CN115887483A - Application of lactobacillus fermentum CECT5716 exopolysaccharide in preparation of medicines for preventing or improving chronic kidney diseases - Google Patents
Application of lactobacillus fermentum CECT5716 exopolysaccharide in preparation of medicines for preventing or improving chronic kidney diseases Download PDFInfo
- Publication number
- CN115887483A CN115887483A CN202211198927.1A CN202211198927A CN115887483A CN 115887483 A CN115887483 A CN 115887483A CN 202211198927 A CN202211198927 A CN 202211198927A CN 115887483 A CN115887483 A CN 115887483A
- Authority
- CN
- China
- Prior art keywords
- exopolysaccharide
- lactobacillus fermentum
- cect5716
- fermentum cect5716
- use according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an application of lactobacillus fermentum (EPS) CET5716 exopolysaccharide in preparing medicines or functional foods for preventing or improving adenine-induced chronic kidney diseases, wherein the lactobacillus fermentum CECT5716 Exopolysaccharide (EPS) is obtained by performing operations such as centrifugation, protein removal, alcohol precipitation, dialysis, glucan gel separation and purification, freeze-drying and the like on lactobacillus fermentum CECT5716 fermentation liquor. Animal in vivo experiments show that mice chronic kidney diseases induced by adenine can be improved by the lactobacillus gasseri CET5716 exopolysaccharide.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to lactobacillus fermentum CECT5716, and particularly relates to application of lactobacillus fermentum CECT5716 exopolysaccharide in preparation of medicines for preventing or improving adenine-induced chronic kidney diseases.
Background
Exopolysaccharides of lactic acid bacteria are high molecular weight carbohydrate polymers secreted outside of the cells by lactic acid bacteria during growth metabolism. Research shows that the chitosan hydrogel not only has physicochemical properties of viscosity increasing, water retention and the like, but also has various biological properties of tumor resistance, oxidation resistance, immunoregulation and the like.
The extracellular polysaccharide of different lactic acid bacteria has different structures, different biological characteristics and strain specificity, so that the extracellular polysaccharide produced by a proper strain is selected for intervention test through the functional characteristics of the extracellular polysaccharide. The invention discloses a Lactobacillus fermentum CECT5716 which is a lactic acid bacterium separated from healthy human milk, at present, the domestic and foreign research on the Lactobacillus fermentum CECT5716 mainly focuses on the research on the probiotic function of the strain, and no research report on extracellular polysaccharide of the Lactobacillus fermentum CECT5716 is found, so that the invention summarizes the relevant research on the separation and preparation of the extracellular polysaccharide, the function of improving CKD and the like.
Through searching, the following two patent publications relevant to the patent application of the invention are found:
1. the lactobacillus plantarum ZDY2013 is applied to preparation of products for relieving kidney injury (CN 112553115A), and the lactobacillus plantarum ZDY2013 is extremely resistant to acid and bile salts, can keep higher survival rate in gastrointestinal fluids, and can inhibit growth of food-borne pathogenic bacteria. In addition, the intestinal flora diversity can be adjusted, so that the gastrointestinal health of a host can be improved. Has the effects of relieving renal fibrosis, improving inflammation appearance symptoms, regulating oxidative stress, regulating and controlling the expression of inflammatory factors and achieving the effect of effectively preventing and treating high-salt diet-induced renal injury.
2. The invention discloses a compound lactobacillus plantarum preparation with a function of reducing blood sugar and application thereof (CN 109136151A), and provides a compound bacterium which consists of lactobacillus plantarum SS18-5 and lactobacillus plantarum SS 18-37. In the compound bacteria, the CFU ratio of the lactobacillus plantarum SS18-5 to the lactobacillus plantarum SS18-37 is 1 (0.01-100). Lactobacillus plantarum SS18-5 with the preservation number of CGMCC No.14917. Lactobacillus plantarum SS18-37 with the preservation number of CGMCC No.14918. The invention also protects the application of the compound bacteria in preparing products; the product is used for treating and/or preventing diabetes. The invention has good effect of reducing blood sugar for diabetes, obviously improves insulin resistance and has protective efficacy for heart, liver, kidney, spleen and pancreas. The invention has wide market application prospect.
By contrast, the present patent application is intrinsically different from the above patent publications.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides application of lactobacillus fermentum CECT5716 exopolysaccharide in preparation of medicines for preventing or improving adenine-induced chronic kidney diseases.
The technical scheme adopted by the invention for solving the technical problem is as follows:
application of Lactobacillus fermentum (Lactobacillus fermentum) CECT5716 exopolysaccharide in preparing medicine or functional food for preventing or improving adenine-induced chronic kidney disease is provided.
Further, the molecular weight of the lactobacillus fermentum CECT5716 exopolysaccharide is about 37000Da;
further, hydrolysis analysis of extracellular polysaccharide is carried out to obtain monosaccharide which is glucose;
further, its structure was analyzed by infrared, methylation and NMR to consist of t-Glcp- (l →, → 4) -Glcp- (1 → and → 4, 6) -Glcp- (1 →), and the relative molar ratio was about 1.
