add saccharifying enzyme (article number: S10018-100mL, 100000U / mL) to the gelatinized starch milk obtained in step (1) at an amount of 200U / g 淀粉 , after enzymatic hydrolysis at 58°C for 30 min, it was treated at 95°C for 20 min and cooled to room temperature to obtain enzymatic hydrolyzate 1.

[0107] (3) After the obtained enzymatic hydrolysate 1 was adjusted to pH 5, pullulanase (catalog number: P875969-100g, 1000U / g) was added to the enzymatic hydrolysate at an addition amount of 30U / g 淀粉 , enzymolyze at 46℃ for 12h, treat at 95℃ for 20min and then cool to obtain enzymolyzate 2.

[0108] (4) Add fungal α-amylase (Cat. No.: G8291, 100000 U / g) to the obtained enzymatic solution 2, with the addition amount being 4 U / g 淀粉 , after enzymatic hydrolysis at 50°C for 15 min, treatment at 95°C for 20 min and cooling, a composite enzymatic hydrolysate was obtained.

[0109] (5) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0110] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0111] The product RS3-5 containing resistant starch was obtained.

[0112] Example 6: Preparation of product (RS3-6) by adding saccharifying enzyme, fungal α-amylase and pullulanase successively

[0113] The specific steps are as follows:

[0114] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 6. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0115] (2) Add saccharifying enzyme (article number: S10018-100mL, 100000U / mL) to the gelatinized starch milk obtained in step (1) at an amount of 200U / g 淀粉 , after enzymatic hydrolysis at 58°C for 30 min, it was treated at 95°C for 20 min and then cooled to obtain enzymatic hydrolyzate 1.

[0116] (3) After the obtained enzymatic hydrolysate 1 was adjusted to pH 5, fungal α-amylase (catalog number: G8291, 100000 U / g) was added to the enzymatic hydrolysate at an amount of 4 U / g 淀粉 , after enzymatic hydrolysis at 50°C for 15 min, treatment at 95°C for 20 min and cooling, enzymatic hydrolyzate 2 was obtained.

[0117] (4) Add pullulanase (catalog number: P875969-100g, 1000U / g) to the obtained enzymatic solution 2, and the addition amount is 30U / g 淀粉 , after being enzymatically hydrolyzed at 46°C for 12 h, it was treated at 95°C for 20 min and cooled to room temperature to obtain a composite enzymatic hydrolyzate.

[0118] (5) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0119] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0120] The product RS3-6 containing resistant starch was obtained.

[0121] Comparative Example 1: Preparation of product (RS3-7) by adding fungal α-amylase and saccharifying enzyme successively

[0122] The specific steps are as follows:

[0123] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 6. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0124] (2) Adding fungal α-amylase (article number: G8291, 100000 U / g) to the gelatinized starch milk obtained in step (1), the addition amount is 4 U / g 淀粉 , after enzymatic hydrolysis at 50°C for 15 min, treatment at 95°C for 20 min and cooling, enzymatic hydrolyzate 1 was obtained.

[0125] (3) After the obtained enzymatic hydrolysate 1 was adjusted to pH 5, saccharifying enzyme (catalog number: S10018-100mL, 100000U / mL) was added to the enzymatic hydrolysate at an amount of 200U / g 淀粉 , enzymolyze at 58℃ for 30min, treat at 95℃ for 20min and then cool to obtain a composite enzymolysis solution.

[0126] (4) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0127] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0128] The product RS3-7 containing resistant starch was obtained.

[0129] Comparative Example 2: Preparation of product by adding saccharifying enzyme and fungal α-amylase successively (RS3-8)

[0130] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 5. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0131] (2) Add saccharifying enzyme (article number: S10018-100mL, 100000U / mL) to the gelatinized starch milk obtained in step (1) at an amount of 200U / g 淀粉 , after enzymatic hydrolysis at 58°C for 30 min, treatment at 95°C for 20 min and cooling, enzymatic hydrolyzate 1 was obtained.

[0132] (3) After the obtained enzymatic hydrolysate 1 was adjusted to pH 6, fungal α-amylase (Cat. No.: G8291, 100000 U / g) was added at an amount of 4 U / g 淀粉 , after enzymatic hydrolysis at 50°C for 15 min, treated at 95°C for 20 min and then cooled to obtain a composite enzymatic hydrolysate.

