阅读说明：本技术 一种降低淀粉消化率的方法及其应用 (Method for reducing starch digestibility and application thereof ) 是由 吴敬 宿玲恰 李玲玲 李娜 于 2019-10-25 设计创作，主要内容包括：本发明公开了一种降低淀粉消化率的方法及其应用,属于酶工程技术领域。本发明提供了一种降低淀粉消化率的方法,此方法可用于制备抗性淀粉,且生产效率十分高；利用此方法将糖原分支酶ChlGBE加入淀粉溶液中改性6h,即可使淀粉溶液中抗性淀粉的含量高达35.06％,较未经改性的淀粉溶液提高9.75％；利用此方法将糖原分支酶HosGBE加入淀粉溶液中改性8h,即可使淀粉溶液中抗性淀粉的含量高达47.36％,较未经改性的淀粉溶液提高22.05％；利用此方法将糖原分支酶VvGBE加入淀粉溶液中改性10h,即可使淀粉溶液中抗性淀粉的含量高达51.4％,较未经改性的淀粉溶液提高26.09％。(The invention discloses a method for reducing starch digestibility and application thereof, belonging to the technical field of enzyme engineering. The invention provides a method for reducing the digestibility of starch, which can be used for preparing resistant starch and has high production efficiency; the glycogen branching enzyme ChlGBE is added into the starch solution for modification for 6 hours by using the method, so that the content of resistant starch in the starch solution can reach 35.06 percent, and is improved by 9.75 percent compared with the unmodified starch solution; by using the method, glycogen branching enzyme HosGBE is added into the starch solution for modification for 8 hours, so that the content of resistant starch in the starch solution can reach 47.36 percent, which is increased by 22.05 percent compared with the unmodified starch solution; the glycogen branching enzyme VvGBE is added into the starch solution for modification for 10 hours by using the method, so that the content of resistant starch in the starch solution can reach 51.4 percent, and is improved by 26.09 percent compared with the unmodified starch solution.)

1. A method for reducing starch digestibility, which is characterized in that starch is modified by one or more glycogen branching enzymes having amino acid sequences shown as SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3.

2. The method for reducing starch digestibility according to claim 1, wherein the starch is added to a buffer solution to obtain a starch solution, the starch solution is gelatinized to obtain a gelatinized starch solution, the gelatinized starch solution is incubated to obtain an incubated starch solution, and finally one or more glycogen branching enzymes having amino acid sequences shown as SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3 are added to the gelatinized starch solution to modify the glycogen branching enzymes.

3. The method for reducing starch digestibility according to claim 2, wherein the glycogen branching enzyme is added in an amount of 100 to 1000U/g starch.

4. The method for reducing the digestibility of starch according to claim 2 or 3, wherein the modification temperature is 25 to 45 ℃, the pH is 5.5 to 8.5, and the modification time is 4 to 12 hours.

5. The method for reducing the digestibility of starch according to any one of claims 2 to 4, wherein the method comprises adding starch to a buffer solution to obtain a starch solution, gelatinizing the starch solution to obtain a gelatinized starch solution, incubating the gelatinized starch solution to obtain an incubated starch solution, adding a glycogen branching enzyme having an amino acid sequence shown in SEQ ID NO. 1 to the gelatinized starch solution, and modifying the glycogen branching enzyme at 30 ℃ and pH7.0 for 6 hours;

or adding starch into a buffer solution to obtain a starch solution, gelatinizing the starch solution to obtain a gelatinized starch solution, incubating the gelatinized starch solution to obtain an incubated starch solution, adding glycogen branching enzyme with an amino acid sequence shown as SEQ ID NO. 2 into the gelatinized starch solution, and modifying for 8 hours at the temperature of 30 ℃ and the pH of 7.0;

or adding starch into a buffer solution to obtain a starch solution, gelatinizing the starch solution to obtain a gelatinized starch solution, incubating the gelatinized starch solution to obtain an incubated starch solution, adding glycogen branching enzyme with an amino acid sequence shown as SEQ ID NO. 3 into the gelatinized starch solution, and modifying for 10 hours at the temperature of 35 ℃ and the pH of 7.5.

6. The method for reducing starch digestibility according to any one of claims 2 to 5, wherein the concentration of starch in the gelatinized starch solution and the incubated starch solution is 10 to 100 g/L.

7. The method of any one of claims 2 to 6, wherein the buffer is a monobasic sodium phosphate-dibasic sodium phosphate buffer, a Tris-HCl buffer, a dibasic sodium phosphate-citric acid buffer or a citric acid buffer.

