Microbial agent for relieving IgA nephropathy

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a microbial agent for relieving IgA nephropathy, the microbial agent comprises Bifidobacterium animalis, Lactobacillus paracasei, Lactococcus lactis subspecies, fucose, fructo-oligosaccharide and resistant dextrin. The microbial agent can reduce IgA, C3 and inflammatory indexes deposited in kidneys, so that related indexes of renal functions are improved; meanwhile, the composition also shows an obvious weight reducing effect and can participate in the development mechanism of relieving the IgA nephropathy.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and in particular relates to a microbial agent for alleviating IgA nephropathy and its application. Background Art

[0002] IgA nephropathy (IgAN) is one of the most common primary glomerulonephritis in the world. In my country, the detection rate of primary glomerulonephritis is about 74%, of which IgAN accounts for about 24%. IgAN is characterized by glomerular mesangial IgA deposition, persistent hematuria, and often accompanied by respiratory or gastrointestinal infections. About 25% of patients diagnosed with IgAN develop end-stage renal disease (ESRD) and require renal replacement therapy 20 years later, which has become an important issue facing global public health.

[0003] However, although IgAN poses a serious threat to human health, its exact pathogenesis has not yet been elucidated. Therefore, the most effective clinical intervention measure for the treatment of IgA nephropathy in my country is symptomatic supportive treatment based on renin-angiotensin system inhibitors. However, it is mainly aimed at delaying the progression of kidney disease, and it is difficult to intervene in the ongoing inflammatory-immune response. Most patients will inevitably enter the end stage of kidney disease and rely on blood filtration and other means to survive. In addition, in recent years, budesonide sustained-release capsules and complement inhibitors such as ipracopan have overcome the serious side effects of systemic hormones and have begun to emerge. However, the limitations of the approval of the my country Food and Drug Administration for the clinical treatment of kidney disease and the high price due to various factors such as patent protection make it difficult for the majority of kidney patients to reach it. Therefore, revealing the pathogenesis of IgAN and finding targeted treatment options for the disease remain the top priorities for the diagnosis and treatment of IgAN.

[0004] The "gut-kidney axis" theory believes that intestinal immune response is a triggering factor of IgAN, which closely links the intestinal microecology with kidney disease. Some studies have shown that intestinal homeostasis imbalance is the key to causing or aggravating autoimmune system diseases. Under pathological conditions, intestinal inflammation can cause increased intestinal permeability and impaired intestinal mucosal barrier function, leading to the penetration of sIgA into the systemic circulation and ultimately the deposition of IgA in tissues. It can be seen that in-depth research on the intestinal flora-induced intestinal mucosal barrier damage involved in the pathogenic mechanism of IgAN and the search for new therapeutic targets are of great scientific and clinical significance.

[0005] There is currently evidence that intestinal flora may play a key initiating role in the development of IgAN. Intervention measures that guide intestinal flora as a new protective treatment for IgAN deserve further study. In view of this, this application is proposed. Summary of the invention

[0006] In order to solve the above technical problems, the applicant improves intestinal flora disorders and relieves kidney inflammation by targeted supplementation of intestinal probiotics and their derivative preparations, thereby achieving the purpose of delaying nephropathy. The study selected an IgAN mouse model induced by mir-23b gene knockout, supplemented three intestinal-specific probiotics and postbiotics, fucose and oligofructose derived therefrom, and dietary fiber, and compared the renal function and inflammatory-immune response changes of IgAN mice before and after supplementation of probiotic-related preparations, to explore whether the specific probiotic-related preparations of this application can alleviate the development of IgAN, and provide new ideas for the use of probiotic-related preparations for the prevention and adjuvant treatment of IgAN. Through research, this application finally obtained a microbial agent or synbiotic preparation that can relieve IgA nephropathy.

[0007] This application specifically includes the following technical solutions:

[0008] The present application first provides a microbial agent capable of alleviating IgA nephropathy, wherein the microbial agent comprises Bifidobacterium animalis, Lactobacillus paracasei and Lactococcus lactis;

[0009] Preferably, the microbial agent further comprises fucose, oligofructose and dietary fiber.

