Method for preparing modified starch by using transglutaminase in conjunction with double-enzyme method

Abstract

A method for preparing modified starch by using a transglutaminase in conjunction with a double-enzyme method, comprising the following steps: pretreatment; directed enzymatic hydrolysis: adding pullulanase into a gelatinized starch emulsion for primary directed enzymatic hydrolysis, and adding isoamylase into a primary enzymatic hydrolysis solution for secondary directed enzymatic hydrolysis; and molecular orientation induction: adding a transglutaminase into the starch emulsion that has undergone the directed enzymatic hydrolysis, so as to carry out molecular orientation induction, carrying out enzyme inactivation after the enzymatic hydrolysis is completed, and carrying out centrifugal drying, thereby obtaining modified starch.

Description

A method for preparing modified starch by glutamine transaminase and double enzyme method Technical Field

[0001] The present invention relates to the technical field of starch adjustment preparation, and in particular to a method for preparing modified starch by using transglutaminase in conjunction with a double enzyme process. Background Art

[0002] Retrogradation of gelatinized starch-based foods during production, storage, and transportation leads to deterioration in the quality of starch-based foods, severely impacting the development of the starch-based food industry and causing food waste. However, moderate retrogradation is beneficial in reducing starch digestibility. On the other hand, full-starch-based materials that can replace plastics have become a research hotspot for biodegradable materials at home and abroad due to their wide availability, ease of degradation, environmental friendliness, safety, and good processability. However, such full-starch-based materials processed from starch still have problems such as brittle cracking, low barrier properties, and poor hydrophobicity, which hinder their promotion and application.

[0003] Extensive research has been conducted on improving starch retrogradation in the prior art. For example, Chinese Patent Publication No. CN110846360B discloses a method for inhibiting starch retrogradation. This method uses starch as a substrate and simultaneously adds cyclodextrin hydrolase and cyclodextrin glucosyltransferase to act synergistically on the starch. The cyclodextrin glucosyltransferase modifies side chains during the starch action process. The cyclodextrin hydrolase is introduced to hydrolyze the cyclodextrin produced during the starch action process into maltooligosaccharides, thereby increasing the efficiency of starch modification and improving the starch's anti-retrogradation properties. Chinese Patent Publication No. CN103859551B discloses a method for inhibiting starch retrogradation using gluten. This method inhibits starch retrogradation by binding gluten to starch, preventing starch molecules from nucleating and reversing, thereby preventing starchy foods from increasing in hardness and improving digestibility. Chinese patent publication number CN109096548B discloses a method for preparing retrograded resistant starch using phycocyanin and its hydrolyzate. The method uses arginine in the protein to form hydrogen bonds with the aldehyde groups of starch to stabilize the mobility of starch molecules. The hydrophobic amino acids in the protein are then used to drive water out of the starch gel, promoting the starch molecular chains to approach each other to form hydrogen bonds, thereby promoting the formation of retrograded starch.

[0004] Prior art also studies the mechanical properties and hydrophobicity of starch. For example, Chinese Patent Publication No. CN103709257B discloses a grafted modified starch as a polylactic acid / starch compatibilizer. This modified starch, prepared under specific process conditions using starch as a raw material and maleic anhydride, polypropylene glycol, and a catalyst as auxiliary materials, can increase the water resistance and mechanical strength of the matrix and reduce the water absorption of the blend, resulting in a low-cost biodegradable plastic. Furthermore, Chinese Patent Publication No. CN112142987B discloses a method for preparing a modified starch, a starch film, and a method for preparing the same. The modified starch is prepared by sequentially modifying starch with an alkali metal hydroxide and then epoxidized soybean oil. The resulting modified starch exhibits both good water resistance and interfacial compatibility, resulting in a starch film prepared using the modified starch having good water resistance and mechanical strength.

[0005] However, prior art research lacks knowledge on how to simultaneously control the degree of starch retrogradation and improve the mechanical, barrier, and hydrophobic properties of starch-based materials. This approach remains a pressing technical challenge. Therefore, the present invention aims to develop a method for preparing modified starch using a dual enzyme process involving transglutaminase to better meet these needs.

[0006] Summary of the Invention

[0007] The technical problem solved by the present invention is to provide a method for preparing modified starch by a dual enzyme method using transglutaminase, so as to accurately control the degree of starch retrogradation while improving the mechanical, barrier and hydrophobic properties of starch-based materials prepared from starch.