[0133] (4) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0134] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0135] The product RS3-8 containing resistant starch was obtained.

[0136] Comparative Example 3: Preparation of product (RS3-9) by adding fungal α-amylase and pullulanase successively

[0137] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 6. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0138] (2) Adding fungal α-amylase (article number: G8291, 100000 U / g) to the gelatinized starch milk obtained in step (1) at an amount of 4 U / g 淀粉 , after enzymatic hydrolysis at 50°C for 15 min, treatment at 95°C for 20 min and cooling, enzymatic hydrolyzate 1 was obtained.

[0139] (3) After the obtained enzymatic hydrolysate 1 was adjusted to pH 5, pullulanase (catalog number: P875969-100g, 1000U / g) was added to the enzymatic hydrolysate at an addition amount of 30U / g 淀粉 , enzymolyze at 46℃ for 12h, treat at 95℃ for 20min and then cool to obtain a composite enzymolysis solution.

[0140] (4) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0141] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0142] The product RS3-9 containing resistant starch was obtained.

[0143] Comparative Example 4: Preparation of product (RS3-10) by adding pullulanase and saccharifying enzyme successively

[0144] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 5. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0145] (2) Add pullulanase (product number: P875969-100g) to the gelatinized starch milk obtained in step (1) at an addition amount of 30 U / g 淀粉 , enzyme activity: 1000U / g, after enzymolysis at 46℃ for 12h, treatment at 95℃ for 20min and cooling, enzymolysis solution 1 was obtained.

[0146] (3) Add saccharifying enzyme (Cat. No.: S10018-100 mL) to the obtained enzymatic solution 1, with the addition amount being 200 U / g 淀粉 , enzyme activity: 100000U / mL, after enzymatic hydrolysis at 58℃ for 30min, treated at 95℃ for 20min and then cooled to obtain a composite enzymatic hydrolysate.

[0147] (4) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0148] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0149] The product RS3-10 containing resistant starch was obtained.

[0150] Comparative Example 5: Preparation of product by adding saccharifying enzyme and pullulanase successively (RS3-11)

[0151] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 5. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0152] (2) Add saccharifying enzyme (article number: S10018-100mL, 100000U / mL) to the gelatinized starch milk obtained in step (1) at an amount of 200U / g 淀粉 , after enzymatic hydrolysis at 58°C for 30 min, treatment at 95°C for 20 min and cooling, enzymatic hydrolyzate 1 was obtained.

[0153] (3) Add pullulanase (catalog number: P875969-100g, 1000U / g) to the obtained enzymatic solution 1, and the addition amount is 30U / g 淀粉 , after enzymatic hydrolysis at 46 °C for 12 h, treated at 95 °C for 20 min and then cooled to obtain a composite enzymatic hydrolysate.

[0154] (4) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0155] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0156] The product RS3-11 containing resistant starch was obtained.

[0157] Comparative Example 6: Preparation of product (RS3-12) by adding pullulanase and fungal α-amylase successively

[0158] (1) Starch and deionized water were mixed to prepare a starch milk with a solid-liquid ratio of 1:4, and the pH was adjusted to 5. The mixture was stirred at 200 rpm at 80° C. for 20 min to obtain a gelatinized starch milk.

[0159] (2) Add pullulanase (product number: P875969-100g, 1000U / g) to the gelatinized starch milk obtained in step (1) at an amount of 30U / g 淀粉 After enzymatic hydrolysis at 46°C for 12 h, the mixture was treated at 95°C for 20 min and then cooled to obtain enzymatic hydrolyzate 1.

[0160] (3) After the obtained enzymatic hydrolysate 1 was adjusted to pH 6, fungal α-amylase (Cat. No.: G8291,

[0161] 100000U / g), the addition amount is 4U / g 淀粉 , after enzymatic hydrolysis at 50°C for 15 min, treated at 95°C for 20 min and then cooled to obtain a composite enzymatic hydrolysate.

