Post-treatment method of single-cell protein fermentation liquor and fermentation method of single-cell protein thalli

Abstract

The invention provides a post-treatment method of single-cell protein fermentation liquor and a fermentation method of single-cell protein thalli. Nuclease and cell wall lytic enzyme are respectively added into fermentation liquor containing single-cell protein thalli to treat the thalli, so that nucleic acid and cell wall residues can be reduced, and the problems that in a traditional fermentation process, the application of the thalli in food is limited due to the high nucleic acid content of the thalli, and the protein extraction efficiency is reduced due to the existence of the cell walls are solved. In addition, by optimizing fermentation conditions, the protein content of the single-cell protein is remarkably increased. The process is simple, low in cost, suitable for industrial production and capable of being widely applied to the fields of feed, food and medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and specifically relates to a post-treatment method for a single-cell protein (SCP) fermentation broth and a fermentation method for single-cell protein bacteria. Background Art

[0002] Traditional protein production methods such as cereal and bean cultivation or dairy farming take months and cannot meet the needs of future human life in terms of quantity, quality, and sustainable supply. There is an urgent need for innovation and development in large-scale, low-cost, sustainable, and high-quality protein production methods.

[0003] Single-cell protein (SCP) is an important protein source and is widely used in fields such as feed, food, and medicine. Candida utilis has become an ideal strain for producing SCP due to its high protein content, rapid growth, and wide substrate utilization ability. The carbon dioxide emissions of an equal amount of yeast protein are approximately 1 / 20 of that of animal protein, the water consumption is only 1 / 200 of animal protein and 1 / 10 of plant protein, and the floor area is also much less than that of animal protein and plant protein, approximately 1 / 100 of animal protein and 1 / 10 of plant protein. Therefore, yeast protein can effectively reduce the dependence on land and water resources and reduce greenhouse gas emissions, having significant environmental protection advantages and being an ideal sustainable protein source.

[0004] However, in traditional fermentation processes, the nucleic acid content in the bacteria is relatively high, which limits its application in food. In addition, the presence of the cell wall also reduces the protein extraction efficiency. Therefore, developing a process for efficiently removing nucleic acids and cell walls is of great significance for improving the quality of single-cell protein. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a post-treatment method for a single-cell protein fermentation broth. In the fermentation broth containing single-cell protein bacteria, nuclease and cell wall-lysing enzyme are respectively added to treat the bacteria, which can reduce the nucleic acid and cell wall residues, and solve the problems in the traditional fermentation process that the high nucleic acid content in the bacteria limits its application in food and the presence of the cell wall reduces the protein extraction efficiency.

[0006] The purpose of the present invention is also to provide a fermentation method for single-cell protein bacteria. By optimizing the fermentation conditions, the protein content of single-cell protein can be significantly increased. Further combining the above post-treatment method and adding nuclease and cell wall-lysing enzyme to its fermentation broth to reduce the nucleic acid and cell wall residues, it is possible to obtain high-purity bacteria rich in single-cell protein efficiently, energy-savingly, and environmentally friendly, and improve the protein content of single-cell protein and its application value in fields such as feed and food.

[0007] To achieve the above object of the invention, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a post-treatment method for a single-cell protein fermentation broth is provided. The method includes adding a cell wall-lysing enzyme to the fermentation broth containing single-cell protein cells to react and hydrolyze the cell wall, and adding a nuclease to react and remove nucleic acids.

[0009] Specifically, a post-treatment method for a single-cell protein fermentation broth includes the following steps:

[0010] 1) Adding a cell wall-lysing enzyme to the fermentation broth containing single-cell protein cells, reacting to hydrolyze the cell wall, and then removing the solution through plate-and-frame filtration to form a filter cake;

[0011] 2) Dissolving the filter cake with water to form a mixture, adding a nuclease and an auxiliary agent thereto, reacting to remove nucleic acids, and then removing the supernatant through centrifugation to obtain a protein concentrate;

[0012] 3) Spray-drying the protein concentrate to obtain single-cell protein cell powder.

[0013] In one embodiment, for the fermentation broth containing single-cell protein cells in step 1), the present invention does not have special requirements for the content of single-cell protein cells therein. Preferably, the dry weight content of the single-cell protein cells is 8-12 wt%, including but not limited to 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt% or the range composed of any two of them;

[0014] In detail, for the fermentation broth containing single-cell protein cells in the present invention, there are no specific requirements for its source and type. For example, it can be obtained through commercial purchase, or can be self-made by any feasible fermentation method, including conventional methods in the art or improved methods based on conventional methods in the art. In specific applications, it can be prepared by the methods disclosed in patents such as CN117363499B and CN117384770B. In specific applications, the relevant operations, process conditions actually adopted, and the devices used can all be selected conventionally in the art, and the present invention has no special requirements.

[0015] In one embodiment, the cell wall-lysing enzyme in step 1) is an ordinary commercially available product that has been publicly disclosed. It is a hydrolase that specifically acts on the cell wall of microorganisms, also known as cell wall-lysing enzyme. For example, 1,4-β-N-acetylmuramidase. In specific applications, the present invention has no special requirements for its source;

[0016] Further, in a specific example, the addition concentration of the cell wall-lysing enzyme in the fermentation broth is 1.5 - 3 mmol / L, including but not limited to 1.5 mmol / L, 1.7 mmol / L, 1.9 mmol / L, 2.0 mmol / L, 2.2 mmol / L, 2.4 mmol / L, 2.6 mmol / L, 2.8 mmol / L, 3 mmol / L, or the range composed of any two of them.

[0017] In one embodiment, for the reaction of hydrolyzing the cell wall in step 1), phosphate is optionally added to the fermentation broth, preferably one or more of potassium phosphate and sodium phosphate, and phosphate radical is an optional component with a buffering effect in this reaction;

[0018] Further, in a specific example, the addition concentration of the phosphate in the fermentation broth is 50 - 75 mmol / L, including but not limited to 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, or the range composed of any two of them.

[0019] In one embodiment, for the reaction in step 1), the pH value is 7.2 - 7.4, including but not limited to 7.2, 7.3, 7.4, or the range composed of any two of them;

[0020] Further, in a specific example, the reaction is carried out under stirring conditions, and the stirring speed is preferably 150 - 250 rpm, including but not limited to 150 rpm, 180 rpm, 200 rpm, 230 rpm, 250 rpm, or the range composed of any two of them.

