Lactobacillus crispatus capable of improving chronic renal failure and metagen thereof

Abstract

The invention discloses lactobacillus crispatus capable of improving chronic renal failure and a metagen thereof, and belongs to the technical field of microbial application, lactobacillus crispatus WZA-LC10 is separated from human excrement, and the biological preservation number of the lactobacillus crispatus WZA-LC10 is CCTCC (China Center for Type Culture Collection) NO: M 20241764. According to the invention, a strain of lactobacillus crispatus WZA-LC10 is screened out and is combined with a medicinal and edible extract, and the lactobacillus crispatus and the metaplasts thereof can effectively promote the recovery of kidney functions and delay the progress of a disease course by improving intestinal health, enhancing immunity and regulating in-vivo metabolism; by adopting the design of replacing viable bacteria with the postbiotics, the problem of flora imbalance or infection possibly caused by the viable bacteria is avoided, the stability of the product is improved, meanwhile, the postbiotics provide various metabolites, have the effects of oxidation resistance, inflammation resistance, immunoregulation and the like, ensure the safety of long-term use, and are suitable for daily health maintenance and prevention of chronic renal failure.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a Lactobacillus crispatus strain capable of improving chronic renal failure and its postbiotics. Background Art

[0002] Chronic renal failure refers to a disease in which kidney function gradually deteriorates. Its main characteristics are continuous damage to renal tubules, glomeruli, and renal interstitium, ultimately leading to the failure of basic functions such as kidney metabolism and detoxification. The causes of chronic renal failure are complex, and common inducing factors include hypertension, diabetes, long-term medication, etc. The early symptoms of chronic renal failure are often not obvious. As the disease progresses, patients often experience a series of symptoms such as edema, hypertension, anemia, uremia, etc., posing a great threat to the quality of life and life safety of patients. Traditional Western medical treatment methods such as drug treatment, dialysis, and kidney transplantation can, to a certain extent, relieve symptoms, but they cannot cure the cause and have relatively large side effects and economic burdens.

[0003] In recent years, with the in-depth research and practice of traditional Chinese medicine theory, the treatment of chronic renal failure has gradually shifted to a holistic regulation approach that treats both the symptoms and the root causes. Traditional Chinese medicine believes that the fundamental causes of chronic renal failure lie in the accumulation of factors such as weak kidney qi, insufficient kidney yin, and declining kidney yang. Therefore, through traditional Chinese medicine conditioning, especially by means of tonifying the kidney, promoting blood circulation, and inducing diuresis, the process of kidney failure can be delayed to a certain extent, and the quality of life of patients can be improved. Substances with homology of medicine and food, as the basis of traditional Chinese medicine conditioning, have the effects of regulating the body, enhancing immunity, and improving metabolism, and are widely used in the prevention and treatment of kidney diseases. At the same time, probiotics and their metabolites (postbiotics), as a hot topic in modern biotechnology research, have been proven to have significant effects in improving the intestinal microecology, regulating immune function, and reducing chronic inflammation. Research shows that probiotics can improve the metabolic function of the kidneys by regulating the balance of the intestinal flora, and help relieve the immune system disorders and abnormal kidney metabolism caused by chronic kidney disease. In addition, probiotics can protect the kidneys by producing metabolic products such as short-chain fatty acids and lactic acid, and improve the self-repair ability of the kidneys.

[0004] However, the current treatment methods for chronic renal failure still face many challenges. Although traditional Chinese medicine theory provides a variety of herbs for tonifying the kidney, how to combine modern technology to improve its efficacy and reduce side effects remains an urgent problem to be solved. In recent years, the combination of probiotics and their postbiotics with substances homologous to medicine and food has provided an innovative direction for the treatment of chronic renal failure. This innovative treatment plan can not only regulate the intestinal microecological balance and improve metabolic function through probiotics, but also form a therapeutic effect of treating both the inside and the outside by combining the kidney-tonifying and regulating effects of traditional Chinese medicine. This innovative way of combining traditional Chinese medicine theory with modern biotechnology is expected to provide a safer, more effective and sustainable treatment plan for patients with chronic renal failure. Summary of the Invention

[0005] In view of the above-mentioned prior art, the object of the present invention is to provide a Lactobacillus crispatus and its postbiotics that can improve chronic renal failure.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a Lactobacillus crispatus WZA-LC10 is provided. This strain was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: inside Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on August 9, 2024, and its biological deposit number is: CCTCC NO: M20241764.

