Method for producing unicellular protein by high-density fermentation

Description

1 / 72 METHOD FOR PRODUCING UNICELLULAR PROTEIN BY MEANS OF HIGH DENSITY FERMENTATION TECHNICAL FIELD

[001] The embodiments of the present application relate to the field of biotechnology, such as a method for producing single-cell protein by means of high-density fermentation. BACKGROUND

[002] Currently, with continuous population growth, the demand for food, especially meat, is constantly increasing. It is estimated that by 2050, global demand for meat will exceed 400 million tons. As the world's most populous country, China's demand for food is also continuously increasing. In 2018, meat consumption in China reached 88,296 million tons, surpassing the combined total of the European Union and the United States. Along with the increase in meat consumption, the demand for feed is also continuously expanding, with feed production exceeding 240 million tons. The main components of feed are primarily starch and protein, with the main protein sources being vegetable protein (e.g., soybean meal) and animal protein (e.g., fishmeal).Soybean meal, a byproduct obtained after soybean oil extraction, has the highest production and most extensive application among 12 types of animal and vegetable oil-based feed products, such as cottonseed meal, peanut meal, and rapeseed meal, etc. Soybean meal production is related to soybean production; however, China's self-sufficiency rate in soybeans is insufficient, with approximately 85% depending on imports. Soybean scarcity has implications for both food security and protein security, requiring the exploration of available alternative feed resources. Furthermore, the... Petition 870250087639, dated 09 / 26 / 2025, page 64 / 146 2 / 72 The supply of animal protein, such as fishmeal, in animal feed is also continuously being restricted. In 2020, global fishmeal consumption was 3 million tons, and it is estimated that by 2025, the fishmeal shortage could reach 1 million tons. China's annual fishmeal production is around 1.2 million tons, with a self-sufficiency rate of about 40% to 50%. In 2020, China's fishmeal imports reached 1.42 million tons, with an import value of 15 billion tons. In addition to the shortage problem, large-scale production and overfishing of fishmeal have also significantly depleted marine fishery resources, resulting in a decrease in the number and population of fish, which has a significant impact on ecological health and security.Based on the above considerations, low-protein feeds and unconventional protein substitutes have become the focus of attention in livestock farming, among which single-cell protein (SCP) is the most important and promising protein substitute.

[003] Single-cell protein, also known as microbial protein, refers to a microbial thallus artificially cultivated from various industrial and agricultural wastes, as well as 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. The chemical composition is generally dominated by proteins and fats.

[004] The application of single-cell protein in feed began around the 1990s. A series of animal experiments were conducted on poultry and cattle, aquatic products, and swine using monosodium glutamate bacteria residues and yeast bacteria residues, etc. The results indicated Petition 870250087639, dated 09 / 26 / 2025, page 65 / 146 3 / 72 that thallus protein had certain benefits for animal health, thus initiating the application of single-cell protein in the field of animal feed. Currently, yeast has been widely used in feed for poultry, livestock, and aquatic products, accounting for about 5% to 10% of all feed production. The proportion of single-cell protein in aquatic feeds is even higher, typically accounting for about 10% to 20%. With the development of technology and processes, proteins derived from Clostridium autoethanogenum and methanogen have also been developed. Although experimental data in animals indicate the safety and bioavailability of these proteins, due to the novelty of the technology, no feed product containing Clostridium autoethanogenum and methanogen has yet been launched on the market. In addition, photosynthetic bacteria and algae also have certain applications in food protein, but production is relatively small and they are not common products.

[005] Single-cell protein is typically prepared using biofermentation technology, which includes the following steps: after the preparation and sterilization of the culture medium is complete, the culture medium is placed in a fermentation tank along with the strain, and fermentation conditions are controlled to allow for rapid strain proliferation; after fermentation is complete, the stems are collected through methods such as centrifugation and precipitation and finally dried to produce the final single-cell protein product. The preparation of single-cell protein through the synthetic biology fermentation method allows for CO2 fixation through biological transformation, yielding stem protein for use in feed, which is beneficial in alleviating resource scarcity caused by the relatively high demand for meat and environmental pollution problems caused by the overuse of fishmeal. Currently, the country is promoting Petition 870250087639, dated 09 / 26 / 2025, page 66 / 146 4 / 72 vigorously promoting low-protein rations and unconventional protein resources, and researchers in agriculture and livestock are also vigorously promoting single-cell protein. Single-cell protein is an important development direction in the field of feed additives in the future. However, the current fermentation and production cycle of single-cell proteins is long, and there are significant deficiencies in both yield and production capacity, hindering the needs of large-scale applications. Therefore, the development of preparation methods for single-cell proteins with greater efficiency and higher yield is a focus of research in the field. SUMMARY OF THE INVENTION

[006] The following is a summary of the subject matter described in detail in this application. This summary is not intended to limit the scope of protection of the claims.

[007] The examples in the present application provide a method for producing single-cell protein by means of high-density fermentation. By designing carbon sources and adopting specific material composition and processes, the method can effect high-density fermentation of stems and efficient synthesis of single-cell proteins in a short time, significantly improving production efficiency and single-cell protein yield.

[008] The examples in the present application provide a method for producing single-cell protein by means of high-density fermentation. The method includes: inoculating a strain of Yarrowia lipolytica into a seed culture medium to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium for high-density fermentation, and supplementing the same with acetic acid and Petition 870250087639, dated 09 / 26 / 2025, page 67 / 146 5 / 72 a nitrogen source during the high-density fermentation process 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.

[009] This application utilizes a strain of Yarrowia lipolytica as the fermentation strain, which is a non-genetically modified strain and is safer than conventional stems. Yarrowia lipolytica is a generally recognized as safe (GRAS) microorganism by the U.S. Food and Drug Administration (FDA), making it an ideal strain for use in the food industry. The method involves designing the carbon source for seed culture and high-density fermentation. The addition of sodium acetate to the seed culture medium enables the promotion of a unique acetic acid metabolic pathway in the stems, establishing the basis for faster growth during subsequent high-density fermentation when acetic acid is added. Meanwhile, sodium acetate is also included in the carbon source of the fermentation culture medium to further induce and promote the acetic acid metabolic pathway in the stems.During the high-density fermentation process, sources of acetic acid and nitrogen are supplemented. On the one hand, acetic acid can neutralize the alkaline substances produced during fermentation, allowing the stems to grow in an ideal neutral pH environment. On the other hand, as a carbon source, acetic acid has a high carbon content, which can effectively promote rapid stem growth and the synthesis of single-cell proteins. Furthermore, acetic acid does not require sterilization and can be added directly. In addition, the culture medium for fermentation contains a ketone fermentation promoter, which exerts catalytic functions on the intermediate products in the process. Petition 870250087639, dated 09 / 26 / 2025, page 68 / 146 6 / 72 acetic acid metabolic pathway, thus promoting rapid stem growth and reducing the fermentation period.

[010] 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 formation of a unique acetic acid metabolic pathway in the stem is induced and promoted, thus establishing the basis for the subsequent supplementation of acetic acid as a carbon source and promoting rapid stem growth during high-density fermentation. Meanwhile, the carbon source of the fermentation culture medium includes a second acetate and / or a ketone compound, which promote rapid stem growth. During the high-density fermentation step, a nitrogen source and acetic acid are supplemented. By regulating the stem so that it is in an ideal neutral pH growth environment, adequate nutrients are supplied to the stem to sustain its continuous growth, thus effectively improving the stem growth rate and the single-cell protein synthesis rate.Through the combination of specific materials and processes, the method allows for high-density fermentation of the stem and efficient synthesis of single-cell proteins in a short period of time. A fermentation product with a higher crude protein content can be obtained, while the cultivation time for high-density fermentation is significantly reduced, and the production efficiency and yield of single-cell proteins are significantly improved.

[011] The preferred technical solution for the present application is as follows, but does not constitute a limitation on the technical solutions provided by the present application. Through the following preferred technical solutions, the objective and beneficial effects of the present application can be better achieved and realized.

[012] Preferably, the first acetate includes either or a Petition 870250087639, dated 09 / 26 / 2025, page 69 / 146 7 / 72 combination of at least two of sodium acetate, potassium acetate and ammonium acetate, additionally, preferably sodium acetate.

[013] Preferably, the mass concentration of the first acetate in the seed culture medium is in the range of 5 g / L to 15 g / L, for example, it could 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.

[014] Preferably, seed culture includes a primary seed culture and a secondary seed culture carried out sequentially.

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

[016] Specifically, the mass concentration of the first acetate (preferably sodium acetate) in the primary seed culture medium is in the range of 5 g / L to 15 g / L, for example, it could 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.

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

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

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

[020] Preferably, the remainder of the primary seed culture medium is water. Petition 870250087639, dated 09 / 26 / 2025, p. 70 / 146 8 / 72

[021] Preferably, the temperature for the primary seed culture is in the range of 30 °C to 33 °C, for example, it could 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., additionally, preferably 31.5 °C to 32.5 °C.

[022] Preferably, the duration of the primary seed culture is in the range of 16 h 18 h, for example, it could be 16.2 h, 16.5 h, 16.8 h, 17 h, 17.2 h, 17.5 h or 17.8 h etc.

[023] Preferably, primary seed culture is carried out under agitation conditions, with the agitation speed varying from 100 rpm to 300 rpm, for example, it could be 120 rpm, 150 rpm, 180 rpm, 200 rpm, 220 rpm, 250 rpm or 280 rpm etc. Additionally, preferably, the agitation speed is 170 rpm to 190 rpm.

[024] Preferably, the primary seed crop is processed to an OD value of 4.2 to 6.8, such as 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., obtaining the primary seed.

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

[026] Specifically, the mass concentration of the first acetate (preferably sodium acetate) in the secondary seed culture medium is in the range of 5 g / L to 20 g / L, for example, it could 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.

[027] The percentage by mass of glucose (anhydrous glucose) in the secondary seed culture medium is in the range of 1% to 3%, for example, it can be 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5% or 2.8% etc. Petition 870250087639, dated 09 / 26 / 2025, page 71 / 146 9 / 72

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

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

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

[031] The percentage by mass of thiamine hydrochloride in the secondary seed culture medium is in the range of 0.2% to 0.8%, for example, it can be 0.3%, 0.4%, 0.5%, 0.6% or 0.7% etc.

[032] Preferably, the secondary seed culture medium additionally comprises a first trace element additive, and the amount of use of the first trace element additive is in the range of 0.8% to 2.6%, for example, it may be 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2% or 2.5% etc. In the present application, the percentages used in the description of the amount of use of the first trace element additive are mass percentages.

[033] Preferably, the remainder in the secondary seed culture medium is water.

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

[035] Specifically, the mass concentration of copper sulfate in Petition 870250087639, dated 09 / 26 / 2025, p. 72 / 146 10 / 72 The first trace element additive is in the range of 4 g / L to 8 g / L, for example, it could 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 etc.

[036] The mass concentration of sodium iodide in the first trace element additive is in the range of 0.12 g / L to 0.16 g / L, for example, it could 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 etc.

[037] The mass concentration of manganese sulfate in the first trace element additive is in the range of 5 g / L to 6 g / L, for example, it may 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 etc.

[038] The mass concentration of sodium molybdate in the first trace element additive is in the range of 0.1 g / L to 0.2 g / L, for example, it may 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 etc.

[039] The mass concentration of boric acid in the first trace element additive is in the range of 0.04 g / L to 0.08 g / L, for example, it could 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 etc.

[040] The mass concentration of cobalt chloride in the first trace element additive is in the range of 0.6 g / L to 0.8 g / L, for example, it could 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 etc.

[041] The mass concentration of zinc chloride in the first trace element additive is in the range of 18 g / L to 21 g / L, for example, it could be 18.5 g / L, 19 g / L, 19.5 g / L, 20 g / L or 20.5 g / L etc.

[042] The mass concentration of ferrous sulfate in the first trace element additive is in the range of 38 g / L to 41 g / L, for example, it could be 38.5 g / L, 39 g / L, 39.5 g / L, 40 g / L or 40.5 g / L etc.

[043] The mass concentration of biotin in the first trace element additive is in the range of 0.3 g / L to 0.5 g / L, for example, it could 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 etc. Petition 870250087639, dated 09 / 26 / 2025, p. 73 / 146 11 / 72

[044] The mass concentration of sulfuric acid in the first trace element additive is in the range of 3 g / L to 5.2 g / L, for example, it could be 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 etc.

