A method for preparing single-cell protein by using a complex bacterial body

Abstract

The application discloses a method for preparing single-cell protein by using a composite bacterium, and belongs to the technical field of microbial fermentation. The method comprises the following steps: continuously culturing Clostridium aceticum and Pichia pastoris respectively, and simultaneously supplementing fresh culture medium and discharging part of the fermentation liquor at the same flow rate to replace the decaying bacterium and maintain the constant volume of the fermentation liquor in the fermentation tank; the fermentation liquor of the Clostridium aceticum and the fermentation liquor of the Pichia pastoris are compounded, separated by a disc centrifuge, and then subjected to enzymolysis and spray drying in sequence to obtain single-cell protein. The application solves the contradiction between high nutrition and low yield or high yield and low absorption of traditional single-cell protein by compounding the Clostridium aceticum with high acid-soluble protein content and the Pichia pastoris with high biomass, and improves the bacterium recovery rate, the equipment operation rate and the proportion of acid-soluble protein, and is easy to mass produce.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for preparing single-cell proteins using complex bacterial cultures. Background Technology

[0002] With the increasing global population, the demand for protein is rising, and traditional protein raw materials can no longer meet the current situation. The global imbalance between protein resource supply and demand has become a rigid pressure: it is estimated that by 2050, the global population will reach 9.3 billion, and the demand for animal protein will increase to 1.25 billion tons, while traditional raw materials such as soybeans and fishmeal are facing supply bottlenecks—my country's dependence on imported soybeans exceeds 80%, fishmeal supply is shrinking due to overfishing, and competition for arable land is intensifying. The Food and Agriculture Organization of the United Nations pointed out the "protein gap" problem as early as 1960. Traditional protein production models are caught in an ecological and efficiency dilemma: animal husbandry contributes 30% of global methane emissions, consumes a large amount of water and land, and more than 65% of the 300 million tons of corn stalks produced annually in my country are not effectively utilized; its land use efficiency is only one-thousandth that of single-cell protein (SCP), and it is easily affected by natural factors, conflicting with sustainable development goals.

[0003] In 2023, global demand for livestock feed increased by 7.8%, and SCP, with its high protein content of 40%-85% and balanced amino acid composition, has become an ideal alternative raw material. my country has listed methanol protein as a key development product, and its potential is also recognized internationally, with synthetic biology and other technologies being included in the research and development plans of many countries. Although it still faces challenges such as cost and nucleic acid control, SCP, as a resource-efficient and environmentally friendly new protein source, has become an inevitable choice to resolve the global protein supply and demand contradiction.

[0004] The single-cell protein obtained from Clostridium fermentation has a high protein content (over 75%) and an excellent amino acid ratio, making it a very good feed protein. However, due to the low biomass of Clostridium aeruginosa, multiple feedings are required in the disc centrifuge during post-processing. Furthermore, the low solid content leads to an unstable separation layer and poor clarification, wasting manpower and time.

[0005] Yeast is a recognized strain for producing single-cell protein. The strain reproduces quickly, has a high cell mass, and the protein content of the single-cell protein obtained is also quite ideal (over 60%). However, there is also a drawback in post-processing. Precisely because of the high cell biomass and high viscosity, it leads to frequent slag discharge or blockage in the disc machine, which also wastes time.

[0006] Therefore, theoretically, Clostridium and yeast can be combined to improve the solid-liquid separation of fermentation broth and reduce industrial production costs. However, there are currently no reports on the successful combination of Clostridium and yeast. Furthermore, addressing bottlenecks such as strain compatibility and substrate utilization to achieve large-scale implementation of this combined preparation remains an open question. Summary of the Invention

[0007] Technical problem solved: To address the above-mentioned technical problems, this invention provides a method for preparing single-cell protein using composite microorganisms. By combining Clostridium with high acid-soluble protein content with yeast with high biomass, the contradiction of "high nutrition but low yield" or "high yield but low absorption" in traditional single-cell protein preparation is resolved. This method improves the microbial recovery rate, equipment operating rate, and acid-soluble protein ratio, and facilitates large-scale production.

