Application of engineered citrobacter freundii in fermentation synthesis of spermine

Abstract

The invention discloses an application of engineered citrobacter freundii in fermentation synthesis of spermine, the engineered citrobacter freundii is citrobacter freundii overexpressing a polyphosphate kinase gene Ppk1, and the engineered citrobacter freundii is inoculated into a fermentation medium for fermentation culture to synthesize spermine. The synthesis and extraction method for producing spermine is simple to operate, high in repeatability and low in cost, the spermine content in thalli obtained through fermentation is high and accounts for 12.42% of the dry weight of bacteria, and the purity of the extracted spermine is as high as 90.26%.

Description

Technical Field

[0001] The present invention relates to the field of biochemical production, and in particular to the application of engineered Citrobacter freundii in the fermentation synthesis of spermine. Background Art

[0002] Spermine is a ubiquitous endogenous polyamine in living organisms, involved in maintaining cellular and neuronal homeostasis. Spermine is a cellular, structurally stable cationic compound containing two amino groups (-NH2), which can interact with negatively charged molecules such as DNA, RNA, and proteins. Spermine is a key factor in many cellular processes, including stabilizing DNA structure, promoting cell division, cell proliferation, and differentiation, and stress responses. As a free radical scavenger and antioxidant, spermine reduces oxidative damage caused by environmental stress. Spermine also acts as a second messenger in cells, extending lifespan, maintaining nucleic acid and cell membrane stability, and controlling cell division and growth. It plays a physiological role in organ development, embryonic development, floral senescence, flower and fruit growth, and responses to biotic and abiotic stresses. Spermine also maintains cellular homeostasis and regulates ion transport across membranes, increasing the bioavailability of arginine, which is required for the synthesis of nitric oxide (NO). Spermine concentrations decrease with aging, potentially affecting the induction of autophagy and contributing to various neurodegenerative diseases. Therefore, spermine prevents memory loss in aging by inducing autophagy. Spermine has multiple pharmacological effects, including anti-inflammatory, anti-antibiotic, anti-tumor, anti-Alzheimer's, and antiviral. Spermine is fully protonated at physiological pH and binds to DNA and RNA macromolecules, mitigating DNA and RNA damage and contributing to its anti-aging effects. Foods rich in spermine can help delay aging, as long-term consumption increases blood spermine levels, promoting longevity, skin rejuvenation, 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. During early embryonic development, spermine promotes cell proliferation and division, maintaining normal embryonic cell function. Studies have shown that spermine deficiency can lead to delayed embryonic growth and development, and even to spontaneous embryonic death. Spermine also participates in the regulation of apoptosis, a normal cell death process that plays an important role in maintaining normal tissue growth and development. Studies have found that spermine can regulate the extent of apoptosis and inhibit excessive cell apoptosis. It can interact with several apoptosis-related factors to regulate the activation and inhibition of apoptotic signaling pathways, thereby maintaining normal cell function and number. Furthermore, spermine is involved in intracellular calcium homeostasis. It can bind to intracellular calcium ions to form a stable complex, regulating the concentration and balance of intracellular calcium ions. Disturbances in intracellular calcium homeostasis are closely associated with the occurrence and development of various diseases. The presence of spermine can maintain intracellular calcium homeostasis and maintain normal cell function.

[0003] Spermine can be used as a health supplement additive to enhance human immunity, resist peroxidation, and slow down aging. Spermine can also be used as an intermediate for organophosphorus pesticides. Currently, spermine is primarily used in the production of acephate, a restricted organophosphorus insecticide. Domestic acephate is the main export product, accounting for approximately 60.0% of total exports. However, in recent years, due to restrictions on the use of acephate in my country, both the supply and demand for acephate have declined, and the demand for spermine in this industry has also decreased. Spermine is a typical anionic surfactant with excellent penetration, emulsification, foaming, and detergency. It is widely used in the chemical, pesticide, fiber, electroplating, mineral processing, and other industries, and is particularly suitable for the manufacture of cosmetics, toothpaste, shampoo, etc. In the textile industry, it is suitable for washing wool and silk.

[0004] Currently, spermine preparation processes are gradually maturing and can be broadly categorized into chemical synthesis, biological extraction, and biosynthesis. Chemical synthesis, with its advantages of low production costs, high production efficiency, and stable product quality, has become the mainstream production process in the industry.