[0008] In the second aspect of the present invention, a Lactobacillus crispatus postbiotic is provided, and the Lactobacillus crispatus postbiotic uses the fermentation metabolites of the above-mentioned Lactobacillus crispatus as the active ingredient.

[0009] Preferably, the Lactobacillus crispatus postbiotic is prepared by the following method:

[0010] Inoculate the above-mentioned Lactobacillus crispatus into a fermentation medium, carry out fermentation culture to obtain a fermentation broth, inactivate the fermentation broth, centrifuge to obtain the supernatant, and dry it to obtain the Lactobacillus crispatus postbiotic;

[0011] The fermentation medium is based on MRS liquid medium, and 1-3% (v / v) of the extract homologous to medicine and food is added to the basic medium;

[0012] The extract homologous to medicine and food is prepared by the following method:

[0013] Mix wolfberries, tribute chrysanthemums and Chinese yams evenly according to the mass ratio of (3-7):(2-4):(0.5-1), crush them to obtain a mixture, add 10-30 times the mass of water of the mixture, decoct for 1-3 h, cool down, and filter to obtain the extract homologous to medicine and food.

[0014] Preferably, the temperature of fermentation culture is 35 - 37 °C, and the time of fermentation culture is 36 - 48 h.

[0015] Preferably, the inoculation amount of Lactobacillus casei is 1% - 5% of the volume of the fermentation medium.

[0016] In the third aspect of the present invention, there is provided the use of the above-mentioned Lactobacillus crispatus or postbiotics of Lactobacillus crispatus in the preparation of a product for improving chronic renal failure.

[0017] Preferably, the product is a food or a drug.

[0018] The mechanism of action of the medicated and edible homologous substances selected in the present invention:

[0019] Wolfberry fruit:

[0020] Wolfberry fruit is rich in various active ingredients, such as polysaccharides and carotenoids, and has the effects of nourishing the liver and kidney, enhancing immunity, and antioxidation. It can enhance physical strength, improve the kidney's metabolic function, and help slow down the decline of renal function and promote the self-repair of the kidney through its rich nutritional components.

[0021] Chrysanthemum morifolium Ramat. cv. Gongju:

[0022] The flavonoid substances in Chrysanthemum morifolium Ramat. cv. Gongju have significant antioxidant and anti-inflammatory effects, which can help relieve the edema and hypertension caused by chronic renal failure and improve blood circulation; Chrysanthemum morifolium Ramat. cv. Gongju reduces the burden on the kidney and promotes the detoxification function of the kidney through its effects of clearing heat and detoxifying, promoting blood circulation and removing blood stasis, and helps slow down the process of renal failure.

[0023] Chinese yam:

[0024] Chinese yam has the effects of benefiting the kidney and strengthening the spleen. The starch substances in Chinese yam may be converted into prebiotic substances during the fermentation process, further promoting the growth of beneficial intestinal flora, improving intestinal health, and indirectly helping the metabolism of the kidney.

[0025] The beneficial effects of the present invention:

[0026] 1. The present invention has screened out a strain of Lactobacillus crispatus WZA-LC10. By combining it with extracts of medicine and food homologous substances, it creatively combines the concept of traditional Chinese medicine conditioning with modern probiotic technology, providing a new solution for the treatment of chronic renal failure. Lactobacillus crispatus and its postbiotics can effectively promote the recovery of kidney function and delay the progression of the disease course by improving intestinal health, enhancing immunity, and regulating metabolism in the body. The design of using postbiotics instead of live bacteria avoids the problems of possible flora imbalance or infection caused by live bacteria, improving the stability of the product. At the same time, postbiotics provide a variety of metabolites with antioxidant, anti-inflammatory, and immunomodulatory effects, ensuring the safety of long-term use and being suitable for daily health maintenance and the prevention of chronic renal failure.

