Method for preparing highly branched starch with different crystal forms from glycogen branching enzyme

Abstract

The invention discloses a method for preparing highly branched starch with different crystal forms from glycogen branching enzyme, and belongs to the field of biologically modified starch. The method comprises the following steps: (1) dispersing starch in a buffer solution to obtain starch milk; (2) carrying out sub-gelatinization pretreatment on the starch milk, and then adding glycogen branching enzyme to carry out enzymolysis reaction; and (3) after the enzymolysis is finished, carrying out alcohol precipitation, washing, freeze drying, grinding and sieving to obtain the branched starch. The conventional modification step of cooling and adding enzyme after high-temperature pre-gelatinization treatment is omitted, the effect that glycogen branching enzyme is directly added at 60 DEG C to act on granular starch with different crystal structures is realized, the complete granular form of the starch is ensured, the post-processing characteristics are ensured, and the effect of synchronously performing enzymolysis reaction and starch granule swelling is realized; the problems caused by treating high viscosity of starch are avoided while the energy consumption is reduced, and the adverse effects of high-temperature gelatinization on the texture and properties of starch products are avoided.

Description

Technical Field

[0001] The invention relates to a method for preparing highly branched starches with different crystal forms by using glycogen branching enzyme, and belongs to the field of biologically modified starch. Background Art

[0002] Starch is the most important carbohydrate for humans and the main source of energy for the human body. Natural starch includes type A, type B and type C. However, natural starch is insoluble in cold water, easily ages and shrinks, and can cause a large fluctuation in blood sugar index within a short period of time after ingestion.

[0003] In order to solve the above problems, physical, chemical and bioenzymatic methods are usually used to modify starch. Among them, enzyme-modified starch has the characteristics of being green and substrate-specific compared to physical and chemical modified starch, and is suitable for large-scale production in factories. The modified branched starch has more branching than natural starch, which can significantly improve the branching degree, amylose content, retrogradation, and digestion characteristics of the original starch. However, in recent years, the research on enzyme-modified starch is mainly through pre-gelatinizing starch (above 90°C) and then cooling and adding enzymes for modification, or directly modifying it at low temperature (about 50°C), but there are problems such as long action time, low substrate concentration, and low action efficiency. In addition, the high-temperature gelatinization system destroys the crystalline structure of starch, and the crystalline structure of starch has an important influence on the processing and application characteristics of food. The application of native starch and its derivatives (chemically modified starch) in industry is mostly in the form of granules, with unique gelatinization properties, gel properties and rheological properties, thus having unique processing properties. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for preparing highly branched starches of different crystal forms by using glycogen branching enzyme, using three different crystal forms of starch (corn starch, potato starch, pea starch) pretreated under sub-gelatinization conditions (60°C) as substrates, and modifying them with glycogen branching enzymes, and the obtained products have high branching degree, low digestibility, and low amylose content. The substrate concentration is increased (10-20%), while saving energy consumption, the granular morphology of starch is guaranteed, its processing and application characteristics such as later gelatinization are guaranteed, and problems such as high viscosity after gelatinization are avoided.

[0005] The present invention provides a method for preparing highly branched starches with different crystalline forms using glycogen branching enzyme, which mainly comprises the following steps:

[0006] (1) dispersing starch in a buffer to obtain starch milk;

[0007] (2) pre-treating the starch milk to sub-gelatinization, and then adding glycogen branching enzyme to perform enzymatic hydrolysis;

[0008] (3) After the enzymatic hydrolysis is completed, alcohol precipitation, washing, freeze-drying, grinding and sieving are carried out to obtain branched starch.

[0009] In one embodiment of the present invention, in step (1), the starch is one of corn starch, potato starch and pea starch.

[0010] In one embodiment of the present invention, in step (1), the buffer solution is a 50 mM sodium citrate solution with a pH of 5 to 7.

[0011] In one embodiment of the present invention, in step (1), the mass concentration of starch milk is 10-20%.

[0012] In one embodiment of the present invention, in step (2), the sub-gelatinization pretreatment is performed under the conditions of preheating at 50-60° C. for 30-60 min.

