Method for producing single-cell protein by means of high-density fermentation

Abstract

Disclosed herein is a method for producing a single-cell protein by means of high-density fermentation. The method comprises: inoculating a Yarrowia lipolytica strain into a seed culture medium for seed culture to obtain a seed liquid; and inoculating the seed liquid into a fermentation culture medium for high-density fermentation, and supplementing same with acetic acid and a nitrogen source during the process of high-density fermentation to obtain the single-cell protein, wherein the carbon source of the seed culture medium comprises a first acetate, and the carbon source of the fermentation culture medium comprises a second acetate and / or a ketone compound. In the present application, the compounding of specific materials and processes effectively increases the growth rate of bacteria and the synthesis rate of single-cell proteins, so that the method can realize the high-density fermentation of bacteria and efficient synthesis of single-cell proteins in a short time, a fermentation product with a higher content of crude proteins can be obtained while the culture time of high-density fermentation is significantly shortened, and the production efficiency and yield of single-cell proteins are greatly improved.

Description

A method for producing single-cell protein through high-density fermentation Technical Field

[0001] The embodiments of the present application relate to the field of biotechnology, for example, a method for producing single-cell protein through high-density fermentation. Background Art

[0002] With the continued growth of the population, people's demand for food, especially meat, is increasing. Global meat demand is projected to exceed 400 million tons by 2050. As the world's most populous country, China's food demand is also growing. In 2018, China's meat consumption reached 88.296 million tons, exceeding the combined total of the European Union and the United States. As meat consumption grows, so does the demand for feed, with feed production exceeding 240 million tons. Feed consists primarily of starch and protein, with the primary sources of protein being soybean meal (a plant-based protein) and fish meal (an animal-based protein). Soybean meal, a byproduct of soybean oil extraction, is the most produced and widely used of 12 animal and plant oil and meal feed products, along with cottonseed meal, peanut meal, and rapeseed meal. Soybean meal production is closely related to soybean production. However, China's soybean self-sufficiency rate is insufficient, with approximately 85% dependent on imports. Soybean shortages are impacting food and protein security, necessitating the use of alternative feed resources. Furthermore, the supply of fishmeal, an animal protein in feed, continues to shrink. Global fishmeal consumption reached 3 million tons in 2020, and the fishmeal shortage is projected to reach 1 million tons by 2025. China's annual fishmeal production is approximately 1.2 million tons, with a self-sufficiency rate of approximately 40%-50%. In 2020, China imported 1.42 million tons of fishmeal, valued at 15 billion yuan. In addition to the shortage issue, the massive production and overfishing of fishmeal have significantly overdrawn marine fishery resources, resulting in a decrease in fish numbers and populations, and significantly impacting ecological health and safety. Based on these considerations, low-protein feeds and unconventional protein substitutes have become a focus of attention in the livestock industry. Among these, single-cell protein (SCP) is one of the most important and promising alternatives.

[0003] Single-cell protein, also known as microbial protein, is a microbial cell cultured from industrial and agricultural waste and petroleum waste. Single-cell protein is not a pure protein, but rather a cytoplasmic mass composed of a mixture of proteins, fats, carbohydrates, nucleic acids, non-protein nitrogenous compounds, vitamins, and inorganic compounds. Its chemical composition is generally dominated by protein and fat.

[0004] The use of single-cell protein in feed began around the 1990s. A series of animal experiments using monosodium glutamate and yeast residues in poultry, livestock, aquaculture, and pigs demonstrated beneficial effects on animal health, thus initiating the use of single-cell protein in feed. Currently, yeast is widely used in finished poultry, livestock, and aquaculture feeds, accounting for approximately 5%-10% of total feed production. Single-cell protein accounts for an even greater proportion in aquaculture feeds, at approximately 10%-20%. With advances in technology and processes, proteins derived from Clostridium alcoholicum and methanogens have also been developed. While animal experimental data demonstrates their safety and bioavailability, these proteins are still relatively new, and no feed products containing them are currently commercially available. Photosynthetic bacteria and algae also have some applications in feed protein, but production volumes are relatively small and they are not mainstream products.

[0005] Single-cell protein (SCP) is typically produced using biofermentation techniques. Specifically, after the culture medium is prepared and sterilized, it and the bacterial strain are placed in a fermenter. Fermentation conditions are controlled to allow the strain to rapidly reproduce. After fermentation, the cells are collected by centrifugation, precipitation, and other methods, and finally dried to produce the final SCP product. Synthetic biology fermentation methods for SCP production allow for CO2 fixation through bioconversion, resulting in bacterial protein for use in feed. This can help alleviate resource shortages caused by the growing demand for meat and environmental pollution caused by excessive use of fish meal. Currently, China is vigorously promoting low-protein feeds and unconventional protein resources, and researchers in the agricultural and animal husbandry sectors are also actively promoting the promotion of SCP. SCP is a key development direction in the future of feed additives. However, the current fermentation production of SCP suffers from long production cycles, significant deficiencies in both yield and production capacity, making it difficult to meet the needs of large-scale applications. Therefore, developing methods for producing SCP with higher efficiency and yield is a research priority in this field.

[0006] Summary of the Invention

[0007] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0008] The embodiments of the present application provide a method for producing single-cell protein by high-density fermentation. By designing a carbon source and combining specific materials and processes, the method can achieve high-density fermentation of bacteria and efficient synthesis of single-cell protein in a short period of time, greatly improving the production efficiency and yield of single-cell protein.

[0009] An embodiment of the present application provides a method for producing single-cell protein by high-density fermentation, the method comprising: inoculating a Yarrowia lipolytica strain into a seed culture medium for seed culture to obtain a seed liquid; inoculating the seed liquid into a fermentation medium for high-density fermentation, and adding acetic acid and a nitrogen source during the high-density fermentation to obtain the single-cell protein; the carbon source of the seed culture medium comprises a first acetate, and the carbon source of the fermentation culture medium comprises a second acetate and / or a ketone compound.

[0010] This application uses Yarrowia lipolytica as a fermentation strain, which is a non-genetically engineered bacterium and is safer than conventional bacteria. It has been certified by the U.S. Food and Drug Administration (FDA) as a generally recognized safe microorganism (GRAS), and is an ideal food industry bacteria. The method designs the carbon source in seed culture and high-density fermentation. Sodium acetate is added to the seed culture medium, which can promote the formation of a unique acetic acid metabolic pathway in the bacteria, laying the foundation for faster growth by adding acetic acid during subsequent high-density fermentation. At the same time, sodium acetate is also included in the carbon source of the fermentation medium to further induce and promote the acetic acid metabolic pathway of the bacteria. Acetic acid and a nitrogen source are added during the high-density fermentation process. On the one hand, the acetic acid can neutralize the alkaline substances produced during the fermentation process, so that the bacteria are in an optimal neutral pH growth environment. On the other hand, acetic acid, as a carbon source, has a high carbon content, which can effectively promote the rapid growth of the bacteria and the synthesis of single-cell proteins, and acetic acid does not require sterilization and can be added directly. Furthermore, the fermentation medium contains a ketone fermentation promoter, which plays a catalytic role in the intermediate product in the acetic acid metabolic pathway, promotes the rapid growth of the bacteria, and shortens the fermentation cycle.

[0011] In summary, in the method provided by the present application, by introducing the first acetate into the carbon source of the seed culture medium, the bacteria are induced and promoted to produce a unique acetic acid metabolic pathway, laying the foundation for adding acetic acid as a carbon source and promoting rapid growth of the bacteria in the subsequent high-density fermentation process; at the same time, the carbon source of the fermentation medium includes a second acetate and / or ketone compounds to promote rapid growth of the bacteria; in the high-density fermentation stage, nitrogen source and acetic acid are added, while regulating the bacteria to be in an optimal neutral pH growth environment, the bacteria are supplemented with appropriate nutrients for continuous growth of the bacteria, effectively improving the growth rate of the bacteria and the synthesis rate of single-cell protein. By compounding specific materials and processes, the method can achieve high-density fermentation of bacteria and efficient synthesis of single-cell protein in a short period of time, while significantly shortening the culture time of high-density fermentation, it can obtain a fermentation product with a higher crude protein content, greatly improving the production efficiency and yield of single-cell protein.

[0012] The following are preferred technical solutions of this application, but are not intended to limit the technical solutions provided in this application. Through the following preferred technical solutions, the objectives and beneficial effects of this application can be better achieved and realized.

[0013] Preferably, the first acetate includes any one of sodium acetate, potassium acetate, and ammonium acetate, or a combination of at least two thereof, more preferably sodium acetate.

[0014] Preferably, the mass concentration of the first acetate in the seed culture medium is 5-15 g / L, for example, it can be 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L or 14 g / L, etc.

[0015] Preferably, the seed culture includes primary seed culture and secondary seed culture performed sequentially.

[0016] Preferably, the primary seed culture medium used for the primary seed culture comprises the following components: 5-15 g / L first acetate, 1%-3% glucose, 0.6%-2% yeast powder, and 1.5%-3.2% casein peptone.

[0017] Specifically, the mass concentration of the first acetate (preferably sodium acetate) in the primary seed culture medium is 5-15 g / L, for example, it can be 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L or 14 g / L, etc.

[0018] The mass percentage of glucose (anhydrous glucose) in the primary seed culture medium is 1%-3%, for example, it can be 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5% or 2.8%, etc.

[0019] The mass percentage of yeast powder in the primary seed culture medium is 0.6%-2%, for example, it can be 0.8%, 1%, 1.2%, 1.5% or 1.8%.

[0020] The mass percentage of casein peptone in the primary seed culture medium is 1.5%-3.2%, for example, it can be 1.6%, 1.8%, 2%, 2.2%, 2.5%, 2.8% or 3%.

[0021] Preferably, the balance in the primary seed culture medium is water.

[0022] Preferably, the temperature of the primary seed culture is 30-33°C, for example, it can be 30.2°C, 30.5°C, 30.8°C, 31°C, 31.2°C, 31.5°C, 31.8°C, 32°C, 32.2°C, 32.5°C or 32.8°C, and more preferably 31.5-32.5°C.

[0023] Preferably, the primary seed culture time is 16-18 hours, for example, it can be 16.2 hours, 16.5 hours, 16.8 hours, 17 hours, 17.2 hours, 17.5 hours or 17.8 hours.

[0024] Preferably, the primary seed culture is carried out under stirring conditions, and the stirring speed is 100-300 rpm, for example, it can be 120 rpm, 150 rpm, 180 rpm, 200 rpm, 220 rpm, 250 rpm or 280 rpm, and more preferably 170-190 rpm.

[0025] Preferably, the primary seeds are cultured to an OD value of 4.2-6.8, for example, an OD value of 4.3, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5 or 6.7, etc., to obtain primary seeds.

[0026] Preferably, the secondary seed culture medium used for the secondary seed culture comprises the following components: 5-20 g / L first acetate, 1%-3% glucose, 0.6%-2% yeast powder, 1.5%-3.2% casein peptone, 0.8%-2.4% magnesium sulfate, and 0.2%-0.8% ammonium sulfate hydrochloride.

[0027] Specifically, the mass concentration of the first acetate (preferably sodium acetate) in the secondary seed culture medium is 5-20 g / L, for example, it can be 6 g / L, 8 g / L, 10 g / L, 11 g / L, 13 g / L, 15 g / L, 17 g / L or 19 g / L, etc.

[0028] The mass percentage of glucose (anhydrous glucose) in the secondary seed culture medium is 1%-3%, for example, it can be 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5% or 2.8%, etc.

[0029] The mass percentage of yeast powder in the secondary seed culture medium is 0.6%-2%, for example, it can be 0.8%, 1%, 1.2%, 1.5% or 1.8%.

[0030] The mass percentage of casein peptone in the secondary seed culture medium is 1.5%-3.2%, for example, it can be 1.6%, 1.8%, 2%, 2.2%, 2.5%, 2.8% or 3%.

[0031] The mass percentage of magnesium sulfate in the secondary seed culture medium is 0.8%-2.4%, for example, it can be 1%, 1.2%, 1.5%, 1.8%, 2% or 2.2%.

