阅读说明：本技术 一种酶法制备凝胶强度可控的热可逆淀粉基凝胶的方法 (Method for preparing thermoreversible starch-based gel with controllable gel strength by enzyme method ) 是由 柏玉香 王禹 金征宇 于 2019-09-20 设计创作，主要内容包括：本发明公开了一种酶法制备凝胶强度可控的热可逆淀粉基凝胶的方法,属于淀粉改性技术领域。本发明通过从热变形菌(Thermoproteus uzoniensis)中克隆到一株4-α-糖基转移酶基因(TuαGT),通过大肠杆菌异源表达,酶学性质表征,确定其为4αGT,并且该酶具有较好的温度稳定性和底物耐受性,使其能够在高温下催化高浓度的淀粉改性,延长其支链淀粉的侧链。本发明的酶法制备凝胶强度可控的热可逆淀粉基凝胶的方法,以淀粉为原料,添加适量的4-α-糖基转移酶,选择合适的添加量和作用时间,来制备高强度抗回生淀粉基凝胶。(The invention discloses a method for preparing thermoreversible starch-based gel with controllable gel strength by an enzyme method, belonging to the technical field of starch modification. The invention clones a 4-alpha-glycosyltransferase gene (Tu alpha GT) from thermomyces (Thermoproteus uzoniensis), and determines the gene to be 4 alpha GT by escherichia coli heterologous expression and enzymological property characterization, and the enzyme has better temperature stability and substrate tolerance, so that the enzyme can catalyze the modification of starch with high concentration at high temperature and prolong the side chain of amylopectin. The method for preparing the thermoreversible starch-based gel with controllable gel strength by the enzyme method takes starch as a raw material, adds a proper amount of 4-alpha-glycosyltransferase, and selects a proper addition amount and action time to prepare the high-strength retrogradation-resistant starch-based gel.)

1. A method for preparing a thermoreversible starch-based gel having controlled gel strength, said method comprising the steps of:

(1) dissolving starch in buffer solution for size mixing, and completely gelatinizing at 70-120 deg.C;

(2) cooling gelatinization temperature to 40-90 deg.C, adjusting pH to 4.0-13.0, adding 4-alpha-glycosyltransferase with amino acid sequence shown in SEQ ID NO.1, inactivating enzyme, drying, and grinding to obtain modified starch;

(3) and dissolving the obtained modified starch, heating and stirring at 70-90 ℃, and solidifying to obtain the modified starch gel.

2. The method of claim 1, wherein the enzymatic hydrolysis time is 1-12 hours.

3. The method according to claim 1, wherein the mass concentration of the starch in the step (1) is 5-10%.

4. The method of claim 1, wherein said starch in step (1) consists essentially of potato starch, corn starch, tapioca starch, wheat starch, pea starch, and rice starch.

5. The method according to claim 1, wherein the 4- α -glycosyltransferase is added in an amount of 0.5 to 10U/g starch in step (2).

6. The method of claim 1, wherein the enzyme deactivation method in step (2) comprises high temperature enzyme deactivation, acid-base enzyme deactivation and ethanol enzyme deactivation.

7. The method according to claim 1, wherein the drying method in the step (2) comprises freeze drying, atmospheric drying, spray drying, roller drying, microwave drying.

8. The method of claim 1, wherein the gel strength is controllable by calculating the gel strength from the time of enzymatic hydrolysis by: -898.39x +6298.3, R²0.9343. Wherein y is the gel strength and x is the modification time; the gel strength is 3000-8000 Pa.

9. A starch-based gel prepared by the method of any one of claims 1 to 8.

10. Use of the starch-based gel according to claim 9 for the manufacture of jelly, yoghurt, tortoise jelly, cheese, confectionery, ice cream.

Technical Field

The invention relates to a method for preparing thermoreversible starch-based gel with controllable gel strength by an enzyme method, belonging to the technical field of starch modification.