[0008] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0009] A method for preparing modified starch by a glutamine transaminase-assisted dual-enzyme process comprises the following steps:

[0010] Pretreatment: Add water to starch to prepare starch emulsion, and heat the starch emulsion to gelatinize for later use;

[0011] Directed enzymatic hydrolysis: The pH value of the gelatinized starch emulsion is adjusted to 5.0-6.0, the temperature is maintained at 55-65°C, and then pullulanase is added for preliminary directed enzymatic hydrolysis for 6-24 hours. The enzyme is inactivated to obtain a primary enzymatic hydrolysate. The pH value of the primary enzymatic hydrolysate is adjusted to 5.5-7.0, the temperature is maintained at 50-55°C, and then isoamylase is added for secondary directed enzymatic hydrolysis for 2-12 hours. After the enzymatic hydrolysis is completed, the enzyme is inactivated at high temperature.

[0012] Inducing Molecular Orientation: After directional enzymatic hydrolysis, the pH of the starch emulsion is adjusted to 5.0-8.0. Transglutaminase is then added to induce molecular orientation. The reaction lasts for 1-3 hours. After enzymatic hydrolysis, the enzyme is inactivated and the starch is centrifuged and dried to obtain the modified starch. During this process, transglutaminase reorients and links the molecules according to specific rules, inducing molecular orientation to form a relatively ideal amylopectin molecular chain structure with an appropriate degree of branching, branch chain length, and distribution.

[0013] Furthermore, during the pretreatment process, the mass concentration of the starch emulsion is 2-8%.

[0014] Furthermore, during the pretreatment process, before the directional enzymatic hydrolysis, the starch emulsion is heated and gelatinized and then treated in a constant temperature water bath at 55 to 65° C. for 10 to 20 minutes.

[0015] Furthermore, during the directed enzymatic hydrolysis process, the enzymatic activity of the pullulanase is 600-1000 U / mL, and the amount of the pullulanase added is 2-10 U / g of dry starch; the enzymatic activity of the isoamylase is 900-1100 U / mL, and the amount of the isoamylase added is 2-10 U / g of dry starch.

[0016] Furthermore, during the directional enzymatic hydrolysis process, a stirrer is used for shear stirring at a stirring rate of 100-400 r / min.

[0017] Furthermore, during the process of inducing molecular orientation, the enzyme activity of the transglutaminase is 80-100 U / g, and the amount of the added transglutaminase is 2-10 U / g starch dry basis.

[0018] Furthermore, stirring is maintained during the process of inducing molecular orientation.

[0019] The present invention also relates to a modified starch prepared by the method described above.

[0020] The use of the modified starch in the preparation of starch-based materials or starch-based products.

[0021] Beneficial Effects: Starch retrogradation is the process by which gelatinized amylose and amylopectin molecules recombine through hydrogen bonds, either within or between them, to form an ordered structure different from the original starch. Starch retrogradation consists of two stages: short-term retrogradation and long-term retrogradation, primarily involving amylose and amylopectin molecules. In the early stages of retrogradation, the gelatinized amylose undergoes molecular rearrangement within a short period of time, forming an ordered double helix structure. Long-term retrogradation, which is slower and dominated by amylopectin, has the greatest impact on the quality of starch-based foods. Amylopectin is the main component of starch, and the higher the content of long B chains in amylopectin, the easier it is to retrograde. The long B chains of amylopectin form the backbone of the macromolecule through α-1,6 bonds. Components of different structural types are distributed along the backbone chain, while short B chains form short branches that serve as bridges between external components and the backbone chain. The method for preparing modified starch by a glutamine transaminase-assisted dual-enzyme process described in the present invention uses pullulanase and isoamylase to act on the long B chains of amylopectin to achieve limited and directional enzymatic hydrolysis of the amylopectin, and further uses TG enzyme to induce molecular orientation to form a relatively ideal amylopectin molecular chain structure with a moderate branching degree, branch chain length and distribution. By controlling the reaction conditions, a moderate degree of retrogradation is obtained, and after being prepared into a starch-based material, the material has excellent mechanical, barrier and hydrophobic properties.