[0005] Because spermine is present in low concentrations in organisms and its components are difficult to separate, direct extraction from biological fluids is generally not recommended. Chemical synthesis of spermine, however, offers simple steps and is amenable to industrial production. Therefore, demand for spermine is met through chemical synthesis. There are two conventional synthesis methods: the first involves alkylation of phenyl bromopropyl ether with putrescine to produce an etheramine compound, which is then ammonolyzed with ammonia to produce spermine. The other involves Machel addition of acrylonitrile to putrescine. The product is then hydrogenated with sodium hydroxide and 95% ethanol solution under Raney nickel at room temperature and 2.7 atmospheres to yield spermine. The overall yield using putrescine as the starting material is 38%.

[0006] Spermine is mainly found in animals and bacteria. Currently, the commonly used biological extraction methods are as follows: (1) Organic solvent extraction method: After crushing the animal and plant tissues, soak, oscillate or ultrasonically treat 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. Acid-base extraction method: Animal and plant tissues are mixed with acid or alkaline solutions to convert spermine into soluble salts. The solution is then diluted with water to precipitate spermine. Finally, through filtering, washing and other steps, the spermine precipitate is obtained. Enzymatic extraction method: Utilize the specific degradation effect of enzymes to decompose proteins in animal and plant tissues into amino acids and polypeptides, thereby increasing the extraction rate of spermine. Commonly used enzymes include proteases, trypsin, etc.

[0007] Currently, there are no reports on the biosynthetic production of spermine, and biosynthetic methods have focused on the production of spermidine. Compared to traditional chemical synthesis, the biosynthesis of spermine offers significant advantages in sustainability, selectivity, and environmental friendliness. Therefore, developing a biosynthetic method for spermine is particularly important. Summary of the Invention

[0008] The purpose of the present invention is to address the problem that the prior art has not yet developed a biosynthetic pathway for synthesizing spermine, and to provide an application of an engineered Citrobacter freundii in the fermentative synthesis of spermine.

[0009] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0010] The invention relates to the use of an engineered Citrobacter freundii in the fermentation synthesis of spermine, wherein the engineered Citrobacter freundii is a Citrobacter freundii that overexpresses the polyphosphate kinase gene Ppk1.

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

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

[0013] Further 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 construction method of the engineered Citrobacter freundii is disclosed in patent CN 104531599 A.

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

[0016] Preferably, the components of the fermentation medium include 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 temperature is 32-42° C., the rotation speed is 200-250 rpm, and the fermentation time is 3.5-4.5 h.

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

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

[0020] The application of the engineered Citrobacter freundii in the fermentation synthesis of spermine comprises the step of extracting spermine from the fermentation liquid obtained by the fermentation synthesis of spermine.

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

[0022] (1) inactivating the fermentation broth, separating the solid and the liquid, and drying the solid to obtain bacterial cells;

[0023] (2) soaking the bacterial cells obtained in step (1) in a trichloroacetic acid aqueous solution, adjusting the pH to 6-7 to obtain a mixed system, refluxing the system with an ethanol aqueous solution, and subjecting the obtained extract to solid-liquid separation to obtain a precipitate and a separated liquid;

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

[0025] In step (1), the inactivation is carried out at 80-90°C for 0.5-1h; the solid-liquid separation is preferably carried out by centrifugation, more preferably by centrifugation at 7000-9000 rpm for 5-15min; the drying temperature is 50-60°C for 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, and further 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; and the pH is adjusted to 6-7 using 20-50 g / L sodium hydroxide aqueous solution.

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

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

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

[0030] Preferably, the second reflux extraction has an extraction temperature of 75-85°C, a time of 2-3 hours, and an ethanol aqueous solution with a concentration of 75%-85% v / v. The amount of ethanol aqueous solution used is 10-20 times the mass of the precipitate obtained in step (2), 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 min.

[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 reduced pressure concentration is 20 to 50 mmHg, and the temperature is 50 to 60°C; the mass ratio of the concentrate to the anhydrous ethanol is 1:2 to 6, preferably 1:5; the solid-liquid separation is preferably centrifugation, more preferably centrifugation at 8000 to 12000 rpm for 5 to 15 minutes; the temperature of the freeze-drying is -40 to -60°C, and the time is 12 to 24 hours.