[0021] In one embodiment, for the reaction in step 1), the temperature is 32 - 38 °C and the time is 2 - 4 h. Specifically, the reaction temperature includes but not limited to 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, or the range composed of any two of them, and the time includes but not limited to 2 h, 2.5 h, 3 h, 3.5 h, 4 h, or the range composed of any two of them.

[0022] In one embodiment, the plate-and-frame filtration in step 1) is a conventional operation means in the field. Specifically, the fermentation broth is separated into single-cell protein thalli and filtrate through a plate-and-frame, and a filter cake is formed under a certain pressure. The pressure is controlled to be 0.2 - 0.4 MPa, including but not limited to 0.2 MPa, 0.25 MPa, 0.3 MPa, 0.35 MPa, 0.4 MPa, or the range composed of any two of them;

[0023] Further, in a specific example, for the plate-and-frame filtration, the aperture of the filter cloth used is 800 - 1200 mesh, including but not limited to 800 mesh, 900 mesh, 1000 mesh, 1100 mesh, 1200 mesh, or the range composed of any two of them;

[0024] Preferably, the material of the filter cloth can be one or more of polyester, polypropylene, and cotton polyamide;

[0025] In specific applications, when the plate-and-frame filter press enters the pressure-holding state, feeding starts. Open the feeding valve to start the feeding pump for feeding. The filtration pressure is controlled at 0.2 - 0.4 Mpa, and the filtration time is 0.5 - 2 h, such as 0.5 h, 1 h, 1.5 h, 2 h, etc. The specific feeding time depends on the liquid flow condition of the valve outlet after filtration. For example, stop the pump in time when 1 / 3 of the liquid flow from the valve outlet does not form a line.

[0026] In one embodiment, in step 2), the filter cake after plate-and-frame filtration is added with water to form a mixed solution. In the mixed solution, the mass ratio of the filter cake to water is 1:2 - 4, including but not limited to 1:2, 1:2.3, 1:2.5, 1:2.8, 1:3, 1:3.3, 1:3.5, 1:3.8, 1:4, or the range composed of any two of them.

[0027] In one embodiment, in step 2), the addition concentration of the nuclease in the mixed solution is 0.2 - 0.6 mmol / L, including but not limited to 0.2 mmol / L, 0.25 mmol / L, 0.3 mmol / L, 0.35 mmol / L, 0.4 mmol / L, 0.45 mmol / L, 0.5 mmol / L, 0.55 mmol / L, 0.6 mmol / L, or the range composed of any two of them.

[0028] In one embodiment, the auxiliary agent in step 2) is selected from one or more of metal salts, cell membrane permeability increasing reagents, and phosphates, preferably one or more of magnesium chloride, DTT (dithiothreitol), 2-mercaptoethanol, and sodium phosphate; introducing the above auxiliary agents into the fermentation broth, the metal ions therein can play a catalytic role on the nuclease, while the cell membrane permeability increasing reagent has the ability to change the cell membrane permeability, making it easier for the nuclease to enter the cell for reaction, and at the same time can also play a role in protecting proteins. The phosphate is a component with a buffering effect; in specific applications, the types of auxiliary agents can all be selected conventionally in the art, and there is no specific requirement for their dosages. Those skilled in the art can determine according to the actual situation;

[0029] Further, in a specific example, the addition concentration of the auxiliary agent in the mixed solution is: magnesium chloride 1 - 2 mmol / L, DTT 60 - 100 mmol / L, 2 - mercaptoethanol 50 - 100 mmol / L, sodium phosphate 5 - 10 mmol / L. Specifically, the addition concentration of magnesium chloride includes but is not limited to 1 mmol / L, 1.2 mmol / L, 1.4 mmol / L, 1.6 mmol / L, 1.8 mmol / L, 2 mmol / L or the range composed of any two of them; the addition concentration of DTT includes but is not limited to 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, 85 mmol / L, 90 mmol / L, 95 mmol / L, 100 mmol / L or the range composed of any two of them; the addition concentration of 2 - mercaptoethanol includes but is not limited to 50 mmol / L, 50 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, 85 mmol / L, 90 mmol / L, 95 mmol / L, 100 mmol / L or the range composed of any two of them; the addition concentration of sodium phosphate includes but is not limited to 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L or the range composed of any two of them.

[0030] In one embodiment, for the reaction in step 2), the pH value is 8.0 - 9.2, including but not limited to 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2 or the range composed of any two of them;

[0031] Further, in a specific example, the reaction is carried out under stirring conditions, and the stirring speed is preferably 150 - 250 rpm, including but not limited to 150 rpm, 180 rpm, 200 rpm, 230 rpm, 250 rpm or the range composed of any two of them.

[0032] In one embodiment, for the reaction in step 2), the temperature is 60 - 85 °C and the time is 20 - 35 min. Specifically, the reaction temperature includes but is not limited to 60 °C, 65 °C, 70 °C, 75 °C, 80 °C, 85 °C or the range composed of any two of them; the time includes but is not limited to 20 min, 22 min, 25 min, 28 min, 30 min, 33 min, 35 min or the range composed of any two of them.

[0033] In specific applications, after the reaction in step 2) is completed, it is cooled to room temperature, and the supernatant is removed by conventional disk centrifugation to obtain a protein concentrate.

[0034] In one embodiment, for the spray drying in step 3), the feeding rate is 4.5 - 6 L / h, the inlet air temperature is 120 - 160 °C, and the frequency is 320 - 380 Hz. Specifically, the feeding rate includes but is not limited to 4.5 L / h, 4.8 L / h, 5 L / h, 5.3 L / h, 5.5 L / h, 5.8 L / h, 6 L / h, or the ranges formed by any two of them; the inlet air temperature includes but is not limited to 120 °C, 125 °C, 130 °C, 135 °C, 140 °C, 145 °C, 150 °C, 155 °C, 160 °C, or the ranges formed by any two of them; the frequency includes but is not limited to 320 Hz, 330 Hz, 340 Hz, 350 Hz, 360 Hz, 370 Hz, 380 Hz, or the ranges formed by any two of them.

[0035] In the second aspect of the present invention, regarding the fermentation broth containing single-cell protein bacteria in step 1), an improved method based on conventional methods in the art is provided. The method improves the protein content of single-cell protein by supplementing specific carbon sources and nitrogen sources during the fermentation process, adjusting the carbon-nitrogen ratio, and controlling the fermentation conditions until the OD600 value is greater than 500 and then stopping the fermentation tank.