[0022] In addition, there are a large number of intramolecular and intermolecular hydrogen bonds in the structural units of ordinary starch, and the content of amylopectin is high, and its glass transition temperature is very close to the decomposition temperature. Therefore, ordinary starch itself does not have plasticity, resulting in the starch-based material prepared from ordinary starch being fragile and having poor mechanical properties. The method of preparing modified starch by glutamine transaminase in conjunction with a dual enzyme method described in the present invention combines physical methods and biological control methods, utilizes stirring and shearing effects and the directional decomposition and induction effects of enzymes to destroy the hydrogen bonds in starch and form a relatively ideal amylopectin molecular chain structure with a moderate degree of branching, branch chain length and distribution. In this process, no organic solvent is required, which is green, environmentally friendly and safe. The preparation process is simple, the nodes are easy to control, and it is suitable for batch processing, production and promotion and application. DETAILED DESCRIPTION

[0023] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments.

[0024] Example 1

[0025] The method for preparing modified starch by a double enzyme method using transglutaminase comprises the following steps:

[0026] Pretreatment: Add water to starch to prepare a starch emulsion with a mass concentration of 5%. Heat the starch emulsion to gelatinize, then keep it in a constant temperature water bath at 60°C for 15 minutes, and then cool it to room temperature for use.

[0027] Directed enzymatic hydrolysis: The gelatinized starch emulsion was adjusted to pH 5.5 with 2 mol / L hydrochloric acid and maintained at 65°C. Pullulanase at 5 U / g starch was then added for preliminary directed enzymatic hydrolysis for 20 hours. The enzyme was then inactivated in a boiling water bath for 15 minutes to obtain a primary enzymatic hydrolysate. The pH of the primary enzymatic hydrolysate was adjusted to 5.5 and the temperature was maintained at 50°C. Isoamylase at 5 U / g starch was then added for secondary directed enzymatic hydrolysis for 2 hours. The enzyme was then inactivated at high temperature. The pullulanase and isoamylase addition ratio was 1:1. An electric stirrer was used for shear stirring during the enzymatic hydrolysis process at a stirring rate of 200 r / min.

[0028] Inducing molecular orientation: The pH of the starch emulsion after the directional debranching enzymatic hydrolysis is adjusted to 7.0, and then transglutaminase is added to induce molecular orientation. The enzyme activity of transglutaminase is 90 U / g, and the amount of transglutaminase added is 6 U / g starch dry basis. The enzymatic hydrolysis temperature is 55°C, and stirring is maintained during the enzymatic hydrolysis. After 1 hour of enzymatic hydrolysis, the reaction is terminated by heating in a boiling water bath, and the modified starch is centrifuged and dried to obtain.

[0029] Example 2

[0030] The method for preparing modified starch by a double enzyme method using transglutaminase comprises the following steps:

[0031] Pretreatment: Add water to starch to prepare a starch emulsion with a mass concentration of 8%. Heat the starch emulsion to gelatinize, then keep the temperature in a constant temperature water bath at 60°C for 20 minutes, and then cool to room temperature for use.

[0032] Directed enzymatic hydrolysis: The gelatinized starch emulsion was adjusted to pH 6.0 with 2 mol / L hydrochloric acid and maintained at 60°C. Pullulanase at 5 U / g starch was then added for preliminary directed enzymatic hydrolysis for 12 hours. The enzyme was then inactivated in a boiling water bath for 15 minutes to obtain a primary enzymatic hydrolysate. The pH of the primary enzymatic hydrolysate was adjusted to 6.5, the temperature was maintained at 55°C, and isoamylase at 5 U / g starch was added for secondary directed enzymatic hydrolysis for 4 hours. After the enzymatic hydrolysis was completed, the enzyme was inactivated at high temperature. The pullulanase and isoamylase addition ratio was 1:1. An electric stirrer was used for shear stirring during the enzymatic hydrolysis process at a stirring rate of 300 r / min.

[0033] Inducing molecular orientation: The pH of the starch emulsion after the directional debranching enzymatic hydrolysis is adjusted to 8.0, and then transglutaminase is added to induce molecular orientation. The enzymatic activity of the transglutaminase is 90 U / g, and the amount of transglutaminase added is 8 U / g starch dry basis. The enzymatic hydrolysis temperature is 55°C, and stirring is maintained during the enzymatic hydrolysis. After 3 hours of enzymatic hydrolysis, the reaction is terminated by heating in a boiling water bath, and the modified starch is centrifuged and dried to obtain.

