Probiotic composition for improving diabetic kidney diseases as well as preparation method and application of probiotic composition

Abstract

The invention aims to provide a probiotic composition for improving diabetic kidney diseases and a preparation method and application thereof, and belongs to the technical field of biological medicine, compound probiotics are creatively adopted to intervene a DKD animal model, and the regulation effect and molecular mechanism of the compound probiotics on the intestinal-renal axis are discussed. According to the composite probiotics disclosed by the invention, the intestinal flora structure is remodeled, the intestinal lipopolysaccharide level is reduced, intestinal toxin entering blood through a portal vein system is reduced, and finally remote regulation and control on the oxidative stress injury of the kidney are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a probiotic composition for improving diabetic kidney disease, its preparation method, and its application. Background Technology

[0002] Diabetic nephropathy (DKD), as one of the most serious microvascular complications of diabetes and the most common cause of end-stage renal disease, has significant clinical implications. According to international studies, approximately 20% to 40% of diabetic patients also have diabetic nephropathy. In my country, the prevalence of DKD among type 2 diabetes patients is as high as 21.8%. This poses a serious threat to public health and also imposes a heavy economic burden on patients.

[0003] The pathogenesis of diabetes mellitus (DKD) is complex, involving multiple pathophysiological processes such as glucose and lipid metabolism disorders, abnormal renal hemodynamics, oxidative stress, and immune inflammatory responses. Its main clinical feature is a progressive decline in renal function accompanied by persistent, massive proteinuria. Although current treatments can slow disease progression to some extent, they cannot completely prevent the occurrence and development of DKD. Therefore, exploring new treatment methods and elucidating the pathogenesis of DKD has become an important direction in current research on diabetes and its complications.

[0004] In recent years, an increasing number of studies have focused on the relationship between gut microbiota homeostasis and systemic metabolic diseases. The "gut-kidney axis" theory provides a new perspective on the pathogenesis of DKD, revealing that the gut microbiota can regulate and influence the process of kidney inflammation and fibrosis through metabolites. Based on this theoretical framework, mainstream prevention and treatment methods mostly focus on the use of probiotics or prebiotics alone. However, practice has shown that using probiotics or prebiotics alone for the prevention and treatment of metabolic diseases has significant limitations. On the one hand, their preventive and treatment effects are unsatisfactory, making it difficult to achieve the ideal therapeutic expectations; on the other hand, the use of such preparations alone carries a high risk of inducing gastrointestinal diseases, especially with long-term use, which can easily cause gut microbiota dysbiosis and disrupt the gut microecological balance. Therefore, compound probiotic intervention is expected to become an important adjunctive treatment for reversing the pathological process of DKD, and its clinical translational value deserves further exploration. Summary of the Invention

[0005] The purpose of this invention is to provide a probiotic composition for improving diabetic nephropathy, its preparation method, and its application. This invention innovatively uses a compound probiotic to intervene in a DKD animal model to explore its regulatory effect on the gut-kidney axis and its molecular mechanism. This invention reveals that the compound probiotic remotely regulates renal oxidative stress damage by reshaping the intestinal flora structure, reducing the level of enterogenic lipopolysaccharides, and decreasing the entry of enterogenic toxins into the bloodstream via the portal vein system.

[0006] The present invention adopts the following technical solution: A probiotic composition for improving diabetic kidney disease comprises the following components in parts by weight: 10 parts of Lactobacillus rhamnosus lyophilized powder, 10 parts of Bifidobacterium animalis lyophilized powder, 0-10 parts of Lactobacillus rhamnosus GG lyophilized powder, 0-10 parts of Bifidobacterium longum lyophilized powder, 0-10 parts of Lactobacillus helveticus lyophilized powder, and 0-10 parts of Lactobacillus acidophilus lyophilized powder.

[0007] Furthermore, a probiotic composition for improving diabetic kidney disease comprises the following components in parts by weight: 10 parts of Lactobacillus rhamnosus lyophilized powder, 10 parts of Bifidobacterium animalis lyophilized powder, 10 parts of Lactobacillus rhamnosus GG lyophilized powder, 10 parts of Bifidobacterium longum lyophilized powder, 10 parts of Lactobacillus helveticus lyophilized powder, and 10 parts of Lactobacillus acidophilus lyophilized powder.