[0036] Specifically, a fermentation method for single-cell protein bacteria includes the following steps:

[0037] (1) Primary seed culture: Activate and culture the strain preserved at low temperature to obtain a primary seed solution;

[0038] (2) Secondary seed expansion culture: Inoculate the primary seed solution into a secondary seed culture medium for expansion culture to obtain a secondary seed solution;

[0039] (3) Fermentation culture: Inoculate the secondary seed solution into a fermentation medium, control appropriate fermentation conditions, and perform single-cell protein bacteria fermentation to obtain a fermentation broth containing single-cell protein bacteria.

[0040] In one embodiment, the strain in step (1) is yeast, selected from any one or several of Yarrowia lipolytica, Saccharomyces cerevisiae, Candida utilis, preferably Yarrowia lipolytica.

[0041] In one embodiment, the primary seed culture in step (1) can adopt conventional methods in the art, and the present invention has no specific requirements for related operations, process conditions, and devices; for example, the activation culture conditions described below can be adopted:

[0042] Among them, the temperature of the activation culture is 30 - 40°C, and the rotation speed is 100 - 200 rpm. Specifically, the temperature includes but is not limited to 30°C, 32°C, 34°C, 36°C, 38°C, 40°C or the ranges formed by any two of them, and the rotation speed includes but is not limited to 100 rpm, 120 rpm, 140 rpm, 160 rpm, 180 rpm, 200 rpm or the ranges formed by any two of them.

[0043] Among them, the OD600 value of the primary seed liquid obtained by activation culture is 2.2 - 4, including but not limited to 2.2, 2.5, 2.8, 3, 3.3, 3.5, 3.8, 4 or the ranges formed by any two of them.

[0044] Among them, the activation culture uses YPD medium;

[0045] Preferably, the volume ratio of the strain to the YPD medium is 2 - 10%, including but not limited to 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or the ranges formed by any two of them.

[0046] In specific applications, a method for culturing the primary seeds is as follows: Take the glycerol tube strain (stored at -20°C) and place it in a 500 mL Erlenmeyer flask containing 50 - 100 mL of YPD medium, and culture it in a reciprocating shaker at 100 - 200 rpm and 30 - 40°C for about 8 - 9 h. When the OD600 is between 2.2 - 4, it indicates that the growth is mature, and the primary seed liquid is obtained.

[0047] In one embodiment, in step (2), the secondary seed expansion culture is to inoculate the activated primary seed liquid in step (1) into a container containing the secondary seed medium for expansion culture. The relevant operations, process conditions, and devices used for the expansion culture can all be selected conventionally in the art; for example, the following expansion culture conditions can be used:

[0048] Among them, the temperature of the expansion culture is 30 - 40°C, including but not limited to 30°C, 32°C, 34°C, 36°C, 38°C, 40°C or the ranges formed by any two of them;

[0049] The reaction is carried out under stirring conditions, and the preferred stirring speed is 150 - 250 rpm, including but not limited to 150 rpm, 180 rpm, 200 rpm, 230 rpm, 250 rpm or the ranges formed by any two of them.

[0050] Among them, the OD600 value of the secondary seed liquid obtained by the expansion culture is 5 - 10, including but not limited to 5, 6, 7, 8, 9, 10 or the ranges formed by any two of them.

[0051] Among them, the formula of the secondary seed culture medium is as follows: glucose 2 - 12 g / L, ammonium chloride 3 - 7 g / L, yeast extract powder 2 - 15 g / L, peptone 5 - 15 g / L, dipotassium hydrogen phosphate 5 - 15 g / L, magnesium sulfate heptahydrate 0.1 - 2 g / L, ferric sulfate 0.1 - 3 g / L, manganese sulfate 0.1 - 1 g / L, copper sulfate 0.1 - 1 g / L, ammonium sulfate 2 - 8 g / L. Specifically, the glucose includes but is not limited to 2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L, 12 g / L or the range composed of any two of them; the ammonium chloride includes but is not limited to 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L or the range composed of any two of them; the yeast extract powder includes but is not limited to 2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L or the range composed of any two of them; the peptone includes but is not limited to 5 g / L, 7 g / L, 9 g / L, 10 g / L, 12 g / L, 14 g / L, 15 g / L or the range composed of any two of them; the dipotassium hydrogen phosphate includes but is not limited to 5 g / L, 7 g / L, 9 g / L, 10 g / L, 12 g / L, 14 g / L, 15 g / L or the range composed of any two of them; the magnesium sulfate heptahydrate includes but is not limited to 0.1 g / L, 0.3 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 1.3 g / L, 1.5 g / L, 1.8 g / L, 2 g / L or the range composed of any two of them; the ferric sulfate includes but is not limited to 0.1 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L or the range composed of any two of them; the manganese sulfate includes but is not limited to 0.1 g / L, 0.3 g / L, 0.5 g / L, 0.7 g / L, 0.9 g / L, 1 g / L or the range composed of any two of them; the copper sulfate includes but is not limited to 0.1 g / L, 0.3 g / L, 0.5 g / L, 0.7 g / L, 0.9 g / L, 1 g / L or the range composed of any two of them; the ammonium sulfate includes but is not limited to 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L or the range composed of any two of them.

[0052] Preferably, the volume ratio of the primary seed liquid inoculated into the secondary seed culture medium is 3 - 10%, including but not limited to 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or the range composed of any two of them.

[0053] In a specific application, one of the secondary seed expansion culture methods includes: inoculating the activated primary seed liquid in step (1) into a 500 mL Erlenmeyer flask containing 50 - 100 mL of secondary seed medium at an inoculation amount of 3 - 10% by volume, and culturing at a constant temperature of 30 - 40 °C for 15 - 20 h. When the OD600 is between 5 and 10, it indicates that the growth is mature, and the secondary seed liquid is obtained.

[0054] In one embodiment, in the fermentation culture process of step (3), the fermentation is aerobic fermentation, and the dissolved oxygen (DO) is controlled at 20 - 40%, including but not limited to 20%, 23%, 25%, 28%, 30%, 33%, 35%, 38%, 40% or the range composed of any two of them.

[0055] In one embodiment, the fermentation conditions in step (3) include:

[0056] Among them, the fermentation temperature is 30 - 40 °C, including but not limited to 30 °C, 32 °C, 34 °C, 36 °C, 38 °C, 40 °C or the range composed of any two of them.

[0057] Among them, the pressure in the fermenter is 0.02 - 0.1 Mpa, including but not limited to 0.02 Mpa, 0.04 Mpa, 0.06 Mpa, 0.08 Mpa, 0.1 Mpa or the range composed of any two of them.

[0058] Among them, the pH value during the fermentation process is controlled at 6 - 7.5, including but not limited to 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.5 or the range composed of any two of them. Preferably, the pH value is controlled by ammonia water.