Further, the extracellular polysaccharide is observed by a scanning electron microscope to show a porous and highly branched structure.
Further, the lactobacillus fermentum CECT5716 fermentation broth is a strain of Lactobacillus fermentum CECT5716, is subjected to static anaerobic culture at MRS 37 ℃ for 12 hours, and is prepared by mixing the following components in a volume ratio of 1-10: inoculating 100 inoculum sizes into MRS liquid culture medium, performing amplification culture to obtain fermentation broth, and performing next experiment.
Further, the lactobacillus fermentum CECT5716 exopolysaccharide is prepared by the following steps:
(1) Pretreatment: heating fermentation broth of Lactobacillus fermentum CECT5716 at 100 deg.C for 20-40min, cooling, centrifuging at 4-10 deg.C and 5000-10000r/min for 10-20min, and collecting supernatant;
(2) And (3) separating the exopolysaccharide: adding 1-3 times volume of 95% absolute ethyl alcohol into the supernatant obtained in the step (1), standing at 2-10 ℃ overnight, centrifuging at 4-10 ℃ at 5000-10000r/min for 10-20min, and removing the supernatant to obtain crude polysaccharide precipitate;
(3) Purification of exopolysaccharide namely protein removal: re-dissolving the precipitate obtained in the step (2) in distilled water, adding trichloroacetic acid to ensure that the final mass concentration of the trichloroacetic acid is 10%, placing the trichloroacetic acid on a magnetic stirrer for ice bath for 3-5h, centrifuging to obtain supernatant, adjusting the pH of the supernatant to be neutral, adding 1-3 times of 95% absolute ethyl alcohol by volume to perform re-ethanol precipitation, standing at 2-10 ℃ overnight, and centrifuging;
(4) And (3) dialysis: re-dissolving the precipitate obtained in the step (3) in distilled water, and dialyzing for 2-3d by selecting a dialysis bag with the interception amount of 8000 Da;
(5) And (3) freeze drying: pre-freezing the dialyzed solution, and freeze-drying for at least 12h to obtain the lactobacillus fermentum CECT5716 exopolysaccharide with the purity of 90-95%.
Further, lactobacillus fermentum CECT5716 exopolysaccharide with purity of 90-95% is separated by column (for example, sepharose CL-6B gel column), and lactobacillus fermentum CECT5716 exopolysaccharide with EPS purity higher than 98% is obtained.
Further, the fermentation broth of the lactobacillus fermentum CECT5716 is prepared by selecting a strain of limolactibacillus fermentum CECT5716 frozen at-80 ℃, streaking and activating the strain on an MRS solid culture medium flat plate, and standing and anaerobically culturing the strain at 37 ℃ for 12 hours until the activity is recovered; and (3) mixing the strains with the ratio of 1-10: inoculating the strain with the volume ratio of 100 into an MRS liquid culture medium, and performing amplification culture.
Furthermore, the medicine or functional food takes lactobacillus fermentum CECT5716 exopolysaccharide as a main active ingredient and is prepared into a pharmaceutically acceptable dosage form and a food acceptable dosage form.
Further, the dosage form includes tablets, powders, granules, capsules or drinks.
Furthermore, the content of the effective active ingredients of the lactobacillus fermentum CECT5716 exopolysaccharide in the dosage form is 1-99% by mass.
Further, the characteristics of adenine-induced chronic kidney disease include weight loss, listlessness, kidney damage, renal interstitial fibrosis, body inflammatory reaction, and impaired intestinal mucosal barrier.
Further, the medicine or functional food comprises lactobacillus fermentum CECT5716 exopolysaccharide as a main active ingredient and auxiliary materials acceptable in medicine or food.
Further, the lactobacillus fermentum CECT5716 exopolysaccharide is effective in intervening treatment and improvement of chronic kidney diseases of mice in animal experiments.
The invention has the advantages and positive effects that:
1. animal in vivo experiments show that the adenine-induced chronic kidney diseases of mice, including appearance, body weight, mental state, kidney injury, inflammatory reaction, intestinal mucosa barrier permeability and the like, can be improved by the lactobacillus fermentum CECT5716 exopolysaccharide. The invention finds a new application of lactobacillus fermentum CECT5716 exopolysaccharide, in particular relates to a new application of lactobacillus fermentum CECT5716 exopolysaccharide in improving chronic kidney diseases, and provides a new method and a new means for intervention and treatment of chronic kidney diseases by high-quality probiotic exopolysaccharide.
2. The invention observes and measures the influence of the preparation form which is prepared by taking lactobacillus fermentum CECT5716 exopolysaccharide as an effective component and can be accepted in medicine or food on the weight, the appearance form, the kidney organ index, the kidney injury, the renal interstitial fibrosis, the expression of inflammatory factors and the intestinal epithelial cell barrier injury of the CKD mouse model by constructing an adenine-induced chronic kidney disease mouse model, and finds that: the exopolysaccharide of the limolactibacillus CECT5716 can obviously improve the appearance and the weight of a mouse.