[0162] (4) The obtained composite enzymatic hydrolysate was stored at 4°C for 24 hours and then purified. The purification steps are as follows:

[0163] The pH of the composite enzymatic hydrolysate was adjusted to 6 with 0.1 mol / L HCI and 0.1 mol / L NaOH, and then high-temperature resistant α-amylase (1400 U / g) was added. After enzymatic hydrolysis at 90°C for 1.5 h, 95% ethanol was added at a ratio of 1:4 and stirred for 4 h; after centrifugation at 8000 r / min for 5 min at room temperature, the supernatant was discarded, the precipitate was retained, washed with 95% ethanol, vacuum freeze-dried at a vacuum degree of 0.05 Pa and -40°C, and sieved with a mesh size of 100.

[0164] The product RS3-12 containing resistant starch was obtained.

[0165] Example 7: Experimental results

[0166] The resistant starch-containing products obtained in each embodiment and comparative example were tested, including RS content, RS3 crystallinity, swelling power and solubility, predicted glycemic index, infrared, etc.

[0167] (1) RS content

[0168] The results are shown in Table 1, which is the RS content in the product obtained in the above example.

[0169] Table 1 RS content

[0170]

[0171] RS content is generally proportional to the proportion of amylose, and the results show that:

[0172] It can be seen from Table 1 that different addition orders have a great influence on RS content. NS has the lowest RS content (9.14%). The RS content of RS3-1 (32.17%), RS3-2 (31.46%), RS3-3 (27.30%), RS3-4 (28.86%), RS3-5 (24.96%) and RS3-6 (25.45%) are all higher than that of native starch (9.14%) and the dual enzyme treatment groups RS3-7 (12.64%), RS3-8 (14.12%), RS-9 (23.85%), RS3-10 (24.16%), RS3-11 (21.36%) and RS3-12 (23.56%) and increased significantly.

[0173] Among them, the two groups RS3-1 and RS3-2, to which α-amylase was added preferentially, had the highest RS content, and RS3-1 had the highest RS content, which was 3.5 times the RS content of 9.14% in natural starch (NS).

[0174] The RS content of the two groups in which the enzyme added preferentially was saccharifying enzyme was the lowest. The RS content of starch increased after being treated with pullulanase and retrogradation, which can be attributed to the leaching of amylose during starch gelatinization. At the same time, the proportion of amylose increased after pullulanase debranched the amylopectin. The amylose formed hydrogen bonds with each other during the retrogradation stage, forming double helix structure crystals in a directional and orderly manner, which effectively reduced the binding sites with digestive enzymes and increased the RS content.

[0175] α-Amylase can randomly hydrolyze the α-1,4 glycosidic bonds of the starch main chain to generate more short chains. Saccharifying enzymes can further shorten the chain length by hydrolyzing from the ends of the starch chains. The increase in the proportion of short straight chains is beneficial to recrystallization in the regeneration stage, and the hydrolysis rate is slower, thus avoiding excessive hydrolysis of starch. The preferential addition of saccharifying enzymes may increase the hydrolysis efficiency of saccharifying enzymes, resulting in excessive hydrolysis after subsequent enzyme treatment.

[0176] Although the RS content in RS4 prepared by chemical cross-linking can reach more than 60%, the safety issues caused by the change in chemical composition have affected its application in the food industry. The preparation of RS3 utilizes the high efficiency and strong substrate specificity of biological enzyme reactions. The structure of starch molecules is changed by biological enzymes, thereby changing the properties of starch, thereby reducing its digestibility. The preparation process does not cause changes in ingredients and is naturally safe, so it is more suitable for use in food. However, it is impossible to prepare extremely high content of RS3 through biological methods.

[0177] (2) The crystallinity of RS3 was tested. The results are shown in Table 2:

[0178] Table 2 Crystallinity

[0179]

