Lactobacillus paracasei Lpc-C66 capable of improving hyperuricemia and application of lactobacillus paracasei Lpc-C66

Abstract

The invention discloses a lactobacillus paracasei Lpc-C66 capable of improving hyperuricemia and application of the lactobacillus paracasei Lpc-C66, the lactobacillus paracasei Lpc-C66 is preserved in the China Center for Type Culture Collection on December 7, 2023, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M20232496. A product prepared from the lactobacillus paracasei Lpc-C66 disclosed by the invention has the effect of improving hyperuricemia, and has good safety.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus paracasei Lpc-C66 having the function of improving hyperuricemia and an application thereof. Background Art

[0002] Hyperuricemia (HUA) is an inflammatory disease caused by purine metabolism disorder, characterized by a persistent increase in the concentration of uric acid (UA) in the blood. Generally, hyperuricemia is diagnosed when the serum uric acid level exceeds 0.42mmol / L (7mg / dl) in men and 0.36mmol / L (6mg / dl) in women. Long-term hyperuricemia not only causes gout, but is also closely related to a variety of metabolic diseases such as chronic kidney disease, diabetes, and cardiovascular disease. In recent years, with the improvement of living standards, the global incidence of hyperuricemia has increased by about 20%, and patients in my country account for 13.3% of the total population. The age of onset tends to be younger, and effective treatment is urgently needed. Traditional treatment methods mainly include drug therapy and diet control, but these methods often have obvious side effects and poor compliance. At present, treatment mainly relies on allopurinol and benzbromarone; but drugs may cause liver and kidney damage and allergic reactions, especially in asymptomatic patients, and the risks and benefits of drugs are controversial. Therefore, the treatment of hyperuricemia has become an important issue that needs to be solved urgently.

[0003] In recent years, with the deepening of probiotic research, people have found that some probiotic strains may have a mitigating effect on hyperuricemia through various mechanisms. For example, some probiotics can inhibit the activity of xanthine oxidase and reduce the production of uric acid; or absorb and degrade purine substances in the intestine to reduce their absorption rate; in addition, probiotics can regulate the homeostasis of intestinal flora and restore intestinal barrier function, thereby reducing the production of endogenous uric acid. As active microorganisms, probiotics can provide health benefits to the host, and because of their low damage to the human body, they are widely accepted as cost-effective treatment methods. Studies have shown that intestinal flora plays an important role in regulating the immune system and protecting the host. Hyperuricemia is closely related to the imbalance of intestinal flora. Probiotics can regulate intestinal microorganisms, enhance intestinal barrier function, and reduce blood uric acid levels by proliferating Lactobacillus and Bifidobacterium. In addition, short-chain fatty acids (such as acetic acid and butyric acid), metabolites of intestinal flora, also play an important role in regulating host metabolism and immunity. Sodium acetate can inhibit the activity of xanthine oxidase and help control uric acid production. Despite this, the application of probiotics in alleviating hyperuricemia is still in the exploratory stage, and there is still a lack of probiotic strains with clear efficacy and mechanism of action. Summary of the invention

[0004] The object of the present invention is to provide a product of Lactobacillus paracasei Lpc-C66 having the effect of improving hyperuricemia and its application. The product prepared by Lactobacillus paracasei Lpc-C66 of the present invention has the effect of improving hyperuricemia and has good safety.

[0005] The technical solution of the present invention is: a Lactobacillus paracasei Lpc-C66, wherein the Lactobacillus paracasei Lpc-C66 has been deposited in the China Center for Type Culture Collection on December 7, 2023, with a deposit number of CCTCC NO: M 20232496.

[0006] The application of the Lactobacillus paracasei Lpc-C66 in the preparation of products for improving hyperuricemia.

[0007] In the aforementioned application, the product is a bacterial agent, food and / or medicine.

[0008] In the aforementioned application, the food is one or more of fermented milk, cheese, milk-containing beverage, solid beverage, and milk powder; the food contains live bacterial liquid and / or inactivated bacterial liquid of Lactobacillus paracasei Lpc-C66.