The test results prove that the lactobacillus fermentum CECT5716 exopolysaccharide is used as an effective component to prepare a pharmaceutically or food acceptable dosage form, and the lactobacillus fermentum CECT5716 exopolysaccharide can obviously improve growth inhibition, listlessness, kidney injury, renal interstitial fibrosis, inflammatory reaction and intestinal mucosa permeability caused by adenine-induced chronic kidney diseases, so that a theoretical basis is provided for clinical research on intervention and treatment of the chronic kidney diseases by using the probiotic exopolysaccharide.
3. The invention provides a medicament or functional food for preventing or improving chronic kidney diseases, which is prepared by using lactobacillus fermentum CECT5716 exopolysaccharide in the preparation of the medicament or functional food for preventing or improving the chronic kidney diseases induced by adenine, has no side effect, and can be used as a safe substitute medicament for treating the chronic kidney diseases in a conventional way.
4. The invention has important significance for researching the lactobacillus fermentum CECT5716 exopolysaccharide for assisting in treating kidney injury and restoring kidney function.
Drawings
FIG. 1 is the elution curve of the crude Lactobacillus fermentum CECT5716 exopolysaccharide product through Sepharose CL-6B chromatographic column;
FIG. 2 is a scanning electron micrograph of the extracellular polysaccharide of Lactobacillus fermentum CECT5716 in accordance with the present invention;
FIG. 3 is a kidney morphology diagram after lactobacillus fermentum CECT5716 exopolysaccharide intervention (wherein, control: blank Control group, gavage normal saline, model: model group, gavage adenine suspension, EPS: lactobacillus fermentum CECT5716 exopolysaccharide group, gavage adenine suspension and lactobacillus fermentum CECT5716 exopolysaccharide);
FIG. 4 is a graph showing the clearance effect of uremia toxin in serum after intervention of extracellular polysaccharide of Lactobacillus fermentum CECT5716 (data are mean values + -S.D; compared with blank group, ## p is less than 0.01; p < 0.05 compared to model group; wherein, control: a blank control group, and normal saline for gastric lavage; model: model group, intragastric adenine suspension; positive: a positive drug group, an intragastric adenine suspension and a positive drug; EPS: lactobacillus fermentum CECT5716 exopolysaccharide group, intragastric adenine suspension and lactobacillus fermentum CECT5716 exopolysaccharide);
FIG. 5 is a dried prognosis renal slice staining diagram of Lactobacillus fermentum CECT5716 (wherein, control: blank Control group, lavage with normal saline; model: model group, lavage with adenine suspension; positive drug: positive drug group, lavage with adenine suspension and positive drug; EPS: lactobacillus fermentum CECT5716 exopolysaccharide group, lavage with adenine suspension, and Lactobacillus fermentum CECT5716 exopolysaccharide);
FIG. 6 is a graph showing the relative expression levels of the renal inflammatory factor IL-1 β in the post-treatment of extracellular polysaccharide-associated fermentation lactobacillus CECT5716 (data are mean. + -. S.D; compared with the blank group, ## p is less than 0.01; wherein, control: a blank control group, and normal saline for gastric lavage; model: model group, intragastric adenine suspension; positive: a positive drug group, a gastric lavage adenine suspension and a positive drug; EPS: lactobacillus fermentum CECT5716 exopolysaccharide group, intragastric adenine suspension and lactobacillus fermentum CECT5716 exopolysaccharide);
FIG. 7 is a graph showing the LPS content in kidney and the relative expression level of claudin-1 protein in colon after the fermentation of Lactobacillus fermentum CECT5716 exopolysaccharide of the present invention (data are mean. + -. S.D; compared with blank group, ## p is less than 0.01; p < 0.05 compared to model group; wherein, control: a blank control group, and normal saline for gastric perfusion; model: model group, intragastric adenine suspension; the positive: a positive drug group, a gastric lavage adenine suspension and a positive drug; EPS: lactobacillus fermentum CECT5716 exopolysaccharide group, intragastric adenine suspension and lactobacillus fermentum CECT5716 exopolysaccharide);
FIG. 8 is a graph of the extracellular polysaccharide-dried GPX and MDA activities of Lactobacillus fermentum CECT5716 in kidney of the present invention (data are mean. + -. S.D; compare blank, ## p is less than 0.01; p < 0.05 compared to model group; wherein, control: a blank control group, and normal saline for gastric lavage; model: model group, intragastric adenine suspension; the positive: a positive drug group, an intragastric adenine suspension and a positive drug; EPS: lactobacillus fermentum CECT5716 exopolysaccharide group, gavage adenine suspension, and lactobacillus fermentum CECT5716 exopolysaccharide).
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.
The various experimental procedures in the specific examples are conventional in the art and are not specifically described herein, and those skilled in the art can refer to various conventional tool books, scientific documents, or related specifications, manuals, etc. before the filing date of the present application.