[0013] In one embodiment of the present invention, in step (2), the glycogen branching enzyme is from Pyrococcus horikoshii OT3 (Ph).

[0014] In one embodiment of the present invention, in step (2), the amount of glycogen branching enzyme added is 100 to 500 U / g dry starch.

[0015] In one embodiment of the present invention, in step (2), the enzymatic hydrolysis conditions are 50-60° C. for 6-16 hours.

[0016] The present invention also provides highly branched starch prepared by the above method.

[0017] The present invention also provides the use of the highly branched starch described above in the preparation of low GI food.

[0018] [Beneficial Effects]

[0019] 1. The present invention utilizes bio-enzyme method to modify starch to prepare branched starch, the raw materials are easily available, the process is simple, the operation is convenient, the product yield is high, and the yield reaches more than 90%.

[0020] 2. The starch is modified by glycogen branching enzyme, which does not introduce other chemical groups and does not produce other types of glycosidic bonds. Only the α-1,4 glycosidic bonds and α-1,6 glycosidic bonds inside the starch molecules are reassembled, so the product is highly safe.

[0021] 3. The optimum reaction temperature of the glycogen branching enzyme provided by the present invention is 60°C. The starch is in a non-gelatinized and swollen state while maintaining a complete granular morphology structure, which solves the problem of starch granule structure destruction under a gelatinization system. The maintenance of the starch granule structure can guarantee its later processing and application characteristics, allowing it to be integrated into traditional staple foods. In addition, the enzymatic reaction and starch swelling are carried out simultaneously, eliminating the high-temperature pre-gelatinization step, reducing energy consumption and reducing the viscosity of the starch product. It was found that glycogen branching enzyme has different branching effects on different crystalline starches. The improvement of production intensity and efficiency is achieved, providing a new green, low-carbon, energy-saving, and environmentally friendly new idea and means for the preparation of branched starch by biological modification.

[0022] 4. The present invention has a short action time and increases the substrate concentration (10-20%), which is much higher than the action concentration of the prior art gelatinization system (generally 5% or less);

[0023] 5. The present invention prepares three types of branched starches. Compared with natural corn starch, potato starch and pea starch, the content of straight chain starch decreases by 12.5%, 9.8% and 13.9% at the highest; the length of branches with DP<13 increases significantly; the fast digestibility of in vitro digestibility decreases by 13.05%, 7.89% and 7.52%; the H value (ΔH) decreases by 5.56 J / g at the highest. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Glycogen branching enzyme purity;

[0025] Figure 2 Relative enzyme activity of glycogen branching enzyme at different pH (A) and temperature (B);

[0026] Figure 3 The starch granule morphology of Examples 1 to 3 and Comparative Examples 1 to 3; natural corn starch (A), natural potato starch (B), natural pea starch (C), Example 1 (D), Example 2 (E), Example 3 (F), Comparative Example 1 (G), Comparative Example 2 (H), Comparative Example 3 (I);

[0027] Figure 4 is the measurement wavelength and reference wavelength of amylose;

[0028] Figure 5 is the standard curve of amylose;

[0029] Figure 6 The amylose content of Examples 1 to 3;

[0030] Figure 7 It is the glycosidic bond ratio of Examples 1 to 3. DETAILED DESCRIPTION

[0031] The present invention will be further described and illustrated below in conjunction with specific implementation methods and accompanying drawings.

[0032] Glycogen branching enzyme: The term "glycogen branching enzyme" refers to an enzyme in class EC 2.4.1.18 as defined in Enzyme Nomenclature.

[0033] Amylose: It is a linear long-chain molecule composed of hundreds to thousands of glucose monomers connected by α-1,4 glycosidic bonds (99%), and contains a small amount of α-1,6 branches (1.0%). The α-1,6 glycosidic bonds at the branch points account for 0.3-0.5% of the total glycosidic bonds, and its degree of polymerization is 324-4920.

