阅读说明：本技术 一种改善牛血清蛋白热稳定性的方法，以及一种透明蛋白胶体及其制备方法 (Method for improving heat stability of bovine serum albumin, transparent protein colloid and preparation method thereof ) 是由 李灿鹏 赵宇婷 于 2019-11-13 设计创作，主要内容包括：一种改善牛血清蛋白热稳定性的方法,涉及蛋白改性领域,其将牛血清蛋白溶解在缓冲溶剂中配置成一定浓度的蛋白溶液,再加入植酸来改善牛血清蛋白的稳定性。该方法的操作简单方便,原料廉价易得,经过其改善后的牛血清蛋白在高温下可稳定存在,基本不会因蛋白质变性而沉淀析出。一种透明蛋白胶体及其制备方法,其同样是将牛血清蛋白溶解在缓冲溶剂中配置成特定浓度的蛋白溶液,再加入植酸以形成胶体。该制备方法的操作简单,条件温和,以牛血清蛋白为原料得到无色透明的蛋白胶体。该透明蛋白胶体无色透明的特性,使其在食品等领域具有较大的应用潜力。(A method for improving the heat stability of bovine serum albumin relates to the field of protein modification, wherein bovine serum albumin is dissolved in a buffer solvent to prepare a protein solution with a certain concentration, and phytic acid is added to improve the stability of the bovine serum albumin. The method has simple and convenient operation and cheap and easily obtained raw materials, and the modified bovine serum albumin can stably exist at high temperature and basically cannot be precipitated due to protein denaturation. A transparent protein colloid is prepared through dissolving bovine serum albumin in buffer solvent to obtain protein solution with specific concentration, and adding phytic acid to obtain colloid. The preparation method is simple to operate and mild in conditions, and the colorless and transparent protein colloid is obtained by taking bovine serum albumin as a raw material. The transparent protein colloid is colorless and transparent, so that the transparent protein colloid has great application potential in the fields of food and the like.)

1. A method of improving the thermal stability of bovine serum albumin, comprising:

mixing bovine serum albumin with a buffer solution to prepare a protein solution with the concentration of 1-10 mg/ml;

mixing the protein solution with phytic acid.

2. The method according to claim 1, wherein the mass ratio of the bovine serum albumin to the phytic acid is 1:0.4 to 2.

3. The method according to claim 2, wherein the concentration of the buffer solution is 30 to 100 mM, and the pH of the buffer solution is 7.0 to 8.0.

4. The method of claim 3, wherein the buffer solution comprises at least one of Tris-HCl buffer, barbiturate sodium-HCl buffer, and PBS buffer; preferably, the buffer solution is Tris-HCl buffer.

5. A method for preparing a transparent protein colloid, comprising:

mixing bovine serum albumin with a buffer solution to prepare a protein solution with the concentration of 100-300 mg/ml;

mixing the protein solution with phytic acid, and heating to 80-100 ℃.

6. The method according to claim 5, wherein the mass ratio of the bovine serum albumin to the phytic acid is 1: 0.05 to 0.25.

7. The method according to claim 5, wherein the concentration of the buffer solution is 30 to 100 mM, and the pH of the buffer solution is 7.2 to 7.6.

8. The method of claim 6, wherein the buffer solution comprises at least one of Tris-HCl buffer, barbiturate sodium-hydrochloric acid buffer, and PBS buffer; preferably, the buffer solution is Tris-HCl buffer.

9. The method according to claim 8, wherein the protein solution is mixed with the phytic acid and then heated for 10 to 30 min.

10. A transparent protein colloid produced by the method for producing a transparent protein colloid according to any one of claims 1 to 9.

Technical Field

The invention relates to the field of protein modification, in particular to a method for improving the heat stability of bovine serum albumin, a transparent protein colloid and a preparation method thereof.

Background

The thermal stability of proteins depends mainly on the three-dimensional structure of proteins and interactions between proteins, and disulfide bonds and hydrophobic interactions play important roles in these reactions. During heating, proteins form polymers of macromolecules via disulfide bonds or other non-covalent bonds, and during acidification, these polymers form the network structure of proteins, in which hydrogen bonds play an important role. Among the factors that influence thermal polymerization of proteins, charge, hydrophobicity, and the content of thiol groups play a crucial role.

