Specially selected strain and targeted blood uric acid reduction application thereof

Abstract

The invention discloses a specially selected strain-prebiotic composition for enhancing and maintaining an ozone high-dose blood purification effect and an application of the specially selected strain-prebiotic composition in reducing blood uric acid. According to the invention, blood cell metabolism and immune system and antioxidant enzyme system can be activated by autologous blood circulation reinfusion; meanwhile, the specially selected targeted strain-prebiotic composition provided by the invention can regulate the balance of intestinal flora, establish an intestinal barrier function, maintain and accelerate metabolism of a human body, and especially promote decomposition and metabolism of intestinal tracts and excretion of purine and uric acid. The composition comprises one or more of lactobacillus plantarum BLP-17 and prebiotics, and after specially selected strains and prebiotics (g / g) are combined according to the proportion of 1: (49-99), the composition has the obvious effect of reducing blood uric acid. According to the combination scheme, the blood purification effect can be remarkably improved and maintained, the index of blood uric acid is remarkably improved, a new technical scheme and means are provided for prevention and treatment of hyperuricemia and gout, and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of food, health care and medical treatment, and specifically relates to the technical field of prevention and treatment of hyperuricemia and gout, and more specifically, to a specially selected bacterial strain composition and its application in prevention and treatment of hyperuricemia and gout. Background Art

[0002] Hyperuricemia, together with hypertension, hyperglycemia, and hyperlipidemia, is collectively known as the "four highs" metabolic disease. It is one of the most common and most harmful chronic diseases in clinical practice. Its typical characteristics are an increase in uric acid content in the body, but a decrease in uric acid excretion. Uric acid in the human body is mainly produced by the metabolism of purine nucleic acids, and the sources of purine nucleic acids are mainly two aspects. One is the intake of food-borne purine nucleic acids; the other is uric acid generated by the degradation of purine nucleic acids after cell apoptosis during cell metabolism in the body.

[0003] The international standard for judging hyperuricemia is the concentration of uric acid in the blood (>420μmol / L for men; >357μmol / L for women). At present, the incidence of hyperuricemia is affected by many factors, such as genetics, gender, lifestyle, eating habits, etc. Surveys show that hyperuricemia is becoming more and more common worldwide, and the number of people with hyperuricemia is increasing rapidly. The prevalence rates in the United Kingdom, the United States, Japan, and Ireland are 18.9%, 20%, 18.5%, and 24.1%, respectively, and the prevalence rates in South Korea, Thailand, Mexico, and Russia are 11.4%, 10.6%, 25.6%, and 16.8%, respectively. The high incidence rate has made hyperuricemia a disease that cannot be ignored. As of 2019, the number of confirmed cases in my country has reached 190 million, and the incidence rates of hyperuricemia in men and women are 20.2% and 8.1%, respectively. The above results show that the incidence rate in men is significantly higher than that in women. At the same time, studies have also shown that the incidence rate of hyperuricemia in the elderly is also increasing.

[0004] Hyperuricemia is a precursor to gout, and long-term hyperuricemia is the most important risk factor for gout. An increase in serum uric acid above a certain threshold is a necessary condition for the formation of uric acid crystals. Although uric acid has an antioxidant effect, when it is present in the cytoplasm or in the environment of atherosclerotic plaques, uric acid is converted into a pro-oxidant that promotes oxidative stress and affects human diseases including cardiovascular disease through this mechanism. Some researchers have pointed out that patients with hyperuricemia have a higher incidence of atrial fibrillation. Increased blood uric acid levels make people more susceptible to colitis. Moreover, uric acid levels have a strong positive correlation with various indicators of metabolic syndrome. In addition, hyperuricemia is also closely related to the development of diseases such as diabetes, hypertension, and kidney disease.

