Halophage mycelium protein and preparation method thereof

Abstract

The invention discloses a halophage mycelium protein and a preparation method thereof.The halophage mycelium protein is prepared from, by mass, 20 million / g of halophage mycelium, 10% of plant protein, 10% of animal protein, 30% of organic matter, 10 million / g of active enzyme, 30% of amino acid and 20% of total nitrogen. The plant protein, the animal protein and the organic matter are used as auxiliary nutrient sources of the halophiles, so that the halophiles can be stimulated to generate more proteins, and the added active enzyme can be used for cracking halophile cells, so that the digestibility of the proteins is improved, and the release of cell contents is promoted; the amino acid can supplement essential amino acid in the culture process of the halophage mycelia, the structural integrity of the protein is ensured, the total nitrogen content serves as a measurement standard, the quality and yield of the protein are conveniently and more accurately evaluated, and meanwhile it is ensured that infectious microbes do not affect normal growth of the halophage mycelia.

Description

Technical Field

[0001] The invention relates to the technical field of microbial products, in particular to a halophilic mycelium protein and a preparation method thereof. Background Art

[0002] Halophilic mycelial proteins refer to proteins produced by microorganisms that survive in high-salt environments. These microorganisms belong to a category of extreme environment microorganisms. Mycelium is a biological structure used for growth and structural support by some microorganisms, such as fungi and some bacteria. In halophilic bacteria, they form mycelium in high-salt environments to adapt to the environment. Halophilic mycelial proteins usually have special structures and functions that can help these microorganisms survive and reproduce in high-salt extreme environments. They can produce a variety of proteins that can help them maintain normal functions in cells under high osmotic pressure environments.

[0003] However, in the actual preparation process of halophilic mycelium protein, due to the lack of auxiliary nutrient sources in its components, more protein cannot be produced during the preparation process of halophilic mycelium protein. At the same time, the lack of total nitrogen content as a measurement standard makes it difficult to accurately evaluate the quality and yield of the protein. Summary of the invention

[0004] The present invention provides a halophilic mycelium protein and a preparation method thereof, which can effectively solve the problem raised in the above background technology that in the actual preparation process of the halophilic mycelium protein, due to the lack of auxiliary nutrient sources in its components, more protein cannot be produced during the preparation process of the halophilic mycelium protein, and the total nitrogen content is not used as a measurement standard, making it difficult to accurately evaluate the quality and yield of the protein.

[0005] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a halophilic mycelium protein, comprising the following components in parts by mass: 10-30 million / g of halophilic mycelium, 5-25% of plant protein, 8-12% of animal protein, 10-40% of organic matter, 5-14 million / g of active enzymes, 20-50% of amino acids and 10-35% of total nitrogen.

[0006] According to the above technical solution, the halophilic mycelium is selected from halophilic bacteria with strong salt tolerance, the plant protein, animal protein and organic matter are used as auxiliary nutrition sources for halophilic bacteria, and the active enzyme refers to an enzyme substance produced by beneficial microorganisms, which is used for the lysis of halophilic bacteria cells and promotes the release of cell contents;

[0007] The amino acids are used to supplement essential amino acids to ensure the structural integrity of the protein, and the total nitrogen content is the total amount of all forms of nitrogen in the mixture, which is used as a measure to evaluate the quality and yield of the protein.

[0008] A method for preparing halophilic mycelial protein comprises the following steps:

[0009] S1. Select and prepare raw materials;

[0010] S2. Cultivate halophilic bacteria by preparing culture medium;

[0011] S3, harvesting and processing the cultured mycelium;

[0012] S4, extracting and purifying the protein;

[0013] S5. Analyze the extracted halophilic mycelium protein products.

[0014] According to the above technical solution, in S1, the raw materials for preparing the halophilic mycelium protein include halophilic mycelium, plant protein, animal protein, organic matter, active enzymes, amino acids and total nitrogen;

[0015] Among them, halophilic mycelium is a halophilic bacterium with strong salt tolerance and high protein production efficiency. Plant protein, animal protein, organic matter and amino acids are basic nutrients that will stimulate halophilic mycelium to produce more protein.

