Application of engineered Citrobacter freundii in fermentation synthesis of spermine

Abstract

This invention discloses the application of engineered *Citrobacter freundii* in the fermentation synthesis of spermine. The engineered *Citrobacter freundii* is a strain overexpressing the polyphosphate kinase gene Ppk1. The engineered *Citrobacter freundii* is inoculated into a fermentation medium for fermentation culture to synthesize spermine. The synthesis and extraction method of this invention for producing spermine is simple to operate, highly reproducible, low in cost, and yields a high spermine content in the fermented bacterial cells, accounting for 12.42% of the bacterial dry weight. The extracted spermine has a purity as high as 90.26%.

Description

Technical Field

[0001] This invention relates to the field of biochemical production, specifically to the application of engineered Citrobacter freundii in the fermentation synthesis of spermine. Background Technology

[0002] Spermine is a ubiquitous endogenous polyamine in living organisms, involved in maintaining cellular and neuronal homeostasis. It is a structurally stable cationic compound consisting of two amino groups (-NH2) that can interact with negatively charged molecules such as DNA, RNA, and proteins. Spermine is a key factor in many cellular processes, including promoting cell division, proliferation, and differentiation, and stress responses by stabilizing DNA structure. As a free radical scavenger and antioxidant, spermine reduces oxidative damage caused by environmental stress. It also acts as a second messenger in cells, extending lifespan, maintaining nucleic acid and cell membrane stability, controlling cell division and growth, and playing a physiological role in organ development, embryonic development, flower senescence, flower and fruit growth, and responses to biotic and abiotic stresses. Spermine also maintains intracellular homeostasis and regulates ion transmembrane transport, enhancing the bioavailability of arginine, which is essential for nitric oxide (NO) synthesis. With age, spermine concentrations decline, which may affect the induction of autophagy, potentially leading to various neurodegenerative diseases. Therefore, spermine prevents age-related memory loss by inducing autophagy. Spermine possesses various pharmacological effects, including anti-inflammatory, anti-antibiotic, anti-tumor, anti-Alzheimer's disease, and antiviral properties. Spermine is fully protonated at physiological pH and binds to DNA and RNA macromolecules, reducing DNA and RNA damage and contributing to its anti-aging effects. Foods rich in spermine are beneficial for delaying aging because long-term consumption leads to increased spermine levels in the blood, promoting longevity, youthful skin, hair and nail growth, and reducing the risk of cardiovascular disease, chronic neurological diseases, chronic inflammatory diseases, rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and cancer. Spermine plays a crucial role in fetal growth and development. In early embryonic development, spermine promotes cell proliferation and division, maintaining normal embryonic cell function. Studies have found that spermine deficiency can lead to delayed embryonic growth and development, and even embryonic death. Furthermore, spermine participates in the regulation of apoptosis, a normal cell death process that plays a crucial role in maintaining normal tissue growth and development. Studies have found that spermine can regulate the degree of apoptosis and inhibit excessive apoptosis. It can interact with some apoptosis-related factors, regulating the activation and inhibition of apoptosis signaling pathways, thereby maintaining normal cell function and number. In addition, spermine is also related to intracellular calcium ion balance. It can bind to intracellular calcium ions to form stable complexes, regulating the concentration and balance of intracellular calcium ions. Disorders of intracellular calcium ion balance are closely related to the occurrence and development of various diseases, while the presence of spermine can maintain intracellular calcium ion balance and normal cell function.

[0003] Spermine can be used as a health supplement additive to enhance immunity, combat oxidation, and delay aging. It can also serve as an intermediate in organophosphorus pesticide production. Currently, spermine is primarily used in the production of acephate, a restricted organophosphorus pesticide. Domestically, acephate is mainly exported, accounting for approximately 60% of the total. However, in recent years, due to restrictions on the use of acephate in my country, both supply and demand have declined, consequently reducing the industry's demand for spermine. Spermine is a typical anionic surfactant with excellent penetration, emulsification, foaming, and detergency properties. It is widely used in chemical, pesticide, fiber, electroplating, and mineral processing industries, and is particularly suitable for the manufacture of cosmetics, toothpaste, and shampoos. In the textile industry, it is suitable for washing wool and silk.

[0004] Currently, the preparation processes for spermine are gradually maturing and can be broadly categorized into chemical synthesis, biological extraction, and biosynthesis. Among these, chemical synthesis has become the mainstream production process in the industry due to its advantages such as low production cost, high production efficiency, and stable product quality.