[0005] At present, the drugs for hyperuricemia and gout at home and abroad mainly include drugs that inhibit uric acid production, drugs that promote renal uric acid excretion, and drugs that metabolize uric acid. The first is drugs that inhibit uric acid production. This type of drug belongs to xanthine oxidase (XOD) inhibitors and is considered the first treatment for gout. The main ones are allopurinol and febuxostat. However, the metabolite of allopurinol, oxypurinol, is excreted by the kidneys, which limits the use of drugs in patients with renal function loss. Allopurinol even has serious side effects, namely fatal allopurinol allergic complications. Febuxostat also has the side effect of increasing the risk of death from cardiovascular disease. Drugs that promote uric acid excretion are considered to be second-line drugs for lowering uric acid to treat gout, mainly probenecid, benzbromarone and lesinad. However, benzbromarone and lesinad have not been widely used due to their side effects on the liver and kidneys, respectively. Metabolic uric acid drugs are considered to be third-line drugs for gout treatment, mainly referring to recombinant uricase, which is usually used in patients with severe gout.

[0006] Since only half of patients with asymptomatic hyperuricemia will develop gout, the side effects caused by drug treatment discourage these patients. As an important member of the human intestinal flora, probiotics have few side effects on the human body, so they have more advantages and application value in the treatment of hyperuricemia than drugs. Summary of the invention

[0007] The present invention is based on the self-activation of blood and the re-infusion of ozone high-dose blood purification, and uses a specially selected strain of probiotics and a prebiotic composition for lowering blood uric acid. The combination of the present invention has the efficacy of effectively lowering blood uric acid content, maintaining the effect of ozone high-dose blood purification and maintaining uric acid metabolic function.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] First, a screening of a selected strain of probiotics for lowering blood uric acid: milk samples from multiple sources were cultured under specific conditions in MRS solid-liquid medium, the bacteria were collected by centrifugation, washed with MSM medium, screened and separated by target substrates, and screened again by continuous enrichment technology, separated again, purified, and obtained a plant lactobacillus (Lactobacillus plantarum) that can effectively degrade uric acid and purine precursors after morphological and molecular biological identification. Lactiplantibacillus plantarum ), named: BLP-17, with a preservation number of CCTCC NO: M 20221833. The total viable count of the freeze-dried powder prepared from this strain is 7×10 10 ~1.3×10 12 CFU / g.

[0010] In a second aspect, a composition for enhancing the effect of lowering blood uric acid is provided, comprising Lactobacillus plantarum BLP-17 and a plurality of prebiotics. The weight ratio of the Lactobacillus plantarum BLP-17 to the prebiotics is 1:(49-99).

[0011] Preferably, in the above technical solution, when the ratio of the added amount of the selected probiotic strain to the prebiotic is within this range, the optimal survival and colonization of the selected probiotic strain can be ensured, thereby optimizing the efficacy of the strain.

[0012] Finally, on the basis of autologous blood activation and re-infusion of ozone high-dose blood purification, the above-mentioned selected strains of Lactobacillus plantarum and prebiotic composition are used, which not only have the ability to degrade blood uric acid, but also the ability to degrade purine precursors, purine precursor substances include but are not limited to inosine, or one or more of guanosine, but also can maintain the effect of high-dose blood purification of ozone, and continuously maintain the normal metabolic function of uric acid in the human body.

[0013] Biodeposit Information:

[0014] Probiotic strain BLP-17, classified and named Lactiplantibacillus plantarumBLP-17 , deposited in the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, Wuhan, China, the deposit date is November 29, 2022, and the deposit number is: CCTCC NO: M 20221833.

[0015] Beneficial effects of the present invention:

[0016] The present invention is based on the self-activation of blood and the re-infusion of ozone high-dose blood purification, and uses a selected strain of probiotics and prebiotics for reducing blood uric acid. The combination has the effects of effectively reducing blood uric acid content, maintaining the effect of ozone high-dose blood purification and maintaining uric acid metabolism function.

[0017] In addition to being able to degrade uric acid, the plant lactobacillus BLP-17 screened in this application can also degrade purine precursors. As a new means of reducing blood uric acid and treating gout, plant lactobacillus BLP-17, which has the ability to degrade purine precursors, can significantly reduce the absorption of food-borne purines without reducing the quality of life (low-purine diet), thereby achieving the effect of a low-purine diet.

[0018] The present invention combines probiotics and prebiotics to prepare a composite bacterial powder which can significantly reduce blood uric acid in gout patients. Compared with clinical chemical drug treatments, the composite bacterial powder is safe, has no toxic side effects, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 . Standard curve of uric acid detection concentration.

[0021] Figure 2 . Standard curve of inosine detection concentration.

[0022] Figure 3 . Standard curve of guanosine detection concentration. Specific implementation plan

[0024] Example 1: Isolation, screening and identification of strains

[0025] 1.1 Isolation and purification of strains

[0026] Multiple milk samples from different sources were diluted and spread on MRS solid medium and cultured at 37°C for 3 days. Single colonies on the plate were picked and further purified by streaking. The purified single bacteria were inoculated into MRS liquid medium, cultured at 37°C and 180rpm for 12 hours, centrifuged and collected, washed 3 times with MSM medium, and added to MSM medium containing 15 mg / L uric acid, guanosine and inosine, respectively. After culture at 37°C for 3 days, they were detected by high performance liquid chromatography (HPLC).

[0027] Through screening and separation, as well as continuous enrichment technology, re-screening, re-isolation and purification, and morphological and molecular biological identification, a strain that can effectively degrade uric acid and purine precursors was obtained and named BLP-17.

[0028] MRS medium formula: 10.0 g peptone, 8.0 g beef extract powder, 4.0 g yeast extract powder, 20.0 g glucose, 2.0 g potassium dihydrogen phosphate, 2.0 g ammonium citrate, 5.0 sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, add deionized water to 1 L. Add 15.0 g agar per liter to the solid medium.

[0029] MSM medium formula: 1.0 g ammonium chloride, 0.5 g potassium dihydrogen phosphate, 1.5 g potassium hydrogen phosphate, 1.0 g sodium chloride, 0.2 g magnesium sulfate heptahydrate, add deionized water to 1 L. Add 15.0 g agar per liter to the solid medium.

[0030] 1.2 Identification of strains

[0031] Phylogenetic analysis of 16S rRNA gene sequences: Bacterial DNA was extracted (Bacterial Genomic DNA Rapid Extraction Kit, Vazyme), and the total DNA of the strain was used as a template for PCR amplification using universal primers for the 16S rRNA gene. The forward primer was 5′-AGAGTTTGATCCTGGCTCAG-3′ and the reverse primer was 5′-TACCTTGTTACGACTT-3′.

[0032] The 50 µL PCR reaction system is: 2.0 µL DNA template, 2.0 µL primers (10 μM) each, 25.0 µL Green Taq Mix, and 19 µL ddH2O.

[0033] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 90 s, for 35 cycles; and complete extension at 72°C for 5 min.

[0034] 4.0 μL of PCR product was electrophoresed on 1% agarose gel. The PCR product was recovered using a recovery kit (Axygen) and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing after TA cloning. The 16S rRNA gene sequence obtained after sequencing was compared with the homology of EzTaxon-e server to identify strain BLP-17 and Lactiplantibacillus plantarum (Lactobacillus plantarum) ATCC 14917 T The sequence similarity is 99.93%.

[0035] In addition, combined with the morphological and physiological and biochemical characteristics of the strain, strain BLP-17 was identified as a Lactobacillus genus ( Lactobacillus sp.). The strain was deposited in China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on November 29, 2022, with the deposit number: CCTCC NO: M 20221833.

[0036] Example 2: Determination of strain tolerance to acid and bile salts

[0037] Lactobacillus plantarum BLP-17 was inoculated at 1% in MRS medium with different pH (pH2, pH3, pH4, pH5) or different bile salt concentrations (0.1%, 0.2%, 0.3%, 0.5%), and the MRS liquid medium without pH adjustment or bile salt addition was used as the control. After culturing at 37°C and 180rpm for 12h, 100μL of bacterial solution was diluted and spread on MRS solid plate, and then cultured at 37°C for 24h, and the number of colonies was counted. The experiment was repeated three times, with three parallels each time. The test results are shown in Tables 1 and 2.

[0038] The results showed that Lactobacillus plantarum BLP-17 had a certain ability to resist acid and bile salts.

[0039] Table 1: Acid resistance test results of strain BLP-17 pH CK 2 3 4 5 Colony count <![CDATA[7.5×10 8 ]]> <![CDATA[3.5×10 8 ]]> <![CDATA[3.9×10 8 ]]> <![CDATA[5.5×10 8 ]]> <![CDATA[6.3×10 8 ]]>

[0040] The strain BLP-17 was inoculated into culture media with different pH values. After culturing for 12 hours, it was observed that the number of live bacteria of strain BLP-17 in MRS culture medium decreased faster as the pH value decreased. At pH 2, the number of colonies was 50% lower than that of the CK group, but there was no change in the order of magnitude. When the pH was 5, there was no significant change in the number of live bacteria compared with CK. This result shows that strain BLP-17 has a good ability to tolerate gastric acid.

[0041] Table 2: Bile salt tolerance test results of strain BLP-17 Bile salt concentration CK 0.1% 0.2% 0.3% 0.5% Colony count <![CDATA[7.8×10 8 ]]> <![CDATA[2.5×10 8 ]]> <![CDATA[1.3×10 8 ]]> <![CDATA[8.6×10 7 ]]> <![CDATA[5.6×10 6 ]]>

[0042] The strain BLP-17 was inoculated in different concentrations of bile salts. As the concentration increased, the number of live bacteria decreased faster. Compared with CK, the number of live bacteria decreased by 2 orders of magnitude at a bile salt concentration of 0.5%. However, at concentrations of 0.1% and 0.2%, the number of live bacteria did not change by an order of magnitude compared with CK, indicating that the strain BLP-17 can tolerate a bile salt concentration of 0.2%. This result shows that the strain BLP-17, as a probiotic, still retains a very high number of live bacteria after being destroyed by gastric acid and bile salts and can enter the intestine to exert its effect.

[0043] Example 3: Determination of the optimal pH and temperature for the strain to degrade uric acid.

[0044] The strain BLP-17 was inoculated into MRS medium and cultured at 37°C and 180 rpm until the logarithmic phase. After centrifugation, the cells were collected and washed three times with MSM medium. The OD 600 The inoculum concentration of strain BLP-17 was 1% and inoculated into MSM medium containing 15 mg / L uric acid. The pH of MSM was adjusted to different values ​​(pH2, pH3, pH4, pH5, pH6, pH7). The culture was carried out at 37°C and 180 rpm for 4 h. The concentration of residual uric acid in the medium was determined and the degradation rate of uric acid was calculated.

[0045] The results showed that the optimal pH for strain BLP-17 to degrade uric acid was 6.5. However, when the pH was between 4 and 5, the degradation rate of uric acid by strain BLP-17 remained above 60%, indicating that it has the ability to degrade uric acid under acidic conditions.

[0046] The above OD 600 The seed solution of strain BLP-17 with a pH of 1.0 was inoculated into MSM medium containing 15 mg / L uric acid at an inoculum size of 1%, and then cultured at 4, 16, 30, 37 and 40 °C, 180 rpm for 4 h. The concentration of residual uric acid in the medium was determined, and the degradation rate of uric acid was calculated.

[0047] Through the test, it was found that the optimal temperature for strain BLP-17 to degrade uric acid is 37°C. At 30°C, the degradation rate of uric acid by strain BLP-17 is still maintained at 80%, but when the temperature is lower than 16°C or higher than 37°C, the efficiency of strain BLP-17 to degrade uric acid is significantly reduced.

[0048] Preparation process of uric acid standard curve: accurately weigh analytically pure (purity> 95%) uric acid, add sterile water to prepare standard products with different gradient concentrations, operate according to the above termination reaction process, and perform HPLC detection to establish the standard curve of each substrate, such as Figure 1 shown.

[0049] The specific detection method of uric acid is: Sepax Bio C 18 Chromatographic column, mobile phase: 20mM potassium dihydrogen phosphate buffer (pH2.5): methanol = (90:10), flow rate 1.0ml / min, detection wavelength 234nm, sample volume 20μl.

[0050] Example 4: Determination of the rate at which strains degrade uric acid and purine precursors.

[0051] The strain BLP-17 was inoculated into MRS medium and cultured at 37°C and 180 rpm until the logarithmic phase. After centrifugation, the cells were collected and washed three times with MSM medium. The OD 600 1.0. The strain BLP-17 was inoculated at 1% in MSM medium (pH 6) containing 15 mg / L uric acid, inosine and guanosine, respectively. After culturing at 37°C and 180 rpm for 4 hours, 1 mL of the medium was centrifuged and filtered. The remaining uric acid, inosine and guanosine in the medium were determined by HPLC, and the degradation rate was calculated. The results are shown in Table 3.

[0052] The preparation process of the standard curve of purine precursor substrate is the same as that of the standard curve of uric acid.

[0053] The specific detection method is: Sepax Bio C 18 Chromatographic column, mobile phase: 20mM potassium dihydrogen phosphate buffer (pH2.5): methanol = (90:10), flow rate 1.0ml / min, detection wavelength 254nm, sample volume 20μl.

[0054] Table 3: Rate of degradation of purine precursors by strain BLP-17 Substrate Uric acid Inosine Guanosine Degradation rate (mg / OD·h·L) 337 97 154

[0055] By measuring the degradation rates of uric acid, inosine and guanosine by strain BLP-17, it was found that strain BLP-17 can degrade uric acid, guanosine and inosine at a relatively fast rate. The degradation rate of uric acid is the fastest, followed by guanosine, and finally inosine.

[0056] Example 5: Preparation of a specially selected bacterial strain composition targeted at lowering uric acid.

[0057] This embodiment provides a method for preparing a uric acid-lowering composition:

[0058] The identified Lactobacillus plantarum BLP-17 was expanded and cultured, centrifuged at 3000 rpm for 10 min, and the supernatant was discarded to obtain the bacteria, which were freeze-dried and crushed to obtain the freeze-dried powder of Lactobacillus plantarum BLP-17, i.e., the freeze-dried powder of the selected strain. The freeze-dried powder was tested and the total viable count was 1.1×10 12 CFU / g.

[0059] Lactobacillus plantarum BLP-17 (viable cell count 1.1×10 12 CFU / g) of freeze-dried powder and prebiotics (oligomannose, stachyose, L-arabinose, xylo-oligosaccharide, inulin, etc.) were sieved through a 60-mesh sieve for later use. The corresponding materials were weighed according to the mass ratio of strain freeze-dried powder to prebiotics in the range of 1: (49-99), mixed evenly, and packaged for later use.

[0060] Example 6: Human body high-dose blood purification and food tasting test

[0061] Autologous blood transfusion is to collect 1000ml of blood, and the blood flows from the blood bag into a quartz glass tank, which is placed in the UBIO treatment device and irradiated with ultraviolet light with a dose of 10J / cm2 and a wavelength of 300nm. The oxygen flow rate is 5 liters / min. After irradiation, the blood is returned to the original sterile blood bag, and then returned to the patient through the original blood infusion device through the vein, once a month.

[0062] A specially selected bacterial strain composition targeting uric acid lowering is prepared into capsules or solid beverage strips, 5-8 g each time, twice a day, for a course of 30 days.

[0063] Example 7: Food tasting test for testing human blood uric acid index

[0064] A human food trial was conducted using the human ozone high-dose blood purification and uric acid-lowering targeted selected strain composition in Example 6 to test blood lipid indicators. All subjects were male, aged between 45 and 70 years old.

[0065] Experimental design and grouping: Two control designs, self-control and inter-group control, were used. 60 subjects were selected, with 15 subjects in each group. According to the requirements of randomized double-blind, the subjects were divided into control group, experimental group 1, experimental group 2 and experimental group 3. The main factors affecting the results, such as age and diet, were considered as much as possible, and a balance test was conducted to ensure the comparability between the groups.

[0066] Experimental group 1 used the human ozone high-dose blood purification and uric acid-lowering targeted specially selected bacterial strain composition in Example 6, experimental group 2 simply used human ozone high-dose blood purification, experimental group 3 simply took the uric acid-lowering targeted specially selected bacterial strain composition, and the control group took a placebo for 30 consecutive days.

[0067] The grouping is shown in Table 4. Before the trial, there was no significant difference in age and blood uric acid among the four groups (P>0.05), which was comparable.

[0068] Table 4 General information of subjects and changes in blood uric acid in the experimental and control groups before and after the trial project Control group Experimental Group 1 Experimental Group 2 Experimental Group 3 Number of cases 15 15 15 15 Average age 55.4±9.4 55.5±9.1 56.2±8.8 57.1±8.9 Serum uric acid before feeding (μmol / L) 512±53 511±56 516±56 510±48 Serum uric acid after feeding (μmol / L) 521±53 417±54 458±53 462±50

[0069] As shown in Table 4, after 30 days, the blood uric acid level in the test group 1 before and after the trial was significantly reduced (P<0.01), and was significantly better than that in the control group and test groups 2-3, and the difference between the groups was extremely significant (P<0.01). Therefore, the combination of the present invention has the effect of regulating blood uric acid, maintaining the high-dose blood purification effect of trioxygen and maintaining the uric acid metabolism function.

Claims

1. A selected bacterial strain-prebiotic composition for enhancing and maintaining the blood purification effect of high-dose ozone and its use in lowering blood uric acid, characterized in that: The selected strain is Lactobacillus plantarum BLP-17, which is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC M 20221833; the selected strain-prebiotic composition is a composition of freeze-dried powder of Lactobacillus plantarum BLP-17 and one or more prebiotics such as oligomannose, stachyose, L-arabinose, oligoxylose, inulin, etc.

2. The selected strain according to claim 1, characterized in that: The identified Lactobacillus plantarum BLP-17 was expanded and cultured, centrifuged at 3000 rpm for 10 min, the supernatant was discarded, the bacteria were obtained, freeze-dried and crushed, and the freeze-dried powder of Lactobacillus plantarum BLP-17 was obtained.

3. The freeze-dried powder of Lactobacillus plantarum BLP-17 according to claim 2, characterized in that The total viable count was 7×10 10 ~1.3×10 12 CFU / g.

4. The composition according to claim 1, characterized in that The weight ratio of the Lactobacillus plantarum BLP-17 freeze-dried powder to one or more prebiotics such as oligomannosaccharides, stachyose, L-arabinose, oligoxylose, inulin, etc. is 1:(49-99).

5. According to claim 1, the high-dose ozone blood purification combined with a specially selected bacterial strain-prebiotic composition and its use in lowering blood uric acid, the specific technical scheme is to use autologous blood transfusion of 150-2000 ml, 1-2 times a month; the specially selected targeted bacterial strain-prebiotic composition is 5-8g each time, taken twice a day, and a course of treatment is 15-30 days.

6. Use of the composition according to any one of claims 1 to 4 in the preparation of food, medicine and health care products for preventing and treating hyperuricemia and / or gout.

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