[0016] Active enzymes are added to improve the properties of the subsequently extracted proteins and to increase the digestibility of the proteins. The total nitrogen content is regulated according to specific needs, so that the proportion of total nitrogen ensures that the miscellaneous bacteria do not affect the growth of halophilic mycelium.

[0017] After the raw materials for preparing the halophilic mycelium protein are determined, each raw material is weighed according to the prescribed mass parts for use.

[0018] According to the above technical solution, S2 specifically includes the configuration of the culture medium and the cultivation of halophilic bacteria;

[0019] In the process of selecting and configuring the culture medium, a suitable halophilic strain is selected according to the halophilic performance, and the culture medium is configured according to the culture conditions, including the nutrient composition and the salt concentration, so that the configured culture medium can support the growth of mycelium under high salinity conditions;

[0020] Secondly, a culture medium that can provide a high-salinity environment is selected, and the salinity of the culture medium is controlled at 2%-8%. The culture medium components include water, nutrient salts, sugars, plant protein, animal protein and organic matter, among which sugars are used as carbon sources, and plant protein, animal protein and organic matter are used as nutrient sources. The components of the culture medium are stirred to ensure that the culture medium is fully mixed.

[0021] According to the above technical scheme, in S2, during the cultivation process of halophilic bacteria, the halophilic bacteria species are first inoculated into the culture medium, and cultured under good ventilation conditions and suitable temperature conditions. The oxygen concentration of the culture medium culture environment is regulated by controlling the ventilation conditions, and the temperature condition of the culture medium culture is 30-37°C.

[0022] According to the above technical solution, in S3, after the halophilic bacteria reach a predetermined culture time in the culture medium and the halophilic bacteria grow to a certain density in the culture medium, the mycelium in the culture medium is harvested. When the mycelium is harvested, it is carried out by centrifugation. Specifically, the culture medium containing the growing mycelium is centrifuged and allowed to stand after centrifugation. The precipitate collected at this time is the mycelium.

[0023] According to the above technical solution, in S3, after the mycelium is harvested by centrifugation, it is necessary to further wash the mycelium and wash it multiple times with a buffer solution to remove residual substances in the culture medium mixed in the mycelium.

[0024] According to the above technical solution, in S4, after the mycelium is processed, protein is extracted and purified. The mycelium harvested in step S3 is first weighed, and then ground and crushed by a grinder. The mycelium is fully ground to break its cell wall and release the protein therein, thereby ensuring complete extraction of the protein.

[0025] After the mycelium is fully ground and crushed, an appropriate solvent is added to it to help release the protein from the cells and maintain its solubility. The protein is then separated from the cell debris by high-speed centrifugation, and the supernatant is collected. The centrifugation time is 20-30 minutes.

[0026] After obtaining the supernatant, the extracted protein needs to be further separated and purified, and one of the salting-out method, chromatography, and ultrafiltration technology is used for separation and purification, wherein the chromatography method includes ion exchange chromatography and gel filtration chromatography, and the halophilic mycelium protein is obtained after purification treatment. The purity of the halophilic mycelium protein can be ensured through multi-step purification treatment, and the impurities inside it can be effectively removed.

[0027] According to the above technical solution, S5 refers to analyzing the halophilic mycelium protein, using spectral analysis, electrophoresis technology and mass spectrometry technology to analyze and identify the separated protein, and verify its protein purity and biological activity;

[0028] After the halophilic mycelium protein is analyzed and identified, a preservative is added therein to prevent protein denaturation. The preservative is one of glycerol or polyethylene glycol. The prepared halophilic mycelium protein is subsequently freeze-dried during storage to maintain its stability.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. In the process of selecting halophilic mycelium, the present invention selects halophilic bacteria with strong salt tolerance and high protein production efficiency, and uses plant protein, animal protein and organic matter as auxiliary nutrient sources for halophilic bacteria, which can stimulate halophilic bacteria to produce more protein, and the added active enzyme can be used for the lysis of halophilic bacteria cells, improve the digestibility of protein, and promote the release of cell contents. Amino acids can supplement the essential amino acids of halophilic mycelium in the culture process to ensure the structural integrity of the protein, and the total nitrogen content is used as a measurement standard to facilitate a more accurate evaluation of the quality and yield of the protein, while ensuring that foreign bacteria do not affect the normal growth of halophilic mycelium. The halophilic mycelium protein prepared by the above-mentioned composition raw materials has higher stability, and is of great significance in improving the stability and functional activity of biotechnology products.

