Use of lactobacillus fermentum cect 5716 exopolysaccharide for the preparation of a medicament for the prevention or amelioration of chronic kidney disease

Abstract

The application discloses application of Limosilactobacillus fermentum CET5716 exopolysaccharide (EPS) in preparation of a drug or a functional food for preventing or improving chronic kidney disease induced by adenine, and the Limosilactobacillus fermentum CET5716 exopolysaccharide (EPS) is obtained through centrifugation, protein removal, alcohol precipitation, dialysis, dextran gel separation and purification, and freeze-drying of fermentation liquor of Limosilactobacillus fermentum CECT5716. Animal in-vivo experiments show that the Limosilactobacillus fermentum CET5716 exopolysaccharide can improve chronic kidney disease of mice induced by adenine through gavage.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to Lactobacillus fermentum CECT 5716, in particular, application of Lactobacillus fermentum CECT 5716 exopolysaccharide in preparation of prevention or improvement of chronic kidney disease induced by adenine. BACKGROUND

[0002] Lactic acid bacteria exopolysaccharide is a high molecular weight carbohydrate polymer secreted by lactic acid bacteria outside the cell in the growth and metabolism process. Studies have shown that it not only has physical and chemical properties such as viscosity and water retention, but also has various biological properties such as anti-tumor, antioxidant and immune regulation.

[0003] Different lactic acid bacteria exopolysaccharides have different structures and biological properties, and have strain specificity, so it is most important to select the appropriate strain exopolysaccharide produced by its functional properties for intervention tests. Limosilactobacillus fermentum CECT 5716 is a lactic acid bacteria isolated from human milk. At present, the research on Lactobacillus fermentum CECT 5716 at home and abroad mainly focuses on the research on the probiotic function of the strain, and there is no research report on Lactobacillus fermentum CECT 5716 exopolysaccharide. Therefore, the isolation and preparation of exopolysaccharide and the role of improving CKD and other related researches thereof are summarized in the present application.

[0004] Through retrieval, the following two patent publications related to the present patent application are found:

[0005] 1. Application of Lactobacillus plantarum ZDY2013 in preparation of products for relieving kidney injury (CN112553115A), the Lactobacillus plantarum ZDY2013 is extremely acid and salt tolerant, can maintain a high survival rate in gastrointestinal fluid, and can inhibit the growth of foodborne pathogenic bacteria. In addition, it can also adjust intestinal flora diversity to improve the health of the host gastrointestinal tract. It has the effects of relieving kidney fibrosis, improving inflammatory symptoms, regulating oxidative stress, regulating the expression of inflammatory factors, and effectively preventing and treating high-salt diet-induced kidney injury.

[0006] 2. A composite Lactobacillus plantarum preparation with blood glucose lowering function and its application (CN109136151A), the present application provides a composite bacteria consisting of Lactobacillus plantarum SS18-5 and Lactobacillus plantarum SS18-37. In the composite bacteria, the CFU ratio of Lactobacillus plantarum SS18-5 and Lactobacillus plantarum SS18-37 is 1:(0.01-100). Lactobacillus plantarum SS18-5 has a preservation number of CGMCC No.14917. Lactobacillus plantarum SS18-37 has a preservation number of CGMCC No.14918. The present application also protects the application of the composite bacteria in the preparation of products; the purpose of the products is to treat and / or prevent diabetes. The present application has good blood glucose lowering effect on diabetes, and significantly improves insulin resistance, and has protective effect on heart, liver, kidney, spleen and pancreas. The present application has broad market application prospect.

[0007] By comparison, the present patent application and the above patent disclosure have essential differences. SUMMARY

[0008] The present application aims to overcome the shortcomings of the prior art, and provides an application of Lactobacillus fermentum CECT 5716 exopolysaccharide in preparing a product for preventing or improving chronic kidney disease induced by adenine.