Application of lactobacillus fermentum (Lactobacillus fermentum) CECT5716 exopolysaccharide in preparing medicine or functional food for preventing or improving adenine-induced chronic kidney disease is provided.
Preferably, the molecular weight of the lactobacillus fermentum CECT5716 exopolysaccharide is about 37000Da;
preferably, the monosaccharide composition obtained by hydrolysis analysis of the exopolysaccharide is glucose;
preferably, the structure is analyzed by infrared, methylation and NMR as consisting of t-Glcp- (l →, → 4) -Glcp- (1 → and → 4, 6) -Glcp- (1 →) with relative molar ratios of about 1.
Preferably, the extracellular polysaccharide is observed to have a porous and highly branched structure by a scanning electron microscope, as shown in fig. 2.
Preferably, the fermentation broth of the lactobacillus fermentum CECT5716 is a strain of Lactobacillus fermentum CECT5716, is subjected to standing anaerobic culture at MRS 37 ℃ for 12 hours, and is subjected to anaerobic culture for 12 hours in a volume ratio of 1-10: inoculating 100 inoculum sizes into MRS liquid culture medium, performing amplification culture to obtain fermentation broth, and performing next experiment.
Preferably, the fermentation broth of the lactobacillus fermentum CECT5716 is prepared by selecting a strain of the lactobacillus fermentum CECT5716 frozen at-80 ℃, streaking and activating on an MRS solid medium plate, standing at 37 ℃ for anaerobic culture for 12h until the activity is recovered; the rejuvenated strain was inoculated with a 3: inoculating the strain with the volume ratio of 100 into an MRS liquid culture medium, and performing scale-up culture.
Preferably, the lactobacillus fermentum CECT5716 exopolysaccharide is prepared by the following steps:
(1) Pretreatment: heating fermentation broth of Lactobacillus fermentum CECT5716 at 100 deg.C for 20-40min, cooling, centrifuging at 4-10 deg.C and 5000-10000r/min for 10-20min, and collecting supernatant;
(2) And (3) separating extracellular polysaccharide: adding 1-3 times volume of 95% absolute ethyl alcohol into the supernatant obtained in the step (1), standing at 2-10 ℃ overnight, centrifuging at 4-10 ℃ at 5000-10000r/min for 10-20min, and removing the supernatant to obtain crude polysaccharide precipitate;
(3) Purification of exopolysaccharide namely protein removal: re-dissolving the precipitate obtained in the step (2) in distilled water, adding trichloroacetic acid to ensure that the final mass concentration of the trichloroacetic acid is 10%, placing the trichloroacetic acid on a magnetic stirrer for ice bath for 3-5h, centrifuging to obtain supernatant, adjusting the pH of the supernatant to be neutral, adding 1-3 times of 95% absolute ethyl alcohol by volume to perform re-ethanol precipitation, standing at 2-10 ℃ overnight, and centrifuging;
(4) And (3) dialysis: re-dissolving the precipitate obtained in the step (3) in distilled water, and dialyzing for 2-3d by selecting a dialysis bag with the interception amount of 8000 Da;
(5) And (3) freeze drying: pre-freezing the dialyzed solution, and freeze-drying for at least 12h to obtain Lactobacillus fermentum CECT5716 exopolysaccharide with purity of 90-95%.
Preferably, the lactobacillus fermentum CECT5716 exopolysaccharide with purity of 90-95% is separated by column (for example, sepharose CL-6B gel column), and the lactobacillus fermentum CECT5716 exopolysaccharide with EPS purity higher than 98% is obtained.
Preferably, the medicament or the functional food takes lactobacillus fermentum CECT5716 exopolysaccharide as a main active ingredient and is prepared into a pharmaceutically acceptable dosage form and a food acceptable dosage form.
Preferably, the dosage form comprises a tablet, a powder, a granule, a capsule or a drink.
Preferably, the content of the effective active ingredients of the lactobacillus fermentum CECT5716 exopolysaccharide in the dosage form is 1-99% by mass.
Preferably, the characteristics induced by adenine-induced chronic kidney disease include weight loss, listlessness, kidney injury, renal interstitial fibrosis, inflammatory response of the body, and impaired intestinal mucosal barrier.
Preferably, the medicine or functional food comprises lactobacillus fermentum CECT5716 exopolysaccharide as a main active ingredient and auxiliary materials acceptable in medicine or food.
Preferably, the lactobacillus fermentum CECT5716 exopolysaccharide is effective in intervening treatment and improvement of chronic kidney diseases of mice in animal experiments.
Preferably, the common uremic toxins are creatinine, urea, uric acid.
Preferably, the chronic kidney disease is characterized by weight loss, listlessness, increased permeability of intestinal mucosa, increase of uremic toxins such as creatinine, urea, uric acid and the like in blood, kidney injury, oxidative stress, renal interstitial fibrosis and the like.
Lactobacillus fermentum CECT5716 (Lactobacillus mucilaginosus CECT 5716) used in the present invention
(Liposilactobacillus cept 5716), the original strain name is: lactobacillus fermentum CECT5716 (lactobacillus fermentum CECT 5716).