[045] Preferably, primary seeds obtained from primary seed culture are inoculated in a secondary seed culture medium at an inoculum size of 5% to 10% for secondary seed culture. The inoculum size of primary seeds can be 6%, 7%, 8% or 9% etc., additionally, preferably 7% to 8%.

[046] It should be noted that the term “inoculum size” refers to the ratio of the volume of seed liquid transferred to the volume of the culture system after inoculation. Illustratively, an inoculum size of 5% to 10% means that the volume fraction of the seed liquid is 5% to 10%. In the following descriptions, “inoculum size” refers to the volume fraction and, for the sake of brevity, will not be discussed individually.

[047] Preferably, air (compressed air) is introduced during the secondary seed culture process.

[048] Preferably, the aeration rate for the secondary seed crop is in the range of 0.1 vvm to 1.3 vvm, for example, it could be 0.2 vvm, 0.3 vvm, 0.5 vvm, 0.7 vvm, 0.9 vvm, 1 vvm or 1.2 vvm etc.

[049] Preferably, secondary seed culture is carried out under agitation conditions, with the agitation speed varying from 150 rpm to 900 rpm, for example, it can be 180 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm or 800 rpm etc.

[050] As the preferred technical solution of the present application, during the secondary seed culture process, the agitation speed and the compressed air aeration rate are gradually increased according to the Petition 870250087639, dated 09 / 26 / 2025, page 74 / 146 12 / 72 continuous decrease in dissolved oxygen.

[051] Illustratively, during the secondary seed culture process, when the initial dissolved oxygen is 100%, the agitation speed is 160 to 200 rpm, and the aeration rate is 0.1 to 0.3 vvm; when 80% < dissolved oxygen < 100%, the agitation speed is 210 to 230 rpm, and the aeration rate is 0.4 to 0.5 vvm; when 60% < dissolved oxygen < 80%, the agitation speed is 260 to 300 rpm, and the aeration rate is 0.3 to 0.5 vvm; when 40% < dissolved oxygen < 60%, the agitation speed is 300 to 400 rpm, and the aeration rate is 0.4 to 0.6 vvm; When dissolved oxygen is 20% < 40%, the agitation speed is 400 to 500 rpm, and the aeration rate is 0.5 to 0.7 vvm; when dissolved oxygen is 10% < 20%, the agitation speed is 500 to 550 rpm, and the aeration rate is 0.6 to 0.8 vvm; when dissolved oxygen is 0% < 10%, the agitation speed is 550 to 650 rpm, and the aeration rate is 0.7 to 0.9 vvm;When the dissolved oxygen is 0, the agitation speed is 700 to 800 rpm, and the aeration rate is 0.9 to 1.2 vvm.

[052] Preferably, the temperature for the secondary seed crop is in the range of 30 °C to 33 °C, for example, it could 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., additionally, preferably 30 °C to 32 °C.

[053] Preferably, the duration of the secondary seed crop is in the range of 8 h 12 h, for example, it could be 8.5 h, 9 h, 9.5 h, 10 h, 10.5 h, 11 h or 11.5 h etc.

[054] Preferably, the secondary seed crop is processed to an OD value of 12 to 16, such as an OD value of 12.5, 13, 13.5, 14, 14.5, 15 or 15.5, obtaining the seed liquid.

[055] Preferably, the carbon source of the fermentation culture medium includes a combination of the second acetate and ketone compound. Petition 870250087639, dated 09 / 26 / 2025, page 75 / 146 13 / 72

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

[057] Preferably, the mass concentration of the second acetate in the fermentation culture medium is in the range of 5 g / L to 15 g / L, for example, it could 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.

[058] Preferably, the mass percentage of ketone compounds in the fermentation culture medium is in the range of 0.1% to 0.6%, for example, it could be 0.2%, 0.3%, 0.4% or 0.5%. The specific content of ketone compounds exerts catalytic functions on the intermediate products of the acetic acid metabolic pathway of the stem during high-density fermentation, which helps to increase the stem growth rate and reduce the fermentation period.

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

[060] Specifically, the mass concentration of the second acetate (preferably sodium acetate) in the fermentation culture medium is in the range of 5 g / L to 15 g / L, for example, it could 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.

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

[062] The percentage by mass of yeast powder in the fermentation culture medium is in the range of 0.6% to 2%, for example, it could be 0.8%, Petition 870250087639, dated 09 / 26 / 2025, page 76 / 146 14 / 72 1%, 1.2%, 1.5% or 1.8% etc.

[063] The percentage by mass of casein peptone in the fermentation culture medium is in the range of 1.5% to 3.2%, for example, it can be 1.6%, 1.8%, 2%, 2.2%, 2.5%, 2.8% or 3% etc.

[064] The percentage by mass of magnesium sulfate in the fermentation culture medium is in the range of 0.8% to 1.2%, for example, it can be 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1% or 1.15% etc.

[065] The percentage by mass of thiamine hydrochloride in the fermentation culture medium is in the range of 0.6% to 1.4%, for example, it can be 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2% or 1.3% etc.

[066] The percentage by mass of betaine in the fermentation culture medium is in the range of 0.5% to 1%, for example, it could be 0.6%, 0.7%, 0.8% or 0.9% etc.

[067] The mass percentage content of ammonium sulfate in the fermentation culture medium is in the range of 3.5% to 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% etc.

[068] The percentage by mass of potassium dihydrogen phosphate in the fermentation culture medium is in the range of 5% to 7%, for example, it may be 5.2%, 5.5%, 5.8%, 6%, 6.2%, 6.5% or 6.8% etc.

[069] The percentage by mass of disodium hydrogen phosphate in the fermentation culture medium is in the range of 2% to 4%, for example, it may be 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5% or 3.8% etc.

[070] The percentage by mass of ketone compounds in the fermentation culture medium is in the range of 0.1% to 0.6%, for example, it can be 0.2%, 0.3%, 0.4% or 0.5% etc.

[071] Preferably, ketone compounds are C3-C10 ketone compounds, for example, ketone compounds can be C3, C4, C5, C6, C7, C8, C9 Petition 870250087639, dated 09 / 26 / 2025, p. 77 / 146 15 / 72 or C10 ketone compounds, including aliphatic ketones and / or alicyclic ketones.

[072] Preferably, the ketone compound includes any one or a combination of at least two of acetone, methyl ethyl ketone, and cyclohexanone; additionally, preferably acetone.

[073] As the preferred technical solution of the present application, the fermentation culture medium contains 0.1% to 0.6% acetone. Since acetone exerts catalytic functions on the intermediate products in the acetic acid metabolic pathway, the introduction of acetone can additionally increase the growth rate of the stems and reduce the fermentation period.

[074] Preferably, in the fermentation culture medium, a second trace element additive is additionally included, and the amount of use of the second trace element additive is in the range of 0.8% to 2.6%, for example, it may be 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2% or 2.5% etc. In the present application, the percentages used in the description of the amount of use of the second trace element additive are mass percentages.

[075] Preferably, the remainder in the fermentation culture medium is water.

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

[077] Specifically, the mass concentration of copper sulfate in the second trace element additive is in the range of 4 g / L to 8 g / L, for example, it could 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 etc.

[078] The mass concentration of sodium iodide in the second additive of Petition 870250087639, dated 09 / 26 / 2025, p. 78 / 146 The 16 / 72 trace element content is in the range of 0.12 g / L to 0.16 g / L, for example, it could 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 etc.

[079] The mass concentration of manganese sulfate in the second trace element additive is in the range of 5 g / L to 6 g / L, for example, it may 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 etc.

[080] The mass concentration of sodium molybdate in the second trace element additive is in the range of 0.1 g / L to 0.2 g / L, for example, it could 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 etc.

[081] The mass concentration of boric acid in the second trace element additive is in the range of 0.04 g / L to 0.08 g / L, for example, it could 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 etc.

[082] The mass concentration of cobalt chloride in the second trace element additive is in the range of 0.6 g / L to 0.8 g / L, for example, it could 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 etc.

[083] The mass concentration of zinc chloride in the second trace element additive is in the range of 18 g / L to 21 g / L, for example, it could be 18.5 g / L, 19 g / L, 19.5 g / L, 20 g / L or 20.5 g / L etc.

[084] The mass concentration of ferrous sulfate in the second trace element additive is in the range of 38 g / L to 41 g / L, for example, it could be 38.5 g / L, 39 g / L, 39.5 g / L, 40 g / L or 40.5 g / L etc.

[085] The mass concentration of biotin in the second trace element additive is in the range of 0.3 g / L to 0.5 g / L, for example, it could 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 etc.

[086] The mass concentration of sulfuric acid in the second trace element additive is in the range of 3 g / L to 5.2 g / L, for example, it could be 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 etc. Petition 870250087639, dated 09 / 26 / 2025, p. 79 / 146 17 / 72

[087] Preferably, the seed liquid is inoculated into a fermentation culture medium at an inoculum size of 3% to 6% for high-density fermentation. The inoculum size of the seed liquid can be 3.5%, 4%, 4.5%, 5% or 5.5% etc.

[088] Preferably, the nitrogen source supplemented during the high-density fermentation process includes any one or a combination of at least two of urea, ammonium sulfate, ammonium nitrate and ammoniacal water; additionally, preferably urea.

[089] As the preferred technical solution of the present application, the carbon source supplemented during the high-density fermentation process is acetic acid, and the nitrogen source supplemented is urea. Since the ideal pH for stem growth is a neutral environment, but alkaline substances are produced during the fermentation process, the pH value will gradually increase. Therefore, supplementation with acetic acid can not only neutralize and stabilize the pH, but also provide a carbon source for growth, and can be added directly without sterilization. Furthermore, acetic acid has a high carbon content and can be supplemented without the need for preparation in aqueous solution, which is more conducive to achieving high-density fermentation.In contrast, if a carbon source requiring preparation in aqueous solution is selected (such as glucose), its supplementation in the fermentation system would simultaneously introduce additional water. This increases the volume of the fermentation broth, leading to its dilution and hindering the achievement of high-density fermentation. The use of urea as a nitrogen source is advantageous because it is more easily absorbed by the stems, thus facilitating protein synthesis. Through the design and combination of the supplemented carbon and nitrogen sources, rapid stem growth and synthesis are achieved. Petition 870250087639, dated 09 / 26 / 2025, page 80 / 146 18 / 72 of protein are promoted more effectively.

[090] Preferably, urea is supplemented in the form of an aqueous urea solution.

[091] Preferably, the mass percentage content of urea in the aqueous urea solution is in the range of 30% to 50%, such as 32%, 35%, 38%, 40%, 42%, 45% or 48% etc., additionally, preferably 40%.

[092] Preferably, the temperature for high-density fermentation ranges from 27 °C to 33 °C, for example, it could 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 etc., additionally, preferably 28 °C to 32 °C.

[093] Preferably, the pH value for high-density fermentation ranges from 6.4 to 7.1, for example, it can be 6.5, 6.6, 6.7, 6.8, 6.9 or 7, etc., additionally, preferably 6.5 to 7.

[094] Preferably, high-density fermentation is carried out under agitation conditions, with the agitation speed varying from 130 rpm to 800 rpm, for example, it could be 150 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm or 750 rpm etc.

[095] Preferably, the aeration rate for high-density fermentation is 0.5 vvm to 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.

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

[097] As the preferred technical solution of the present application, regulating the composition and flow rate of the gas introduced during the high-density fermentation process is more conducive to promoting stem growth and protein synthesis. Specifically: Petition 870250087639, dated 09 / 26 / 2025, page 81 / 146 19 / 72 when the OD value is less than 300, air (compressed air) is introduced, with an aeration rate of 0.6 vvm to 1 vvm; When 300 < DO value < 500, a mixed gas of air and oxygen is introduced, with an air to oxygen ratio of (1.5 to 2.5:1, for example, the ratio can be 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, etc., preferably 2:1; the aeration rate is 0.8 vvm to 1.2 vvm; When the DO value is in the range of 500 to 700, a mixed gas of air and oxygen is introduced, with an air to oxygen ratio of (0.8 to 1.2):1, for example, the ratio can be 0.9:1, 1:1, 1.1:1, etc., preferably 1:1; the aeration rate is 1.2 vvm to 1.8 vvm; When the DO value is greater than 700, a mixed gas of air and oxygen is introduced, with an air to oxygen ratio of 1: (1.5 to 2.5), for example, the ratio may be 1:1.6, 1:1.8, 1:2, 1:2.2 or 1:2.4, etc., preferably 1:2; the aeration rate is 1.8 vvm to 2.4 vvm.