[0031] 2. Centrifugation is convenient for centrifuging the culture medium containing growing mycelium, which is convenient for effectively harvesting the mycelium in the culture medium, and the mycelium is washed with a salt-free or low-salt buffer to remove residual substances in the culture medium mixed with the mycelium, thereby ensuring that the harvested mycelium has a higher purity and preventing impurities from affecting the quality of subsequent protein extraction and purification;

[0032] By analyzing the extracted protein, it is convenient to detect the purity and activity of the protein and ensure the preparation quality of the halophilic mycelium protein. Then, freeze-drying treatment and the addition of preservatives can effectively ensure the stability of the halophilic mycelium protein during storage and prevent it from denaturing. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0034] In the attached picture:

[0035] Figure 1 It is a flow chart of the steps of the preparation method of the present invention. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0037] Example: Figure 1As shown, the present invention provides a technical solution, a halophilic mycelium protein, comprising the following components in parts by mass: 20 million / g halophilic mycelium, 10% plant protein, 10% animal protein, 30% organic matter, 10 million / g active enzyme, 30% amino acid and 20% total nitrogen.

[0038] Based on the above technical scheme, halophilic mycelium is selected from halophilic bacteria with strong salt tolerance, plant protein, animal protein and organic matter are used as auxiliary nutrition sources for halophilic bacteria, and active enzyme refers to enzyme substances produced by beneficial microorganisms, which are used for the lysis of halophilic bacteria cells and promote the release of cell contents;

[0039] Amino acids are used to supplement essential amino acids to ensure the structural integrity of proteins. Total nitrogen content is the total amount of all forms of nitrogen in the mixture. As a measurement standard, it is mainly used to evaluate the quality and yield of proteins.

[0040] A method for preparing halophilic mycelial protein comprises the following steps:

[0041] S1. Select and prepare raw materials;

[0042] S2. Cultivate halophilic bacteria by preparing culture medium;

[0043] S3, harvesting and processing the cultured mycelium;

[0044] S4, extracting and purifying the protein;

[0045] S5. Analyze the extracted halophilic mycelium protein products.

[0046] Based on the above technical scheme, in S1, the raw materials for preparing the halophilic mycelium protein mainly include halophilic mycelium, plant protein, animal protein, organic matter, active enzyme, amino acid and total nitrogen;

[0047] Among them, halophilic mycelium is a halophilic bacterium with strong salt tolerance and high protein production efficiency. Plant protein, animal protein, organic matter and amino acids are basic nutrients that will stimulate halophilic mycelium to produce more protein.

[0048] The addition of active enzymes is mainly used to improve the properties of the subsequently extracted protein and to improve the digestibility of the protein. The total nitrogen content is regulated according to specific needs, so that the proportion of total nitrogen ensures that the miscellaneous bacteria do not affect the growth of halophilic mycelium.

[0049] After the raw materials for preparing the halophilic mycelium protein are determined, each raw material is weighed according to the prescribed mass parts for use.

[0050] Based on the above technical solution, S2 specifically includes the configuration of the culture medium and the cultivation of halophilic bacteria;

[0051] In the process of selecting and configuring the culture medium, the appropriate halophilic strain is selected mainly based on the halophilic performance, and the culture medium is configured according to the culture conditions, which include the nutrient composition and salt concentration, so that the configured culture medium can support the growth of mycelium under high salinity conditions;

[0052] Secondly, a culture medium that can provide a high-salinity environment is selected, and the salinity of the culture medium is controlled to 5%. The main components of the culture medium include water, nutrient salts, sugars, plant protein, animal protein and organic matter, among which the nutrient salts are nitrates, sugars are used as carbon sources, and plant protein, animal protein and organic matter are used as nutrient sources. The main components of the culture medium are stirred to ensure that the culture medium is fully mixed.