[0027] 2. The present invention adopts a method of treating both the symptoms and the root causes. By directly acting on the kidneys with traditional Chinese medicine and food homologous substances, it improves metabolic and immune functions. At the same time, postbiotics provide comprehensive conditioning and support for chronic renal failure by regulating the intestinal microecology and enhancing endocrine balance. Detailed implementation mode

[0028] It should be noted that the following detailed description is illustrative and aims to provide further explanation for the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0029] As described in the background art, chronic renal failure is a gradually developing pathological process, usually accompanied by various complications such as edema, hypertension, anemia, and uremia. These symptoms greatly affect the quality of life of patients. Although current treatment methods such as drug treatment, dialysis, and kidney transplantation can relieve symptoms to a certain extent, these methods usually have relatively large side effects, are difficult to cure the cause of the disease, and also have a heavy economic burden. Traditional Chinese medicine provides another treatment idea through overall conditioning and treating both the symptoms and the root causes, emphasizing treatment means such as nourishing the kidney qi and promoting blood circulation to remove blood stasis. However, its curative effect is often relatively slow.

[0030] Based on this, the present invention proposes an innovative solution that combines modern probiotic technology with traditional Chinese medicine and food homologous substances. By screening a probiotic with the potential to improve kidney function - Lactobacillus crispatus WZA-LC10, and combining its fermentation metabolites (postbiotics) with medicine and food homologous substances (such as wolfberry fruits, tribute chrysanthemums, and Chinese yams), through the method of treating both internally and externally, it integrates the advantages of traditional Chinese medicine and modern biotechnology, forming an innovative, efficient, and safe treatment system, which can provide a continuous and effective health management solution for patients with chronic renal failure, breaking through the limitations of existing treatment means and having significant clinical application value.

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0032] The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0033] Example 1: Isolation and identification of strains:

[0034] The Lactobacillus crispatus WZA-LC10 strain of the present invention was isolated from the feces of healthy adults.

[0035] 1. Isolation of strains:

[0036] After pretreatment of the fecal sample, it was stored in a -80°C refrigerator in 30% glycerol. After taking it out and thawing, the sample was mixed well, 0.5 g of the sample was taken and added to 4.5 mL, and gradient dilution was carried out with 0.9% physiological saline. 100 μL of the bacterial liquid with a suitable dilution gradient was applied to a culture dish containing sterile MRS solid medium and cultured at 37°C for 48 h. The typical colonies of Lactobacillus crispatus were picked and streaked and purified on the MRS solid medium multiple times until the colony morphology on the entire plate was consistent. Single colonies were picked and transferred to MRS liquid medium for enrichment and stored frozen at -80°C in 30% glycerol to obtain strain WZA-LC10.

[0037] 2. Strain identification:

[0038] The WZA-LC10 strain was sent to the Henan Engineering Technology Research Center for Industrial Microbial Strains for 16S rDNA gene sequence identification. The identification results showed that the strain belongs to Lactobacillus crispatus. And the strain was deposited, and the deposit information is as follows:

[0039] Strain name: Lactobacillus crispatus WZA-LC10

[0040] Latin name: Lactobacillus crispatus

[0041] Depositary institution: China Center for Type Culture Collection

[0042] Abbreviation of depositary institution: CCTCC

[0043] Address: Inside Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province

[0044] Deposit date: August 9, 2024

[0045] Preservation number: CCTCC NO: M 20241764.

[0046] Example 2: Preparation of Lactobacillus crispatus WZA-LC10 bacterial agent:

[0047] Inoculate Lactobacillus crispatus WZA-LC10 into MRS solid medium for activation. After continuous activation for two generations, the activated Lactobacillus crispatus is obtained. The activated Lactobacillus crispatus WZA-LC10 is inoculated into MRS liquid medium at an inoculation amount of 2% (volume fraction) and cultured at 37 °C for 24 h to obtain a bacterial solution. The bacterial solution is centrifuged at 8000 g for 10 min, the supernatant is discarded, the precipitated bacteria are collected, and the bacteria are resuspended with sterile physiological saline (0.85% NaCl), and the concentration of the bacterial suspension is adjusted to 1×10 8 cfu / mL.