[0032] The mass percentage of ammonium sulfate hydrochloride in the secondary seed culture medium is 0.2%-0.8%, for example, it can be 0.3%, 0.4%, 0.5%, 0.6% or 0.7%.

[0033] Preferably, the secondary seed culture medium further includes a first trace element additive, and the amount of the first trace element additive is 0.8%-2.6%, for example, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2% or 2.5%. In this application, the percentages used in the description of the amount of the first trace element additive are by mass.

[0034] Preferably, the balance in the secondary seed culture medium is water.

[0035] Preferably, the first trace element additive comprises the following components: copper sulfate 4-8 g / L, sodium iodide 0.12-0.16 g / L, manganese sulfate 5-6 g / L, sodium molybdate 0.1-0.2 g / L, boric acid 0.04-0.08 g / L, cobalt chloride 0.6-0.8 g / L, zinc chloride 18-21 g / L, ferrous sulfate 38-41 g / L, biotin 0.3-0.5 g / L, and sulfuric acid 3-5.2 g / L.

[0036] Specifically, the mass concentration of copper sulfate in the first trace element additive is 4-8 g / L, for example, it can be 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L or 7.5 g / L.

[0037] The mass concentration of sodium iodide in the first trace element additive is 0.12-0.16 g / L, for example, it can be 0.125 g / L, 0.13 g / L, 0.135 g / L, 0.14 g / L, 0.145 g / L, 0.15 g / L or 0.155 g / L.

[0038] The mass concentration of manganese sulfate in the first trace element additive is 5-6 g / L, for example, it can be 5.1 g / L, 5.2 g / L, 5.3 g / L, 5.4 g / L, 5.5 g / L, 5.6 g / L, 5.7 g / L, 5.8 g / L or 5.9 g / L.

[0039] The mass concentration of sodium molybdate in the first trace element additive is 0.1-0.2 g / L, for example, it can be 0.11 g / L, 0.12 g / L, 0.13 g / L, 0.14 g / L, 0.15 g / L, 0.16 g / L, 0.17 g / L, 0.18 g / L or 0.19 g / L.

[0040] The mass concentration of boric acid in the first trace element additive is 0.04-0.08 g / L, for example, it can be 0.045 g / L, 0.05 g / L, 0.055 g / L, 0.06 g / L, 0.065 g / L, 0.07 g / L or 0.075 g / L.

[0041] The mass concentration of cobalt chloride in the first trace element additive is 0.6-0.8 g / L, for example, it can be 0.62 g / L, 0.65 g / L, 0.68 g / L, 0.7 g / L, 0.72 g / L, 0.75 g / L or 0.78 g / L.

[0042] The mass concentration of zinc chloride in the first trace element additive is 18-21 g / L, for example, 18.5 g / L, 19 g / L, 19.5 g / L, 20 g / L or 20.5 g / L.

[0043] The mass concentration of ferrous sulfate in the first trace element additive is 38-41 g / L, for example, 38.5 g / L, 39 g / L, 39.5 g / L, 40 g / L or 40.5 g / L.

[0044] The mass concentration of biotin in the first trace element additive is 0.3-0.5 g / L, for example, it can be 0.32 g / L, 0.35 g / L, 0.38 g / L, 0.4 g / L, 0.42 g / L, 0.45 g / L or 0.48 g / L.

[0045] The mass concentration of sulfuric acid in the first trace element additive is 3-5.2 g / L, for example, 3.2 g / L, 3.5 g / L, 3.8 g / L, 4 g / L, 4.2 g / L, 4.5 g / L, 4.8 g / L or 5 g / L.

[0046] Preferably, the first-level seeds obtained from the first-level seed culture are inoculated into the second-level seed culture medium at an inoculation rate of 5%-10% for second-level seed culture. The inoculation rate of the first-level seeds can be 6%, 7%, 8% or 9%, etc., and more preferably 7%-8%.

[0047] It should be noted that the term "inoculum size" refers to the ratio of the volume of the seed solution transferred to the volume of the culture system after inoculation. For example, an inoculum size of 5%-10% represents a 5%-10% volume fraction of the seed solution. For simplicity, all references to "inoculum size" will refer to volume fractions, and for clarity, individual descriptions will not be given.

[0048] Preferably, air (compressed air) is introduced during the cultivation of the secondary seeds.

[0049] Preferably, the ventilation rate of the secondary seed culture is 0.1-1.3 vvm, for example, it can be 0.2 vvm, 0.3 vvm, 0.5 vvm, 0.7 vvm, 0.9 vvm, 1 vvm or 1.2 vvm, etc.

[0050] Preferably, the secondary seed culture is carried out under stirring conditions, and the stirring speed is 150-900 rpm, for example, it can be 180 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm or 800 rpm.

[0051] As a preferred technical solution of the present application, during the secondary seed cultivation process, the stirring speed and the compressed air ventilation speed are gradually increased according to the continuous decrease of dissolved oxygen.

[0052] For example, during the secondary seed cultivation process, the initial dissolved oxygen is 100%, the stirring speed is 160-200 rpm, and the ventilation speed is 0.1-0.3 vvm; 80% ≤ dissolved oxygen <100%, the stirring speed is 210-230 rpm, and the ventilation speed is 0.4-0.5 vvm; 60% ≤ dissolved oxygen <80%, the stirring speed is 260-300 rpm, and the ventilation speed is 0.3-0.5 vvm; 40% ≤ dissolved oxygen <60%, the stirring speed is 300-400 rpm, and the ventilation speed is 0. The concentration of dissolved oxygen is 0.4-0.6vvm; 20% ≤ dissolved oxygen < 40%, the stirring speed is 400-500rpm, and the ventilation speed is 0.5-0.7vvm; 10% ≤ dissolved oxygen < 20%, the stirring speed is 500-550rpm, and the ventilation speed is 0.6-0.8vvm; 0 < dissolved oxygen < 10%, the stirring speed is 550-650rpm, and the ventilation speed is 0.7-0.9vvm; the dissolved oxygen is 0, the stirring speed is 700-800rpm, and the ventilation speed is 0.9-1.2vvm.

[0053] Preferably, the temperature of the secondary seed culture is 30-33°C, for example, it can be 30.2°C, 30.5°C, 30.8°C, 31°C, 31.2°C, 31.5°C, 31.8°C, 32°C, 32.2°C, 32.5°C or 32.8°C, etc., and more preferably 30-32°C.

[0054] Preferably, the secondary seed culture time is 8-12 hours, for example, it can be 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours or 11.5 hours.

[0055] Preferably, the secondary seeds are cultured to an OD value of 12-16, for example, an OD value of 12.5, 13, 13.5, 14, 14.5, 15 or 15.5, to obtain the seed solution.

[0056] Preferably, the carbon source of the fermentation medium comprises a combination of a second acetate and a ketone compound.

[0057] Preferably, the second acetate includes any one of sodium acetate, potassium acetate, and ammonium acetate, or a combination of at least two thereof, preferably sodium acetate.

[0058] Preferably, the mass concentration of the second acetate in the fermentation medium is 5-15 g / L, for example, it can be 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L or 14 g / L, etc.

[0059] Preferably, the mass percentage of the ketone compound in the fermentation medium is 0.1%-0.6%, for example, 0.2%, 0.3%, 0.4%, or 0.5%. A specific amount of ketone compound catalyzes the intermediates of the acetic acid metabolic pathway of the microorganism during high-density fermentation, thereby helping to increase the growth rate of the microorganism and shorten the fermentation cycle.

[0060] Preferably, the fermentation medium comprises the following components: 5-15 g / L of second acetate, 1%-3% of glucose, 0.6%-2% of yeast powder, 1.5%-3.2% of casein peptone, 0.8%-1.2% of magnesium sulfate, 0.6%-1.4% of ammonium sulfate hydrochloride, 0.5%-1% of betaine, 3.5%-6.8% of ammonium sulfate, 5%-7% of potassium dihydrogen phosphate, 2%-4% of disodium hydrogen phosphate, and 0.1%-0.6% of ketone compounds.

[0061] Specifically, the mass concentration of the second acetate (preferably sodium acetate) in the fermentation medium is 5-15 g / L, for example, it can be 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L or 14 g / L, etc.

[0062] The mass percentage of glucose (anhydrous glucose) in the fermentation medium is 1%-3%, for example, it can be 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5% or 2.8%.

[0063] The mass percentage of yeast powder in the fermentation medium is 0.6%-2%, for example, 0.8%, 1%, 1.2%, 1.5% or 1.8%.

[0064] The mass percentage of casein peptone in the fermentation medium is 1.5%-3.2%, for example, it can be 1.6%, 1.8%, 2%, 2.2%, 2.5%, 2.8% or 3%.

[0065] The mass percentage of magnesium sulfate in the fermentation medium is 0.8%-1.2%, for example, it can be 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1% or 1.15%.

[0066] The mass percentage of ammonium sulfate hydrochloride in the fermentation medium is 0.6%-1.4%, for example, it can be 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2% or 1.3%.

[0067] The mass percentage of betaine in the fermentation medium is 0.5%-1%, for example, 0.6%, 0.7%, 0.8% or 0.9%.

[0068] The mass percentage of ammonium sulfate in the fermentation medium is 3.5%-6.8%, for example, it can be 3.8%, 4%, 4.2%, 4.5%, 4.8%, 5%, 5.2%, 5.5%, 5.8%, 6%, 6.2% or 6.5%.

[0069] The mass percentage of potassium dihydrogen phosphate in the fermentation medium is 5%-7%, for example, it can be 5.2%, 5.5%, 5.8%, 6%, 6.2%, 6.5% or 6.8%.

[0070] The mass percentage of disodium hydrogen phosphate in the fermentation medium is 2%-4%, for example, it can be 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5% or 3.8%.

[0071] The mass percentage of ketone compounds in the fermentation medium is 0.1%-0.6%, for example, 0.2%, 0.3%, 0.4% or 0.5%.

[0072] Preferably, the ketone compound is a C3-C10 ketone compound, for example, it can be a C3, C4, C5, C6, C7, C8, C9, C10 ketone compound, including aliphatic ketone and / or alicyclic ketone.

[0073] Preferably, the ketone compound includes any one of acetone, methyl ethyl ketone, and cyclohexanone, or a combination of at least two thereof, with acetone being more preferred.

[0074] As a preferred technical solution of the present application, the fermentation medium contains 0.1%-0.6% acetone. Since acetone acts as a catalytic intermediate in the acetic acid metabolic pathway, the introduction of acetone can further increase the growth rate of the bacteria and shorten the fermentation cycle.

[0075] Preferably, the fermentation medium further includes a second trace element additive, and the amount of the second trace element additive is 0.8%-2.6%, for example, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2% or 2.5%. In this application, the percentages used in the description of the amount of the second trace element additive are by mass percentage.

[0076] Preferably, the balance of the fermentation medium is water.

[0077] Preferably, the second trace element additive comprises the following components: copper sulfate 4-8 g / L, sodium iodide 0.12-0.16 g / L, manganese sulfate 5-6 g / L, sodium molybdate 0.1-0.2 g / L, boric acid 0.04-0.08 g / L, cobalt chloride 0.6-0.8 g / L, zinc chloride 18-21 g / L, ferrous sulfate 38-41 g / L, biotin 0.3-0.5 g / L, and sulfuric acid 3-5.2 g / L.

[0078] Specifically, the mass concentration of copper sulfate in the second trace element additive is 4-8 g / L, for example, it can be 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L or 7.5 g / L.

[0079] The mass concentration of sodium iodide in the second trace element additive is 0.12-0.16 g / L, for example, it can be 0.125 g / L, 0.13 g / L, 0.135 g / L, 0.14 g / L, 0.145 g / L, 0.15 g / L or 0.155 g / L.

[0080] The mass concentration of manganese sulfate in the second trace element additive is 5-6 g / L, for example, 5.1 g / L, 5.2 g / L, 5.3 g / L, 5.4 g / L, 5.5 g / L, 5.6 g / L, 5.7 g / L, 5.8 g / L or 5.9 g / L.

[0081] The mass concentration of sodium molybdate in the second trace element additive is 0.1-0.2 g / L, for example, it can be 0.11 g / L, 0.12 g / L, 0.13 g / L, 0.14 g / L, 0.15 g / L, 0.16 g / L, 0.17 g / L, 0.18 g / L or 0.19 g / L.

