Application of AKK bacteria in preparation of medicine for inhibiting neuronal cell senescence after peripheral nerve injury

Abstract

The invention relates to the technical field of microbial medicines, and particularly discloses application of AKK bacteria in preparation of a medicine for inhibiting neuronal cell senescence after peripheral nerve injury. The AKK bacteria can be prepared into a microbial inoculum through the processes of culturing, fermenting, centrifuging, freeze drying and the like. The microbial agent can inhibit aging of dorsal root ganglion neuronal cells induced by peripheral nerve injury and reduce expression of aging-related proteins such as P16 and P21, so that aging of the dorsal root ganglion neuronal cells is inhibited. The nerve function recovery can be promoted; the dysfunction caused by nerve injury is relieved. The peripheral nerve injury is intervened from the perspective of cell senescence regulation, a new intervention way is provided for treatment of the peripheral nerve injury, and the method has the advantages of being high in safety, simple in preparation process, wide in application prospect and the like.

Description

Technical Field

[0001] This invention relates to the field of microbial pharmaceutical technology, and in particular to the application of AKK bacteria in the preparation of drugs that inhibit neuronal cell senescence after peripheral nerve injury. Background Technology

[0002] Peripheral nerve injury (PNI) is a common traumatic disease in clinical practice, often caused by factors such as trauma, compression, or metabolic abnormalities. Following PNI, patients frequently experience a range of pathological changes, including sensory dysfunction, motor impairment, and neuropathic pain, significantly reducing their quality of life. Although the peripheral nervous system possesses a certain degree of self-regenerative capacity, the slow rate and poor quality of nerve regeneration mean that relying solely on natural repair often fails to restore satisfactory nerve function. Therefore, developing treatment strategies that effectively promote nerve repair is of significant clinical importance.

[0003] Recent studies have revealed that neuronal senescence plays a crucial role in the pathological process following peripheral nerve injury. After nerve injury, damaged nerve cells and surrounding supporting cells (such as Schwann cells) exhibit a pronounced senescent phenotype, characterized by cell cycle arrest, enhanced activity of senescence-associated β-galactosidase (SA-β-gal), and increased expression of senescence-related proteins (such as p16 and p21). These senescent cells secrete various inflammatory substances and chemokines, forming an senescence-associated secretory phenotype. This not only exacerbates local inflammation at the injury site but also inhibits nerve regeneration through mechanisms such as suppressing axonal growth. Therefore, targeting and inhibiting cellular senescence after nerve injury is becoming an important new research direction for promoting nerve repair.

[0004] Currently, clinical treatments for peripheral nerve injuries mainly include nerve suturing, nerve transplantation, and conservative treatment using a combination of neurotrophic factors, anti-inflammatory drugs, and antioxidants. However, although existing surgical and drug treatments can alleviate symptoms to some extent, their ability to intervene in the key pathological process of cellular senescence after nerve injury remains limited, often resulting in unsatisfactory recovery of nerve function. Therefore, finding novel treatment strategies that can effectively inhibit neuronal aging and promote nerve regeneration is an urgent technical task.

[0005] The gut microbiota, often referred to as the human body's "second genome," plays a crucial role in maintaining health and influencing disease development. As a complete "virtual endocrine organ" and a key remote regulator, the gut microbiota exerts a central influence on host physiological and pathological processes, and responds to bodily damage. Furthermore, with the emergence and refinement of the "gut-brain-peripheral nerve axis" concept, it has been confirmed that gut microbiota and their metabolites can profoundly affect peripheral nervous system function and disease progression through immune, neural, and endocrine pathways.

[0006] However, existing research has focused less on the relationship between neuronal senescence caused by peripheral nerve injury and the gut microbiota, and the relevant molecular and cellular mechanisms remain unclear. Therefore, developing a strategy that utilizes gut symbiotic bacteria or their metabolites to inhibit neuronal and supporting cell senescence after peripheral nerve injury, thereby promoting nerve regeneration and recovery of nerve function, has significant research value and application prospects for enriching the treatment options for peripheral nerve injury and improving clinical efficacy. Summary of the Invention

[0007] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing an application of AKK bacteria in the preparation of drugs that inhibit neuronal cell senescence after peripheral nerve injury. This intervention addresses peripheral nerve injury from the perspective of cell senescence regulation, providing a new intervention approach for the treatment of peripheral nerve injury.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention is to provide the use of AKK bacteria in the preparation of a medicament for inhibiting neuronal cell senescence after peripheral nerve injury, characterized in that the AKK bacteria... Akkermansia muciniphila .