[0180] The starch granules are semi-crystalline and divided into crystalline and amorphous regions. The XRD patterns of NS, RS3-1, RS3-2, RS3-3, RS3-4, RS3-5, RS3-6, RS3-7, RS3-8, RS3-9, RS3-10, RS3-11, and RS3-12 are shown in Figure 2. Figure 2 As shown. The natural starch sample exhibits a typical A-type crystalline structure, which has obvious diffraction peaks at 2θ=15°, 17.0°, 18.0° and 23.0°. After treatment, the RS3 sample prepared in the embodiment shows a mixed characteristic of B-type and V-type crystals, with main peaks located at 15°, 17°, 20°, 22° and 24°. These results show that pullulanase alone and compound enzyme treatment can change the crystal type of starch and increase its ability to resist the action of digestive enzymes. The relative crystallinity and crystal type of NS and RS3 samples are shown in Table 2. The crystallinity of NS, RS3-1, RS3-2, RS3-3, RS3-4, RS3-5, RS3-6, RS3-7, RS3-8, RS3-9, RS3-10, RS3-11 and RS3-12 are 30.15%, 57.16%, 56.48%, 51.21%, 51.09%, 44.24%, 45.98%, 32.42%, 33.14%, 43.12%, respectively. %, 43.86%, 41.02% and 43.57% respectively. The crystallinity of the prepared RS3 samples was higher than that of natural starch. Table 2 shows that the RS3-1 and RS3-2 groups with the priority addition of α-amylase in the three-enzyme composite group have the highest crystallinity, while the RS3-5 and RS3-6 groups with the priority addition of saccharifying enzyme have the lowest crystallinity. Among them, RS3-1 has the highest crystallinity and RS3-5 has the lowest. The priority addition of saccharifying enzyme may have over-hydrolyzed the starch chain. The crystallinity is positively correlated with the enzymatic resistance of RS3. The higher the crystallinity, the stronger the enzymatic resistance of RS3. The reason for the increase in crystallinity is that the activity of starch chains and the double helix structure in starch granules increase during the preparation of RS3, resulting in changes in the structure of the crystalline and amorphous regions of starch. The content of amylose is significantly increased during the debranching process of pullulanase, thereby promoting the formation of a more ordered double helix structure of starch during the retrogradation process. The exo-cutting property of saccharifying enzymes may increase the proportion of starch molecules with appropriate chain lengths, thereby promoting the formation of new double helical structures, and the crystallinity of starch is highly correlated with resistance to digestive enzymes. In summary, the RS3 sample prepared by the three-enzyme composite method has a significant change in crystal type compared to NS, and the crystallinity is significantly increased compared to NS and the double enzyme composite group, and the structure is more compact, which improves the anti-enzymatic effect.

[0181] (3) The swelling power and solubility of the products prepared in the examples and comparative examples were tested. The results are shown in Table 3.

[0182] Table 3 Swelling power and solubility

[0183]

[0184]

[0185] The swelling capacity of starch reflects the water absorption capacity of amylose during the swelling process. As shown in Table 3, the swelling capacity of RS3-1 (5.61 g / g), RS3-2 (5.12 g / g), RS3-3 (6.32 g / g), RS3-4 (6.24 g / g), RS3-5 (8.92 g / g) and RS3-6 (8.60 g / g) was significantly higher than that of NS (13.21 g / g) and the double enzyme treatment groups RS3-7 (11.12 g / g), RS3-8 (10 .03g / g), RS3-9 (6.94g / g), RS3-10 (6.42g / g), RS3-11 (7.86g / g) and RS3-12 (7.12g / g), which were negatively correlated with the RS content. Among the three enzyme treatment groups, RS3-1 and RS3-2, which preferentially added α-amylase, had the lowest swelling power, and RS3-5 and RS3-6, which preferentially added saccharifying enzymes, had the lowest swelling power. The swelling power of RS3 samples was lower than that of NS, which may be due to the production of more amylose after debranching. Amylose may, to a certain extent, prevent the destruction of the double helix structure of amylopectin and inhibit the expansion of starch granules. The swelling power is usually reflected in the texture of the product after adding RS3, which is negatively correlated with the RS content. After adding the same amount of RS3 but with different swelling power, the group with low swelling power has a harder texture and a crispier taste. The appropriate addition of RS3 when preparing biscuits and other foods can effectively improve the taste of the product.