[0008] Technical solution: A method for preparing single-cell protein using composite bacterial cells, comprising the following steps: S1. Perform anaerobic continuous fermentation of Clostridium aeruginosa, while adding fresh culture medium for Clostridium aeruginosa and discharging part of the fermentation liquid at the same flow rate to replace the decaying bacteria and maintain a constant volume of fermentation liquid in the fermenter. S2. Conduct continuous fermentation culture of Pichia pastoris, while adding fresh culture medium of Pichia pastoris and draining part of the fermentation liquid at the same flow rate to replace the decaying cells and maintain a constant volume of fermentation liquid in the fermenter. S3. The fermentation broth of Clostridium aeruginosa and the fermentation broth of Pichia pastoris were combined, separated by a disc centrifuge, and then subjected to enzymatic hydrolysis and spray drying to obtain single-cell protein.

[0009] Preferably, the Clostridium aeruginosa is 羧基还原梭菌 GG6, accession number CGMCC No. 34975; the described Pichia pastoris is Pichia pastoris. 毕赤酵母 GG7, with accession number CGMCCNo.39081.

[0010] Preferably, in step S1, syngas is used as a raw material for fermentation. The syngas, by volume fraction, includes 30% CO, 55% CO2 and 15% H2, and the gas flow rate of the syngas is controlled at 50~150 mL / min.

[0011] Preferably, in step S1, the fermentation temperature is controlled at 35~38℃ and the stirring speed is controlled at 80~120rpm.

[0012] Preferably, the dilution ratio is defined as the ratio of the volume of fresh culture medium added to the volume of fermentation broth in the fermenter, and the dilution ratio in S1 and S2 is controlled to be 0.3~1.0.

[0013] Preferably, the fresh culture medium for the Clostridium aeruginosa comprises: 0.5 g / L yeast extract, 1 g / L ammonium chloride, 0.2 g / L L-cysteine ​​hydrochloride, 0.2 g / L sodium sulfide, 0.5 g / L potassium dihydrogen phosphate, 5 mL / L trace element stock solution, 5 mL / L vitamin solution stock solution, and 1 mL / L phosphate.

[0014] Furthermore, the trace element mother liquor comprises: 10 g / L nitric acid triacetic acid, 0.5 g / L cobalt chloride hexahydrate, 2 g / L manganese sulfate, 0.1 g / L nickel chloride hexahydrate, 0.2 g / L ferric sulfate, 0.1 g / L sodium selenite, 1 g / L zinc sulfate, 0.1 g / L copper chloride, 0.1 g / L sodium molybdate, and 0.1 g / L sodium tungstate.

[0015] Furthermore, the vitamin solution stock solution comprises: calcium pantothenate 50 mg / L, lipoic acid 50 mg / L, vitamin B6 100 mg / L, thiamine 50 mg / L, vitamin B2 50 mg / L, biotin 20 mg / L, folic acid 20 mg / L, para-aminobenzoic acid 50 mg / L, nicotinic acid 50 mg / L, and vitamin B12 50 mg / L.

[0016] Preferably, the fresh culture medium of Pichia pastoris comprises: 30 g / L phosphate, 6 g / L magnesium sulfate heptahydrate, 0.1 g / L calcium sulfate dihydrate, 5.2 g / L potassium sulfate, 4 g / L 48wt% potassium hydroxide, 20 g / L glycerol and 3.6 mL / L PTM1 trace element solution.

[0017] Furthermore, the PTM1 trace element solution comprises: zinc sulfate heptahydrate 42 g / L, copper sulfate pentahydrate 6 g / L, ferrous sulfate heptahydrate 65 g / L, manganese sulfate monohydrate 3 g / L, cobalt chloride hexahydrate 0.5 g / L, sodium iodide 0.08 g / L, 95 wt% sulfuric acid 5 mL / L, and biotin 0.2 g / L.

[0018] Beneficial effects: This invention combines anaerobic and aerobic bacterial fermentation broths and separates them using a disc centrifuge. Compared with treating low biomass fermentation broths separately, the residual bacterial count in the clear liquid can be reduced by a certain percentage, and the bacterial recovery rate is significantly improved.

[0019] Due to the improved feeding properties, the number of times the disc machine discharges slag is reduced, which not only reduces power consumption but also reduces the amount of high-pressure cleaning water used, extending the continuous operation time (i.e., improving equipment operating rate).