[098] Preferably, the method for determining the endpoint of high-density fermentation is as follows: monitor the DO value of the high-density fermentation system. If the increase in DO value per hour is less than or equal to 20 in two consecutive hours, indicating slow stem growth, it is considered that no further growth will occur and the endpoint of high-density fermentation is reached. The fermentation broth is then released from the fermentation tank.

[099] In the present application, “OD value” refers to the DO value of the fermentation broth (culture system), which is obtained by ultraviolet spectrophotometry, specifically the DO value at 600 nm.

[100] Preferably, high-density fermentation is processed to an OD value of 780 to 820, such as 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 Petition 870250087639, dated 09 / 26 / 2025, page 82 / 146 20 / 72 completed to obtain the single-cell protein.

[101] Preferably, high-density fermentation includes a first fermentation stage, a second fermentation stage, and a third fermentation stage, with DO values ​​increasing sequentially.

[102] The DO value of the first fermentation stage is less than 500, and the carbon to nitrogen ratio of the supplemented acetic acid and the nitrogen source in the first fermentation stage is greater than 18, such as a carbon to nitrogen ratio of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 100, etc.

[103] The DO value of the second fermentation stage is in the range of 500 to 700, and the carbon to nitrogen ratio of the supplemented acetic acid and the nitrogen source in the second fermentation stage is in the range of 5 to 18, such as a carbon to nitrogen ratio of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17, etc.

[104] The DO value of the third fermentation stage is greater than 700, and the carbon to nitrogen ratio of the supplemented acetic acid and the nitrogen source in the third fermentation stage is less than 5, such as a carbon to nitrogen ratio of 1, 1.5, 2, 2.5, 3, 3.5, 4 or 4.5, etc.

[105] As the preferred technical solution of the present application, the carbon-to-nitrogen ratio of the carbon sources and supplemented nitrogen sources during the high-density fermentation process is regulated. In the initial stage, with a relatively low DO value, the carbon-to-nitrogen ratio is adjusted to be greater than 18, which favors vigorous stem growth. When the DO value is 500 to 700, the carbon-to-nitrogen ratio is adjusted to be 5 to 18, which is beneficial for both stem growth and protein synthesis simultaneously. In the final stage of Petition 870250087639, dated 09 / 26 / 2025, page 83 / 146 In 21 / 72 high-density fermentation, the carbon-to-nitrogen ratio is adjusted to be less than 5, delaying stem growth and increasing the rate of protein synthesis. This synthesizes proteins completely and contributes to improving the crude protein content in the fermentation product. The present application allows for faster stem growth and higher crude protein content by adjusting the carbon-to-nitrogen ratio, which reduces the fermentation culture period and increases protein yield, simultaneously improving production efficiency and single-cell protein yield.

[106] In the present application, the feed rate of supplemented acetic acid and the nitrogen source can be regulated according to the carbon to nitrogen ratio at different stages of high-density fermentation; illustratively, the feed rate of acetic acid is 0.6 g / L to 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., additionally, preferably 0.8 g / L to 2.5 g / L; The nitrogen source feed rate is 0.1 g / L to 3.2 g / L, for example, it could 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.

[107] Preferably, during the initial stage of high-density fermentation, the feed rates of acetic acid and nitrogen sources are regulated, the carbon-to-nitrogen ratio is adjusted to be greater than 18 to promote vigorous stem growth. As fermentation progresses, the feed rates of acetic acid and nitrogen sources are adjusted to maintain a carbon-to-nitrogen ratio of 5 to 18, simultaneously facilitating rapid stem growth and rapid protein synthesis. Further adjustments to the feed rates of acetic acid and nitrogen sources are made to maintain a carbon-to-nitrogen ratio less than 5, thus facilitating single-cell protein synthesis and increasing the content of Petition 870250087639, dated 09 / 26 / 2025, page 84 / 146 22 / 72 crude protein. Throughout the high-density fermentation stage, the ideal effect for achieving the fastest stem growth rate and the highest crude protein content is achieved by regulating and balancing the carbon-to-nitrogen ratio.

[108] As the preferred technical solution of the present application, by regulating the processes during different stages of high-density fermentation, including pH range, temperature, rotation speed, carbon to nitrogen ratio, dissolved oxygen and gas flow rate, the thallus growth environment is adjusted to achieve ideal thallus growth conditions and the best state for single-cell protein synthesis.

[109] Preferably, the first fermentation stage includes a stage A, a stage B, and a stage C carried out sequentially, while high-density fermentation includes a stage A, a stage B, a stage C, a second fermentation stage, and a third fermentation stage carried out sequentially, specifically as follows: In stage A, the dissolved oxygen (DO) value is less than 100, with a temperature ranging from 31°C to 33°C, a pH value of 6.4 to 6.6, an aeration rate of 0.5 vvm to 0.7 vvm, a rotation speed of 130 rpm to 170 rpm, and a carbon-to-nitrogen ratio greater than 18 for both the supplemented acetic acid and the nitrogen source; Additionally, preferably in stage A, air (compressed air) is introduced at a controlled temperature of 32 °C, a pH value of 6.5, an aeration rate of 0.6 v / m, a rotation speed of 150 rpm, an acetic acid feed rate of 0.8 L / h, and a controlled carbon-to-nitrogen ratio greater than 18. Under these conditions, the stalks grow rapidly.

[110] In stage B, 100 < OD value < 300, with temperature Petition 870250087639, dated 09 / 26 / 2025, p. 85 / 146 23 / 72 ranging from 30 °C to 32 °C, a pH value of 6.5 to 6.7, an aeration rate of 0.9 vvm to 1.1 vvm, a rotation speed of 180 rpm to 220 rpm, and a carbon to nitrogen ratio greater than 18 for the supplemented acetic acid and nitrogen source; Additionally, preferably in stage B, air (compressed air) is introduced at a controlled temperature of 31 °C, a pH of 6.6, an aeration rate of 1 v / m, a rotation speed of 200 rpm, an acetic acid feed rate of 1.2 L / h, and a controlled carbon-to-nitrogen ratio greater than 18. Under these conditions, the stalks grow rapidly.

[111] In stage C, 300 < DO value < 500, with a temperature ranging from 29 °C to 31 °C, a pH value of 6.6 to 6.8, an aeration rate of 1.1 vvm to 1.3 vvm, a rotation speed of 280 rpm to 320 rpm, and a carbon to nitrogen ratio greater than 18 for the supplemented acetic acid and nitrogen source; Additionally, preferably at stage C, a mixed gas of air and oxygen is introduced, with an air to oxygen ratio of (1.5 to 2.5:1), more preferably 2:1. The temperature is controlled at 30 °C, the pH value at 6.7, the aeration rate at 1.2 v / m, the rotation speed at 300 rpm, the acetic acid feed rate at 1.6 L / h, and the carbon to nitrogen ratio is controlled to be greater than 18. Under these conditions, the thalli grow rapidly.

[112] The DO value of the second fermentation stage is in the range of 500 to 700, with a temperature ranging from 28 °C to 30 °C, a pH value of 6.7 to 6.9, an aeration rate of 1.5 vvm to 1.7 vvm, a rotation speed of 450 rpm to 550 rpm, and a carbon to nitrogen ratio of 5 to 18 for the supplemented acetic acid and nitrogen source; Additionally, preferably in the second stage of fermentation, Petition 870250087639, dated 09 / 26 / 2025, p. 86 / 146 24 / 72 A mixed gas of air and oxygen is introduced, with an air to oxygen ratio of (0.8 to 1.2):1, more preferably 1:1. The temperature is controlled at 29 °C, the pH value at 6.8, the aeration rate at 1.6 v / m, the rotation speed at 500 rpm, the acetic acid feed rate at 2.2 L / h, and the carbon to nitrogen ratio is controlled at 5 to 18. Under these conditions, the growth rate of the thalli is normal, and the single-cell protein is synthesized simultaneously.

[113] The DO value of the third fermentation stage is greater than 700, with a temperature ranging from 27 °C to 29 °C, a pH value of 6.9 to 7.1, an aeration rate of 1.9 vvm to 2.1 vvm, a rotation speed of 650 rpm to 750 rpm, and a carbon to nitrogen ratio of less than 5 for the supplemented acetic acid and nitrogen source; Additionally, preferably in the third fermentation stage, a mixed gas of air and oxygen is introduced, with an air to oxygen ratio of 1: (1.5 to 2.5), more preferably 1:2. The temperature is controlled at 28 °C, the pH value at 7, the aeration rate at 2.0 vvm, the rotation speed at 700 rpm, the acetic acid feed rate at 2.5 L / h, and the carbon to nitrogen ratio is controlled to be less than 5. Under these conditions, the growth rate of the stems is slow, allowing sufficient synthesis of single-cell protein.

[114] When the OD value reaches 780 to 820 and stem growth is slow, fermentation can be completed by emptying the fermentation tank to obtain the fermentation broth.

[115] Preferably, after the high-density fermentation is complete, the method additionally includes a post-treatment step.

[116] Preferably, the post-treatment method includes: subjecting the fermentation broth obtained from high-density fermentation to solid-liquid separation, washing and drying in sequence to obtain the Petition 870250087639, dated 09 / 26 / 2025, page 87 / 146 25 / 72 single-cell protein.

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

[118] Preferably, the detergent used for washing is water.

[119] Preferably, the drying method includes spray drying.

[120] Preferably, the method specifically includes the following steps: (1) Inoculate the Yarrowia lipolytica strain into the primary seed culture medium, and cultivate for 16 ha 18 ha at a temperature of 30 °C to 33 °C and a rotation speed of 100 rpm to 300 rpm until the OD value reaches 4.2 to 6.8, thus obtaining the primary seed; The primary seed culture medium includes the following components: 5 to 15 g / L of sodium acetate, 1% to 3% glucose, 0.6% to 2% yeast powder, and 1.5% to 3.2% casein peptone; (2) Inoculate the primary seed in the secondary seed culture medium at an inoculum size of 5% to 10%, and cultivate for 8 ha to 12 ha at a temperature of 30 °C to 33 °C, a rotation speed of 150 rpm to 900 rpm and an aeration rate of 0.1 vvm to 1.3 vvm until the DO value reaches 12 to 16, thus obtaining the secondary seed, i.e., the seed liquid; The secondary seed culture medium includes the following components: 5 to 20 g / L of sodium acetate, 1% to 3% glucose, 0.6% to 2% yeast powder, 1.5% to 3.2% casein peptone, 0.8% to 2.4% magnesium sulfate, 0.2% to 0.8% thiamine hydrochloride, and 0.8% to 2.6% first trace element additive; The first trace element additive includes the following components: 4 to 8 g / L of copper sulfate, 0.12 to 0.16 g / L of sodium iodide, 5 to 6 g / L of sulfate. Petition 870250087639, dated 09 / 26 / 2025, page 88 / 146 26 / 72 of manganese, 0.1 to 0.2 g / L of sodium molybdate, 0.04 to 0.08 g / L of boric acid, 0.6 to 0.8 g / L of cobalt chloride, 18 to 21 g / L of zinc chloride, 38 to 41 g / L of ferrous sulfate, 0.3 to 0.5 g / L of biotin, and 3 to 5.2 g / L of sulfuric acid; (3) Inoculate the seed liquid into the fermentation culture medium at an inoculum size of 3% to 6% for high-density fermentation, and supplement with acetic acid and urea during the high-density fermentation process until the OD value reaches 780 to 820, thus obtaining the single-cell protein; The fermentation culture medium includes the following components: 5 to 15 g / L of sodium acetate, 1% to 3% glucose, 0.6% to 2% yeast powder, 1.5% to 3.2% casein peptone, 0.8% to 1.2% magnesium sulfate, 0.6% to 1.4% thiamine hydrochloride, 0.5% to 1% betaine, 3.5% to 6.8% ammonium sulfate, 5% to 7% potassium dihydrogen phosphate, 2% to 4% disodium hydrogen phosphate, 0.1% to 0.6% acetone, and 0.8% to 2.6% of a second trace element additive; The second trace element additive includes the following components: 4 to 8 g / L of copper sulfate, 0.12 to 0.16 g / L of sodium iodide, 5 to 6 g / L of manganese sulfate, 0.1 to 0.2 g / L of sodium molybdate, 0.04 to 0.08 g / L of boric acid, 0.6 to 0.8 g / L of cobalt chloride, 18 to 21 g / L of zinc chloride, 38 to 41 g / L of ferrous sulfate, 0.3 to 0.5 g / L of biotin, and 3 to 5.2 g / L of sulfuric acid; High-density fermentation includes a first fermentation stage, a second fermentation stage, and a third fermentation stage, with the dissolved oxygen (DO) value increasing sequentially; The dissolved oxygen (DO) value of the first fermentation stage is less than 500, with a temperature ranging from 29°C to 33°C, a pH value of 6.5 to 6.8, an aeration rate of 0.5 vvm to 1.3 vvm, a rotation speed of 280 rpm to 320 rpm, and a carbon-to-nitrogen ratio greater than 18 for acetic acid. Petition 870250087639, dated 09 / 26 / 2025, page 89 / 146 27 / 72 supplemented with urea; The dissolved oxygen (DO) value of the second fermentation stage is in the range of 500 to 700, with a temperature ranging from 28 °C to 30 °C, a pH value of 6.7 to 6.9, an aeration rate of 1.5 vvm to 1.7 vvm, a rotation speed of 450 rpm to 550 rpm, and a carbon to nitrogen ratio of 5 to 18 for the supplemented acetic acid and nitrogen source; The dissolved oxygen (DO) value of the third fermentation stage is greater than 700, with a temperature ranging from 27°C to 29°C, a pH value of 6.9 to 7.1, an aeration rate of 1.9 vvm to 2.1 vvm, a rotation speed of 650 rpm to 750 rpm, and a carbon-to-nitrogen ratio of less than 5 for the supplemented acetic acid and nitrogen source.

