Soluble microbial protein and preparation method thereof

Abstract

The invention belongs to the technical field of microbial protein separation and extraction, and particularly relates to soluble microbial protein and a preparation method thereof. The method comprises the following steps: fermenting and culturing microbial thalli to a logarithmic phase, and centrifuging to obtain mycelia; the mycelium is freeze-dried and then smashed, and mycelium powder is obtained; the mycelium powder is placed in water to be dispersed and homogenized, and homogenized homogenate is obtained; finally, alkaline protease is added into the homogenate for enzymolysis and enzyme deactivation, and the soluble microbial protein is obtained. On one hand, microbial cell walls are directly destroyed through mechanical shearing force and collision action generated by high-speed rotation of the wall breaking machine, and intracellular proteins and active ingredients are released; on the other hand, high-pressure homogenization is combined, mechanical shear force and cavitation effect are utilized to destroy a protein compact structure and expose restriction enzyme cutting sites, and alkaline protease is combined for directional hydrolysis, so that the enzymolysis time is greatly shortened, the efficiency is improved, and the extraction rate is greatly improved.

Description

Technical Field

[0001] The present application belongs to the technical field of separation and extraction of microbial proteins, and specifically relates to a soluble microbial protein and a preparation method thereof. Background Art

[0002] Protein is an essential nutrient for the human body. Traditional plant and animal proteins face bottlenecks such as resource shortages and high carbon emissions in production, necessitating the urgent need for sustainable alternatives. Microbial protein, as an alternative protein source, contains all essential amino acids, complements plant proteins, and approaches the nutritional value of animal proteins. Mycelium (e.g., edible fungi) is an important source of microbial protein, offering advantages such as high protein (10-63% dry weight), high fiber content, low fat, and no cholesterol. However, protein extraction is limited by dense cell walls and inefficient enzymatic hydrolysis processes. Traditional methods rely on physical disruption or chemical treatments, which can easily destroy heat-sensitive nutrients, produce residual reagents, and result in low extraction yields. Conventional mechanical disruption methods (e.g., ball milling and sonication) incompletely disrupt dense protein structures (e.g., plant cell walls and microbial membranes), resulting in low release rates of target proteins (typically <60%) and high energy consumption. Traditional enzymatic hydrolysis technologies rely on single or broad-spectrum enzymes, lack precise recognition of target proteins, and are prone to forming insoluble aggregates and poor solubility. Summary of the Invention

[0003] The purpose of the present invention is to address the deficiencies of the prior art and provide a soluble microbial protein and a method for preparing the same, specifically adopting the following technical solutions: In a first aspect, the present invention provides a method for preparing soluble microbial protein, comprising the following steps: S1. Fermenting and culturing the microbial cells to the logarithmic growth phase, and centrifuging to obtain mycelium; S2. freeze-drying the mycelium, and then crushing the mycelium to obtain mycelium powder; S3, dispersing the mycelium powder in water, and then homogenizing it under a pressure of 70 MPa to 90 MPa to obtain a homogenous slurry; S4. Adding alkaline protease to the homogenized slurry for enzymatic hydrolysis and inactivating the enzyme to obtain the soluble microbial protein.

[0004] As a further preferred embodiment, the microbial cells include at least one of Fusarium brevicaulis, Fusarium graminearum, Fusarium oxysporum, and Fusarium venezuelae.

[0005] As a further preferred embodiment, the pulverization time is 1-4 minutes, and the pulverized particle size is 20-50 mesh. In the above preparation method, the smaller the particle size, the larger the specific surface area. Small particles are more easily broken by shear forces during high-pressure homogenization, and the collision frequency between enzyme molecules and substrate binding sites is higher, which is more conducive to subsequent operations.

[0006] As a further preferred embodiment, the ratio of the mycelium powder to water in S3 is 1:20 to 1:30.

[0007] As a further preferred embodiment, the rotation speed during the dispersion is 8000 rpm / min to 10000 rpm / min.

[0008] As a further preferred embodiment, the homogenization treatment time is 20 min to 30 min.