[0010] Furthermore, the dietary fiber is resistant dextrin.

[0011] Furthermore, the bacterial activity concentrations of Bifidobacterium animalis, Lactobacillus paracasei and Lactococcus lactis in the microbial agent are 1-4*10 9 cfu / g.

[0012] In some embodiments, the Bifidobacterium animalis is Bifidobacterium animalis SF-B-31, the Lactobacillus paracasei is Lactobacillus paracasei SF-L31, and the Lactococcus lactis is Lactococcus lactis subspecies SF-Q-53.

[0013] Furthermore, the microbial agent further includes postbiotics; the postbiotics include inactivated Bifidobacterium animalis, inactivated Lactobacillus paracasei and inactivated Lactococcus lactis.

[0014] Furthermore, the mass ratio of the components is: animal bifidobacterium: Lactobacillus paracasei: Lactococcus lactis: inactivated animal bifidobacterium: inactivated Lactobacillus paracasei: inactivated Lactococcus lactis: fucose: oligofructose: dietary fiber = (1-2): (1-2): (1-2): (0-1.5): (0-1.5): (0-1.5): (2-2.5): (0.5-1): (0.5-1).

[0015] In some embodiments, the postbiotics are prepared by processing Bifidobacterium animalis, Lactobacillus paracasei and Lactococcus lactis.

[0016] Furthermore, the preparation process of probiotics such as animal Bifidobacterium, Lactobacillus paracasei, Lactococcus lactis, etc. is as follows:

[0017] 1) Inoculation and primary fermentation: After inoculation, each strain was subjected to conventional fermentation;

[0018] 2) Secondary fermentation and drying / embedding:

[0019] The bacterial liquids obtained from the initial fermentation are fermented again, and a protective agent is added to the fermentation product, which is processed in sequence by vacuum freeze crystal drying, covalent cross-linking embedding and microcapsule coating to obtain freeze-dried powder.

[0020] Furthermore, the preparation process of the postbiotics includes:

[0021] After the above-mentioned secondary fermentation and drying / encapsulation, the obtained freeze-dried powder is homogenized after high-pressure wall breaking, and the culture conditions for the secretion of active factors of the inactivated bacteria are precisely controlled. The aseptic spray-drying process is used to seal the active factors of the bacteria in the bacteria. The crushing and packaging processes of the product are strictly controlled by gas control technology to ensure that the microbial indicators of the product meet international standards before being put into storage.

[0022] The present application also provides a method for preparing a microbial agent for alleviating IgA nephropathy, comprising: obtaining freeze-dried powders of animal Bifidobacterium, Lactobacillus paracasei and Lactococcus lactis respectively, mixing them and then adding postbiotics, fucose, oligofructose and dietary fiber.

[0023] The present application also provides the use of the aforementioned microbial agent in alleviating IgA nephropathy.

[0024] The present application also provides the use of the aforementioned microbial agent in preparing a pharmaceutical composition for alleviating IgA nephropathy.

[0025] The present application also provides a method for alleviating IgA nephropathy, comprising the step of administering any of the aforementioned microbial agents to a patient.

[0026] Compared with the prior art, this application has at least the following advantages:

[0027] 1) The present application provides a new microbial preparation or synbiotic preparation, which proves its use in the treatment and relief of IgA nephropathy.

[0028] 2) The present application selects three different probiotics, namely Bifidobacterium animalis, Lactobacillus paracasei, and Lactococcus lactis subspecies, and adds fucose and oligofructose, dietary fiber, and postbiotics derived from the inactivated probiotics to further enhance the probiotics' effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 , the test results and scores of albumin, urea nitrogen and blood creatinine concentrations under different concentration groups in the preliminary test;

[0031] Figure 2 , Pre-experimental PAS pathological results;

[0032] Figure 3 , IgA mouse weight trend results;

[0033] Figure 4 , improvement trend chart of mouse blood creatinine;

[0034] Figure 5 , Improvement trend chart of mouse urea nitrogen table;

[0035] Figure 6 , 24-hour urine microalbumin index trend of mice;

[0036] Figure 7 , the changing trend of TNF-α, an inflammatory index in the blood of mice;

[0037] Figure 8 , the changing trend of inflammatory index Il-1β in mouse blood;

[0038] Fig. 9 , HE staining of mouse kidney;

[0039] Fig.10 , the trend of mouse kidney pathology scores;

[0040] Fig.11 , trend of immunofluorescence IgA scores in mouse kidney.