[0008] A method for preparing a probiotic composition for improving diabetic kidney disease includes the following steps: S1. Preparation of Lactobacillus rhamnosus lyophilized powder: Lactobacillus rhamnosus strain was inoculated into MRS medium and cultured in an anaerobic environment at 37℃ for 48 hours. The bacterial cells were collected by centrifugation at 4000g and 4℃ for 5 minutes. The cells were then washed three times with sterile PBS. The washed bacterial cells were collected, mixed with a lyophilization protectant, and then freeze-dried under vacuum to obtain Lactobacillus rhamnosus lyophilized powder. S2. Preparation of Bifidobacterium animalis lyophilized powder: Bifidobacterium animalis strain was inoculated into MRS medium and cultured under anaerobic conditions at 37℃ for 72 hours; the bacterial cells were collected by centrifugation at 4℃ with a relative centrifugal force of 4000g for 10 minutes using a refrigerated centrifuge, the supernatant was discarded, the bacterial cells were gently resuspended in pre-cooled sterile PBS, and the washing was repeated 3 times. The collected bacterial cells were resuspended in lyophilization protectant, dispensed, and then placed in a vacuum freeze dryer for freeze drying to obtain Bifidobacterium animalis lyophilized powder; S3. Preparation of Lactobacillus rhamnosus GG lyophilized powder: Lactobacillus rhamnosus GG strain was inoculated into MRS medium and cultured in an anaerobic environment at 37℃ for 24 hours. The bacterial cells were collected by centrifugation at 4000g and 4℃ for 10 minutes, the supernatant was discarded, and the bacterial cells were gently resuspended in pre-cooled sterile PBS. The washing was repeated 3 times, the bacterial cells were collected and resuspended in lyophilization protectant, dispensed and freeze-dried to obtain Lactobacillus rhamnosus GG lyophilized powder. S4. Preparation of Bifidobacterium longum lyophilized powder: Bifidobacterium longum strain was inoculated into MRS medium and cultured under anaerobic conditions at 37℃ for 24 hours; the bacterial cells were collected by centrifugation at 4℃ with a relative centrifugal force of 4000g for 10 minutes using a refrigerated centrifuge, the supernatant was discarded, the bacterial cells were gently resuspended in pre-cooled sterile PBS, and the washing was repeated 3 times. The collected bacterial cells were resuspended in a lyophilization protectant, dispensed, and then placed in a vacuum freeze dryer for lyophilization to obtain Bifidobacterium animalis lyophilized powder; S5. Preparation of Lactobacillus helveticus lyophilized powder: Lactobacillus helveticus was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 18 hours. The bacterial cells were collected by centrifugation at 4°C and 4000g for 10 minutes using a refrigerated centrifuge. The bacterial cells were washed three times with sterile PBS buffer. The collected bacterial cells were suspended in a lyophilization protectant, dispensed, and then placed in a freeze dryer for lyophilization to obtain Lactobacillus helveticus lyophilized powder. S6. Preparation of Lactobacillus acidophilus lyophilized powder: Lactobacillus acidophilus was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 48 hours. The bacteria were collected by centrifugation at 4000g and 4°C for 5 minutes, washed three times with sterile PBS, and suspended in lyophilization protectant. Finally, the bacteria were freeze-dried under vacuum to obtain Lactobacillus acidophilus lyophilized powder. S7. Mix Lactobacillus rhamnosus lyophilized powder, Bifidobacterium animalis lyophilized powder, Lactobacillus rhamnosus GG lyophilized powder, Bifidobacterium longum lyophilized powder, Lactobacillus helveticus lyophilized powder and Lactobacillus acidophilus lyophilized powder in a certain proportion to obtain the probiotic composition.