[0009] Furthermore, the active ingredient of the drug is selected from live AKK bacteria, inactivated AKK bacteria, AKK bacteria fermentation broth, or AKK bacteria extract.

[0010] Furthermore, the drug exerts its effect by reducing the expression of neuronal cell senescence markers.

[0011] Furthermore, the aging markers include P16 and P21.

[0012] Furthermore, the peripheral nerve injury includes at least one of the following: sciatic nerve injury, nerve compression injury, or nerve ischemia-reperfusion injury.

[0013] Furthermore, the drug is administered via intravenous injection or oral administration. Furthermore, the dosage form of the drug is any one of capsules, tablets, oral preparations, microcapsule preparations, or injections.

[0014] A second aspect of the invention is to provide the use of a pharmaceutical composition comprising the above-described AKK bacterium and its pharmaceutically acceptable excipients in the preparation of a medicament for inhibiting neuronal cell senescence following peripheral nerve injury.

[0015] Furthermore, the pharmaceutical composition is in unit dose form, wherein the concentration of AKK bacteria in each unit dose form is not less than 1 × 10⁻⁶. 6 CFU / mL.

[0016] Further, the pharmaceutical composition is a bacterial agent, wherein the bacterial agent is prepared by inoculating the AKK bacteria of any one of claims 1-2 into a culture medium and activating and fermenting it sequentially to obtain a fermentation broth; the fermentation broth is centrifuged, mixed with a protectant, and then freeze-dried to obtain AKK bacteria strain powder; the AKK bacteria strain powder is formulated according to the live bacteria count ratio to obtain the bacterial agent.

[0017] Compared with the prior art, the beneficial effects of the present invention are: (1) The present invention discovers that the intestinal bacteria AKK or its metabolites can be used to inhibit neuronal cell senescence after peripheral nerve injury. This is an intervention on peripheral nerve injury from the perspective of cell senescence regulation, providing a new intervention approach for the treatment of peripheral nerve injury and expanding the application scope of intestinal bacteria in the treatment of nervous system diseases.

[0018] (2) The AKK bacteria or its metabolites provided by the present invention can significantly reduce the expression of aging-related proteins such as P16 and P21 in dorsal root ganglion neurons after peripheral nerve injury, thereby inhibiting the aging of dorsal root ganglion neurons.

[0019] (3) Experimental results show that the intervention of the AKK bacteria or its metabolites in this invention significantly enhances the regenerative potential and structural repair quality of the sciatic nerve after injury in mice, indicating that the scheme can promote the recovery of nerve function and reduce the functional impairment caused by nerve damage.

[0020] (4) The AKK bacteria provided by the present invention are probiotics with high safety. The bacterial agent prepared by fermentation culture and freeze drying has good stability and simple production process. It has good biological safety and industrial application prospects and can be used to develop drugs or functional preparations for treating peripheral nerve injury-related diseases. Attached Figure Description

[0021] Figure 1 This is an immunofluorescence staining result of the aging marker P21 in the mouse dorsal root ganglion according to an embodiment of the present invention. In the figure, a is before modeling and b is after modeling. Scale bar, 20μm. It was found that after sciatic nerve transection modeling, the expression level of the aging marker P21 in the mouse dorsal root ganglion was significantly increased.

[0022] Figure 2 This is an immunofluorescence staining statistical diagram of the aging marker P21 in the mouse dorsal root ganglion according to an embodiment of the present invention. The data are expressed as mean ± SEM. p < 0.05 p < 0.01, indicating a significant difference between the two sets of data.