[0009] The technical solution adopted by the present application to solve its technical problems is:

[0010] An application of Lactobacillus fermentum (Limosilactobacillus fermentum) CECT5716 exopolysaccharide in preparing a drug for preventing or improving chronic kidney disease induced by adenine.

[0011] Further, the molecular weight of the Lactobacillus fermentum CECT 5716 exopolysaccharide is about 37000 Da;

[0012] Further, the monosaccharide composition obtained by exopolysaccharide hydrolysis analysis is glucose;

[0013] Further, the structure is analyzed by infrared, methylation and NMR to be composed of t-Glcp-(l→,→4)-Glcp-(1→ and→

[0014] 4,6)-Glcp-(1→) with a relative molar ratio of about 1:7:1.

[0015] Further, it is found by scanning electron microscopy observation that the exopolysaccharide has a porous and highly branched structure.

[0016] Further, the Lactobacillus fermentum CECT 5716 fermentation broth is a Lactobacillus fermentum CECT 5716 strain, which is inoculated into MRS liquid medium at a volume ratio of 1-10:100 at 37°C for 12h anaerobic static culture, and the fermentation broth obtained by expanding culture is used for the next experiment.

[0017] Further, the Lactobacillus fermentum CECT 5716 exopolysaccharide is prepared by the following steps:

[0018] (1) Pretreatment: the Lactobacillus fermentum CECT 5716 fermentation broth is heated at 100°C for 20-40min, and after cooling, centrifuged at 4-10°C and 5000-10000r / min for 10-20min, and the supernatant is taken;

[0019] (2) Isolation of exopolysaccharide: 1-3 times volume of 95% anhydrous ethanol is added to the supernatant obtained in step (1), and after standing overnight at 2-10°C, centrifuged at 4-10°C and 5000-10000r / min for 10-20min, the supernatant is discarded, and the crude polysaccharide precipitate is obtained;

[0020] (3) Purification of exopolysaccharide, i.e. protein removal: the precipitate obtained in step (2) is redissolved in distilled water, and trichloroacetic acid is added to a final concentration of 10% by mass, and placed in an ice bath on a magnetic stirrer for 3-5h, and the supernatant is obtained by centrifugation, and the pH is adjusted to neutral, and 1-3 times volume of 95% anhydrous ethanol is added for re-alcohol precipitation, and after standing overnight at 2-10°C, centrifuged;

[0021] (4) Dialysis: the precipitate obtained in step (3) is redissolved in distilled water, and a dialysis bag with a cut-off of 8000Da is selected for dialysis for 2-3d;

[0022] (5) Freeze-drying: the dialyzed solution is pre-cooled and freeze-dried for at least 12h to obtain Lactobacillus fermentum CECT 5716 exopolysaccharide with a purity of 90-95%.

[0023] Further, the Lactobacillus fermentum CECT 5716 exopolysaccharide with a purity of 90-95% is column-separated (for example, using a Sepharose CL-6B gel column) to obtain Lactobacillus fermentum CECT 5716 exopolysaccharide with a purity of more than 98%.

[0024] Further, the Limosilactobacillus fermentum CECT 5716 fermentation broth is a Limosilactobacillus fermentum CECT 5716 strain picked up from -80 DEG C frozen storage, streaked and activated on MRS solid culture medium plate, and incubated at 37 DEG C for 12 hours under anaerobic culture until recovery of activity; the recovered strain is inoculated into MRS liquid culture medium at a volume ratio of 1-10:100, and expanded culture is carried out to obtain the Limosilactobacillus fermentum CECT 5716 fermentation broth.

[0025] Further, the medicine is prepared by taking the Limosilactobacillus fermentum CECT 5716 exopolysaccharide as a main active ingredient, and forming a medically acceptable dosage form.

[0026] Further, the dosage form includes tablets, powders, granules, capsules or drinks.

[0027] Further, the content of the effective active ingredient of the Limosilactobacillus fermentum CECT 5716 exopolysaccharide in the dosage form is 1-99%.