[0009] Further, the MRS medium comprises the following components: 10g casein peptone, 10g beef extract, 5g yeast powder, 5g glucose, 5g sodium acetate, 2g diammonium citrate, 1g Tween 80, 2g K2HPO4, 0.2g MgSO4·7H2O, 0.05g MnSO4·H2O, 20g CaCO3, 15g agar, and 1L distilled water; the pH of the MRS medium is 6.8.

[0010] Furthermore, the freeze-drying protectant described in S1-S6 comprises a mixture of skim milk powder, sucrose, and trehalose in a mass ratio of 2:1:1.

[0011] The use of a probiotic composition in the preparation of a drug to improve diabetic kidney disease.

[0012] The beneficial effects of this invention are as follows: This invention uses a compound probiotic intervention in a DKD animal model to explore its regulatory effect on the gut-kidney axis and its molecular mechanism. This study reveals that the compound probiotic remotely regulates renal oxidative stress damage by reshaping the gut microbiota structure, reducing the level of enterogenic lipopolysaccharides, and decreasing the entry of enterogenic toxins into the bloodstream via the portal venous system.

[0013] This invention discovered the use of 10 9 Different probiotic combinations with CFU / g were used to treat DKD mice for 10 weeks. The results showed that the probiotics were effective in improving kidney damage in DKD mice. Among them, the combination of 6 probiotics had the most significant effect on improving kidney damage in DKD mice. Attached Figure Description

[0014] Figure 1 The effect of different probiotic combinations on fasting blood glucose and body weight in DKD mice is shown in the figure. Figure 2 Figure showing the effect of different probiotic combinations on renal function loss in DKD mice; Figure 3 Figure showing the effects of different probiotic combinations on the morphology of kidney and colon tissues in DKD mice (scale bar is 50 μm). Figure 4 Figure showing the effect of probiotic composition on the gut microbiota of DKD; Figure 5 The figure shows the effect of different probiotic formulations on apoptosis in the kidney tissue of DKD mice. Detailed Implementation

[0015] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0016] The strains of Lactobacillus rhamnosus, Bifidobacterium animalis, Lactobacillus rhamnosus GG, Bifidobacterium longum, Lactobacillus helveticus, and Lactobacillus acidophilus provided in this invention are all laboratory-preserved strains.

[0017] Example 1: Formulation and preparation process of probiotic composition Prepare different probiotic compositions according to the proportions shown in Table 1. All raw materials in the table are by weight (1 kg is 1 part by weight).

[0018] Table 1. Probiotic compositions with different formulations The MRS medium comprises the following components: 10g casein peptone, 10g beef extract, 5g yeast extract, 5g glucose, 5g sodium acetate, 2g diammonium citrate, 1g Tween 80, 2g K2HPO4, 0.2g MgSO4·7H2O, 0.05g MnSO4·H2O, 20g CaCO3, 15g agar, and 1L distilled water; the pH of the MRS medium is 6.8.

