Method for preparing highly branched starch of different crystal forms by using glycogen branching enzyme

Abstract

A method for preparing a highly branched starch of different crystal forms by using a glycogen branching enzyme. The method comprises: (1) dispersing starch in a buffer to obtain starch milk; (2) performing a sub-gelatinization pretreatment on the starch milk, and then adding a glycogen branching enzyme for enzymolysis; and (3) after the enzymolysis is completed, performing ethanol precipitation, washing, freeze-drying, grinding and sieving to obtain branched starch. By means of omitting the conventional modification step of high-temperature pre-gelatinization followed by cooling and enzyme addition, the glycogen branching enzyme is directly added at 60°C to process granular starch with different crystal form structures, which maintains the intact granular morphology of the starch, thereby achieving the effects of simultaneous enzymolysis and starch granule swelling.

Description

A method for preparing highly branched starch of different crystal forms using glycogen branching enzymes Technical Field

[0001] This invention relates to a method for preparing highly branched starch of different crystal forms using glycogen branching enzymes, belonging to the field of biomodified starch. Background Technology

[0002] Starch, as the most important carbohydrate for humans, is a major source of energy. Natural starch includes types A, B, and C. However, natural starch is insoluble in cold water, easily ages and shrinks, and can cause significant fluctuations in glycemic index shortly after ingestion.

[0003] To address the aforementioned issues, physical, chemical, and enzymatic methods are commonly used to modify starch. Among these, enzymatically modified starch, compared to physically and chemically modified starch, offers advantages such as being environmentally friendly and having substrate specificity, making it suitable for large-scale industrial production. Modified branched starch exhibits greater branching than natural starch, significantly improving the branching degree, amylose content, retrogradation, and digestibility of native starch. However, recent research on enzymatically modified starch has primarily focused on pregelatinized starch (above 90°C) followed by cooling and enzyme modification, or direct modification at low temperatures (around 50°C). These methods suffer from long reaction times, low substrate concentrations, and low efficiency. Furthermore, high-temperature gelatinization systems disrupt the crystalline structure of starch, which significantly impacts its processing and application characteristics in food. Native starch and its derivatives (chemically modified starch) are primarily used in industrial applications in granular form, possessing unique gelatinization, gelling, and rheological properties, thus exhibiting unique processing performance. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for preparing highly branched starches of different crystalline forms using glycogen branching enzymes. Three different crystalline starches (corn starch, potato starch, and pea starch) pretreated under sub-gelatinization conditions (60°C) are used as substrates. The glycogen branching enzyme modifies these substrates, resulting in products with high branching degree, low digestibility, and low amylose content. This method increases the substrate concentration (10-20%), saving energy while maintaining the starch's granule morphology and ensuring its subsequent processing and application characteristics, such as gelatinization, and avoiding problems such as high viscosity after gelatinization.

[0005] This invention provides a method for preparing highly branched starch of different crystal forms using glycogen branching enzymes, mainly comprising the following steps:

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

[0007] (2) The starch milk is pretreated by subgelatinization, and then glycogen branching enzyme is added for enzymatic hydrolysis.

[0008] (3) After enzymatic hydrolysis, the starch is subjected to alcohol precipitation, washing, freeze drying, grinding and sieving 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 the starch milk is 10-20%.

[0012] In one embodiment of the present invention, in step (2), the pretreatment conditions for sub-gelatinization are 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 derived from Pyrococcus horikoshii OT3(Ph).

[0014] In one embodiment of the present invention, in step (2), the amount of glycogen branching enzyme added is 100-500 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 method described above.

[0017] The present invention also provides the application of the above-described highly branched starch in the preparation of low-GI foods. [Beneficial Effects]

[0018] 1. This invention utilizes a bio-enzymatic method to modify starch to prepare branched starch. The raw materials are readily available, the process is simple and easy to operate, and the product yield is high, reaching over 90%.

[0019] 2. By using glycogen branching enzymes to modify starch, no other chemical groups are introduced, nor are other types of glycosidic bonds generated. Only the α-1,4 glycosidic bonds and α-1,6 glycosidic bonds within the starch molecule are reassembled, thus the product has high safety.

