Preparation method of compound enzyme of functional pea resistant starch

Abstract

The present invention relates to the field of starch production, and discloses a functional pea resistant starch compound enzyme preparation method, which comprises: S1, uniformly mixing pea starch and a buffer solution, and adjusting the pH value to prepare a starch suspension; s2, adding alpha-amylase into the starch suspension to carry out glucosidic bond enzymolysis, and then inactivating the enzyme to obtain enzymatic hydrolysate; s3, carrying out disproportionation, cyclization, coupling and hydrolysis reaction on the enzymatic hydrolysate by using 4-alpha-glycosyl transferase, and then inactivating enzyme to obtain further enzymatic hydrolysate; s4, adding pullulanase into the enzymatic hydrolysate, carrying out debranching treatment, and then inactivating the enzyme to obtain further enzymatic hydrolysate; and S5, centrifuging and washing the final enzymatic hydrolysate, and freeze-drying the precipitate to obtain the enzyme modified pea starch. The chemical properties such as crystallinity, short-range order, double helix degree, branching degree, resistant starch content and the like of the enzyme modified pea starch prepared by the method are close to or superior to the chemical properties of original pea starch.

Description

Technical Field

[0001] The invention relates to the field of starch production, and more specifically to a method for preparing a composite enzyme of functional pea resistant starch. Background Art

[0002] my country is very rich in starch resources, which are used for thickening, bonding, stabilization and other purposes in the food industry. In addition, starch plays a special role in the food and medical fields. Modified starch can resist decomposition by digestive enzymes, and after entering the large intestine, it is fermented by beneficial intestinal bacteria to produce short-chain fatty acids such as butyric acid. Butyric acid can enhance the intestinal barrier function, inhibit the growth of harmful bacteria, and prevent intestinal inflammation and disease. In specific dietary interventions, resistant starch helps to improve the structure of intestinal microbial communities, regulate indicators related to metabolic syndrome, and provide new options for dietary management of patients with obesity, diabetes, etc., showing its unique value in health maintenance. However, the characteristics of native starch itself limit its application. For a long time, countries around the world have attached great importance to the development and utilization of starch resources. In particular, the modification of starch by enzymes to improve the performance of starch has received widespread attention recently.

[0003] Pea starch is rich in protein, carotene, etc. After ingestion, it can effectively enhance the body's immunity, replenish qi, promote the production of body fluids and quench thirst, harmonize the body's defense system, and has a good auxiliary therapeutic effect on dizziness and headache caused by qi and blood deficiency. However, pea starch has a relatively high calorie content and is prone to obesity. This is mainly attributed to the molecular structure of starch and its digestibility. Therefore, changing the molecular structure of pea starch to reduce its calories and avoid obesity is an effective solution to the development of functional pea starch. Resistant starch is a type of starch that cannot be digested and absorbed in the human small intestine, but can be fermented and utilized by microorganisms in the colon. It has physiological functions similar to dietary fiber, such as reducing blood sugar response, improving intestinal health, low calories, and increasing satiety, so it has important application value in the food industry.

[0004] Enzymatic modification is to treat starch with various enzyme preparations, thereby changing the molecular size and structure of starch, chain length distribution and the properties of starch suspension, forming specific particles or molecular forms. However, the precise control of the enzyme modification process is difficult and further exploration is needed to achieve more ideal results. In addition, some enzyme modification processes are relatively complex and are not conducive to large-scale industrial production.

[0005] 4-α-glycosyltransferase (4αGtase) is an amylase that can catalyze a variety of reactions, including disproportionation, cyclization, coupling and hydrolysis reactions. Amylose is used to extend the outer chain of amylopectin, which can be used to modify starch to produce thermoreversible starch gels, and can form different products such as large cyclodextrins and slowly digestible starch. During the modification process, the amylose content is reduced, the molecular weight of starch is reduced, and various components such as long side chain amylopectin, medium relative molecular weight amylopectin, large cyclodextrins, oligosaccharides and monosaccharides are produced. Among them, the retrogradation of amylopectin is conducive to the formation of the slow digestion characteristics of starch. A new type of amylopectin cluster with slow digestion and resistance characteristics can be formed to enhance the slow digestion performance of starch. Therefore, the present invention is to modify pea starch using a composite enzyme method of α-amylase, 4-α-glycosyltransferase and pullulanase, and develop an enzyme modification method that can significantly increase the content of resistant starch in peas, which will promote the sustainable and healthy development of my country's green modified starch industry. Summary of the invention

[0006] The invention provides a composite enzyme preparation method for functional pea resistant starch, which has simple process, is safe and pollution-free, and can significantly increase the content of resistant starch in peas.