[0028] Further, the characteristics caused by the chronic kidney disease induced by adenine include weight loss, mental depression, kidney damage, renal interstitial fibrosis, body inflammation, and damaged intestinal mucosal barrier.

[0029] Further, the medicine includes the Limosilactobacillus fermentum CECT 5716 exopolysaccharide as a main active ingredient and a medically acceptable excipient.

[0030] Further, the Limosilactobacillus fermentum CECT 5716 exopolysaccharide can effectively intervene in and improve the chronic kidney disease of mice in animal experiments.

[0031] The advantages and positive effects obtained by the present application are as follows:

[0032] 1. The present application is proved by animal in-vivo experiments that the Limosilactobacillus fermentum CECT 5716 exopolysaccharide can improve the chronic kidney disease of mice induced by adenine, including appearance, weight, mental state, kidney damage, inflammation and intestinal mucosal barrier permeability. The present application finds a new use of the Limosilactobacillus fermentum CECT 5716 exopolysaccharide, especially a new use in improving chronic kidney disease, and provides a new method and new means for high-quality probiotic exopolysaccharide to intervene in and treat chronic kidney disease.

[0033] 2, The application observes and measures the influence of the extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716 as an effective component on the body weight, appearance, kidney organ index, kidney injury, renal interstitial fibrosis, expression of inflammatory factors and intestinal epithelial cell barrier damage of the CKD mouse model by constructing an adenine-induced chronic kidney disease mouse model, and finds that the extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716 can significantly improve the appearance and body weight of mice.

[0034] The above test results prove that the extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716 as an effective component, which is prepared into a medically acceptable dosage form, can significantly improve the growth inhibition, mental debilitation, kidney injury, renal interstitial fibrosis, inflammation and intestinal mucosal permeability caused by adenine-induced chronic kidney disease, and provides a theoretical basis for the clinical research of applying the extracellular polysaccharide of probiotics to intervene and treat chronic kidney disease.

[0035] 3, The extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716 is used in the preparation of a drug for preventing or improving adenine-induced chronic kidney disease, and a drug for preventing or improving chronic kidney disease is provided, which has no side effects and can be used as a safe alternative drug for conventional treatment of chronic kidney disease.

[0036] 4, The application has important significance for studying the extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716 in the auxiliary treatment of kidney injury and the recovery of kidney function. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is the elution curve of the crude product of the extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716 in the application through Sepharose CL-6B chromatographic column;

[0038] Figure 2 It is a scanning electron microscope photo of the extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716 in the application;

[0039] Figure 3 It is a kidney morphology graph after intervention of the extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716 (wherein, Control: blank control group, intragastrically administered with physiological saline; Model: model group, intragastrically administered with adenine suspension; EPS: extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716 group, intragastrically administered with adenine suspension and extracellular polysaccharide of Limosilactobacillus fermentum CECT 5716);

[0040] Figure 4Figure of uremic toxins clearance in serum after intervention of Lactobacillus fermentum CECT 5716 exopolysaccharide in the present application (data is mean ± S.D; compared with blank group, ## p<0.01; compared with model group, **p<0.05; wherein, Control: blank control group, intragastric normal saline; Model: model group, intragastric adenine suspension; Postive: positive drug group, intragastric adenine suspension and positive drug; EPS: Lactobacillus fermentum CECT 5716 exopolysaccharide group, intragastric adenine suspension and Lactobacillus fermentum CECT 5716 exopolysaccharide;

[0041] Figure 5 Figure of kidney section staining after intervention of Lactobacillus fermentum CECT 5716 exopolysaccharide in the present application (wherein, Control: blank control group, intragastric normal saline; Model: model group, intragastric adenine suspension; Postive: positive drug group, intragastric adenine suspension and positive drug; EPS: Lactobacillus fermentum CECT 5716 exopolysaccharide group, intragastric adenine suspension and Lactobacillus fermentum CECT 5716 exopolysaccharide);