[0034] Branched starch: "Branched starch" and "modified starch" can be used interchangeably. It is a multi-branched soluble polymer carbohydrate composed of D-glucose. The main chain is composed of D-glucose groups connected by α-1,4 glycosidic bonds. There is a branch for every 24 to 30 D-glucose groups, and the branch point is connected to the main chain by α-1,6 glycosidic bonds.

[0035] The degree of polymerization (DP) refers to the average number of anhydroglucose units in a molecule.

[0036] Fast-digesting starch refers to the part of starch that causes blood sugar levels to rise quickly after ingestion.

[0037] Slowly digestible starch refers to the starch component that is fermented in the large intestine.

[0038] The main reagents involved in the following examples: isoamylase (EC 3.2.1.68, 200 U / mL) was purchased from Megazyme, USA, pepsin (P7000, EC 3.4.23.1, 250 U / mg) and saccharifying enzyme (A7095, EC 3.2.1.3, 260 U / mL) were purchased from SigmaAldrich, USA, porcine pancreatic enzyme (P110505, EC 232-468-9) and ordinary corn starch were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., glycogen branching enzyme was from Pyrococcus horikoshii OT3 (Ph), and the enzyme preparation method was referenced to the literature: Jing Zhu, Jie Long, Xingfei Li, et al. Improving the thermalstability and branching efficiency of Pyrococcus horikoshii OT3 glycogenbranching enzyme [J]. International Journal of Biological Macromolecules 255(2024)128010, and other commonly used reagents were of domestic analytical grade.

[0039] In the following examples, the enzyme activity determination method is as follows: Under the optimal reaction conditions, the amount of enzyme required to consume 1 mg of amylose per minute is measured in U. A 1 mg / mL amylose solution is placed in a 5 mL centrifuge tube and placed in a water bath shaker for 5 min. 10 μg of enzyme is added, shaken evenly, and reacted in a water bath shaker at 200 rpm for 10 min. 0.5 mL of 0.5 mol / L hydrochloric acid solution is added for inactivation, 1 mL of iodine solution is added for color development for 5 min, and the absorbance change at 660 nm is measured and the enzyme activity is calculated.

[0040] Solution configuration required for the reaction:

[0041] Sodium citrate solution: Accurately weigh 12.9 g of anhydrous sodium citrate (anhydrous) and dilute to 1 L (pH 5.0).

[0042] Amylose solution: Accurately weigh 10 mg of amylose, add 1 mL of 0.5 mol / L NaOH solution, and then add ultrapure water to make up to 10 mL.

[0043] Iodine solution preparation: 0.5 mL of stock iodine solution (containing 0.26 g I2 and 2.6 g KI in water) and 1 mL of 1 mol / L HCl. Use ultrapure water to make up to 130 mL.

[0044] Amylopectin solution: Accurately weigh 10 mg of amylopectin, add 1 mL of 0.5 mol / L NaOH solution, and then add ultrapure water to make up to 10 mL.

[0045] Iodine solution: Accurately weigh 0.1 g of I2 and 1 g of KI, add ultrapure water to make up to 50 mL.

[0046] The detection methods involved in the following examples are as follows:

[0047] Determination of starch granule morphology:

[0048] Take the enzyme-modified starch sample, fix it on the sample stage of a scanning electron microscope (SEM) with black double-sided tape, then coat a thin layer of gold film, and then place the sample stage under the scanning electron microscope to magnify and observe the granule morphology characteristics of the three crystalline forms of starch. The acceleration voltage is 3.0 kV.

[0049] Determination of amylose content:

[0050] The amylose content in the modified starch sample was determined by the double-wavelength method. Respectively take 10 mg of amylose and amylopectin to prepare a stock solution of 1 mg / mL. Take 3 mL of amylose solution and 6 mL of amylopectin solution, use 0.2 mol / L HCl to adjust the pH to 3.0, then add 1 mL of iodine solution, make up to 50 mL, and let it stand for 10 min. Using the acid water added with the iodine reagent as the blank, scan in the wavelength range of 400 - 800 nm to draw the amylose absorption curve. Select the amylose determination wavelengths λ1 and λ2. Respectively take 0.5, 1.0, 1.5, 2.0, 2.5 mL of the amylose stock solution, use 0.2 mol / L HCl to adjust the pH to 3.0, add 1 mL of iodine solution, make up to 50 mL, and let it stand for 10 min. Measure the absorbance at wavelengths λ1 and λ2, △A straight = A λ1 -A λ2 , with the amylose concentration as the abscissa (μg / mL) and △A straight as the ordinate, draw the amylose standard curve, and calculate the amylose content of the sample according to the amylose standard curve.