[0007] A composite enzyme preparation method for functional pea resistant starch, using pea starch as raw material, and modifying the pea starch by a composite enzyme method of α-amylase, 4-α-glycosyltransferase and pullulanase, specifically comprising the following steps:

[0008] S1: Mix pea starch and buffer evenly, and adjust the pH to prepare a starch suspension;

[0009] S2: adding α-amylase to the starch suspension to hydrolyze the glycosidic bonds, then inactivating the enzyme to obtain an enzymatic hydrolyzate;

[0010] S3: using 4-α-glycosyltransferase to carry out disproportionation, cyclization, coupling and hydrolysis reactions on the enzymatic hydrolysate, and then inactivating the enzyme to obtain a further enzymatic hydrolysate;

[0011] S4: adding pullulanase to the enzymatic hydrolyzate to perform debranching treatment, and then inactivating the enzyme to obtain a further enzymatic hydrolyzate;

[0012] S5: centrifuging and washing the final enzymatic hydrolyzate, and freeze-drying the precipitate to obtain enzyme-modified pea starch.

[0013] The invention adopts α-amylase to partially hydrolyze α-1,4-glycosidic bonds inside starch, and the hydrolysis products are dextrin, oligoglucan, hexose and the like; then 4-α-glycosyltransferase is used to carry out disproportionation, cyclization, coupling and hydrolysis reactions on starch chains, and various components such as long side chain amylopectin, amylopectin with medium relative molecular mass, macrocyclic dextrin, oligosaccharides and monosaccharides are produced; finally, pullulanase is used to specifically hydrolyze α-1,6-glycosidic bonds in amylopectin and other polysaccharides, so that the branch chains of amylopectin are separated from the main chain to form a series of linear starches with different chain lengths, and the final product is maltotriose.

[0014] The chemical properties of these modified starches, such as crystallinity, short-range order, degree of double helix, and resistant starch content, are superior to those of native pea starch.

[0015] A better technical solution of the present invention is:

[0016] In step S1: weigh 10-30 g of starch, add 200 mL-400 ml of 0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to adjust the pH to 4.0-6.0, place in a 90-100° C. water bath and stir to prepare a starch suspension.

[0017] Further preferably, 20 g of starch is weighed, 200 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer is added to adjust the pH to 6.0, and the mixture is placed in a 100° C. water bath and stirred to prepare a starch suspension.

[0018] In step S2: add 20-80u / g (starch dry basis) of α-amylase to the starch suspension, place in a 20-40°C water bath and stir to react for 30-120min; then add 1mol / L sodium hydroxide solution to adjust the pH to 9-10 to terminate the enzyme reaction.

[0019] Further preferably, 40 u / g (starch dry basis) of α-amylase is added to the starch suspension, and the mixture is stirred in a water bath at 20-40° C. for 60 min; and then sodium hydroxide solution is added to adjust the pH to 9.5 to terminate the enzyme reaction.

[0020] In step S3: adjust the pH to 4.0-6.0 with 0.01M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, add 50-80u / g (starch dry basis) of 4-α-glycosyltransferase to the enzymatic hydrolyzate, place in a 20-40°C water bath and stir to react for 1-3h, and after the reaction is completed, add 1mol / L sodium hydroxide solution to adjust the pH to 9-10 to terminate the enzyme reaction.

[0021] Further preferred is: adjusting the pH to 5.0 with 0.01M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, adding 80u / g (starch dry basis) of 4-α-glycosyltransferase to the enzymatic hydrolyzate, placing it in a 40°C water bath and stirring the reaction for 1h, and after the reaction is completed, adding 1mol / L sodium hydroxide solution to adjust the pH to 10 to terminate the enzyme reaction.