[0020] The composite hydrolysate obtained by this invention can increase the proportion of acid-soluble protein (>60%), making it easier to digest as feed and thus more practical. Attached Figure Description

[0021] Figure 1 This invention utilizes composite bacterial cells to prepare single-cell protein hydrolysates. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0023] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] All strains were purchased from a microbial culture center, with Clostridium aeruginosa being the most abundant. 梭菌属 羧基还原菌 GG6, accession number CGMCC No. 34975; the described Pichia pastoris is Pichia pastoris. 毕赤酵母 GG7, with accession number CGMCC No.39081.

[0026] The fresh culture medium for *Clostridium aerogenes* comprises: 0.5 g / L yeast extract, 1 g / L ammonium chloride, 0.2 g / L L-cysteine ​​hydrochloride, 0.2 g / L sodium sulfide, 0.5 g / L potassium dihydrogen phosphate, 5 mL / L trace element stock solution, 5 mL / L vitamin solution stock solution, and 1 mL / L phosphate. The trace element stock solution comprises: 10 g / L nitric acid triacetic acid, 0.5 g / L cobalt chloride hexahydrate, 2 g / L manganese sulfate, 0.1 g / L nickel chloride hexahydrate, 0.2 g / L ferric sulfate, 0.1 g / L sodium selenite, 1 g / L zinc sulfate, 0.1 g / L copper chloride, 0.1 g / L sodium molybdate, and 0.1 g / L sodium tungstate. The vitamin solution stock solution comprises: calcium pantothenate 50 mg / L, lipoic acid 50 mg / L, vitamin B6 100 mg / L, thiamine 50 mg / L, vitamin B2 50 mg / L, biotin 20 mg / L, folic acid 20 mg / L, para-aminobenzoic acid 50 mg / L, nicotinic acid 50 mg / L, and vitamin B12 50 mg / L.

[0027] The fresh culture medium (basal fermentation medium) for Pichia pastoris includes: 30 g / L phosphate, 6 g / L magnesium sulfate heptahydrate, 0.1 g / L calcium sulfate dihydrate, 5.2 g / L potassium sulfate, 4 g / L 48wt% potassium hydroxide, 20 g / L glycerol, and 3.6 mL / L PTM1 trace element solution. The PTM1 trace element solution includes: 42 g / L zinc sulfate heptahydrate, 6 g / L copper sulfate pentahydrate, 65 g / L ferrous sulfate heptahydrate, 3 g / L manganese sulfate monohydrate, 0.5 g / L cobalt chloride hexahydrate, 0.08 g / L sodium iodide, 5 mL / L 95wt% sulfuric acid, and 0.2 g / L biotin. YPD medium includes: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 2% agar powder added to the solid medium. Example 1

[0028] 1. Experimental Methods (1) Culture of strains Take 1 mL of frozen Clostridium aeruginosa from -80℃. 羧基还原梭菌 GG6 (CGMCC No. 34975) was transferred entirely into 4 mL test tubes and anaerobically cultured at 37°C for 2–3 days. The grown strain was then inoculated at a 3% inoculation rate into a blue glass bottle containing 200 mL of culture medium and anaerobically cultured at 37°C for 16–18 h.

[0029] Take the frozen strain (Pichia pastoris) from the -80℃ freezer. 毕赤酵母 (GG7) was streaked onto YPD plates and incubated at 30°C for 2 days. A single colony was picked and inoculated into a 4 mL YPD tube, and incubated at 30°C and 220 rpm for 16–18 h to obtain primary seed culture. This was then transferred at a 1% inoculum to 50 mL YPD (250 mL Erlenmeyer flask) and incubated under the same conditions to obtain secondary seed culture. (2) Constructing a fermentation system Clostridium aeruginosa: In a 3 L fermentation system, add 0.5 g yeast powder, 1 g ammonium chloride, 0.2 g L-cysteine ​​hydrochloride, 0.2 g sodium sulfide, 0.5 g potassium dihydrogen phosphate, 5 mL trace element stock solution, 5 mL vitamin solution stock solution, and 1 mL phosphate, and sterilize at 121℃ for 20 min.

[0030] Trace element stock solution (1 L): 10 g nitrilotriacetic acid, 0.5 g cobalt chloride hexahydrate, 2 g manganese sulfate, 0.1 g nickel chloride hexahydrate, 0.2 g ferric sulfate, 0.1 g sodium selenite, 1 g zinc sulfate, 0.1 g copper chloride, 0.1 g sodium molybdate, and 0.1 g sodium tungstate.