Background

Starch is a pure natural raw material with wide source and low price. Starch colloid produced by using starch as a raw material is widely applied to various industries such as food, chemical industry and the like. The starch gel is a high molecular compound, and is transparent or semitransparent gel with certain elasticity and hardness formed after starch is gelatinized. In the food industry, compared with other food colloids, the starch colloid is easier to digest and absorb by human bodies, is not easy to generate adverse reactions such as flatulence and nephrotoxicity, can be added into infant food, and has wide market prospect. In other industries, starch colloids have the advantages of light weight, no corrosion, no pollution, low cost, and the like. However, the native starch gels have a number of drawbacks, among which the starch gel strength is too high and uncontrollable, which makes the starch gel products difficult to apply. Therefore, the method has very important industrial value for the modification of starch gel.

Many factors influence the gel strength of starch, such as amylose content and size, polysaccharide content in starch, lipid content, etc. The content and size of amylose therein have been the focus of research. The starch gel is a mixed system formed by amylose and amylopectin through hydrogen bond interaction in the cooling process, wherein amylose is released and mutually wound to form a three-dimensional network structure after the starch is gelatinized. The strength of the gel formed by starch is affected when the amylose content is low or the double helix structure of amylose is affected by complexation with lipids. Therefore, the formation of starch gel is closely related to the content of amylose in starch. The amylose content and the molecular weight of starch from different sources are different, so that the gel strength of the starch is greatly different under the same conditions. Therefore, the function of controlling the gel strength of the starch can be achieved by controlling the content of amylose.

Therefore, how to modify starch, change the content of amylose in the starch while keeping the whole structure of the starch undamaged, improve the strength of starch-based gel and ensure good characteristics of the prepared starch gel is a technical problem to be solved urgently.

Disclosure of Invention

In order to solve the existing technical problems, the invention provides a method for modifying starch so as to realize starch-based gel thermoreversible and controllable gel strength, which clones a 4-alpha-glycosyltransferase gene (Tu alpha GT) from thermomyces (Thermoproteus uzonicensis), determines the gene to be 4 alpha GT by escherichia coli heterologous expression and enzymological property characterization, and has better temperature stability and substrate tolerance, so that the enzyme can catalyze high-concentration starch modification at high temperature and prolong the side chain of amylopectin. The invention mainly adopts three key aspects of control to realize the preparation of the high-strength retrogradation-resistant starch-based gel: 1) screening starch types; 2) optimizing the addition amount of the 4-alpha-glycosyltransferase; 3) optimizing the action time of the 4-alpha-glycosyltransferase. The method for preparing the thermoreversible starch-based gel with controllable gel strength by the enzyme method takes starch as a raw material, adds a proper amount of 4-alpha-glycosyltransferase, and selects a proper addition amount and action time to prepare the high-strength retrogradation-resistant starch-based gel. The invention has simple preparation process, simple equipment and good product effect.

The first purpose of the invention is to provide a preparation method of heat reversible starch-based gel with controllable gel strength, which comprises the following steps:

(1) dissolving starch in buffer solution for size mixing, and completely gelatinizing at 70-120 deg.C;

(2) cooling to 40-90 deg.C, adjusting pH to 4.0-13.0, adding 4-alpha-glycosyltransferase with amino acid sequence shown in SEQ ID NO.1, inactivating enzyme, drying, and grinding to obtain modified starch;

(3) and dissolving the obtained modified starch, heating and stirring at 70-90 ℃, and solidifying to obtain the modified starch gel.

In one embodiment of the invention, the time of enzymatic hydrolysis is 1-12 hours.

In one embodiment of the present invention, the mass concentration of the starch in the step (1) is 5% to 10%.

In one embodiment of the present invention, the starch in step (1) mainly comprises potato starch, corn starch, tapioca starch, wheat starch, pea starch and rice starch.

In one embodiment of the present invention, preferably, the starch is potato starch.

In one embodiment of the present invention, the 4- α -glycosyltransferase is added in an amount of 0.5 to 10U/g 4- α -glycosyltransferase/starch in step (2).