Specifically, the preparation and detection are as follows:
example 1: preparation of lactobacillus fermentum CECT5716 exopolysaccharide
The formula of the culture medium is as follows: 10g of MRS culture medium, namely 10g of beef extract, 5g of yeast extract, 10g of peptone, 20g of glucose, 2g of dipotassium phosphate, 5g of anhydrous sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 2g of diammonium hydrogen citrate, 801mL of Tween and 1000mL of distilled water, and adjusting the pH value to 6.2.
(1) Selecting single colony of Lactobacillus fermentum CECT5716, inoculating to seed culture medium, and culturing at 37 deg.C for 12 hr to obtain activated seed fermentation liquid with bacteria content of 5 × 10 9 CFU/mL。
(2) And (3) amplification culture: inoculating the seed fermentation broth into a fermentation medium according to the volume ratio of 3.
(3) Inactivating enzyme and degerming body: the fermentation broth of Lactobacillus fermentum CECT5716 was boiled at 100 ℃ for 30min, cooled and centrifuged (4 ℃,8000r/min,20min, unless otherwise stated, the centrifugation conditions described below are used as parameters) to obtain the supernatant.
(4) Alcohol precipitation: adding two times of volume of absolute ethyl alcohol into the supernatant, standing overnight at 4 ℃, and centrifuging to obtain a precipitate.
(5) Removing protein: and (5) re-dissolving the precipitate obtained in the step (4) in a proper amount of distilled water, adding trichloroacetic acid to make the final concentration of the trichloroacetic acid to be 10%, stirring the solution for 4 hours under an ice bath condition, centrifuging the solution, collecting supernatant, and adjusting the pH of the supernatant to be neutral.
(6) And (3) alcohol precipitation: adding anhydrous ethanol with volume twice of the supernatant, standing at 4 deg.C overnight, centrifuging to obtain precipitate, dissolving the precipitate with water, dialyzing with dialysis bag with cut-off of 8000Da for 3d, and freeze drying the dialysate to obtain crude extracellular polysaccharide with purity of (92.2 + -1.8)%.
Example 2: purification of lactobacillus fermentum CECT5716 exopolysaccharide
Dissolving the crude exopolysaccharide prepared in example 1 with distilled water to prepare a solution of 10mg/mL, wherein the sample loading amount is 1mL, separating by a Sepharose CL-6B gel column, eluting with distilled water, collecting eluent, collecting 1mL in each tube, tracking and detecting the polysaccharide content by a phenol-sulfuric acid method to draw an elution curve, combining main peak (peak 2) filtrates as shown in figure 1, and freeze-drying to prepare purified exopolysaccharide, which is named as pEPS and pEPS as a scanning electron microscope image as shown in figure 2. The purity of pEPS is up to 98% measured by phenol-sulfuric acid method.
Example 3
The polysaccharide of example 2 was weighed and dissolved in 1mL of edible solute at a concentration of 200mg/kg/d to obtain a sample for preventing or ameliorating chronic kidney disease as described in the present invention.
Example 4
Male BALB/C mice (body weight about 20-23 g) of SPF grade 7 weeks old were used as experimental material and randomly divided into 3 groups of 10 mice each, and the treatment of each group was as follows: continuously gavage aseptic normal saline (the mass volume concentration of the sterile normal saline is 0.85%) for 6 weeks in a blank control group; model group: performing continuous gavage with adenine/body weight of 100mg/kg adenine for 6 weeks; EPS group: the samples for preventing or improving chronic kidney disease (administered 1 time a day at 200 mg/kg/d) described in example 3 were administered for 6 weeks with continuous gavage of 100mg/kg adenine/body weight. Mice were weighed once a week and observed to record changes in appearance morphology according to table 1, and mice were dissected for the last week, observed for kidney morphology and weighed.
TABLE 1 Scoring method
| Characterization phenomena | Health care | Mild (fur is slightly wrinkled) | Moderate wrinkle and lethargy of fur | Fur is seriously wrinkled and thin | Is very serious |
| Score value | 4-5 | 3-4 | 2-3 | 1-2 | 0-1 |
Body weight change, appearance morphology, and kidney morphology and kidney index score during the mouse experiment, wherein kidney morphology is shown in figure 3. The results show that after the exopolysaccharide of the lactobacillus fermentum CECT5716 is used for gastric perfusion, the weight of a mouse is recovered, the appearance of the mouse is relieved, the mental state is improved, and the renal edema is reduced, which indicates that the exopolysaccharide of the lactobacillus fermentum CECT5716 can obviously improve the growth inhibition, the appearance of the mouse is upright, the mental depression and the renal edema caused by the adenine-induced chronic kidney diseases.