[0082] The mass concentration of boric acid in the second trace element additive is 0.04-0.08 g / L, for example, it can be 0.045 g / L, 0.05 g / L, 0.055 g / L, 0.06 g / L, 0.065 g / L, 0.07 g / L or 0.075 g / L.

[0083] The mass concentration of cobalt chloride in the second trace element additive is 0.6-0.8 g / L, for example, it can be 0.62 g / L, 0.65 g / L, 0.68 g / L, 0.7 g / L, 0.72 g / L, 0.75 g / L or 0.78 g / L.

[0084] The mass concentration of zinc chloride in the second trace element additive is 18-21 g / L, for example, 18.5 g / L, 19 g / L, 19.5 g / L, 20 g / L or 20.5 g / L.

[0085] The mass concentration of ferrous sulfate in the second trace element additive is 38-41 g / L, for example, 38.5 g / L, 39 g / L, 39.5 g / L, 40 g / L or 40.5 g / L.

[0086] The mass concentration of biotin in the second trace element additive is 0.3-0.5 g / L, for example, it can be 0.32 g / L, 0.35 g / L, 0.38 g / L, 0.4 g / L, 0.42 g / L, 0.45 g / L or 0.48 g / L.

[0087] The mass concentration of sulfuric acid in the second trace element additive is 3-5.2 g / L, for example, 3.2 g / L, 3.5 g / L, 3.8 g / L, 4 g / L, 4.2 g / L, 4.5 g / L, 4.8 g / L or 5 g / L.

[0088] Preferably, the seed liquid is inoculated into the fermentation medium at an inoculation rate of 3%-6% for high-density fermentation. The inoculation rate of the seed liquid can be 3.5%, 4%, 4.5%, 5% or 5.5%, etc.

[0089] Preferably, the nitrogen source added during the high-density fermentation process includes any one of urea, ammonium sulfate, ammonium nitrate, and ammonia water, or a combination of at least two of them, with urea being more preferred.

[0090] As the preferred technical solution of the present application, the carbon source added during the high-density fermentation is acetic acid, and the nitrogen source added is urea. Since the optimal growth pH of the thalline is a neutral environment, but its fermentation process produces alkaline substances, the pH will gradually increase. Therefore, adding acetic acid can both neutralize and stabilize the pH, and can provide a carbon source for growth, and does not require sterilization and can be directly added; Moreover, the carbon content in acetic acid is high, and it is not necessary to add water to prepare a solution when adding, which is more conducive to achieving high-density fermentation. If a carbon source (such as glucose) that needs to be prepared in an aqueous solution is selected, more water will be added while adding the carbon source to the fermentation system, increasing the volume of the fermentation liquid and diluting the fermentation liquid, making it difficult to achieve high-density fermentation. Using urea as a nitrogen source, it is more easily absorbed by the thalline, which is conducive to protein synthesis. By designing and compounding the carbon source and nitrogen source added, the rapid growth of the thalline and the synthesis of protein are more effectively promoted.

[0091] Preferably, the urea is added in the form of a urea aqueous solution.

[0092] Preferably, the mass percentage of urea in the urea aqueous solution is 30-50%, such as 32%, 35%, 38%, 40%, 42%, 45% or 48%, and more preferably 40%.

[0093] Preferably, the temperature of the high-density fermentation is 27-33°C, for example, it can be 27.5°C, 28°C, 28.5°C, 29°C, 29.5°C, 30°C, 30.5°C, 31°C, 31.5°C, 32°C or 32.5°C, and more preferably 28-32°C.

[0094] Preferably, the pH value of the high-density fermentation is 6.4-7.1, for example, it can be 6.5, 6.6, 6.7, 6.8, 6.9 or 7, and more preferably 6.5-7.

[0095] Preferably, the high-density fermentation is carried out under stirring conditions, and the stirring speed is 130-800 rpm, for example, it can be 150 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm or 750 rpm.

[0096] Preferably, the aeration rate of the high-density fermentation is 0.5-2.4 vvm, for example, it can be 0.6 vvm, 0.8 vvm, 1 vvm, 1.2 vvm, 1.5 vvm, 1.8 vvm, 2 vvm or 2.2 vvm, etc.

[0097] Preferably, the gas introduced during the high-density fermentation process includes air and optionally oxygen (pure oxygen).

[0098] As a preferred technical solution of the present application, by regulating the gas components and the introduction rate during the high-density fermentation process, it is more conducive to promoting bacterial growth and protein synthesis. Specifically:

[0099] When the OD value is less than 300, air (compressed air) is introduced at a ventilation rate of 0.6-1vvm;

[0100] When 300≤OD value<500, a mixture of air and oxygen is introduced, with the ratio of air to oxygen being (1.5-2.5):1, for example, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, etc., preferably 2:1; the ventilation speed is 0.8-1.2 vvm;

[0101] When the OD value is 500-700, a mixture of air and oxygen is introduced, with the ratio of air to oxygen being (0.8-1.2):1, for example, 0.9:1, 1:1, 1.1:1, etc., preferably 1:1, and the ventilation rate is 1.2-1.8 vvm;

[0102] When the OD value is greater than 700, a mixture of air and oxygen is introduced, with the ratio of air to oxygen being 1:(1.5-2.5), for example, 1:1.6, 1:1.8, 1:2, 1:2.2 or 1:2.4, preferably 1:2, and the ventilation rate being 1.8-2.4 vvm.

[0103] Preferably, the endpoint judgment method of the high-density fermentation is: monitoring the OD value of the high-density fermentation system, and if the increase in the OD value per hour for 2 consecutive hours is ≤20, it indicates that the bacteria grow slowly, then it is judged that there will be no further growth, and the endpoint of high-density fermentation is reached, and the fermentation liquid is removed from the tank to obtain the fermentation liquid.

[0104] In this application, "OD value" refers to the OD value of the fermentation broth (culture system), which is obtained by ultraviolet spectrophotometry, specifically the OD value at 600 nm.

[0105] Preferably, the high-density fermentation is performed to an OD value of 780-820, for example, an OD value of 782, 785, 788, 790, 792, 795, 798, 800, 802, 805, 808, 810, 812, 815 or 818, etc., and the fermentation is terminated to obtain the single-cell protein.

[0106] Preferably, the high-density fermentation includes a first fermentation stage, a second fermentation stage, and a third fermentation stage in which the OD values ​​increase sequentially;

[0107] The OD value of the first fermentation stage is less than 500, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source in the first fermentation stage is greater than 18, for example, the carbon-nitrogen ratio is 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100, etc.

[0108] The OD value of the second fermentation stage is 500-700, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source in the second fermentation stage is 5-18, for example, the carbon-nitrogen ratio is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.

[0109] The OD value of the third fermentation stage is greater than 700, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source in the third fermentation stage is less than 5, for example, the carbon-nitrogen ratio is 1, 1.5, 2, 2.5, 3, 3.5, 4 or 4.5.

[0110] As the preferred technical solution of the present application, the carbon-nitrogen ratio of the carbon source and nitrogen source added during high-density fermentation is regulated. In the early stage when the OD value is low, the carbon-nitrogen ratio is set to be >18, and this stage is conducive to vigorous bacterial growth; when the OD value is 500-700, the carbon-nitrogen ratio is set to be 5-18, which is conducive to synthesizing protein while bacterial growth; to the later stage of high-density fermentation, the carbon-nitrogen ratio is set to be <5, bacterial growth slows down, and the speed of protein synthesis increases at the same time, fully synthesizing protein, which is conducive to improving the content of crude protein in the fermentation product. The present application can achieve the fastest bacterial growth and the highest crude protein content by adjusting the carbon-nitrogen ratio, and improve the yield of protein while shortening the fermentation culture cycle, thereby achieving the simultaneous improvement of the production efficiency and yield of single-cell protein.

[0111] In the present application, the feeding rate of acetic acid and nitrogen source can be regulated according to the carbon-nitrogen ratio of high-density fermentation at different stages; illustratively, the feeding rate of acetic acid is 0.6-2.8 g / L, for example, it can be 0.8 g / L, 1 g / L, 1.2 g / L, 1.5 g / L, 1.8 g / L, 2 g / L, 2.2 g / L, 2.5 g / L or 2.7 g / L, etc., further preferably 0.8-2.5 g / L; the feeding rate of nitrogen source is 0.1-3.2 g / L, for example, it can be 0.2 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 1.2 g / L, 1.5 g / L, 1.8 g / L, 2 g / L, 2.2 g / L, 2.5 g / L, 2.8 g / L or 3 g / L, etc.

[0112] Preferably, in the early stages of high-density fermentation, the feed rates of acetic acid and nitrogen source are regulated to set a carbon-nitrogen ratio greater than 18 to promote vigorous bacterial growth. As fermentation progresses, the feed rates of acetic acid and nitrogen source are adjusted to maintain a carbon-nitrogen ratio of 5-18, promoting rapid bacterial growth and simultaneous protein synthesis. The feed rates of acetic acid and nitrogen source are further adjusted to maintain a carbon-nitrogen ratio less than 5, thereby facilitating single-cell protein synthesis and increasing crude protein content. By regulating and balancing the carbon-nitrogen ratio throughout the high-density fermentation stage, the optimal results of achieving the fastest bacterial growth and the highest crude protein content are achieved.

[0113] As the preferred technical solution of the present application, by regulating the processes of different periods of high-density fermentation, including the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen and ventilation volume, the bacterial growth environment is adjusted to achieve the optimal growth conditions of the bacteria and the optimal state of single-cell protein synthesis.

[0114] Preferably, the first fermentation stage includes stage A, stage B and stage C performed sequentially, thereby making the high-density fermentation include stage A, stage B, stage C, the second fermentation stage and the third fermentation stage performed sequentially, specifically as follows:

[0115] In the stage A, the OD value is less than 100, the temperature is 31-33° C., the pH value is 6.4-6.6, the aeration rate is 0.5-0.7 vvm, the rotation speed is 130-170 rpm, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source is greater than 18;

[0116] Further preferably, air (compressed air) is introduced into stage A, the temperature is controlled at 32°C, the pH value is 6.5, the ventilation rate is 0.6vvm, the rotation speed is 150rpm, the acetic acid feeding rate is 0.8L / h, and the carbon-nitrogen ratio is controlled to be greater than 18. Under these conditions, the bacteria grow rapidly.

[0117] In the stage B, 100≤OD value<300, temperature is 30-32°C, pH value is 6.5-6.7, aeration rate is 0.9-1.1vvm, rotation speed is 180-220rpm, and carbon-nitrogen ratio of acetic acid and nitrogen source is >18;

[0118] Further preferably, air (compressed air) is introduced into the stage B, the temperature is controlled at 31°C, the pH value is 6.6, the ventilation rate is 1vvm, the rotation speed is 200rpm, the acetic acid feeding rate is 1.2L / h, and the carbon-nitrogen ratio is controlled to be greater than 18. Under these conditions, the bacteria grow rapidly.

[0119] In the stage C, 300≤OD value<500, temperature is 29-31°C, pH value is 6.6-6.8, aeration rate is 1.1-1.3vvm, rotation speed is 280-320rpm, and carbon-nitrogen ratio of acetic acid and nitrogen source is >18;

[0120] Further preferably, in stage C, a mixture of air and oxygen is introduced, the ratio of air to oxygen is (1.5-2.5):1, more preferably 2:1, the temperature is controlled at 30°C, the pH value is 6.7, the ventilation rate is 1.2vvm, the rotation speed is 300rpm, the acetic acid feeding rate is 1.6L / h, and the carbon-nitrogen ratio is controlled to be greater than 18. Under these conditions, the bacteria grow rapidly.

[0121] The OD value of the second fermentation stage is 500-700, the temperature is 28-30°C, the pH value is 6.7-6.9, the aeration rate is 1.5-1.7 vvm, the rotation speed is 450-550 rpm, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source is 5-18;

[0122] Further preferably, a mixture of air and oxygen is introduced into the second fermentation stage, the ratio of air to oxygen is (0.8-1.2):1, more preferably 1:1, the temperature is controlled at 29°C, the pH value is 6.8, the ventilation rate is 1.6vvm, the rotation speed is 500rpm, the acetic acid feeding rate is 2.2L / h, and the carbon-nitrogen ratio is controlled at 5-18. Under these conditions, the bacterial growth rate is normal and single-cell protein is synthesized at the same time.