[0042] Figure 6 Figure of relative expression level of kidney inflammatory factor IL-1β after intervention of Lactobacillus fermentum CECT 5716 exopolysaccharide in the present application (data is mean ± S.D; compared with blank group, ## p<0.01; wherein, Control: blank control group, intragastric normal saline; Model: model group, intragastric adenine suspension; Postive: positive drug group, intragastric adenine suspension and positive drug; EPS: Lactobacillus fermentum CECT 5716 exopolysaccharide group, intragastric adenine suspension and Lactobacillus fermentum CECT 5716 exopolysaccharide);

[0043] Figure 7 Figure of LPS content in kidney and relative expression level of claudin-1 protein in colon after intervention of Lactobacillus fermentum CECT 5716 exopolysaccharide in the present application (data is mean ± S.D; compared with blank group, ## p<0.01; compared with model group, **p<0.05; wherein, Control: blank control group, intragastric normal saline; Model: model group, intragastric adenine suspension; Postive: positive drug group, intragastric adenine suspension and positive drug; EPS: Lactobacillus fermentum CECT 5716 exopolysaccharide group, intragastric adenine suspension and Lactobacillus fermentum CECT 5716 exopolysaccharide);

[0044] Figure 8Figure 2: Graph of GPX and MDA activity in the kidney after intervention with Lactobacillus fermentum CECT 5716 exopolysaccharide (data is expressed as mean ± S.D; compared to the blank group, ## p<0.01; compared to the model group, **p<0.05; wherein, Control: blank control group, intragastrically administered with physiological saline; Model: model group, intragastrically administered with adenine suspension; Positive: positive drug group, intragastrically administered with adenine suspension and positive drug; EPS: Lactobacillus fermentum CECT 5716 exopolysaccharide group, intragastrically administered with adenine suspension and Lactobacillus fermentum CECT 5716 exopolysaccharide. DETAILED DESCRIPTION

[0045] The present application is further illustrated below with reference to the examples, which are illustrative and not limiting, and cannot be used to limit the scope of protection of the present application.

[0046] The various experimental operations involved in the specific embodiments are all routine techniques in the art, and the parts not specifically annotated herein can be implemented by referring to various commonly used toolbooks, scientific and technical literature or related instructions, manuals, etc. before the filing date of the present application.

[0047] A Lactobacillus fermentum CECT 5716 exopolysaccharide for use in the preparation of a medicament for preventing or improving chronic kidney disease induced by adenine.

[0048] Preferably, the molecular weight of the Lactobacillus fermentum CECT 5716 exopolysaccharide is about 37000 Da;

[0049] Preferably, the monosaccharide composition obtained by hydrolysis analysis of the exopolysaccharide is glucose;

[0050] Preferably, the structure is analyzed by infrared, methylation and NMR to be composed of t-Glcp-(l→,→4)-Glcp-(1→ and→

[0051] 4,6)-Glcp-(1→) with a relative molar ratio of about 1:7:1.

[0052] Preferably, scanning electron microscopy is used to observe that the exopolysaccharide presents a porous and highly branched structure, as shown in Figure 2

[0053] ​More preferably, the Lactobacillus fermentum CECT 5716 fermentation broth is obtained by inoculating the Lactobacillus fermentum CECT 5716 strain in MRS liquid medium at a volume ratio of 1-10: 100, and carrying out expansion culture for 12 h at 37°C under anaerobic static culture.

[0054] More preferably, the Lactobacillus fermentum CECT 5716 fermentation broth is obtained by inoculating the Lactobacillus fermentum CECT 5716 strain in MRS liquid medium at a volume ratio of 3: 100, and carrying out expansion culture for 12 h at 37°C under anaerobic static culture.