[0186] The solubility of starch reflects the ability of amylose molecules to escape from starch granules. In Table 3, the solubility of RS3-1 (37.94%), RS3-2 (36.25%), RS3-3 (28.12%), RS3-4 (28.87%), RS3-5 (21.71%) and RS3-6 (22.47%) were significantly improved compared with NS (17.32%) and the double enzyme treatment groups RS3-7 (18.71%), RS3-8 (19.14%), RS3-9 (21.01%), RS3-10 (21.98%), RS3-11 (20.16%) and RS3-12 (21.54%). Among the triple enzyme treatment groups, RS3-1 and RS3-2, which preferentially added α-amylase, had the highest solubility, while RS3-5 and RS3-6, which preferentially added saccharifying enzyme, had the lowest solubility, among which RS3-1 had the most obvious effect (37.94%). Solubility is usually positively correlated with the proportion of short straight chains in starch, and the higher the proportion of short straight chains, the easier it is to form dense crystals during the retrogradation stage. This may be because pullulanase debranching can significantly increase the content of amylose; α-amylase and saccharifying enzyme can effectively shorten the chain length of amylose and increase the proportion of amylose, and amylose and branched starch with small molecular weight are more easily degraded into soluble molecules. At the same time, enzyme treatment and modification of the amorphous region of starch can significantly improve the swelling power and solubility of starch. When multiple enzymes are used in combination, the solubility of starch is significantly improved, and the degree of degradation of starch granules is greater.

[0187] (4) The predicted glycemic index of the products prepared in the examples and comparative examples was tested. The results are shown in Table 4:

[0188] Table 4 Prediction of glycemic index

[0189] Table 4 Expected Glycemic Index(eGI)

[0190]

[0191]

[0192] Table 4 lists the eGI of native starch and RS3 prepared. The eGI of the 13 starches measured in this invention are in ascending order as follows: RS3-1 < RS3-2 < RS3-3 < RS3-4 < RS3-6 < RS3-5 < RS3-9 < RS3-12 < RS3-10 < RS3-11 < RS3-8 < RS3-7 < NS. eGI represents the ability to relatively raise blood glucose levels. According to the glycemic index value, foods can be classified into low glycemic index foods (less than 55), medium glycemic index foods (56 - 69), and high glycemic index foods (higher than 70). Compared with native starch, the eGI of the composite enzyme treatment group decreased significantly. As can be seen from Table 4, the eGI of RS3-1 and RS3-2 groups with α-amylase added first in the three-enzyme treatment group decreased significantly, while the decrease in RS3-5 and RS3-6 groups with glucoamylase added first was the least. Among them, RS3-1 (26.57) had the lowest eGI, and RS3-5 had the highest eGI among RS3 samples. The glycemic index is closely related to starch structure, amylose content, and crystallinity. A high RS content will reduce the rate of in vitro digestion of starch, resulting in a decrease in the glycemic index; there is a corresponding relationship between the eGI of RS3 samples and indicators such as infrared spectroscopy and X-ray diffraction.

[0193] (5) FT-IR can well reflect the changes in functional groups, helical structure, crystallinity, and short-range structural order of starch molecules. The FT-IR pattern of the RS3 sample is as Figure 1 shown. No new absorption peaks appeared in the infrared spectra of NS, RS3-1, RS3-2, RS3-3, RS3-4, RS3-5, RS3-6, RS3-7, RS3-8, RS3-9, RS3-10, RS3-11, and RS3-12, indicating that the chain-breaking treatment with α-amylase and glucoamylase and the pullulan debranching treatment did not form new chemical groups. 3400 cm -1 is related to -OH stretching and hydrogen bond vibration. The smooth peak at 3400 cm -1 in RS3-1 indicates that the sample formed more hydrogen bonds. Among them, RS3-1 had the smoothest absorption peak, indicating that the sample formed the tightest connection during the retrogradation stage; 2930 cm -1 is related to C-H asymmetric stretching and vibration. The absorption peak of the RS3 sample became weaker at this position, indicating that C-H was partially destroyed after gelatinization and retrogradation treatment; 1645 cm -1 is related to the water bending in the amorphous region of starch granules, reflecting the crystalline water in starch. The absorption peak of the RS3 sample became significantly weaker, indicating that the aldehyde group of starch was partially destroyed after gelatinization and retrogradation treatment; 1155 cm -1The left and right are C=O absorption peaks, representing the α-1,4 glycosidic bond absorption peaks. The weakening absorption peak of RS3 sample represents the reduction in the number of α-1,4 glycosidic bonds, indicating that the chain length of RS3 sample is effectively shortened by enzyme treatment, among which RS3-1 absorption peak is the weakest; 1080cm -1 It is mainly related to the bending vibration of the C-H bond, and the RS3 sample is located at 1080 cm -1 The absorption peak intensity increased, indicating that the hydrogen bonding ability of starch molecules became stronger, and the hydrogen bonding force is the embodiment of the ordered structure of starch molecules. In summary, the chemical structures of NS and RS3 samples prepared by enzymatic method are similar and there is no difference in groups, while the crystal order of RS3 samples is significantly improved. Among them, RS3-1 and RS3-2, which are preferentially added with α-amylase, have the highest crystal order, while RS3-5 and RS3-6, which are preferentially added with saccharifying enzyme, have the lowest.