[0059] Among them, the OD600 value of the fermentation broth containing single-cell protein bacteria obtained by fermentation is above 500, including but not limited to 500, 520, 540, 560, 580, 600, 620, 640, 660 or the range composed of any two of them. Preferably, it is 520 - 580.

[0060] Among them, the formula of the fermentation medium is the same as that of the secondary seed medium, that is, the formula of the fermentation medium is: glucose 2 - 12 g / L, ammonium chloride 3 - 7 g / L, yeast extract powder 2 - 15 g / L, peptone 5 - 15 g / L, dipotassium hydrogen phosphate 5 - 15 g / L, magnesium sulfate heptahydrate 0.1 - 2 g / L, ferric sulfate 0.1 - 3 g / L, manganese sulfate 0.1 - 1 g / L, copper sulfate 0.1 - 1 g / L, ammonium sulfate 2 - 8 g / L;

[0061] Preferably, the volume ratio of the secondary seed liquid inoculated into the fermentation medium is 3-10%, including but not limited to 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or the range composed of any two of them.

[0062] In one embodiment, in step (3) during the fermentation process, an operation of supplementing a carbon source and a nitrogen source is included, and by supplementing a specific carbon source and nitrogen source, the carbon-nitrogen ratio is adjusted;

[0063] Furthermore, in a specific example, during the fermentation process, as the bacteria gradually grow, when 300 > OD600 ≥ 100, the carbon source is started to be supplemented at a flow rate of 0.01-0.02 wt% per minute; and at the same time, the rapid-acting nitrogen source is supplemented to adjust the carbon-nitrogen ratio to (5-15):1, and the flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio; when OD600 ≥ 300, the carbon source is supplemented at a flow rate of 0.02 - 0.04 wt% per minute, and at the same time, the slow-acting nitrogen source is supplemented to adjust the carbon-nitrogen ratio to (20-50):1, and the flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio, where,

[0064] The said flow rate is calculated based on the mass of the fermentation broth that can be accommodated by the maximum volume of the fermenter (i.e., the maximum liquid loading).

[0065] Among them, the carbon source is selected from one or more combinations of soybean oil, rapeseed oil, lard, cottonseed oil, corn oil, linseed oil, olive oil.

[0066] Among them, the rapid-acting nitrogen source is selected from one or more combinations of corn steep liquor, peptone.

[0067] Among them, the slow-acting nitrogen source is selected from one or more combinations of an aqueous solution of cottonseed powder and soybean meal powder with a concentration of 200-400 g / L.

[0068] In specific applications, during the fermentation culture process in step (3), the OD value of the fermentation broth is detected every hour. When the OD of the fermentation broth no longer increases for two consecutive hours, the fermentation can be stopped to obtain a fermentation broth containing single-cell protein bacteria;

[0069] For the said fermentation broth containing single-cell protein bacteria, the dry weight content of the single-cell protein bacteria is above 10 wt%, usually about 10-12 wt%.

[0070] The single-cell protein bacteria powder obtained by the method of the present invention has a protein content of up to more than 85 wt%, and the yield of the bacteria protein is up to more than 96%.

[0071] The single-cell protein bacteria powder obtained by the method of the present invention has a nucleic acid residue of less than 1.5 wt% and a cell wall residue of less than 1.2 wt%.

[0072] The single-cell protein bacterial powder obtained by the method of the present invention is provided for application in the feed field: as a high-protein feed additive, it is used in livestock, poultry and aquaculture.

[0073] The single-cell protein bacterial powder obtained by the method of the present invention is provided for application in the food field: as a substitute for plant protein, it is used in vegetarian foods, protein beverages, etc.

[0074] The single-cell protein bacterial powder obtained by the method of the present invention is provided for application in the pharmaceutical field: as a functional protein raw material, it is used in nutritional supplements and foods for special medical purposes.

[0075] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0076] 1. By optimizing the carbon-nitrogen ratio and fermentation conditions in the fermentation process, the bacterial cell yield and protein content are significantly increased.

[0077] 2. The fermentation broth is post-treated with nuclease and cell wall-lysing enzyme, effectively reducing the nucleic acid and cell wall residues, and improving the purity and digestibility of the protein.

[0078] 3. The process is simple and the cost is low, which is suitable for industrial production. Detailed implementation manners

[0079] For the convenience of understanding the present invention, the present invention will be further described below in conjunction with embodiments. It should be understood that the following embodiments are only for better understanding of the present invention, and do not mean that the present invention is limited only to the following embodiments.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The term "and / or" as used herein may include any and all combinations of one or more of the related listed items.

[0081] The main analysis methods used in the embodiments and comparative examples of the present invention:

[0082] (1) OD detection

[0083] In the present invention, the term "OD value" refers to the OD value of the fermentation broth, which is obtained by ultraviolet spectrophotometry. Specifically: use an ultraviolet spectrophotometer (Alpha-1106, Shanghai Puyuan Instrument Co., Ltd.) to detect the OD value of the fermentation broth at 600 nm.

[0084] (2) Protein content

[0085] According to the national standard GB / T 6432-2018 "Determination of Crude Protein in Feeds - Kjeldahl Method", the crude protein content (mass fraction) is obtained through digestion, ammonia distillation, and titration, with the unit of wt%.

[0086] (3) Bacterial protein yield:

[0087] Bacterial protein yield = protein content after spray drying * weight / (fermentation liquid output * dry weight of bacteria at the end of the tank * bacterial protein content) * 100%.

[0088] (4) Nucleic acid residues

[0089] Refer to the national standard "GB / T 38485-2021 Droplet Digital PCR Method for Determination of Trace Gene Residues in Microorganisms".

[0090] (5) Cell wall residues

[0091] Refer to the national standard "GB / T 20886.2-2021 Yeast Product Quality Requirements Part 2: Yeast Processed Products Appendix I Determination of Polysaccharides in Yeast Cell Wall".