[0009] In the above application, the medicine comprises live bacterial liquid and / or inactivated bacterial liquid of Lactobacillus paracasei Lpc-C66, and / or other medicines compatible with the live bacterial liquid and / or inactivated bacterial liquid of Lactobacillus paracasei Lpc-C66, as well as pharmaceutically acceptable carriers and / or excipients.

[0010] In the aforementioned application, the Lactobacillus paracasei Lpc-C66 is used to prepare a bacterial agent, food and / or medicine for reducing plasma uric acid and / or plasma xanthine enzyme activity.

[0011] In the above application, the Lactobacillus paracasei Lpc-C66 is used to prepare a bacterial agent, food and / or medicine for reducing the activity of liver adenosine deaminase and / or liver xanthine enzyme.

[0012] In the aforementioned application, the Lactobacillus paracasei Lpc-C66 is used to prepare a bacterial agent, food and / or medicine for reducing the level of alanine aminotransferase and / or aspartate aminotransferase in plasma.

[0013] The aforementioned application, the Lactobacillus paracasei Lpc-C66 is used to prepare a bacterial agent, food and / or medicine for reducing urea nitrogen and creatinine levels in the liver

[0014] In the aforementioned application, the inactivated bacterial solution of Lactobacillus paracasei Lpc-C66 is prepared as follows:

[0015] 1 g of Lpc-C66 bacterial powder was dissolved in 500 mL of LB liquid culture medium, and after culturing at 37°C for 24 h, the bacterial suspension was inactivated at 121°C for 30 min under high temperature and high pressure, and centrifuged at 3000 rpm for 15 min at room temperature. After the centrifugation, the supernatant was removed, and an appropriate amount of PBS was added to wash away the residual culture medium, and the mixture was centrifuged again to remove the supernatant to obtain the inactivated animal Bifidobacterium lactis subsp. Lpc-C66 bacterial cells. 1 mL of physiological saline was added to every 50 mg of the bacterial cells to dissolve the inactivated Lactobacillus paracasei Lpc-C66 bacterial liquid.

[0016] Compared with the prior art, Lactobacillus paracasei Lpc-C66 of the present invention can alleviate hyperuricemia by reducing plasma uric acid, reducing plasma xanthine enzyme activity and reducing liver xanthine enzyme activity, can effectively reduce blood uric acid level, and has good safety. Experiments have shown that the live bacteria liquid and the inactivated bacteria liquid of Lactobacillus paracasei Lpc-C66 of the present invention all have the ability of improving hyperuricemia well. In addition, Bifidobacterium lactis subspecies BA-C9 of the present invention has good gastrointestinal tolerance, and therefore is easier to survive by stomach and small intestine after product is taken orally or eaten. The present invention not only provides a new probiotic strain, but also provides a scientific basis for its application in functional foods and pharmaceutical preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The Gram-stained bacterial morphology of Lactobacillus paracasei Lpc-C66 of the present invention;

[0018] Figure 2 The invention discloses the effect of Lactobacillus paracasei Lpc-C66 on the activity of plasma uric acid (UA) and xanthine oxidase (XOD) in hyperuricemia mice.

[0019] Figure 3 The invention relates to the effect of Lactobacillus paracasei Lpc-C66 on the activities of adenosine deaminase (ADA) and xanthine oxidase (XOD) in the liver of hyperuricemia mice;

[0020] Figure 4 The invention relates to the effect of Lactobacillus paracasei Lpc-C66 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in the plasma of hyperuricemia mice;

[0021] Figure 5 The invention discloses the influence of Lactobacillus paracasei Lpc-C66 on blood urea nitrogen (BUN) and creatinine (Cr) in the plasma of hyperuricemia mice.

[0022] Figure 6 The invention discloses the effect of the inactivated Lactobacillus paracasei Lpc-C66 on the plasma uric acid (UA) and xanthine oxidase (XOD) activity in hyperuricemia mice;

[0023] Figure 7 The invention discloses the effect of the inactivated Lactobacillus paracasei Lpc-C66 on the activities of adenosine deaminase (ADA) and xanthine oxidase (XOD) in the liver of hyperuricemia mice;

[0024] Figure 8 The invention discloses the effect of the inactivated Lactobacillus paracasei Lpc-C66 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in the plasma of hyperuricemia mice;

[0025] Fig. 9 The invention discloses the influence of the inactivated Lactobacillus paracasei Lpc-C66 on blood urea nitrogen (BUN) and creatinine (Cr) in the plasma of hyperuricemia mice. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0027] Embodiment 1, the screening and identification of Lactobacillus paracasei Lpc-C66:

[0028] 1. Sample source;

[0029] The strain used in the present invention was isolated from the feces of a six-year-old child.