[121] As the preferred technical solution of the present application, the duration for high-density fermentation (i.e., fermentation period) is in the range of 25 h to 60 h, for example, it could be 26 h, 28 h, 30 h, 32 h, 35 h, 38 h, 40 h, 42 h, 45 h, 48 h, 50 h, 52 h, 55 h, 58 h etc. Additionally, preferably 28 to 36 h, and more preferably 28 to 32 h.

[122] Preferably, the total yield (dry weight) of single-cell protein is greater than 150 g / L, for example, it could 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. Additionally, preferably greater than or equal to 160 g / L, and even more preferably 200 to 250 g / L.

[123] Preferably, the single-cell protein yield is greater than 2.5 g / (Lh), for example, it could be 2.6 g / (Lh), 2.8 g / (Lh), 3 g / (Lh), 3.5 g / (Lh), 4 g / (Lh), 5 g / (Lh), 6 g / (Lh), 7 g / (bh), 8 g / (bh), 8.5 g / g / (Lh), 9 g / (L«h) or 10 g / (bh), etc. Additionally, preferably greater than or equal to 3 g / (L^h), and even more preferably, 6 to 9 g / (L^h).

[124] Preferably, the percentage by mass of protein content Petition 870250087639, dated 09 / 26 / 2025, pp. 90 / 146 28 / 72 crude protein in single-cell protein is greater than or equal to 35%, for example, it could be 38%, 40%, 42%, 45%, 48%, 50%, 52%, 55%, 58%, 60%, 62%, 65% or 68% etc. Additionally, preferably greater than or equal to 55%, and even more preferably greater than or equal to 60%.

[125] Compared with related technologies, examples of the present invention exhibit the following beneficial effects: (1) In the method for producing single-cell protein by means of high-density fermentation provided by the embodiments of the present application, the introduction of the first acetate into the carbon source of the seed culture medium induces and promotes the formation of a unique acetic acid metabolic pathway in the stalk, thus establishing the basis for subsequent acetic acid supplementation as a carbon source and promoting rapid stalk growth during high-density fermentation. Meanwhile, the fermentation culture medium contains a second acetate and / or ketone compounds, which promote rapid stalk growth.During the high-density fermentation stage, nitrogen and acetate sources are supplemented to maintain the thallus in an ideal pH-neutral growth environment, and the carbon-to-nitrogen ratio is regulated to provide the thallus with the appropriate nutrients for its continuous growth, effectively increasing the thallus growth rate and the single-cell protein synthesis rate. Through the combination of specific materials and processes, the method allows for high-density thallus fermentation and efficient single-cell protein synthesis in a short period of time, obtaining a fermentation product with a higher crude protein content, significantly reducing the high-density fermentation cultivation time and significantly increasing the efficiency and yield of single-cell protein production. (2) Through further optimization and design of material and process, Petition 870250087639, dated 09 / 26 / 2025, pp. 91 / 146 29 / 72 of the adaptive composition of the medium, specific feeding methods, and gradual process regulation strategies during high-density fermentation, the examples in the present application achieve rapid stem growth and rapid single-cell protein synthesis. This results in a high-density fermentation time (fermentation period) of 28 to 30 hours, which is reduced to one-third compared to the single-cell protein production time in related technologies. Meanwhile, the dry weight of the single-cell protein is 200 to 243 g / L, and the yield is greater than or equal to 6 g / (L^h), potentially 20 times higher than the yield of the same type. The crude protein content is greater than or equal to 60%, potentially increased by 50% compared to related technologies, significantly improving the yield and production capacity of the single-cell protein.

[126] After reading and understanding the attached detailed drawings and descriptions, other aspects can be understood. DESCRIPTION OF THE DRAWINGS

[127] The attached drawings are used to facilitate understanding of the technical solutions presented in this application and form an integral part of the descriptive report. They are used in conjunction with the embodiments of this application to explain the technical solution presented in this application and do not constitute limitations to the technical solution of this application.

[128] Figure 1 is a growth trend graph of the high-density fermentation process in the method given in Example 1.

[129] Figure 2 is a test diagram of the fermentation broth obtained using the method given in Example 1. DETAILED DESCRIPTION

[130] The technical solution to the present application will be explained further. Petition 870250087639, dated 09 / 26 / 2025, page 92 / 146 30 / 72 through specific modalities. Subject matter experts should understand that the examples described are intended only to assist in understanding this application and should not be considered as specific limitations thereof.

[131] The terms “comprise”, “include”, “have”, “contain” or any other variations thereof used in this article are intended to encompass non-exclusive inclusion. For example, a composition, step, method, product or device comprising the listed elements is not necessarily limited to those elements, but may also include other elements not explicitly listed or the elements inherent in such composition, step, method, product or device.

[132] In this application, attributes designated as “first” and “second” may include, explicitly or implicitly, one or more such attributes, used to distinguish and describe the attributes, without any distinction of order or importance. In the description of this application, unless otherwise indicated, the meaning of “multiple” is two or more of two.

[133] In this application, the term “OD value” refers to the DO value of the fermentation broth, which is obtained by ultraviolet spectrophotometry. Specifically, an ultraviolet spectrophotometer (Alpha-1106, Shanghai labspectrum Instrument Co., Ltd.) is used to measure the DO value of the fermentation broth at 600 nm.

[134] In the following specific embodiments of this application, the test methods for dry weight, yield and crude protein content are as follows: (1) Dry weight of single-cell protein: the unit is g / L

[135] Weigh 1.00 L of fermentation broth and centrifuge at 8,000 revolutions per minute (rpm) for 10 min. After the stalks are obtained, add 1 L of water. Petition 870250087639, dated 09 / 26 / 2025, page 93 / 146 31 / 72 is then added for uniform resuspension. The mixture is centrifuged again at 8,000 rpm for 10 minutes to obtain the wet stalk, the mass of which is weighed and recorded as m1. 5,000 g of wet stalk are removed from the mixture and dried at 103 ± 2 °C according to the method described in the national standard GB / T 64352014 “Determination of Moisture in Feeds” to obtain the dry stalk, the mass of which is weighed as m2 (with an accuracy of 4 decimal places).

[136] Cell dry weight (DCW) = m2 / 5,000 x m1 / 1.00. (2) Yield: the unit is g / (Lh)

[137] Yield = Dry weight of single-cell protein ^ Duration of high-density fermentation (fermentation period). (3) Crude protein content

[138] 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 by digestion, ammonia distillation and titration.

[139] In the following specific embodiments of the present application, the Yarrowia lipolytica strain was acquired from the China Industrial Culture Collection Center, with accession number CICC 32291; several components used for the preparation of the culture medium were commercially available chemicals.

[140] In the following specific embodiments of the present application, the method for determining the endpoint of high-density fermentation is as follows: if the increase in DO value per hour is less than 20 in two consecutive hours, indicating very slow stem growth, it is considered that no further growth will occur and the endpoint of high-density fermentation is reached. The fermentation broth is then released from the fermentation tank. Example 1

[141] A method for producing single-cell protein by means of Petition 870250087639, dated 09 / 26 / 2025, pp. 94 / 146 32 / 72 high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: 200 mL of primary seed culture medium were prepared, comprising the following components: 8 g / L sodium acetate, 1.8% anhydrous glucose, 1.2% yeast powder, and 2.2% casein peptone; the primary seed culture medium was sterilized at 121 °C for 20 min in a sterilizer. After sterilization, the primary seed culture medium was placed on a super clean bench. After returning to room temperature, the cryopreservation solution of the thawed Yarrowia lipolytica strain was added to the primary seed culture medium and placed in a shaker, then cultured for 16 hours at a temperature of 32 °C and a rotation speed of 180 rpm, with an OD value of approximately 4.2, thus obtaining the primary seed; (2) Secondary seed crop: L of secondary seed culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.2% anhydrous glucose, 1.5% yeast powder, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% thiamine hydrochloride; The secondary seed culture medium was added to a 5 L fermentation tank and sterilized at 121 °C for 20 min in a sterilizer. After cooling to 32 °C, the primary seed was added to the fermentation tank of the secondary seed culture medium at an inoculum size of 7.5% via flame inoculation. mL of trace element additive after sterilization and filtration were then added, with the following formula: 4 g / L copper sulfate, 0.12 g / L sodium iodide, 5 g / L manganese sulfate, 0.1 g / L molybdate Petition 870250087639, dated 09 / 26 / 2025, pp. 95 / 146 33 / 72 of sodium, 0.04 g / L of boric acid, 0.6 g / L of cobalt chloride, 18 g / L of zinc chloride, 38 g / L of ferrous sulfate, 0.3 g / L of biotin, and 3 g / L of sulfuric acid.

[142] In the secondary seed culture, when the initial dissolved oxygen was at 100%, the stirring speed was set to 180 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.2 vvm; when the dissolved oxygen ranged from 80% to 100%, the stirring speed was set to 220 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.3 vvm; when the dissolved oxygen ranged from 60% to 80%, the stirring speed was set to 280 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.4 vvm; when the dissolved oxygen ranged from 40% to 60%, the stirring speed was set to 350 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.5 vvm; When the dissolved oxygen ranged from 20% to 40%, the stirring speed was set to 440 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.6 vvm;When dissolved oxygen ranged from 10% to 20%, the stirring speed was set to 520 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.7 vvm; when dissolved oxygen ranged from 0% to 10%, the stirring speed was set to 600 rpm, the temperature was 30 °C, and the compressed air aeration rate was 0.8 vvm; when dissolved oxygen was at 0%, the stirring speed was set to 700 rpm, the temperature was 30 °C, and the compressed air aeration rate was 0.9 vvm.

[143] The duration for the secondary seed culture was 8 h, with an OD value of approximately 12, resulting in the secondary seed. (3) High-density fermentation

[144] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% Petition 870250087639, dated 09 / 26 / 2025, pp. 96 / 146 34 / 72 magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[145] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 40% urea solution.