[0019] The method for preparing the bacterial powder of the present invention is as follows: (1) Preparation of freeze-dried Lactobacillus rhamnosus powder: Lactobacillus rhamnosus strain was inoculated into MRS medium and cultured in an anaerobic environment at 37°C for 48 hours. The bacterial cells were collected by centrifuging at 4000g and 4°C for 5 minutes using a refrigerated centrifuge. The cells were then washed three times with sterile PBS and freeze-dried under vacuum to obtain freeze-dried Lactobacillus rhamnosus powder. (2) Preparation of Bifidobacterium animalis lyophilized powder: Bifidobacterium animalis strain was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 72 hours; the bacterial cells were collected by centrifugation at 4°C with a relative centrifugal force of 4000g for 10 minutes using a refrigerated centrifuge, the supernatant was discarded, the bacterial cells were gently resuspended with pre-cooled sterile PBS, and the washing was repeated 3 times. The collected bacterial cells were resuspended in lyophilization protectant, dispensed and placed in a vacuum freeze dryer for freeze drying to obtain Bifidobacterium animalis lyophilized powder; (3) Preparation of Lactobacillus rhamnosus GG lyophilized powder: Lactobacillus rhamnosus GG strain was inoculated into MRS medium and cultured in an anaerobic environment at 37℃ for 24 hours. The bacterial cells were collected by centrifugation at 4000g and 4℃ for 10 minutes. The supernatant was discarded, and the bacterial cells were gently resuspended in pre-cooled sterile PBS. The washing was repeated 3 times. The collected bacterial cells were resuspended in lyophilization protectant, dispensed, and then freeze-dried to obtain Lactobacillus rhamnosus GG lyophilized powder. (4) Preparation of Bifidobacterium longum lyophilized powder: Bifidobacterium longum strain was inoculated into MRS medium and cultured under anaerobic conditions at 37℃ for 24 hours; the bacterial cells were collected by centrifugation at 4℃ with a relative centrifugal force of 4000g for 10 minutes using a refrigerated centrifuge, the supernatant was discarded, the bacterial cells were gently resuspended with pre-cooled sterile PBS, and the washing was repeated 3 times. The collected bacterial cells were resuspended in the lyophilization protectant, dispensed and placed in a vacuum freeze dryer for freeze drying to obtain Bifidobacterium animalis lyophilized powder; (5) Preparation of Lactobacillus helveticus lyophilized powder: Lactobacillus helveticus was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 18 hours. The cells were collected by centrifuging at 4°C and 4000g for 10 minutes using a refrigerated centrifuge. The cells were washed three times with sterile PBS buffer. The collected cells were suspended in a lyophilization protectant, dispensed, and then placed in a freeze dryer for lyophilization to obtain Lactobacillus helveticus lyophilized powder. (6) Preparation of Lactobacillus acidophilus lyophilized powder: Lactobacillus acidophilus was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 48 hours. The bacteria were collected by centrifugation at 4000g and 4°C for 5 minutes. The bacteria were washed three times with sterile PBS and suspended in lyophilization protectant. Finally, the bacteria were freeze-dried under vacuum to obtain Lactobacillus acidophilus lyophilized powder. (7) Mix the freeze-dried powder according to the different formulas in Table 1 and set aside.

[0020] Example 2: Establishment of an animal model of diabetic nephropathy (DKD) and experimental treatment protocol (1) 1. Establishment of a mouse model of diabetic nephropathy Fifty four-week-old male C57BL / 6 mice were housed in an SPF-grade environment with a temperature of 21℃-26℃, humidity of 50%-60%, and a light / dark cycle every 12 hours. After one week of acclimatization, the mice were randomly divided into five groups: control group (N group, n=10), model group (D group, n=10), formula A intervention group (DA group, n=10), formula B intervention group (DB group, n=10), and formula C intervention group (DC group, n=10). Throughout the experiment, mice in group N were fed a low-fat control diet, while the other mice were fed a 60% high-fat model diet. After six weeks of continuous housing, insulin resistance was observed in the high-fat diet mice using the insulin tolerance test (ITT) and glucose tolerance test (GTT). After fasting for 12 hours, we administered a low dose of streptozotocin (STZ, 65 mg / kg) to mice with insulin resistance for two consecutive days. We then measured the fasting blood glucose (FBG) of each group of mice. If the two measurements were both higher than 11.1 mmol / L, the mouse model of type 2 diabetes mellitus (T2DM) was considered to have been successfully established.

[0021] (2) Treatment of different formulation groups of probiotic compositions The concentration to be prepared is 1×10 9 After the DKD model was successfully established, the probiotic combination solutions of each formulation group (CFU / mouse) were administered to mice in groups DA, DB, and DC by gavage for 10 weeks according to Table 1. Simultaneously, mice in groups N and D were given corresponding doses of physiological saline. The daily gavage volume for each mouse was 2 mL.

[0022] Example 3: Investigating the effects of different probiotic formulations on the general condition of DKD mice Experimental methods: Throughout the mouse rearing period, body weight and fasting blood glucose were measured weekly. Mice were fasted for 8 hours before blood collection. A special fixture for tail vein blood collection was used to restrain the mice. The tail was wiped with 75% alcohol, and the lancet was inserted into the tail vein. The first drop of blood was discarded, and the second drop was collected using a blood glucose meter, with the blood glucose level recorded. Feed, water, and bedding were changed every two days, and the condition of each group of mice was observed and recorded. The experimental results are shown in Table 2 and... Figure 1 As shown.