Example 5
SPF grade 7 week old BALB/C male mice (body weight about 20-23 g) were used as experimental material and randomly divided into 3 groups of 10 mice each, and the treatment of each group was as follows: the blank control group was continuously subjected to intragastric administration of sterile normal saline (the concentration by mass volume thereof was 0.85%) for 6 weeks; model group: performing continuous gavage with adenine/body weight of 100mg/kg adenine for 6 weeks; positive drug group: continuously performing intragastric administration of 100mg/kg adenine and administration of 200mg/kg Haikun Shenxi capsule according to adenine/body weight mode; EPS group: the stomach was drenched with 100mg/kg adenine and the drug for preventing or improving chronic kidney disease described in example 3 (1 time daily, 200mg/kg/d for each dose) for 6 weeks in a adenine/body weight manner. Mice were dissected for the last week and serum, kidney, colon were collected.
The enzyme-linked immunosorbent assay kit IS adopted to detect the creatinine, urea, uric acid, IS, pCS and TMAO levels in mouse serum, and the result shows that as shown in figure 4, compared with a blank group, the adenine suspension induction enables the serum creatinine, urea, uric acid, IS, pCS and TMAO levels of mice in a CKD model group to be obviously increased and respectively reach 345%, 255%, 124%, 122%, 70.84% and 96.64%, and after the extracellular polysaccharide intervention treatment of lactobacillus fermentum CECT5716, the serum creatinine, urea, uric acid, IS, pCS and TMAO levels are obviously decreased and respectively reach 36.38%, 38.72%, 30.71%, 51.62%, 33.59% and 46.47%. It IS demonstrated that lactobacillus fermentum CECT5716 exopolysaccharide can alleviate CKD by degrading creatinine, urea nitrogen, uric acid, IS, pCS, TMAO.
The results of the observation of the degree of renal injury and renal interstitial fibrosis and the degree of renal apoptosis were obtained by HE staining, masson staining and caspase-3 immunohistochemical staining, respectively, and are shown in FIG. 5. Fig. 5 shows that compared with the blank group, the kidney of the model group mice has obvious renal tubular dilatation, vacuolation, renal interstitial fibrosis, collagen fiber deposition and visible caspase-3 accumulation in the renal tubular, glomerular and renal interstitial tissues, and after the treatment of lactobacillus fermentum CECT5716 exopolysaccharide intervention, the phenomena of renal fibrosis, renal tubular dilatation, caspase-3 accumulation and the like of the model mouse CKD mice are obviously improved, which indicates that the intervention of lactobacillus fermentum CECT5716 exopolysaccharide can alleviate kidney injury and renal interstitial fibrosis and renal apoptosis.
The real-time fluorescent quantitative PCR detection shows that the expression of the inflammatory factor IL-1 beta in the kidney tissue is shown in figure 6. Fig. 6 shows that the relative expression level of IL-1 β in the kidney of the mouse model group is significantly increased compared to the blank group, and the relative expression level of IL-1 β in the kidney of the mouse model CKD is significantly decreased after intervention treatment with the extracellular polysaccharide of lactobacillus fermentum CECT5716, which indicates that the extracellular polysaccharide of lactobacillus fermentum CECT5716 can effectively improve the renal inflammatory response induced by adenine-induced CKD mouse model to prolong the renal function of CKD mouse.
The expression of the claudin-1 protein in the colon tissue is detected by real-time fluorescent quantitative PCR and the content of LPS is detected by a mouse LPS ELISA kit, and the result is shown in figure 7. Fig. 7 shows that the relative expression level of claudin-1 protein in colon of mice in model group is significantly reduced, after intervention treatment with extracellular polysaccharide of lactobacillus fermentum CECT5716, the relative expression level of claudin-1 protein in colon of CKD mice is increased, and the content of LPS is reduced, which indicates that extracellular polysaccharide of lactobacillus fermentum CECT5716 can restore the integrity of adenine-induced intestinal mucosal barrier of CKD mice model by increasing the relative expression level of claudin-1 protein, thereby reducing LPS from entering blood circulation.
The enzyme linked immunosorbent assay kit is adopted to detect the GSH-PX and MDA activities in the kidney of the mouse, and the result is shown in figure 8. Figure 8 shows that the model group mice had significantly reduced GSH-PX activity and significantly increased MDA activity in the kidney, and that after treatment with lactobacillus fermentum CECT5716 exopolysaccharide intervention, GSH-PX activity and significantly decreased MDA activity in the CKD mouse model kidney. The lactobacillus fermentum CECT5716 exopolysaccharide can improve the oxidative stress degree of adenine-induced CKD mouse model to relieve CKD by regulating the GSH-PX and MDA activities in the kidney.
And (4) conclusion: according to characterization indexes such as appearance form, body weight, kidney form and kidney index of mice, physiological and biochemical indexes such as creatinine, urea and uric acid levels in serum, relative expression level of inflammatory factors in kidney, permeability of intestinal mucosa barrier and the like, it can be obtained that the lactobacillus fermentum CECT5716 exopolysaccharide can obviously relieve adverse effects such as weight loss, listlessness, kidney injury, renal interstitial fibrosis and inflammatory reaction caused by adenine-induced chronic kidney diseases, and provides a basis for clinical treatment of kidney injury.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, alterations and modifications are possible without departing from the spirit and scope of this disclosure and appended claims, and accordingly, the scope of this disclosure is not limited to the embodiments disclosed.