[0092] (6) In vitro protein digestibility

[0093] Weigh 1.000g (accurate to 0.001g) of the test sample and place it in a 50mL centrifuge tube, add 10mL of 1mg / mL porcine pepsin solution (pH=2.0) prepared in advance, and then add 0.5mL of chloramphenicol solution to destroy the environment for microbial growth. After sealing, incubate at 37°C in a constant temperature water bath shaker for 4h; after digestion with pepsin, neutralize with 0.2mol / LNaOH, then add 10mL of prepared phosphate buffer (0.2mol / L, pH=6.8), and then adjust its pH value to 6.8 with prepared 1mol / L HCl or 1mol / LNaOH; then add 50mg / mL 1mL porcine pancreatin solution, seal the mouth, and then place in a constant temperature water bath shaker at 39°C for digestion for 4, 8, 12, 16, 20, 24, and 28h respectively; after digestion with trypsin, proceed to the next step, add 5mL of the prepared 20% sulfosalicylic acid solution, 15 The mixture was centrifuged at 000 r / min for 15 min, the supernatant was removed, and the precipitate was placed in a drying oven at 80°C overnight. The protein digestibility to be tested = (content in the original sample - content in the precipitate) / content in the original sample × 100%.

[0094] In the embodiments and comparative examples of the present invention, the sources of the main raw materials used are as follows. Unless otherwise specified, other raw materials and reagents were purchased from commercial sources:

[0095] Yarrowia lipolytica was purchased from China Industrial Microbiological Culture Collection Center, with the collection number: CICC32291;

[0096] The Saccharomyces cerevisiae strain was purchased from the China Center of Industrial Culture Collection, with the preservation number: CICC 32883;

[0097] The Candida utilis strain was purchased from the China Center of Industrial Culture Collection, with the preservation number: CICC31593;

[0098] Cell wall lytic enzyme (1,4-β-N-acetylmuramidase): Purchased from Vazyme Biotech Co., Ltd., product number M0253;

[0099] Nuclease: Purchased from Supbio Biotechnology Co., Ltd., product number S10068;

[0100] All other raw materials and each component for preparing the culture medium are common commercially available chemicals.

[0101] Example 1

[0102] 1) Preparation of the fermentation broth containing single-cell protein bacteria:

[0103] (1) Primary seed culture: Take 1 mL of the Yarrowia lipolytica strain in a glycerol tube (stored at -20°C) and transfer it to a 500 mL Erlenmeyer flask containing 50 mL of YPD medium. Incubate it on a reciprocating shaker at 150 rpm and 32°C for constant activation culture until the OD600 is between 2.5 and 4 to obtain the primary seed liquid.

[0104] (2) Secondary seed expansion culture: Inoculate the primary seed fermentation broth into a shake flask containing the secondary seed medium at an inoculation amount of 3% by volume, and perform constant expansion culture at 32°C and 200 rpm until the OD600 is between 5 and 10 to obtain the secondary seed fermentation broth;

[0105] Among them, the formula of the secondary seed medium is: glucose 2 g / L, ammonium chloride 3 g / L, yeast extract powder 2 g / L, peptone 5 g / L, dipotassium hydrogen phosphate 5 g / L, magnesium sulfate heptahydrate 0.1 g / L, ferric sulfate 0.1 g / L, manganese sulfate 0.1 g / L, copper sulfate 0.1 g / L, ammonium sulfate 2 g / L.

[0106] (3) Fermentation culture: Prepare a 50 L fermenter with an initial liquid filling volume of 25 L. Inoculate the secondary seed fermentation broth into the sterile fermentation medium at an inoculation amount of 5% by volume for aerobic fermentation. Control the dissolved oxygen content at 30%, the fermentation temperature at 32°C, control the pH at 6.8 with ammonia water, and the tank pressure at 0.02 - 0.1 Mpa;

[0107] Among them, the formula of the sterile fermentation medium is the same as that of the secondary seed medium in step (2);

[0108] As the bacterial cells gradually grow, when 300 > OD600 ≥ 100, soybean oil is added as a carbon source at a flow rate of 0.01 wt% per minute; at the same time, corn steep liquor is added as a readily available nitrogen source to adjust the carbon-nitrogen ratio to 5:1, and the flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio.

[0109] When OD600 ≥ 300, soybean oil is added as a carbon source at a flow rate of 0.02 wt% per minute, and at the same time, an aqueous solution of cottonseed cake powder with a concentration of 200 g / L is added as a slowly available nitrogen source to adjust the carbon-nitrogen ratio to 20:1, and the flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio.

[0110] When the OD600 of the fermentation broth rises to 500 and does not increase for two consecutive hours, the fermentation is stopped to obtain a fermentation broth containing single-cell protein bacterial cells.

[0111] The obtained fermentation broth containing single-cell protein bacterial cells has a dry weight content of single-cell protein bacterial cells of 12 wt%.

[0112] 2) Post-treatment of the fermentation broth containing single-cell protein bacterial cells:

[0113] (1) Removal of cell walls: The above-mentioned fermentation broth containing single-cell protein bacterial cells is added with cell wall-lysing enzyme at a final concentration of 1.5 mmol / L and potassium phosphate at 50 mmol / L. The reaction conditions are a temperature of 32 °C, a pH of 7.2, a rotation speed of 150 rpm, and a reaction time of 2 hours.

[0114] After the reaction, plate and frame filtration is carried out to remove the solution. The filter cloth pore size is 800 mesh, the filter cloth material is polypropylene, the filtration pressure is 0.4 Mpa, and the filtration time is 2 h to obtain a filter cake.

[0115] (2) Removal of nucleic acids: The filter cake is dissolved by adding 3 times its weight of water, and then nuclease at a final concentration of 0.2 mmol / L, magnesium chloride at 1 mmol / L, DTT at 60 mmol / L, 2-mercaptoethanol at 50 mmol / L, and sodium phosphate at 5 mmol / L are added. The reaction temperature is adjusted to 60 °C, the pH is 8.0, the rotation speed is 150 rpm, and the reaction time is 20 min.

[0116] The solution after nucleic acid removal is subjected to disc centrifugation to remove the supernatant to obtain a protein concentrate.

[0117] (3) Spray drying: The protein concentrate is spray-dried. The spray-drying feed rate is 4.5 L / h, the inlet air temperature is 120 °C, the frequency is 320 Hz, and single-cell protein powder is harvested every half hour to obtain single-cell protein bacterial cell powder rich in high protein.

[0118] The prepared single-cell protein bacterial cell powder is detected to have a protein content of 86 wt% and a yield of bacterial cell protein of 96%.

[0119] Single-cell protein bacterial powder, with nucleic acid residue of 1.2 wt%, cell wall residue of 1.1 wt%, and digestibility of 97%.

[0120] Example 2

[0121] 1) Prepare a fermentation broth containing single-cell protein bacteria:

[0122] (1) Primary seed culture: Take 1 mL of Saccharomyces cerevisiae glycerol tube strain (stored at -20°C) into a 500 mL Erlenmeyer flask containing 50 mL of YPD medium, and culture it at a constant temperature of 34°C with a reciprocating shaker at 150 rpm until the OD600 is between 2.5 and 4 to obtain the primary seed liquid.