In one embodiment of the present invention, the enzyme deactivation method in step (2) comprises high temperature enzyme deactivation, acid-base enzyme deactivation and ethanol enzyme deactivation.

In one embodiment of the present invention, the drying method in step (2) comprises freeze drying, atmospheric drying, spray drying, roller drying, and microwave drying.

In one embodiment of the present invention, the gel strength is controllable by calculating the gel strength according to the enzymolysis time, and the calculation method includes: -898.39x +6298.3, R²0.9343. Wherein y is the gel strength and x is the modification time; the gel strength is 3000-8000 Pa.

The second object of the present invention is to provide a starch-based gel prepared by the above method.

The third purpose of the invention is to provide the application of the starch-based gel in jelly, old yoghourt, tortoise jelly, cheese, candy and ice cream.

The invention has the beneficial effects that:

(1) the source of the 4-alpha-glycosyltransferase is Thermoproteus uzoniensis. The highest similarity of the enzyme sequence and other 4-alpha-glycosyltransferase sequences is only 70 percent through sequence alignment; the optimal reaction temperature of the 4-alpha-glycosyltransferase is 50-80 ℃, and the optimal reaction pH is 5.0-8.0; the minimal acting substrate unit of 4-alpha-glycosyltransferase is maltose, which is not active against monosaccharides such as glucose.

(2) The 4-alpha-glycosyltransferase of the invention has the relative enzyme activity of more than 90 percent at 65-80 ℃, and has better temperature stability; the starch can be catalyzed and modified at the high temperature of 60-90 ℃, and the starch conversion rate is up to more than 80%; can catalyze 10-30% (w/v) high-concentration starch modification, and the starch conversion rate is up to more than 90%.

(3) Compared with the prior art, the invention can realize higher gel strength in a shorter time.

(4) The gel strength of the gel prepared by adjusting the enzymolysis time is in the range of 2000-8000 Pa, so that the method is suitable for preparing jelly, old yogurt, tortoise jelly, cheese, candy and ice cream, and has a wider application range.

Drawings

FIG. 1: and (4) nickel column purification of the 4-alpha-glycosyltransferase.

FIG. 2: optimum reaction temperature of the enzyme.

FIG. 3: optimum reaction pH of the enzyme.

FIG. 4: 4-alpha-glycosyltransferase time of action versus gel strength.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

(1)4 alpha GTase enzyme activity determination method

mu.L of an appropriately diluted enzyme solution was added to a 1% maltotriose (G3) solution (50mM sodium citrate buffer, pH6.0) and reacted at 75 ℃ for 6 hours. The reaction was stopped by boiling water bath for 10 min. And determining the glucose content in the reaction solution by adopting a glucose oxidase kit method. Disproportionation activity was defined as the amount of enzyme required to produce 1 μmol glucose per minute.

The enzyme activity unit is defined as: the amount of enzyme required to produce 1. mu. mol glucose per minute.

(2) Calculation formula of starch conversion rate:

in the formula: x is the starch conversion rate; c. C₀Is the amylose content in the original starch; c. C₁Is the amylose content of the starch after enzyme modification.

(3) Method for measuring gel strength:

the gel strength of the samples was determined using a texture analyzer. The gel samples were cut into cylinders 25mm in diameter and 10mm in height. The texture properties of the starch gels were determined at 25 ℃ using a texture analyser of the TA-XTplus type, UK. The height was measured at 15mm using a P36R probe, the speed before measurement was 0.5mm/s, the speed after measurement was 0.5mm/s, and the trigger force was 5 g.

(4) Method for measuring thermal reversible performance:

the thermoreversibility of the samples was determined using a rotational rheometer. The gel sample was placed evenly on the rheometer test platform. Selecting a stainless steel flat plate clamp with the diameter of 40mm, setting the distance to be 1000 mu m, scanning the strain value to be 1 percent, oscillating the strain value to be 1Hz, heating the temperature from 5 ℃ to 90 ℃ at a constant speed of 10 ℃/min, and measuring the change condition of the storage modulus G 'and the loss modulus G' of the starch gel along with the temperature rise.