[0055] More preferably, the Lactobacillus fermentum CECT 5716 exopolysaccharide is prepared by the following steps:

[0056] (1) Pretreatment: The Lactobacillus fermentum CECT 5716 fermentation broth is heated at 100°C for 20-40 min, and after cooling, centrifuged at 4-10°C and 5000-10000 r / min for 10-20 min, and the supernatant is taken;

[0057] (2) Isolation of exopolysaccharide: 1-3 times the volume of 95% anhydrous ethanol is added to the supernatant obtained in step (1), and after standing overnight at 2-10°C, centrifuged at 4-10°C and 5000-10000 r / min for 10-20 min, the supernatant is discarded, and a crude polysaccharide precipitate is obtained;

[0058] (3) Purification of exopolysaccharide, i.e. protein removal: The precipitate obtained in step (2) is dissolved in distilled water, and trichloroacetic acid is added to a final concentration of 10% by mass, and placed in an ice bath on a magnetic stirrer for 3-5 h, and the supernatant is obtained by centrifugation, and the pH is adjusted to neutral, and 1-3 times the volume of 95% anhydrous ethanol is added for re-alcohol precipitation, and after standing overnight at 2-10°C, centrifugation is carried out;

[0059] (4) Dialysis: The precipitate obtained in step (3) is dissolved in distilled water, and a dialysis bag with a cutoff of 8000 Da is selected for dialysis for 2-3 days;

[0060] (5) Freeze-drying: The dialyzed solution is pre-cooled and freeze-dried for at least 12 h to obtain Lactobacillus fermentum CECT 5716 exopolysaccharide with a purity of 90-95%.

[0061] Preferably, the Lactobacillus fermentum CECT 5716 exopolysaccharide with a purity of 90-95% is column isolated (e.g., using a Sepharose CL-6B gel column) to obtain a Lactobacillus fermentum CECT 5716 exopolysaccharide with a purity of more than 98%.

[0062] Preferably, the drug is prepared in a pharmaceutically acceptable dosage form with the Lactobacillus fermentum CECT 5716 exopolysaccharide as the main active ingredient.

[0063] Preferably, the dosage form includes tablets, powders, granules, capsules or drinks.

[0064] Preferably, the dosage form includes tablets, powders, granules, capsules or drinks.

[0065] Preferably, the characteristics of the chronic kidney disease induced by adenine include weight loss, mental depression, kidney damage, renal interstitial fibrosis, body inflammation, and impaired intestinal mucosal barrier.

[0066] Preferably, the drug includes the Lactobacillus fermentum CECT 5716 exopolysaccharide as the main active ingredient and pharmaceutically acceptable excipients.

[0067] Preferably, the Lactobacillus fermentum CECT 5716 exopolysaccharide effectively intervenes in the treatment and improves chronic kidney disease in mice in animal experiments.

[0068] Preferably, the common uremic toxins are creatinine, urea and uric acid.

[0069] Preferably, the characteristics of the chronic kidney disease include weight loss, mental depression, increased intestinal mucosal permeability, elevated uremic toxins such as creatinine, urea and uric acid in the blood, kidney damage, oxidative stress and renal interstitial fibrosis.

[0070] The Lactobacillus fermentum CECT 5716 used in the present application is Lactobacillus fermentum CECT 5716

[0071] (Limosilactobacillus fermentum CECT 5716), which is originally named Lactobacillus fermentum CECT 5716 (Lactobacillus fermentum CECT 5716).

[0072] Specifically, the related preparation and detection are as follows:

[0073] Example 1: Preparation of Lactobacillus fermentum CECT 5716 exopolysaccharide

[0074] The medium formula is: MRS medium, i.e. beef extract 10 g, yeast extract 5 g, peptone 10 g, glucose 20 g, potassium phosphate dibasic 2 g, sodium acetate anhydrous 5 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, citric acid diammonium 2 g, Tween 80 1 mL, distilled water 1000 mL, and the pH value is adjusted to 6.2.