[0034] Comparative Example 1

[0035] The method for preparing modified starch by the dual enzyme method using transglutaminase is different from Example 2 in that isoamylase is first added for preliminary directional enzymatic hydrolysis, and then pullulanase is added for secondary directional enzymatic hydrolysis, which specifically includes the following steps:

[0036] Pretreatment: Add water to starch to prepare a starch emulsion with a mass concentration of 8%. Heat the starch emulsion to gelatinize, then keep the temperature in a constant temperature water bath at 60°C for 20 minutes, and then cool to room temperature for use.

[0037] Directed enzymatic hydrolysis: The pH value of the gelatinized starch emulsion was adjusted to 6.5 with 2 mol / L hydrochloric acid and the temperature was maintained at 55°C. Then, 5 U / g of starch was added for preliminary directed enzymatic hydrolysis. The enzymatic hydrolysis time was 4 hours. The enzyme was then inactivated in a boiling water bath for 15 minutes to obtain a primary enzymatic hydrolysate. The pH value of the primary enzymatic hydrolysate was adjusted to 6.0 and the temperature was maintained at 60°C. Then, 5 U / g of starch was added for secondary directed enzymatic hydrolysis. The enzymatic hydrolysis time was 12 hours. After the enzymatic hydrolysis was completed, the enzyme was inactivated at high temperature. The addition ratio of pullulanase to isoamylase was 1:1. During the enzymatic hydrolysis process, an electric stirrer was used for shear stirring at a stirring rate of 300 r / min.

[0038] Inducing molecular orientation: The pH of the starch emulsion after the directional debranching enzymatic hydrolysis is adjusted to 8.0, and then transglutaminase is added to induce molecular orientation. The enzymatic activity of the transglutaminase is 90 U / g, and the amount of transglutaminase added is 8 U / g starch dry basis. The enzymatic hydrolysis temperature is 55°C, and stirring is maintained during the enzymatic hydrolysis. After 3 hours of enzymatic hydrolysis, the reaction is terminated by heating in a boiling water bath, and the modified starch is centrifuged and dried to obtain.

[0039] Comparative Example 2

[0040] The method for preparing modified starch by glutamine transaminase and dual enzyme method, the difference between control example 2 and example 2 is that continuous stirring is not maintained, and the addition ratio of pullulanase and isoamylase is 1:0.5, specifically comprising the following steps:

[0041] Pretreatment: Add water to starch to prepare a starch emulsion with a mass concentration of 8%. Heat the starch emulsion to gelatinize, then keep the temperature in a constant temperature water bath at 60°C for 20 minutes, and then cool to room temperature for use.

[0042] Directed enzymatic hydrolysis: The gelatinized starch emulsion was adjusted to pH 6.0 with 2 mol / L hydrochloric acid and maintained at 60°C. Pullulanase at 5 U / g starch was then added for preliminary directed enzymatic hydrolysis for 12 hours. The enzyme was then inactivated in a boiling water bath for 15 minutes to obtain a primary enzymatic hydrolysate. The pH of the primary enzymatic hydrolysate was adjusted to 6.5 and the temperature was maintained at 55°C. Isoamylase at 2.5 U / g starch was then added for secondary directed enzymatic hydrolysis for 4 hours. The enzymes were then inactivated at high temperature. The ratio of pullulanase to isoamylase was 1:0.5.

[0043] Inducing molecular orientation: The pH of the starch emulsion after the directional debranching enzymatic hydrolysis is adjusted to 8.0, and then transglutaminase is added to induce molecular orientation. The enzyme activity of transglutaminase is 90 U / g, and the amount of transglutaminase added is 8 U / g starch dry basis. The enzymatic hydrolysis temperature is 55°C. After enzymatic hydrolysis for 3 hours, the reaction is terminated by heating in a boiling water bath, and the modified starch is centrifuged and dried to obtain.

[0044] The modified starch and native starch obtained in Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were subjected to performance testing and comparison.

[0045] The mechanical properties of starch are tested by cooling the starch gelatinized to room temperature and then using a micrometer and texture analyzer to measure tensile strength, elongation at break, starch gel strength, and starch gel elasticity. A rapid viscometer is used to test the retrogradation properties of starch-based materials.

[0046] The barrier and hydrophobicity testing methods for starch are as follows: starch is made into a film, and the air permeability or moisture permeability of the starch film is tested. The transmittance is measured at a wavelength of 600nm using a UV-visible spectrophotometer. The moisture permeability of the film is tested using the pseudo-cup method. The hydrophobicity of the starch-based material can be tested based on the solubility of starch in water.