[0123] The third fermentation stage has an OD value greater than 700, a temperature of 27-29° C., a pH of 6.9-7.1, an aeration rate of 1.9-2.1 vvm, a rotation speed of 650-750 rpm, and a carbon-nitrogen ratio of less than 5 for the added acetic acid and nitrogen source;

[0124] Further preferably, a mixture of air and oxygen is introduced into the third fermentation stage, the ratio of air to oxygen is 1:(1.5-2.5), more preferably 1:2, the temperature is controlled at 28°C, the pH value is 7, the ventilation rate is 2.0vvm, the rotation speed is 700rpm, the acetic acid feeding rate is 2.5L / h, and the carbon-nitrogen ratio is controlled to be less than 5. Under these conditions, the bacterial growth rate is slow and single-cell protein is fully synthesized.

[0125] When the OD value reaches 780-820 and the bacterial growth slows down, the culture can be put into the tank to complete high-density fermentation and obtain fermentation liquid.

[0126] Preferably, the high-density fermentation further includes a post-processing step after completion.

[0127] Preferably, the post-treatment method comprises: sequentially performing solid-liquid separation, washing and drying on the fermentation liquid obtained by the high-density fermentation to obtain the single-cell protein.

[0128] Preferably, the solid-liquid separation method is centrifugal separation.

[0129] Preferably, the washing detergent is water.

[0130] Preferably, the drying method comprises spray drying.

[0131] Preferably, the method specifically comprises the following steps:

[0132] (1) inoculating Yarrowia lipolytica into a primary seed culture medium, culturing at a temperature of 30-33° C. and a rotation speed of 100-300 rpm for 16-18 hours to an OD value of 4.2-6.8, and obtaining a primary seed;

[0133] The primary seed culture medium comprises the following components: 5-15 g / L sodium acetate, 1%-3% glucose, 0.6%-2% yeast powder, and 1.5%-3.2% casein peptone;

[0134] (2) inoculating the primary seeds into a secondary seed culture medium at an inoculum size of 5%-10%, and culturing the culture medium at a temperature of 30-33° C., a rotation speed of 150-900 rpm, and an aeration rate of 0.1-1.3 vvm for 8-12 hours until the OD value reaches 12-16, thereby obtaining secondary seeds, i.e., the seed solution;

[0135] The secondary seed culture medium comprises the following components: 5-20 g / L sodium acetate, 1%-3% glucose, 0.6%-2% yeast powder, 1.5%-3.2% casein peptone, 0.8%-2.4% magnesium sulfate, 0.2%-0.8% ammonium sulfate hydrochloride, and 0.8%-2.6% of a first trace element additive;

[0136] The first trace element additive comprises the following components: copper sulfate 4-8 g / L, sodium iodide 0.12-0.16 g / L, manganese sulfate 5-6 g / L, sodium molybdate 0.1-0.2 g / L, boric acid 0.04-0.08 g / L, cobalt chloride 0.6-0.8 g / L, zinc chloride 18-21 g / L, ferrous sulfate 38-41 g / L, biotin 0.3-0.5 g / L, and sulfuric acid 3-5.2 g / L;

[0137] (3) inoculating the seed liquid into a fermentation medium at an inoculum size of 3%-6% for high-density fermentation, and adding acetic acid and urea during the high-density fermentation until the OD value reaches 780-820, thereby obtaining the single-cell protein;

[0138] The fermentation medium comprises the following components: 5-15 g / L sodium acetate, 1%-3% glucose, 0.6%-2% yeast powder, 1.5%-3.2% casein peptone, 0.8%-1.2% magnesium sulfate, 0.6%-1.4% ammonium sulfate hydrochloride, 0.5%-1% betaine, 3.5%-6.8% ammonium sulfate, 5%-7% potassium dihydrogen phosphate, 2%-4% disodium hydrogen phosphate, 0.1%-0.6% acetone, and 0.8%-2.6% of a second trace element additive;

[0139] The second trace element additive comprises the following components: copper sulfate 4-8 g / L, sodium iodide 0.12-0.16 g / L, manganese sulfate 5-6 g / L, sodium molybdate 0.1-0.2 g / L, boric acid 0.04-0.08 g / L, cobalt chloride 0.6-0.8 g / L, zinc chloride 18-21 g / L, ferrous sulfate 38-41 g / L, biotin 0.3-0.5 g / L, and sulfuric acid 3-5.2 g / L;

[0140] The high-density fermentation includes a first fermentation stage, a second fermentation stage, and a third fermentation stage with OD values ​​increasing in sequence;

[0141] The first fermentation stage has an OD value of <500, a temperature of 29-33°C, a pH of 6.5-6.8, an aeration rate of 0.5-1.3 vvm, a rotation speed of 280-320 rpm, and a carbon-nitrogen ratio of >18 for the added acetic acid and urea;

[0142] The OD value of the second fermentation stage is 500-700, the temperature is 28-30°C, the pH value is 6.7-6.9, the aeration rate is 1.5-1.7 vvm, the rotation speed is 450-550 rpm, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source is 5-18;

[0143] The OD value of the third fermentation stage is greater than 700, the temperature is 27-29° C., the pH value is 6.9-7.1, the ventilation speed is 1.9-2.1 vvm, the rotation speed is 650-750 rpm, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source is less than 5.

[0144] As a preferred technical solution of the present application, the time of high-density fermentation (i.e., fermentation cycle) is 25-60h, for example, it can be 26h, 28h, 30h, 32h, 35h, 38h, 40h, 42h, 45h, 48h, 50h, 52h, 55h, 58h, etc., further preferably 28-36h, and even more preferably 28-32h.

[0145] Preferably, the total yield (dry weight) of the single cell protein is >150 g / L, for example, it can be 155 g / L, 160 g / L, 170 g / L, 180 g / L, 190 g / L, 200 g / L, 210 g / L, 220 g / L, 230 g / L, 240 g / L, 250 g / L or 260 g / L, etc., more preferably ≥160 g / L, and even more preferably 200-250 g / L.

[0146] Preferably, the single-cell protein yield is >2.5 g / (L·h), for example, it can be 2.6 g / (L·h), 2.8 g / (L·h), 3 g / (L·h), 3.5 g / (L·h), 4 g / (L·h), 5 g / (L·h), 6 g / (L·h), 7 g / (L·h), 8 g / (L·h), 8.5 g / (L·h), 9 g / (L·h) or 10 g / (L·h), etc., more preferably ≥3 g / (L·h), and even more preferably 6-9 g / (L·h).

[0147] Preferably, the mass percentage of crude protein in the single cell protein is ≥35%, for example, it can be 38%, 40%, 42%, 45%, 48%, 50%, 52%, 55%, 58%, 60%, 62%, 65% or 68%, etc., more preferably ≥55%, and even more preferably ≥60%.

[0148] Compared with the related art, the embodiments of the present application have the following beneficial effects:

[0149] (1) In the method for producing single-cell protein by high-density fermentation provided in the embodiment of the present application, by adding a first acetate to the carbon source of the seed culture medium, the bacteria are induced and promoted to produce a unique acetic acid metabolic pathway, laying the foundation for adding acetic acid as a carbon source in the subsequent high-density fermentation and promoting the rapid growth of the bacteria; at the same time, the fermentation medium contains a second acetate and / or ketone compounds to promote the rapid growth of the bacteria; in the high-density fermentation stage, nitrogen source and acetic acid are added to put the bacteria in the optimal neutral pH growth environment, and the carbon-nitrogen ratio is regulated to supplement the bacteria with appropriate nutrients for continuous growth of the bacteria, effectively improving the growth rate of the bacteria and the synthesis rate of single-cell protein. By compounding specific materials and processes, the method can achieve high-density fermentation of bacteria and efficient synthesis of single-cell protein in a short time, while significantly shortening the culture time of high-density fermentation, and can obtain fermentation products with higher crude protein content, greatly improving the production efficiency and yield of single-cell protein.

[0150] (2) The embodiments of the present application achieve rapid growth of bacteria and rapid synthesis of single-cell protein by further optimizing the design of materials and processes, adapting the culture medium composition, specific feeding method and step-by-step process control strategy during high-density fermentation, so that the time of high-density fermentation (fermentation cycle) is 28-30 hours, which is shortened to 1 / 3 of the single-cell protein production time of related technologies. At the same time, the dry weight of single-cell protein is 200-243g / L, and the output is ≥6g / (L·h), which is 20 times higher than the output of the same type; the crude protein content is ≥60%, which is 50% higher than that of related technologies, thereby significantly improving the output and production capacity of single-cell protein.

[0151] Still other aspects will become apparent upon reading and understanding the accompanying drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] The accompanying drawings are used to provide a further understanding of the technical solution of this article and constitute a part of the specification. Together with the embodiments of this application, they are used to explain the technical solution of this article and do not constitute a limitation on the technical solution of this article.

[0153] FIG1 is a growth trend diagram of the high-density fermentation process in the method provided in Example 1;

[0154] FIG2 is a test diagram of the fermentation broth obtained by the method provided in Example 1. DETAILED DESCRIPTION

[0155] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0156] As used herein, the terms "comprises," "including," "having," "containing" or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a listed element is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0157] In this application, features defined as "first" or "second" may explicitly or implicitly include one or more of the features, and are used to distinguish and describe features, without distinction of order or importance. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0158] In this application, the term "OD value" refers to the OD value of the fermentation broth, which is obtained by ultraviolet spectrophotometry. Specifically, the OD value of the fermentation broth at 600 nm is detected using an ultraviolet spectrophotometer (Alpha-1106, Shanghai Spectron Instrument Co., Ltd.).

[0159] In the following specific embodiments of the present application, the test methods for dry weight, yield and crude protein content are as follows:

[0160] (1) Dry weight of single cell protein: g / L

[0161] Weigh 1.00 L of fermentation liquid, centrifuge at 8000 rpm for 10 min, add 1 L of water to obtain the bacteria, resuspend evenly, and centrifuge again at 8000 rpm for 10 min to obtain wet bacteria, and weigh its mass as m1; take 5.000 g from m1 and dry it at 103±2°C according to the national standard GB / T6435-2014 "Determination of Water in Feed" to obtain dry bacteria, and weigh its weight m2 (accurate to 4 decimal places).

[0162] Dry cell weight DCW = m2 / 5.000 × m1 / 1.00

[0163] (2) Output: Unit: g / (L·h)

[0164] Yield = dry weight of single-cell protein ÷ time of high-density fermentation (fermentation cycle).

[0165] (3) Crude protein content

[0166] 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.

[0167] In the following specific embodiments of the present application, the Yarrowia lipolytica strain was purchased from the China Industrial Microbiological Culture Collection Center with the collection number CICC 32291; and all components of the culture medium were commercially available chemicals.

[0168] In the following specific embodiments of the present application, the method for judging the endpoint of the high-density fermentation is: if the increase in the OD value per hour is less than 20 for 2 consecutive hours, it indicates that the bacterial growth is very slow, and it is judged that there will be no further growth, and the end point of high-density fermentation is reached, and the fermentation liquid is obtained by tank removal.

[0169] Example 1

[0170] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0171] (1) Primary seed culture:

[0172] Prepare 200 mL of primary seed culture medium consisting of 8 g / L sodium acetate, 1.8% anhydrous glucose, 1.2% yeast extract, and 2.2% casein peptone; sterilize in an autoclave at 121°C for 20 min, place in a clean bench after sterilization, and after returning to room temperature, add the thawed frozen stock of Yarrowia lipolytica to the primary seed culture medium. Place in a shaker and incubate at 32°C and 180 rpm for 16 h. The OD value is approximately 4.2, thus obtaining primary seeds.

[0173] (2) Secondary seed culture:

[0174] Prepare 2 L of secondary seed culture medium, consisting of: 15 g / L sodium acetate, 2.2% anhydrous glucose, 1.5% yeast extract, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% ammonium sulfate hydrochloride;

[0175] The secondary seed culture medium was added to a 5L fermenter, sterilized in an autoclave at 121°C for 20 min, and after cooling to 32°C, the primary seeds were added to the secondary seed culture medium fermenter at a rate of 7.5% by fire ring inoculation.