[146] In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon to nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: Under the conditions of OD < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; Petition 870250087639, dated 09 / 26 / 2025, page 97 / 146 35 / 72 under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; Under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 v / m, a rotation speed of 300 rpm, a carbon source feed rate of 1.6 L / h, and a controlled carbon to nitrogen ratio of 20, the thallus grew rapidly; Under conditions of an OD value of 500 to 700, a controlled temperature of 29 °C, a pH of 6.8, an aeration rate of a mixed gas (air: pure oxygen in a 1:1 ratio) of 1.6 v / m, a rotation speed of 500 rpm, a carbon source feed rate of 2.2 L / h, and a controlled carbon to nitrogen ratio of 10, the thallus growth rate was normal and single-cell protein was synthesized simultaneously; Under conditions of an OD value greater than 700, a controlled temperature of 28 °C, a pH of 7, an aeration rate of a mixed gas (air: pure oxygen in a 1:2 ratio) of 2.0 v / m, a rotation speed of 700 rpm, a carbon source feed rate of 2.5 L / h, and a controlled carbon to nitrogen ratio of 3, the thallus growth rate was slow, allowing sufficient synthesis of single-cell protein.

[147] After 30 h of cultivation, the OD value reached 790, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[148] The fermentation broth was centrifuged and washed with water, being Petition 870250087639, dated 09 / 26 / 2025, pp. 98 / 146 36 / 72 then 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%.

[149] During the high-density fermentation process of this example, samples were collected every hour. Based on the peak absorption of the DO value of the fermentation broth at 600 nm, the DO of the stem was calculated after conversion and correction. The average value was obtained from three measurements. A growth trend graph was plotted based on growth time and DO value. The resulting graph is shown in Figure 1.

[150] A sample of the fermentation broth obtained from the high-density fermentation in this example was collected for microscopic examination. The test instrument was a digital microscope (longbase 610D, Qingdao Longbase Medical Equipment Co., Ltd.). The test image obtained is shown in Figure 2. Example 2

[151] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: 200 mL of primary seed culture medium were prepared, comprising the following components: 8 g / L sodium acetate, 1.8% anhydrous glucose, 1.2% yeast powder, and 2.2% casein peptone; the primary seed culture medium was sterilized at 121 °C for 20 min in a sterilizer. After sterilization, the primary seed culture medium was placed on a super clean bench. After returning to room temperature, the cryopreservation solution of the thawed Yarrowia lipolytica strain was added to the primary seed culture medium and placed in a shaker, then cultured for 17 hours at a temperature of 32 °C and a Petition 870250087639, dated 09 / 26 / 2025, p. 99 / 146 37 / 72 rotation speed of 180 rpm, with an OD value around 4.8, thus obtaining the primary seed; (2) Secondary seed crop: L of secondary seed culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.2% anhydrous glucose, 1.5% yeast powder, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% thiamine hydrochloride; The secondary seed culture medium was added to a 5 L fermentation tank and sterilized at 121 °C for 20 min in a sterilizer. After cooling to 32 °C, the primary seed was added to the fermentation tank of the secondary seed culture medium at an inoculum size of 7.5% via flame inoculation. mL of trace element additive after sterilization and filtration were then added, with the following formula: 4 g / L copper sulfate, 0.12 g / L sodium iodide, 5 g / L manganese sulfate, 0.1 g / L sodium molybdate, 0.04 g / L boric acid, 0.6 g / L cobalt chloride, 18 g / L zinc chloride, 38 g / L ferrous sulfate, 0.3 g / L biotin, and 3 g / L sulfuric acid.

[152] In the secondary seed culture, when the initial dissolved oxygen was at 100%, the stirring speed was set to 180 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.2 vvm; when the dissolved oxygen ranged from 80% to 100%, the stirring speed was set to 220 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.3 vvm; when the dissolved oxygen ranged from 60% to 80%, the stirring speed was set to 280 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.4 vvm; when the dissolved oxygen ranged from 40% to 60%, the stirring speed was set to 350 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.5 vvm; when the dissolved oxygen Petition 870250087639, dated 09 / 26 / 2025, pp. 100 / 146 When the dissolved oxygen content (38 / 72) varied from 20% to 40%, the stirring speed was set to 440 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.6 vvm; when the dissolved oxygen content varied from 10% to 20%, the stirring speed was set to 520 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.7 vvm; when the dissolved oxygen content varied from 0% to 10%, the stirring speed was set to 600 rpm, the temperature was 30 °C, and the compressed air aeration rate was 0.8 vvm; when the dissolved oxygen content was at 0%, the stirring speed was set to 750 rpm, the temperature was 30 °C, and the compressed air aeration rate was 1 vvm.

[153] The duration for the secondary seed culture was 10 h, with an OD value of approximately 14, resulting in the secondary seed. (3) High-density fermentation

[154] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate. Petition 870250087639, dated 09 / 26 / 2025, pp. 101 / 146 39 / 72 0.45 g / L of biotin, and 5.2 g / L of sulfuric acid.

[155] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 40% urea solution.

[156] In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon to nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: Under the conditions of OD < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon to nitrogen ratio of 30, the stalk grew rapidly; Under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 30, the stalk grew rapidly; Under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 v / m, a rotation speed of 300 rpm, a carbon source feed rate of 1.6 L / h, and a controlled carbon to nitrogen ratio of 30, the thallus grew rapidly; under the conditions of an OD value of 500 to 700, a controlled temperature of 29 °C, a pH of 6.8, an aeration rate of a mixed gas (air: Petition 870250087639, dated 09 / 26 / 2025, pp. 102 / 146 40 / 72 pure oxygen in a 1:1 ratio) of 1.6 vvm, a rotation speed of 500 rpm, a carbon source feed rate of 2.2 L / h, and a controlled carbon to nitrogen ratio of 12, the thallus growth rate was normal, while single-cell protein was synthesized simultaneously; Under conditions of an OD value greater than 700, a controlled temperature of 28 °C, a pH of 7, an aeration rate of a mixed gas (air: pure oxygen in a 1:2 ratio) of 2.0 v / m, a rotation speed of 700 rpm, a carbon source feed rate of 2.5 L / h, and a controlled carbon to nitrogen ratio of 2, the thallus growth rate was slow, allowing sufficient synthesis of single-cell protein.

[157] After 30 h of cultivation, the OD value reached 786, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[158] The fermentation broth was centrifuged and washed with water, then spray dried to obtain single-cell protein with a dry weight of 238 g / L, a yield of 7.9 g / (Lh), and a crude protein content of 60%. Example 3

[159] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: 200 mL of primary seed culture medium were prepared, comprising the following components: 8 g / L sodium acetate, 1.8% anhydrous glucose, 1.2% yeast powder, and 2.2% casein peptone; the primary seed culture medium was sterilized at 121 °C for 20 min in a sterilizer. After sterilization, the primary seed culture medium was Petition 870250087639, dated 09 / 26 / 2025, pp. 103 / 146 41 / 72 placed on a super clean bench. After returning to room temperature, the cryopreservation solution of the thawed Yarrowia lipolytica strain was added to the primary seed culture medium and placed in a shaker, then cultured for 18 ha at a temperature of 32 °C and a rotation speed of 180 rpm, with an OD value of approximately 5.8, while the primary seed was obtained; (2) Secondary seed crop: L of secondary seed culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.2% anhydrous glucose, 1.5% yeast powder, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% thiamine hydrochloride; The secondary seed culture medium was added to a 5 L fermentation tank and sterilized in a sterilizer at 121 °C for 20 min. After cooling to 32 °C, the primary seed was added to the fermentation tank of the secondary seed culture medium at an inoculum size of 7.5% via flame inoculation. mL of trace element additive after sterilization and filtration were then added, with the following formula: 4 g / L copper sulfate, 0.12 g / L sodium iodide, 5 g / L manganese sulfate, 0.1 g / L sodium molybdate, 0.04 g / L boric acid, 0.6 g / L cobalt chloride, 18 g / L zinc chloride, 38 g / L ferrous sulfate, 0.3 g / L biotin, and 3 g / L sulfuric acid; In the secondary seed culture, when the initial dissolved oxygen was at 100%, the stirring speed was set to 180 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.2 vvm; when the dissolved oxygen ranged from 80% to 100%, the stirring speed was set to 220 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.3 vvm; when the dissolved oxygen ranged from 60% to 80%, the speed of Petition 870250087639, dated 09 / 26 / 2025, pp. 104 / 146 42 / 72 agitation was set to 280 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.4 vvm; when dissolved oxygen ranged from 40% to 60%, the agitation speed was set to 350 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.5 vvm; when dissolved oxygen ranged from 20% to 40%, the agitation speed was set to 440 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.6 vvm; when dissolved oxygen ranged from 10% to 20%, the agitation speed was set to 520 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.7 vvm; When dissolved oxygen ranged from 0% to 10%, the stirring speed was set to 600 rpm, the temperature was 30 °C, and the compressed air aeration rate was 0.8 vvm; when dissolved oxygen was at 0%, the stirring speed was set to 800 rpm, the temperature was 30 °C, and the compressed air aeration rate was 1.2 vvm.

[160] The duration for the secondary seed culture was 12 h, with an OD value of approximately 16, resulting in the secondary seed. (3) High-density fermentation

[161] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements after sterilization and filtration were Petition 870250087639, dated 09 / 26 / 2025, pp. 105 / 146 43 / 72 were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[162] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 40% urea solution; In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon-to-nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: under the conditions of O < DO value < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 vvm, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon-to-nitrogen ratio of 50, the stem grew rapidly; Under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 50, the stalk grew rapidly; Under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 vvm, a rotation speed of 300 rpm, and a carbon source feed rate of 1.6 Petition 870250087639, dated 09 / 26 / 2025, pp. 106 / 146 44 / 72 At a controlled carbon-to-nitrogen ratio of 50 L / h, the stalk grew rapidly; Under conditions of an OD value of 500 to 700, a controlled temperature of 29 °C, a pH of 6.8, an aeration rate of a mixed gas (air: pure oxygen in a 1:1 ratio) of 1.6 v / m, a rotation speed of 500 rpm, a carbon source feed rate of 2.2 L / h, and a controlled carbon to nitrogen ratio of 18, the thallus growth rate was normal, while single-cell protein was synthesized simultaneously; Under conditions of an OD value greater than 700, a controlled temperature of 28 °C, a pH of 7, a mixed gas aeration rate (air: pure oxygen in a 1:2 ratio) of 2.0 vvm, a rotation speed of 700 rpm, a carbon source feed rate of 2.5 L / h, and a controlled carbon to nitrogen ratio of 1, the thallus growth rate was slow, allowing sufficient single-cell protein synthesis.

[163] After 30 h of cultivation, the OD value was 792, indicating slow thallus growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[164] The fermentation broth was centrifuged and washed with water, then 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%. Example 4

[165] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: same as step (1) in Example 1, Petition 870250087639, dated 09 / 26 / 2025, pp. 107 / 146 45 / 72 to obtain the primary seed. (2) Secondary seed culture: same as step (2) in Example 1, to obtain the secondary seed. (3) High-density fermentation.

[166] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[167] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 30% ammonium sulfate solution.

[168] In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon to nitrogen ratio, dissolved oxygen, and Petition 870250087639, dated 09 / 26 / 2025, pp. 108 / 146 46 / 72 gas flow rate during different stages of the process. The specifications were as follows: Under the conditions of OD < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; Under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; Under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 v / m, a rotation speed of 300 rpm, a carbon source feed rate of 1.6 L / h, and a controlled carbon to nitrogen ratio of 20, the thallus grew rapidly; Under conditions of an OD value of 500 to 700, a controlled temperature of 29 °C, a pH of 6.8, an aeration rate of a mixed gas (air: pure oxygen in a 1:1 ratio) of 1.6 v / m, a rotation speed of 500 rpm, a carbon source feed rate of 2.2 L / h, and a controlled carbon to nitrogen ratio of 10, the thallus growth rate was normal, while single-cell protein was synthesized simultaneously; After 48 hours of cultivation, the dissolved oxygen (DO) value was 523, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[169] The fermentation broth was centrifuged and washed with water, then Petition 870250087639, dated 09 / 26 / 2025, pp. 109 / 146 4Ί / Ί2 spray-dried to obtain single-cell protein with a dry weight of 180 g / L, a yield of 3.Ί5 g / (Lh), and a crude protein content of 41%. Example 5 [1Ί0] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: same as step (1) in Example 1, to obtain the primary seed. (2) Secondary seed culture: same as step (2) in Example 1, to obtain the secondary seed. (3) High-density fermentation. [1Ί1] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, Ί% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid. Petition 870250087639, dated 09 / 26 / 2025, pp. 110 / 146 48 / 72

[172] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 20% ammonium nitrate solution; In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon-to-nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: under the conditions of O < DO value < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 vvm, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon-to-nitrogen ratio of 20, the stem grew rapidly; Under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; Under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 v / m, a rotation speed of 300 rpm, a carbon source feed rate of 1.6 L / h, and a controlled carbon to nitrogen ratio of 20, the thallus grew rapidly; After 52 hours of cultivation, the dissolved oxygen (DO) value was approximately 478, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. Petition 870250087639, dated 09 / 26 / 2025, pp. 111 / 146 49 / 72 (4) Post-treatment

[173] The fermentation broth was centrifuged and washed with water, then 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%. Example 6

[174] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: same as step (1) in Example 1, to obtain the primary seed. (2) Secondary seed culture: same as step (2) in Example 1, to obtain the secondary seed. (3) High-density fermentation.