[0041] Fig.12 , trend of immunofluorescence C3 score in mouse kidney. DETAILED DESCRIPTION

[0042] The technical solution of the present application will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0043] The following terms or definitions are provided only to help understand the present application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0044] Unless otherwise defined below, the meaning of all technical terms and scientific terms used in the specific embodiments of the present application is intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present application.

[0045] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

[0046] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0047] The terms "approximately" and "substantially" in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10% deviation from the indicated value, preferably ±5%.

[0048] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0049] The bacterial composition of the present application

[0050] The present application confirms that a microbial agent comprising Bifidobacterium animalis, Lactobacillus paracasei and Lactococcus lactis has the efficacy of alleviating IgA nephropathy.

[0051] In some embodiments, the bacterial agent also includes fucose, oligofructose and dietary fiber.

[0052] In some specific embodiments, the dietary fiber is resistant dextrin.

[0053] In some specific embodiments, the animal Bifidobacterium herein is selected from animal Bifidobacterium SF-B-31, the Lactobacillus paracasei herein is selected from Lactobacillus paracasei SF-L31, and the Lactococcus lactis herein is selected from Lactococcus lactis subspecies SF-Q-53.

[0054] It can be seen from the data of the specific examples that the bacterial activity concentrations of Bifidobacterium animalis, Lactobacillus paracasei and Lactococcus lactis in the microbial agent of the present application are between 1 and 4*10 9 cfu / g, the effect advantage is significant.

[0055] In some embodiments, the microbial agent of the present application further includes postbiotic components; these postbiotic components include inactivated Bifidobacterium animalis, inactivated Lactobacillus paracasei and inactivated Lactococcus lactis.

[0056] According to the experimental data of the present application, the preferred range of the mass ratio of each group in the bacterial agent of the present application is as follows: Bifidobacterium animalis: Lactobacillus paracasei: Lactococcus lactis: inactivated Bifidobacterium animalis: inactivated Lactobacillus paracasei: inactivated Lactococcus lactis: fucose: oligofructose: dietary fiber = (1-2): (1-2): (1-2): (0-1.5): (0-1.5): (0-1.5): (2-2.5): (0.5-1): (0.5-1). Within this range, good effects are achieved.

[0057] In some more specific embodiments, the preparation process of probiotics such as Bifidobacterium animalis, Lactobacillus paracasei, Lactococcus lactis, etc. includes the following basic steps:

[0058] 1) Inoculation and primary fermentation: After inoculation, each strain was subjected to conventional fermentation;

[0059] 2) Secondary fermentation and freeze drying: The bacterial liquid obtained from the primary fermentation is fermented again, and a protective agent is added to the fermentation product and freeze-dried using vacuum freeze crystal drying technology, covalent cross-linking embedding technology or microencapsulation technology.

[0060] Furthermore, the preparation process of postbiotics may specifically include: after the above-mentioned secondary fermentation and freeze-drying, the obtained freeze-dried powder is homogenized after high-pressure wall breaking, the culture conditions for the secretion of active factors of the inactivated bacteria are precisely controlled, the aseptic spray-drying process is used to seal the active factors of the bacteria in the bacteria, and the crushing and packaging processes of the product are strictly controlled through gas control technology to ensure that the microbial indicators of the product meet the international standards before being put into storage.

[0061] In some embodiments, the basic preparation of the microbial agent for alleviating IgA nephropathy of the present application includes: obtaining freeze-dried powders of Bifidobacterium animalis, Lactobacillus paracasei and Lactococcus lactis respectively, mixing them and then adding postbiotics, fucose, oligofructose and dietary fiber.