[0009] As a further preferred embodiment, the concentration of the alkaline protease is 2% to 4%. In the above preparation process, an alkaline protease concentration higher than 4% tends to produce bitter peptides <500 Da, while an alkaline protease concentration lower than 2% produces large fragments >3000 Da. Therefore, both too high and too low concentrations are not conducive to subsequent operations.

[0010] As a further preferred embodiment, the parameters during the enzymatic hydrolysis are as follows: The pH is 7.0-7.5; the temperature is 45-50℃; and the time is 2 h-3 h.

[0011] As a further preferred embodiment, the specific process of enzyme inactivation is as follows: After adding alkaline protease to the homogenate for enzymatic hydrolysis, the homogenate is placed in a 100° C. water bath for 10 minutes to inactivate the enzyme, and centrifuged for 10 minutes to obtain the soluble microbial protein.

[0012] In a second aspect, the present invention provides a soluble microbial protein prepared by the above-mentioned preparation method.

[0013] The beneficial effects of the present invention are: On the one hand, the present invention directly destroys the microbial cell wall through the mechanical shear force and collision effect generated by the high-speed rotation of the wall breaking machine, releasing intracellular proteins and active ingredients; on the other hand, combined with high-pressure homogenization, the mechanical shear force and cavitation effect are used to destroy the dense structure of the protein, exposing the enzyme cleavage site, and combined with the directional hydrolysis of alkaline protease, the enzymatic hydrolysis time is greatly shortened and the efficiency is improved, and the extraction rate is greatly improved; this synergistic effect overcomes the problem of insufficient contact of the enzymatic hydrolysis site caused by protein structure shielding in the traditional enzymatic hydrolysis process, thereby improving the extraction rate of soluble protein, and no additional chemical reagents are required, avoiding the burden of subsequent separation and purification, providing a solution for efficient and low-cost extraction of highly soluble microbial protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 Shown is a graph showing the content of soluble microbial protein prepared in Examples 1 to 4 and Comparative Examples 1 to 5. DETAILED DESCRIPTION

[0016] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0017] Example 1 A method for preparing soluble microbial protein comprises the following steps: (1) Ferment the Fusarium graminearum until the logarithmic growth phase, collect the mycelium by centrifugation, and wash it with PBS three times; (2) freeze-drying the mycelial liquid obtained in step (1) and crushing it with a wall-breaking machine for 2 minutes to a particle size of 20-50 mesh; (3) The mycelium powder obtained in step (2) was dispersed in water with a solid-liquid ratio of 1:20. After mixing evenly, the mixture was dispersed in a disperser at a speed of 10,000 rpm / min for 3 minutes. (4) The dispersed system obtained in step (3) was homogenized at a homogenization pressure of 90 MPa and a homogenization time of 30 min; (5) Add 4% of the mass of mycelium alkaline protease (purchased from Yuanye Company, the activity of alkaline protease is 200 U / mg) to the homogenate obtained in step (4) for enzymatic hydrolysis. Use sodium bicarbonate solution to adjust the pH of the homogenate to 7.0, adjust the temperature to 50°C, and hydrolyze for 3 h under stirring. After the enzymatic hydrolysis is completed, heat in a water bath at 100°C for 10 min to inactivate the enzyme, and centrifuge for 10 min to obtain soluble microbial protein.

[0018] Example 2 A method for preparing soluble microbial protein comprises the following steps: (1) Ferment the Fusarium graminearum until the logarithmic growth phase, collect the mycelium by centrifugation, and wash it with PBS three times; (2) freeze-drying the mycelial liquid obtained in step (1) and crushing it with a wall-breaking machine for 2 minutes to a particle size of 20-50 mesh; (3) The mycelium powder obtained in step (2) was dispersed in water with a solid-liquid ratio of 1:20. After mixing evenly, the mixture was dispersed in a disperser at a speed of 10,000 rpm / min for 3 minutes. (4) The dispersed system obtained in step (3) was homogenized at a homogenization pressure of 70 MPa and a homogenization time of 30 min; (5) Add 3% of the mass of mycelium to the homogenate obtained in step (4) with alkaline protease (purchased from Yuanye Company, with an activity of 200 U / mg) for enzymatic hydrolysis. After adjusting the pH of the homogenate to 7.5, adjust the temperature to 45°C and perform enzymatic hydrolysis for 3 h under stirring. After the enzymatic hydrolysis is completed, heat in a water bath at 100°C for 10 min to inactivate the enzyme, and centrifuge for 10 min to obtain soluble microbial protein.