[0051] Determination of the branch chain length distribution:

[0052] The chain length distribution was determined by high performance anion exchange chromatography (HPAEC-PAD). 10 mg (dry basis) starch sample was accurately weighed and dissolved in 5 mL sodium acetate solution (50 mmol / L, pH 4.5). 2 mL of the sample solution was vortexed in a boiling water bath for 30 min, and then the sample was kept at 40 ° C for 10 min. 10 μL of isoamylase (180 U / mg) was added and reacted at 40 ° C for 24 h. After that, the reaction was terminated in a boiling water bath for 20 min, and centrifuged at 10000 rpm for 10 min. The supernatant was passed through a 0.22 μm water membrane, and the filtrate (200 μL) was injected into the HPAEC-PAD system. The system was equipped with a caropac PA200 column, the column temperature was 30 ° C, the mobile phase was ultrapure water containing 0.25 M sodium hydroxide and 1 M sodium acetate, and the flow rate was 0.5 mL / min.

[0053] In vitro digestibility test:

[0054] Prepare 0.75mol / L HCl, take 75mL and add 5mg pepsin and 25mg guar gum to form a pepsin solution, prepare and use immediately, and store at 4℃. Dissolve 2g porcine pancreatin in 40mL ultrapure water, stir magnetically for 20min, centrifuge at 8000rpm for 10min, and take 30mL of supernatant. Add 0.4mL saccharifying enzyme and mix thoroughly to prepare a mixed enzyme solution, prepare and use immediately, and store at 4℃. Take 200mg (dry basis) sample and add it to 15mL sodium acetate buffer (0.2mol / L, pH 5.0), place in a 95℃ water bath, vortex gelatinize for 30min, place in a 37℃ water bath shaker (200rpm) for 15min, and add 10 glass beads. Add 2mL pepsin solution and react for 30min. Then, 1 mL of mixed enzyme solution was added, and 0.2 mL of the reaction solution was taken at 20 min and 120 min, respectively, and 5 times anhydrous ethanol was added to inactivate the enzyme. The obtained mixture was centrifuged at 9000 rpm for 5 min, and 0.1 mL of the supernatant was taken.

[0055] The specific formula is as follows:

[0056] RDS(%)=(G20-FG)×0.9 / TS

[0057] SDS (%) = (G120-G20) × 0.9 / TS

[0058] RS(%)=100-RDS(%)-SDS(%)

[0059] in:

[0060] FG-free glucose content in starch before enzymatic hydrolysis (mg);

[0061] G20-glucose content produced after 20 min of amylase hydrolysis (mg);

[0062] G120-glucose content produced after 120 min of amylase hydrolysis (mg);

[0063] TS-total starch content in the sample (mg).

[0064] Determination of thermodynamic properties:

[0065] Use differential scanning calorimetry (DSC) to measure the changes in the thermodynamic properties of modified starch. Accurately weigh 2 mg of modified starch in an aluminum crucible, add deionized water at a ratio of 1:2 (w / w), and seal the crucible immediately. Place the crucible in a sealed bag and place it in a 4°C refrigerator to balance the moisture for 12 hours. Raise the temperature from 20°C to 100°C at a heating rate of 10°C / min, and keep the nitrogen flow rate constant at 20mL / min. Use a sealed empty crucible as a control. Each sample is measured three times in parallel, and the starting temperature (T O ), peak temperature (T P ), termination temperature (T C ) and enthalpy (ΔH).