[0005] Because spermine content in organisms is low and its components are difficult to separate, spermine is generally not extracted directly from bodily fluids. However, chemical synthesis of spermine is simple and can be industrially produced, thus meeting the demand for spermine through chemical synthesis. Two conventional synthetic methods are as follows: The first involves alkylating phenylbromopropyl ether with putrescine to generate an ether amine compound, followed by ammonolysis with ammonia to produce spermine. The second method involves Machel addition of acrylonitrile with putrescine. The product is then hydrogenated at room temperature and 2.7 atm using sodium hydroxide and 95% ethanol solution under Raney nickel catalysis to obtain spermine, with a total yield of 38% using putrescine as a starting material.

[0006] Spermine is mainly found in animals and bacteria. Currently, the commonly used biological extraction methods are as follows: (1) Organic solvent extraction: After crushing animal and plant tissues, soaking, shaking or sonicating them with organic solvents (such as methanol, ethanol, etc.) to dissolve spermine in the solvent. Then, the solvent is recovered by distillation, evaporation, etc., to obtain an extract containing spermine. (2) Acid-base extraction: Mixing animal and plant tissues with acid or alkali solutions to convert spermine into soluble salts. Then, diluting the solution with water to precipitate spermine. Finally, through filtration, washing and other steps, spermine precipitate is obtained. (3) Enzymatic extraction: Utilizing the specific degradation effect of enzymes, the proteins in animal and plant tissues are decomposed into amino acids and polypeptides, thereby improving the extraction rate of spermine. Commonly used enzymes include proteases, trypsin, etc.

[0007] Currently, there are no reports of biosynthesizing spermine; biosynthetic methods are mainly focused on producing spermidine. Compared with traditional chemical synthesis, the biosynthesis of spermine has significant advantages in terms of sustainability, selectivity, and environmental friendliness. Therefore, developing a method for the biosynthesis of spermine is particularly important. Summary of the Invention

[0008] The purpose of this invention is to address the problem that existing technologies have not yet developed a biosynthetic pathway for the synthesis of spermine, and to provide the application of engineered Citrobacter freundii in the fermentation synthesis of spermine.

[0009] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0010] The application of engineered Citrobacter freundii in the fermentation synthesis of spermine, wherein the engineered Citrobacter freundii is Citrobacter freundii overexpressing the polyphosphate kinase gene Ppk1.

[0011] Specifically, engineered Citrobacter freundii was expanded and then inoculated into a fermentation medium for fermentation culture to synthesize spermine.

[0012] Preferably, the engineered Citrobacter freundii uses Citrobacter freundii as a host and introduces the host's own polyphosphate kinase gene Ppk1.

[0013] More preferably, the *Citrobacter freundii* is *Citrobacter freundii* ATCC 8090, and the GenBank number of the polyphosphate kinase gene Ppk1 is ANAVO1000007.1.

[0014] Most preferably, the method for constructing the engineered Citrobacter freundii is disclosed in patent CN 104531599 A.

[0015] The fermentation medium comprises glucose, sodium chloride, ammonium chloride, and dipotassium hydrogen phosphate; the pH of the fermentation medium is neutral.

[0016] Preferably, the fermentation medium comprises 0.1-0.3 g / L glucose, 0.1-0.2 g / L sodium chloride, 0.1-0.2 g / L ammonium chloride, and 0.04-0.05 g / L dipotassium hydrogen phosphate; the pH of the fermentation medium is 7.0-7.5, preferably 7.0.

[0017] The fermentation culture is carried out at a temperature of 32–42°C, a rotation speed of 200–250 rpm, and a time of 3.5–4.5 h.

[0018] Preferably, the engineered Citrobacter freundii is inoculated into the fermentation medium in the form of a seed culture for fermentation; OD 600The engineered Citrobacter freundii culture medium with a pH of 1.6–1.7 is inoculated into the seed culture medium at a volume ratio of 1:8000–10000 and cultured at 35–40°C and 200–250 rpm for 12–14 h to obtain the engineered Citrobacter freundii seed culture. The seed culture medium comprises 10–20 g / L tryptone, 7–8 g / L yeast extract, 3–5 g / L glucose, and 25–75 g / mL kanamycin, and has a pH of 7.0–7.5, preferably 7.0.