[0176] Then add 20 mL of sterile filtered trace element additives with the following formula: copper sulfate 4 g / L, sodium iodide 0.12 g / L, manganese sulfate 5 g / L, sodium molybdate 0.1 g / L, boric acid 0.04 g / L, cobalt chloride 0.6 g / L, zinc chloride 18 g / L, ferrous sulfate 38 g / L, biotin 0.3 g / L, and sulfuric acid 3 g / L;

[0177] In the secondary seed culture, the initial dissolved oxygen is 100%, the stirring speed is set to 180 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.2 vvm; the dissolved oxygen is 80-100%, the stirring speed is set to 220 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.3 vvm; the dissolved oxygen is 60-80%, the stirring speed is set to 280 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.4 vvm; the dissolved oxygen is 40-60%, the stirring speed is set to 350 rpm, the temperature is 32°C, and the ventilation speed of compressed air is The speed is 0.5vvm; when the dissolved oxygen is 20-40%, the stirring speed is set to 440rpm, the temperature is 31°C, and the ventilation speed of compressed air is 0.6vvm; when the dissolved oxygen is 10-20%, the stirring speed is set to 520rpm, the temperature is 31°C, and the ventilation speed of compressed air is 0.7vvm; when the dissolved oxygen is 0-10%, the stirring speed is set to 600rpm, the temperature is 30°C, and the ventilation speed of compressed air is 0.8vvm; when the dissolved oxygen is 0%, the stirring speed is set to 700rpm, the temperature is 30°C, and the ventilation speed of compressed air is 0.9vvm.

[0178] The secondary seeds were cultured for 8 h, with an OD value of about 12, and the secondary seeds were obtained.

[0179] (3) High-density fermentation

[0180] Prepare 40 L of fermentation medium, consisting of: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast extract, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% ammonium sulfate hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone;

[0181] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0182] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0183] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 40% urea solution;

[0184] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0185] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0186] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0187] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, control carbon-nitrogen ratio at 20, under these conditions the bacteria grow rapidly;

[0188] The OD value was 500-700, the temperature was controlled at 29°C, the pH was 6.8, the mixed gas (air: pure oxygen at a ratio of 1:1) was aerated at a rate of 1.6 vvm, the rotation speed was 500 rpm, the carbon source feed rate was 2.2 L / h, and the carbon-nitrogen ratio was controlled at 10. Under these conditions, the bacterial cell growth rate was normal and single-cell protein was synthesized.

[0189] OD value > 700, temperature controlled at 28°C, pH 7, mixed gas (air: pure oxygen 1:2) ventilation rate 2.0vvm, rotation speed 700rpm, carbon source feeding rate 2.5L / h, controlled carbon-nitrogen ratio of 3. Under these conditions, the bacterial growth rate is slow and single-cell protein is fully synthesized.

[0190] After culturing for 30 hours, the OD value is 790, and the bacteria grow slowly, then the cells can be put into the tank to obtain the fermentation liquid.

[0191] (4) Post-processing

[0192] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 240 g / L, a yield of 8 g / (L·h), and a crude protein content of 62%.

[0193] During the high-density fermentation process of this embodiment, samples were taken every 1 h, and the bacterial OD was calculated after conversion and correction based on the absorption peak of the OD value of the fermentation liquid at 600 nm. The average value of three measurements was taken, and a growth trend graph was drawn based on the growth time and OD value, as shown in Figure 1.

[0194] The fermentation broth obtained by the high-density fermentation in this example was sampled for microscopic examination using a digital microscope (Longbase 610D, Qingdao Changji Medical Instrument Co., Ltd.). The obtained test graph is shown in FIG2 .

[0195] Example 2

[0196] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0197] (1) Primary seed culture:

[0198] Prepare 200 mL of primary seed culture medium consisting of 8 g / L sodium acetate, 1.8% anhydrous glucose, 1.2% yeast extract, and 2.2% casein peptone; sterilize in an autoclave at 121°C for 20 min, place in a clean bench after sterilization, and after returning to room temperature, add the thawed frozen stock of Yarrowia lipolytica to the primary seed culture medium. Place in a shaker and incubate at 32°C and 180 rpm for 17 h. The OD value is approximately 4.8, thus obtaining primary seeds.

[0199] (2) Secondary seed culture:

[0200] Prepare 2 L of secondary seed culture medium, consisting of: 15 g / L sodium acetate, 2.2% anhydrous glucose, 1.5% yeast extract, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% ammonium sulfate hydrochloride;

[0201] The secondary seed culture medium was added to a 5L fermenter, sterilized in an autoclave at 121°C for 20 min, and after cooling to 32°C, the primary seeds were added to the secondary seed culture medium fermenter at a rate of 7.5% by fire ring inoculation.

[0202] Then add 20 mL of sterile filtered trace element additives with the following formula: copper sulfate 4 g / L, sodium iodide 0.12 g / L, manganese sulfate 5 g / L, sodium molybdate 0.1 g / L, boric acid 0.04 g / L, cobalt chloride 0.6 g / L, zinc chloride 18 g / L, ferrous sulfate 38 g / L, biotin 0.3 g / L, and sulfuric acid 3 g / L;

[0203] In the secondary seed culture, the initial dissolved oxygen is 100%, the stirring speed is set to 180 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.2 vvm; the dissolved oxygen is 80-100%, the stirring speed is set to 220 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.3 vvm; the dissolved oxygen is 60-80%, the stirring speed is set to 280 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.4 vvm; the dissolved oxygen is 40-60%, the stirring speed is set to 350 rpm, the temperature is 32°C, and the ventilation speed of compressed air is The air velocity is 0.5vvm; when the dissolved oxygen is 20-40%, the stirring speed is set to 440rpm, the temperature is 31°C, and the ventilation speed of the compressed air is 0.6vvm; when the dissolved oxygen is 10-20%, the stirring speed is set to 520rpm, the temperature is 31°C, and the ventilation speed of the compressed air is 0.7vvm; when the dissolved oxygen is 0-10%, the stirring speed is set to 600rpm, the temperature is 30°C, and the ventilation speed of the compressed air is 0.8vvm; when the dissolved oxygen is 0%, the stirring speed is set to 750rpm, the temperature is 30°C, and the ventilation speed of the compressed air is 1vvm.

[0204] The secondary seeds were cultured for 10 h, and the OD value was about 14, thus obtaining the secondary seeds.

[0205] (3) High-density fermentation

[0206] Prepare 40 L of fermentation medium, the components of which are: sodium acetate 15 g / L, anhydrous glucose 2.6%, yeast extract 1.8%, casein peptone 2.8%, magnesium sulfate 1.2%, ammonium sulfate hydrochloride 1.4%, betaine 1%, ammonium sulfate 6.8%, potassium dihydrogen phosphate 7%, disodium hydrogen phosphate 4%, and acetone 0.2%;

[0207] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0208] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0209] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 40% urea solution;

[0210] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0211] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 30. Under these conditions, the bacteria grow rapidly;

[0212] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 30. Under these conditions, the bacteria grow rapidly;

[0213] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, control carbon-nitrogen ratio at 30, under these conditions the bacteria grow rapidly;

[0214] The OD value was 500-700, the temperature was controlled at 29°C, the pH was 6.8, the mixed gas (air: pure oxygen at a ratio of 1:1) was aerated at a rate of 1.6 vvm, the rotation speed was 500 rpm, the carbon source feed rate was 2.2 L / h, and the carbon-nitrogen ratio was controlled at 12. Under these conditions, the bacterial cell growth rate was normal and single-cell protein was synthesized.

[0215] OD value>700, control temperature at 28℃, pH at 7, mixed gas (air: pure oxygen at 1:2) ventilation rate at 2.0vvm, rotation speed at 700rpm, carbon source feeding rate at 2.5L / h, control carbon-nitrogen ratio at 2. Under these conditions, bacterial growth rate is slow and single-cell protein is fully synthesized.

[0216] After culturing for 30 hours, the OD value was 786, and the bacteria grew slowly. The culture was put into a tank to obtain the fermentation liquid.

[0217] (4) Post-processing

[0218] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 238 g / L, a yield of 7.9 g / (L·h), and a crude protein content of 60%.

[0219] Example 3

[0220] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0221] (1) Primary seed culture:

[0222] Prepare 200 mL of primary seed culture medium consisting of 8 g / L sodium acetate, 1.8% anhydrous glucose, 1.2% yeast extract, and 2.2% casein peptone; sterilize in an autoclave at 121°C for 20 min, place in a clean bench after sterilization, and after returning to room temperature, add the thawed frozen stock of Yarrowia lipolytica to the primary seed culture medium. Place in a shaker and incubate at 32°C and 180 rpm for 18 h. The OD value is approximately 5.8, thus obtaining primary seeds.

[0223] (2) Secondary seed culture:

[0224] Prepare 2 L of secondary seed culture medium, consisting of: 15 g / L sodium acetate, 2.2% anhydrous glucose, 1.5% yeast extract, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% ammonium sulfate hydrochloride;

[0225] The secondary seed culture medium was added to a 5L fermenter, sterilized in an autoclave at 121°C for 20 min, and after cooling to 32°C, the primary seeds were added to the secondary seed culture medium fermenter at a rate of 7.5% by fire ring inoculation.

[0226] Then add 20 mL of sterile filtered trace element additives with the following formula: copper sulfate 4 g / L, sodium iodide 0.12 g / L, manganese sulfate 5 g / L, sodium molybdate 0.1 g / L, boric acid 0.04 g / L, cobalt chloride 0.6 g / L, zinc chloride 18 g / L, ferrous sulfate 38 g / L, biotin 0.3 g / L, and sulfuric acid 3 g / L;

[0227] In the secondary seed culture, the initial dissolved oxygen is 100%, the stirring speed is set to 180 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.2 vvm; the dissolved oxygen is 80-100%, the stirring speed is set to 220 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.3 vvm; the dissolved oxygen is 60-80%, the stirring speed is set to 280 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.4 vvm; the dissolved oxygen is 40-60%, the stirring speed is set to 350 rpm, the temperature is 32°C, and the ventilation speed of compressed air is The speed is 0.5vvm; when the dissolved oxygen is 20-40%, the stirring speed is set to 440rpm, the temperature is 31°C, and the ventilation speed of compressed air is 0.6vvm; when the dissolved oxygen is 10-20%, the stirring speed is set to 520rpm, the temperature is 31°C, and the ventilation speed of compressed air is 0.7vvm; when the dissolved oxygen is 0-10%, the stirring speed is set to 600rpm, the temperature is 30°C, and the ventilation speed of compressed air is 0.8vvm; when the dissolved oxygen is 0%, the stirring speed is set to 800rpm, the temperature is 30°C, and the ventilation speed of compressed air is 1.2vvm.

[0228] The secondary seeds were cultured for 12 h, and the OD value was about 16, thus obtaining the secondary seeds.

[0229] (3) High-density fermentation

[0230] Prepare 40 L of fermentation medium, consisting of: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast extract, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% ammonium sulfate hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone;

[0231] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0232] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0233] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 40% urea solution;

[0234] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0235] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 50. Under these conditions, the bacteria grow rapidly;

[0236] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 50. Under these conditions, the bacteria grow rapidly;

[0237] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen at 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, control carbon-nitrogen ratio at 50, under these conditions the bacteria grow rapidly;

[0238] The OD value was 500-700, the temperature was controlled at 29°C, the pH was 6.8, the mixed gas (air: pure oxygen at a ratio of 1:1) was aerated at a rate of 1.6 vvm, the rotation speed was 500 rpm, the carbon source feed rate was 2.2 L / h, and the carbon-nitrogen ratio was controlled at 18. Under these conditions, the bacterial cell growth rate was normal and single-cell protein was synthesized.

[0239] OD value>700, temperature controlled at 28℃, pH at 7, mixed gas (air: pure oxygen at 1:2) ventilation rate at 2.0vvm, rotation speed at 700rpm, carbon source feeding rate at 2.5L / h, carbon-nitrogen ratio controlled at 1. Under these conditions, bacterial growth rate is slow and single-cell protein is fully synthesized.