[0023] Figure 3 This describes the phylum-level changes in gut microbiota differences among the sciatic nerve transection groups and the sham-operated group (Sham) at 1 day (d), 4 days, 7 days, 14 days, and 28 days after sciatic nerve transection, according to embodiments of the present invention. A significant increase in the expression level of the Verrucous microbiota was found on day 7.

[0024] Figure 4 This describes the species-level changes in gut microbiota differences at the genus level in the sciatic nerve transect at 1 day (d), 4 days, 7 days, 14 days, 28 days, and the sham-operated group (Sham) according to embodiments of the present invention. A significant increase in Akkermansia expression was found in the Verrucous microbes phylum on day 7.

[0025] Figure 5 According to the Mean Decrease Accuracy of the present invention, it is indicated that there is a change in the expression of Akkermansia.

[0026] Figure 6 According to the embodiment of the present invention, Diff in mean abundanc indicates that there is a significant difference in the expression level of Akkermansia on day 7.

[0027] Figure 7 According to the LDA SCORE in the embodiments of the present invention, Akkermansia showed the greatest difference in expression compared to other bacteria on day 7.

[0028] Figure 8 This is a schematic diagram of the gavage procedure for mice two weeks prior to the invention, which is used to establish a sciatic nerve transect model, and three groups of gavage experiments.

[0029] Figure 9This image shows the immunofluorescence staining results of two aging markers, P16 and P21, in the mouse dorsal root ganglion according to an embodiment of the present invention. Scale bar, 20 μm. (Sham: Sham operation; PBS: Phosphate Buffered Saline; AKK: Akkermansia; FMT: fecal microbiota transplantation). The results show that the expression of aging markers in the two groups of mice receiving AKK and FMT by gavage was significantly decreased compared to the Sham group and the PBS group, indicating a significant improvement in the aging status of the mouse dorsal root ganglion.

[0030] Figure 10 and Figure 11 The images show the immunofluorescence staining statistics of P16 and P21 of the mouse dorsal root ganglia according to the embodiments of the present invention. The data are expressed as mean ± SEM; *p<0.05, **p<0.01 (Sham: Sham operation; PBS: Phosphate Buffered Saline; AKK: Akkermansia; FMT: fecal microbiotatransplantation). It was found that the statistical results of aging markers in the two groups of mice receiving AKK and FMT by gavage were significantly different from those in the Sham group, indicating that the aging of the mouse dorsal root ganglia was significantly improved.

[0031] Figure 12 The image shows fluorescence staining of axonal growth, with a representing the control group and b representing the experimental group.

[0032] Figure 13 A statistical graph showing the expression level of SCG10. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the present invention clearer, embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0034] Terms and definitions in this invention The term "AKK bacteria" refers to an important intestinal bacterium, a genus belonging to a Gram-negative family, one of whose known species is fully named "AKK". Akkermansia muciniphila ““ is a harsh anaerobic bacterium. The bacterial culture used in the embodiments of this invention has the preservation number ATCC BAA-835 and is commercially available.

[0035] While peripheral nerves possess a certain capacity for spontaneous regeneration after injury, the regeneration efficiency is often low and the recovery of nerve function is limited in actual repair processes. Notably, injury to the fragile peripheral axons of dorsal root ganglion (DRG) neurons leads to a higher incidence of somatosensory dysfunction (such as pain) in the elderly. Furthermore, studies have shown that the neural expression of IL6, a SASP component in the DRG, increases with age, and this expression further increases after peripheral injury, indicating that peripheral nerve injury can negatively impact DRG neurons to some extent. In our research, we observed that alterations in gut microbiota composition were common in animal models of peripheral nerve injury, and changes in the abundance of specific microbiota were closely related to neuronal aging. This suggests that gut microbiota may delay neuronal aging caused by injury by regulating host immune and metabolic pathways. However, existing research has focused less on the interaction between neuronal aging caused by peripheral nerve injury and the gut microbiota, and the relevant molecular and cellular mechanisms remain unclear. Therefore, in-depth research into its regulatory mechanisms, exploring its signaling pathways and key metabolites, holds promise for providing new intervention strategies and therapeutic targets for delaying aging after nerve injury.

[0036] Example 1 A mouse model of peripheral nerve injury was constructed.