[0022] In step S4: 20-60u / g (starch dry basis) of pullulanase is added to the enzymatic hydrolyzate, and the solution is placed in a 20-50°C water bath and stirred for reaction for 6-12h. After the reaction is completed, 1mol / L sodium hydroxide solution is added to adjust the pH to 9-10 to terminate the enzyme reaction.

[0023] Further preferably, 40 u / g (starch dry basis) of pullulanase is added to the enzymatic hydrolyzate, and the solution is placed in a 47° C. water bath and stirred for reaction for 12 h. After the reaction is completed, 1 mol / L sodium hydroxide solution is added to adjust the pH to 10 to terminate the enzyme reaction.

[0024] In step S5: after the final enzymatic hydrolysate is centrifuged at 5000-8000 rpm for 10-20 min, three times the volume of water of the final enzymatic hydrolysate is added to wash the precipitate, and after washing three times with water, the precipitate is centrifuged again at 5000-8000 rpm for 10-20 min. After centrifugation, the precipitate is freeze-dried to obtain enzyme-modified pea starch.

[0025] Further preferably, after the final enzymatic hydrolysate is centrifuged at 6000 rpm for 10 min, three times the amount of water is added to wash the precipitate, which is washed three times with water and centrifuged again at 6000 rpm for 10 min. The precipitate after centrifugation is freeze-dried to obtain enzyme-modified pea starch.

[0026] The beneficial effects of the present invention are:

[0027] The present invention improves the resistant starch content in pea starch, utilizes α-amylase, 4-α-glycosyltransferase and pullulanase composite enzyme method to modify pea starch, solves the problem that the resistant starch content of pea starch itself is not high, develops high value-added, high-performance, environmentally friendly and safe green enzyme modification method which can significantly improve the resistant starch content of pea starch, and will promote the sustainable and healthy development of my country's green enzyme modified starch industry.

[0028] The chemical properties of the enzyme-modified pea starch prepared by the present invention, such as crystallinity, short-range order, double helix degree, branching degree, resistant starch content, etc., are close to or better than those of the original pea starch. The preparation process is relatively simple, the cycle is short, it is green and safe, the production cost is low, the equipment is simple, it is suitable for industrial production, and it is safe and pollution-free during production. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1is a data table of structural properties of native starch and enzyme-modified starch of the present invention;

[0030] Figure 2 is a data table of glucose and resistant starch contents of native pea starch and enzyme-modified pea starch of the present invention;

[0031] Figure 3 It is the SEM electron microscope picture of the native starch and the enzyme-modified starch of the present invention;

[0032] Figure 3 In: a is: pea native starch; b is: Example 4; c is: Comparative Example 1; d is: Comparative Example 2;

[0033] Figure 4 It is the XRD diagram of the native starch and the enzyme-modified starch of the present invention. DETAILED DESCRIPTION

[0034] The subject matter described herein will now be discussed with reference to example embodiments. It should be understood that the discussion of these embodiments is only to enable those skilled in the art to better understand and implement the subject matter described herein, and the functions and arrangements of the elements discussed may be changed without departing from the scope of protection of the contents of this specification. Each example may omit, replace or add various processes or components as needed. In addition, the features described relative to some examples may also be combined in other examples.

[0035] Example 1

[0036] In this embodiment, a composite enzyme preparation method for functional pea resistant starch is proposed, using pea starch as a raw material, and modifying the pea starch by a composite enzyme method of α-amylase, 4-α-glycosyltransferase and pullulanase, which specifically includes the following steps:

[0037] S1: Mix pea starch and buffer evenly, and adjust the pH to prepare a starch suspension;

[0038] Specifically, 10 g of starch was weighed, 200 mL ml of 0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was added to adjust the pH to 4.0, and the mixture was placed in a 90° C. water bath and stirred to prepare a starch suspension.