[0062] In some embodiments, since the bacterial agent of the present application can be used to alleviate IgA nephropathy, it can be prepared into a pharmaceutical composition for alleviating IgA nephropathy, and the pharmaceutical composition can further contain a pharmaceutically acceptable carrier, etc. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient and / or its salt is chemically and / or physically compatible with other ingredients in the preparation and physiologically compatible with the recipient.

[0063] In other ways, in view of the fact that the microbial agent of the present application can be used to alleviate IgA nephropathy, therefore, the present application also includes a method or strategy for treating IgA nephropathy, which includes the step of administering the aforementioned effective dose of the microbial agent or pharmaceutical composition to the subject or the experimenter. The term "effective dose" or "effective amount" is any amount that protects the experimenter from the onset of the disease or promotes the regression of the disease when used alone or in combination with another drug, and the evidence of the regression of the disease is the reduction in the severity of the disease symptoms, the increase in the frequency and duration of the disease asymptomatic period, or the prevention of damage or disability caused by the disease. The term "subject" or "subject" is a mammal. Mammals include primates (e.g., humans and non-human primates such as monkeys) and rodents (e.g., mice and rats). In certain embodiments, the individual or experimenter is a human. "Subject" can be a patient, and such patients are human subjects in need of treatment, such as individuals suffering from IgA nephropathy.

[0064] The following are specific embodiments, through which the present application is explained.

[0065] Example 1. Screening of microorganisms for bacterial agents

[0066] In previous studies, the applicant confirmed that there is a strong correlation between human genetic background, intestinal microecology and the pathogenesis of IgAN. Through genetic association analysis of 136 genetic polymorphisms that affect the difference in intestinal bacteria composition in 1511 cases of IgAN and 4469 healthy controls, 9 SNPs were found to be associated with the onset of IgAN; among them, the risk type genotype is associated with a decrease in beneficial intestinal bacteria (Dialister / Bacilli) and an increase in harmful bacteria (Erysipelotrichaceae / Lachnobacterium); in IgAN patients, the abundance of bacteria such as Ruminococcus, Lachnospiraceae and Streptococcaeae increased significantly. Combined with the "General Rules for Food Probiotics" promulgated in 2022, this application screens three probiotics that have significantly improved the body's immunity as basic bacteria for the treatment of IgA nephropathy: Bifidobacterium animalis subsp. lactis, Lactobacillus paracasei, and Lactococcus lactis subsp. lactis. Although the relationship between the three bacteria and IgA nephropathy is unclear, based on previous studies and combined with their basic characteristics, the applicant has reason to suspect that the combination of the three bacteria may be used to alleviate IgAN nephropathy.

[0067] Subsequently, the applicant used freeze crystal drying, covalent cross-linking encapsulation and microencapsulation encapsulation technology to treat the three types of bacteria, respectively. While maintaining the probiotics' high-efficiency activity and tolerance to the external environment, it promoted the activity of the bacterial flora to reach the intestine directly, and explored the important role of four groups of freeze-dried powders with different doses / active raw materials in intervening in the occurrence and development of the disease in IgA nephropathy model mice.

[0068] In addition, to ensure the follow-up experiments, the applicant also established the high and low doses of the bacterial agent through preliminary experiments. The applicant learned through analysis that most probiotics used in mouse experiments are in the order of 10 9 CFU / Kg is the most effective. According to the "Regulations on the Application and Review of Probiotic Health Foods", the number of live bacteria in probiotic health foods should not be less than 10 during their shelf life. 6 CFU / Kg, and finally the number of live bacteria in the experiment was initially set at 10 9 CFU / Kg, and set 1-6*10 9 Preliminary experiments were carried out at six different concentration gradients of CFU / Kg, specifically:

[0069] SPF-level IgA nephropathy model mice were selected as the experimental group, with 3 mice in each group. Six concentration gradients were set in the experimental group, namely: 1*10 9 CFU / Kg, 2*10 9 CFU / Kg, 3*10 9 CFU / Kg, 4*10 9 CFU / Kg, 5*10 9 CFU / Kg, 6*10 9 CFU / Kg, and the corresponding probiotic preparations were given by gavage, with each mouse in each group receiving a dosage of 100 mg / Kg / day; at the same time, two control groups were set up, including C57 wild mice and IgA nephropathy model mice, and the dosage was 1 mL of normal saline. The gavage time was 4 weeks, and the frequency was once / every other day. Each group of mice was given regular feed to ensure free and sufficient water intake. After 4 weeks, the 24-hour urine protein of the mice was collected using a sterile metabolic cage, and the 24-hour urine microalbumin concentration was measured. The specific results are shown in Figure 1 c. After that, anesthesia was performed and blood samples were collected by eyeball sampling to test urea nitrogen concentration and serum creatinine concentration. The results are shown in Figure 1 a and 1b.

[0070] The mice were perfused by cardiac perfusion until the kidneys turned white. The abdominal cavity was then fully exposed. The bilateral kidneys were freed. The kidney capsule was gently removed and rinsed with saline. The kidneys were cut longitudinally along the hilum with a blade. Half of the kidneys were placed in 4% paraformaldehyde for dehydration and used for paraffin section preparation. The sections were then mounted and dried in a baking machine for 30 minutes. PAS staining was started according to the instructions of the periodic acid-Schiff (PAS) staining kit. The specific results are shown in the PAS pathology diagram of the pre-experiment. Figure 2 .

[0071] In order to comprehensively evaluate the renal function remission degree of mice, the renal remission score of the pre-experimental mice was scored according to the Oxford pathological staging of IgA nephropathy (MEST-C classification), where the renal pathological remission result was set at 0-3 points (the pathological score was to reduce objective factors, and this experiment set 3 different scorers for evaluation), the serum creatinine remission was set at 0-2 points, the urea nitrogen remission was set at 0-2 points, and the 24-hour urine microalbumin remission was set at 0-3 points. The renal function remission score was summarized after adding up the final data. See the pre-experimental figure. Figure 1 d.

[0072] According to the renal function remission score chart, within a certain range, as the concentration of live bacteria increases, the renal function remission score gradually increases, and the specific range is concentrated in 1-4*10 9CFU / Kg, and when the concentration of live bacteria continued to increase, the renal function of mice gradually showed a downward trend. Therefore, in order to continue to explore the disease-alleviating effect of the microbial agent on IgA nephropathy model mice, this application used 1*10 9 CFU / Kg and 4*10 9 CFU / Kg is differentiated into high and low doses to meet the needs of different populations, and will continue to be explored in formal experiments.

[0073] Example 2: Design and preparation of bacterial agent

[0074] 1. Design of microbial agents

[0075] The applicant obtained Bifidobacterium animalis SF-B-41, Lactobacillus paracasei SF-L-31 (preservation number: CGMCC23492.), and Lactococcus lactis subspecies SF-Q-53 (the above strains were provided by Shandong Sunflower Bioengineering Co., Ltd., and the numbers were provided by the company), and mixed them to prepare bacterial agents. On this basis, further attempts were made to add their derivative preparations, including postbiotic freeze-dried agents of the three bacteria, as well as components such as fucose, oligofructose and dietary fiber.

[0076] Therefore, this application sets up a total of four different groups of bacterial mixtures to meet the needs of intestinal microecology of different bodies. The specific components, activities and dosages are shown in Table 1 below.

[0077] Table 1. Components, activities and dosages of the four groups

[0078]

[0079]

[0080] 2. Production of microbial agents

[0081] 1) Probiotics preparation process:

[0082] Inoculation, fermentation and cultivation: For the three types of probiotics, sterile four-valve group transplanting technology is used to accurately control the fermentation and cultivation conditions, such as fermentation temperature, pressure, speed, stirring degree, pH value, oxygen / carbon dioxide content, strain concentration, etc. This application adopts a complete automatic cleaning and sterilization system, local precise heat exchange and feeding temperature control system, automatic pressure maintenance and pipeline leakage self-inspection device, cooling system, all transplanting pipelines are fully automated, one-button cleaning and sterilization, to solve the contamination risks caused by traditional flame inoculation and negative pressure needle inoculation, to ensure zero error and high purity inoculation.