[0020] 3. The optimal reaction temperature of the glycogen branching enzyme provided by this invention is 60℃. The starch remains in a non-gelatinized, swollen state while maintaining its complete granular structure, solving the problem of starch granule structure destruction in gelatinization systems. Maintaining the starch granule structure ensures its subsequent processing and application characteristics, allowing it to be incorporated into traditional staple foods. Furthermore, the enzymatic hydrolysis reaction and starch swelling occur simultaneously, eliminating the need for high-temperature pregelatinization, reducing energy consumption, and lowering the viscosity of the starch product. The invention also revealed differences in branching effects of the glycogen branching enzyme on different starch crystalline forms. This improves production intensity and efficiency, providing a green, low-carbon, energy-saving, and environmentally friendly new approach and method for the bio-modification and preparation of branched starch.

[0021] 4. The present invention has a short reaction time and increases the substrate concentration (10-20%), which is much higher than the reaction concentration in the existing gelatinization system (generally 5% or less).

[0022] 5. The present invention prepared three types of branched starch. Compared with natural corn starch, potato starch and pea starch, the amylose content decreased by up to 12.5%, 9.8% and 13.9% respectively; the branch length of DP<13 increased significantly; the rapid digestibility decreased by 13.05%, 7.89% and 7.52% in vitro; and the glutathione value (ΔH) decreased by up to 5.56 J / g. Attached Figure Description

[0023] Figure 1. Purity of glycogen branching enzyme;

[0024] Figure 2 shows the relative enzyme activities of glycogen branching enzyme at different pH (A) and temperature (B);

[0025] Figure 3 shows the starch granule morphology of Examples 1-3 and Comparative Examples 1-3; 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), and Comparative Example 3 (I);

[0026] Figure 4 shows the determination wavelength and reference wavelength of amylose.

[0027] Figure 5 shows the standard curve of amylose;

[0028] Figure 6 shows the amylose content in Examples 1-3;

[0029] Figure 7 shows the glycosidic bond ratios in Examples 1-3. Detailed Implementation

[0030] The present invention will be further described and illustrated below with reference to specific implementation methods and accompanying drawings.

[0031] Glycogen branching enzyme: The term “glycogen branching enzyme” refers to an enzyme in class EC 2.4.1.18 as defined by enzyme nomenclature.

[0032] Amylose is a linear long-chain molecule composed of hundreds to thousands of glucose monomers linked 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 to 0.5% of the total glycosidic bonds, and its degree of polymerization is 324 to 4920.

[0033] Branched starch: "Branched starch" and "modified starch" can be used interchangeably. It is a multi-branched soluble polycarbohydrate composed of D-glucose. The main chain is composed of D-glucose groups linked by α-1,4 glycosidic bonds. There is one branch for every 24 to 30 D-glucose groups. The branching point is connected to the main chain by α-1,6 glycosidic bonds.

[0034] The degree of polymerization (DP) refers to the average number of anhydrous glucose units in a molecule.

[0035] Rapidly digestible starch refers to the portion of starch that causes a rapid rise in blood sugar levels after ingestion.

[0036] Slow-digesting starch refers to the starch component that ferments in the large intestine.

[0037] The main reagents used in the following examples were: isoamylase (EC 3.2.1.68, 200 U / mL) 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) purchased from Sigma-Aldrich, USA; porcine pancreatin (P110505, EC 232-468-9) and ordinary corn starch purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and glycogen branching enzyme derived from Pyrococcus horikoshii OT3 (Ph). The enzyme preparation method was based on the literature: Jing Zhu, Jie Long, Xingfei Li, et al. Improving the thermal stability and branching efficiency of Pyrococcus horikoshii OT3 glycogen branching enzyme[J]. International Journal of Biological Macromolecules 255(2024)128010, other commonly used reagents are all domestically produced analytical grade.

[0038] Enzyme activity assay method: Under optimal reaction conditions, the amount of enzyme required to consume 1 mg of amylose per minute, expressed in units of U. Add 1 mg / mL amylose solution to a 5 mL centrifuge tube and incubate in a water bath for 5 min. Add 10 μg of enzyme, shake well, and react in a water bath at 200 rpm for 10 min. Inactivate the enzyme by adding 0.5 mL of 0.5 mol / L hydrochloric acid solution, and develop the color with 1 mL of iodine solution for 5 min. Measure the absorbance change at 660 nm and calculate the enzyme activity.