[0066] Determination of α-1,6 glycosidic bond ratio:

[0067] Weigh 10 mg of modified starch sample and disperse it in 0.5 mL of D2O. Heat it in a boiling water bath to make it evenly dispersed. Vortex and shake it every 10 minutes until heating is stopped after 1 hour. After freezing at -80°C overnight, put it in a freeze dryer and dry it in vacuum for 2 days. When measuring, the sample is redissolved in D2O with internal standard. The nuclear magnetic resonance (1HNMR) spectrum is recorded using a nuclear magnetic resonance spectrometer with a spectral width of 8000 Hz, and the data is processed using Mestre Nova 12.0.3 software.

[0068] In order to further explore the characteristics of glycogen branching enzyme, the enzymatic properties of glycogen branching enzyme were further studied. 1 mg / mL straight-chain starch substrate was prepared with 0.05 mol / L sodium acetate buffer (pH 6.0), 10 μg enzyme was added, and the enzyme activity was measured after reacting at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 10 min. The optimal reaction temperature of glycogen branching enzyme was 60°C ( Figure 2 ).

[0069] In order to further explore the characteristics of glycogen branching enzyme, 1 mg / mL amylose substrate solution with different pH values ​​(pH 3, 4, 5, 6, 7, 8) was prepared and the enzyme activity was measured after reacting at 60°C for 10 min. The optimal reaction pH of glycogen branching enzyme was 5.0 ( Figure 2 ).

[0070] Example 1

[0071] 10% (w / w) corn starch milk was prepared with 0.05 mol / L sodium citrate solution (pH 5.0), preheated at 60°C for 30 min, 300 U glycogen branching enzyme was added per g starch (dry basis) for 10 h, three times anhydrous ethanol was added, and the mixture was allowed to stand for 30 min. The mixture was centrifuged at 10000 rpm for 10 min, washed three times with deionized water, placed in a -80°C refrigerator for 24 h, freeze-dried, ground and sieved, and passed through a 100-mesh sieve to obtain branched starch for use, and the sample was named CS-G.

[0072] Example 2

[0073] The preparation method is the same as that in Example 1, except that the starch is potato starch and the sample is named PS-G.

[0074] Example 3

[0075] The preparation method is consistent with that in Example 1, except that the starch is pea starch and the sample is named PBS-G.

[0076] Embodiment 4-7

[0077] The preparation method is the same as that in Example 1, except that the addition amount of glycogen branching enzyme is 100 U / g dry starch, 200 U / g dry starch, 400 U / g dry starch, and 500 U / g dry starch, respectively.

[0078] Embodiment 8-11

[0079] The preparation method is the same as that in Example 1, except that the enzymatic hydrolysis time is 8 h, 12 h, 14 h, and 16 h, respectively.

[0080] Comparative Example 1

[0081] 10% (w / w) corn starch milk was prepared with 0.05 mol / L sodium citrate solution (pH 5.0), preheated at 70°C for 30 min, 300 U glycogen branching enzyme was added per g starch (dry basis) for 10 h, three times anhydrous ethanol was added, and the mixture was allowed to stand for 30 min. The mixture was centrifuged at 10000 rpm for 10 min, washed three times with deionized water, placed in a -80°C refrigerator for 24 h, freeze-dried, ground and sieved, and passed through a 100-mesh sieve to obtain branched starch for use.

[0082] Comparative Example 2

[0083] The preparation method is the same as that of Comparative Example 1, except that the starch is potato starch.

[0084] Comparative Example 3

[0085] The preparation method is the same as that of Comparative Example 1, except that the starch is pea starch.

[0086] Native corn starch (CS), potato starch (PS) and pea starch (PBS) were used as controls.

[0087] (1) Starch granule morphology

[0088] from Figure 3 It can be seen that at 60°C, modified corn starch, potato starch and pea starch all maintained complete granular morphology, and at 70°C, the corn starch granules were almost completely broken. The non-gelatinization system ensured the later processing and application characteristics of the modified starch.

[0089] (2) Amylose content

[0090] like Figure 6 As shown, at 60°C, compared with natural starch, the amylose content of modified corn starch, potato starch and pea starch decreased by 12.5%, 9.8% and 13.9%, respectively.