[0123] (2) Seed expansion culture: Inoculate the primary seed fermentation broth into a shake flask containing the secondary seed medium at an inoculation amount of 5% by volume, and perform aerobic expansion culture at a constant temperature of 32°C and 200 rpm until the OD600 is between 5 and 10 to obtain the secondary seed fermentation broth;

[0124] Among them, the formula of the secondary seed medium is: glucose 5 g / L, ammonium chloride 6 g / L, yeast extract powder 8 g / L, peptone 10 g / L, dipotassium hydrogen phosphate 12 g / L, magnesium sulfate heptahydrate 0.4 g / L, ferric sulfate 0.3 g / L, manganese sulfate 0.6 g / L, copper sulfate 0.5 g / L, ammonium sulfate 4 g / L.

[0125] (3) Fermentation culture: Prepare a 50 L fermenter with an initial liquid volume of 25 L. Inoculate the secondary seed fermentation broth into the sterile fermentation medium at an inoculation amount of 6% by volume for aerobic fermentation. Control the dissolved oxygen content at 40%, the fermentation temperature at 34°C, control the pH at 6.8 with ammonia water, and the tank pressure at 0.02 - 0.1 Mpa;

[0126] Among them, the formula of the sterile fermentation medium is the same as that of the secondary seed medium in step (2);

[0127] As the bacteria gradually grow, when 300 > OD600 ≥ 100, start to supplement soybean oil as the carbon source at a flow rate of 0.02 wt% per minute; and at the same time, supplement corn steep liquor as the quick-acting nitrogen source to adjust the carbon-nitrogen ratio to 10:1, and adjust the flow rate of the nitrogen source according to the carbon-nitrogen ratio.

[0128] When OD600 ≥ 300, supplement soybean oil as the carbon source at a flow rate of 0.03 wt% per minute, and at the same time, supplement an aqueous solution of soybean meal powder with a concentration of 300 g / L as the slow-acting nitrogen source to adjust the carbon-nitrogen ratio to 30:1, and adjust the flow rate of the nitrogen source according to the carbon-nitrogen ratio.

[0129] When the OD600 of the fermentation broth rises to 520 and no longer increases for two consecutive hours, the fermentation can be stopped to obtain a fermentation broth containing single-cell protein bacteria.

[0130] The obtained fermentation broth containing single-cell protein thalli has a dry weight content of single-cell protein thalli of 11.5 wt%.

[0131] 2) Post-treatment of the fermentation broth containing single-cell protein thalli

[0132] (1) Removal of cell wall: The above-mentioned fermentation broth containing single-cell protein thalli is added with cell wall-lysing enzyme with a final concentration of 2 mmol / L and potassium phosphate with a concentration of 60 mmol / L. The reaction conditions are a temperature of 35 °C, a pH of 7.2, a rotation speed of 200 rpm, and a reaction time of 3 hours.

[0133] After the reaction, plate-and-frame filtration is carried out to remove the solution. The aperture of the filter cloth is 1000 mesh, the filter cloth material is polyester, the filtration pressure is 0.3 Mpa, and the filtration time is 1.5 h to obtain a filter cake.

[0134] (2) Removal of nucleic acid: The filter cake is dissolved by adding 3 times its weight of water, and then nuclease with a final concentration of 0.4 mmol / L, magnesium chloride with a concentration of 1.5 mmol / L, DTT with a concentration of 80 mmol / L, 2-mercaptoethanol with a concentration of 60 mmol / L, and sodium phosphate with a concentration of 6 mmol / L are added. The reaction temperature is adjusted to 70 °C, the pH is 8.5, the rotation speed is 200 rpm, and the reaction time is 25 minutes.

[0135] The solution after nucleic acid removal is subjected to disc centrifugation to remove the supernatant to obtain a protein concentrate.

[0136] (3) Spray drying: The protein concentrate is spray-dried. The feed rate of spray drying is 5 L / h, the inlet air temperature is 140 °C, the frequency is 350 Hz, and single-cell protein powder is harvested every half hour to obtain single-cell protein thalli powder rich in high protein.

[0137] The prepared single-cell protein thalli powder is detected to have a protein content of 88 wt% and a thalli protein yield of 86%.

[0138] The single-cell protein thalli powder has a nucleic acid residue of 0.8 wt%, a cell wall residue of 0.9 wt%, and a digestibility of 98.4%.

[0139] Example 3

[0140] 1) Preparation of a fermentation broth containing single-cell protein thalli:

[0141] (1) Primary seed culture: Take 1 mL of Candida utilis glycerol tube strain (stored at -20 °C) and place it in a 500 mL Erlenmeyer flask containing 50 mL of YPD medium. Incubate it at a constant temperature of 36 °C with a reciprocating shaker at 150 rpm until the OD600 is between 2.5 and 4 to obtain a primary seed solution.

[0142] (2) Seed expansion culture: Inoculate the primary seed fermentation broth into a shake flask containing the secondary seed culture medium at an inoculation amount of 7% by volume, and perform constant-temperature expansion culture at 36 °C and 200 rpm until the OD600 is between 5 and 10 to obtain the secondary seed fermentation broth;

[0143] Among them, the formula of the secondary seed culture medium is: glucose 10 g / L, ammonium chloride 7 g / L, yeast extract powder 15 g / L, peptone 15 g / L, dipotassium hydrogen phosphate 14 g / L, magnesium sulfate heptahydrate 2 g / L, ferric sulfate 2 g / L, manganese sulfate 1 g / L, copper sulfate 0.8 g / L, ammonium sulfate 7 g / L.

[0144] (3) Fermentation culture: Prepare a 50 L fermenter with an initial liquid loading of 25 L. Inoculate the secondary seed fermentation broth into the sterile fermentation medium at an inoculation amount of 8% by volume for aerobic fermentation. Control the dissolved oxygen content at 35%, the fermentation temperature at 36 °C, control the pH at 6.8 with ammonia water, and the tank pressure at 0.02 - 0.1 Mpa;

[0145] Among them, the formula of the sterile fermentation medium is the same as that of the secondary seed culture medium in step (2);

[0146] As the bacteria gradually grow, when 300 > OD600 ≥ 100, start to supplement soybean oil as the carbon source at a flow rate of 0.01 wt% per minute; and at the same time supplement corn steep liquor as the quickly available nitrogen source to adjust the carbon-nitrogen ratio to 15:1, and adjust the flow rate of the nitrogen source according to the carbon-nitrogen ratio.