[0048] Example 3: Preparation of postbiotics of Lactobacillus crispatus:

[0049] (1) Preparation of fermentation medium:

[0050] Add 1% (volume fraction) of traditional Chinese medicine extract to MRS liquid medium to prepare the fermentation medium;

[0051] The medicine and food homology extract is prepared by the following method:

[0052] Mix wolfberry fruits, chrysanthemums and Chinese yams evenly according to the mass ratio of 3:2:0.5, crush them to obtain a mixed material, add 20 times the mass of water of the mixed material, decoct for 2 h, cool down, filter, and obtain the medicine and food homology extract;

[0053] (2) Inoculate the activated Lactobacillus crispatus seed liquid (the viable bacteria count is 1×10 8 cfu / mL) into the fermentation medium at an inoculation amount of 1% (volume fraction) and perform fermentation culture at 37 °C for 48 h to obtain a fermentation broth;

[0054] (3) Treat the fermentation broth at 110 °C for 30 min, centrifuge to take the supernatant, and dry to obtain postbiotics of Lactobacillus crispatus.

[0055] Example 4: Preparation of postbiotics of Lactobacillus crispatus:

[0056] (1) Preparation of fermentation medium:

[0057] Add 2% (volume fraction) of traditional Chinese medicine extract to MRS liquid medium to prepare the fermentation medium;

[0058] The medicine and food homology extract is prepared by the following method:

[0059] Mix wolfberries, chrysanthemums, and Chinese yams evenly according to a mass ratio of 5:3:0.75, pulverize to obtain a mixed material, add water 20 times the mass of the mixed material, decoct for 2 h, cool down, and filter to obtain a homologous extract of medicine and food;

[0060] (2) Inoculate the activated Lactobacillus crispatus seed liquid (the viable cell count is 1×10 8 cfu / mL) into the fermentation medium at an inoculation amount of 3% (volume fraction), and perform fermentation culture at 37 °C for 48 h to obtain a fermentation broth;

[0061] (3) Treat the fermentation broth at 110 °C for 30 min, centrifuge to obtain the supernatant, and dry to obtain Lactobacillus crispatus postbiotics.

[0062] Example 5: Preparation of Lactobacillus crispatus postbiotics:

[0063] (1) Preparation of the fermentation medium:

[0064] Add the Chinese medicine extract with a volume fraction of 3% to the MRS liquid medium to prepare the fermentation medium;

[0065] The homologous extract of medicine and food is prepared by the following method:

[0066] Mix wolfberries, chrysanthemums, and Chinese yams evenly according to a mass ratio of 7:4:1, pulverize to obtain a mixed material, add water 20 times the mass of the mixed material, decoct for 2 h, cool down, and filter to obtain a homologous extract of medicine and food;

[0067] (2) Inoculate the activated Lactobacillus crispatus seed liquid (the viable cell count is 1×10 8 cfu / mL) into the fermentation medium at an inoculation amount of 5% (volume fraction), and perform fermentation culture at 37 °C for 48 h to obtain a fermentation broth;

[0068] (3) Treat the fermentation broth at 110 °C for 30 min, centrifuge to obtain the supernatant, and dry to obtain Lactobacillus crispatus postbiotics.