[0023] Table 2. Effects of different probiotic formulations on mouse body weight and fasting blood glucose at the end of the experiment. (n=10, *Compared with the DKD group, *P<0.05, **P<0.01, ***P<0.001) like Figure 1 As shown in Figure A, the body weight of mice in group N increased slowly with age, while the body weight of mice in group D gradually decreased. Compared to mice in group D, mice in group DC showed some relief from weight loss symptoms, with a significant difference at the end of the experiment (P < 0.01). The body weight changes of the other formulation groups were somewhat lower than those of group D, but the differences were not statistically significant. Figure 1 As shown in B, we found that the fasting blood glucose of mice in group N remained at a normal level, while the fasting blood glucose of mice in group D continued to rise. Compared with group D, the fasting blood glucose of mice in group DC decreased significantly at week 4, the fasting blood glucose of mice in group DB decreased significantly at week 6, and the fasting blood glucose of mice in group DA only decreased to a certain extent in the later stage of treatment, with no statistical difference.

[0024] Furthermore, in the later stages of feeding, mice in group D exhibited typical symptoms of polydipsia, polyphagia, and polyuria, and their mental state was poor; these symptoms were alleviated in mice in groups DB and DC. This suggests that the combined use of six probiotics can, to some extent, alleviate the general condition of diabetic nephropathy.

[0025] Example 4: Effects of different probiotic formulations on kidney damage in DKD mice Experimental methods: One week before euthanasia, 24-hour urine samples were collected from each group of mice to detect 24-hour proteinuria. At the end of the experiment, mice were anesthetized and their eyeballs were quickly removed. Blood was collected, allowed to stand at room temperature for 3 hours, and then centrifuged at 3000 rpm for 10 minutes at 4°C. Serum was collected and stored at -80°C for subsequent serological testing. After euthanasia, kidney tissue was collected, weighed, and the kidney index was calculated.

[0026] To verify the effects of different probiotic compositions on kidney damage in DKD mice, the kidney index, 24-hour urinary protein (24h-UP), serum creatinine (Scr), and blood urea nitrogen (BUN) levels were compared among the groups of mice. The results are as follows: Figure 2 As shown in Table 3.

[0027] Table 3. Effects of different probiotic formulations on mouse kidneys. (n=10, *Compared with the DKD group, *P<0.05, **P<0.01, ***P<0.001) like Figure 2 As shown in Table 3, compared with group N, all indicators of mice in group D were significantly elevated (P<0.05), indicating kidney damage. However, all indicators of DKD mice treated with different probiotic formulations improved, indicating that the probiotic composition of this invention can improve kidney damage in DKD mice to a certain extent. Furthermore, we found that group DC showed a better effect in improving kidney function (P<0.05).

[0028] Example 5: Effects of different probiotic formulations on the morphology of kidney and colon tissues in DKD mice Experimental methods: Mouse kidney and colon tissues were fixed in 4% paraformaldehyde for 24-48 hours and then dehydrated using an automated dehydrator. The mouse kidney and ileum tissues were then embedded using a semi-automatic embedding machine. The embedded paraffin blocks were then sectioned on a microtome to a thickness of 4 μm and placed on anti-detachment slides to bake at 60°C for 1 hour. The sections were stained with hematoxylin and eosin (HE), observed under a microscope, and photographed.

[0029] The results are as follows Figure 3 As shown, HE staining of the kidneys revealed that the kidney tissue structure of mice in group N was intact, with normal glomerular and tubular structures; mice in group D showed glomerular hypertrophy and thickening of the tubular basement membrane; after probiotic intervention in different formulation groups, kidney lesions in group DC were significantly improved, indicating that the combined use of 5 probiotics can reverse kidney damage caused by diabetes.