[0075] (1) A single colony of Lactobacillus fermentum CECT 5716 was inoculated into a seed culture medium and incubated at 37°C for 12 h to obtain an activated seed fermentation broth, and the bacterial content of the seed broth was 5×10 9 CFU / mL.

[0076] (2) Scale-up culture: the seed fermentation broth was inoculated into a fermentation medium at a volume ratio of 3:100, and incubated at 37°C for 16 h to obtain a Lactobacillus fermentum CECT 5716 fermentation broth.

[0077] (3) Enzyme inactivation and removal of bacterial cells: the Lactobacillus fermentum CECT 5716 fermentation broth was boiled at 100°C for 30 min, and after cooling, centrifugation was performed (4°C, 8000 r / min, 20 min, unless otherwise specified, the centrifugation conditions described below are this parameter), and the supernatant was taken.

[0078] (4) Alcohol precipitation: two volumes of anhydrous ethanol were added to the supernatant, which was allowed to stand overnight at 4°C, and then centrifuged to obtain a precipitate.

[0079] (5) Protein removal: the precipitate obtained in step (4) was dissolved in an appropriate amount of distilled water, and trichloroacetic acid was added to a final concentration of 10%, which was stirred in an ice bath for 4 h, centrifuged, and the supernatant was collected and adjusted to neutral pH.

[0080] (6) Re-alcohol precipitation: two volumes of anhydrous ethanol were added to the supernatant, which was allowed to stand overnight at 4°C, and then centrifuged to obtain a precipitate, which was dissolved in water and dialyzed for 3 days using a dialysis bag with a cutoff of 8000 Da. After freeze-drying of the dialysate, a crude extracellular polysaccharide was obtained, with a purity of (92.2±1.8) %.

[0081] Example 2: Purification of extracellular polysaccharide of Lactobacillus fermentum CECT 5716

[0082] The crude extracellular polysaccharide obtained in Example 1 was dissolved in distilled water to prepare a 10 mg / mL solution, and 1 mL of the solution was loaded onto a Sepharose CL-6B gel column for separation, and distilled water was used for elution. The eluate was collected at a rate of 1 mL per tube, and the polysaccharide content was detected by the phenol-sulfuric acid method to draw an elution curve, as shown in Figure 1 The filtrate of the main peak (peak 2) was combined and freeze-dried to obtain purified extracellular polysaccharide, designated as pEPS. The scanning electron microscope image of pEPS is shown in Figure 2The purity of pEPS was up to 98% as measured by phenol-sulfuric acid method.

[0083] Example 3

[0084] The polysaccharide in Example 2 was weighed and dissolved in 1 mL of edible solute to a concentration of 200 mg / kg / d to obtain the sample for preventing or improving chronic kidney disease as described in the present application.

[0085] Example 4

[0086] SPF level 7-week-old BALB / C male mice (body weight about 20-23 g) were used as experimental materials and randomly divided into 3 groups, 10 in each group, and each group was treated as shown below: the blank control group was continuously gavaged with sterile normal saline (mass concentration 0.85%) for 6 weeks; the model group was continuously gavaged with 100 mg / kg of adenine per body weight for 6 weeks; the EPS group was continuously gavaged with 100 mg / kg of adenine per body weight and fed with the sample for preventing or improving chronic kidney disease as described in Example 3 (fed once a day, and the amount of each feeding was 200 mg / kg / d) for 6 weeks. The body weight of the mice was measured once a week, and the appearance and morphology of the mice were observed and recorded according to Table 1. At the end of the experiment, the mice were dissected, and the kidney morphology was observed and weighed.

[0087] Table 1 Scoring method

[0088] Characterization of the phenomenon Healthy Mild (slightly ruffled coat) Moderately ruffled coat, lethargic Severely ruffled coat, thin Very severe Score 4-5 3-4 2-3 1-2 0-1

[0089] The body weight change, appearance and morphology, and kidney morphology and kidney index score of the mice during the experiment are shown in Table 1, and the kidney morphology is shown in Figure 3 The results show that after gavaging with L. fermentum CECT 5716 exopolysaccharide, the body weight of the mice was restored, the erect appearance was alleviated, the mental state was improved, and the kidney edema was reduced, indicating that L. fermentum CECT 5716 exopolysaccharide can significantly improve the growth inhibition, erect appearance, mental depression, and kidney edema caused by chronic kidney disease induced by adenine.

