Preparation method and application of lactobacillus paracasei for improving hyperuricemia

Abstract

The invention relates to the field of lactobacillus paracasei, in particular to a preparation method and application of lactobacillus paracasei for improving hyperuricemia, and the preparation method comprises the following steps: activating a lactobacillus paracasei Jlus66 strain through an MRS liquid culture medium at 36-38 DEG C, then adding the activated Jlus66 strain into the MRS liquid culture medium, and culturing at 36-38 DEG C to obtain the lactobacillus paracasei for improving hyperuricemia. And then centrifuging and abandoning supernate to obtain thalli, and dissolving the thalli with normal saline until the concentration of a bacterial suspension is 1 * 10 < 9 > cfu / ml. The invention provides a method for regulating uric acid metabolism by regulating intestinal flora through the lactobacillus paracasei Jlus66 so as to improve hyperuricemia, side effects can be effectively avoided, and the method is a more natural and safer way.

Description

Technical Field

[0001] The invention relates to the field of Lactobacillus paracasei, and in particular to a preparation method and application of Lactobacillus paracasei for improving hyperuricemia. Background Art

[0002] Hyperuricemia is one of the most important metabolic diseases caused by high uric acid. The incidence rate is high in many countries, and the trend is obviously younger. The gradual increase in the intake of high-fat, high-purine foods (seafood, beer and red meat) and the sub-healthy physical state under excessive stress can easily lead to elevated uric acid levels in the body. Purine metabolism disorders, excessive uric acid production or reduced uric acid excretion can lead to abnormally high uric acid levels in the blood, causing hyperuricemia and eventually developing into gout. Gout is a crystal-related joint disease caused by the deposition of monosodium uric acid crystals in or around the joints. In addition, hyperuricemia and gout also increase the probability of cardiovascular disease, chronic kidney disease, diabetes, metabolic syndrome and neurodegenerative diseases.

[0003] In the human body, endogenous and exogenous nucleotides are converted into nucleosides, which are further converted into purine bases. Uric acid is the terminal metabolite of purine compounds. A moderate amount of uric acid is beneficial, and hyperuricemia without gout is usually "asymptomatic", but multiple factors such as genetic diversity, environmental exposure, gene-environment interaction and intrinsic risk factors (including age, gender and weight) increase the risk of gout. At present, the main drugs for the clinical treatment of hyperuricemia are allopurinol, colchicine, benzbromarone, etc., but they are usually accompanied by adverse reactions such as gastrointestinal irritation or liver and kidney damage. In terms of non-drug intervention, low-purine diet as a clinical nutritional strategy to reduce serum uric acid levels and gout risk has limited effectiveness and poor compliance. The key to uric acid metabolism in the body is the balance between uric acid produced by the liver and uric acid excreted through the kidneys and intestines. About two-thirds of the uric acid in the body is excreted mainly through the kidneys, while the remaining one-third is excreted through the intestines. For patients with renal insufficiency, intestinal secretion of uric acid has also become an important pathway, and transport proteins that promote uric acid excretion are present in various parts of the intestine. Intestinal dysbiosis can lead to increased serum uric acid concentrations, damaged intestinal barriers, increased permeability, and excessive lipopolysaccharide entering the blood. Lipopolysaccharide binds to the TLR4 receptor, causing upregulation of proinflammatory cytokines. When renal function is impaired due to inflammation, it can lead to abnormal uric acid metabolism in the kidneys. Therefore, the use of more natural, effective, and safe functional ingredients is of great significance for the future treatment of hyperuricemia. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a preparation method of Lactobacillus paracasei for improving hyperuricemia.

[0005] To achieve the above object, the present invention provides the following technical solutions: A method for preparing Lactobacillus paracasei for improving hyperuricemia comprises the following steps: The Lactobacillus paracasei Jlus66 strain was activated by MRS liquid medium at 36-38°C, and then the activated Jlus66 strain was added to the MRS liquid medium and cultured at 36-38°C, and then centrifuged and the supernatant was discarded to obtain the bacterial cells and dissolved with physiological saline to make the bacterial suspension concentration of 1×10 9 cfu / ml is enough, the Lactobacillus paracasei Jlus66 strain is preserved in the General Microbiology Center of China Microbiological Culture Collection Administration, with the preservation number CGMCC No.22101. The strain was obtained after a series of screening experiments in "natural fermented milk" in Jilin City, Jilin Province.

[0006] As a further solution of the present invention: the Lactobacillus paracasei Jlus66 strain is activated in the MRS liquid culture medium for three consecutive times.

[0007] As a further solution of the present invention: the volume ratio of the activated Jlus66 strain to the MRS liquid culture medium is 1:50.

[0008] As a further embodiment of the present invention, the activation and culture temperature of Lactobacillus paracasei in MRS liquid culture medium are both 37°C.

[0009] As a further solution of the present invention: the centrifugal frequency is 3500-4500 r / min, and the centrifugal time is 20 min.

[0010] The present invention also provides the use of the bacterial suspension obtained by the above preparation method in preparing a drug for improving hyperuricemia.

[0011] Compared with the prior art, the beneficial effect of the present invention is that the method proposed by the present invention of regulating intestinal flora to regulate uric acid metabolism by Lactobacillus paracasei Jlus66 and thus improving hyperuricemia can effectively avoid side effects and is a more natural and safe approach. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Graph showing the uric acid content in the serum of each group of mice in the examples of the present invention.

[0013] Figure 2 Graph showing the urea nitrogen content in the serum of each group of mice in the examples of the present invention.

[0014] Figure 3 Graph showing the creatinine content in the serum of each group of mice in the examples of the present invention.

[0015] Figure 4Graph showing the content of pro-inflammatory factor TNF-α in the serum of each group of mice in the examples of the present invention.

[0016] Figure 5 Graph showing the content of pro-inflammatory factor IL-1β in the serum of each group of mice in the examples of the present invention.

[0017] Figure 6 Graph showing the content of pro-inflammatory factor IL-6 in the serum of each group of mice in the examples of the present invention.

[0018] Figure 7 Graph showing xanthine oxidase levels in the serum of each group of mice in the examples of the present invention.

[0019] Figure 8 Graph showing xanthine oxidase content in the liver of each group of mice in the examples of the present invention.

[0020] Fig. 9 Graph showing the ABCG2 and URAT1 levels in the kidneys of each group of mice in the examples of the present invention. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Probiotics are human-synthesized nutrients and functional metabolites, and have made rapid progress in preventing or treating a variety of metabolic syndromes such as obesity, diabetes, inflammatory bowel disease, and chronic kidney disease. Adding the Lactobacillus paracasei Jlus66 prepared by the present invention to food or medicine can regulate intestinal flora, restore the host's purine metabolism, reduce uric acid content and xanthine oxidase activity, regulate uric acid transporter expression, and effectively prevent uric acid accumulation. Improving hyperuricemia includes reducing the content of related uric acid metabolic factors and proinflammatory factors in serum, reducing the activity of key enzymes for uric acid synthesis in serum and liver, up-regulating the expression of uric acid reabsorption transporters in the kidneys, and down-regulating the expression of uric acid secretion transporters, and the application includes medicines.

[0023] Embodiment: A method for preparing Lactobacillus paracasei for improving hyperuricemia comprises the following steps: The Lactobacillus paracasei Jlus66 strain with the deposit number of CGMCC No.22101 was activated three times in MRS liquid medium at 37°C. The activated Jlus66 strain was added to MRS liquid medium at a volume ratio of 2% of the strain and MRS liquid medium, and cultured at 37°C for 24 h. The strain was centrifuged at 4000 r / min for 20 min to obtain bacterial precipitation, the supernatant was discarded, the bacterial cells were separated, and dissolved with physiological saline to adjust the concentration of the bacterial suspension to 1×10 9 cfu / ml is enough.

[0024] Experimental mice grouping and intervention Six-week-old male C57BL / 6J mice (18 ± 2 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Liaoning, China) and acclimatized for 7 days at 22 ± 2 °C, 55 ± 5% relative humidity, and a fixed 12 h light-dark cycle with free access to laboratory standard chow and purified water.

[0025] After the adaptation period, the mice were randomly divided into 4 groups (n=10): (1) blank control group (CON), which ate and drank normal water, received intraperitoneal injection of 0.2 ml of normal saline daily, and gavage of 0.1 ml / 10 g of normal saline daily; (2) positive control group (AC), which ate a high uric acid diet supplemented with 20% yeast by mass fraction, drank normal water, received intraperitoneal injection of 0.2 ml of 300 mg / kg potassium oxonate suspension (OXO) dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na) by mass volume ratio daily, and gavage of 0.1 ml / 10 g of allopurinol aqueous solution; (3) hyperuric acid model group (HUA), which ate a high uric acid diet, drank normal water, received intraperitoneal injection of OXO daily, and gavage of 0.1 ml / 10 g of normal saline daily; (4) probiotic experimental group (Jlus66), which ate a high uric acid diet, drank normal water, received intraperitoneal injection of OXO daily, and gavage of 0.1 ml / 10 g of the product of the above embodiment.

[0026] After 8 weeks, follow-up testing was performed.

[0027] Effects of Lactobacillus paracasei Jlus66 on related uric acid metabolic factors in serum See also Figure 1 The serum UA (uric acid) level in the HUA group was significantly higher than that in the CON group, indicating that the mouse model was successfully established. In contrast, the serum uric acid level in the Jlus66 group and the AC group decreased.

[0028] See Figure 2-Figure 3CRE (creatinine content) and BUN (urea nitrogen content) are two main indicators for clinical evaluation of renal impairment. When renal function is impaired or damaged, serum CRE and BUN levels will increase. Compared with the CON group, BUN and CRE increased in the HUA group after feeding, while they decreased significantly in the Jlus66 group.

[0029] See Figure 7-Figure 8 , overactivation of key urea synthases in the liver, such as XOD (Xanthine Oxidase), can lead to increased uric acid secretion and further to the occurrence of hyperuricemia. The XOD activity in the serum of the HUA group was higher, and it returned to normal levels after supplementation with Jlus66. The XOD level in the liver also underwent the same changes, and the changes were even more obvious.

[0030] See Figure 4-Figure 6 , the levels of serum pro-inflammatory factors TNF-α, IL-1β, and IL-6 were significantly upregulated in the HUA group and returned to normal after supplementation with Jlus66.

[0031] See Fig. 9 ABCG2 and URAT1 are important uric acid secretion transporters and uric acid reabsorption transporters for uric acid transport. After Jlus66 intervention, the expression of ABCG2 was significantly upregulated and the expression of URAT1 was downregulated.

[0032] Therefore, the above results show that Lactobacillus paracasei Jlus66 can regulate uric acid metabolism in hyperuricemia model mice, and has the function of reducing uric acid and improving hyperuricemia in mice. Specifically, Lactobacillus paracasei Jlus66 can significantly reduce the content of uric acid, urea nitrogen, creatinine, proinflammatory factors TNF-α, IL-1β, and IL-6 in serum, reduce the activity of xanthine oxidase in serum and liver, upregulate the expression of uric acid reabsorption transporter in kidney, and downregulate the expression of uric acid secretion transporter. It provides theoretical reference and basis for Lactobacillus paracasei Jlus66 in the development of products to improve hyperuricemia, and overcome the defects of adverse reactions of existing therapeutic drugs.

[0033] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for preparing a Lactobacillus paracasei for improving hyperuricemia, characterized in that: The following steps are involved: The Lactobacillus paracasei Jlus66 strain was activated by MRS liquid medium at 36-38°C, and then the activated Jlus66 strain was added to the MRS liquid medium and cultured at 36-38°C, and then centrifuged and the supernatant was discarded to obtain the bacterial cells and dissolved with physiological saline to make the bacterial suspension concentration of 1×10 9 cfu / ml, the Lactobacillus paracasei Jlus66 strain is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and the deposit number is CGMCC No.22101.

2. The preparation method of the Lactobacillus paracasei for improving hyperuricemia according to claim 1, wherein The activation method of the Lactobacillus paracasei Jlus66 strain in the MRS liquid culture medium is three consecutive activations.

3. The preparation method of the Lactobacillus paracasei for improving hyperuricemia according to claim 1 or 2, characterized in that, The volume ratio of the activated Jlus66 strain to the MRS liquid culture medium is 1:

50.

4. The preparation method of Lactobacillus paracasei for improving hyperuricemia according to claim 1, characterized in that, The Lactobacillus paracasei was activated and cultured in MRS liquid culture medium at a temperature of 37°C.

5. The preparation method of Lactobacillus paracasei for improving hyperuricemia according to claim 1, characterized in that, The centrifugal frequency was 3500-4500 r / min and the centrifugal time was 20 min.

6. Use of a bacterial suspension obtained by the method for preparing Lactobacillus paracasei for improving hyperuricemia as claimed in any one of claims 1 to 5 in preparing a drug for improving hyperuricemia.

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