[0019] Preferably, the engineered Citrobacter freundii seed culture is inoculated into the fermentation medium at a volume ratio of 1:10 to 30 for fermentation culture; more preferably, the engineered Citrobacter freundii seed culture is inoculated into the fermentation medium at a volume ratio of 1:20 for fermentation culture.

[0020] The above-mentioned engineered Citrobacter freundii is used in the fermentation synthesis of spermine, including the step of extracting spermine from the fermentation broth obtained from the fermentation synthesis of spermine.

[0021] Specifically, the step of extracting spermine includes:

[0022] (1) Inactivate the fermentation broth, separate the solid and liquid, dry the solid, and obtain bacterial cells;

[0023] (2) Soak the bacterial cells obtained in step (1) in a trichloroacetic acid aqueous solution, adjust the pH to 6-7 to obtain a mixed system, and reflux extract it with an ethanol aqueous solution. The resulting extract is subjected to solid-liquid separation to obtain a precipitate and a separation liquid.

[0024] (3) Filter the separation liquid obtained in step (2), concentrate the filtrate under reduced pressure to obtain a concentrate, add anhydrous ethanol to it, mix well, separate the solid and liquid, freeze dry the supernatant to obtain the product.

[0025] In step (1), the inactivation is performed at 80-90°C for 0.5-1h; the solid-liquid separation is preferably centrifugation, more preferably centrifugation at 7000-9000 rpm for 5-15min; the drying temperature is 50-60°C and the time is 0.5-1h, preferably drying at 55°C for 1h.

[0026] In step (2), the concentration of the trichloroacetic acid aqueous solution is 20-50 g / L. Preferably, the mass ratio of the bacterial cells to the trichloroacetic acid aqueous solution is 1:50-150. More preferably, the mass ratio of the bacterial cells to the trichloroacetic acid aqueous solution is 1:100. The soaking time is 0.5-1 h, during which stirring is performed. The pH is adjusted to 6-7 using a 20-50 g / L sodium hydroxide aqueous solution.

[0027] In step (2), the reflux extraction is carried out at a temperature of 75-85°C for 2-3 hours. The concentration of the ethanol aqueous solution used is 60%-80% v / v, and the amount of ethanol aqueous solution used is 1-10 times the volume of the mixed system, preferably 5 times.

[0028] Preferably, in step (2), the solid-liquid separation is centrifugation, and more preferably centrifugation at 7000-9000 rpm for 5-15 min.

[0029] Preferably, in order to improve the yield of spermine from the fermentation broth containing spermine, when extracting spermine using the above method for extracting spermine from the fermentation broth, the precipitate obtained in step (2) is subjected to a second reflux extraction with an aqueous ethanol solution, the second extract is subjected to a second solid-liquid separation, the second separated liquid is collected, and the second separated liquid is combined with the separated liquid obtained in step (2) and then the operation in step (3) is performed (i.e., the separated liquid obtained in step (2) is combined with the second separated liquid, filtered, the filtrate is concentrated under reduced pressure to obtain a concentrate, anhydrous ethanol is added to it, mixed evenly, solid-liquid separation is performed, and the supernatant is freeze-dried).

[0030] Preferably, the second reflux extraction is carried out at an extraction temperature of 75-85°C for 2-3 hours, and the concentration of the ethanol aqueous solution used is 75%-85% v / v. The amount of ethanol aqueous solution used is 10-20 times the mass of the precipitate obtained in step (2) above, preferably 10 times.

[0031] Preferably, the second solid-liquid separation is centrifugation.

[0032] More preferably, the second solid-liquid separation is performed by centrifugation at 7000-9000 rpm for 5-15 minutes.

[0033] In step (3), the filtration is microfiltration, preferably using a filter membrane with a pore size of 0.45 μm; the working pressure of the vacuum concentration is 20-50 mmHg and the temperature is 50-60℃; the mass ratio of the concentrate to the anhydrous ethanol is 1:2-6, preferably 1:5; the solid-liquid separation is preferably centrifugation, more preferably centrifugation at 8000-12000 rpm for 5-15 min; the freeze-drying temperature is -40 to -60℃ and the time is 12-24 h.

[0034] Beneficial effects:

[0035] (1) The present invention uses engineered Citrobacter freundii to ferment and synthesize spermine. The operation is simple, highly reproducible, and low in cost. The fermented cells have a high spermine content, accounting for 12.42% of the bacterial dry weight.