[0030] Meanwhile, HE staining of the ileum showed that, compared to group N, group D mice had fewer goblet cells and a relatively increased number of Paneth cells in the colonic mucosa, with more common inflammatory cell infiltration and disordered structural arrangement in the lamina propria. The colonic structure improved after intervention with probiotic compositions in all different formulations (P < 0.001). Interestingly, group DC had more colonic goblet cells than group N.

[0031] Example 6: Effects of probiotic composition on gut microbiota in DKD Experimental methods: One day before euthanasia, feces from each group of mice were collected in a biosafety cabinet, placed in sterile 1.5 mL Eppendorf tubes, and stored at -80°C. The tubes were then transported via a cold chain to Shanghai Zhongke New Life Technology Co., Ltd. for 16S rDNA gene sequencing. The experimental results are shown in Table 4. Figure 4 As shown.

[0032] Table 4. Effects of probiotic compositions in the DC group on the α-diversity of gut microbiota in DKD mice. From Table 4 and Figure 4 It was found that the ACE index, Chao index, and Shannon index of the DC group increased significantly, while the Simpson index decreased. In conclusion, the combined use of six probiotics can significantly increase the α-diversity of gut microbiota in DKD mice, indicating that it can improve gut microbiota.

[0033] Example 7: Effects of different probiotic formulations on apoptosis in the kidney tissue of DKD mice Kidney tissue was homogenized in RIPA lysis buffer containing PMSF. Protein concentration was determined using a BCA protein quantification kit. Equal volumes of protein were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a PVDF membrane using wet transfer. The membrane was blocked with 5% skim milk at room temperature for 1 hour, followed by overnight incubation at 4°C with primary antibodies Bcl2, Bax, Clevered caspase-3, and β-actin. The PVDF membrane was then incubated at room temperature with HRP-labeled goat anti-mouse IgG or HRP-labeled goat anti-rabbit IgG for 1–2 hours. ECL imaging was used to visualize specific bands. Protein levels were quantified using ImageJ analysis software.

[0034] Experimental results: The expression of apoptosis-related markers at the protein level was detected by Western blotting, and the results are as follows: Figure 5 As shown, the expression of the anti-apoptotic gene Bcl-2 was significantly decreased in group D and significantly increased in group DC (P < 0.01); while the expression levels of the pro-apoptotic genes Bax and Cleaved Caspase-3 were significantly increased in group D, and decreased after intervention with different probiotic combinations, especially in group DC. This suggests that the combined use of five probiotics can improve renal apoptosis in DKD mice.

Claims

1. A probiotic composition for improving diabetic kidney disease, characterized in that: The product comprises the following components in parts by weight: 10 parts of Lactobacillus rhamnosus lyophilized powder, 10 parts of Bifidobacterium animalis lyophilized powder, 0-10 parts of Lactobacillus rhamnosus GG lyophilized powder, 0-10 parts of Bifidobacterium longum lyophilized powder, 0-10 parts of Lactobacillus helveticus lyophilized powder, and 0-10 parts of Lactobacillus acidophilus lyophilized powder.

2. The probiotic composition for improving diabetic kidney disease according to claim 1, characterized in that: The product comprises the following components in parts by weight: 10 parts of Lactobacillus rhamnosus lyophilized powder, 10 parts of Bifidobacterium animalis lyophilized powder, 10 parts of Lactobacillus rhamnosus GG lyophilized powder, 10 parts of Bifidobacterium longum lyophilized powder, 10 parts of Lactobacillus helveticus lyophilized powder, and 10 parts of Lactobacillus acidophilus lyophilized powder.