[0039] Preparation of the solution required for the reaction:

[0040] Sodium citrate solution: Accurately weigh 12.9g of anhydrous sodium citrate and dilute to 1L (pH 5.0).

[0041] 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.

[0042] Preparation of iodine solution: 0.5 mL of stock iodine solution (an aqueous solution containing 0.26 g I₂ and 2.6 g KI) and 1 mL of 1 mol / L HCl. Bring the volume to 130 mL with ultrapure water.

[0043] 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.

[0044] Iodine solution: Accurately weigh 0.1g I2 and 1g KI, and add ultrapure water to make up to 50mL.

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

[0046] Starch granule morphology determination:

[0047] Take an enzyme-modified starch sample, fix it on the sample stage of a scanning electron microscope (SEM) with black double-sided tape, then coat it with a thin layer of gold film, and place the sample stage under the scanning electron microscope to observe the granular morphology characteristics of the three crystalline starches under magnification. The accelerating voltage is 3.0 kV.

[0048] Amylose content determination:

[0049] The content of amylose in modified starch samples was determined using a dual-wavelength method. 10 mg of amylose and 10 mg of amylopectin were used to prepare 1 mg / mL stock solutions. 3 mL of the amylose solution and 6 mL of the amylopectin solution were taken, and the pH was adjusted to 3.0 using 0.2 mol / L HCl. Then, 1 mL of iodine solution was added, and the volume was brought to 50 mL. The solutions were allowed to stand for 10 min. Using the acidic water with added iodine as a blank, the absorption curve of amylose was plotted by scanning in the 400–800 nm wavelength range. Wavelengths λ1 and λ2 were selected for amylose determination. 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the amylose stock solution were taken, and the pH was adjusted to 3.0 using 0.2 mol / L HCl. 1 mL of iodine solution was added, and the volume was brought to 50 mL. The solutions were allowed to stand for 10 min. The absorbance was measured at wavelengths λ1 and λ2. ΔAamylose = Aamylose. λ1 -A λ2 A standard curve for amylose was plotted with amylose concentration as the x-axis (μg / mL) and ΔA as the y-axis. The amylose content of the sample was then calculated based on the standard curve.

[0050] Branch length distribution determination:

[0051] Chain length distribution was determined using high-performance anion exchange chromatography (HPAEC-PAD). 10 mg (dry basis) of starch sample was accurately weighed and dissolved in 5 mL of 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, then incubated at 40 °C for 10 min. 10 μL of isoamylase (180 U / mg) was added, and the reaction was continued at 40 °C for 24 h. The reaction was then terminated by boiling in a water bath for 20 min. The sample was centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm aqueous membrane. 200 μL of the filtrate was injected into the HPAEC-PAD system. This system was equipped with a Caropac PA200 column at 30 °C, with an ultrapure water mobile phase containing 0.25 M sodium hydroxide and 1 M sodium acetate, and a flow rate of 0.5 mL / min.

[0052] In vitro digestibility assay:

[0053] Prepare a 0.75 mol / L HCl solution. Take 75 mL of the solution and add 5 mg of pepsin and 25 mg of guar gum to form a pepsin solution. Prepare and use immediately, and store at 4℃. Dissolve 2 g of porcine pancreatin in 40 mL of ultrapure water, stir magnetically for 20 min, and centrifuge at 8000 rpm for 10 min. Take 30 mL of the supernatant. Add 0.4 mL of saccharifying enzyme and mix thoroughly to prepare a mixed enzyme solution. Prepare and use immediately, and store at 4℃. Take 200 mg (dry basis) of the sample and add it to 15 mL of sodium acetate buffer (0.2 mol / L, pH 5.0). Incubate at 95℃ in a water bath and vortex for 30 min. Then, incubate at 37℃ in a shaker (200 rpm) for 15 min and add 10 glass beads. Add 2 mL of pepsin solution and react for 30 min. Then add 1 mL of mixed enzyme solution, and take 0.2 mL of reaction solution at 20 min and 120 min respectively. Add 5 times the amount of anhydrous ethanol to inactivate the enzyme. After centrifuging the mixture at 9000 rpm for 5 min, take 0.1 mL of supernatant.

[0054] The specific formula is as follows:

[0055] RDS(%)=(G20-FG)×0.9 / TS;

[0056] SDS(%)=(G120-G20)×0.9 / TS;

[0057] RS(%)=100-RDS(%)-SDS(%);

[0058] in:

[0059] Free glucose content (mg) in starch before FG-enzymatic hydrolysis treatment;

[0060] The glucose content (mg) produced after hydrolysis of G20-amylase for 20 min;

[0061] The glucose content (mg) produced after hydrolysis of G120-amylase for 120 min;

[0062] Total starch content (mg) in TS sample.

[0063] Thermodynamic property determination:

[0064] The thermodynamic property changes of modified starch were determined using differential scanning calorimetry (DSC). 2 mg of modified starch was accurately weighed into an aluminum crucible, and deionized water was added at a ratio of 1:2 (w / w). The crucible was immediately sealed. The crucible was then placed in a sealed bag and stored in a 4°C refrigerator for 12 hours to equilibrate the moisture. The temperature was increased from 20°C to 100°C at a rate of 10°C / min, with a constant nitrogen flow rate of 20 mL / min. A sealed empty crucible was used as a control. Each sample was measured in triplicate. The initial temperature (T0) was calculated using the instrument's built-in software. OPeak temperature (T) P ), termination temperature (T) C ) and enthalpy (ΔH).

[0065] Determination of the α-1,6 glycosidic bond ratio:

[0066] 10 mg of modified starch sample was weighed and dispersed in 0.5 mL of D2O. The mixture was heated in a boiling water bath to ensure uniform dispersion, with vortexing every 10 min until 1 h, after which heating was stopped. After freezing overnight at -80℃, the sample was vacuum dried in a freeze dryer for 2 days. For analysis, the sample was redissolved in D2O containing an internal standard. Nuclear magnetic resonance (¹H NMR) spectra were recorded using a nuclear magnetic resonance spectrometer with a spectral width of 8000 Hz, and data processing was performed using Mestre Nova 12.0.3 software.

[0067] To further investigate the characteristics of glycogen branching enzyme, its enzymatic properties were studied. A 1 mg / mL amylose substrate was prepared using 0.05 mol / L sodium acetate buffer (pH 6.0), and 10 μg of enzyme was added. The enzyme activity was measured after reacting at 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 10 min. The optimal reaction temperature for glycogen branching enzyme was 60℃ (Figure 2).

[0068] To further investigate the characteristics of glycogen branching enzyme, 1 mg / mL amylose substrate solutions with different pH values ​​(pH 3, 4, 5, 6, 7, 8) were prepared and reacted at 60℃ for 10 min before enzyme activity was measured. The optimal reaction pH for glycogen branching enzyme was 5.0 (Figure 2).

[0069] Example 1

[0070] A 10% (w / w) corn starch slurry was prepared using 0.05 mol / L sodium citrate solution (pH 5.0), preheated at 60℃ for 30 min, and 300 U of glycogen branching enzyme was added per gram of starch (dry basis) for 10 h. Three times the volume of 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, frozen at -80℃ for 24 h, freeze-dried, ground, and sieved through a 100-mesh sieve to obtain branched starch for later use. The sample was named CS-G.

[0071] Example 2

[0072] The preparation method is the same as in Example 1, except that the starch is potato starch and the sample is named PS-G.

[0073] Example 3

[0074] The preparation method is the same as in Example 1, except that the starch is pea starch and the sample is named PBS-G.

[0075] Example 4

[0076] The preparation method is the same as in Example 1, except that the amount of glycogen branching enzyme added is 100 U / g dry starch.

[0077] Example 5

[0078] The preparation method is the same as in Example 1, except that the amount of glycogen branching enzyme added is 200 U / g dry starch.

[0079] Example 6

[0080] The preparation method is the same as in Example 1, except that the amount of glycogen branching enzyme added is 400 U / g dry starch.

[0081] Example 7

[0082] The preparation method is the same as in Example 1, except that the amount of glycogen branching enzyme added is 500 U / g dry starch.

[0083] Example 8

[0084] The preparation method is the same as in Example 1, except that the enzymatic hydrolysis time is 8 hours.

[0085] Example 9

[0086] The preparation method is the same as in Example 1, except that the enzymatic hydrolysis time is 12 hours.

[0087] Example 10

[0088] The preparation method is the same as in Example 1, except that the enzymatic hydrolysis time is 14 hours.

[0089] Example 11

[0090] The preparation method is the same as in Example 1, except that the enzymatic hydrolysis time is 16 hours.

[0091] Comparative Example 1

[0092] A 10% (w / w) corn starch slurry was prepared using 0.05 mol / L sodium citrate solution (pH 5.0), preheated at 70℃ for 30 min, and 300 U of glycogen branching enzyme was added per gram of starch (dry basis) for 10 h. Three times the volume of 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, frozen at -80℃ for 24 h, freeze-dried, ground, and sieved through a 100-mesh sieve to obtain branched starch for later use.

[0093] Comparative Example 2

[0094] The preparation method is the same as that of Comparative Example 1, the only difference being that the starch is potato starch.

[0095] Comparative Example 3

[0096] The preparation method is the same as that of Comparative Example 1, the only difference being that the starch is pea starch.

[0097] Natural corn starch (CS), potato starch (PS), and pea starch (PBS) were used as controls.

[0098] (1) Starch granule morphology

[0099] As shown in Figure 3, the modified corn starch, potato starch, and pea starch all maintained their intact granular morphology at 60℃, while the corn starch granules almost completely ruptured at 70℃. The non-gelatinized system ensured the processing and application characteristics of the modified starch in the later stages.

[0100] (2) Amylose content

[0101] As shown in Figure 6, at 60℃, 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.

[0102] (3) Branch chain length distribution

[0103] 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, while the chain lengths with DP > 25 decreased by 2.61%, 0.83%, and 1.41%, respectively.

[0104] Table 1. Chain length distribution ratios in Examples 1-3

[0105] (4) In vitro digestibility

[0106] As shown in Table 2, compared with natural starch, the RDS content of modified corn starch was significantly reduced by 13.05%, while the RDS content of modified potato starch and pea starch decreased by 7.89% and 7.52%, respectively.

[0107] Table 2. RDS, SDS, and RS content in Examples 1-3

[0108] (5) Analysis of thermodynamic properties

[0109] The thermal stability of different crystalline starches all showed an increasing trend, while the degree of orderliness of the starch particles decreased. The ΔH 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.

[0110] Table 3 Thermal stability of Examples 1-3

[0111] (6) Glycosidic bond ratio

[0112] The α-1,6 glycosidic bond content of modified corn starch increased from 4.45% to 12.19%, representing a 173.93% increase compared to 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), representing increases of 91.94% and 71.37% compared to unmodified starch.

[0113] This invention modifies starch at 60℃, with starch granule swelling and enzymatic hydrolysis occurring simultaneously. This reduces energy consumption and production costs, and avoids problems such as high viscosity after gelatinization.

[0114] Direct high-temperature enzymatic modification of ungelatinized starch allows for simultaneous enzymatic action and starch gelatinization, saving industrial resources and simplifying the modification process. This has significant value in the industrial production of branched starch. Compared to native starch, it reduces amylose content and increases amylopectin chain length (DP < 13). It also improves branching degree, digestibility, and the proportion of glycosidic bonds. Modified starch and its derivatives can be used to encapsulate bioactive preparations, sweeteners, and prebiotics. Resistant starch or slow-digesting starch can be obtained, which is valuable for developing low-GI foods and assisting obese or diabetic patients. Due to the presence of slow-digesting components, it can also be used in slow-release and controlled-release nutrient systems or drug delivery.

[0115] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A process for the preparation of branched starch, characterized in that, The method mainly comprises the following steps: (1) dispersing starch in buffer to obtain starch milk; (2) performing sub-paste pre-treatment on the starch milk, and then adding glycogen branching enzyme to perform enzymolysis reaction; the sub-paste pre-treatment is pre-heated at 50-60℃ for 30-60 min; (3) after the enzymolysis is completed, alcohol precipitation, washing, freeze-drying, and grinding and sieving are performed to obtain branched starch.

2. A process for the preparation of a 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 is a 50 mM sodium citrate solution with pH of 5-7.

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

5. The method for preparing branched starch according to claim 1, characterized in that, In step (2), the sub-paste pre-treatment is pre-heated at 60℃ for 30 min.

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

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

8. The method for preparing branched starch according to claim 1, characterized in that, In step (2), the enzymolysis condition is reaction at 50-60℃ for 6-16 h.

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

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

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