[175] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid. Petition 870250087639, dated 09 / 26 / 2025, pp. 112 / 146 50 / 72 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[176] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a 28% ammonia solution; In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon-to-nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: under the conditions of O < DO value < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 vvm, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon-to-nitrogen ratio of 20, the stem grew rapidly; Under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; Under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 v / m, a rotation speed of 300 rpm, a carbon source feed rate of 1.6 L / h, and a controlled carbon to nitrogen ratio of 20, the thallus grew rapidly; under conditions of a dissolved oxygen (DO) value of 500 to 700, a temperature Petition 870250087639, dated 09 / 26 / 2025, pp. 113 / 146 Under controlled conditions of 29 °C, a pH of 6.8, a mixed gas aeration rate (air: pure oxygen in a 1:1 ratio) of 1.6 v / m, a rotation speed of 500 rpm, a carbon source feed rate of 2.2 L / h, and a controlled carbon to nitrogen ratio of 10, the thallus growth rate was normal, while single-cell protein was synthesized simultaneously; After 50 hours of cultivation, the dissolved oxygen (DO) value was approximately 585, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[177] The fermentation broth was centrifuged and washed with water, then 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%.

[178] A comparison of Example 1 with Examples 4 to 6 demonstrates that in the high-density fermentation process of Example 1, supplementation with urea as a nitrogen source and acetic acid as a carbon source can more effectively promote rapid stem growth and protein synthesis. In this case, urea, as a nitrogen source, is more readily absorbed by the stem, which favors protein synthesis. The nitrogen sources supplemented in Examples 4 to 6 do not favor absorption by the stem, resulting in slow growth, which not only prolongs the fermentation period but also reduces the single-cell protein yield obtained. Example 7

[179] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: Petition 870250087639, dated 09 / 26 / 2025, pp. 114 / 146 52 / 72 (1) Primary seed culture: same as step (1) in Example 1, to obtain the primary seed. (2) Secondary seed culture: same as step (2) in Example 1, to obtain the secondary seed. (3) High-density fermentation.

[180] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[181] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 40% urea solution.

[182] In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, and Petition 870250087639, dated 09 / 26 / 2025, pp. 115 / 146 53 / 72 rotational speed, carbon to nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: Under the conditions of OD < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and maintaining a constant carbon to nitrogen ratio of 20, the stalk grew rapidly; Under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and maintaining a constant carbon to nitrogen ratio of 20, the thallus grew rapidly; Under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 v / m, a rotation speed of 300 rpm, a carbon source feed rate of 1.6 L / h, and maintaining a constant carbon to nitrogen ratio of 20, the thallus grew rapidly; Under conditions of an OD value of 500 to 700, a controlled temperature of 29 °C, a pH of 6.8, an aeration rate of a mixed gas (air: pure oxygen in a 1:1 ratio) of 1.6 v / m, a rotation speed of 500 rpm, a carbon source feed rate of 2.2 L / h, and maintaining a constant carbon to nitrogen ratio of 20, the thallus growth rate was normal, while single-cell protein was synthesized simultaneously; After 36 hours of cultivation, the dissolved oxygen (DO) value reached approximately 641, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. Petition 870250087639, dated 09 / 26 / 2025, pp. 116 / 146 54 / 72 (4) Post-treatment

[183] ​​The fermentation broth was centrifuged and washed with water, then 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%.

[184] A comparison of Example 1 with Example 7 demonstrates that in the high-density fermentation process of Example 1, the carbon-to-nitrogen ratio was adjusted according to the fermentation stage. When the carbon-to-nitrogen ratio was greater than 18, stalk growth was vigorous, and when the carbon-to-nitrogen ratio was less than 5, stalk growth slowed, while the rate of protein synthesis increased. By adjusting the carbon-to-nitrogen ratio, faster stalk growth and higher crude protein content were achieved. In Example 7, the high-density fermentation process maintained a high and constant carbon-to-nitrogen ratio, resulting in a slower rate of protein synthesis and, ultimately, a lower crude protein content. Example 8

[185] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: same as step (1) in Example 1, to obtain the primary seed. (2) Secondary seed culture: same as step (2) in Example 1, to obtain the secondary seed. (3) High-density fermentation.

[186] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% Petition 870250087639, dated 09 / 26 / 2025, pp. 117 / 146 55 / 72 magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[187] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 40% urea solution.

[188] In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon to nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: Under the conditions of OD < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and maintaining a constant carbon to nitrogen ratio of 12, the stalk grew rapidly; Petition 870250087639, dated 09 / 26 / 2025, pp. 118 / 146 56 / 72 under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and maintaining a constant carbon to nitrogen ratio of 12, the stalk grew rapidly; Under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 v / m, a rotation speed of 300 rpm, a carbon source feed rate of 1.6 L / h, and maintaining a constant carbon to nitrogen ratio of 12, the thallus grew rapidly; Under conditions of an OD value of 500 to 700, a controlled temperature of 29 °C, a pH of 6.8, an aeration rate of a mixed gas (air: pure oxygen in a 1:1 ratio) of 1.6 v / m, a rotation speed of 500 rpm, a carbon source feed rate of 2.2 L / h, and maintaining a constant carbon to nitrogen ratio of 12, the thallus growth rate was normal, while single-cell protein was synthesized simultaneously.

[189] After 46 h of cultivation, the OD value was approximately 536, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[190] The fermentation broth was centrifuged and washed with water, then 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%.

[191] A comparison of Example 1 with Example 8 demonstrates that in the high-density fermentation process of Example 1, the carbon-to-nitrogen ratio was regulated according to the fermentation stage. When the carbon-to-nitrogen ratio was greater than 18, stem growth was vigorous, and Petition 870250087639, dated 09 / 26 / 2025, pp. 119 / 146 57 / 72 when the carbon-to-nitrogen ratio was less than 5, stem growth slowed, while the rate of protein synthesis increased. By adjusting the carbon-to-nitrogen ratio, faster stem growth and higher crude protein content were achieved. In Example 8, the high-density fermentation process maintained a constant carbon-to-nitrogen ratio of 12, resulting in lower stem growth rate and lower protein synthesis rate, as well as lower yield and efficiency. Example 9

[192] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: same as step (1) in Example 1, to obtain the primary seed. (2) Secondary seed culture: same as step (2) in Example 1, to obtain the secondary seed. (3) High-density fermentation.

[193] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements after sterilization and filtration were Petition 870250087639, dated 09 / 26 / 2025, pages 120 / 146 58 / 72 were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[194] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 40% urea solution.

[195] In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon to nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: Under the conditions of OD < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and maintaining a constant carbon to nitrogen ratio of 2, the thallus grew rapidly; Under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and maintaining a constant carbon to nitrogen ratio of 2, the thallus grew rapidly; under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a ratio of 2:1) of 1.2 vvm, a rotation speed of 300 rpm, a Petition 870250087639, dated 09 / 26 / 2025, pp. 121 / 146 59 / 72 carbon source feeding rate of 1.6 L / h, and maintaining a constant carbon to nitrogen ratio of 2, the stalk grew rapidly; Under conditions of an OD value of 500 to 700, a controlled temperature of 29 °C, a pH of 6.8, an aeration rate of a mixed gas (air: pure oxygen in a 1:1 ratio) of 1.6 v / m, a rotation speed of 500 rpm, a carbon source feed rate of 2.2 L / h, and maintaining a constant carbon to nitrogen ratio of 2, the thallus growth rate was normal, while single-cell protein was synthesized simultaneously; After 64 hours of cultivation, the dissolved oxygen (DO) value was approximately 528, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[196] The fermentation broth was centrifuged and washed with water, then 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%.

[197] A comparison of Example 1 with Example 9 demonstrates that in the high-density fermentation process of Example 1, the carbon-to-nitrogen ratio was regulated according to the fermentation stage. When the carbon-to-nitrogen ratio was greater than 18, stalk growth was vigorous, and when the carbon-to-nitrogen ratio was less than 5, stalk growth slowed, while the rate of protein synthesis increased. By adjusting the carbon-to-nitrogen ratio, faster stalk growth and higher crude protein content were achieved. In Example 9, the high-density fermentation process maintained a low and constant carbon-to-nitrogen ratio, resulting in a slow stalk growth rate and a long fermentation period. Comparative Example 1 Petition 870250087639, dated 09 / 26 / 2025, pp. 122 / 146 60 / 72

[198] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: 200 mL of primary seed culture medium were prepared, comprising the following components: 1.8% anhydrous glucose, 1.2% yeast powder, and 2.2% casein peptone; the primary seed culture medium was sterilized at 121 °C for 20 min in a sterilizer. After sterilization, the primary seed culture medium was placed on a super clean bench. After returning to room temperature, the cryopreservation solution of the thawed Yarrowia lipolytica strain was added to the primary seed culture medium and placed in a shaker, and then cultured for 16 ha at a temperature of 32 °C and a rotation speed of 180 rpm, with an OD value of approximately 4.2, while the primary seed was obtained; (2) Secondary seed crop: L of secondary seed culture medium were prepared, comprising the following components: 2.2% anhydrous glucose, 1.5% yeast powder, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% thiamine hydrochloride; The secondary seed culture medium was added to a 5 L fermentation tank and sterilized in a sterilizer at 121 °C for 20 min. After cooling to 32 °C, the primary seed was added to the fermentation tank of the secondary seed culture medium at an inoculum size of 7.5% via flame inoculation. mL of trace element additive after sterilization and filtration were then added, with the following formula: 4 g / L of copper sulfate, Petition 870250087639, dated 09 / 26 / 2025, pp. 123 / 146 61 / 72 0.12 g / L sodium iodide, 5 g / L manganese sulfate, 0.1 g / L sodium molybdate, 0.04 g / L boric acid, 0.6 g / L cobalt chloride, 18 g / L zinc chloride, 38 g / L ferrous sulfate, 0.3 g / L biotin, and 3 g / L sulfuric acid.

[199] In the secondary seed culture, when the initial dissolved oxygen was at 100%, the stirring speed was set to 180 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.2 vvm; when the dissolved oxygen ranged from 80% to 100%, the stirring speed was set to 220 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.3 vvm; when the dissolved oxygen ranged from 60% to 80%, the stirring speed was set to 280 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.4 vvm; when the dissolved oxygen ranged from 40% to 60%, the stirring speed was set to 350 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.5 vvm; When the dissolved oxygen ranged from 20% to 40%, the stirring speed was set to 440 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.6 vvm;When dissolved oxygen ranged from 10% to 20%, the stirring speed was set to 520 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.7 vvm; when dissolved oxygen ranged from 0% to 10%, the stirring speed was set to 600 rpm, the temperature was 30 °C, and the compressed air aeration rate was 0.8 vvm; when dissolved oxygen was at 0%, the stirring speed was set to 700 rpm, the temperature was 30 °C, and the compressed air aeration rate was 0.9 vvm.

[200] The duration for the secondary seed culture was 8 h, with an OD value of approximately 12, resulting in the secondary seed. (3) High-density fermentation

[201] 40 L of fermentation culture medium were prepared, comprising the following components: 2.6% anhydrous glucose, 1.8% powder Petition 870250087639, dated 09 / 26 / 2025, pp. 124 / 146 62 / 72 of yeast, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[202] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 40% urea solution.