[0091] (3) Branch chain length distribution

[0092] As shown in Table 1, the chain lengths of modified corn starch, potato starch, and pea starch with DP < 13 increased by 2.34%, 3.89%, and 0.81%, respectively, and the chain lengths of modified corn starch, potato starch, and pea starch with DP > 25 decreased by 2.61%, 0.83%, and 1.41%, respectively.

[0093] Table 1 Chain length distribution ratio of Examples 1-3

[0094]

[0095] (4) In vitro digestibility

[0096] As shown in Table 2, compared with natural starch, the RDS content of modified corn starch was significantly reduced by 13.05%, and the RDS content of modified potato starch and pea starch decreased by 7.89% and 7.52%, respectively.

[0097] Table 2 RDS, SDS, RS content of Examples 1-3

[0098]

[0099] (5) Thermodynamic property analysis

[0100] The thermal stability of different crystalline starches showed an upward trend, and the degree of order of starch internal particles decreased. The △H values ​​of modified corn starch, potato starch, and pea starch decreased to 10.38 J / g, 10.77 J / g, and 7.38 J / g, respectively.

[0101] Table 3 Thermal stability of Examples 1-3

[0102]

[0103] (6) Glycosidic bond ratio

[0104] The α-1,6 glycosidic bond content of modified corn starch increased from 4.45% to 12.19%, an increase of 173.93% compared with unmodified starch. The α-1,6 glycosidic bond content of modified potato starch and pea starch increased from 3.85% and 5.66% to 7.39% and 9.68% respectively ( Figure 7 ), which was increased by 91.94% and 71.37% compared with the unmodified starch.

[0105] The invention is based on modifying starch at 60°C, and the starch granules swell and enzymolysis reactions are carried out simultaneously, thereby reducing energy consumption and production costs, and avoiding problems such as high viscosity after gelatinization.

[0106] The direct high-temperature enzymatic modification of unpregelatinized starch enables the simultaneous enzymatic action and starch gelatinization, which saves industrial resources, simplifies the modification steps, and has great value in the industrial production of branched starch. Compared with native starch, the content of amylose is reduced and the length of branched chains is increased (DP < 13). The degree of branching is increased, digestibility is improved, and the proportion of glycosidic bonds is increased. Modified starch and its derivatives can be used to encapsulate bioactive preparations, sweeteners and prebiotics. Resistant starch or slowly digestible starch can be obtained, which is of great value for developing low GI foods and helping obese or diabetic patients. Due to the presence of slowly digestible components, it can also be used for slow and controlled release of nutrient systems or transportation of drugs.

[0107] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing branched starch, characterized in that: The main steps are as follows: (1) dispersing starch in a buffer to obtain starch milk; (2) pre-treating the starch milk to sub-gelatinization, and then adding glycogen branching enzyme to perform enzymatic hydrolysis; (3) After the enzymatic hydrolysis is completed, alcohol precipitation, washing, freeze-drying, grinding and sieving are carried out to obtain branched starch.

2. The method for preparing branched starch according to claim 1, characterized in that: In step (1), the starch is one of corn starch, potato starch and pea starch.

3. The method for preparing branched starch according to claim 1, characterized in that: In step (1), the buffer solution is a 50 mM sodium citrate solution with a pH of 5 to 7.

4. The method for preparing branched starch according to claim 1, characterized in that: In step (1), the mass concentration of starch milk is 10-20%.

5. The method for preparing branched starch according to claim 1, characterized in that: In step (2), the sub-gelatinization pretreatment condition is preheating at 50-60°C for 30-60 minutes.

6. The method for preparing branched starch according to claim 1, characterized in that: In step (2), the glycogen branching enzyme is derived from Pyrococcus horikoshii OT3 (Ph).

7. The method for preparing branched starch according to claim 1, characterized in that: In step (2), the added amount of glycogen branching enzyme is 100 to 500 U / g dry starch.

8. The method for preparing branched starch according to claim 1, characterized in that: In step (2), the enzymatic hydrolysis conditions are 50-60° C. for 6-16 hours.

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

10. Use of the branched starch according to claim 9 in preparing low GI food.

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