[0083] Secondary fermentation and drying / encapsulation: In order to ensure the concentration of the bacteria, the bacterial solution obtained from the first fermentation is fermented and concentrated again, and a protective agent is added to prevent it from being damaged by freezing, drying, and thawing during the drying process. It is then dried, emulsified, and embedded. Vacuum freeze-crystallization drying, covalent cross-linking embedding, and microencapsulation are used to further reduce the death of the bacteria and enhance the activity of the strain.

[0084] Crushing, mixing and packaging: Based on the design concept of raw materials and combined with mixing technology, the bacteria are crushed to eliminate the shelf life decay caused by mechanical stress damage. The whole process is carried out in a purification environment with overall 10,000-level plus 100-level protection. The crushing and packaging processes of the products are strictly controlled through gas control technology. Multiple inspections are carried out to ensure that the microbial indicators of the products meet international standards before they are put into storage.

[0085] 2) Postbiotic preparation process:

[0086] Continuing with the probiotic production process steps, after the above-mentioned secondary fermentation and drying / encapsulation, the obtained freeze-dried powder is homogenized after high-pressure wall breaking, and the culture conditions for the secretion of active factors of the inactivated bacteria are precisely controlled. The aseptic spray-drying process is used to seal the active factors of the bacteria in the bacteria, and the gas control technology is used to strictly control the crushing and packaging process of the product. The product is inspected multiple times to ensure that the microbial indicators meet the international standards before being put into storage.

[0087] Example 3: Evaluation of bacterial agents in alleviating IgA nephropathy

[0088] 1. Animal grouping

[0089] 28 SPF-level IgA nephropathy model mice were selected as the experimental group, C57 wild mice as the negative control group, and mir-23b knockout mice as the positive control group. The experimental groups included high probiotics group, low probiotics group, low probiotics group + postbiotics, and high probiotics group + postbiotics. The control group included an equal amount of saline (C57 wild mice) and an equal amount of saline (mir-23b knockout mice). Each group had 7 mice, and the corresponding probiotic-related preparations were gavaged. The specific dosing formula of the experimental group can be found in the table of live bacteria of the four types of freeze-dried powder and raw materials 3-3. The dosage for each mouse in each group was 100 mg / Kg / day, and the composite probiotic freeze-dried powder was dissolved in 1 mL of saline. The dosage for mice in the control group was 1 mL of saline. All drugs were prepared and used immediately. After unsealing, the drugs were placed in a -80°C refrigerator for freezing and storage. The gavage time was 8 weeks, and the frequency was 1 time / every other day. The weight of mice was measured once a week, and the dosage of mice was adjusted at any time according to the changes in their weight. Each group of mice was given regular feed to ensure free and sufficient water intake.

[0090] 2. Specimen Collection

[0091] Before oral administration, a sterile metabolic cage was used to collect urine samples from mice for 24 hours, the urine volume for 24 hours was recorded, and the urinary protein for 24 hours was tested. After oral administration for 8 weeks, the mice were stopped from oral administration, and fasted but not watered for 12 hours. After weighing each mouse, 24-hour urine samples were collected from the mice again, the urine volume for 24 hours was recorded, and the urinary protein for 24 hours was tested. After that, anesthesia was performed (cotton balls soaked in ether and mice were sealed in a glass container), and after deep anesthesia, at least 1 mL of blood samples were collected by eye sampling in a sterilized EP tube, and then placed in a high-speed centrifuge for centrifugation (set The temperature was set at 4°C and the speed was 3000r / min) for 10 minutes, and the supernatant, i.e., serum, was aspirated with a pipette into an EP tube and placed in a -80°C refrigerator for use in the detection of blood creatinine and urea nitrogen; the mice were perfused by the mouse heart perfusion method until the mouse kidneys turned white, and then the mouse abdominal cavity was fully exposed, the bilateral mouse kidneys were freed, the kidney capsule was gently removed and rinsed with physiological saline, and a blade was used to make a longitudinal incision along the renal hilum, half of which was placed in 4% paraformaldehyde for dehydration and used for the preparation of paraffin sections; the other half was placed in a -80°C refrigerator for immunofluorescence.