[0019] Example 3 A method for preparing soluble microbial protein comprises the following steps: (1) Ferment the Fusarium graminearum until the logarithmic growth phase, collect the mycelium by centrifugation, and wash with PBS 2-3 times; (2) freeze-drying the mycelial liquid obtained in step (1) and crushing it with a wall-breaking machine for 2 minutes to a particle size of 20-50 mesh; (3) Add water to the mycelium powder obtained in step (2) to disperse it at a solid-liquid ratio of 1:30. After mixing evenly, use a disperser to disperse it for 3 minutes at a disperser speed of 10,000 rpm / min. (4) The dispersed system obtained in step (3) was homogenized at a homogenization pressure of 80 MPa and a homogenization time of 30 min; (5) Add 2% of the mass of mycelium to the homogenate obtained in step (4) with alkaline protease (purchased from Yuanye Company, with an activity of 200 U / mg) for enzymatic hydrolysis. After adjusting the pH of the homogenate to 7.5 with sodium bicarbonate solution, adjust the temperature to 50°C and perform enzymatic hydrolysis for 3 h under stirring. After the enzymatic hydrolysis is completed, heat in a water bath at 100°C for 10 min to inactivate the enzyme, and centrifuge for 10 min to obtain soluble microbial protein.

[0020] Example 4 A method for preparing soluble microbial protein comprises the following steps: (1) Ferment the Fusarium graminearum until the logarithmic growth phase, collect the mycelium by centrifugation, and wash with PBS 2-3 times; (2) freeze-drying the mycelial liquid obtained in step (1) and crushing it with a wall-breaking machine for 2 minutes to a particle size of 20-50 mesh; (3) The mycelium powder obtained in step (2) was dispersed in water with a solid-liquid ratio of 1:20. After mixing evenly, the mixture was dispersed in a disperser at a speed of 10,000 rpm / min for 3 minutes. (4) The dispersed system obtained in step (3) was homogenized at a homogenization pressure of 90 MPa and a homogenization time of 20 min; (5) Add 3% of the mass of mycelium to the homogenate obtained in step (4) with alkaline protease (purchased from Yuanye Company, with an activity of 200 U / mg) for enzymatic hydrolysis. After adjusting the pH of the homogenate to 7.5 with sodium bicarbonate solution, adjust the temperature to 45°C and perform enzymatic hydrolysis for 3 h under stirring. After the enzymatic hydrolysis is completed, heat in a water bath at 100°C for 10 min to inactivate the enzyme, centrifuge for 10 min, and obtain soluble microbial protein. The crushed particle size is 20-50 mesh.

[0021] Comparative Example 1 A method for preparing soluble microbial protein is similar to that of Example 1, except that step (4) of homogenization is omitted and 4% alkaline protease is directly added to proceed to step (5).

[0022] Comparative Example 2 A method for preparing soluble microbial protein is similar to that of Example 1, except that alkaline protease is not added in step (5). The pH of the slurry is adjusted to 7.0 using sodium bicarbonate solution, the temperature is adjusted to 50°C, and enzymatic hydrolysis is carried out under stirring for 3 h. After the enzymatic hydrolysis is completed, the enzyme is inactivated by heating in a water bath at 100°C for 10 min, and the soluble microbial protein is obtained by centrifugation for 10 min.

[0023] Comparative Example 3 A method for preparing soluble microbial protein is similar to that of Example 1, except that the 4% alkaline protease added in step (5) is replaced by 1% alkaline protease.