[0240] After culturing for 30 hours, the OD value was 792, and the bacteria grew slowly. The culture was put into a tank to obtain the fermentation liquid.

[0241] (4) Post-processing

[0242] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 243 g / L, a yield of 8.1 g / (L·h), and a crude protein content of 61%.

[0243] Example 4

[0244] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0245] (1) Primary seed culture: The same as step (1) of Example 1 is used to obtain primary seeds.

[0246] (2) Secondary seed culture: The same as step (2) of Example 1 is used to obtain secondary seeds.

[0247] (3) High-density fermentation

[0248] Prepare 40 L of fermentation medium, the components of which are: sodium acetate 15 g / L, anhydrous glucose 2.6%, yeast extract 1.8%, casein peptone 2.8%, magnesium sulfate 1.2%, ammonium sulfate hydrochloride 1.4%, betaine 1%, ammonium sulfate 6.8%, potassium dihydrogen phosphate 7%, disodium hydrogen phosphate 4%, and acetone 0.2%;

[0249] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0250] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0251] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 30% ammonium sulfate solution;

[0252] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0253] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0254] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0255] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, control carbon-nitrogen ratio at 20, under these conditions the bacteria grow rapidly;

[0256] The OD value was 500-700, the temperature was controlled at 29°C, the pH was 6.8, the mixed gas (air: pure oxygen at a ratio of 1:1) was aerated at a rate of 1.6 vvm, the rotation speed was 500 rpm, the carbon source feed rate was 2.2 L / h, and the carbon-nitrogen ratio was controlled at 10. Under these conditions, the bacterial cell growth rate was normal and single-cell protein was synthesized.

[0257] After culturing for 48 hours, the OD value was 523, and the bacteria grew slowly. The culture was put into a tank to obtain a fermentation liquid.

[0258] (4) Post-processing

[0259] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 180 g / L, a yield of 3.75 g / (L·h), and a crude protein content of 41%.

[0260] Example 5

[0261] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0262] (1) Primary seed culture: The same as step (1) of Example 1 is used to obtain primary seeds.

[0263] (2) Secondary seed culture: The same as step (2) of Example 1 is used to obtain secondary seeds.

[0264] (3) High-density fermentation

[0265] Prepare 40 L of fermentation medium, consisting of: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast extract, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% ammonium sulfate hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone;

[0266] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0267] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0268] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 20% ammonium nitrate solution;

[0269] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0270] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0271] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0272] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, control carbon-nitrogen ratio at 20, under these conditions the bacteria grow rapidly;

[0273] After culturing for 52 hours, the OD value was about 478, and the bacteria grew slowly. The culture was put into the tank to obtain the fermentation liquid.

[0274] (4) Post-processing

[0275] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 160 g / L, a yield of 3.08 g / (L·h), and a crude protein content of 38%.

[0276] Example 6

[0277] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0278] (1) Primary seed culture: The same as step (1) of Example 1 is used to obtain primary seeds.

[0279] (2) Secondary seed culture: The same as step (2) of Example 1 is used to obtain secondary seeds.

[0280] (3) High-density fermentation

[0281] Prepare 40 L of fermentation medium, consisting of: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast extract, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% ammonium sulfate hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone;

[0282] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0283] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0284] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is a sterilized 28% ammonia solution;

[0285] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0286] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0287] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0288] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0289] The OD value was 500-700, the temperature was controlled at 29°C, the pH was 6.8, the mixed gas (air: pure oxygen at a ratio of 1:1) was aerated at a rate of 1.6 vvm, the rotation speed was 500 rpm, the carbon source feed rate was 2.2 L / h, and the carbon-nitrogen ratio was controlled at 10. Under these conditions, the bacterial cell growth rate was normal and single-cell protein was synthesized.

[0290] After culturing for 50 hours, the OD value was about 585, and the bacteria grew slowly. The culture was put into a tank to obtain the fermentation liquid.

[0291] (4) Post-processing

[0292] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 190 g / L, a yield of 3.8 g / (L·h), and a crude protein content of 42%.

[0293] Comparing Example 1 with Examples 4-6, it can be seen that the addition of urea as a nitrogen source and acetic acid as a carbon source during the high-density fermentation process in Example 1 can more effectively promote rapid bacterial growth and protein synthesis. Urea, as a nitrogen source, is more easily absorbed by the bacteria, which is beneficial for protein synthesis. The addition of nitrogen sources in Examples 4-6 is not conducive to bacterial absorption, resulting in slow growth, prolonged fermentation cycles, and reduced single-cell protein yields.

[0294] Example 7

[0295] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0296] (1) Primary seed culture: The same as step (1) of Example 1 is used to obtain primary seeds.

[0297] (2) Secondary seed culture: The same as step (2) of Example 1 is used to obtain secondary seeds.

[0298] (3) High-density fermentation

[0299] Prepare 40 L of fermentation medium, the components of which are: sodium acetate 15 g / L, anhydrous glucose 2.6%, yeast extract 1.8%, casein peptone 2.8%, magnesium sulfate 1.2%, ammonium sulfate hydrochloride 1.4%, betaine 1%, ammonium sulfate 6.8%, potassium dihydrogen phosphate 7%, disodium hydrogen phosphate 4%, and acetone 0.2%;

[0300] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0301] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0302] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 40% urea solution;

[0303] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0304] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and maintain a constant carbon-nitrogen ratio of 20. Under these conditions, the bacteria grow rapidly;

[0305] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and maintain a constant carbon-nitrogen ratio of 20. Under these conditions, the bacteria grow rapidly;

[0306] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, maintain a constant carbon-nitrogen ratio of 20, under these conditions the bacteria grow rapidly;

[0307] The OD value was 500-700, the temperature was controlled at 29°C, the pH was 6.8, the mixed gas (air: pure oxygen at a ratio of 1:1) was aerated at a rate of 1.6 vvm, the rotation speed was 500 rpm, the carbon source feed rate was 2.2 L / h, and a constant carbon-nitrogen ratio of 20 was maintained. Under these conditions, the bacterial cell growth rate was normal and single-cell protein was synthesized.

[0308] After culturing for 36 hours, the OD value was about 641, and the bacteria grew slowly. The culture was put into the tank to obtain the fermentation liquid.

[0309] (4) Post-processing

[0310] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 210 g / L, a yield of 5.8 g / (L·h), and a crude protein content of 35%.

[0311] Comparing Example 1 with Example 7, it can be seen that in Example 1, the carbon-nitrogen ratio is regulated according to the fermentation stage during the high-density fermentation process. When the carbon-nitrogen ratio is greater than 18, the bacterial growth is vigorous, and when the carbon-nitrogen ratio is less than 5, the bacterial growth slows down, and the rate of protein synthesis increases. By adjusting the carbon-nitrogen ratio, the fastest bacterial growth and the highest crude protein content are achieved. In Example 7, the high-density fermentation process maintains a constant high carbon-nitrogen ratio, resulting in a slower protein synthesis rate and a lower crude protein content.

[0312] Example 8

[0313] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0314] (1) Primary seed culture: The same as step (1) of Example 1 is used to obtain primary seeds.

[0315] (2) Secondary seed culture: The same as step (2) of Example 1 is used to obtain secondary seeds.

[0316] (3) High-density fermentation

[0317] Prepare 40 L of fermentation medium, the components of which are: sodium acetate 15 g / L, anhydrous glucose 2.6%, yeast extract 1.8%, casein peptone 2.8%, magnesium sulfate 1.2%, ammonium sulfate hydrochloride 1.4%, betaine 1%, ammonium sulfate 6.8%, potassium dihydrogen phosphate 7%, disodium hydrogen phosphate 4%, and acetone 0.2%;

[0318] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0319] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0320] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 40% urea solution;

[0321] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0322] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and maintain a constant carbon-nitrogen ratio of 12. Under these conditions, the bacteria grow rapidly;

[0323] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, maintain a constant carbon-nitrogen ratio of 12, under these conditions the bacteria grow rapidly;

[0324] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen at 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, maintain a constant carbon-nitrogen ratio of 12, under these conditions the bacteria grow rapidly;

[0325] The OD value was 500-700, the temperature was controlled at 29°C, the pH was 6.8, the mixed gas (air: pure oxygen at a ratio of 1:1) was aerated at a rate of 1.6 vvm, the rotation speed was 500 rpm, the carbon source feed rate was 2.2 L / h, and a constant carbon-nitrogen ratio of 12 was maintained. Under these conditions, the bacterial cell growth rate was normal and single-cell protein was synthesized.

[0326] After culturing for 46 hours, the OD value was about 536, and the bacteria grew slowly. The culture was put into a tank to obtain the fermentation liquid.

[0327] (4) Post-processing

[0328] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 186 g / L, a yield of 4.04 g / (L·h), and a crude protein content of 55%.

[0329] Comparing Example 1 with Example 8, it can be seen that in Example 1, the carbon-nitrogen ratio is regulated according to the fermentation stage during the high-density fermentation process. When the carbon-nitrogen ratio is greater than 18, the bacterial growth is vigorous, and when the carbon-nitrogen ratio is less than 5, the bacterial growth slows down, while the rate of protein synthesis increases. By adjusting the carbon-nitrogen ratio, the fastest bacterial growth and the highest crude protein content are achieved. In Example 8, the high-density fermentation process maintains a constant carbon-nitrogen ratio of 12, resulting in slower bacterial growth and protein synthesis rates, and reduced yield and efficiency.

[0330] Example 9

[0331] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0332] (1) Primary seed culture: The same as step (1) of Example 1 is used to obtain primary seeds.

[0333] (2) Secondary seed culture: The same as step (2) of Example 1 is used to obtain secondary seeds.

[0334] (3) High-density fermentation

[0335] Prepare 40 L of fermentation medium, consisting of: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast extract, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% ammonium sulfate hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone;

[0336] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0337] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0338] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 40% urea solution;

[0339] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0340] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, maintain a constant carbon-nitrogen ratio of 2, under which conditions the bacteria grow rapidly;

[0341] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, maintain a constant carbon-nitrogen ratio of 2, under which conditions the bacteria grow rapidly;

[0342] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, maintain a constant carbon-nitrogen ratio of 2, under which conditions the bacteria grow rapidly;

[0343] The OD value was 500-700, the temperature was controlled at 29°C, the pH was 6.8, the mixed gas (air: pure oxygen at a ratio of 1:1) was aerated at a rate of 1.6 vvm, the rotation speed was 500 rpm, the carbon source feed rate was 2.2 L / h, and a constant carbon-nitrogen ratio of 2 was maintained. Under these conditions, the bacterial cell growth rate was normal and single-cell protein was synthesized.

[0344] After culturing for 64 hours, the OD value was about 528, and the bacteria grew slowly. The culture was put into a tank to obtain the fermentation liquid.

[0345] (4) Post-processing

[0346] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 172 g / L, a yield of 2.68 g / (L·h), and a crude protein content of 58%.

[0347] Comparing Example 1 with Example 9, it can be seen that in Example 1, the carbon-nitrogen ratio is regulated according to the fermentation stage during the high-density fermentation process. When the carbon-nitrogen ratio is greater than 18, the bacterial growth is vigorous, and when the carbon-nitrogen ratio is less than 5, the bacterial growth slows down, and the rate of protein synthesis increases. By adjusting the carbon-nitrogen ratio, the fastest bacterial growth and the highest crude protein content are achieved. In Example 9, the high-density fermentation process maintains a constant low carbon-nitrogen ratio, resulting in slow bacterial growth and a long fermentation cycle.

[0348] Comparative Example 1

[0349] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0350] (1) Primary seed culture:

[0351] Prepare 200 mL of primary seed culture medium consisting of 1.8% anhydrous glucose, 1.2% yeast powder, and 2.2% casein peptone; sterilize in an autoclave at 121°C for 20 min, place in a clean bench after sterilization, and after returning to room temperature, add the thawed frozen stock of Yarrowia lipolytica to the primary seed culture medium. Place in a shaker and incubate at 32°C and 180 rpm for 16 h. The OD value is approximately 4.2, thus obtaining primary seeds.