[0030] 2. Isolation and purification of strains;

[0031] Take out the stored strain from the -80℃ refrigerator and inoculate it onto fresh LB medium at pH 6 by streaking. Invert and incubate in a 37℃ constant temperature incubator for 2-3 days. When a single clone grows out, pick it with an inoculation loop and inoculate it onto fresh LB medium at pH 6 by streaking. After a single clone grows out, store it in a 4℃ refrigerator for later use.

[0032] 3. Identification of Lactobacillus paracasei Lpc-C66;

[0033] 3.1. Colony characteristics;

[0034] After culturing the isolated and purified Lactobacillus paracasei Lpc-C66 in LB solid medium for 48 hours, the colonies become white or light yellow, viscous, sometimes creamy, with neat edges and a diameter of up to 1-2 mm.

[0035] 3.2、Morphology under microscope;

[0036] Colony smear of Lactobacillus paracasei Lpc-C66: Gram-positive, rod-shaped, square at both ends, arranged singly or in chains, without flagella, non-motile, see Figure 1 , Figure 1The figure is a Gram-stained bacterial morphology of Lactobacillus paracasei Lpc-C66 of the present invention.

[0037] 3.3, 16S rDNA identification;

[0038] The sequencing results of Lactobacillus paracasei Lpc-C66 are as follows:

[0039]

[0040] The strain of the present invention is deposited under the name of Lactobacillus paracasei Lpc-C66, the depository unit is China Center for Type Culture Collection, the depository address is Wuhan University, Wuhan, China, the depository number is CCTCC NO: M 20232496, and the depository date is December 7, 2023.

[0041] Example 2: Lactobacillus paracasei Lpc-C66 reduces uric acid in hyperuricemia mice:

[0042] 1. Experimental materials;

[0043] Thirty-two C57BL / 6 male mice were purchased from Shanghai Slake Experimental Animal Center (company license number: SCXK(Shanghai)2013-0016) and were kept in the SPF-level animal breeding room of the Experimental Animal Center of Zhejiang Chinese Medical University.

[0044] 2. Reagents: ALT kit (Cat. No.: C009-2 Nanjing Jiancheng Bioengineering Institute), AST kit (Cat. No.: C010-2 Nanjing Jiancheng Bioengineering Institute), TG kit (Cat. No.: A110-1-1 Nanjing Jiancheng Bioengineering Institute), TC kit (Cat. No.: A111-1-1 Nanjing Jiancheng Bioengineering Institute), urea nitrogen kit (Cat. No.: C013-2-1 Nanjing Jiancheng Bioengineering Institute), creatinine kit (Cat. No.: C011-2-1 Nanjing Jiancheng Bioengineering Institute), mouse xanthine oxidase (XOD) ELISA kit (Cat. No.: CB10361-Muco Abogen), mouse adenosine deaminase (ADA) ELISA kit (Cat. No.: CB10711-Muco Abogen).

[0045] 3. Rearing environment: room temperature 20-25℃, light and dark alternation every 12 hours, relative humidity about 50%, feed, drinking water, etc. are uniformly matched, keep the indoor environment well ventilated, clean every day, and maintain a good hygienic environment in the cage. All experiments comply with the Animal Ethics Code and the relevant regulations of the Laboratory Animal Ethics Committee, and are specifically carried out in accordance with IAC and other relevant standard operating procedures (SOP). After the experiment, blood is collected from the abdominal aorta under anesthesia, and the bodies are uniformly handled harmlessly.