8. The method for reducing starch digestibility according to any one of claims 2 to 7, wherein the pasting is performed by stirring the starch solution in a boiling water bath at a speed of 100 to 200r/min for 20 to 40 min.

9. The method for reducing the digestibility of starch according to any one of claims 2 to 8, wherein the incubation is performed by stirring the gelatinized starch solution at a speed of 100 to 200r/min at a temperature of 25 to 45 ℃ for 10 to 20 min.

10. Use of a method of reducing starch digestibility according to any one of claims 1 to 9 in the production of resistant starch.

Technical Field

The invention relates to a method for reducing starch digestibility and application thereof, belonging to the technical field of enzyme engineering.

Background

Starch is the main carbon and energy storage polysaccharide in nature, can provide 70-80% of energy for human bodies, and is the most important dietary energy source for the human bodies. Depending on the digestibility, starch can be classified into three types, namely Rapidly Digestible Starch (RDS), Slowly Digestible Starch (SDS) and Resistant Starch (RS). Wherein, the fast digestion starch (RDS) is starch which is completely digested and absorbed in the small intestine, the slow digestion starch is starch which is completely digested and absorbed in the small intestine within 20-120 min, and the resistant starch is starch which can not be digested and absorbed by the small intestine after 120 min.

Research shows that the resistant starch has the characteristics of low digestibility, specific absorption by intestinal probiotics and the like, can regulate blood sugar and reduce blood fat, the important effects of promoting mineral absorption and protecting intestinal tract are described (see in particular "Glycemi c and intestinal nutrient of subjects with type 2diabetes after conversion of microorganisms of organic energy bara [ J ]. Journal of American dietary Association,2002,102(8): 1139. 1142", "Han K H, Sekikawa M, Shimada K I, et al. nutrient of microorganisms of. Therefore, it is important to reduce the digestibility of starch to produce more resistant starch.

At present, three methods for preparing resistant starch are available, namely physical modification, chemical modification and enzymatic modification. Among them, both physical modification and chemical modification have problems of many byproducts, low production efficiency, poor safety, etc. (refer to "Goesart H, Bijtebiera, Delcor JA. hydrolysises of amylopectic polysaccharides: level of amino chains as an organic differential starch [ J ]. Carbohydrate Research,2010,345(3): 397-401."), and thus, enzymatic modification is attracting more attention in the field of preparing resistant starch.

However, the enzyme-method modification has the problem of low production efficiency when the resistant starch is prepared by the enzyme-method modification because the enzyme such as the existing maltogenic amylase, β -amylase, amylosucrase and/or glucosyltransferase has low efficiency of cutting the starch and generating the digestion-resistant chemical bond in the starch, and thus, a method for efficiently reducing the digestion rate of the starch is still needed to improve the production efficiency of the resistant starch.

Disclosure of Invention

[ problem ] to

The invention aims to provide a method for efficiently reducing the digestibility of starch.

[ solution ]

In order to solve the technical problems, the invention provides a method for reducing the digestibility of starch, which is to modify starch by using one or more glycogen branching enzymes (glycogenin branching enzymes) with amino acid sequences shown as SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO: 3. The digestibility is the rate at which the starch is digested by the human body to release glucose.

In one embodiment of the invention, the method comprises the steps of adding starch into a buffer solution to obtain a starch solution, gelatinizing the starch solution to obtain a gelatinized starch solution, incubating the gelatinized starch solution to obtain an incubated starch solution, and adding one or more glycogen branching enzymes with amino acid sequences shown as SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3 into the gelatinized starch solution for modification.

In one embodiment of the present invention, the glycogen branching enzyme is added in an amount of 100 to 1000U/g starch.

In one embodiment of the present invention, the glycogen branching enzyme is added in an amount of 500U/g starch.

In one embodiment of the invention, the temperature of the modification is 25-45 ℃, the pH is 5.5-8.5, and the time is 4-12 h.