[0037] Following sciatic nerve injury, the injury signal can be transmitted retrogradely to the dorsal root ganglion (DRG). Disruption of axoplasmic transport and inflammatory response can cause DRG neuronal damage, abnormal excitability, and neuropathic pain. Although a regeneration process can be initiated, the regenerative capacity is limited and complete repair is difficult.

[0038] Dorsal root ganglion tissue samples were obtained from C57BL / 6 mice.

[0039] 20g ± SPF grade C57BL / 6 mice were selected, and their weight was uniformly measured to be around 20g before surgery. The mice were first anesthetized or euthanized by carbon dioxide asphyxiation. An incision was made in the pelvis using surgical scissors, and the entire piece of skin was torn away. An incision was made in the ventral wall muscle. Along this incision, the spinal cord was dissected parallel to the spine. After further trimming to remove the spinal cord, the nerve bundles of the dorsal root ganglia were grasped with microforceps, and the dorsal root ganglia of the C57BL / 6 mice were separated and placed in pre-cooled PBS for immunofluorescence experiments.

[0040] Establishment of a mouse model of peripheral nerve injury.

[0041] A mouse sciatic nerve transection model was established. Before surgery, the mice were weighed to approximately 20g. A compound anesthetic was administered intraperitoneally at a dose of 0.3ml / 100g. After anesthesia, the hair on the thigh side was shaved, and the area was disinfected with 75% alcohol. The skin was cut open with surgical scissors to separate the fascia from the muscle and expose the sciatic nerve. The sciatic nerve was sutured after exposure in the Sham group. In the experimental group, the sciatic nerve was transected by surgical scissors to approximately 5mm, then sutured and disinfected with iodine. After the C57BL / 6 mice woke up, they were transferred to the mouse room.

[0042] Example 2 After peripheral nerve injury, the expression of aging markers increased in the dorsal root ganglia of mice.

[0043] In this case, the animals were divided into two groups of five each. They were fed with pure water for seven days, and then the dorsal root ganglia were extracted for immunofluorescence staining of the aging marker P21 to observe the expression of P21 before and after injury (e.g., Figure 1 (As shown).

[0044] Statistical results of P21 expression level data are as follows: Figure 2 As shown, the results indicate that the expression of the aging marker P21 was significantly increased in the dorsal root ganglia of mice with sciatic nerve injury, which demonstrates that peripheral nerve injury does indeed lead to aging of neurons in the dorsal root ganglia of mice.

[0045] Example 3. After peripheral nerve injury, the Akkermansia gradient in the gut microbiota of mice increases.

[0046] In this embodiment, fecal samples from the peripheral nerve injury groups at 1d, 4d, 7d, 14d, 28d and Sham groups were sequenced using 16sDNA to analyze the differential bacterial communities among the groups.

[0047] The results showed that, compared with the Sham group, the Verrucomicrobiota flora was significantly elevated at the phylum level on day 7 in the peripheral nerve injury groups at 1d, 3d, and 7d (e.g., Figure 3 As shown), the changes at the genus level were most pronounced in Akkermansia on day 7 (e.g. Figure 4 As shown), we further improve the Mean Decrease Accuracy (as shown). Figure 5 As shown), Diff inmean abundance (as shown) Figure 6 (as shown) and LDA SCOR (as shown) Figure 7 As shown in the figure, Akkermansia was affected by peripheral nerve damage, which was particularly pronounced on the seventh day.

[0048] Example 4 Akkermansia can significantly improve neuronal senescence in the dorsal root ganglion.

[0049] In this study, mice were divided into four groups of five mice each. All mice were pre-treated with a mixed antibiotic regimen (neomycin sulfate 1 g / L, streptomycin sulfate 1 g / L, and ampicillin 1 g / L) for 14 days before peripheral nerve injury was induced and administered via gavage. Since 16S DNA sequencing showed that Akkermansia levels were highest 7 days after peripheral nerve injury, fecal microbiota from mice with 7 days of peripheral nerve injury was administered via gavage to these mice once daily for 7 consecutive days. The fecal microbiota was diluted with physiological saline at a concentration of 100 mg / ml, centrifuged at 500g for 5 min, and the supernatant was collected. 0.2 ml of the supernatant was administered to each mouse via gavage. The Akkermansia concentration used for gavage was 5 × 10⁻⁶. 7 CFU / mL, 0.2 ml per mouse was administered by gavage. Seven days later, the dorsal root ganglion senescence was compared between the naturally fed Sham group, the control group administered PBS by gavage, the FMT group administered fecal microorganisms by gavage, and the AKK group administered Akkermansia by gavage (e.g., CFU / mL). Figure 8 (As shown).