[0024] Comparative Example 4 A method for preparing soluble microbial protein is similar to that of Example 1, except that the homogenization pressure in step (4) is replaced by 20 MPa from 90 MPa.

[0025] Comparative Example 5 A method for preparing soluble microbial protein, the preparation method is similar to that of Example 1, the only difference being that in step (2), the pulverization by a wall-breaking machine for 2 minutes is not performed, and step (3) is performed directly after freeze-drying.

[0026] Example 5 The extraction rate of the soluble microbial protein prepared in Examples 1 to 4 and Comparative Examples 1 to 5 was determined by the following method: The protein extraction rate was measured by Coomassie Brilliant Blue G-250. The results were as follows Figure 1 As shown. Figure 1 It can be seen that the soluble microbial protein extracted by the method described in the present invention has a high extraction rate. As shown in Example 1, Comparative Example 1 and Comparative Example 4, high-pressure homogenization pretreatment exposes enzyme cleavage sites, combined with alkaline protease directional hydrolysis, greatly shortens the enzymatic hydrolysis time and improves efficiency, and the soluble microbial protein extraction rate can reach more than 20%; As shown in Example 1, Comparative Example 2 and Comparative Example 3, adding an appropriate amount of alkaline protease can significantly enhance the extraction rate of soluble microbial protein. In addition, in combination with Example 1 and Comparative Example 5, it can be seen that the wall breaking machine can directly destroy the microbial cell wall for 2 minutes, significantly improving the subsequent enzymatic hydrolysis or extraction efficiency. In contrast, the extraction rate of the soluble microbial protein prepared in Examples 1-4 is higher, among which the extraction rate of the soluble microbial protein prepared in Example 1 is the highest, which shows that the additive ratio and processing conditions described in this patent are scientific and appropriate.

[0027] The embodiments of the present application are described above in conjunction with the accompanying drawings. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the core idea of the present application, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present application and the claims, all of which are within the protection of the present application.

Claims

1. A method for preparing soluble microbial protein, characterized in that: The following steps are involved: S1. Fermenting and culturing the microbial cells to the logarithmic growth phase, and centrifuging to obtain mycelium; S2. freeze-drying the mycelium, and then crushing the mycelium to obtain mycelium powder; S3, dispersing the mycelium powder in water, and then homogenizing it under a pressure of 70 MPa to 90 MPa to obtain a homogenous slurry; S4. Adding alkaline protease to the homogenized slurry for enzymatic hydrolysis and inactivating the enzyme to obtain the soluble microbial protein.

2. The preparation method according to claim 1, characterized in that The microbial cells include at least one of Fusarium brevicaulis, Fusarium graminearum, Fusarium oxysporum, and Fusarium venezuelae.

3. The preparation method according to claim 1, characterized in that The pulverization time is 1 min-4 min, and the pulverized particle size is 20-50 meshes.

4. The preparation method according to claim 1, characterized in that The ratio of the mycelium powder to water in S3 is 1:20 to 1:

30.

5. The preparation method according to claim 1, characterized in that The rotation speed during the dispersion is 8000 rpm / min to 10000 rpm / min.

6. The preparation method according to claim 1, characterized in that The homogenization time is 20 min to 30 min.

7. The preparation method according to claim 1, characterized in that The concentration of the alkaline protease is 2% to 4%.

8. The preparation method according to claim 1, characterized in that The parameters during the enzymatic hydrolysis are as follows: The pH is 7.0-7.5; the temperature is 45-50℃; and the time is 2 h-3 h.

9. The preparation method according to claim 1, characterized in that The specific process of enzyme inactivation is as follows: After adding alkaline protease to the homogenate for enzymatic hydrolysis, the homogenate is placed in a water bath at 80-100° C. for 5-10 minutes to inactivate the enzyme, and centrifuged for 10 minutes to obtain the soluble microbial protein.

10. A soluble microbial protein, characterized in that The invention is prepared by the preparation method according to any one of claims 1 to 9.

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