In one embodiment of the invention, the method comprises the steps of firstly adding starch into a buffer solution to obtain a starch solution, then gelatinizing the starch solution to obtain a gelatinized starch solution, then incubating the gelatinized starch solution to obtain an incubated starch solution, finally adding glycogen branching enzyme with an amino acid sequence shown as SEQ ID NO. 1 into the gelatinized starch solution, and modifying for 6 hours at the temperature of 30 ℃ and the pH of 7.0;

or adding starch into a buffer solution to obtain a starch solution, gelatinizing the starch solution to obtain a gelatinized starch solution, incubating the gelatinized starch solution to obtain an incubated starch solution, adding glycogen branching enzyme with an amino acid sequence shown as SEQ ID NO. 2 into the gelatinized starch solution, and modifying for 8 hours at the temperature of 30 ℃ and the pH of 7.0;

or adding starch into a buffer solution to obtain a starch solution, gelatinizing the starch solution to obtain a gelatinized starch solution, incubating the gelatinized starch solution to obtain an incubated starch solution, adding glycogen branching enzyme with an amino acid sequence shown as SEQ ID NO. 3 into the gelatinized starch solution, and modifying for 10 hours at the temperature of 35 ℃ and the pH of 7.5.

In one embodiment of the invention, the concentration of starch in the gelatinized starch solution and the incubated starch solution is 10-100 g/L.

In one embodiment of the invention, the concentration of starch in the gelatinized starch solution and the incubated starch solution is 25 g/L.

In one embodiment of the invention, the glycogen branching enzyme whose amino acid sequence is shown in SEQ ID NO. 1 is derived from Chlorella (Chlorella kessleri).

In one embodiment of the present invention, the glycogen branching enzyme whose amino acid sequence is shown in SEQ ID NO:2 is derived from Homo sapiens (Homo sapiens).

In one embodiment of the present invention, the glycogen branching enzyme whose amino acid sequence is shown in SEQ ID NO. 3 is derived from Vibrio vulnificus (Vibrio vulgaris).

In one embodiment of the present invention, the buffer is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, a Tris-HCl buffer, a disodium hydrogen phosphate-citric acid buffer, or a citric acid buffer.

In one embodiment of the present invention, the buffer is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

In one embodiment of the present invention, the concentration of the buffer solution is 20 to 100 mmol/L.

In one embodiment of the present invention, the concentration of the buffer solution is 50mmol/L

In one embodiment of the invention, the pasting is to stir the starch solution in a boiling water bath at a speed of 100-200 r/min for 20-40 min.

In one embodiment of the invention, the pasting is carried out by stirring the starch solution in a boiling water bath at a speed of 150r/min for 30 min.

In one embodiment of the invention, the incubation is to stir the gelatinized starch solution at 25-45 ℃ for 10-20 min at a speed of 100-200 r/min.

In one embodiment of the invention, the incubation is performed by stirring the gelatinized starch solution at 30 ℃ for 10min at a speed of 150 r/min.

In one embodiment of the invention, the starch is corn starch, potato starch, tapioca starch, or sweet potato starch.

The invention provides the application of the method for reducing the digestibility of the starch in producing the resistant starch. The slowly digestible starch is starch which is completely digested and absorbed in the small intestine within 20-120 min. The resistant starch refers to starch which cannot be digested and absorbed by the small intestine after 120 min.

[ advantageous effects ]

The invention provides a method for reducing the digestibility of starch, which can be used for preparing resistant starch and has high production efficiency; the glycogen branching enzyme (ChlGBE) with the amino acid sequence shown as SEQ ID NO. 1 is added into the starch solution for modification for 6 hours by using the method, so that the content of resistant starch in the starch solution can reach 35.06 percent, and is improved by 9.75 percent compared with the unmodified starch solution; the glycogen branching enzyme (HosGBE) with an amino acid sequence shown as SEQ ID NO. 2 is added into the starch solution for modification for 8 hours by using the method, so that the content of resistant starch in the starch solution can reach 47.36 percent, and is increased by 22.05 percent compared with the unmodified starch solution; by using the method, glycogen branching enzyme (VvGBE) with an amino acid sequence shown as SEQ ID NO. 3 is added into the starch solution for modification for 10 hours, so that the content of resistant starch in the starch solution can reach 51.4 percent, and is improved by 26.09 percent compared with the unmodified starch solution.

Detailed Description

The invention is further illustrated with reference to specific examples.