[0031] Vitamin stock solution (1 L): Calcium pantothenate 50 mg, lipoic acid 50 mg, vitamin B6 100 mg, thiamine 50 mg, vitamin B2 50 mg, biotin 20 mg, folic acid 20 mg, para-aminobenzoic acid 50 mg, nicotinic acid 50 mg, vitamin B12 50 mg.

[0032] After sterilization, nitrogen gas is introduced through the air inlet to replace the gas in the fermenter and anaerobic culture medium storage tank. When the temperature drops to about 37°C, the stirring controller and pH electrode are installed. The nitrogen gas connected to the fermenter is turned off, and simulated synthesis gas (volume fraction: 30% CO, 55% CO2, 15% H2) is introduced into the first fermenter through the air inlet. The gas flow rate is controlled at 100 mL / min, the fermentation temperature is controlled at 37°C, and the stirring speed is controlled at 100 rpm.

[0033] Pichia pastoris: 3.6 L of basal fermentation medium was loaded into a 5 L fermenter, sterilized and cooled, and then the pH was adjusted to 5.0 with ammonia.

[0034] (3) After completing the construction of the fermentation system, 100 mL of the above-mentioned Clostridium aeruginosa cultured to the logarithmic growth phase was introduced into a 5 L Clostridium aeruginosa fermenter. Samples were taken at regular intervals to determine the cell biomass and fermentation products. The Pichia pastoris tank was inoculated with a 10% inoculum of secondary seed, with the temperature controlled at 30℃, the rotation speed at 500 rpm, and the dissolved oxygen (DO) maintained at around 30%. After the initial glycerol was depleted (DO rose sharply), glycerol was added to maintain DO between 20% and 30% to promote cell growth until the cell wet weight reached approximately 200 g / L. Glycerol addition was stopped, and the cell was starved until DO rose again, until all the glycerol in the tank was consumed again. Starvation culture was then performed for 2 h, followed by methanol induction, with methanol as the sole carbon source for fed-batch fermentation. With the addition of methanol, dissolved oxygen decreased significantly, indicating that the yeast was utilizing methanol. Adjust the methanol flow rate, starting with a low flow rate and gradually increasing it to 9% of the flow rate that the cells can tolerate. During this stage, the DO is controlled at around 20%, and the cell wet weight is stabilized at about 214 g / L.

[0035] (4) 200L scale-up culture Clostridium aeruginosa: Once the strain achieved stable growth and steady increase in metabolites in a 5 L fermenter, it was transferred to a 200 L reactor for further propagation. Similarly, the culture medium (120 L) was first sterilized, then syngas was introduced at a flow rate of 100 mL / min, the fermentation temperature was maintained at 37°C, and the stirring speed was controlled at 100 rpm. After preparation, the 5 L fermenter seed was inoculated into the 200 L reactor, and samples were taken at regular intervals to determine the cell biomass and fermentation products.

[0036] Pichia pastoris: Once the Pichia pastoris had adapted to methanol as a carbon source, it was transferred to a 200L reactor for propagation. The culture medium (120L) was first sterilized, and after cooling, the pH was adjusted to 5.0 with ammonia. Subsequently, 5L of seed culture from the fermenter was inoculated into the 200L reactor, and samples were taken at regular intervals to determine the cell biomass and fermentation products.

[0037] Biomass was determined using a UV spectrophotometer, with absorbance measured at 600 nm. Fermentation products were analyzed using an Amine HPX-87H (7.8 mm × 300 mm) liquid chromatography column purchased from Bio-Rad. Chromatographic conditions were as follows: mobile phase: 5 mM dilute sulfuric acid; injection volume: 10 µL; column temperature: 30 °C; sample chamber temperature: 25 °C; flow rate: 0.9 mL / min; elution time: 30 min; elution method: isocratic elution.

[0038] (5) After the Clostridium aeruginosa strain in the reactor reaches the stationary phase, the discharge is initiated while fresh culture medium is added at a dilution of 0.3 to start continuous fermentation. Pichia pastoris is also started in continuous fermentation mode: fresh fermentation basal culture medium is added at a dilution of 0.6 while the fermentation broth is discharged, and the methanol flow rate is maintained at 21.6 mL / h to stabilize DO at 20% and maintain the cell wet weight at approximately 200 g / L. The dilution is defined as the ratio of the volume of fresh culture medium added to the volume of the fermentation broth in the fermenter.

