A method for improving the stability and solubility of fish myofibrillar proteins

Abstract

The application provides a method for improving the stability and solubility of fish myofibrillar protein, and belongs to the technical field of aquatic protein processing. The method comprises the following steps: mixing fish myofibrillar protein solution with enzymes and carrying out enzymolysis; the enzymes include one or more of glutamine enzyme, protein glutamine enzyme, chymotrypsin and papain. The method provided by the application improves the stability and low-salt solubility of fish myofibrillar protein.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquatic protein processing, and particularly relates to a method for improving the stability and solubility of fish myofibrillar protein. BACKGROUND

[0002] Tilapia is an important freshwater fish in the global aquaculture industry, with a short growth cycle and low breeding cost, providing about 15% of animal protein for 4.3 billion people and being one of the main sources of animal protein in the future. At present, tilapia is the third largest freshwater fish in China, with a yield of 165.5 million tons in 2020, ranking first in the world. Guangdong Province has a rich yield of tilapia. At the present stage, the tilapia industry is facing problems such as low processing technology (single product form, mainly fresh live fish, frozen whole fish, frozen fish fillets, and surimi gel products), few high-value-added products, the nutritional mechanism of protein not being fully utilized, increasing breeding and processing costs, decreasing profits, and insufficient development of the domestic market. Therefore, it is urgent to expand the processing application of tilapia and improve the economic value-added and quality of fish protein products.

[0003] With the development of production technology and the improvement of economic level, consumers pay more and more attention to the nutritional and health problems caused by the intake of high-sodium salt and fat. Under this background, the development of new low-sodium fluid food based on the structure and functional properties of fish myofibrillar protein can meet the needs of more people, fill the gap of fish myofibrillar protein in protein beverages or liquid food, and meet the needs of special groups (such as the elderly or people with difficulty chewing) in high-quality low-allergic meat protein, which has good application prospects and important significance, and conforms to the future development trend.

[0004] Due to the low salt insolubility of tilapia myofibrillar protein (TMP), its application range in production is greatly limited, so it is necessary to modify the myofibrillar protein to improve its functional properties and broaden its application range and increase the processing added value. Domestic and foreign scholars have tried a series of chemical, physical and enzymatic methods including glycosylation, ultrasonic, heating, ultra-high pressure and protease enzymolysis to improve the functional properties of fish myofibrillar protein, but the above methods have some problems, such as poor protein stability and solubility after treatment. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a method for improving the stability and low-sodium solubility of fish myofibrillar protein, which improves the stability and solubility of fish myofibrillar protein.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] The application provides a method for improving the stability and solubility of fish myofibrillar protein, comprising: mixing fish myofibrillar protein solution with enzymes, and enzymolysis.

[0008] The enzymes comprise one or more of glutaminase, protein glutaminase, chymotrypsin and papain.

[0009] Preferably, the mass concentration of fish myofibrillar protein in the fish myofibrillar protein solution is 2%.

[0010] Preferably, the enzyme addition amount is 100 U per gram of fish myofibrillar protein.

[0011] Preferably, the enzymolysis conditions comprise: the enzymolysis time is 3-24 h, the enzymolysis pH value is 7.0-9.0, and the enzymolysis temperature is 37-50 DEG C.

[0012] Preferably, the source of the fish myofibrillar protein comprises freshwater fish or seawater fish.

[0013] Preferably, the freshwater fish comprises tilapia.

[0014] The application utilizes glutaminase to catalyze the deamidation reaction of fish myofibrillar protein to form protein with high solubility and high stability. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Hydrolysis degree and deamidation degree of TMP after treatment with different enzymes;

[0016] Figure 2 Changes of solubility and deamidation degree of TMP after treatment with glutaminase for different time;

[0017] Figure 3 Appearance diagram of TMP after centrifugation after treatment with glutaminase for different time;

[0018] Figure 4 SDS-PAGE band changes of TMP after treatment with glutaminase for different time;

[0019] Figure 5 Potential changes of TMP after treatment with glutaminase for different time; DETAILED DESCRIPTION

[0020] The application provides a method for improving the stability and solubility of fish myofibrillar protein, comprising: mixing fish myofibrillar protein solution with enzymes, and enzymolysis; the enzymes comprise glutaminase, protein glutaminase, chymotrypsin and papain.

[0021] In this invention, the preferred mass concentration of fish myofibrillar protein in the fish myofibrillar protein solution is 2%. The preferred amount of fish myofibrillar protein added per gram is 100 U. The preferred conditions for enzymatic hydrolysis in this invention include: a hydrolysis time of 0–24 h, a hydrolysis pH of 7.0–9.0, and a hydrolysis temperature of 50 °C.

[0022] In this invention, the source of the fish myofibrillar protein preferably includes freshwater fish or saltwater fish. In this invention, the freshwater fish preferably includes tilapia.