Coli BL21(DE3) referred to in the following examples was purchased from precious bioengineering (gangong) ltd; the corn starch referred to in the examples below was obtained from Shandong Linyi practice Co., Ltd;

the media involved in the following examples are as follows:

LB liquid medium: yeast powder 5.0 g.L^-1Tryptone 10.0 g.L^-1、NaCl 10.0g·L^-1Kanamycin 100 mg. L^-1Ampicillin 100 mg.L^-1。

LB solid medium: yeast powder 5.0 g.L^-1Tryptone 10.0 g.L^-1、NaCl 10.0g·L^-115 g.L agar powder^-1Kanamycin 100 mg. L^-1Ampicillin 100 mg.L^-1。

TB liquid medium: yeast powder 24 g.L^-1Tryptone 12 g. L^-15 g.L of glycerin^-1、K₂HPO₄12.54g·L^-1、KH₂PO₄2.31g·L^-1Kanamycin 100 mg. L^-1Ampicillin 100 mg.L^-1。

The detection methods referred to in the following examples are as follows:

the method for measuring the content of the resistant starch comprises the following steps:

α -amylase (2g, available from Sigma chemical Co., Ltd.) from pig pancreas was added to distilled water (24mL) and sufficiently stirred for 10min, 1500g was centrifuged for 10min, the supernatant (20mL) was transferred to a beaker and then mixed with amyloglucosidase (0.4mL, available from Sigma chemical Co., Ltd.) and distilled water (3.6mL) to obtain a digestive enzyme solution, the reaction solution was taken, a part of the reaction solution was centrifuged at 12000rpm for 15min to take the supernatant, the content of free glucose was detected with a GOD-POD kit, the other part of the reaction solution was boiled to inactivate the enzyme to adjust pH to 5.2, incubated at 37 ℃ for 10min, and then 0.75mL of the digestive enzyme solution was added, samples were taken at reaction times of 20min and 120min respectively while heating to boil inactivate the enzyme for 10min, the content of glucose was measured with a GOD-POD kit, and the contents of fast-digested starch (RDS), slow-digested starch (SDS) and Resistant Starch (RS) in the reaction solution were calculated.

Calculation of content of fast-digestible starch (RDS), slow-digestible starch (SDS) and Resistant Starch (RS):

RDS(％)＝(G20-G0)×0.9×100；

SDS(％)＝(G120-G20)×0.9×100；

RS(％)＝100％-RDS(％)-SDS(％)；

wherein G0 is the free glucose content in units (mg); g20 is the amount of glucose released in units (mg) within 20min of digestion; g120 is the amount of glucose released in units (mg) within 120min of digestion.

The method for measuring the enzyme activity of the branching enzyme comprises the following steps:

accurately weighing 0.5g (till +/-0.001 g) of potato amylopectin (purchased from Shanghai Rongsheng biological and pharmaceutical industries, Ltd.) into a beaker filled with a buffer solution, placing the beaker in a boiling water bath, heating and boiling for 30min until a substrate solution is clear and transparent, and then fixing the volume by using a 100mL volumetric flask to obtain the substrate solution; weighing 0.26g of iodine and 2.6g of potassium iodide, adding the iodine and the potassium iodide into a beaker filled with distilled water, uniformly stirring, and then carrying out constant volume by using a 10mL brown volumetric flask to obtain a Lugos solution (the solution is prepared at least 3 days before use to ensure that all iodine is dissolved, and the solution is placed in a shade and a cool place in a dark place and can keep the effective period of half a year); mixing 100 μ L Lugols solution, 50 μ L hydrochloric acid (2M), and 26mL distilled water to obtain termination solution (the solution is placed in dark place and is ready for use); setting a control group and an experimental group, wherein the control group comprises: 200. mu.L substrate solution + 200. mu.L distilled water; experimental groups: 200. mu.L substrate + 200. mu.L crude enzyme solution; mixing control group and experimental group without enzyme solution, and incubating at optimum temperature of ChlGBE and HosGBE (optimum temperature of 30 deg.C and optimum temperature of VvGBE of 35 deg.C) for 10 min; after 10min, add to the experimental group200 mu L of diluted crude enzyme solution is placed at the optimal temperature of glycogen branching enzyme for reaction for 15 min; after 15min, respectively adding 200 μ L of the product from the control group and the experimental group into 4mL of stop solution, and standing in the dark for about 20min until the color is stable; after 20min, the absorbance (A) was determined in a spectrophotometer₅₃₀) And calculating the activity of the glycogen branching enzyme.

Definition of branching enzyme activity:

the amount of enzyme required per 1mg of amylopectin reduction per unit volume of time is defined as 1 enzyme activity unit (1U).

Calculating the enzyme activity of the branching enzyme:

wherein A is the enzyme activity of branching enzyme, unit (U/mL); m is₀Amylopectin content, in units (mg) in the control group; m is₁Is the amylopectin content in units (mg) in the experimental group; d is the dilution multiple of the crude enzyme solution; t is the reaction time of the crude enzyme solution and the substrate in unit (min); v is the volume of the crude enzyme solution in units (mL).