[0147] When OD600 ≥ 300, supplement soybean oil as the carbon source at a flow rate of 0.03 wt% per minute, and at the same time supplement an aqueous solution of cottonseed cake powder with a concentration of 400 g / L as the slowly available nitrogen source to adjust the carbon-nitrogen ratio to 40:1, and adjust the flow rate of the nitrogen source according to the carbon-nitrogen ratio.

[0148] When the OD600 of the fermentation broth rises to 515 and no longer increases for two consecutive hours, the fermentation can be stopped to obtain the fermentation broth containing single-cell protein bacteria.

[0149] The obtained fermentation broth containing single-cell protein bacteria has a dry weight content of single-cell protein bacteria of 10.5 wt%.

[0150] 2) Post-treatment of the fermentation broth containing single-cell protein bacteria

[0151] (1) Removal of cell wall: Add cell wall-lysing enzyme with a final concentration of 3 mmol / L and potassium phosphate with a concentration of 70 mmol / L to the above-mentioned fermentation broth containing single-cell protein bacteria. The reaction conditions are a temperature of 35 °C, a pH of 7.2, a rotation speed of 200 rpm, and a reaction time of 4 hours.

[0152] After the reaction, plate-and-frame filtration was carried out to remove the solution. The pore size of the filter cloth was 1200 mesh, the filter cloth material was polyamide, the filtration pressure was 0.35 Mpa, the filtration time was 1.8 h, and the filter cake was obtained.

[0153] (2) Nucleic acid removal: After dissolving the filter cake in 3 times the weight of water, nuclease with a final concentration of 0.6 mmol / L, magnesium chloride of 2 mmol / L, DTT of 90 mmol / L, 2-mercaptoethanol of 90 mmol / L, and sodium phosphate of 9 mmol / L were added. The reaction temperature was adjusted to 80 °C, pH 8.7, the rotation speed was 200 rpm, and the reaction time was 30 minutes.

[0154] The solution after nucleic acid removal was subjected to disc centrifugation to remove the supernatant, and a protein concentrate was obtained.

[0155] (3) Spray drying: The protein concentrate was spray-dried. The feed rate of the spray drying was 6 L / h, the inlet air temperature was 160 °C, the frequency was 380 Hz, and the single-cell protein powder was harvested every half hour, and the single-cell protein bacterial powder rich in high protein could be obtained.

[0156] For the prepared single-cell protein bacterial powder, the protein content was detected to be 85.6 wt%, and the yield of bacterial protein was 85%.

[0157] For the single-cell protein bacterial powder, the nucleic acid residue was 1.3 wt%, the cell wall residue was 1.1 wt%, and the digestibility was 96.2%.

[0158] Comparative Example 1

[0159] Fermentation was carried out with reference to the steps of Example 1. When the fermentation broth was post-treated in step 2), the difference was only that in the operation of removing the cell wall in step (1), the cell wall-lysing enzyme was not added: 50 mmol / L potassium phosphate was added to the fermentation broth, and the reaction conditions were a temperature of 32 °C, pH 7.2, a rotation speed of 150 rpm, and a reaction time of 2 hours. After the reaction, plate-and-frame filtration was carried out to remove the solution, and a filter cake was obtained.

[0160] Other operations and conditions remained unchanged.

[0161] For the prepared single-cell protein bacterial powder, the protein content was detected to be 52.8 wt%, and the yield of bacterial protein was 16.8%.

[0162] For the single-cell protein bacterial powder, the nucleic acid residue was 8.6 wt%, the cell wall residue was 25.3 wt%, and the digestibility was 54%.

[0163] Comparative Example 2

[0164] Fermentation was carried out according to the steps of Example 1. When the fermentation broth was post-treated in step 2), the only difference was that no nuclease was added in the operation of removing nucleic acid in step (2): after adding the filter cake to 3 times its weight of water for dissolution, 1 mmol / L magnesium chloride, 60 mmol / L DTT, 50 mmol / L 2-mercaptoethanol, and 5 mmol / L sodium phosphate were added, the reaction temperature was adjusted to 60 °C, pH 8.0, the rotation speed was 150 rpm, and the nuclease reaction time was 20 minutes. The solution after removing nucleic acid was centrifuged by a disc centrifuge to remove the supernatant, and a protein concentrate was obtained.

[0165] Other operations and conditions remained unchanged.

[0166] The obtained single-cell protein cell powder was detected to have a protein content of 45.5 wt% and a cell protein yield of 24.2%.

[0167] The cell protein cell powder had a nucleic acid residue of 24.7 wt%, a cell wall residue of 4.8 wt%, and a digestibility of 52%.

[0168] Comparative Example 3

[0169] Fermentation was carried out according to the steps of Example 1. When the fermentation broth was post-treated in step 2), the only differences were as follows:

[0170] No cell wall-lysing enzyme was added in the operation of removing the cell wall in step (1): 50 mmol / L potassium phosphate was added to the fermentation broth, and the reaction conditions were a temperature of 32 °C, pH 7.2, a rotation speed of 150 rpm, and a reaction time of 2 hours. After the reaction, plate-and-frame filtration was carried out to remove the solution, and a filter cake was obtained.

[0171] At the same time, no nuclease was added in the operation of removing nucleic acid in step (2): after adding the filter cake to 3 times its weight of water for dissolution, 1 mmol / L magnesium chloride, 60 mmol / L DTT, 50 mmol / L 2-mercaptoethanol, and 5 mmol / L sodium phosphate were added, the reaction temperature was adjusted to 60 °C, pH 8.0, the rotation speed was 150 rpm, and the reaction time was 20 minutes. The solution after removing nucleic acid was centrifuged by a disc centrifuge to remove the supernatant, and a protein concentrate was obtained.

[0172] Other operations and conditions remained unchanged.

[0173] The obtained single-cell protein cell powder was detected to have a protein content of 40.2 wt% and a cell protein yield of 12.6%.

[0174] The cell protein cell powder had a nucleic acid residue of 28.3 wt%, a cell wall residue of 30.4 wt%, and a digestibility of 32%.

[0175] It is easy to understand that the above embodiments are merely examples for clear illustration, and do not mean that the present invention is limited thereto. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A post-treatment method for a single-cell protein fermentation broth, characterized in that, The method includes the steps of adding a cell wall-lysing enzyme to a fermentation broth containing single-cell protein bacteria to react and hydrolyze the cell wall, and adding a nuclease to react and remove nucleic acids.