[0352] (2) Secondary seed culture:

[0353] Prepare 2 L of secondary seed culture medium, the components of which are: 2.2% anhydrous glucose, 1.5% yeast extract, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% ammonium sulfate hydrochloride;

[0354] The secondary seed culture medium was added to a 5L fermenter, sterilized in an autoclave at 121°C for 20 min, and after cooling to 32°C, the primary seeds were added to the secondary seed culture medium fermenter at a rate of 7.5% by fire ring inoculation.

[0355] Then add 20 mL of sterile filtered trace element additives with the following formula: copper sulfate 4 g / L, sodium iodide 0.12 g / L, manganese sulfate 5 g / L, sodium molybdate 0.1 g / L, boric acid 0.04 g / L, cobalt chloride 0.6 g / L, zinc chloride 18 g / L, ferrous sulfate 38 g / L, biotin 0.3 g / L, and sulfuric acid 3 g / L;

[0356] In the secondary seed culture, the initial dissolved oxygen is 100%, the stirring speed is set to 180 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.2 vvm; the dissolved oxygen is 80-100%, the stirring speed is set to 220 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.3 vvm; the dissolved oxygen is 60-80%, the stirring speed is set to 280 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.4 vvm; the dissolved oxygen is 40-60%, the stirring speed is set to 350 rpm, the temperature is 32°C, and the ventilation speed of compressed air is The speed is 0.5vvm; when the dissolved oxygen is 20-40%, the stirring speed is set to 440rpm, the temperature is 31°C, and the ventilation speed of compressed air is 0.6vvm; when the dissolved oxygen is 10-20%, the stirring speed is set to 520rpm, the temperature is 31°C, and the ventilation speed of compressed air is 0.7vvm; when the dissolved oxygen is 0-10%, the stirring speed is set to 600rpm, the temperature is 30°C, and the ventilation speed of compressed air is 0.8vvm; when the dissolved oxygen is 0%, the stirring speed is set to 700rpm, the temperature is 30°C, and the ventilation speed of compressed air is 0.9vvm.

[0357] The secondary seeds were cultured for 8 h, with an OD value of about 12, and the secondary seeds were obtained.

[0358] (3) High-density fermentation

[0359] Prepare 40 L of fermentation medium, the components of which are: 2.6% anhydrous glucose, 1.8% yeast extract, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% ammonium sulfate hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone;

[0360] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0361] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0362] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 40% urea solution;

[0363] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0364] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0365] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grows relatively fast;

[0366] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, and control carbon-nitrogen ratio at 20;

[0367] After culturing for 68 hours, the OD value was about 372, and the bacteria grew slowly. The culture was put into a tank to obtain the fermentation liquid.

[0368] (4) Post-processing

[0369] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 120 g / L, a yield of 1.7 g / (L·h), and a crude protein content of 60%.

[0370] Since the carbon sources of the primary seed culture medium, secondary seed culture medium and fermentation culture medium of Comparative Example 1 do not contain sodium acetate, the bacteria will grow slowly when encountering acetic acid during the high-density fermentation stage. It takes more time to adapt to the environment of acetate ions to form the acetic acid metabolic pathway. The growth time is longer and it is difficult to reach high density, resulting in low yield and efficiency.

[0371] Comparative Example 2

[0372] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0373] (1) Primary seed culture: The same as step (1) of Example 1 is used to obtain primary seeds.

[0374] (2) Secondary seed culture: The same as step (2) of Example 1 is used to obtain secondary seeds.

[0375] (3) High-density fermentation

[0376] Prepare 40 L of fermentation medium, consisting of: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast extract, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% ammonium sulfate hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone;

[0377] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0378] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0379] Prepare the supplementary carbon source and nitrogen source, the carbon source is a sterilized 40% sodium acetate aqueous solution, and the nitrogen source is a sterilized 40% urea solution;

[0380] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0381] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0382] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 20;

[0383] After 42 hours of cultivation, the OD value was about 172, the fermentation liquid was saponified, the bacteria were broken and died, and the fermentation liquid was centrifuged, washed with water, and spray-dried to obtain a single-cell protein with a dry weight of 60 g / L, a yield of 1.4 g / (L·h), and a crude protein content of 38%.

[0384] Since alkaline substances are produced during the fermentation process, the pH of the fermentation tank will gradually increase. In Comparative Example 2, sodium acetate (alkaline) is added as a carbon source during the high-density fermentation process, the pH becomes more alkaline, and sodium ions accumulate, causing saponification reaction and inhibiting bacterial growth.

[0385] Comparative Example 3

[0386] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0387] (1) Primary seed culture:

[0388] Prepare 200 mL of primary seed culture medium consisting of 8 g / L sodium acetate, 1.8% anhydrous glucose, 1.2% yeast extract, and 2.2% casein peptone; sterilize in an autoclave at 121°C for 20 min, place in a clean bench, and after returning to room temperature, add thawed frozen stock of Schizochytrium sp. (accession number ATCC 20888, purchased from the American Type Culture Collection) to the primary seed culture medium. Incubate in a shaker at 32°C and 180 rpm for 16 h, achieving an OD value of approximately 4.2, thereby obtaining primary seeds.

[0389] (2) Secondary seed culture:

[0390] Prepare 2 L of secondary seed culture medium, consisting of: 15 g / L sodium acetate, 2.2% anhydrous glucose, 1.5% yeast extract, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% ammonium sulfate hydrochloride;

[0391] The secondary seed culture medium was added to a 5L fermenter, sterilized in an autoclave at 121°C for 20 min, and after cooling to 32°C, the primary seeds were added to the secondary seed culture medium fermenter at a rate of 7.5% by fire ring inoculation.

[0392] Then add 20 mL of sterile filtered trace element additives with the following formula: copper sulfate 4 g / L, sodium iodide 0.12 g / L, manganese sulfate 5 g / L, sodium molybdate 0.1 g / L, boric acid 0.04 g / L, cobalt chloride 0.6 g / L, zinc chloride 18 g / L, ferrous sulfate 38 g / L, biotin 0.3 g / L, and sulfuric acid 3 g / L;

[0393] In the secondary seed culture, the initial dissolved oxygen is 100%, the stirring speed is set to 180 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.2 vvm; the dissolved oxygen is 80-100%, the stirring speed is set to 220 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.3 vvm; the dissolved oxygen is 60-80%, the stirring speed is set to 280 rpm, the temperature is 32°C, and the ventilation speed of compressed air is 0.4 vvm; the dissolved oxygen is 40-60%, the stirring speed is set to 350 rpm, the temperature is 32°C, and the ventilation speed of compressed air is The speed is 0.5vvm; when the dissolved oxygen is 20-40%, the stirring speed is set to 440rpm, the temperature is 31°C, and the ventilation speed of compressed air is 0.6vvm; when the dissolved oxygen is 10-20%, the stirring speed is set to 520rpm, the temperature is 31°C, and the ventilation speed of compressed air is 0.7vvm; when the dissolved oxygen is 0-10%, the stirring speed is set to 600rpm, the temperature is 30°C, and the ventilation speed of compressed air is 0.8vvm; when the dissolved oxygen is 0%, the stirring speed is set to 700rpm, the temperature is 30°C, and the ventilation speed of compressed air is 0.9vvm.

[0394] The secondary seeds were cultured for 8 h, with an OD value of about 12, and the secondary seeds were obtained.

[0395] (3) High-density fermentation

[0396] Prepare 40 L of fermentation medium, consisting of: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast extract, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% ammonium sulfate hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone;

[0397] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0398] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0399] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 40% urea solution;

[0400] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0401] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0402] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 20;

[0403] After 96 hours of culture, the OD value was about 224, and the bacteria grew slowly. The culture was put into the tank to obtain the fermentation liquid.

[0404] (4) Post-processing

[0405] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 86 g / L, a yield of 0.9 g / (L·h), and a crude protein content of 28%.

[0406] Comparing Example 1 with Comparative Example 3, it can be seen that the present application uses Yarrowia lipolytica as the strain, a non-genetically engineered bacterium that is safer than conventional bacteria. It is a generally recognized as safe (GRAS) microorganism certified by the FDA and widely used in the food industry. This application uses a specific strain and a specific fermentation method to quickly obtain high yields of single-cell protein. Comparative Example 3 uses Schizochytrium, which not only has a long fermentation cycle but also produces low single-cell protein yields.

[0407] Comparative Example 4

[0408] A method for producing single-cell protein by high-density fermentation, comprising the following steps:

[0409] (1) Primary seed culture: The same as step (1) of Example 1 is used to obtain primary seeds.

[0410] (2) Secondary seed culture: The same as step (2) of Example 1 is used to obtain secondary seeds.

[0411] (3) High-density fermentation

[0412] Prepare 40 L of fermentation medium, the components of which are: sodium acetate 15 g / L, anhydrous glucose 2.6%, yeast extract 1.8%, casein peptone 2.8%, magnesium sulfate 1.2%, ammonium sulfate hydrochloride 1.4%, betaine 1%, ammonium sulfate 6.8%, potassium dihydrogen phosphate 7%, and disodium hydrogen phosphate 4%;

[0413] The fermentation medium was added to a 100 L fermenter, and the fermenter was steam sterilized. After cooling to 32°C, the secondary seeds were added to the 100 L fermenter at a 5% inoculum rate.

[0414] Then add 400 mL of trace elements after sterilization and filtration to the fermentation tank culture medium. The formula is: copper sulfate 8 g / L, sodium iodide 0.16 g / L, manganese sulfate 6 g / L, sodium molybdate 0.2 g / L, boric acid 0.08 g / L, cobalt chloride 0.8 g / L, zinc chloride 21 g / L, ferrous sulfate 41 g / L, biotin 0.45 g / L, and sulfuric acid 5.2 g / L.

[0415] Prepare the supplementary carbon source and nitrogen source, wherein the carbon source is acetic acid and the nitrogen source is sterilized 40% urea solution;

[0416] During high-density fermentation, the pH range, temperature, rotation speed, carbon-nitrogen ratio, dissolved oxygen, and ventilation volume are controlled at different stages to adjust the bacterial growth environment to achieve optimal conditions for bacterial growth and single-cell protein synthesis, as follows:

[0417] 0≤OD value<100, control temperature at 32℃, pH at 6.5, compressed air ventilation rate at 0.6vvm, rotation speed at 150rpm, carbon source feeding rate at 0.8L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0418] 100≤OD value<300, control temperature at 31℃, pH at 6.6, compressed air ventilation rate at 1vvm, rotation speed at 200rpm, carbon source feeding rate at 1.2L / h, and control carbon-nitrogen ratio at 20. Under these conditions, the bacteria grow rapidly;

[0419] 300≤OD value<500, control temperature at 30℃, pH at 6.7, mixed gas (air:pure oxygen 2:1) ventilation rate at 1.2vvm, rotation speed at 300rpm, carbon source feeding rate at 1.6L / h, control carbon-nitrogen ratio at 20, under these conditions the bacteria grow rapidly;

[0420] The OD value was 500-700, the temperature was controlled at 29°C, the pH was 6.8, the mixed gas (air: pure oxygen at a ratio of 1:1) was aerated at a rate of 1.6 vvm, the rotation speed was 500 rpm, the carbon source feed rate was 2.2 L / h, and the carbon-nitrogen ratio was controlled at 10. Under these conditions, the bacterial cell growth rate was normal and single-cell protein was synthesized.

[0421] After culturing for 51 hours, the OD value was about 647, and the bacteria grew slowly. The culture was put into the tank to obtain the fermentation liquid.

[0422] (4) Post-processing

[0423] The fermentation broth was centrifuged, washed with water, and spray-dried to obtain single-cell protein with a dry weight of 216 g / L, a yield of 4.23 g / (L·h), and a crude protein content of 60%.

[0424] Comparison of Example 1 and Comparative Example 4 shows that the fermentation medium of the present application contains a ketone compound (preferably acetone), which catalyzes the intermediate product in the acetic acid metabolic pathway, promotes rapid bacterial growth, and shortens the fermentation cycle. The fermentation medium of Comparative Example 4 does not contain a ketone compound, resulting in slow bacterial growth and a prolonged fermentation cycle.