[0069] Comparative Example 1:

[0070] (1) Preparation of the fermentation medium:

[0071] Add the Chinese medicine extract with a volume fraction of 1% to the MRS liquid medium to prepare the fermentation medium;

[0072] The homologous extract of medicine and food is prepared by the following method:

[0073] Pulverize wolfberries to obtain a mixed material, add water 20 times the mass of the mixed material, decoct for 2 h, cool down, and filter to obtain a homologous extract of medicine and food;

[0074] (2) Inoculate the activated Lactobacillus crispatus seed solution (with a viable count of 1×10 8 cfu / mL) into the fermentation medium at an inoculation amount of 1% (volume fraction), and perform fermentation culture at 37°C for 48 h to obtain a fermentation broth;

[0075] (3) Treat the fermentation broth at 110°C for 30 min, centrifuge to obtain the supernatant, and dry it to obtain Lactobacillus crispatus postbiotics.

[0076] Comparative Example 2:

[0077] (1) Preparation of the fermentation medium:

[0078] Add the traditional Chinese medicine extract at a volume fraction of 1% to the MRS liquid medium to prepare the fermentation medium;

[0079] The medicine and food homologous extract is prepared by the following method:

[0080] Crush Gongju to obtain a mixture, add water 20 times the mass of the mixture, decoct for 2 h, cool down, and filter to obtain the medicine and food homologous extract;

[0081] (2) Inoculate the activated Lactobacillus crispatus seed solution (with a viable count of 1×10 8 cfu / mL) into the fermentation medium at an inoculation amount of 1% (volume fraction), and perform fermentation culture at 37°C for 48 h to obtain a fermentation broth;

[0082] (3) Treat the fermentation broth at 110°C for 30 min, centrifuge to obtain the supernatant, and dry it to obtain Lactobacillus crispatus postbiotics.

[0083] Comparative Example 3:

[0084] (1) Preparation of the fermentation medium:

[0085] Add the traditional Chinese medicine extract at a volume fraction of 1% to the MRS liquid medium to prepare the fermentation medium;

[0086] The medicine and food homologous extract is prepared by the following method:

[0087] Crush Chinese yam to obtain a mixture, add water 20 times the mass of the mixture, decoct for 2 h, cool down, and filter to obtain the medicine and food homologous extract;

[0088] (2) Inoculate the activated Lactobacillus crispatus seed solution (with a viable count of 1×10 8 cfu / mL) into the fermentation medium at an inoculation amount of 1% (volume fraction), and perform fermentation culture at 37°C for 48 h to obtain a fermentation broth;

[0089] (3) Treat the fermentation broth at 110 °C for 30 min, centrifuge to obtain the supernatant, and dry it to obtain the postbiotic of Lactobacillus crispatus.

[0090] Comparative Example 4:

[0091] (1) Inoculate the activated Lactobacillus crispatus seed liquid (the viable cell count is 1×10 8 cfu / mL) into MRS liquid medium at an inoculation amount of 1% (volume fraction), and perform fermentation culture at 37 °C for 48 h to obtain the fermentation broth;

[0092] (2) Treat the fermentation broth at 110 °C for 30 min, centrifuge to obtain the supernatant, and dry it to obtain the postbiotic of Lactobacillus crispatus.

[0093] Experimental Example 1: Effects of Lactobacillus crispatus and its postbiotics on rats with chronic renal failure model:

[0094] 1. Construction of rats with chronic renal failure model:

[0095] Refer to the method 1.4 in the literature "[1] Li Xiuwen, Li Dali. Protective effect of luteolin on rats with chronic renal failure [J]. Guiding Journal of Traditional Chinese Medicine, 2021" to construct rats with chronic renal failure model:

[0096] Prepare 2.5% adenine suspension, and gavage SPF-grade rats (200 - 250 g) at a dose of 200 mg / (kg·d) for 30 consecutive days to establish a rat chronic renal failure model.

[0097] 2. Grouping and administration:

[0098] Randomly divide the rats that meet the chronic renal failure model into 6 treatment groups and 1 model group, with 10 rats in each group.