[0039] (6) Collect the two fermentation broths discharged from continuous fermentation respectively, determine the biomass of each fermentation broth, and then mix the fermentation broths according to different volume ratios of Clostridium aeruginosa: aerobic protein: 4:1, 5:1 and 6:1.

[0040] (7) The well-mixed fermentation broth is continuously fed into a disc centrifuge (model SWDB-608) with a speed of 8000 rpm to achieve solid-liquid separation and obtain the cell concentrate and supernatant.

[0041] (8) The bacterial concentrate is processed in a spray dryer to obtain single-cell protein, with a feed rate of 1.5m. 3 The feed pressure was 105 bar, the inlet air temperature was 180℃, and the outlet air temperature was 98℃. After drying, composite single-cell protein was obtained. The product content detection steps are as follows: a. The determination of moisture content in the composite single-cell protein in this invention refers to the People's Republic of China National Standard GB / T6435-2014 Method for Determination of Moisture Content in Feed.

[0042] b. The determination of crude ash in the composite single-cell protein in this invention refers to the People's Republic of China National Standard GB / T6438-2007 Method for Determination of Crude Ash in Feed.

[0043] c. The determination of crude protein in the composite single-cell protein in this invention refers to the People's Republic of China National Standard GB / T6432-2018 Determination of Crude Protein in Feed - Kjeldahl Method.

[0044] 2. Experimental Results Different compound ratios were used to obtain composite single cells via disc centrifugation. A final compound ratio of 6:1 was selected. This ratio reduced the disc centrifugation slag discharge cycle, and the cell recovery rate reached 98%. Relevant test indicators are shown in Table 2. When the proportion of *Clostridium aeruginosa* increased, the crude protein content increased, which is related to the *Clostridium aeruginosa* protein content (>75%). *Clostridium* is rich in protein, which can increase the mixed protein content. However, the acid-soluble protein content was less than 60%. To increase the amount of acid-soluble protein, further enzymatic hydrolysis was used to obtain composite cell hydrolysate.

[0045] Comparative Example 1 Comparative Example 1 uses Clostridium aeruginosa fermentation broth in a disc centrifuge. The experimental methods, strain cultivation, and scale-up fermentation are the same as in Example 1, except that the continuously discharged mash is directly fed into the disc centrifuge for separation to obtain Clostridium aeruginosa single-cell protein.

[0046] Comparative Example 2 Comparative Example 2 uses Pichia pastoris fermentation broth in a disc centrifuge. The experimental methods, strain cultivation, and scale-up fermentation are the same as in Example 1, except that the continuously discharged mash is directly fed into the disc centrifuge for separation to obtain Pichia pastoris single-cell protein.

[0047] Table 1. Comparison of parameters between Example 1 (compound ratio 6:1) and Comparative Examples 1 and 2

[0048] Table 2. Detection results of composite single-cell proteins prepared at different ratios. Example 2

[0049] The experimental method is the same as in Example 1. The only difference is that after the material is separated by a disc centrifuge, a compound enzyme is added for enzymatic hydrolysis. That is, in step (8) of the experimental method, the bacterial cell concentrate is added to the compound enzyme for enzymatic hydrolysis. The compound enzyme includes 0.1 wt% glucanase, 0.1 wt% mannanase and 0.1 wt% neutral protease based on the dry weight of the concentrate. The compound enzymatic hydrolysis temperature is 55℃, the enzymatic hydrolysis pH is 6.0, and the enzymatic hydrolysis time is 8 h.

[0050] After complex enzymatic hydrolysis, the acid-soluble protein content of the complex bacterial protein hydrolysate increased to 43%, thus further increasing its proportion of crude protein to over 60%. Through enzymatic hydrolysis and other processes, the hydrolysate "crushes" large single-cell protein molecules into small peptides and amino acids, naturally and significantly increasing the content of the acid-soluble portion.

[0051] Table 3 Detection results of Example 1 and Example 2

[0052] This invention solves the contradiction of traditional single-cell protein’s “high nutrition but low yield” or “high yield but low absorption” by combining Clostridium with high acid-soluble protein content with yeast with high biomass. It improves cell recovery rate, equipment operation rate and acid-soluble protein ratio, and is easy to produce on a large scale.

Claims

1. A method for preparing single-cell protein using complex bacterial cells, characterized in that, The steps include the following: S1. Perform anaerobic continuous fermentation of Clostridium aeruginosa, while adding fresh culture medium for Clostridium aeruginosa and discharging part of the fermentation liquid at the same flow rate to replace the decaying bacteria and maintain a constant volume of fermentation liquid in the fermenter. S2. Conduct continuous fermentation culture of Pichia pastoris, while adding fresh culture medium of Pichia pastoris and draining part of the fermentation liquid at the same flow rate to replace the decaying cells and maintain a constant volume of fermentation liquid in the fermenter. S3. The fermentation broth of Clostridium aeruginosa and the fermentation broth of Pichia pastoris were combined, separated by a disc centrifuge, and then subjected to enzymatic hydrolysis and spray drying to obtain single-cell protein.

2. The method for preparing single-cell protein using composite bacterial cells according to claim 1, characterized in that, The Clostridium aeruginosa is Clostridium carboxidivorans GG6, accession number CGMCC No. 34975; the described Pichia pastoris is Pichia pastoris. Komagataella phaffii GG7, with accession number CGMCC No.39081.

3. The method for preparing single-cell protein using composite bacterial cells according to claim 1, characterized in that, In step S1, syngas is used as a raw material for fermentation. The syngas, by volume fraction, includes 30% CO, 55% CO2 and 15% H2, and the gas flow rate of the syngas is controlled at 50~150 mL / min.

4. The method for preparing single-cell protein using composite bacterial cells according to claim 1, characterized in that, In S1, the fermentation temperature is controlled at 35~38℃, and the stirring speed is controlled at 80~120rpm.

5. The method for preparing single-cell protein using composite bacterial cells according to claim 1, characterized in that, The dilution ratio is defined as the ratio of the volume of fresh culture medium added to the volume of fermentation broth in the fermenter. Therefore, the dilution ratio in S1 and S2 is controlled at 0.3~1.

0.

6. The method for preparing single-cell protein using composite bacterial cells according to claim 1, characterized in that, The fresh culture medium for the Clostridium aeruginosa includes: 0.5 g / L yeast extract, 1 g / L ammonium chloride, 0.2 g / L L-cysteine ​​hydrochloride, 0.2 g / L sodium sulfide, 0.5 g / L potassium dihydrogen phosphate, 5 mL / L trace element stock solution, 5 mL / L vitamin solution stock solution, and 1 mL / L phosphate.

7. The method for preparing single-cell protein using composite bacterial cells according to claim 6, characterized in that, The trace element mother liquor comprises: 10 g / L nitric acid, 0.5 g / L cobalt chloride hexahydrate, 2 g / L manganese sulfate, 0.1 g / L nickel chloride hexahydrate, 0.2 g / L ferric sulfate, 0.1 g / L sodium selenite, 1 g / L zinc sulfate, 0.1 g / L copper chloride, 0.1 g / L sodium molybdate, and 0.1 g / L sodium tungstate.

8. The method for preparing single-cell protein using composite bacterial cells according to claim 6, characterized in that, The vitamin solution stock solution comprises: calcium pantothenate 50 mg / L, lipoic acid 50 mg / L, vitamin B6 100 mg / L, thiamine 50 mg / L, vitamin B2 50 mg / L, biotin 20 mg / L, folic acid 20 mg / L, para-aminobenzoic acid 50 mg / L, nicotinic acid 50 mg / L, and vitamin B12 50 mg / L.

9. The method for preparing single-cell protein using composite bacterial cells according to claim 1, characterized in that, The fresh culture medium for Pichia pastoris comprises: 30 g / L phosphate, 6 g / L magnesium sulfate heptahydrate, 0.1 g / L calcium sulfate dihydrate, 5.2 g / L potassium sulfate, 4 g / L 48 wt% potassium hydroxide, 20 g / L glycerol, and 3.6 mL / L PTM1 trace element solution.

10. A method for preparing single-cell protein using composite bacterial cells according to claim 9, characterized in that, The PTM1 trace element solution comprises: zinc sulfate heptahydrate 42 g / L, copper sulfate pentahydrate 6 g / L, ferrous sulfate heptahydrate 65 g / L, manganese sulfate monohydrate 3 g / L, cobalt chloride hexahydrate 0.5 g / L, sodium iodide 0.08 g / L, 95 wt% sulfuric acid 5 mL / L, and biotin 0.2 g / L.

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