[0425] The applicant declares that while the above-mentioned examples illustrate the method for producing single-cell protein by high-density fermentation, the present application is not limited to the above-mentioned process steps, which does not imply that the present application must rely on the above-mentioned process steps for implementation. Those skilled in the art should understand that any improvements to the present application, equivalent replacements for the raw materials used in the present application, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present application.

Claims

1. A method for producing single-cell protein by high-density fermentation, comprising: inoculating Yarrowia lipolytica strains into a seed culture medium for seed culture to obtain a seed solution; inoculating the seed liquid into a fermentation medium for high-density fermentation, and adding acetic acid and a nitrogen source during the high-density fermentation to obtain the single-cell protein; The carbon source of the seed culture medium includes a first acetate, and the carbon source of the fermentation culture medium includes a second acetate and / or a ketone compound.

2. The method according to claim 1, wherein: The first acetate includes any one of sodium acetate, potassium acetate, and ammonium acetate, or a combination of at least two thereof, preferably sodium acetate.

3. The method according to claim 2, wherein: The mass concentration of the first acetate in the seed culture medium is 5-15 g / L.

4. The method according to claim 2 or 3, wherein: The seed culture includes primary seed culture and secondary seed culture which are carried out in sequence.

5. The method according to claim 4, wherein: The primary seed culture medium used in the primary seed culture comprises the following components: 5-15 g / L of first acetate, 1%-3% of glucose, 0.6%-2% of yeast powder, and 1.5%-3.2% of casein peptone; Preferably, the primary seed culture temperature is 30-33°C and the time is 16-18h; Preferably, the primary seed culture is carried out under stirring conditions, and the stirring speed is 100-300 rpm; Preferably, the primary seeds are cultured to an OD value of 4.2-6.8 to obtain primary seeds.

6. The method according to claim 4 or 5, wherein: The secondary seed culture medium used in the secondary seed culture comprises the following components: 5-20 g / L of first acetate, 1%-3% of glucose, 0.6%-2% of yeast powder, 1.5%-3.2% of casein peptone, 0.8%-2.4% of magnesium sulfate, and 0.2%-0.8% of ammonium sulfate hydrochloride; Preferably, the secondary seed culture medium further comprises a first trace element additive, wherein the first trace element additive The dosage of the element additive is 0.8%-2.6%; Preferably, the first trace element additive comprises the following components: copper sulfate 4-8 g / L, sodium iodide 0.12-0.16 g / L, manganese sulfate 5-6 g / L, sodium molybdate 0.1-0.2 g / L, boric acid 0.04-0.08 g / L, cobalt chloride 0.6-0.8 g / L, zinc chloride 18-21 g / L, ferrous sulfate 38-41 g / L, biotin 0.3-0.5 g / L, and sulfuric acid 3-5.2 g / L.

7. The method according to any one of claims 4 to 6, wherein: The first-level seeds obtained by the first-level seed culture are inoculated in a second-level seed culture medium at an inoculation rate of 5%-10% for second-level seed culture; Preferably, the ventilation rate of the secondary seed culture is 0.1-1.3 vvm; Preferably, the secondary seed culture is carried out under stirring conditions, and the stirring speed is 150-900 rpm; Preferably, the temperature of the secondary seed culture is 30-33°C and the time is 8-12h; Preferably, the secondary seeds are cultured to an OD value of 12-16 to obtain the seed solution.

8. The method according to any one of claims 1 to 7, wherein: The carbon source of the fermentation medium includes a combination of a second acetate and a ketone compound; Preferably, the second acetate includes any one of sodium acetate, potassium acetate, and ammonium acetate, or a combination of at least two thereof, and sodium acetate is further preferred; Preferably, the mass concentration of the second acetate in the fermentation medium is 5-15 g / L; Preferably, the mass percentage of ketone compounds in the fermentation medium is 0.1%-0.6%; Preferably, the fermentation medium comprises the following components: 5-15 g / L of second acetate, 1%-3% of glucose, 0.6%-2% of yeast powder, 1.5%-3.2% of casein peptone, 0.8%-1.2% of magnesium sulfate, 0.6%-1.4% of ammonium sulfate hydrochloride, 0.5%-1% of betaine, 3.5%-6.8% of ammonium sulfate, 5%-7% of potassium dihydrogen phosphate, 2%-4% of disodium hydrogen phosphate, and 0.1%-0.6% of ketone compounds; Preferably, the ketone compound is a C3-C10 ketone compound; Preferably, the ketone compound includes any one of acetone, methyl ethyl ketone, and cyclohexanone, or a combination of at least two thereof, and acetone is further preferred; Preferably, the fermentation medium further comprises a second trace element additive, and the dosage of the second trace element additive is 0.8%-2.6%; Preferably, the second trace element additive comprises the following components: copper sulfate 4-8 g / L, sodium iodide 0.12-0.16 g / L, manganese sulfate 5-6 g / L, sodium molybdate 0.1-0.2 g / L, boric acid 0.04-0.08 g / L, cobalt chloride 0.6-0.8 g / L, zinc chloride 18-21 g / L, ferrous sulfate 38-41 g / L, biotin 0.3-0.5 g / L, and sulfuric acid 3-5.2 g / L.

9. The method according to any one of claims 1 to 8, wherein: The seed liquid is inoculated into the fermentation medium at an inoculation rate of 3%-6% to perform high-density fermentation; Preferably, the nitrogen source added during the high-density fermentation process includes any one of urea, ammonium sulfate, ammonium nitrate, and ammonia water, or a combination of at least two of them, and urea is more preferred.

10. The method according to any one of claims 1 to 9, wherein: The temperature of the high-density fermentation is 27-33°C, preferably 28-32°C; Preferably, the pH value of the high-density fermentation is 6.4-7.1, more preferably 6.5-7; Preferably, the high-density fermentation is carried out under stirring conditions, and the stirring speed is 130-800 rpm; Preferably, the aeration rate of the high-density fermentation is 0.5-2.4 vvm; Preferably, the high-density fermentation is performed until the OD value reaches 780-820, and then the fermentation is terminated to obtain the single-cell protein.

11. The method according to any one of claims 1 to 10, wherein: The high-density fermentation includes a first fermentation stage, a second fermentation stage, and a third fermentation stage with OD values ​​increasing in sequence; The OD value of the first fermentation stage is less than 500, and the acetic acid and nitrogen source are added in the first fermentation stage. Carbon-nitrogen ratio>18; The OD value of the second fermentation stage is 500-700, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source in the second fermentation stage is 5-18; The OD value of the third fermentation stage is greater than 700, and the carbon-nitrogen ratio of the supplemented acetic acid and nitrogen source in the third fermentation stage is less than 5.

12. The method according to claim 11, wherein: The first fermentation stage includes stage A, stage B and stage C which are performed sequentially; In the stage A, the OD value is less than 100, the temperature is 31-33° C., the pH value is 6.4-6.6, the aeration rate is 0.5-0.7 vvm, the rotation speed is 130-170 rpm, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source is greater than 18; In the stage B, 100≤OD value<300, temperature is 30-32°C, pH value is 6.5-6.7, aeration speed is 0.9-1.1vvm, rotation speed is 180-220rpm, and carbon-nitrogen ratio of supplemented acetic acid and nitrogen source is>18; In the stage C, 300≤OD value<500, temperature is 29-31°C, pH value is 6.6-6.8, aeration rate is 1.1-1.3vvm, rotation speed is 280-320rpm, and carbon-nitrogen ratio of supplemented acetic acid and nitrogen source is>18; The OD value of the second fermentation stage is 500-700, the temperature is 28-30°C, the pH value is 6.7-6.9, the aeration rate is 1.5-1.7vvm, the rotation speed is 450-550rpm, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source is 5-18; The OD value of the third fermentation stage is greater than 700, the temperature is 27-29° C., the pH value is 6.9-7.1, the ventilation speed is 1.9-2.1 vvm, the rotation speed is 650-750 rpm, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source is less than 5.

13. The method according to any one of claims 1 to 12, wherein: After the high-density fermentation is completed, a post-processing step is also included; Preferably, the post-treatment method comprises: sequentially performing solid-liquid separation, washing and drying on the fermentation liquid obtained by the high-density fermentation to obtain the single-cell protein; Preferably, the solid-liquid separation method is centrifugal separation.

14. The method according to any one of claims 1 to 13, wherein: The method specifically comprises the following steps: (1) inoculating the Yarrowia lipolytica strain into a primary seed culture medium, culturing for 16-18 hours at a temperature of 30-33° C. and a rotation speed of 100-300 rpm until the OD value is 4.2-6.8, thereby obtaining a primary seed; The primary seed culture medium comprises the following components: 5-15 g / L sodium acetate, 1%-3% glucose, 0.6%-2% yeast powder, and 1.5%-3.2% casein peptone; (2) inoculating the primary seeds in a secondary seed culture medium at an inoculum amount of 5%-10%, and culturing for 8-12 hours at a temperature of 30-33° C., a rotation speed of 150-900 rpm, and a ventilation speed of 0.1-1.3 vvm until the OD value is 12-16, thereby obtaining secondary seeds, i.e., the seed solution; The secondary seed culture medium comprises the following components: 5-20 g / L sodium acetate, 1%-3% glucose, 0.6%-2% yeast powder, 1.5%-3.2% casein peptone, 0.8%-2.4% magnesium sulfate, 0.2%-0.8% ammonium sulfate hydrochloride, and 0.8%-2.6% first trace element additive; The first trace element additive comprises the following components: 4-8 g / L copper sulfate, 0.12-0.16 g / L sodium iodide, 5-6 g / L manganese sulfate, 0.1-0.2 g / L sodium molybdate, 0.04-0.08 g / L boric acid, 0.6-0.8 g / L cobalt chloride, 18-21 g / L zinc chloride, 38-41 g / L ferrous sulfate, 0.3-0.5 g / L biotin, 3-5.2 g / L sulfuric acid; (3) inoculating the seed liquid into a fermentation medium at an inoculation rate of 3%-6% for high-density fermentation, and adding acetic acid and urea during the high-density fermentation until the OD value is 780-820 to obtain the single-cell protein; The fermentation medium comprises the following components: 5-15 g / L sodium acetate, 1%-3% glucose, 0.6%-2% yeast powder, 1.5%-3.2% casein peptone, 0.8%-1.2% magnesium sulfate, 0.6%-1.4% ammonium sulfate hydrochloride, 0.5%-1% betaine, 3.5%-6.8% ammonium sulfate, 5%-7% potassium dihydrogen phosphate, 2%-4% disodium hydrogen phosphate, 0.1%-0.6% acetone, and 0.8%-2.6% of a second trace element additive; The second trace element additive includes the following components: copper sulfate 4-8g / L, sodium iodide 0.12-0.16g / L, manganese sulfate 5-6g / L, sodium molybdate 0.1-0.2g / L, boric acid 0.04-0.08g / L, cobalt chloride 0.6-0.8g / L, Zinc chloride 18-21g / L, ferrous sulfate 38-41g / L, biotin 0.3-0.5g / L, sulfuric acid 3-5.2g / L; The high-density fermentation includes a first fermentation stage, a second fermentation stage, and a third fermentation stage with OD values ​​increasing in sequence; The OD value of the first fermentation stage is less than 500, the temperature is 29-33°C, the pH value is 6.5-6.8, the aeration rate is 0.5-1.3vvm, the rotation speed is 280-320rpm, and the carbon-nitrogen ratio of the added acetic acid and urea is greater than 18; The OD value of the second fermentation stage is 500-700, the temperature is 28-30°C, the pH value is 6.7-6.9, the aeration rate is 1.5-1.7vvm, the rotation speed is 450-550rpm, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source is 5-18; The OD value of the third fermentation stage is greater than 700, the temperature is 27-29° C., the pH value is 6.9-7.1, the ventilation speed is 1.9-2.1 vvm, the rotation speed is 650-750 rpm, and the carbon-nitrogen ratio of the added acetic acid and nitrogen source is less than 5.

15. The method according to any one of claims 1 to 14, wherein: The single cell protein yield is >2.5 g / (L·h), preferably 6-9 g / (L·h); Preferably, the mass percentage of crude protein in the single cell protein is ≥35%, more preferably ≥55%.

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