[0039] S2: adding α-amylase to the starch suspension to hydrolyze the glycosidic bonds, then inactivating the enzyme to obtain an enzymatic hydrolyzate;

[0040] Specifically, 20u / g (starch dry basis) of α-amylase was added to the starch suspension, and the mixture was placed in a 20°C water bath and stirred for reaction for 30 minutes; then 1mol / L sodium hydroxide solution was added to adjust the pH to 9 to terminate the enzyme reaction.

[0041] S3: using 4-α-glycosyltransferase to carry out disproportionation, cyclization, coupling and hydrolysis reactions on the enzymatic hydrolysate, and then inactivating the enzyme to obtain a further enzymatic hydrolysate;

[0042] Specifically: adjust the pH to 4.0 with 0.01M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, add 50u / g (starch dry basis) 4-α-glycosyltransferase to the enzymatic hydrolyzate, place it in a 20°C water bath and stir the reaction for 1h. After the reaction is completed, add 1mol / L sodium hydroxide solution to adjust the pH to 9 to terminate the enzyme reaction.

[0043] S4: adding pullulanase to the enzymatic hydrolyzate to perform debranching treatment, and then inactivating the enzyme to obtain a further enzymatic hydrolyzate;

[0044] Specifically, 20u / g (starch dry basis) of pullulanase was added to the enzymatic hydrolyzate, and the solution was placed in a 20°C water bath and stirred for 6 hours. After the reaction was completed, 1mol / L sodium hydroxide solution was added to adjust the pH to 9 to terminate the enzyme reaction.

[0045] S5: centrifuging and washing the final enzymatic hydrolyzate, and freeze-drying the precipitate to obtain enzyme-modified pea starch.

[0046] Specifically, after the final enzymatic hydrolysate was centrifuged at 5000 rpm for 10 min, three times the volume of water of the final enzymatic hydrolysate was added to wash the precipitate, and after washing three times with water, the precipitate was centrifuged again at 5000 rpm for 10 min. After centrifugation, the precipitate was freeze-dried to obtain enzyme-modified pea starch.

[0047] Example 2

[0048] In this embodiment, a composite enzyme preparation method for functional pea resistant starch is proposed, using pea starch as a raw material, and modifying the pea starch by a composite enzyme method of α-amylase, 4-α-glycosyltransferase and pullulanase, which specifically includes the following steps:

[0049] S1: Mix pea starch and buffer evenly, and adjust the pH to prepare a starch suspension;

[0050] Specifically, 20 g of starch was weighed, 300 ml of 0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was added to adjust the pH to 5.0, and the mixture was placed in a 95° C. water bath and stirred to prepare a starch suspension.

[0051] S2: adding α-amylase to the starch suspension to hydrolyze the glycosidic bonds, then inactivating the enzyme to obtain an enzymatic hydrolyzate;

[0052] Specifically, 50u / g (starch dry basis) of α-amylase was added to the starch suspension, and the mixture was placed in a 30°C water bath and stirred for 75 minutes; then 1mol / L sodium hydroxide solution was added to adjust the pH to 9.5 to terminate the enzyme reaction.

[0053] S3: using 4-α-glycosyltransferase to carry out disproportionation, cyclization, coupling and hydrolysis reactions on the enzymatic hydrolysate, and then inactivating the enzyme to obtain a further enzymatic hydrolysate;

[0054] Specifically: adjust the pH to 5.0 with 0.01M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, add 65u / g (starch dry basis) 4-α-glycosyltransferase to the enzymatic hydrolyzate, place it in a 30°C water bath and stir for 2h. After the reaction is completed, add 1mol / L sodium hydroxide solution to adjust the pH to 9.5 to terminate the enzyme reaction.

[0055] S4: adding pullulanase to the enzymatic hydrolyzate to perform debranching treatment, and then inactivating the enzyme to obtain a further enzymatic hydrolyzate;

[0056] Specifically, 40u / g (starch dry basis) of pullulanase was added to the enzymatic hydrolyzate, and the solution was placed in a 35°C water bath and stirred for 9 hours. After the reaction was completed, 1mol / L sodium hydroxide solution was added to adjust the pH to 9.5 to terminate the enzyme reaction.