[0053] Based on the above technical solution, in S2, during the cultivation of halophilic bacteria, the halophilic bacteria species are first inoculated into the culture medium and cultured under good ventilation conditions and suitable temperature conditions. The oxygen concentration of the culture medium culture environment is mainly regulated by controlling the ventilation conditions, and the temperature condition of the culture medium culture is 34°C.

[0054] Based on the above technical solution, in S3, the mycelium in the culture medium is harvested after the halophilic bacteria reaches a predetermined culture time in the culture medium and the halophilic bacteria grow to a certain density in the culture medium. When the mycelium is harvested, centrifugation is mainly used. Specifically, the culture medium containing the growing mycelium is centrifuged and allowed to stand after centrifugation. The precipitate collected at this time is the mycelium.

[0055] Based on the above technical solution, in S3, after the mycelium is harvested by centrifugation, the mycelium needs to be further cleaned and washed multiple times with a buffer solution to remove residual substances in the culture medium mixed in the mycelium, and the buffer solution is a low-salt solution.

[0056] Based on the above technical solution, in S4, after the mycelium is processed, protein is extracted and purified. The mycelium harvested in step S3 is first weighed, and then ground and crushed by a grinder. The mycelium is fully ground to break its cell wall and release the protein therein, thereby ensuring complete extraction of the protein.

[0057] After the mycelium is fully ground and crushed, an appropriate solvent is added to the mycelium as a pH adjustment solution to help release the protein from the cells and maintain its solubility. The protein is then separated from the cell debris by high-speed centrifugation, and the supernatant is collected. The centrifugation time is 25 minutes.

[0058] After obtaining the supernatant, the extracted protein needs to be further separated and purified, and chromatography is mainly used for separation and purification, wherein the chromatography includes gel filtration chromatography, and the halophilic mycelium protein is obtained after purification treatment. The purity of the halophilic mycelium protein can be ensured through multi-step purification treatment, and the impurities inside it can be effectively removed.

[0059] Based on the above technical scheme, S5 mainly refers to the analysis of halophageal mycelium proteins, mainly using spectral analysis, electrophoresis technology and mass spectrometry technology to analyze and identify the separated proteins, and verify their protein purity and biological activity;

[0060] After the halophilic mycelium protein is analyzed and identified, a preservative is added therein to prevent protein denaturation. The preservative is glycerol, and the prepared halophilic mycelium protein is subsequently freeze-dried during storage to maintain its stability.

[0061] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A halophilic mycelial protein, characterized in that: The invention comprises the following components in parts by mass: 10-30 million / gram of halophilic mycelium, 5-25% of plant protein, 8-12% of animal protein, 10-40% of organic matter, 5-14 million / gram of active enzyme, 20-50% of amino acid and 10-35% of total nitrogen.

2. The halophilic mycelial protein according to claim 1, characterized in that: The halophilic mycelium is made of halophilic bacteria with strong salt tolerance, the plant protein, animal protein and organic matter are used as auxiliary nutrition sources for the halophilic bacteria, and the active enzyme refers to an enzyme substance produced by beneficial microorganisms, which is used for the lysis of halophilic bacteria cells and promotes the release of cell contents; The amino acids are used to supplement essential amino acids to ensure the structural integrity of the protein, and the total nitrogen content is the total amount of all forms of nitrogen in the mixture, which is used as a measure to evaluate the quality and yield of the protein.

3. A method for preparing a halophilic mycelium protein, according to the method for preparing a halophilic mycelium protein according to any one of claims 1-2, characterized in that: The specific preparation steps include: S1. Select and prepare raw materials; S2. Cultivate halophilic bacteria by preparing culture medium; S3, harvesting and processing the cultured mycelium; S4, extracting and purifying the protein; S5. Analyze the extracted halophilic mycelium protein products.