[203] In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon to nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: Under the conditions of OD < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon to nitrogen ratio of 20, the thallus grew. Petition 870250087639, dated 09 / 26 / 2025, pages 125 / 146 63 / 72 quickly; Under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 v / m, a rotation speed of 300 rpm, a carbon source feed rate of 1.6 L / h, and a controlled carbon to nitrogen ratio of 20; After 68 hours of cultivation, the dissolved oxygen (DO) value reached approximately 372, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[204] The fermentation broth was centrifuged and washed with water, then 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%.

[205] Because sodium acetate was not included in the carbon sources of the primary seed culture medium, the secondary seed culture medium, and the fermentation culture medium of Comparative Example 1, the stalk grew slowly when exposed to acetic acid during the high-density fermentation stage. Additional time was required for adaptation to the acetate environment in order to establish an acetic acid metabolic pathway, resulting in longer growth duration, difficulty in achieving high density, and low yield and efficiency. Comparative Example 2 Petition 870250087639, dated 09 / 26 / 2025, pages 126 / 146 64 / 72

[206] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: same as step (1) in Example 1, to obtain the primary seed. (2) Secondary seed culture: same as step (2) in Example 1, to obtain the secondary seed. (3) High-density fermentation.

[207] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[208] The carbon source and the supplemented nitrogen source were prepared. The carbon source was a sterile 40% aqueous sodium acetate solution, and the nitrogen source was a sterile 40% urea solution; Petition 870250087639, dated 09 / 26 / 2025, pp. 127 / 146 65 / 72 In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon-to-nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: Under the conditions of OD < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 20; After 42 h of cultivation, the dissolved oxygen (DO) value was approximately 172. The fermentation broth was saponified, and the stems were lysed and inactivated. The fermentation broth was then centrifuged, washed with water, and spray-dried. The resulting single-cell protein had a dry weight of 60 g / L, a yield of 1.4 g / (L^h), and a crude protein content of 38%.

[209] Due to the generation of alkaline substances during the fermentation process, the pH in the fermentation tank gradually increased. In Comparative Example 2, sodium acetate (alkaline) was supplemented as a carbon source during the high-density fermentation process, resulting in a more alkaline pH and accumulation of sodium ions, which led to Petition 870250087639, dated 09 / 26 / 2025, pp. 128 / 146 66 / 72 saponification reactions and inhibited stem growth. Comparative Example 3

[210] A method for producing single-cell protein by means of high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: 200 mL of primary seed culture medium were prepared, comprising the following components: 8 g / L sodium acetate, 1.8% anhydrous glucose, 1.2% yeast powder, and 2.2% casein peptone; the primary seed culture medium was sterilized at 121 °C for 20 min in a sterilizer. After sterilization, the primary seed culture medium was placed on a super clean bench. After returning to room temperature, the cryopreservation solution of the thawed Schizochytrium strain (ATCC accession number 20888, acquired from the American Type Culture Collection) was added to the primary seed culture medium and placed in a shaker, and cultured for 16 ha at a temperature of 32 °C and a rotation speed of 180 rpm, with an OD value of approximately 4.2, while the primary seed was obtained; (2) Secondary seed crop: L of secondary seed culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.2% anhydrous glucose, 1.5% yeast powder, 2.5% casein peptone, 1.2% magnesium sulfate, and 0.6% thiamine hydrochloride; The secondary seed culture medium was added to a 5 L fermentation tank and sterilized in a sterilizer at 121 °C for 20 min. After cooling to 32 °C, the primary seed was added to the fermentation tank of the secondary seed culture medium in a size of Petition 870250087639, dated 09 / 26 / 2025, pages 129 / 146 67 / 72 7.5% inoculum via flame inoculation; mL of trace element additive after sterilization and filtration were then added, with the following formula: 4 g / L copper sulfate, 0.12 g / L sodium iodide, 5 g / L manganese sulfate, 0.1 g / L sodium molybdate, 0.04 g / L boric acid, 0.6 g / L cobalt chloride, 18 g / L zinc chloride, 38 g / L ferrous sulfate, 0.3 g / L biotin, and 3 g / L sulfuric acid; In the secondary seed culture, when the initial dissolved oxygen was at 100%, the stirring speed was set to 180 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.2 vvm; when the dissolved oxygen ranged from 80% to 100%, the stirring speed was set to 220 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.3 vvm; when the dissolved oxygen ranged from 60% to 80%, the stirring speed was set to 280 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.4 vvm; when the dissolved oxygen ranged from 40% to 60%, the stirring speed was set to 350 rpm, the temperature was 32 °C, and the compressed air aeration rate was 0.5 vvm; When the dissolved oxygen ranged from 20% to 40%, the stirring speed was set to 440 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.6 vvm;When dissolved oxygen ranged from 10% to 20%, the stirring speed was set to 520 rpm, the temperature was 31 °C, and the compressed air aeration rate was 0.7 vvm; when dissolved oxygen ranged from 0% to 10%, the stirring speed was set to 600 rpm, the temperature was 30 °C, and the compressed air aeration rate was 0.8 vvm; when dissolved oxygen was at 0%, the stirring speed was set to 700 rpm, the temperature was 30 °C, and the compressed air aeration rate was 0.9 vvm.

[211] The duration for the secondary seed culture was 8 h, with an OD value of approximately 12, resulting in the secondary seed. Petition 870250087639, dated 09 / 26 / 2025, pp. 130 / 146 68 / 72 (3) High-density fermentation

[212] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, 4% disodium hydrogen phosphate, and 0.2% acetone; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[213] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 40% urea solution.

[214] In high-density fermentation, the stem growth environment was adjusted to achieve ideal conditions for stem growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon to nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: under conditions of OD < OD value < 100, a controlled temperature of Petition 870250087639, dated 09 / 26 / 2025, pp. 131 / 146 At 69 / 72 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 20; After 96 hours of cultivation, the dissolved oxygen (DO) value reached approximately 224, indicating slow stem growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[215] The fermentation broth was centrifuged and washed with water, then 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%.

[216] A comparison of Example 1 with Comparative Example 3 demonstrates that the present application uses Yarrowia lipolytica as the strain, which is a non-genetically modified strain and safer than conventional stems. Yarrowia lipolytica is a microorganism generally recognized as safe (GRAS) by the FDA and widely used in the food industry. The present application uses a specific strain and adopts a specific fermentation method to rapidly obtain high-yield single-cell protein. In Comparative Example 3, Schizochytrium is used, which not only results in a long fermentation period but also results in low single-cell protein production. Comparative Example 4

[217] A method for producing single-cell protein by means of Petition 870250087639, dated 09 / 26 / 2025, pages 132 / 146 70 / 72 high-density fermentation, specifically comprising the following steps: (1) Primary seed culture: same as step (1) in Example 1, to obtain the primary seed. (2) Secondary seed culture: same as step (2) in Example 1, to obtain the secondary seed. (3) High-density fermentation.

[218] 40 L of fermentation culture medium were prepared, comprising the following components: 15 g / L sodium acetate, 2.6% anhydrous glucose, 1.8% yeast powder, 2.8% casein peptone, 1.2% magnesium sulfate, 1.4% thiamine hydrochloride, 1% betaine, 6.8% ammonium sulfate, 7% potassium dihydrogen phosphate, and 4% disodium hydrogen phosphate; The fermentation culture medium was added to a 100 L fermentation tank and sterilized with steam through the fermentation tank. After cooling to 32 °C, the secondary seed was added to the 100 L fermentation tank at an inoculum size of 5%. 400 mL of trace elements, after sterilization and filtration, were then added to the culture medium in the fermentation tank, with the following formula: 8 g / L copper sulfate, 0.16 g / L sodium iodide, 6 g / L manganese sulfate, 0.2 g / L sodium molybdate, 0.08 g / L boric acid, 0.8 g / L cobalt chloride, 21 g / L zinc chloride, 41 g / L ferrous sulfate, 0.45 g / L biotin, and 5.2 g / L sulfuric acid.

[219] The carbon source and the supplemented nitrogen source were prepared. The carbon source was acetic acid, and the nitrogen source was a sterilized 40% urea solution.

[220] In high-density fermentation, the growth environment of Petition 870250087639, dated 09 / 26 / 2025, pages 133 / 146 The 71 / 72 thallus was adjusted to achieve ideal conditions for thallus growth and single-cell protein synthesis by regulating the pH range, temperature, rotation speed, carbon-to-nitrogen ratio, dissolved oxygen, and gas flow rate during different stages of the process. The specifications were as follows: Under the conditions of OD < 100, a controlled temperature of 32 °C, a pH of 6.5, a compressed air aeration rate of 0.6 v / m, a rotation speed of 150 rpm, a carbon source feed rate of 0.8 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; Under the conditions of 100 < DO value < 300, a controlled temperature of 31 °C, a pH of 6.6, a compressed air aeration rate of 1 v / m, a rotation speed of 200 rpm, a carbon source feed rate of 1.2 L / h, and a controlled carbon to nitrogen ratio of 20, the stalk grew rapidly; Under the conditions of 300 < DO value < 500, a controlled temperature of 30 °C, a pH of 6.7, an aeration rate of a mixed gas (air: pure oxygen in a 2:1 ratio) of 1.2 v / m, a rotation speed of 300 rpm, a carbon source feed rate of 1.6 L / h, and a controlled carbon to nitrogen ratio of 20, the thallus grew rapidly; Under conditions of an OD value of 500 to 700, a controlled temperature of 29 °C, a pH of 6.8, an aeration rate of a mixed gas (air: pure oxygen in a 1:1 ratio) of 1.6 v / m, a rotation speed of 500 rpm, a carbon source feed rate of 2.2 L / h, and a controlled carbon to nitrogen ratio of 10, the thallus growth rate was normal, while single-cell protein was synthesized simultaneously; After 51 hours of cultivation, the DO value reached approximately 647, indicating Petition 870250087639, dated 09 / 26 / 2025, pages 134 / 146 72 / 72 slow stem growth. The fermentation broth was then released from the fermentation tank. (4) Post-treatment

[221] The fermentation broth was centrifuged and washed with water, then 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%.

[222] A comparison of Example 1 with Comparative Example 4 demonstrates that the fermentation culture medium of the present application contains ketone compounds (preferably acetone), which exert catalytic functions on the intermediate products in the acetic acid metabolic pathway, thus promoting rapid stem growth and reducing the fermentation period. The fermentation culture medium of Comparative Example 4 does not contain ketone compounds, resulting in slow stem growth and a prolonged fermentation period.

[223] The applicant declares that the present application illustrates the method for the production of single-cell protein by means of high-density fermentation through the Examples mentioned, but the present application is not limited to the process steps mentioned, nor does it imply that the present application must be based on the process steps mentioned for its implementation. Those skilled in the art should understand that any improvements to the present application, equivalent substitutions of raw materials used in the present application, addition of auxiliary components and selection of specific methods are all within the scope of protection and disclosure of the present application. Petition 870250087639, dated 09 / 26 / 2025, pages 135 / 146

Claims

1 / 9 CLAIMS 1. Method for producing single-cell protein by means of high-density fermentation, characterized in that it includes: inoculating a strain of Yarrowia lipolytica into a seed culture medium to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium for high-density fermentation, and supplementing the same with acetic acid and a nitrogen source during the high-density fermentation process to obtain the single-cell protein; wherein a carbon source of the seed culture medium comprises a first acetate, and a carbon source of the fermentation culture medium comprises a second acetate and / or a ketone compound.

2. Method according to claim 1, characterized in that the first acetate includes any one or a combination of at least two of sodium acetate, potassium acetate and ammonium acetate, preferably sodium acetate.

3. Method,According to claim 2, characterized in that the mass concentration of the first acetate in the seed culture medium is in the range of 5 g / L to 15 g / L.

4. Method, according to claim 2 or 3, characterized in that the seed culture includes a primary seed culture and a secondary seed culture carried out sequentially.