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] The extracted tilapia myofibrillar protein was dispersed in ultrapure water (4 samples) to a mass concentration of 2%. The pH was adjusted to 8.0, and 100 U / g (based on protein) of glutaminase, protein glutaminase, chymotrypsin, and papain were added respectively. After treatment at 50°C for 9 hours, the pH was adjusted to 7.0, and the degree of hydrolysis was determined by formaldehyde titration. The degree of deamidation was determined and calculated using an ammonia assay kit. Figure 1 As shown, TMP exhibited low degrees of hydrolysis after treatment with the four enzymes: 1.13% (glutaminase), 0.95% (protein glutaminase), 1.33% (chymotrypsin), and 1.50% (papain). TMP also showed high degrees of deamidation after enzyme treatment: 6.49% (glutaminase), 4.77% (protein glutaminase), 7.19% (chymotrypsin), and 7.06% (papain). This indicates that under these conditions, the enzyme modification of TMP mainly involves the decomposition of the side-chain amide groups, rather than the cleavage of protein peptide bonds. Based on this, the low-salt solubility of myofibrillar proteins can be initially increased.

[0026] Example 2

[0027] The extracted tilapia myofibrillar protein was dispersed in ultrapure water to a concentration of 2%, the pH was adjusted to 8.0, 100 U / g glutaminase was added, and the mixture was treated at 50°C for different times. The pH was then adjusted to 7.0, and the solubility and deamidation degree of TMP at different treatment times were measured. Figure 2As shown, the deamidation of untreated TMP was only 0.37%, and after glutaminase treatment, the deamidation degree showed a trend of first increasing and then tending to be stable with the extension of treatment time (0h: 0.81%, 3h: 2.85%, 6h: 5.21%, 9h: 6.85%, 24h: 6.64%). The solubility of TMP before and after treatment showed significant difference when dissolved in 0.05M sodium chloride solution. The solubility of untreated TMP was low, only 19.21%. After glutaminase deamidation treatment, the solubility of TMP increased significantly, and the solubility increased with the extension of treatment time (0h: 20.12%, 3h: 31.91%, 6h: 42.13%, 9h: 54.52%, 24h: 64.83%). The above results showed that the low salt solubility of TMP could be significantly improved after enzyme treatment.

[0028] Example 3

[0029] The extracted tilapia muscle fiber protein was dispersed in ultrapure water to make its mass concentration 2%, the pH was adjusted to 7.0, 100 U / g glutaminase was added, and after treatment at 50°C for different time, the pH was adjusted to 7.0, and then the centrifugal treatment (3000xg, 10min) was carried out, and the appearance state was observed. It can be seen from Figure 3 that the solubility of the original TMP is low, and the solubility of TMP gradually increases with the extension of treatment time, and when the treatment time is 9h, the solution shows a uniform dispersion state, indicating that glutaminase treatment significantly improves the solubility and dispersibility of TMP, and the solution finally shows a stable state.

[0030] Example 4

[0031] The deamidated muscle fiber protein (deamidated TMP) obtained in Example 2 was mixed with SDS-PAGE loading buffer (5x) at a ratio of 3:1 (v / v), the mixture was boiled in boiling water bath for 5min, 10μg of the mixed system was taken and loaded on 12% Bis-Tris precast gel for analysis, electrophoresis voltage was 120V, electrophoresis time was about 1h, and then staining, decoloring and scanning of protein bands were carried out for analysis. The SDS-PAGE reduced electrophoretogram revealed the basic subunit composition of deamidated TMP. According to Figure 4 it can be seen that the characteristic bands of TMP, MHC (myosin heavy chain), Actin (actin) and MLC (myosin light chain) did not change significantly before and after glutaminase treatment, and glutaminase treatment did not significantly change the shape of SDS-PAGE band, which indicated that glutaminase treatment did not change the primary structure and subunit composition of TMP, and there was no large number of peptide bond breakage.

[0032] Example 5

[0033] The extracted tilapia myofibrillar protein was dispersed in ultrapure water to a concentration of 2%, the pH was adjusted to 9.0, 100 U / g glutaminase was added, and the mixture was treated at 50°C for different times. The pH was then adjusted to 7.0, and the potential of the treated samples was measured using a Malvern laser particle size analyzer. Figure 5 The untreated TMP exhibited a relatively low net negative charge (-18.81 mV). With prolonged treatment time, the charge of TMP showed an increasing trend (0h: -21.83 mV, 3h: -34.12 mV, 6h: -35.25 mV, 9h: -35.01 mV, 24h: 41.72 mV), and the negative charge increased significantly. Deamidation resulted in a higher charge density in proteins deamidinated by glutaminase (through the generation of more COO- groups in the protein molecule), and the charge of myofibrillar proteins after enzyme treatment was all above -30 mV. This indicates that glutaminase deamidation enhances electrostatic repulsion and inhibits the assembly of filamentous myosin by resisting electrostatic interactions, thereby promoting protein-water interactions and improving the solubility and stability of TMP.

[0034] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for improving the stability and low-salt solubility of fish myofibrillar protein, characterized in that, include: Fish myofibrillar protein solution was mixed with enzymes and then enzymatically hydrolyzed. The enzyme is glutaminase; The source of the fish myofibril protein is tilapia.

2. The method according to claim 1, characterized in that, The fish myofibrillar protein solution has a mass concentration of 2%.

3. The method according to claim 1, characterized in that, The amount of enzyme added per gram of fish myofibril protein is 100U.

4. The method according to claim 1, characterized in that, The conditions for enzymatic hydrolysis include: a hydrolysis time of 3 to 24 hours, a hydrolysis pH of 7.0 to 9.0, and a hydrolysis temperature of 37°C to 50°C.

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