Claims (10)
1. Application of lactobacillus fermentum (Lactobacillus fermentum) CECT5716 exopolysaccharide in preparing medicine or functional food for preventing or improving adenine-induced chronic kidney disease is provided.
2. Use according to claim 1, characterized in that: the molecular weight of the lactobacillus fermentum CECT5716 exopolysaccharide is 37000Da; the monosaccharide is glucose; the structure of the compound consists of t-Glcp- (l → 4) -Glcp- (1 → and → 4, 6) -Glcp- (1 →) and the relative molar ratio is 1; the extracellular polysaccharide is observed by a scanning electron microscope to show a porous and highly branched structure.
3. Use according to claim 1, characterized in that: the fermentation broth of the lactobacillus fermentum CECT5716 is a strain of the lactobacillus fermentum CECT5716, is subjected to standing anaerobic culture at the temperature of MRS 37 ℃ for 12 hours, and is prepared by mixing the following components in a volume ratio of 1-10: inoculating 100 inoculum sizes into MRS liquid culture medium, and performing amplification culture to obtain fermentation broth.
4. Use according to any one of claims 1 to 3, characterized in that: the lactobacillus fermentum CECT5716 exopolysaccharide is prepared by the following steps:
(1) Pretreatment: heating fermentation broth of Lactobacillus fermentum CECT5716 at 100 deg.C for 20-40min, cooling, centrifuging at 4-10 deg.C and 5000-10000r/min for 10-20min, and collecting supernatant;
(2) And (3) separating the exopolysaccharide: adding 1-3 times volume of 95% absolute ethyl alcohol into the supernatant obtained in the step (1), standing overnight at 2-10 ℃, centrifuging for 10-20min at 4-10 ℃ at 5000-10000r/min, and removing the supernatant to obtain crude polysaccharide precipitate;
(3) Purification of exopolysaccharide namely protein removal: re-dissolving the precipitate obtained in the step (2) in distilled water, adding trichloroacetic acid to ensure that the final mass concentration of the trichloroacetic acid is 10%, placing the trichloroacetic acid on a magnetic stirrer for ice bath for 3-5h, centrifuging to obtain supernatant, adjusting the pH of the supernatant to be neutral, adding 1-3 times of 95% absolute ethyl alcohol by volume to perform re-ethanol precipitation, standing at 2-10 ℃ overnight, and centrifuging;
(4) And (3) dialysis: re-dissolving the precipitate obtained in the step (3) in distilled water, and dialyzing for 2-3d by selecting a dialysis bag with the interception amount of 8000 Da;
(5) And (3) freeze drying: pre-freezing the dialyzed solution, and freeze-drying for at least 12h to obtain Lactobacillus fermentum CECT5716 exopolysaccharide with purity of 90-95%.
5. The use according to claim 4, wherein: and separating the lactobacillus fermentum CECT5716 exopolysaccharide with the purity of 90-95% by using a Sepharose CL-6B gel chromatography column, and drying to obtain the lactobacillus fermentum CECT5716 exopolysaccharide with the EPS purity higher than 98%.
6. Use according to claim 1, characterized in that: the medicine or functional food takes lactobacillus fermentum CECT5716 exopolysaccharide as a main active ingredient, and is prepared into a pharmaceutically acceptable dosage form and a food acceptable dosage form.
7. Use according to claim 6, characterized in that: the dosage form comprises tablets, powder, granules, capsules or drinks.
8. Use according to claim 7, characterized in that: the effective active ingredients of the lactobacillus fermentum CECT5716 exopolysaccharide in the dosage form have the mass percentage content of 1-99%;
or the medicine or functional food comprises lactobacillus fermentum CECT5716 exopolysaccharide as a main active ingredient and pharmaceutically or food acceptable adjuvants.
9. Use according to claim 1, characterized in that: the characteristics caused by adenine-induced chronic kidney disease include weight loss, listlessness, kidney injury, renal interstitial fibrosis, body inflammatory response, and impaired intestinal mucosal barrier.