[0057] S5: centrifuging and washing the final enzymatic hydrolyzate, and freeze-drying the precipitate to obtain enzyme-modified pea starch.

[0058] Specifically, after the final enzymatic hydrolysate was centrifuged at 6500 rpm for 15 minutes, three times the volume of water of the final enzymatic hydrolysate was added to wash the precipitate, and after washing three times with water, the precipitate was centrifuged again at 6500 rpm for 15 minutes. After centrifugation, the precipitate was freeze-dried to obtain enzyme-modified pea starch.

[0059] Example 3

[0060] In this embodiment, a composite enzyme preparation method for functional pea resistant starch is proposed, using pea starch as a raw material, and modifying the pea starch by a composite enzyme method of α-amylase, 4-α-glycosyltransferase and pullulanase, which specifically includes the following steps:

[0061] S1: Mix pea starch and buffer evenly, and adjust the pH to prepare a starch suspension;

[0062] Specifically, 30 g of starch was weighed, 400 ml of 0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer was added to adjust the pH to 6.0, and the mixture was placed in a 100° C. water bath and stirred to prepare a starch suspension.

[0063] S2: adding α-amylase to the starch suspension to hydrolyze the glycosidic bonds, then inactivating the enzyme to obtain an enzymatic hydrolyzate;

[0064] Specifically, 80u / g (starch dry basis) of α-amylase was added to the starch suspension, and the mixture was placed in a 40°C water bath and stirred for reaction for 120min; then 1mol / L sodium hydroxide solution was added to adjust the pH to 10 to terminate the enzyme reaction.

[0065] S3: using 4-α-glycosyltransferase to carry out disproportionation, cyclization, coupling and hydrolysis reactions on the enzymatic hydrolysate, and then inactivating the enzyme to obtain a further enzymatic hydrolysate;

[0066] Specifically: adjust the pH to 6.0 with 0.01M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, add 80u / g (starch dry basis) of 4-α-glycosyltransferase to the enzymatic hydrolyzate, place it in a 40°C water bath and stir the reaction for 3 hours. After the reaction is completed, add 1mol / L sodium hydroxide solution to adjust the pH to 10 to terminate the enzyme reaction.

[0067] S4: adding pullulanase to the enzymatic hydrolyzate to perform debranching treatment, and then inactivating the enzyme to obtain a further enzymatic hydrolyzate;

[0068] Specifically, 60u / g (starch dry basis) of pullulanase was added to the enzymatic hydrolyzate, and the solution was placed in a 50°C water bath and stirred for 12 hours. After the reaction was completed, 1mol / L sodium hydroxide solution was added to adjust the pH to 10 to terminate the enzyme reaction.

[0069] S5: centrifuging and washing the final enzymatic hydrolyzate, and freeze-drying the precipitate to obtain enzyme-modified pea starch.

[0070] Specifically, after the final enzymatic hydrolysate was centrifuged at 8000 rpm for 20 minutes, three times the volume of water of the final enzymatic hydrolysate was added to wash the precipitate, and after washing three times with water, the precipitate was centrifuged again at 8000 rpm for 20 minutes. After centrifugation, the precipitate was freeze-dried to obtain enzyme-modified pea starch.

[0071] Example 4

[0072] In this embodiment, a composite enzyme preparation method for functional pea resistant starch is proposed, which specifically comprises the following steps:

[0073] S1: Weigh 20 g of pea starch, add 200 ml of 0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to adjust the pH to 6.0, and place in a 100° C. water bath and stir to prepare a starch suspension;

[0074] S2: Add α-amylase (40u / g) and react for 60min. Then add 1mol / L sodium hydroxide solution to adjust the pH to 9.5 to terminate the reaction;

[0075] S3: Use 0.01M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to adjust the pH of the enzymatic solution to 5.0, add 4-α-glycosyltransferase (80u / g), place in a 40℃ water bath and stir for 1h. After the reaction is completed, add 1mol / L sodium hydroxide solution to adjust the pH to 10 to terminate the reaction;

[0076] S4: Add pullulanase (40u / g) to the enzymatic hydrolyzate and place in a 47°C water bath for stirring and reaction for 12h. After the reaction is completed, add 1mol / L sodium hydroxide solution to adjust the pH to 10 to terminate the reaction;

[0077] S5: After the enzymatic hydrolysate was centrifuged at 6000r for 10min, three times the volume of water of the final enzymatic hydrolysate was added to wash the precipitate, and after washing three times, the precipitate was centrifuged at 6000r for 10min again. The centrifugal precipitate was freeze-dried to obtain enzyme-modified pea starch.