[0090] Example 5

[0091] SPF-grade 7-week-old male BALB / c mice (approximately 20-23g in weight) were randomly divided into three groups of 10 mice each. The treatments for each group were as follows: The blank control group received sterile saline (0.85% by volume) by gavage for 6 weeks; the model group received 100 mg / kg adenine by gavage for 6 weeks at a rate of adenine / body weight; the positive control group received 100 mg / kg adenine by gavage and 200 mg / kg of Haikun Shenxi capsules orally; and the EPS group received 100 mg / kg adenine by gavage and the drug for preventing or improving chronic kidney disease described in Example 3 (200 mg / kg / day, once daily) for 6 weeks. In the last week, the mice were dissected, and serum, kidneys, and colons were collected.

[0092] The levels of creatinine, urea, uric acid, IS, pCS, and TMAO in mouse serum were detected using an enzyme-linked immunosorbent assay (ELISA) kit. The results showed that... Figure 4 Compared with the control group, adenine suspension induction significantly increased serum creatinine, urea, uric acid, IS, pCS, and TMAO levels in CKD model mice, by 345%, 255%, 124%, 122%, 70.84%, and 96.64%, respectively. After treatment with Lactobacillus fermentum CECT 5716 extracellular polysaccharide, serum creatinine, urea, uric acid, IS, pCS, and TMAO levels significantly decreased, by 36.38%, 38.72%, 30.71%, 51.62%, 33.59%, and 46.47%, respectively. This indicates that Lactobacillus fermentum CECT 5716 extracellular polysaccharide can alleviate CKD by degrading creatinine, urea nitrogen, uric acid, IS, pCS, and TMAO.

[0093] HE staining, Masson staining, and caspase-3 immunohistochemical staining were used to observe the degree of renal injury, renal interstitial fibrosis, and renal cell apoptosis, respectively. The results are shown in the table below. Figure 5 . Figure 5 This indicates that, compared with the control group, the model group mice showed significant renal tubular dilation, vacuolation, renal interstitial fibrosis, collagen fiber deposition, and visible caspase-3 accumulation in the renal tubules, glomeruli, and renal interstitium. After treatment with Lactobacillus fermentum CECT 5716 extracellular polysaccharide, the renal fibrosis, renal tubular dilation, and caspase-3 accumulation in the CKD mouse model were significantly improved, indicating that Lactobacillus fermentum CECT 5716 extracellular polysaccharide intervention can reduce kidney damage and alleviate renal interstitial fibrosis and renal cell apoptosis.

[0094] The expression of the inflammatory cytokine IL-1β in kidney tissue was detected by real-time quantitative PCR. Results are shown in [Figure number missing]. Figure 6 . Figure 6The results show that compared with the blank group, the relative expression level of IL-1β in the kidney of the model group of mice is significantly increased, and after the intervention treatment of Lactobacillus fermentum CECT 5716 exopolysaccharide, the relative expression level of IL-1β in the kidney of the CKD mouse model is significantly decreased, which shows that Lactobacillus fermentum CECT 5716 exopolysaccharide can effectively improve the inflammatory response of the kidney caused by the adenine-induced CKD mouse model to prolong the kidney function of the CKD mouse.