3. A method for preparing a probiotic composition for improving diabetic kidney disease as described in claim 1 or 2, characterized in that: Includes the following steps: S1. Preparation of Lactobacillus rhamnosus lyophilized powder: Lactobacillus rhamnosus strain was inoculated into MRS medium and cultured in an anaerobic environment at 37℃ for 48 hours. The bacterial cells were collected by centrifugation at 4000g and 4℃ for 5 minutes. The cells were then washed three times with sterile PBS. The washed bacterial cells were collected, mixed with a lyophilization protectant, and then freeze-dried under vacuum to obtain Lactobacillus rhamnosus lyophilized powder. S2. Preparation of Bifidobacterium animalis lyophilized powder: Bifidobacterium animalis strain was inoculated into MRS medium and cultured under anaerobic conditions at 37℃ for 72 hours; the bacterial cells were collected by centrifugation at 4℃ with a relative centrifugal force of 4000g for 10 minutes using a refrigerated centrifuge, the supernatant was discarded, the bacterial cells were gently resuspended in pre-cooled sterile PBS, and the washing was repeated 3 times. The collected bacterial cells were resuspended in lyophilization protectant, dispensed, and then placed in a vacuum freeze dryer for freeze drying to obtain Bifidobacterium animalis lyophilized powder; S3. Preparation of Lactobacillus rhamnosus GG lyophilized powder: Lactobacillus rhamnosus GG strain was inoculated into MRS medium and cultured in an anaerobic environment at 37℃ for 24 hours. The bacterial cells were collected by centrifugation at 4000g and 4℃ for 10 minutes, the supernatant was discarded, and the bacterial cells were gently resuspended in pre-cooled sterile PBS. The washing was repeated 3 times, the bacterial cells were collected and resuspended in lyophilization protectant, dispensed and freeze-dried to obtain Lactobacillus rhamnosus GG lyophilized powder. S4. Preparation of Bifidobacterium longum lyophilized powder: Bifidobacterium longum strain was inoculated into MRS medium and cultured under anaerobic conditions at 37℃ for 24 hours; the bacterial cells were collected by centrifugation at 4℃ with a relative centrifugal force of 4000g for 10 minutes using a refrigerated centrifuge, the supernatant was discarded, the bacterial cells were gently resuspended in pre-cooled sterile PBS, and the washing was repeated 3 times. The collected bacterial cells were resuspended in a lyophilization protectant, dispensed, and then placed in a vacuum freeze dryer for lyophilization to obtain Bifidobacterium animalis lyophilized powder; S5. Preparation of Lactobacillus helveticus lyophilized powder: Lactobacillus helveticus was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 18 hours. The bacterial cells were collected by centrifugation at 4°C and 4000g for 10 minutes using a refrigerated centrifuge. The bacterial cells were washed three times with sterile PBS buffer. The collected bacterial cells were suspended in a lyophilization protectant, dispensed, and then placed in a freeze dryer for lyophilization to obtain Lactobacillus helveticus lyophilized powder. S6. Preparation of Lactobacillus acidophilus lyophilized powder: Lactobacillus acidophilus was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 48 hours. The bacteria were collected by centrifugation at 4000g and 4°C for 5 minutes, washed three times with sterile PBS, and suspended in lyophilization protectant. Finally, the bacteria were freeze-dried under vacuum to obtain Lactobacillus acidophilus lyophilized powder. S7. Mix Lactobacillus rhamnosus lyophilized powder, Bifidobacterium animalis lyophilized powder, Lactobacillus rhamnosus GG lyophilized powder, Bifidobacterium longum lyophilized powder, Lactobacillus helveticus lyophilized powder and Lactobacillus acidophilus lyophilized powder in a certain proportion to obtain the probiotic composition.

4. The method for preparing a probiotic composition for improving diabetic kidney disease according to claim 3, characterized in that: The MRS medium comprises the following components: 10g casein peptone, 10g beef extract, 5g yeast extract, 5g glucose, 5g sodium acetate, 2g diammonium citrate, 1g Tween 80, 2g K2HPO4, 0.2g MgSO4·7H2O, 0.05g MnSO4·H2O, 20g CaCO3, 15g agar, and 1L distilled water; the pH of the MRS medium is 6.

8.

5. The method for preparing a probiotic composition for improving diabetic kidney disease according to claim 3, characterized in that: The freeze-drying protectant described in S1-S6 comprises a mixture of skim milk powder, sucrose, and trehalose in a mass ratio of 2:1:

1.

6. The use of the probiotic composition as described in claim 1 in the preparation of a medicament for improving diabetic kidney disease.

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