[0078] The crystallinity of the enzyme-modified pea starch prepared in this example is 37.21%, which is significantly greater than the crystallinity of the original starch (25.77%). The absorbance ratio of 1047cm-1 / 1022cm-1 (R1) and 995cm-1 / 1022cm-1 (R2) reflects the short-range order and double helix degree of the starch. Comparing the R1 and R2 of the enzyme-modified pea starch (R1=3.016, R2=4.109) and the original pea starch (R1=0.722, R2=1.391), it can be clearly seen that the enzyme modification treatment increases the short-range order and double helix degree of the starch. It can be seen from the chain length distribution and branching degree data that the branch chain distribution is flatter, and the branching degree is reduced from 10.300% to 4.240%. The resistant starch content of native pea starch was 1.55%. After the pea starch was modified by a composite enzyme method of α-amylase, 4-α-glycosyltransferase and pullulanase, the resistant starch content increased to 56.67%, and the glucose content after 120 minutes of digestion was 33 mg / dL. This shows that the enzyme modification treatment has a significant change in the structure of the native pea starch, making the enzyme-modified pea starch more resistant to enzymolysis than the native starch.

[0079] The modified starch granules obtained in this example were subjected to application tests:

[0080] (1) Adding it to baked goods and dairy products can help lower the glycemic index (GI value) of food, providing more suitable dietary options for diabetic patients; at the same time, it can increase dietary fiber content, improve intestinal function, and prevent intestinal problems such as constipation;

[0081] (2) Applied in the cosmetics industry as an additive to foundation;

[0082] (3) In the medical field, resistant starch can be used as a drug carrier to control the rate of drug release and improve drug efficacy; the short-chain fatty acids produced by its fermentation have a protective effect on the intestinal mucosa and help prevent intestinal diseases;

[0083] (4) It is used as a functional ingredient in a variety of foods and can significantly reduce the digestion rate, thereby reducing the amount of blood sugar released.

[0084] Comparative Example 1: A method for preparing a composite enzyme of functional pea resistant starch, which specifically comprises the following steps:

[0085] (1) Weigh 20 g of pea starch, add 200 ml of 0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to adjust the pH to 6.0, and stir in a 50° C. water bath to prepare a starch suspension;

[0086] (2) Add α-amylase (40u / g) and react for 60min. Then add 1mol / L sodium hydroxide solution to adjust the pH to 9.5 to terminate the reaction;

[0087] (3) Use 0.01M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to adjust the pH of the enzymatic solution to 5.0, add 4-α-glycosyltransferase (80u / g), place in a 40°C water bath and stir for 1h. After the reaction is completed, add 1mol / L sodium hydroxide solution to adjust the pH to 10 to terminate the reaction;

[0088] (4) Add pullulanase (40u / g) to the enzymatic hydrolyzate and place in a 47°C water bath for stirring and reaction for 12 hours. After the reaction is completed, add 1 mol / L sodium hydroxide solution to adjust the pH to 10 to terminate the reaction;

[0089] (5) After the enzymatic hydrolysate was centrifuged at 6000 r for 10 min, three times the volume of water of the final enzymatic hydrolysate was added to wash the precipitate, and after washing three times with water, the precipitate was centrifuged at 6000 r for 10 min again. The centrifugal precipitate was freeze-dried to obtain enzyme-modified pea starch.