[0047] The results are shown in Table 1.

[0048] Table 1 Mechanical, barrier, hydrophobic and retrogradation properties of starch before and after enzyme treatment

[0049] As shown in Table 1, compared with native starch, the modified starch prepared by Example 1 and Example 2 has greatly improved mechanical properties, barrier properties and retrogradation, better processability, and retrogradation is controlled within a preferred range, which is beneficial for reducing starch digestibility while reducing product quality deterioration. However, when the enzymatic hydrolysis order of pullulanase and isoamylase is changed in Control Example 1, when the ratio of pullulanase and isoamylase is improper in Example 2, and when the stirring control is improper during enzymatic hydrolysis and induction of molecular orientation, the modified starch obtained is not significantly improved compared with the native starch, and the effect is not ideal. This shows that the technical solution of the present invention is the result of the synergistic effect of each step.

[0050] A lower starch retrogradation value isn't necessarily better. A high retrogradation value, such as a native starch value of 2387, results in foods with a hard texture and poor rehydration. However, foods with a starch retrogradation value below 1000, such as low-retrogradation starch, have high viscosity and low gel strength and elasticity. Therefore, moderate retrogradation is crucial for starch-based foods. The starch foods prepared with moderate retrogradation, as in Examples 1 and 2, maintain a certain hardness while also exhibiting good gel strength and elasticity, resulting in excellent edible and processing qualities.

[0051] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing modified starch by using glutamine aminotransferase and dual enzyme method, characterized in that: The steps include: Pretreatment: Add water to starch to prepare starch emulsion, and heat the starch emulsion for gelatinization for later use; Directed enzymatic hydrolysis: adjust the pH value of the gelatinized starch emulsion to 5.0-6.0, maintain the temperature at 55-65°C, then add pullulanase for preliminary directed enzymatic hydrolysis for 6-24 hours, inactivate the enzyme to obtain a primary enzymatic hydrolysate, adjust the pH value of the primary enzymatic hydrolysate to 5.5-7.0, maintain the temperature at 50-55°C, then add isoamylase for secondary directed enzymatic hydrolysis for 2-12 hours, inactivate the enzyme at high temperature after the enzymatic hydrolysis is completed; Inducing molecular orientation: The pH value of the starch emulsion after directional enzymatic hydrolysis is adjusted to 5.0-8.0, and then glutamine aminotransferase is added to induce molecular orientation. The reaction time is 1-3 hours. After the enzymatic hydrolysis is completed, the enzyme is inactivated and the modified starch is obtained by centrifugation and drying.

2. The method for preparing modified starch by glutamine aminotransferase-assisted dual enzyme method according to claim 1, characterized in that: During the pretreatment process, the mass concentration of the starch emulsion is 2-8%.

3. The method for preparing modified starch by glutamine aminotransferase-assisted dual enzyme method according to claim 1, characterized in that: During the pretreatment process, before the directional enzymatic hydrolysis, the starch emulsion is heated and gelatinized and then treated in a constant temperature water bath at 55 to 65°C for 10 to 20 minutes.

4. The method for preparing modified starch by glutamine aminotransferase-assisted dual enzyme method according to claim 1, characterized in that: In the directional enzymatic hydrolysis process, the enzymatic activity of the pullulanase is 600-1000U / mL, and the added amount of the pullulanase is 2-10U / g starch dry basis; the enzymatic activity of the isoamylase is 900-1100U / mL, and the added amount of the isoamylase is 2-10U / g starch dry basis.

5. The method for preparing modified starch by glutamine aminotransferase and dual enzyme method according to claim 1, characterized in that: During the directional enzymatic hydrolysis process, a stirrer is used for shear stirring at a stirring rate of 100-400 r / min.

6. The method for preparing modified starch by glutamine aminotransferase-assisted dual enzyme method according to claim 1, characterized in that: During the process of inducing molecular orientation, the enzyme activity of the transglutaminase is 80-100 U / g, and the added amount of the transglutaminase is 2-10 U / g starch dry basis.

7. The method for preparing modified starch by glutamine aminotransferase and dual enzyme method according to claim 1, characterized in that: Stirring was maintained during the process of inducing molecular orientation.

8. A modified starch, characterized in that The method is prepared by any one of claims 1 to 7.

9. Use of the modified starch according to claim 8 in preparing starch-based materials or starch-based products.

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