4. The method for preparing a halophilic mycelial protein according to claim 3, characterized in that: In S1, the raw materials for preparing the halophilic mycelium protein include halophilic mycelium, plant protein, animal protein, organic matter, active enzymes, amino acids and total nitrogen; Among them, halophilic mycelium is a halophilic bacterium with strong salt tolerance and high protein production efficiency. Plant protein, animal protein, organic matter and amino acids are basic nutrients that will stimulate halophilic mycelium to produce more protein. Active enzymes are added to improve the properties of the subsequently extracted proteins and to increase the digestibility of the proteins. The total nitrogen content is regulated according to specific needs, so that the proportion of total nitrogen ensures that the miscellaneous bacteria do not affect the growth of halophilic mycelium. After the raw materials for preparing the halophilic mycelium protein are determined, each raw material is weighed according to the prescribed mass parts for use.

5. The method for preparing a halophilic mycelial protein according to claim 3, characterized in that: Said S2 specifically includes the configuration of the culture medium and the cultivation of halophilic bacteria; In the process of selecting and configuring the culture medium, a suitable halophilic strain is selected according to the halophilic performance, and the culture medium is configured according to the culture conditions, including the nutrient composition and the salt concentration, so that the configured culture medium can support the growth of mycelium under high salinity conditions; Secondly, a culture medium that can provide a high-salinity environment is selected, and the salinity of the culture medium is controlled at 2%-8%. The culture medium components include water, nutrient salts, sugars, plant protein, animal protein and organic matter, among which sugars are used as carbon sources, and plant protein, animal protein and organic matter are used as nutrient sources. The components of the culture medium are stirred to ensure that the culture medium is fully mixed.

6. The method for preparing a halophilic mycelial protein according to claim 5, characterized in that: In S2, during the culture process of halophilic bacteria, the halophilic bacteria are first inoculated into the culture medium and cultured under ventilation conditions and suitable temperature conditions. The oxygen concentration of the culture medium environment is regulated by controlling the ventilation conditions, and the temperature condition of the culture medium is 30-37°C.

7. The method for preparing a halophilic mycelial protein according to claim 3, characterized in that: In the S3, after the halophilic bacteria have reached a predetermined culture time in the culture medium and the halophilic bacteria have grown to a certain density in the culture medium, the mycelium in the culture medium is harvested. When the mycelium is harvested, it is carried out by centrifugation. Specifically, the culture medium containing the growing mycelium is centrifuged and allowed to stand after centrifugation. The precipitate collected at this time is the mycelium.

8. The method for preparing a halophilic mycelial protein according to claim 7, characterized in that: In S3, after the mycelia are harvested by centrifugation, they need to be further cleaned by washing with a buffer solution for multiple times to remove residual substances in the culture medium mixed in the mycelia.

9. The method for preparing a halophilic mycelial protein according to claim 7, characterized in that: In S4, after the mycelium is processed, protein is extracted and purified. The mycelium harvested in step S3 is first weighed, and then ground and crushed by a grinder. The mycelium cell walls are broken by fully grinding the mycelium to release the protein therein, thereby ensuring complete extraction of the protein. After the mycelium is fully ground and crushed, an appropriate solvent is added to it to help release the protein from the cells and maintain its solubility. The protein is then separated from the cell debris by high-speed centrifugation, and the supernatant is collected. The centrifugation time is 20-30 minutes. After obtaining the supernatant, the extracted protein needs to be further separated and purified, and one of the salting-out method, chromatography, and ultrafiltration technology is used for separation and purification, wherein the chromatography method includes ion exchange chromatography and gel filtration chromatography, and the halophilic mycelium protein is obtained after purification treatment, and the purity of the halophilic mycelium protein can be ensured through multi-step purification treatment.

10. The method for preparing halophilic mycelial protein according to claim 3, characterized in that: In said S5, it refers to analyzing the halophilic mycelium protein, using spectral analysis, electrophoresis technology and mass spectrometry technology to analyze and identify the separated protein, and verify its protein purity and biological activity; After the halophilic mycelium protein is analyzed and identified, a preservative is added therein to prevent protein denaturation. The preservative is one of glycerol or polyethylene glycol, and the prepared halophilic mycelium protein is subsequently freeze-dried during storage.

Images