[0194] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing resistant starch with high RS content, high crystallinity, low swelling power, high solubility and low eGI value, characterized in that: The method comprises the following steps: (1) adding fungal α-amylase to the gelatinized starch milk, performing enzymolysis at 45 to 55° C. for 15 to 20 minutes, and then cooling to obtain an enzymolysis solution 1; (2) adding pullulanase to the enzymolysis solution 1 obtained in step (1), performing enzymolysis at 45-50° C. for 12 hours, and cooling to obtain enzymolysis solution 2; adding saccharifying enzyme to the enzymolysis solution 2, performing enzymolysis at 55-60° C. for 25-30 minutes, and cooling to obtain a composite enzymolysis solution; or adding saccharifying enzyme to the enzymolysis solution 1 obtained in step (1), performing enzymolysis at 55-60° C. for 25-30 min, and then cooling to obtain enzymolysis solution 2; adding pullulanase to the enzymolysis solution 2, performing enzymolysis at 45-50° C. for 12 hours, and then cooling to obtain a composite enzymolysis solution; (3) After adjusting the pH of the composite enzymatic hydrolyzate prepared in step (2), adding a thermostable α-amylase for enzymatic hydrolysis, and then precipitating with alcohol and drying to obtain resistant starch.

2. The method according to claim 1, characterized in that In step (1), the gelatinized starch milk is prepared by mixing starch and water to obtain a starch milk with a solid-liquid ratio of 1 to 1.5:4 to 4.5, adjusting the pH to 5.5 to 6.5, and gelatinizing at 80 to 85° C. and 200 to 300 rpm for 20 to 25 minutes to obtain the gelatinized starch milk.

3. The method according to claim 1 or 2, characterized in that: In step (1), the starch includes but is not limited to: corn starch, tapioca starch, wheat starch, rice starch or potato starch.

4. The method according to any one of claims 1 to 3, characterized in that: The added amount of the fungal alpha-amylase is 2-4 U / g starch.

5. The method according to any one of claims 1 to 4, characterized in that: In step (2), after adjusting the pH of the enzymatic hydrolyzate 1 obtained in step (1) to 5.8-6.2, pullulanase or saccharifying enzyme is added; The added amount of the pullulanase is 25-35 U / g starch; the added amount of the saccharifying enzyme is 200-300 U / g starch.

6. The method according to any one of claims 1 to 5, characterized in that: In step (3), after adjusting the pH of the composite enzymatic hydrolysate to 5.8-6.2, a thermostable α-amylase is added, and enzymatic hydrolysis is carried out at 85-95° C. for 1.5-2 min, and then ethanol is added, stirred, and centrifuged; the obtained precipitate is dried to obtain resistant starch.

7. The method according to claim 6, characterized in that The addition amount of the thermostable α-amylase is 2-4 U / g starch; the drying includes but is not limited to normal pressure drying, vacuum freeze drying, and microwave drying.

8. The method according to any one of claims 1 to 7, characterized in that: The fungal α-amylase includes but is not limited to the fungal α-amylase with the catalog number G8291; the saccharifying enzyme includes but is not limited to the saccharifying enzyme with the catalog number S10018-100mL; the pullulanase includes but is not limited to the pullulanase with the catalog number P875969-100g.

9. The resistant starch prepared by the method according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 or the resistant starch according to claim 9 in the preparation of low eGI products, or in food and pharmaceutical products.

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