[0092] 3. Various indicators testing

[0093] Quantitative detection of 24-hour urine protein: Strictly follow the instructions of the 24-hour urine microalbumin ELISA kit (Cat. No. H127-1-2) of Nanjing Jiancheng Bioengineering Co., Ltd.;

[0094] Serum creatinine test: Strictly follow the instructions of the creatinine (CRE) assay kit (Cat. No. C011-2-1) of Nanjing Jiancheng Bioengineering Co., Ltd.;

[0095] Urea nitrogen detection: Strictly follow the instructions of the urea nitrogen (BUN) detection kit (urease method) (Cat. No. C013-2-1) of Nanjing Jiancheng Bioengineering Co., Ltd.;

[0096] Preparation of paraffin sections:

[0097] The kidney specimens fixed overnight with paraformaldehyde solution were taken out and rinsed with circulating tap water for 2 hours; dehydration with different concentrations of alcohol gradient: 75% alcohol, 85% alcohol, 95% alcohol, each dehydration for 15 minutes, 100% anhydrous alcohol dehydration for 15 minutes, and repeated 3 times; xylene (transparent agent) was completely transparent: xylene I was transparent for 15 minutes, xylene (Ⅱ, Ⅲ) was transparent for 30 minutes each; paraffin embedding: the kidney specimens were immersed in 58℃ paraffin, and then embedded in paraffin with an embedding machine. After the paraffin solidified, paraffin blocks of 4cm×2cm were made; sectioning: the embedded kidney specimens were sectioned with a slicer, the section thickness was 3μm, 3 slices were cut for each kidney specimen, and the slides were mounted. After drying, the slices were baked in a baking machine (58℃) for 30 minutes for HE staining.

[0098] PAS staining: Strictly follow the instructions of the Periodic Acid-Schiff (PAS) staining kit (Cat. No. C0124) of Nanjing Jiancheng Bioengineering Co., Ltd.

[0099] 4. Specific experimental results

[0100] 1) Weight:

[0101] Compared with the control group, all IgAN mice showed a trend of weight loss. Compared with the non-administered group, the weight of mice in the drug-treated group also showed a downward trend, and there was a significant difference (p < 0.05). Figure 3 shown.

[0102] 2) Renal function evaluation:

[0103] Compared with the non-administered group, the serum creatinine and urea nitrogen of the mice in the administered group showed an improved trend, among which the low-dose probiotics + postbiotics group had a significant difference compared with the 23b non-administered group, p < 0.05. Figure 4 and Figure 5 .

[0104] 3) Changes in 24-hour urine microalbumin:

[0105] Compared with the non-administered group, the 24-hour urine microalbumin index of the administered group mice showed a significant downward trend, among which the high-dose probiotic group and the high-probiotic group + postbiotic group were significantly different from the 23b non-administered group, p < 0.05. Figure 6 .

[0106] 4) Changes in inflammatory indicators:

[0107] Compared with the non-administered group, the inflammatory indicators (especially TNF-α) in the blood of the treated group mice showed a significant downward trend. Among them, the low-dose probiotic group and the low-probiotic group + postbiotic group were significantly different from the 23b non-administered group (p < 0.05). Figure 7 and Figure 8 .

[0108] 5) Renal pathological changes: Compared with the non-administered group, the pathological scores of the kidneys (cytoplasm and nucleus HE staining) of the mice in the administered group showed a significant downward trend, specifically, the proliferation of glomerular mesangial cells was alleviated. Among them, the low-dose probiotic group and the low-probiotic group + postbiotic group were significantly different from the 23b non-administered group (p < 0.05). For details, see Fig. 9 and Fig.10 .

[0109] 6) Changes in renal immunofluorescence IgA and C3: Compared with the non-administered group, the renal immunofluorescence IgA and C3 scores of the administered group mice showed a significant downward trend, among which the low-dose probiotic group, low-probiotic group + postbiotic group and 23b non-administered group showed significant differences, as shown in detail. Fig.11 and Fig.12 .

[0110] Based on the above index results, it can be concluded that the four groups of composite microbial agents set in this application can reduce the IgA, C3 and inflammatory indicators deposited in the kidneys to varying degrees, and improve the indicators related to renal function; at the same time, this product also shows a significant weight loss effect, which may be involved in alleviating the development mechanism of IgA nephropathy. Among them, the low-dose probiotics + postbiotics group showed obvious advantages compared with other groups, and the comprehensive effects are as follows: low probiotics + postbiotics > low probiotics > high probiotics + postbiotics > high probiotics.

[0111] The foregoing description of the specific exemplary embodiments of the present application is for the purpose of illustration and illustration. These descriptions are not intended to limit the present application to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present application and its practical application, so that those skilled in the art can realize and utilize the various exemplary embodiments of the present application and various selections and changes. The scope of the present application is intended to be limited by the claims and their equivalents.

Claims

1. A microbial agent for alleviating IgA nephropathy, characterized in that: The microbial agents include Bifidobacterium animalis, Lactobacillus paracasei and Lactococcus lactis.

2. The microbial agent according to claim 1, characterized in that The microbial agent also includes fucose, oligofructose and dietary fiber.

3. The microbial agent according to claim 2, characterized in that: The dietary fiber is resistant dextrin.

4. The microbial agent according to any one of claims 1 to 3, characterized in that: The bacterial activity concentrations of Bifidobacterium animalis, Lactobacillus paracasei and Lactococcus lactis in the microbial agent are 1-4*10 9 cfu / g.

5. The microbial agent according to any one of claims 1-2, characterized in that: The microbial agent further includes postbiotics; the postbiotics include inactivated Bifidobacterium animalis, inactivated Lactobacillus paracasei and inactivated Lactococcus lactis.

6. The microbial agent according to any one of claims 1 to 3, characterized in that: The mass ratio of each group is: animal bifidobacterium: Lactobacillus paracasei: Lactococcus lactis: inactivated animal bifidobacterium: inactivated Lactobacillus paracasei: inactivated Lactococcus lactis: fucose: oligofructose: dietary fiber = (1-2): (1-2): (1-2): (0-1.5): (0-1.5): (0-1.5): (2-2.5): (0.5-1): (0.5-1).

7. The microbial agent according to any one of claims 1 to 5, characterized in that: The preparation process of the animal bifidobacterium, Lactobacillus paracasei and Lactococcus lactis is as follows: 1) Inoculation and primary fermentation: After inoculation, each strain was subjected to conventional fermentation; 2) Secondary fermentation and drying / embedding: The bacterial liquids obtained from the initial fermentation are fermented again, a protective agent is added to the fermented product, and vacuum freeze crystal drying, covalent cross-linking embedding and microencapsulation are sequentially processed to obtain freeze-dried powder; Preferably, the preparation process of the postbiotics is as follows: The freeze-dried powder obtained by secondary fermentation and drying / encapsulation is homogenized after high-pressure wall breaking, and the culture conditions for the secretion of active factors of the inactivated bacteria are precisely controlled. The aseptic spray-drying process is adopted to seal the active factors of the bacteria inside the bacteria. The crushing and packaging processes of the product are strictly controlled by gas control technology to ensure that the microbial indicators of the product meet the international standards before being put into storage.

8. The method for preparing the microbial agent according to any one of claims 1 to 7, characterized in that: Freeze-dried powders of Bifidobacterium animalis, Lactobacillus paracasei and Lactococcus lactis were obtained respectively, mixed and then postbiotics, fucose, oligofructose and dietary fiber were added.

9. Use of the microbial agent according to any one of claims 1 to 6 in alleviating IgA nephropathy, or use in preparing a pharmaceutical composition for alleviating IgA nephropathy.

10. A method for alleviating IgA nephropathy, characterized in that: The step of administering the microbial agent according to any one of claims 1 to 6 to a subject.

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