The three-dimensional structure of proteins is maintained by weak interaction forces, such as hydrophobic interactions, electrostatic interactions, hydrogen bonds, van der waals forces, and disulfide bonds. Their free energy of stabilization Δ Gstab is generally lower than 60kJ/mol, and therefore the conformational stability of proteins is low. Thus, treatment of proteins with acids, bases, concentrated saline solutions, heat, and radiation can alter the conformation of the protein to varying degrees. Denaturation of a protein is any change in its conformation (secondary, tertiary or quaternary), which is not accompanied by cleavage of peptide bonds in the primary structure. Heat is the most common physical factor causing protein denaturation. In general, most proteins are stable between 0-4 ℃ and most thermal denaturation is irreversible. The rate of denaturation depends on temperature, and for most reactions the temperature is increased by 10 ℃ and the reaction rate is increased by about 2 times. However, in a typical denaturation temperature range, the temperature is increased by 10 ℃ and the reaction rate is increased by about 600 times for protein denaturation. This is a result of the lower energy of each interaction that stabilizes the secondary, tertiary, and quaternary structures. Heat treatment is widely applied to the separation, purification, processing and preservation processes of proteins, and the physical and chemical properties of the proteins are greatly influenced by heat denaturation of the proteins caused by temperature rise. During the processing, the functional properties of the protein can be changed to different degrees by heat denaturation, so that the heat-denatured protein can increase the functional properties of the protein and does not form precipitates to influence the application of the protein, and therefore, the improvement of the heat stability of the protein is urgently needed.

BSA (bovine serum albumin), which is a major component of bovine plasma protein and mainly affects the functional properties of bovine plasma protein, the production of whey and casein in plasma or cheese, which are well known potential sources of nutritional and functional proteins, is a relatively small component of whey protein, which contributes to the improvement of the functional properties of whey protein, and therefore, effective utilization of plasma and whey protein is essential to improve the functional properties of BSA. At present, few reports on improving the heat stability of BSA (bovine serum albumin) exist, and many reports on heat induction form opaque protein colloid, and one of the main purposes of the protein is to use the protein in food, so that the application range of the protein in the food field and other fields is limited.

Disclosure of Invention

The first purpose of the invention is to provide a method for improving the heat stability of bovine serum albumin, which has simple and convenient operation, cheap and easily-obtained raw materials, and can effectively improve the heat stability of bovine serum albumin, so that the bovine serum albumin is not easy to denature in a high-temperature environment.

The second objective of the present invention is to provide a method for preparing a transparent protein colloid, which is simple in operation and mild in conditions, and can be used for quickly and efficiently preparing a transparent protein colloid from bovine serum albumin as a raw material.

The third purpose of the invention is to provide a transparent protein colloid prepared by the preparation method of the transparent protein colloid, the transparent protein colloid has the advantages of easily available raw materials, simple generation conditions, colorless and transparent property, and good application potential in the fields of food and the like.

The embodiment of the invention is realized by the following steps:

a method of improving the thermal stability of bovine serum albumin comprising:

mixing bovine serum albumin with a buffer solution to prepare a protein solution with the concentration of 1-10 mg/ml;

the protein solution is mixed with phytic acid.

A method of preparing a clear protein colloid comprising:

mixing bovine serum albumin with a buffer solution to prepare a protein solution with the concentration of 100-300 mg/ml;

mixing the protein solution with phytic acid, and heating to 80-100 ℃.

A transparent protein colloid is prepared by the preparation method of the transparent protein colloid.

The embodiment of the invention has the beneficial effects that:

the embodiment of the invention provides a method for improving the heat stability of bovine serum albumin, which is characterized in that bovine serum albumin is dissolved in a buffer solvent to prepare a protein solution with a certain concentration, and phytic acid is added to improve the stability of the bovine serum albumin. The method has simple and convenient operation and cheap and easily obtained raw materials, and the modified bovine serum albumin can stably exist at high temperature and basically cannot be precipitated due to protein denaturation.

The embodiment of the invention also provides a transparent protein colloid and a preparation method thereof, which also comprises the steps of dissolving bovine serum albumin in a buffer solvent to prepare a protein solution with a specific concentration, and adding phytic acid to form the colloid. The preparation method is simple to operate and mild in conditions, and the colorless and transparent protein colloid is obtained by taking bovine serum albumin as a raw material. The transparent protein colloid is colorless and transparent, so that the transparent protein colloid has great application potential in the fields of food and the like.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic diagram of a hyaline colloid provided in example 8 of the present invention;

FIG. 2 is a schematic diagram of protein behavior at different temperatures and different ratios according to an exemplary embodiment of the present invention;

FIG. 3 is a graph showing the protein solubility curves at different temperatures and different ratios according to the experimental examples of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following describes a method for improving the heat stability of bovine serum albumin, a transparent protein colloid, and a preparation method thereof according to embodiments of the present invention.

A method of improving the thermal stability of bovine serum albumin comprising:

s1, mixing bovine serum albumin with a buffer solution to prepare a protein solution with the concentration of 1-10 mg/ml;

s2, mixing the protein solution with phytic acid.

Bovine serum protein is the main component of bovine plasma protein, mainly affecting the functional properties of bovine plasma protein, the production of whey and casein in plasma or cheese, and is a well-known potential source of nutritional and functional proteins. Bovine serum albumin is denatured at the temperature of more than 70 ℃, turbidity occurs and precipitation begins, few reports are made on improving the heat stability of bovine serum albumin at present, only a small number of reports show that the used materials are complex, and the operation is not very convenient.