[0034] Beneficial effects:

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

[0036] (2) The method for extracting spermine from a fermentation broth of the present invention is simple to operate, highly reproducible, and has high spermine yield and purity. Compared with methods for extracting spermine from other biological tissues, the method is safe, controllable, low-cost, time-saving, and highly efficient. The trichloroacetic acid used in step (2) can prevent a large amount of protein dissolution while breaking the bacterial cell wall, thereby facilitating the free release of spermine; the ethanol reflux further enhances the dissolution and extraction effect of spermine. The spermine extract obtained by the method of the present invention has few impurity peaks detected by HPLC, and the spermine purity is as high as 90.26%. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

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

[0039] Figure 2 This is a diagram of the spermine product produced by engineering Citrobacter freundii in Example 1.

[0040] Figure 3 The HPLC spectrum of the spermine extract obtained in Example 1 is shown. DETAILED DESCRIPTION

[0041] The present invention is further described below with reference to the following examples. It should be understood that the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0042] The sources of raw materials used in the following examples are as follows:

[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 potassium dihydrogen phosphate were all domestically produced analytical grade or biological grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0045] The components of the seed liquid culture medium used in the following examples are 15 g / L tryptone, 7.5 g / L yeast extract, 4 g / L glucose, 50 g / mL karatomycin, and a pH of 7.0-7.5.

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

[0047] Example 1

[0048] The engineered Citrobacter freundii is used to ferment and synthesize spermine, and spermine is extracted from the fermentation broth. The specific steps are as follows:

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

[0050] S2. The seed solution obtained in S1 was inoculated into the fermentation medium at a volume ratio of 1:20, and the fermentation was carried out at 32°C and 200 rpm for 4 h to obtain a fermentation broth;

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

[0052] S4. Soak the cells obtained in S3 in a 30 g / L trichloroacetic acid aqueous solution (100 times their mass) and stir for 1 hour. Neutralize with 40 g / L sodium hydroxide to a pH of 7 to obtain a mixed system. Add 70% v / v ethanol aqueous solution (5 times the volume of the mixed system) and reflux extract at 80°C for 2 hours. Centrifuge at 8000 rpm for 10 minutes to obtain a precipitate and a first separated liquid.

[0053] S5. To the precipitate obtained in S4, 10 times its mass of 80% v / v ethanol aqueous solution was added, refluxed at 80 ° C for 2h, centrifuged at 8000 rpm for 10min, and the second separated liquid was collected;

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

[0055] S7. Add 5 times the mass of anhydrous ethanol to the concentrate obtained in S6, mix well, centrifuge at 10,000 rpm for 10 min, and freeze-dry the supernatant at -50°C for 18 h to obtain the spermine extract.

[0056] Example 2

[0057] The engineered Citrobacter freundii was used to ferment and synthesize spermine, and spermine was extracted from the fermentation broth. Except for step S2, the remaining steps were the same as those in Example 1. Step S2 of this embodiment was as follows: the seed liquid obtained in S1 was inoculated into the fermentation medium at a volume ratio of 1:10, and the fermentation culture was carried out at 32°C and 200 rpm for 4 hours to obtain a fermentation broth.

[0058] Example 3

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

[0060] Example 4

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

[0062] Example 5

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

[0064] Example 6

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

[0068] Example 8

[0069] Prepare 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 spermine concentrations of 1000, 500, 300, 100, and 0 ppm, respectively.

[0070] The spermine extracts obtained in Examples 1 to 7 were redissolved in water to prepare samples 1 to 7 with a concentration of 0.5 mg / mL. 500 μL of each of samples 1 to 7 and a 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 seconds, the mixture was reacted in a 40°C water bath for 30 minutes. 100 μL of ammonia water was added to terminate the reaction. After standing in the dark at room temperature for 30 minutes, the mixture was filtered through a 0.22 μm membrane. Liquid chromatography was used to determine the concentration of the spermine standard solution. A standard curve was drawn with the peak area of ​​the series of spermine standard solutions as the ordinate and the concentration as the ordinate. The actual content of spermine in samples 1 to 7 (the spermine extracts obtained in Examples 1 to 7) was calculated based on the peak area obtained from the standard curve and the detection of samples 1 to 7.