[0036] (2) The method for extracting spermine from fermentation broth according to the present invention is simple to operate, highly reproducible, and yields and has high spermine purity. Compared with methods for extracting spermine from other biological tissues, it is safer, more controllable, less expensive, faster, and more efficient. The trichloroacetic acid used in step (2) can break down bacterial cell walls while preventing the dissolution of large amounts of protein, thus facilitating the release of spermine; ethanol reflux further enhances the dissolution and extraction effect of spermine. The spermine extract obtained using the method of the present invention has few impurity peaks in HPLC detection and a spermine purity as high as 90.26%. Attached Figure Description

[0037] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0038] Figure 1 This is a photograph of the engineered Citrobacter freundii fermentation broth from Example 1.

[0039] Figure 2 The image shows the synthesis and preparation of spermine product using engineered Citrobacter freundii in Example 1.

[0040] Figure 3 The image shows the HPLC chromatogram of the spermine extract obtained in Example 1. Detailed Implementation

[0041] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0042] The raw materials used in the following examples are from the following sources:

[0043] The engineered Citrobacter freundii was obtained from our laboratory and constructed using the method disclosed in patent CN 104531599 A.

[0044] Tryptone, yeast extract, sodium chloride, glucose, ammonium chloride, and dipotassium hydrogen phosphate were all domestically produced analytical grade or biological grade, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0045] The seed culture medium used in the following examples consisted of 15 g / L tryptone, 7.5 g / L yeast extract, 4 g / L glucose, 50 g / mL kanamycin, and a pH of 7.0–7.5.

[0046] The fermentation medium used in the following examples consisted of 0.2 g / L glucose, 0.15 g / L sodium chloride, 0.15 g / L ammonium chloride, 0.046 g / L dipotassium hydrogen phosphate, and a pH of 7.0–7.5.

[0047] Example 1

[0048] The engineered Citrobacter freundii fermentation method synthesizes spermine, and the spermine is extracted from the fermentation broth. The specific steps are as follows:

[0049] S1. OD 600 The engineered Citrobacter freundii culture medium of 1.67 was inoculated into a seed culture medium with a volume of 10,000 (v / v) and cultured at 37°C and 200 rpm for 12 h.

[0050] S2. The seed culture obtained in S1 was inoculated into the fermentation medium at a volume ratio of 1:20, and fermented at 32℃ and 200rpm for 4h to obtain the fermentation broth.

[0051] S3. The fermentation broth obtained in S2 was inactivated at 90℃ for 1 h, centrifuged at 8000 rpm for 10 min, and the precipitate was dried in an oven at 55℃ for 0.5 h to obtain the bacterial cells;

[0052] S4. The bacterial cells obtained in S3 were soaked in 30 g / L trichloroacetic acid aqueous solution at 100 times their weight and stirred for 1 h. The solution was neutralized to pH 7 with 40 g / L sodium hydroxide to obtain a mixed system. Five times the volume of the mixed system was added to a 70% v / v ethanol aqueous solution, and the mixture was refluxed at 80 °C for 2 h. The mixture was then centrifuged at 8000 rpm for 10 min to obtain the precipitate and the first separation solution.

[0053] S5. Add 10 times its mass of 80% v / v ethanol aqueous solution to the precipitate obtained in S4, reflux extract at 80℃ for 2 h, centrifuge at 8000 rpm for 10 min, and collect the second separation liquid.

[0054] S6. Combine the first separated liquid obtained in S4 with the second separated liquid obtained in S5, filter the combined liquid through a 0.45μm microporous filter, and concentrate the filtrate under reduced pressure (30mmHg, 55℃) to obtain a concentrate.

[0055] S7. Add 5 times its mass of anhydrous ethanol to the concentrate obtained in S6, mix well, centrifuge at 10000 rpm for 10 min, and freeze-dry the supernatant at -50℃ for 18 h to obtain the spermine extract.

[0056] Example 2

[0057] Engineered Citrobacter freundii was used to ferment and synthesize spermine, and spermine was extracted from the fermentation broth. Except for step S2, the other steps were the same as in Example 1. Step S2 in this example is: the seed liquid obtained in S1 is inoculated into the fermentation medium at a volume ratio of 1:10, and fermented at 32°C and 200 rpm for 4 hours to obtain the fermentation broth.