[0095] The expression of claudin-1 protein in colon tissue was detected by real-time fluorescent quantitative PCR, and the content of LPS was detected by mouse LPS ELISA enzyme-linked immunosorbent assay kit, and the results are shown in Figure 7 . Figure 7 The results show that compared with the blank group, the relative expression level of claudin-1 protein in the colon of the model group of mice is significantly reduced, and after the intervention treatment of Lactobacillus fermentum CECT5716 exopolysaccharide, the relative expression level of claudin-1 protein in the colon of the CKD mouse is increased, and the content of LPS is reduced, which shows that Lactobacillus fermentum CECT5716 exopolysaccharide can restore the integrity of the intestinal mucosal barrier of the adenine-induced CKD mouse model by increasing the relative expression level of claudin-1 protein to reduce LPS into the blood circulation.

[0096] The activities of GSH-PX and MDA in the kidney of mice were detected by enzyme-linked immunosorbent assay (ELISA) kit, and the results are shown in Figure 8 . Figure 8 The results show that compared with the blank group, the activity of GSH-PX in the kidney of the model group of mice is significantly reduced, and the activity of MDA is significantly increased, and after the intervention treatment of Lactobacillus fermentum CECT5716 exopolysaccharide, the activity of GSH-PX in the kidney of the CKD mouse model is significantly increased, and the activity of MDA is significantly reduced. It is proved that Lactobacillus fermentum CECT 5716 exopolysaccharide can improve the oxidative stress degree of adenine-induced CKD mouse model by regulating the activities of GSH-PX and MDA in the kidney to relieve CKD.

[0097] Conclusion: Through the characterization indexes of mouse appearance, body weight, kidney morphology, kidney index, and physiological and biochemical indexes such as the levels of creatinine, urea, uric acid in serum, the relative expression level of inflammatory factors in kidney, and the permeability of intestinal mucosal barrier, it can be concluded that Lactobacillus fermentum CECT 5716 exopolysaccharide can significantly reduce the adverse effects of adenine-induced chronic kidney disease, such as weight loss, mental debilitation, kidney damage, renal interstitial fibrosis, and inflammatory response, which provides a basis for clinical treatment of kidney damage.

[0098] Although the embodiments of the present application have been disclosed for illustrative purposes, those skilled in the art can understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present application and the appended claims, and thus the scope of the present application is not limited to the disclosed embodiments.

Claims

1. Use of an exopolysaccharide produced by Lactobacillus fermentum (L. fermentum) CECT 5716 in the manufacture of a medicament for preventing or ameliorating chronic kidney disease induced by adenine. Limosilactobacillus fermentum ) CECT 5716 in the manufacture of a medicament for preventing or ameliorating chronic kidney disease induced by adenine. The molecular weight of the exopolysaccharide of the Lactobacillus fermentum CECT 5716 is 37,000 Da, the monosaccharide composition is glucose and the structure consists of t-Glc p -(1→,→4)-Glc p -(1→ and →4,6)-Glc p -(1→) in a relative molar ratio of 1 :7:1 and it was observed by scanning electron microscopy that the exopolysaccharide presents a porous and highly branched structure; The improvement is degradation of creatinine, urea nitrogen, uric acid, IS, pCS and TMAO; The prevention or improvement is alleviation of kidney injury, relief of renal interstitial fibrosis and kidney cell apoptosis; The medicine takes extracellular polysaccharide of Lactobacillus fermentum CECT5716 as the main active ingredient and is prepared into a medically acceptable dosage form.

2. Use according to claim 1, characterized in that: The purity of the extracellular polysaccharide of Lactobacillus fermentum CECT5716 is 90%-95%.

3. Use according to claim 1, characterized in that: The purity of the extracellular polysaccharide of Lactobacillus fermentum CECT5716 is higher than 98%.

4. Use according to claim 1, characterized in that: The dosage form is selected from tablets, powders, granules, capsules or drinks.

5. Use according to claim 4, characterized in that: The mass percentage content of the extracellular polysaccharide of Lactobacillus fermentum CECT5716 in the dosage form is 1%-99%. Alternatively, the medicine comprises the extracellular polysaccharide of Lactobacillus fermentum CECT5716 as the main active ingredient and a medically acceptable adjuvant.

Images