[0099] Start administering drugs on the 1st day after the end of modeling. The drug administration situations of each treatment group are as follows:

[0100] Treatment group 1: Gavage the Lactobacillus crispatus WZA-LC10 bacterial agent prepared in Example 2 at 50 mg / Kg of rats;

[0101] Treatment group 2: Gavage the postbiotic of Lactobacillus crispatus WZA-LC10 prepared in Example 3 at 50 mg / Kg of rats;

[0102] Treatment group 3: Gavage the postbiotic of Lactobacillus crispatus WZA-LC10 prepared in Comparative Example 1 at 50 mg / Kg of rats;

[0103] Treatment group 4: Gavage the postbiotic of Lactobacillus crispatus WZA-LC10 prepared in Comparative Example 2 at 50 mg / Kg of rats;

[0104] Treatment group 5: Intragastrically administered Lactobacillus crispatus WZA-LC10 postbiotics prepared in Comparative Example 3 to rats at a dose of 50 mg / Kg;

[0105] Treatment group 6: Intragastrically administered Lactobacillus crispatus WZA-LC10 postbiotics prepared in Comparative Example 4 to rats at a dose of 50 mg / Kg;

[0106] Model group: Intragastrically administered an equal volume of normal saline;

[0107] Another 10 unmodeled rats were used as the normal control group and intragastrically administered an equal volume of normal saline.

[0108] Each treatment group was medicated once a day for 30 consecutive days.

[0109] 3. Test indicators:

[0110] At the end of the 4th week of treatment, after fasting for 24 h, blood was collected from the abdominal aorta, and ELISA was used to detect the contents of IL-6, TNF-α, serum creatinine (Scr), and blood urea nitrogen (BUN) in the rat serum.

[0111] 4. Test results:

[0112] Table 1 Contents of IL-6, TNF-α, Scr, and BUN in the sera of rats with different treatments

[0113]

[0114]

[0115] As can be seen from Table 1, compared with the model group, the contents of IL-6, TNF-α, BUN, and Scr in the sera of rats intragastrically administered with Lactobacillus crispatus WZA-LC10 bacterium agent and postbiotics of Lactobacillus crispatus with different treatments were significantly decreased. Among them, the differences in the contents of IL-6, TNF-α, BUN, and Scr in the sera of rats intragastrically administered with the postbiotics of Lactobacillus crispatus prepared in Example 3 and the normal control group were the smallest, indicating that the combination of the postbiotics of Lactobacillus crispatus and the medicine and food homologous extract in this application has a significant curative effect on the treatment of rats with chronic renal failure model.

[0116] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A strain of Lactobacillus crispatus WZA-LC10, characterized in that: Its biological deposit number is: CCTCC NO: M 20241764.

2. A Lactobacillus crispatus postbiotic, characterized in that: The Lactobacillus crispatus postbiotics uses the fermentation metabolites of the Lactobacillus crispatus according to claim 1 as active ingredients.

3. The Lactobacillus crispatus postbiotic according to claim 2, characterized in that The Lactobacillus crispatus postbiotic is prepared by the following method: The Lactobacillus crispatus of claim 1 is inoculated into a fermentation medium for fermentation culture to obtain a fermentation broth, the fermentation broth is inactivated, the supernatant is taken out by centrifugation, and the supernatant is dried to obtain a Lactobacillus crispatus postbiotic; The fermentation medium uses MRS liquid medium as the basic medium, and adds 1-3% of the medicinal and edible extract into the basic medium; The medicinal and edible extract is prepared by the following method: Evenly mix wolfberry, tribute chrysanthemum and yam in a mass ratio of (3-7): (2-4): (0.5-1), grind to obtain a mixture, add water 10-30 times the mass of the mixture, boil for 1-3 hours, cool, filter and obtain a medicinal and edible extract.

4. The Lactobacillus crispatus postbiotic according to claim 3, characterized in that The fermentation temperature is 35-37°C, and the fermentation time is 36-48h.

5. The Lactobacillus crispatus postbiotic according to claim 3, characterized in that The inoculation amount of Lactobacillus casei is 1%-5% of the volume of the fermentation medium.

6. Use of the Lactobacillus crispatus according to claim 1 or the Lactobacillus crispatus postbiotic according to any one of claims 2 to 5 in the preparation of a product for improving chronic renal failure 1.

7. The use according to claim 6, characterized in that: The product is a food or a medicine.

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