[0090] As shown in the accompanying drawings, the crystallinity of the enzyme-modified pea starch prepared in this example is 30.89%, which is significantly greater than the crystallinity of the original starch (25.77%). The absorbance ratios 1047cm-1 / 1022cm-1 (R1) and 995cm-1 / 1022cm-1 (R2) reflect the short-range order and double helicity of the starch. From the chain length distribution and branching degree data, it can be seen that the branch chain distribution is flatter, and the branching degree is reduced from 10.300% to 5.510%. Comparison of R1 and R2 of enzyme-modified pea starch (R1=1.421, R2=2.015) and pea original starch (R1=0.722, R2=1.391) shows that the enzyme modification treatment increases the short-range order and double helicity of the starch. The resistant starch content of pea starch is 1.55%. After the pea starch is modified by the complex enzyme method of α-amylase, 4-α-glycosyltransferase and pullulanase, the resistant starch content is increased to 35.94%, and the glucose content after digestion for 120 minutes is 45 mg / dL. (See Table 1 and Table 2 in the attached figure)

[0091] Comparative Example 2: A method for preparing a composite enzyme of functional pea resistant starch, which specifically comprises the following steps:

[0092] (1) Weigh 20 g of pea starch, add 200 ml of 0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to adjust the pH to 5.0, and place in a 100° C. water bath and stir to prepare a starch suspension;

[0093] (2) Add 4-α-glycosyltransferase (80u / g) and place in a 40°C water bath, stirring for 1 hour. After the reaction is completed, add 1 mol / L sodium hydroxide solution to adjust the pH to 10 to terminate the reaction;

[0094] (3) Add pullulanase (40u / g) to the enzymatic hydrolyzate and place in a 47°C water bath for stirring and reaction for 12 hours. After the reaction is completed, add 1 mol / L sodium hydroxide solution to adjust the pH to 10 to terminate the reaction;

[0095] (4) After the enzymatic hydrolysate was centrifuged at 6000r for 10 min, three times the volume of water of the final enzymatic hydrolysate was added to wash the precipitate, and after washing three times with water, the precipitate was centrifuged at 6000r for 10 min again. The centrifugal precipitate was freeze-dried to obtain enzyme-modified pea starch.

[0096] As shown in the accompanying drawings, the crystallinity of the enzyme-modified pea starch prepared in this example is 33.31%, which is significantly greater than the crystallinity of the original starch (25.77%). The absorbance ratios 1047cm-1 / 1022cm-1 (R1) and 995cm-1 / 1022cm-1 (R2) reflect the short-range order and double helix degree of the starch. From the chain length distribution and branching degree data, it can be seen that the branch chain distribution is flatter, and the branching degree is reduced from 10.300% to 6.190%. Comparing the R1 and R2 of the enzyme-modified pea starch (R1=0.773, R2=2.402) and the original pea starch (R1=0.722, R2=1.391), it can be clearly seen that the enzyme modification treatment increases the short-range order and double helix degree of the starch. The resistant starch content of native pea starch is 1.55%. After the pea starch is modified by a complex enzyme method of α-amylase, 4-α-glycosyltransferase and pullulanase, the resistant starch content is increased to 45.32%, and the glucose content after digestion for 120 minutes is 38 mg / dL.

[0097] The present invention utilizes a composite enzyme method of alpha-amylase, 4-alpha-glycosyltransferase and pullulanase to modify pea starch, thereby increasing the content of resistant starch in pea starch. The chemical properties of the prepared enzyme-modified pea starch, such as crystallinity, short-range order, double helix degree, branching degree, and resistant starch content, are superior to those of native pea starch. Generally, the methods currently used to increase the content of resistant starch in starch are relatively single, and the degree of increase in the content of resistant starch is not high. The alpha-amylase, 4-alpha-glycosyltransferase and pullulanase used in the present invention, in addition to the main product of enzyme-modified pea starch, only have a small amount of by-products such as glucose, maltose, maltobiose, and maltotriose. The process operation is simple, green and safe, and the use performance is stable.

[0098] The above describes an embodiment of the present embodiment, but the present embodiment is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present embodiment, ordinary technicians in this field can also make many forms, all of which are within the protection of the present embodiment.