[0050] The results showed that the expression of the aging marker P21 in dorsal root ganglion neurons of mice receiving fecal microbial gavage and Akkermansia gavage was significantly decreased compared with the control group receiving PBS gavage, indicating that aging was effectively inhibited (e.g., Figure 9-11 As shown in the figure, Akkermansia metabolites are involved in inhibiting dorsal root ganglion neuronal senescence induced by peripheral nerve injury. These data indicate that Akkermansia can inhibit dorsal root ganglion neuronal senescence induced by peripheral nerve injury.

[0051] Example 5 To verify whether AKK can promote nerve regeneration after injury, an experiment was designed to compare the sciatic nerve growth and recovery in mice of the Control and AKK groups. The experiment consisted of two groups of five mice each. Following the method described in Example 1, the sciatic nerve was transected, and a silicone catheter was sutured in place, connecting the two ends of the transected nerve to the catheter to allow the nerve to grow inside. The Control group was fed normally, while the AKK group received normal feeding plus AKK via gavage (intermittent gavage three times a week, as referenced in the literature), at the same dosage as in Example 4. After eight weeks, the silicone catheters were removed from the mice, and immunofluorescence experiments were performed to compare the nerve growth.

[0052] The results are as follows Figure 12As shown in the figure, DAPI staining is used to display the distribution of all cells in the tissue; SCG10 labels newly formed axons. The comparison revealed that axonal growth in the AKK group was significantly higher than that in the control group, indicating that AKK can have a beneficial effect on nerve repair.

[0053] like Figure 13 As shown, Figure 12 The statistical bar chart shows that the SCG10 expression level in the AKK group was significantly higher than that in the Control group. As a key regulatory protein for axonal growth, the significant upregulation of SCG10 expression directly reflects enhanced nerve regeneration capacity. Through quantitative analysis of the regeneration marker SCG10, this invention confirms that AKK intervention can significantly improve the axonal repair capacity of damaged nerve tissue. The experimental group showed a highly significant increase in SCG10 expression (…). (P<0.0001) This not only confirms the extension of regenerated axons in terms of spatial distribution, but also reveals at the molecular level that specific gut microbiota and their metabolites may accelerate the functional repair of peripheral nerves by activating the endogenous regeneration program of neurons and overcoming the inhibitory environment after injury. This signifies that gut microbiota intervention can effectively enhance the ability of neurons to synthesize cytoskeletal components, providing a direct chain of evidence from gut intervention to nerve regeneration for peripheral nerve injury repair. It also lays a solid experimental foundation for developing clinical nerve repair strategies based on probiotics or their metabolites.

[0054] Example 6 The preparation method of the microbial agent provided in this embodiment is as follows: (1) Store in a glycerin preservation tube at -80℃ Akkermansia muciniphila The bacterial strain was removed under aseptic conditions, rapidly thawed at room temperature, and inoculated into a liquid culture medium for activation culture. The culture medium can be suitable for... Akkermansia muciniphila The culture medium for bacterial growth, such as mucin basal medium, is used for anaerobic incubation at 37°C for 12–24 h to allow the bacteria to regain their growth activity and enter the logarithmic growth phase.

[0055] (2) The activated bacterial solution is inoculated into fresh culture medium at a certain inoculation ratio for expansion and fermentation. Preferably, the inoculation amount is 1%~10% (volume percentage), the culture temperature is 30~37℃, and the culture time is 18~36 h. Culture is carried out under anaerobic conditions to allow the bacteria to grow fully and reach a high viable cell concentration. After fermentation, the culture is ready. Akkermansia muciniphila Fermentation broth.