[0058] Example 3

[0059] Engineered Citrobacter freundii was used to ferment and synthesize spermine, and spermine was extracted from the fermentation broth. Except for step S2, the other steps were the same as in Example 1. Step S2 in this example is: the seed liquid obtained in S1 is inoculated into the fermentation medium at a volume ratio of 1:30, and fermented at 32°C and 200 rpm for 4 hours to obtain the fermentation broth.

[0060] Example 4

[0061] Engineered Citrobacter freundii was used to ferment and synthesize spermine, and spermine was extracted from the fermentation broth. Except for step S2, the other steps were the same as in Example 1. Step S2 in this example is: the seed liquid obtained in S1 is inoculated into the fermentation medium at a volume ratio of 1:20, and fermented at 37°C and 200 rpm for 4 hours to obtain the fermentation broth.

[0062] Example 5

[0063] Engineered Citrobacter freundii was used to ferment and synthesize spermine, and spermine was extracted from the fermentation broth. Except for step S2, the other steps were the same as in Example 1. Step S2 in this example is: the seed liquid obtained in S1 is inoculated into the fermentation medium at a volume ratio of 1:20, and fermented at 42°C and 200 rpm for 4 hours to obtain the fermentation broth.

[0064] Example 6

[0065] Engineered Citrobacter freundii was used to synthesize spermine via fermentation, and spermine was extracted from the fermentation broth. Except for step S4, the remaining steps were the same as in Example 1. Step S4 in this example was as follows: The bacterial cells obtained in S3 were soaked in 100 times their weight of a 3% trichloroacetic acid aqueous solution and stirred for 1 hour. The solution was neutralized to pH 7 with 4% sodium hydroxide, and 5 times (v / v) 60% v / v ethanol aqueous solution was added. The mixture was refluxed at 80°C for 2 hours and centrifuged at 8000 rpm for 10 minutes to obtain the precipitate and the first separation liquid.

[0066] Example 7

[0067] The synthesis and extraction of spermine by *Citrobacter freundii* was performed in the same manner as in Example 1, except for step S4. Step S4 in this example was as follows: the bacterial cells obtained in S3 were soaked in 100 times their weight of a 3% trichloroacetic acid aqueous solution and stirred for 1 hour. The solution was neutralized to pH 7 with 4% sodium hydroxide, and 5 times (v / v) of an 80% v / v ethanol aqueous solution was added. The mixture was then refluxed at 80°C for 2 hours and centrifuged at 8000 rpm for 10 minutes to obtain the precipitate and the first separation liquid.

[0068] Example 8

[0069] Preparation of spermine stock solution: Accurately weigh 0.01 g of spermine standard, dissolve in water, and dilute to 10 mL. The concentration of this spermine stock solution is 1000 ppm. Dilute this spermine stock solution to prepare a series of spermine standard solutions with concentrations of 1000, 500, 300, 100, and 0 ppm.

[0070] The spermine extracts obtained in Examples 1-7 were reconstituted in water to prepare samples 1-7 with a concentration of 0.5 mg / mL. 500 μL of each of samples 1-7 and the series of spermine standard solutions were placed in 2 mL plastic centrifuge tubes. 500 μL of dansyl chloride derivatization solution (10 mg / mL, dissolved in acetone) was added to each tube. After vortexing for 20 s, the mixture was reacted in a water bath at 40 °C for 30 min. The reaction was terminated by adding 100 μL of ammonia water. After standing at room temperature in the dark for 30 min, the mixture was filtered through a 0.22 μm membrane. The results were analyzed by liquid chromatography. A standard curve was plotted with the peak area of ​​the series of spermine standard solutions as the ordinate and the concentration as the ordinate. The actual spermine content in samples 1-7 (the spermine extracts obtained in Examples 1-7) was calculated based on the standard curve and the peak areas of samples 1-7.

[0071] The experimental results are shown in Table 1. Using the method of Example 1, 13.76 mg of spermine extract could be obtained from 0.1 g of fermented dried bacteria (the bacterial cells obtained after drying in step S3). The actual spermine content in the extract, as detected by high-performance liquid chromatography, was 12.42 mg. Therefore, the spermine content in the bacterial cells obtained by the fermentation synthesis method of Example 1 was 12.42%, with an extraction purity of 90.26%. The spermine content in general animal tissue is approximately 0.23%, and the purity of spermine extracted from animal tissue is generally about 80%. Using the same synthesis method, but changing the inoculation ratio of the bacterial seed liquid and the fermentation temperature, the spermine content in the bacterial cells was 11.69% in Example 2, 7.42% in Example 3, 11.08% in Example 4, and 9.87% in Example 5. Using the same extraction method, but changing the concentration of the ethanol-water solution obtained by reflux extraction in step S4, the spermine purity was measured to be 76.76% in Example 6 and 80.51% in Example 7. It is evident that the spermine synthesized by the method of the present invention has a high content in the bacterial cells, and the spermine extracted by the extraction and separation method has high purity.