5. Method, according to claim 4, characterized in that a primary seed culture medium used for the primary seed culture includes the following components: 5 to 15 g / L of first acetate, 1% to 3% glucose, 0.6% to 2% yeast powder and 1.5% to 3.2% casein peptone; preferably, the temperature for the primary seed culture Petition 870250087639, dated 09 / 26 / 2025, p. 136 / 146 2 / 9 varies from 30 °C to 33 °C, and the duration varies from 16 to 18 hours; preferably, primary seed culture is carried out under agitation conditions.with the agitation speed varying from 100 rpm to 300 rpm; preferably, the primary seed culture is processed to an OD value of 4.2 to 6.8, obtaining a primary seed.

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

7. Method, according to any one of claims 4 to 6, characterized in that primary seeds obtained from the primary seed culture are inoculated into the secondary seed culture medium at an inoculum size of 5% to 10% for the secondary seed culture; preferably, the aeration rate for the seed culture Petition 870250087639, dated 09 / 26 / 2025, p. 137 / 146 3 / 9 secondary varies from 0.1 vvm to 1.3 vvm; preferably, secondary seed cultivation is carried out under agitation conditions, with the agitation speed varying from 150 rpm to 900 rpm; preferably, the temperature for secondary seed cultivation varies from 30 °C to 33 °C, and the duration varies from 8 h to 12 h; preferably,The secondary seed culture is processed to an OD value of 12 to 16, obtaining the seed liquid.

8. Method, according to any one of claims 1 to 7, characterized in that the carbon source of the fermentation culture medium includes a combination of the second acetate and the ketone compound; preferably, the second acetate includes any one or a combination of at least two of sodium acetate, potassium acetate and ammonium acetate, additionally, preferably sodium acetate; preferably, the mass concentration of the second acetate in the fermentation culture medium is in the range of 5 g / L to 15 g / L; preferably, the mass percentage content of ketone compounds in the fermentation culture medium is in the range of 0.1% to 0.6%; Preferably, the fermentation culture medium includes the following components: 5 to 15 g / L of second acetate, 1% to 3% glucose, 0.6% to 2% yeast powder, 1.5% to 3.2% casein peptone, 0,8% to 1.2% magnesium sulfate, 0.6% to 1.4% thiamine hydrochloride, 0.5% to 1% betaine, 3.5% to 6.8% ammonium sulfate, 5% to 7% potassium dihydrogen phosphate, 2% to 4% disodium hydrogen phosphate and 0.1% to 0.6% ketone compounds; preferably, the ketone compounds are C3-C10 ketone compounds; preferably, the ketone compound includes any one or a combination of at least two of acetone, methyl ethyl ketone and cyclohexanone, additionally, preferably acetone; Preferably, the fermentation culture medium additionally includes a second trace element additive, and the amount of use of the second trace element additive is in the range of 0.8% to 2.6%; preferably, the second trace element additive includes the following components: 4 to 8 g / L of copper sulfate, 0.12 to 0.16 g / L of sodium iodide, 5 to 6 g / L of manganese sulfate, 0.1 to 0.2 g / L of sodium molybdate, 0.04 to 0.0.8 g / L of boric acid, 0.6 to 0.8 g / L of cobalt chloride, 18 to 21 g / L of zinc chloride, 38 to 41 g / L of ferrous sulfate, 0.3 to 0.5 g / L of biotin and 3 to 5.2 g / L of sulfuric acid.

9. Method, according to any one of claims 1 to 8, characterized in that the seed liquid is inoculated into the fermentation culture medium at an inoculum size of 3% to 6% for high-density fermentation; preferably, the nitrogen source supplemented during the high-density fermentation process includes any one or a combination of at least two of urea, ammonium sulfate, ammonium nitrate and ammoniacal water, additionally, preferably urea.

10. Method, according to any one of claims 1 to 9, characterized in that the temperature for high-density fermentation varies from 27 °C to 33 °C, preferably 28 °C to 32 °C; preferably, the pH value for high-density fermentation varies from 6.4 to 7.1, additionally,Preferably 6.5 to 7; preferably, high-density fermentation is carried out under agitation conditions, with the agitation speed varying from 130 rpm to 800 rpm; preferably, the aeration rate for high-density fermentation Petition 870250087639, dated 09 / 26 / 2025, pp. 139 / 146 5 / 9 varies from 0.5 vvm to 2.4 vvm; preferably, high-density fermentation is processed to an OD value of 780 to 820, and the fermentation is terminated to obtain the single-cell protein.

11. Method, according to any one of claims 1 to 10, characterized in that the high-density fermentation includes a first fermentation stage, a second fermentation stage, and a third fermentation stage, with the OD values ​​increasing sequentially; the OD value of the first fermentation stage is less than 500,and the carbon-to-nitrogen ratio of the supplemented acetic acid and the nitrogen source in the first fermentation stage is greater than 18; the DO value of the second fermentation stage is in the range of 500 to 700, and the carbon-to-nitrogen ratio of the supplemented acetic acid and the nitrogen source in the second fermentation stage ranges from 5 to 18; the DO value of the third fermentation stage is greater than 700, and the carbon-to-nitrogen ratio of the supplemented acetic acid and the nitrogen source in the third fermentation stage is less than 5.

12. Method, according to claim 11, characterized in that the first fermentation stage includes a stage A, a stage B, and a stage C carried out sequentially; In stage A, the dissolved oxygen (DO) value is less than 100, with a temperature ranging from 31°C to 33°C, a pH value of 6.4 to 6.6, an aeration rate of 0.5 vvm to 0.7 vvm, and a rotation speed of 130 rpm to 170 rpm.and a carbon-to-nitrogen ratio greater than 18 for the supplemented acetic acid and nitrogen source; in stage B, 100 < DO value < 300, with a temperature ranging from Petition 870250087639, dated 09 / 26 / 2025, page 140 / 146 6 / 9 30 °C to 32 °C, a pH value of 6.5 to 6.7, an aeration rate of 0.9 vvm to 1.1 vvm, a rotation speed of 180 rpm to 220 rpm, and a carbon-to-nitrogen ratio greater than 18 for the supplemented acetic acid and nitrogen source; In stage C, the dissolved oxygen (DO) value is 300 < 500, with a temperature ranging from 29°C to 31°C, a pH value from 6.6 to 6.8, an aeration rate of 1.1 vvm to 1.3 vvm, a rotation speed of 280 rpm to 320 rpm, and a carbon-to-nitrogen ratio greater than 18 for the supplemented acetic acid and nitrogen source; the DO value of the second fermentation stage is in the range of 500 to 700, with a temperature ranging from 28°C to 30°C, a pH value from 6.7 to 6.9, and an aeration rate of 1.5 vvm to 1.7 vvm.a rotation speed of 450 rpm to 550 rpm, and a carbon-to-nitrogen ratio of 5 to 18 for the supplemented acetic acid and nitrogen source; the DO value of the third fermentation stage is greater than 700, with a temperature ranging from 27 °C to 29 °C, a pH value of 6.9 to 7.1, an aeration rate of 1.9 vvm to 2.1 vvm, a rotation speed of 650 rpm to 750 rpm, and a carbon-to-nitrogen ratio less than 5 for the supplemented acetic acid and nitrogen source.

13. Method, according to any one of claims 1 to 12, characterized in that after the completion of high-density fermentation, it additionally includes a post-treatment step; Preferably, a post-treatment method includes subjecting a fermentation broth obtained from high-density fermentation to solid-liquid separation, washing, and drying in sequence to obtain the single-cell protein; preferably,A method of solid-liquid separation is centrifugal separation. Petition 870250087639, dated 09 / 26 / 2025, p. 141 / 146 7 / 9.

14. Method, according to any one of claims 1 to 13, characterized in that it specifically includes the following steps: (1) inoculating the Yarrowia lipolytica strain into the primary seed culture medium and cultivating for 16 ha 18 ha at a temperature of 30 °C to 33 °C and a rotation speed of 100 rpm to 300 rpm until the OD value reaches 4.2 to 6.8, obtaining the primary seed; The primary seed culture medium includes the following components: 5 to 15 g / L of sodium acetate, 1% to 3% glucose, 0.6% to 2% yeast powder, and 1.5% to 3.2% casein peptone; (2) Inoculate the primary seed in the secondary seed culture medium at an inoculum size of 5% to 10%, and cultivate for 8 ha to 12 ha at a temperature of 30 °C to 33 °C, a rotation speed of 150 rpm to 900 rpm and an aeration rate of 0.1 vvm to 1.3 vvm until the OD value reaches 12 to 16, obtaining the secondary seed, i.e., the seed liquid; The secondary seed culture medium includes the following components: 5 to 20 g / L of sodium acetate, 1% to 3% glucose, 0.6% to 2% yeast powder, 1.5% to 3.2% casein peptone, 0.8% to 2.4% magnesium sulfate, 0.2% to 0.8% thiamine hydrochloride and 0.8% to 2.6% first trace element additive;The first trace element additive includes the following components: 4 to 8 g / L of copper sulfate, 0.12 to 0.16 g / L of sodium iodide, 5 to 6 g / L of manganese sulfate, 0.1 to 0.2 g / L of sodium molybdate, 0.04 to 0.08 g / L of boric acid, 0.6 to 0.8 g / L of cobalt chloride, 18 to 21 g / L of zinc chloride, 38 to 41 g / L of ferrous sulfate, 0.3 to 0.5 g / L of biotin and 3 to 5.2 g / L of sulfuric acid; (3) inoculate the seed liquid into the fermentation culture medium at an inoculum size of 3% to 6% for high-density fermentation, and Petition 870250087639, dated 09 / 26 / 2025, pp. 142 / 146 8 / 9 supplement the same with acetic acid and urea during the high-density fermentation process until the OD value reaches 780 to 820, obtaining the single-cell protein; The fermentation culture medium includes the following components: 5 to 15 g / L of sodium acetate, 1% to 3% glucose, 0.6% to 2% yeast powder, 1.5% to 3.2% casein peptone, 0.8% to 1.2% magnesium sulfate, 0.6% to 1.4% thiamine hydrochloride, 0.5% to 1% betaine, 3.5% to 6.8% ammonium sulfate, 5% to 7% potassium dihydrogen phosphate, 2% to 4% disodium hydrogen phosphate, 0.1% to 0.6% acetone and 0.8% to 2.6% of a second trace element additive;The second trace element additive includes the following components: 4 to 8 g / L of copper sulfate, 0.12 to 0.16 g / L of sodium iodide, 5 to 6 g / L of manganese sulfate, 0.1 to 0.2 g / L of sodium molybdate, 0.04 to 0.08 g / L of boric acid, 0.6 to 0.8 g / L of cobalt chloride, 18 to 21 g / L of zinc chloride, 38 to 41 g / L of ferrous sulfate, 0.3 to 0.5 g / L of biotin, and 3 to 5.2 g / L of sulfuric acid; high-density fermentation includes a first fermentation stage, a second fermentation stage, and a third fermentation stage, with the DO value increasing sequentially; The dissolved oxygen (DO) value of the first fermentation stage is less than 500, with a temperature ranging from 29 °C to 33 °C, a pH value of 6.5 to 6.8, an aeration rate of 0.5 vvm to 1.3 vvm, a rotation speed of 280 rpm to 320 rpm, and a carbon to nitrogen ratio greater than 18 for the supplemented acetic acid and urea;The dissolved oxygen (DO) value of the second fermentation stage is in the range of 500 to 700, with a temperature ranging from 28 °C to 30 °C, a pH value of 6.7 to 6.9, an aeration rate of 1.5 vvm to 1.7 vvm, a rotation speed of 450 rpm to 550 rpm, and a carbon to nitrogen ratio of 5 to 18 for the supplemented acetic acid and nitrogen source; The dissolved oxygen (DO) value of the third fermentation stage is greater than 700, with a temperature ranging from 27 °C to 29 °C, a pH value of 6.9 to 7.1, an aeration rate of 1.9 vvm to 2.1 vvm, a rotation speed of 650 rpm to 750 rpm, and a carbon-to-nitrogen ratio of less than 5 for the supplemented acetic acid and nitrogen source.

15. Method, according to any one of claims 1 to 14, characterized in that the single-cell protein yield is greater than 2.5 g / (L·h), preferably 6 to 9 g / (L·h);Preferably, the percentage by mass of crude protein in single-cell protein is greater than or equal to 35%, additionally, preferably greater than or equal to 55%. Petition 870250087639, dated 09 / 26 / 2025, pp. 144 / 146;

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