Claims

1. A method for preparing a composite enzyme of functional pea resistant starch, characterized in that: The steps include: S1: Mix pea starch and buffer evenly, and adjust the pH to prepare a starch suspension; S2: adding α-amylase to the starch suspension to hydrolyze the glycosidic bonds, then inactivating the enzyme to obtain an enzymatic solution; S3: using 4-α-glycosyltransferase to carry out disproportionation, cyclization, coupling and hydrolysis reactions on the enzymatic hydrolysate, and then inactivating the enzyme to obtain a further enzymatic hydrolysate; S4: adding pullulanase to the enzymatic hydrolyzate to perform debranching treatment, and then inactivating the enzyme to obtain a further enzymatic hydrolyzate; S5: centrifuging and washing the final enzymatic hydrolyzate, and freeze-drying the precipitate to obtain enzyme-modified pea starch.

2. The method for preparing a composite enzyme of functional pea resistant starch according to claim 1, characterized in that: In step S1: weigh 10-30 g of starch, add 200 mL-400 ml of 0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to adjust the pH to 4.0-6.0, place in a 90-100° C. water bath and stir to prepare a starch suspension.

3. The method for preparing a composite enzyme of functional pea resistant starch according to claim 2, characterized in that: Weigh 20 g of starch, add 200 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer to adjust the pH to 6.0, and place in a 100° C. water bath and stir to prepare a starch suspension.

4. The method for preparing a composite enzyme of functional pea resistant starch according to claim 1, characterized in that: In step S2: add 20-80u / g α-amylase to the starch suspension, place it in a 20-40°C water bath and stir to react for 30-120min; then add 1mol / L sodium hydroxide solution to adjust the pH to 9-10 to terminate the enzyme reaction.

5. The method for preparing a composite enzyme of functional pea resistant starch according to claim 4, characterized in that: Add 40u / g α-amylase to the starch suspension, place it in a water bath at 20-40°C, stir and react for 60 minutes; then add sodium hydroxide solution to adjust the pH to 9.5 to terminate the enzyme reaction.

6. The method for preparing a composite enzyme of functional pea resistant starch according to claim 1, characterized in that: In step S3: adjust the pH to 4.0-6.0 with 0.01M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, add 50-80u / g 4-α-glycosyltransferase to the enzymatic solution, place it in a 20-40°C water bath and stir to react for 1-3h. After the reaction is completed, add 1mol / L sodium hydroxide solution to adjust the pH to 9-10 to terminate the enzyme reaction.

7. The method for preparing a composite enzyme of functional pea resistant starch according to claim 6, characterized in that: The pH value was adjusted to 5.0 with 0.01 M disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, 80 u / g 4-α-glycosyltransferase was added to the enzymatic solution, and the solution was stirred in a 40°C water bath for 1 h. After the reaction was completed, 1 mol / L sodium hydroxide solution was added to adjust the pH value to 10 to terminate the enzyme reaction.

8. The method for preparing a composite enzyme of functional pea resistant starch according to claim 1, characterized in that: In step S4: 20-60u / g of pullulanase is added to the enzymatic hydrolyzate, and the solution is placed in a 20-50°C water bath and stirred for reaction for 6-12h. After the reaction is completed, 1mol / L sodium hydroxide solution is added to adjust the pH to 9-10 to terminate the enzyme reaction.

9. The method for preparing a composite enzyme of functional pea resistant starch according to claim 8, characterized in that: 40u / g of pullulanase was added to the enzymatic hydrolyzate, and the solution was placed in a 47°C water bath and stirred for 12h. After the reaction was completed, 1mol / L sodium hydroxide solution was added to adjust the pH to 10 to terminate the enzyme reaction.

10. The method for preparing a composite enzyme of functional pea resistant starch according to claim 1, characterized in that: In step S5: after the final enzymatic hydrolysate is centrifuged at 5000-8000 rpm for 10-20 min, three times the volume of water of the final enzymatic hydrolysate is added to wash the precipitate, and after washing three times with water, the precipitate is centrifuged again at 5000-8000 rpm for 10-20 min. After centrifugation, the precipitate is freeze-dried to obtain enzyme-modified pea starch.

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