[0072] Table 1. Spermine content in fermented cells and purity of extracted spermine from Examples 1-7

[0073]

[0074] The formulas for calculating the spermine content and spermine purity are as follows:

[0075]

[0076]

[0077] This invention provides a concept and method for the application of engineered Citrobacter freundii in the fermentation synthesis of spermine. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. Application of engineered Citrobacter freundii in the fermentation synthesis of spermine, wherein the engineered Citrobacter freundii is an overexpressing polyphosphate kinase gene. Ppk1 Citrobacter freundii; in, The *Citrobacter freundii* mentioned is *Citrobacter freundii* ATCC 8090, and the polyphosphate kinase gene mentioned is... Ppk1 The GenBank number is ANAVO1000007.

1.

2. The application according to claim 1, characterized in that, Engineered Citrobacter freundii was inoculated into a fermentation medium for fermentation culture to synthesize spermine.

3. The application according to claim 2, characterized in that, The fermentation medium comprises glucose, sodium chloride, ammonium chloride, and dipotassium hydrogen phosphate; and / or the fermentation culture is carried out at a temperature of 32-42°C, a rotation speed of 200-250 rpm, and a time of 3.5-4.5 h.

4. The application according to claim 3, characterized in that, The fermentation medium comprises 0.1-0.3 g / L glucose, 0.1-0.2 g / L sodium chloride, 0.1-0.2 g / L ammonium chloride, 0.04-0.05 g / L dipotassium hydrogen phosphate, and is neutral in pH.

5. The application according to any one of claims 1 to 4, characterized in that, This includes the step of extracting spermine from the fermentation broth obtained from the fermentation synthesis of spermine.

6. The application according to claim 5, characterized in that, The steps for extracting spermine include: (1) Inactivate the fermentation broth, separate the solid and liquid, dry the solid, and obtain the bacterial cells; (2) Soak the bacterial cells obtained in step (1) in a trichloroacetic acid aqueous solution, adjust the pH to 6-7 to obtain a mixed system, and reflux extract it with an ethanol aqueous solution. Separate the solid and liquid components of the extract to obtain a precipitate and a separation solution. (3) Filter the separation liquid obtained in step (2), concentrate the filtrate under reduced pressure to obtain a concentrate, add anhydrous ethanol to it, mix well, separate the solid and liquid, freeze dry the supernatant to obtain the product.

7. The application according to claim 6, characterized in that, In step (1), the inactivation is performed at 80-90°C for 0.5-1 h; the drying temperature is 50-60°C and the time is 0.5-1 h.

8. The application according to claim 6, characterized in that, In step (2), the concentration of the trichloroacetic acid aqueous solution is 20-50 g / L, and the mass ratio of trichloroacetic acid to bacterial cells is 1:1-150; the soaking time is 0.5-1 h, during which stirring is performed; the reflux extraction is performed at an extraction temperature of 75-85℃ for 2-3 h, and the concentration of the ethanol aqueous solution used is 60-80% v / v, and the amount of ethanol aqueous solution used is 1-10 times the volume of the mixed system.

9. The application according to claim 6, characterized in that, The precipitate obtained in step (2) is subjected to a second reflux extraction with an ethanol aqueous solution. The resulting second extract is subjected to a second solid-liquid separation. The second separated liquid is collected. The second separated liquid is combined with the separated liquid obtained in step (2) and then the operation in step (3) is performed.

10. The application according to claim 9, characterized in that, The second reflux extraction is carried out at a temperature of 75-85°C for 2-3 hours. The concentration of the ethanol aqueous solution used is 75%-85% v / v, and the amount of ethanol aqueous solution used is 10-20 times the mass of the precipitate obtained in step (2).

11. The application according to claim 6, characterized in that, In step (3), the filtration is microfiltration; the mass ratio of the concentrate to the anhydrous ethanol is 1:2 to 6.

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