10. Use according to any one of claims 1 to 9, characterized in that: the lactobacillus fermentum CECT5716 exopolysaccharide is used for effectively intervening and treating chronic kidney diseases of mice in animal experiments.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211198927.1A CN115887483A (en) | 2022-09-29 | 2022-09-29 | Application of lactobacillus fermentum CECT5716 exopolysaccharide in preparation of medicines for preventing or improving chronic kidney diseases |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211198927.1A CN115887483A (en) | 2022-09-29 | 2022-09-29 | Application of lactobacillus fermentum CECT5716 exopolysaccharide in preparation of medicines for preventing or improving chronic kidney diseases |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115887483A true CN115887483A (en) | 2023-04-04 |
Family
ID=86473351
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211198927.1A Pending CN115887483A (en) | 2022-09-29 | 2022-09-29 | Application of lactobacillus fermentum CECT5716 exopolysaccharide in preparation of medicines for preventing or improving chronic kidney diseases |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115887483A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116590373A (en) * | 2023-06-29 | 2023-08-15 | 广东海洋大学 | Fermented lactobacillus mucilaginosus extracellular polysaccharide, preparation method and application |
| CN116585360A (en) * | 2023-05-24 | 2023-08-15 | 微康益生菌(苏州)股份有限公司 | Probiotic agent for improving chronic kidney disease and application thereof |
| CN116769673A (en) * | 2023-08-04 | 2023-09-19 | 云南农业大学 | Lactic acid bacteria with high extracellular polysaccharide yield and application thereof in preparation of wiredrawing yoghourt |
-
2022
- 2022-09-29 CN CN202211198927.1A patent/CN115887483A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116585360A (en) * | 2023-05-24 | 2023-08-15 | 微康益生菌(苏州)股份有限公司 | Probiotic agent for improving chronic kidney disease and application thereof |
| CN116585360B (en) * | 2023-05-24 | 2023-11-14 | 微康益生菌(苏州)股份有限公司 | Probiotic agent for improving chronic kidney disease and application thereof |
| CN116590373A (en) * | 2023-06-29 | 2023-08-15 | 广东海洋大学 | Fermented lactobacillus mucilaginosus extracellular polysaccharide, preparation method and application |
| CN116590373B (en) * | 2023-06-29 | 2023-11-17 | 广东海洋大学 | Fermented lactobacillus mucilaginosus extracellular polysaccharide, preparation method and application |
| CN116769673A (en) * | 2023-08-04 | 2023-09-19 | 云南农业大学 | Lactic acid bacteria with high extracellular polysaccharide yield and application thereof in preparation of wiredrawing yoghourt |
| CN116769673B (en) * | 2023-08-04 | 2023-12-29 | 云南农业大学 | Lactic acid bacteria with high extracellular polysaccharide yield and application thereof in preparation of wiredrawing yoghourt |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115887483A (en) | Application of lactobacillus fermentum CECT5716 exopolysaccharide in preparation of medicines for preventing or improving chronic kidney diseases | |
| CN114507621B (en) | Lactobacillus plantarum and application thereof in reducing uric acid, weight and inflammation | |
| CN112999261A (en) | Natto fermented composition capable of relieving arteriosclerosis and preparation method and application thereof | |
| CN111040044B (en) | Cordyceps militaris intracellular polysaccharide, preparation method and application thereof in regulating intestinal flora | |
| CN115414391B (en) | Application of lactobacillus plantarum MA2 in preparation of medicine for preventing or improving adenine-induced chronic kidney disease | |
| CN114717147A (en) | Metazoan prepared from Lactobacillus rhamnosus and used for relieving fatty liver and obesity, and application thereof | |
| CN112662581A (en) | Lactobacillus acidophilus producing extracellular polysaccharide with anti-tumor, blood sugar reducing and blood fat reducing activities and application thereof | |
| CN114437989A (en) | Lactobacillus fermentum LF028 with blood sugar reducing effect and application thereof | |
| CN115895966B (en) | Bifidobacterium bifidum BL002 for assisting in relieving gout and application thereof | |
| CN112831444A (en) | Lactobacillus rhamnosus LR2Y and application thereof | |
| CN117264840A (en) | Lactobacillus brevis XY8 and application thereof in preparation of food and medicine for resisting aging and improving gout | |
| JP3995733B2 (en) | Immunostimulatory composition | |
| CN114437997B (en) | Lactobacillus plantarum SF-L38 and application thereof in preparation of blood sugar control product | |
| CN113209141B (en) | Composition based on brown algae extract and lactobacillus reuteri and application thereof | |
| CN111548964B (en) | Lactobacillus casei and application thereof in preventing and treating nephritis | |
| KR102048434B1 (en) | A composition as a prebiotic for improving intestinal microflora containing High-molecular fraction from radish leave | |
| CN112043723B (en) | Application of bacillus amyloliquefaciens exopolysaccharide | |
| CN111713659B (en) | Preparation method of sargassum fusiforme product for inhibiting activity of alpha-glucosidase and sargassum fusiforme product | |
| CN117099850B (en) | Fermented plant-based yoghurt and preparation method and application thereof | |
| CN117721033B (en) | Lactobacillus mucilaginosus KS6 and application thereof in preparation of anti-inflammatory and sleep-aiding foods and medicines | |
| CN112336847B (en) | Use of probiotic compositions in the manufacture of a medicament | |
| CN112870235B (en) | Application of hericium erinaceus powder in preparation of product for preventing or treating hyperuricemia-related diseases | |
| CN114921351B (en) | Pichia pastoris DPUY-F1 with probiotics function and application thereof in relieving colonitis symptoms | |
| CN117384788B (en) | Saliva combined lactobacillus SM4 and application thereof in preparation of whitening and cholesterol lowering foods and medicines | |
| CN112675292B (en) | Use of probiotic-containing formulations for the treatment of diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |