A method for preparing highly branched starch using double enzymes

Abstract

The application discloses a method for preparing high-branched starch by using double enzymes and belongs to the field of biologically modified starches. The method mainly uses glycogen branching enzyme and cyclodextrin glucosyltransferase to cooperatively modify different crystal forms of starch in a non-gelatinized swelling system (60 DEG C) to prepare high-branched starch, guarantees the reaction under the premise of guaranteeing the starch grain morphology, guarantees the application characteristics in the later processing, enables the high-branched starch to be fused with traditional starch-based staple carriers, realizes the synchronous enzyme modification and swelling reaction of the starch, reduces the step of cooling after high-temperature gelatinization, improves the production efficiency, avoids the adverse effect of high-temperature gelatinization on the starch-based products, and has the advantages of high yield, high branching degree, low digestibility, high glycosidic bond proportion and the like, and provides a healthy and green new idea for the field of deep processing of starch.

Description

Technical Field

[0001] This invention relates to a method for preparing highly branched starch using two 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, prone to aging and shrinkage, and can cause significant fluctuations in glycemic index shortly after ingestion. Enzyme-modified starch can replace fat in some food industries and also provides transport protection for some sensitive components and bioactive substances.

[0003] Modified branched starch exhibits greater branching than natural starch, significantly improving its branching degree, amylose content, retrogradation, and digestibility. This is significant for developing low-GI foods that are beneficial for obese or diabetic patients. However, the effects of single-enzyme modification of starch are limited. Therefore, methods using dual enzymes to modify starch and obtain branched starch have been developed. For example, the article "Pre-treatment of granular rice starch to enhance branching enzyme catalysis" discloses a method for preparing branched starch using dual enzymes in a gelatinized state, primarily through the treatment of rice starch with α-amylase and glycogen branching enzyme. However, the resulting branched starch still suffers from low branching degree and low glycosidic bond ratio, limiting its application in the food industry. Furthermore, to achieve effective bond conversion rates, enzymatic modification of starch, both domestically and internationally, has focused on gelatinization conditions. This process disrupts the starch's granular structure and impairs its unique processing properties, such as gelatinization characteristics, gelling properties, and rheological properties. As a result, it can only be consumed by dissolving or dispersing in beverages and paste-like foods, and cannot be integrated with traditional starch-based staple food carriers. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for preparing highly branched starch. This method primarily involves the synergistic modification of starches of different crystalline forms using glycogen branching enzymes and cyclodextrin glucosyltransferases in a non-gelatinized swelling system (60°C). This invention utilizes the non-gelatinized swelling state of starch for enzymatic hydrolysis, achieving simultaneous starch heating and swelling with enzymatic hydrolysis. This maintains a relatively intact starch granule structure, ensuring its subsequent processing and application characteristics, such as gelatinization. It reduces reaction energy consumption while avoiding the problems caused by the high viscosity of gelatinized starch, shortens the processing steps required for cooling after gelatinization, and yields a product with high yield, high branching degree, low digestibility, high glycosidic bond ratio, and low RDS content.

[0005] This invention provides a method for preparing branched starch, which mainly includes the following steps:

[0006] (1) Disperse starch in buffer solution to obtain starch milk, and preheat at 50-60℃;

[0007] (2) Add cyclodextrin glucosyltransferase and glycogen branching enzyme to carry out enzymatic hydrolysis;

[0008] (3) After the enzymatic hydrolysis is completed, the enzyme is inactivated, centrifuged, washed, dried, ground and sieved to obtain branched starch.

[0009] In one embodiment of the present invention, in step (1), the mass concentration of starch milk is 10-20%; the buffer solution is a 0.05 mol / L sodium citrate buffer solution.

[0010] In one embodiment of the present invention, in step (1), the starch is starch with a straight-chain starch content of more than 1%, including one or more of corn starch, potato starch, and pea starch.

[0011] In one embodiment of the present invention, in step (2), the glycogen branching enzyme is derived from Pyrococcus horikoshii OT3(Ph), and the cyclodextrin glucosyltransferase is derived from Bacillus licheniformis.

[0012] In one embodiment of the present invention, in step (2), the amount of cyclodextrin glucosyltransferase added is 0.05-0.15 mL / g dry starch.

[0013] In one embodiment of the present invention, in step (2), the amount of glycogen branching enzyme added is 100-500 U / g dry starch.

[0014] In one embodiment of the present invention, in step (2), cyclodextrin glucosyltransferase and glycogen branching enzyme are added for enzymatic hydrolysis. Specifically, after adding cyclodextrin glucosyltransferase, the pH is adjusted to 11-12, the enzyme is allowed to stand to inactivate, the pH is then adjusted to 5-7, and finally glycogen branching enzyme is added for the reaction.

[0015] In one embodiment of the present invention, in step (2), cyclodextrin glucosyltransferase and glycogen branching enzyme are added for enzymatic hydrolysis. Specifically, after adding glycogen branching enzyme, the pH is adjusted to 11-12, the enzyme is allowed to stand to inactivate, the pH is then adjusted to 5-7, and cyclodextrin glucosyltransferase is added for further reaction.

[0016] In one embodiment of the present invention, in step (2), cyclodextrin glucosyltransferase and glycogen branching enzyme are added for enzymatic hydrolysis, specifically including: simultaneously adding cyclodextrin glucosyltransferase and glycogen branching enzyme for reaction.

[0017] In one embodiment of the present invention, in step (3), anhydrous ethanol is used to inactivate the enzyme.

[0018] The present invention provides a branched starch prepared by the method described above.

[0019] The present invention also provides the application of the branched starch described above in the food industry.

[0020] [Beneficial Effects]

[0021] (1) This invention utilizes a bio-enzymatic method to modify starch to prepare branched starch. Cyclodextrin glucosyltransferase, a type of α-amylase, undergoes three reactions—cyclization, disproportionation, and coupling—accompanying the production of cyclodextrin. Cyclodextrin is readily soluble in water and is washed away. Glycogen branching enzymes can specifically act on starch molecules, increasing the degree of branching of starch by rearranging α-1,4 and α-1,6 glycosidic bonds. This enzymatic recombination process does not introduce any foreign chemical groups, thus the resulting modified starch has higher safety.

[0022] (2) This invention, based on a reaction at 60℃, maintains a relatively intact starch granule structure, ensuring its subsequent gelatinization and other processing characteristics, enabling it to integrate with traditional starch-based staple food carriers. Simultaneously, it avoids the problem of high viscosity after high-temperature starch treatment, achieving simultaneous starch swelling and enzymatic hydrolysis, shortening the reaction steps, and improving industrial production efficiency. It provides an innovative, green, and low-carbon solution for the production of branched starch. This method is not only energy-saving and consumption-reducing but also environmentally friendly, representing a new direction for sustainable development.

[0023] (3) Compared with the prior art, the present invention solves the problems of low conversion rate, long reaction time and low product yield of single glycogen branching enzyme. Porous starch is prepared by pretreatment with cyclodextrin glucosyltransferase, which forms a pore structure of varying sizes, opens up the original tightly arranged starch chains, provides more possible substrates for glycogen branching enzyme, and reduces the steric hindrance between the action site of glycogen branching enzyme and the substrate binding, thereby increasing the product yield (more than 75%) and the degree of branching.

[0024] (4) The branched starch obtained in this invention has the highest increase of 14.9% in α-1,6 bond content compared with natural starch; the rapid digestibility in in vitro digestibility is reduced by 36.14%; and in terms of thermodynamic properties, the gelatinization enthalpy (ΔH) is reduced by up to 7.59 J / g; and the starch viscoelasticity is significantly reduced. Attached Figure Description

[0025] Figure 1 The starch granule morphology of Examples 1-9;

[0026] Figure 2 The viscoelastic properties of the branched corn starch prepared in Examples 1, 4, and 7;

[0027] Figure 3 The viscoelastic properties of potato branched starch prepared in Examples 2, 5, and 8;

[0028] Figure 4 The viscoelastic properties of pea branched starch prepared in Examples 3, 6, and 9;

[0029] Figure 5 The ratio of branched starch glycosidic bonds obtained in Examples 1-9. Detailed Implementation

[0030] In the following examples, the cyclodextrin glucosyltransferase was derived from Bacillus licheniformis, with an enzyme activity of 3 KNU-CP / g, and was purchased from Novozymes (China) Biotechnology Co., Ltd.; the glycogen branching enzyme was derived from Pyrococcus horikoshii OT3 (Ph), and 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. Corn starch, potato starch, and pea starch belong to type A starch, type B starch, and type C starch, respectively.

[0031] The detection methods involved in the following embodiments are as follows:

[0032] Starch granule morphology determination:

[0033] 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.

[0034] In vitro digestibility assay:

[0035] 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 pancreatic amylase 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.

[0036] The specific formula is as follows:

[0037] RDS(%) = (G20 - FG) × 0.9 / TS

[0038] SDS (%) = (G120 - G20) × 0.9 / TS

[0039] RS(%)=100-RDS(%)-SDS(%)

[0040] in:

[0041] Free glucose content (mg) in starch before FG-enzymatic hydrolysis treatment;

[0042] The glucose content (mg) produced after hydrolysis of G20-amylase for 20 min;

[0043] The glucose content (mg) produced after hydrolysis of G120-amylase for 120 min;

[0044] Total starch content (mg) in TS sample.

[0045] Thermodynamic property determination:

[0046] 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).

[0047] Gelatinization property determination:

[0048] Take 2g of the sample before and after modification and add 16.5mL of deionized water. Place it in the aluminum box of the Rapid Viscosity Analyzer (RVA). To prevent starch granule sedimentation, manually stir the starch suspension continuously with the matching plastic stirrer before testing. The specific heating procedure is as follows: the sample is first held at 50℃ for 1min, then heated to 95℃ at a rate of 5.4℃ / min, and held at 95℃ for 5min. Then, the temperature is cooled from 95℃ to 50℃ at a rate of 5.4℃ / min, and held at 50℃ for 2min. For the first 10s of the experiment, the stirrer speed is 960rpm to fully disperse the sample; then the speed is reduced and maintained at 160rpm. The RVA instrument automatically records the change in sample viscosity with heating time and temperature during the above heating-holding-cooling process.

[0049] Determination of the α-1,6 glycosidic bond ratio:

[0050] Weigh 10 mg of the modified starch sample and disperse it in 0.5 mL of D2O. Heat in a boiling water bath to ensure uniform dispersion, vortexing every 10 min until 1 h, then stop heating. Freeze overnight at -80℃, then freeze-dry under vacuum for 2 days. For analysis, reconstitute the sample in D2O containing an internal standard. Record nuclear magnetic resonance (NMR) using a nuclear magnetic resonance spectrometer. 1 ¹H NMR spectrum with a spectral width of 8000 Hz was obtained and data was processed using Mestre Nova 12.0.3 software.

[0051] Example 1

[0052] Corn starch was dispersed in a 0.05 mol / L sodium citrate buffer solution at pH 5.0 to prepare a 10% wt starch slurry. The mixture was preheated at 60°C for 30 min, and 0.1 mL of cyclodextrin glucosyltransferase (per g of dry starch) was added and reacted for 6 h. The pH was adjusted to 11.5, and the mixture was allowed to stand for 15 min to inactivate the enzyme. The pH was then adjusted to 6.0, and glycogen branching enzyme (500 U / g of dry starch) was added and reacted for 20 h. Three times the volume of anhydrous ethanol was added to inactivate the enzyme, and the mixture was allowed to stand for 30 min. The mixture was centrifuged at 10,000 rpm for 10 min, and then washed three times with deionized water. The precipitate was frozen at -80°C for 24 h, freeze-dried under vacuum, and ground through a 100-mesh sieve to obtain a highly branched starch sample, named CS-C / G.

[0053] Example 2

[0054] The preparation method is the same as in Example 1, except that corn starch is replaced with potato starch, and the resulting highly branched starch is named PS-C / G.

[0055] Example 3

[0056] The preparation method is the same as in Example 1, except that corn starch is replaced with pea starch. The resulting highly branched starch is named PBS-C / G.

[0057] Example 4

[0058] Corn starch was dispersed in a 0.05 mol / L sodium citrate buffer solution at pH 5.0 to prepare a 10% wt starch slurry. The slurry was preheated at 60°C for 30 min, and 300 U / g dry starch glycogen branching enzyme was added and reacted for 10 h. The pH was adjusted to 11.5, and the enzyme was inactivated by standing for 15 min. The pH was then adjusted to 7.0, and 0.05 mL (per g dry starch) of cyclodextrin glucosyltransferase was added and reacted for 18 h. The enzyme was inactivated by adding 3 times anhydrous ethanol, and the slurry was allowed to stand for 30 min. The slurry was centrifuged at 10,000 rpm for 10 min, and then washed three times with deionized water. The precipitate was frozen at -80°C for 24 h, freeze-dried under vacuum, and ground through a 100-mesh sieve to obtain a highly branched starch sample, which was named CS-G / C.

[0059] Example 5

[0060] The preparation method is the same as in Example 4, except that corn starch is replaced with potato starch, and the resulting highly branched starch is named PS-G / C.

[0061] Example 6

[0062] The preparation method is the same as in Example 4, except that corn starch is replaced with pea starch, and the resulting highly branched starch is named PBS-G / C.

[0063] Example 7

[0064] Corn starch was dispersed in a 0.05 mol / L sodium citrate buffer solution at pH 5.0 to prepare a 10% wt starch slurry. The slurry was preheated at 60°C for 30 min, and 0.05 mL of cyclodextrin glucosyltransferase (per g of dry starch) was added. 300 U / g of dry starch glycogen branching enzyme was added and the mixture was reacted for 10 h. The enzyme was inactivated by adding 3 times the volume of anhydrous ethanol, allowed to stand for 30 min, centrifuged at 10,000 rpm for 10 min, and then washed 3 times with deionized water. The precipitate was frozen at -80°C for 24 h, freeze-dried under vacuum, and ground through a 100-mesh sieve to obtain a highly branched starch sample, named CS-C+G.

[0065] Example 8

[0066] The preparation method is the same as in Example 7, except that corn starch is replaced with potato starch, and the resulting highly branched starch is named PS-C+G.

[0067] Example 9

[0068] The preparation method is the same as in Example 7, except that corn starch is replaced with pea starch, and the resulting highly branched starch is named PBS-C+G.

[0069] Starch granule morphology:

[0070] from Figure 1 It can be seen that the dual-enzyme modified corn starch, potato starch, and pea starch (Examples 1-9) all maintained their complete granular morphology at 60℃. The non-gelatinization system of the present invention ensures the processing and application characteristics of the modified starch in the later stage.

[0071] Gelatinization property analysis:

[0072] Depend on Figure 2-4 As can be seen, corn starch, potato starch, and pea starch all exhibit the common RVA test curves, with viscosity first increasing and then decreasing. The viscosity of the products is significantly lower than that of the starch before modification.

[0073] In vitro digestibility analysis:

[0074] In in vitro simulated digestion assays, the results (as shown in Table 1) indicate that the proportion of rapidly digestible starch in the branched starch obtained by dual-enzyme modification in this invention decreased to below 70%. After pretreatment with cyclodextrin glucosyltransferase followed by the addition of glycogen branching enzyme, the rapidly digestible starch content of corn starch, potato starch, and pea starch decreased by 36.14%, 20.03%, and 19.62%, respectively, compared to the original starch. The slowly digestible starch content of corn starch increased by 20.14%, and the resistant starch content increased by 21.07%.

[0075] Table 1. RDS, SDS, and RS content of Examples 1-9

[0076]

[0077] Thermodynamic property analysis:

[0078] As shown in Table 2, the gelatinization enthalpy (ΔH) of the branched starch obtained by dual-enzyme modification was significant. Among them, the corn starch treated with cyclodextrin glucosyltransferase and then reacted with glycogen branching enzyme showed the highest decrease of 7.59 J / g.

[0079] Table 2 Thermodynamic properties of Examples 1-9

[0080]

[0081] Analysis of the proportion of α-1,6 glycosidic bonds:

[0082] like Figure 5 As shown in Table 3, the branched starch obtained by dual-enzyme modification has a significantly increased α-1,6 glycosidic bond ratio compared to native starch. Among them, the α-1,6 bond ratio of CS-C / G is 19.35%, which is 4.34 times higher than that of natural corn starch. PS-C / G and PBS-C / G have 2.36 times and 2.82 times higher ratios than natural starch, respectively.

[0083] Table 3. Glycosidic bond ratios in Examples 1-9

[0084]

[0085] 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 method for preparing branched starch, characterized in that, Includes the following steps: (1) Disperse starch in a buffer solution to obtain starch milk, and preheat it at 50-60 °C; the mass concentration of the starch milk is 10-20%; the buffer solution is 0.05 mol / L sodium citrate buffer solution; The starch is corn starch; (2) After adding cyclodextrin glucosyltransferase, adjust the pH to 11-12, let it stand to inactivate the enzyme, then adjust the pH to 5-7, and finally add glycogen branching enzyme to react. The amount of cyclodextrin glucosyltransferase added is 0.05-0.15 mL / g dry starch; the amount of glycogen branching enzyme added is 100-500 U / g dry starch. The glycogen branching enzyme was derived from Pyrococcus horikoshii OT3, and the cyclodextrin glucosyltransferase was derived from Bacillus licheniformis; (3) After the enzymatic hydrolysis is completed, the enzyme is inactivated, centrifuged, washed, dried, ground and sieved to obtain branched starch.

2. The preparation method according to claim 1, characterized in that, In step (1), the starch is starch with a straight-chain starch content of more than 1%.

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