[0071] Experimental result is as shown in Table 1, can extract and obtain 13.76mg spermine extract in 0.1g fermentation dry bacteria (step S3 dried thalline) by the method for embodiment 1, and in the spermine extract detected by HPLC, spermine actual content is 12.42mg, therefore spermine content is 12.42% in the thalline obtained by the fermentation synthesis method of embodiment 1, extraction purity is 90.26%, and the spermine content of general animal tissue is about 0.23%, and the purity of spermine extracted from animal tissue is generally about 80%.Use the same synthesis method, change thalline seed liquid inoculation ratio and fermentation culture temperature, in embodiment 2 thalline, spermine content is 11.69%, embodiment 3 is 7.42%, embodiment 4 is 11.08%, and embodiment 5 is 9.87%.Use the same extraction method, change the concentration of the ethanolic aqueous solution of step S4 reflux extraction, recording spermine purity in embodiment 6 is 76.76%, and embodiment 7 spermine purity is 80.51%. It can be seen that the spermine synthesized by the method of the present invention has a high content in the bacteria, and the spermine extracted by the extraction and separation method has a high purity.

[0072] Table 1 Spermine content in the fermented cells obtained in Examples 1 to 7 and the purity of the extracted spermine

[0073]

[0074] The calculation formulas for the above-mentioned spermine content and spermine purity are as follows:

[0075]

[0076]

[0077] The present invention provides a concept and method for applying engineered Citrobacter freundii to the fermentation and synthesis of spermine. Numerous methods and approaches exist for implementing this technical solution. The foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. Application of an engineered Citrobacter freundii in the fermentation synthesis of spermine, wherein the engineered Citrobacter freundii is a Citrobacter freundii that overexpresses the polyphosphate kinase gene Ppk1.

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

3. The use according to claim 2, characterized in that The components of the fermentation medium include glucose, sodium chloride, ammonium chloride and dipotassium hydrogen phosphate; preferably, the components of the fermentation medium include 0.1-0.3 g / L of glucose, 0.1-0.2 g / L of sodium chloride, 0.1-0.2 g / L of ammonium chloride, 0.04-0.05 g / L of dipotassium hydrogen phosphate, and a neutral pH; and / or, the fermentation culture is performed 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 use according to any one of claims 1 to 3, characterized in that The method comprises the steps of extracting spermine from the fermentation liquid obtained by fermenting and synthesizing spermine.

5. The use according to claim 4, characterized in that The step of extracting spermine comprises: (1) inactivating the fermentation broth, separating the solid and the liquid, and drying the solid to obtain bacterial cells; (2) soaking the bacterial cells obtained in step (1) in a trichloroacetic acid aqueous solution, adjusting the pH to 6-7 to obtain a mixed system, refluxing the system with an ethanol aqueous solution, and subjecting the obtained extract to solid-liquid separation to obtain a precipitate and a separated liquid; (3) Filter the separated liquid obtained in step (2), and concentrate the filtrate under reduced pressure to obtain a concentrate, add anhydrous ethanol to the concentrate, mix well, separate the solid and liquid, and freeze-dry the supernatant to obtain the concentrate.

6. The use according to claim 5, characterized in that In step (1), the inactivation is carried out at 80-90° C. for 0.5-1 h; the drying temperature is 50-60° C. for 0.5-1 h.

7. The use according to claim 5, characterized in that In step (2), the concentration of the trichloroacetic acid aqueous solution is 20-50 g / L, and the mass ratio of the trichloroacetic acid to the bacterial cells is 1:1-150; the soaking time is 0.5-1 hour, during which stirring is performed; the reflux extraction is performed at a temperature of 75-85° C. and for 2-3 hours, and the concentration of the ethanol aqueous solution used is 60-80% v / v, and the amount of the ethanol aqueous solution used is 1-10 times the volume of the mixed system.

8. The use according to claim 5, characterized in that The precipitate obtained in step (2) is subjected to a second reflux extraction with an ethanol aqueous solution, the obtained 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 of step (3) is performed.

9. The use according to claim 8, characterized in that The second reflux extraction has an extraction temperature of 75-85°C, a time of 2-3 hours, an ethanol aqueous solution with a concentration of 75%-85% v / v, and an amount of ethanol aqueous solution used that is 10-20 times the mass of the precipitate obtained in step (2).

10. The use according to claim 5, 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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