[0056] (3) Centrifuge the obtained fermentation broth at low temperature to collect the cell precipitate. Centrifugation conditions can be 3000~8000 rpm for 5~15 min. After centrifugation, discard the supernatant to obtain the cell precipitate. The obtained cell precipitate can be washed 1~2 times with sterile physiological saline or phosphate buffer to remove residual components of the culture medium.

[0057] (4) After obtaining the bacterial cell precipitate, the bacterial cells are mixed with a cryoprotectant to improve the survival rate of the bacterial cells during the freeze-drying process. The cryoprotectant can be a commonly used freeze-drying cryoprotectant in the art, such as skim milk, trehalose, sucrose, lactose, glycerol, or a combination thereof. Preferably, the mass concentration of the cryoprotectant is 5% to 20%. The bacterial cell precipitate and the cryoprotectant solution are thoroughly mixed to obtain a uniform bacterial cell suspension.

[0058] (5) The bacterial suspension is pretreated, for example, by pre-freezing at -40°C to -80°C for 4 to 12 hours to completely freeze it. Then the pre-frozen sample is placed in a freeze dryer for vacuum freeze-drying. The freeze-drying temperature is generally -40°C to -20°C, the vacuum degree is preferably 10 to 50 Pa, and the drying time is 12 to 36 hours to obtain Lactobacillus freeze-dried bacterial powder.

[0059] (6) After obtaining the freeze-dried bacterial powder, its viable count can be determined by plate counting and formulated according to a certain viable count ratio. For example, freeze-dried bacterial powders of different strains can be mixed according to the viable count ratio to make the final bacterial agent have a viable count concentration of 10. 6 ~10 12 CFU / g. Pharmaceutically acceptable excipients, such as maltodextrin, starch, or microcrystalline cellulose, may also be added to improve the stability and processability of the microbial agent.

[0060] The microbial agent described in this invention can be obtained through the above steps. This microbial agent can be further prepared into various pharmaceutical dosage forms, such as capsules, tablets, granules, or oral powders, to inhibit neuronal cell aging after peripheral nerve injury and promote nerve function recovery.

[0061] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0063] For any points not covered above, existing technologies shall apply.

[0064] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of AKK bacteria in the preparation of drugs that inhibit neuronal cell senescence after peripheral nerve injury, characterized in that, The AKK bacteria is Akkermansia muciniphila .

2. The application according to claim 1, characterized in that, The active ingredient of the drug is selected from live AKK bacteria, inactivated AKK bacteria, AKK bacteria fermentation broth, or AKK bacteria extract.

3. The application according to claim 1 or 2, characterized in that, The drug works by reducing the expression of neuronal cell senescence markers.

4. The application according to claim 3, characterized in that, The aging markers include P16 and P21.

5. The application according to claim 1, characterized in that, The peripheral nerve injury includes at least one of the following: sciatic nerve injury, nerve compression injury, or nerve ischemia-reperfusion injury.

6. The application according to claim 1, characterized in that, The drug is administered via intravenous injection or oral administration.

7. The application according to claim 1, characterized in that, The dosage form of the drug is any one of capsules, tablets, oral preparations, microcapsules, or injections.

8. Use of a pharmaceutical composition comprising the AKK bacterium as described in claim 1 and pharmaceutically acceptable excipients thereto in the preparation of a medicament for inhibiting neuronal cell senescence following peripheral nerve injury.

9. The application according to claim 8, characterized in that, The pharmaceutical composition is in unit dose form, wherein the concentration of AKK bacteria in each unit dose form is not less than 1×10⁻⁶. 6 CFU / mL.

10. The application according to claim 8, characterized in that, The pharmaceutical composition is a bacterial agent, wherein the bacterial agent is prepared by inoculating the AKK bacteria of any one of claims 1-2 into a culture medium and activating and fermenting it sequentially to obtain a fermentation broth; the fermentation broth is centrifuged, mixed with a protectant, and then freeze-dried to obtain AKK bacteria strain powder; the AKK bacteria strain powder is formulated according to the live bacteria count ratio to obtain the bacterial agent.

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