Based on the above, the invention provides a method for improving the heat stability of bovine serum albumin, which adopts phytic acid and a buffer solution to be matched for use, and has an obvious improvement effect on the heat stability of the bovine serum albumin solution within a certain concentration range (1-10 mg/ml). The method is simple to operate, and the used materials are simple and easy to obtain, so that the method has a good use value.

Optionally, the mass ratio of bovine serum albumin to phytic acid is 1:0.4 to 2. Through a great deal of creative work of the inventor, the mass ratio of the phytic acid to the bovine serum albumin is at least 0.4:1, and the phytic acid can show thermal stability at a high temperature of more than 70 ℃. And with the further increase of the dosage of the phytic acid, the improvement effect is also improved to a certain extent. Better results have been obtained when the mass ratio of phytic acid to bovine serum albumin is 1: 1. The increase of the dosage of the phytic acid is not obvious any more for improving the effect. Considering the improvement effect and the loss of phytic acid, the mass ratio of the bovine serum albumin to the phytic acid is defined as 1: preferably in the range of 0.4 to 2. Preferably, the mass ratio of the bovine serum albumin to the phytic acid is 1:0.4 to 1.4.

Furthermore, the concentration of the buffer solution adopted in the embodiment of the invention is 30-100 mM, and the pH of the buffer solution is 7.0-8.0. Specifically, the buffer solution includes at least one of Tris-HCl buffer, barbiturate sodium-hydrochloric acid buffer, and PBS buffer. Preferably, the buffer solution is Tris-HCl buffer. The type of the buffer solution can be selected according to the application environment of the bovine serum albumin. In the embodiment of the present invention, the desired effect of improving the thermal stability can be obtained only by ensuring the pH range of the buffer solution.

The embodiment of the invention also provides a preparation method of the transparent protein colloid, which is characterized by comprising the following steps:

s1, mixing bovine serum albumin with a buffer solution to prepare a protein solution with the concentration of 100-300 mg/ml;

s2, mixing the protein solution with phytic acid, and heating to 80-100 ℃.

As mentioned above, bovine serum albumin is a relatively small component of whey protein, which contributes to the improvement of the functional properties of whey protein, and effective utilization of plasma and whey protein is essential for improving the functional properties of bovine serum albumin. However, in the prior art, bovine serum albumin is often formed into opaque protein colloid through heat induction. In the case of proteins, one of the main uses is in food, and the opaque nature limits the range of applications of proteins in the food and other fields.

Based on this, the inventor develops a method for thermally inducing bovine serum albumin to generate transparent protein colloid by using phytic acid and buffer solution on the basis of improving the bovine serum albumin thermal stability by using phytic acid and buffer solution. Compared with the method for improving the bovine serum albumin heat stability, the method is more suitable for the protein solution with the protein concentration of 100-300 mg/ml when the transparent protein colloid is prepared, and the stable colloid is difficult to form when the protein concentration is too low or too high.

Further, a great deal of creative work of the inventor finds that, in order to form a stable transparent protein colloid, the mass ratio of bovine serum albumin to phytic acid is 1: 0.05 to 0.25. If the dosage of the phytic acid is too small, only opaque protein colloid is formed; if the phytic acid is used in an excessive amount, the colloid is difficult to exist stably, and finally turbidity and even precipitation occur.

Optionally, when preparing the transparent protein colloid, the concentration of the buffer solution is 30-100 mM, and the pH of the buffer solution is 7.2-7.6. The buffer solution comprises at least one of Tris-HCl buffer solution, barbital sodium-hydrochloric acid buffer solution and PBS buffer solution; preferably, the buffer solution is Tris-HCl buffer. Likewise, the kind of the buffer solution can be selected according to the application environment of the hyaline colloid. When the transparent protein colloid is prepared, the pH value range of the buffer solution is only required to be ensured.

Further, the transparent protein colloid can be formed only by heating the mixed protein solution and phytic acid, wherein the heating temperature is 80-100 ℃, and the heating time is 10-30 min. And cooling to room temperature after heating is finished, thus obtaining the stable transparent protein colloid. The heating temperature is not suitable to be too high, the heating time is not suitable to be too long, otherwise, the bovine serum albumin has the possibility of further deforming and precipitating, and thus, the transparent protein colloid is difficult to obtain. Meanwhile, if the heating temperature is too low and the heating time is too short, the bovine serum albumin cannot form a stable colloidal structure, and the bovine serum albumin returns to the solution form again after being cooled to room temperature.

The embodiment of the invention also provides a transparent protein colloid which is prepared by the preparation method of the transparent protein colloid. The transparent protein colloid has the advantages of easily obtained raw materials, simple generation conditions, colorless and transparent property, and good application potential in the fields of food and the like.

The features and properties of the present invention are described in further detail below with reference to examples.