2. The post-processing method according to claim 1, characterized in that the step Including: 1) Adding a cell wall-lysing enzyme to a fermentation broth containing single-cell protein bacteria, reacting to hydrolyze the cell wall, and then removing the solution through plate-and-frame filtration to form a filter cake; 2) Dissolving the filter cake with water to form a mixture, adding a nuclease and an auxiliary agent thereto, reacting to remove nucleic acids, and then removing the supernatant through centrifugation to obtain a protein concentrate; 3) Spray-drying the protein concentrate to obtain single-cell protein bacteria powder.

3. The post-treatment method according to claim 2, characterized in that, The fermentation broth containing single-cell protein bacteria in step 1), wherein the dry weight content of single-cell protein bacteria is 8-12 wt%; and / or The addition concentration of the cell wall-lysing enzyme in the fermentation broth in step 1) is 1.5-3 mmol / L; and / or For the reaction of hydrolyzing the cell wall in step 1), phosphate is optionally added to the fermentation broth, preferably one or more of potassium phosphate and sodium phosphate; Preferably, the addition concentration of the phosphate in the fermentation broth is 50-75 mmol / L; and / or For the reaction in step 1), the pH value is 7.2-7.4; and / or For the reaction in step 1), the temperature is 32-38 °C and the time is 2-4 h; and / or The reaction in step 1) is carried out under stirring conditions, and the stirring speed is preferably 150-250 rpm.

4. The post-treatment method according to claim 2, wherein, In the mixture in step 2), the mass ratio of the filter cake to water is 1:2-4; and / or The addition concentration of the nuclease in the mixture in step 2) is 0.2-0.6 mmol / L; and / or The auxiliary agent in step 2) is selected from one or more of metal salts, cell membrane permeability increasing reagents, and phosphates, preferably one or more of magnesium chloride, DTT, 2-mercaptoethanol, and sodium phosphate; Preferably, the addition concentrations of the auxiliary agent in the mixture are: magnesium chloride 1-2 mmol / L, DTT 60-100 mmol / L, 2-mercaptoethanol 50-100 mmol / L, and sodium phosphate 5-10 mmol / L; and / or For the reaction in step 2), the pH value is 8.0-9.2; and / or For the reaction in step 2), the temperature is 60-85 °C and the time is 20-35 min; and / or The reaction in step 2) is carried out under stirring conditions, and the stirring speed is preferably 150-250 rpm; and / or For the spray-drying in step 3), the feeding speed is 4.5-6 L / h, the inlet air temperature is 120-160 °C, and the frequency is 320-380 Hz.

5. A fermentation method for single-cell protein bacteria, characterized in that the steps Including: (1) Activating and culturing the strain stored at low temperature to obtain a primary seed liquid; (2) Inoculating the primary seed liquid into a secondary seed culture medium for scale-up culture to obtain a secondary seed liquid; (3) Inoculating the secondary seed liquid into a fermentation medium, controlling appropriate fermentation conditions, and performing single-cell protein bacteria fermentation to obtain a fermentation broth containing single-cell protein bacteria.

6. The fermentation method according to claim 5, wherein The strain in step (1) is yeast, selected from any one or several of Yarrowia lipolytica, Saccharomyces cerevisiae, and Candida utilis, preferably Yarrowia lipolytica; and / or The activation culture described in step (1) includes at least one of the following conditions: The temperature of the activation culture is 30 - 40°C, and the rotation speed is 100 - 200 rpm; The activation culture uses YPD medium; preferably, the volume ratio of the strain to the YPD medium is 2 - 10%; The OD600 value of the primary seed liquid is 2.2 - 4.

7. The fermentation method according to claim 5, characterized in that, The scale-up culture described in step (2) includes at least one of the following conditions: The temperature of the scale-up culture is 30 - 40°C; The reaction is carried out under stirring conditions, and the stirring speed is preferably 150 - 250 rpm; The formula of the secondary seed medium is: glucose 2 - 12 g / L, ammonium chloride 3 - 7 g / L, yeast extract powder 2 - 15 g / L, peptone 5 - 15 g / L, dipotassium hydrogen phosphate 5 - 15 g / L, magnesium sulfate heptahydrate 0.1 - 2 g / L, ferric sulfate 0.1 - 3 g / L, manganese sulfate 0.1 - 1 g / L, copper sulfate 0.1 - 1 g / L, ammonium sulfate 2 - 8 g / L; The volume ratio of the primary seed liquid inoculated into the secondary seed medium is 3 - 10%; The OD600 value of the secondary seed liquid is 5 - 10.

8. The fermentation method according to claim 5, wherein The fermentation described in step (3) uses aerobic fermentation, and the dissolved oxygen (DO) is controlled at 20 - 40%; and / or In one embodiment, the fermentation described in step (3) includes at least one of the following conditions: The fermentation temperature is 30 - 40°C; The pressure in the fermenter is 0.02 - 0.1 Mpa; The pH value during the fermentation process is controlled at 6 - 7.5, preferably, the pH value is controlled by ammonia water; The formula of the fermentation medium is the same as that of the secondary seed medium; The volume ratio of the secondary seed liquid inoculated into the fermentation medium is 3 - 10%; The OD600 value of the fermentation broth containing single-cell protein bacteria is above 500, preferably 520 - 580.

9. The fermentation method according to claim 5, characterized in that, During the fermentation process in step (3), it includes the operation of supplementing carbon source and nitrogen source, and the carbon-nitrogen ratio is adjusted by supplementing carbon source and nitrogen source; Preferably, during the fermentation process, when 300 > OD600 ≥ 100, the carbon source is started to be supplemented at a flow rate of 0.01 - 0.02 wt% per minute; and at the same time, the quick-acting nitrogen source is supplemented to adjust the carbon-nitrogen ratio to (5 - 15):1, and the flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio; when OD600 ≥ 300, the carbon source is supplemented at a flow rate of 0.02 - 0.04 wt% per minute, and at the same time, the slow-acting nitrogen source is supplemented to adjust the carbon-nitrogen ratio to (20 - 50):1, and the flow rate of the nitrogen source is adjusted according to the carbon-nitrogen ratio.

10. The fermentation method according to claim 9, wherein, The carbon source is selected from one or more combinations of soybean oil, rapeseed oil, lard, cottonseed oil, corn oil, linseed oil, olive oil; and / or The quick-acting nitrogen source is selected from one or more combinations of corn steep liquor, peptone; and / or The slow-acting nitrogen source is selected from one or more combinations of aqueous solutions of cottonseed meal and soybean meal powder with a concentration of 200 - 400 g / L.