[0046] 4. Methods:

[0047] Laboratory animal husbandry;

[0048] Rearing environment: room temperature 20-25℃, light and dark alternation every 12 hours, relative humidity about 50%, feed, drinking water, etc. are uniformly matched, keep the indoor environment well ventilated, clean every day, and maintain a good hygienic environment in the cage. All experiments comply with the Animal Ethics Code and the relevant regulations of the Experimental Animal Ethics Committee, and are specifically carried out in accordance with IAC and other relevant standard operating procedures (SOP). After the experiment, blood was collected from the abdominal aorta under anesthesia, and the bodies were uniformly handled harmlessly.

[0049] After one week of adaptation feeding in an SPF animal laboratory, C57BL / 6 mice were randomly divided into a normal control group (CON group) (n=10); a hyperuric acid model group (MOD group) (n=10); an Lpc-C66+ model group (Lpc-C66 group) (n=10); and an allopurinol+ model group (Allopurinol group) (n=10). The CON group was fed a normal diet and water, while the other groups were fed a high-purine diet. To induce hyperuricemia (HUA), mice were intraperitoneally injected with 200 mg / kg of potassium oxonate dissolved in 0.5% CMC-Na solution, and the injection lasted for 2 weeks. After successful modeling, oral administration began. The mice in the Lactobacillus paracasei group were given Lpc-C66 by oral administration (4×10 9 CFU / mL, 0.25mL / day / mouse), and mice in the allopurine group were given allopurine by gavage (40mg / kg, 0.25mL / day / mouse). The intervention lasted for 2 weeks, and all mice were allowed to eat and drink freely, and their body weight was measured every week. The feed consumed by the mice was replaced with new feed every 5 days, and the body weight of the mice was recorded every week to detect changes. After the feeding, the mice were anesthetized with 1% pentobarbital intraperitoneal injection, and blood was taken from the inferior vena cava to measure biochemical indicators such as ALT and AST.

[0050] Index determination:

[0051] 4.1. Detection of plasma uric acid (UA) level;

[0052] Serum or plasma samples can be measured directly. If there are suspended solids in the sample, they can be removed by centrifugation. Depending on the expected uric acid concentration in the sample, the sample may need to be appropriately diluted. The diluent is usually physiological saline (0.9% NaCl) or PBS (0.01M, pH 7.4). In a 96-well microplate, add 25 μL of different concentrations of standard to each standard tube, and add 25 μL of the sample to be tested to each assay tube. Add 250 μL of reagent 2 to each well. Vortex mix for 3 seconds, let stand at room temperature for 5 minutes, then centrifuge at 2000×g for 5 minutes and take the supernatant. Transfer 160 μL of supernatant to a new microplate well. Add 50 μL of reagent 3 and reagent 4 to each well. Use an ELISA reader to vibrate the plate for 10 seconds and let stand at room temperature for 15 minutes. Use an ELISA reader to measure the absorbance (OD) value of each well at a wavelength of 690 nm. Calculate the uric acid content in the sample using the standard curve. The standard curve is drawn by measuring the OD values ​​of a series of standards with known concentrations.

[0053] 4.2. Detection of plasma and liver xanthine (XOD) enzyme activity;

[0054] Dilute the standard according to the instructions. Then set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50μL of the standard on the enzyme-labeled plate, add 40μL of sample diluent to the test sample wells, and then add 10μl of the test sample (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix. Seal the plate with a sealing film and incubate at 37℃ for 30min. Dilute the 30-fold concentrated washing solution with distilled water 30 times for use. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30s and discard it, repeat this 5 times, and pat dry. Add 50μL of enzyme-labeled reagent to each well, except for the blank well. Once again, seal the plate with a sealing film and incubate at 37°C for 30 minutes. Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard it. Repeat this 5 times and pat dry. First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color at 37°C in the dark for 10 minutes. Add 50μL of stop solution to each well to stop the reaction (the blue color immediately turns yellow). Use the blank air conditioner as zero and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0055] 4.3. Plasma ALT detection;

[0056] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding ALT / GPT activity unit.

[0057] 4.4. Plasma AST detection;

[0058] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding AST / GPT activity unit.

[0059] 4.5. Detection of liver ADA content;

[0060] Take 20 mg of mouse liver and grind the liver with PBS as solvent according to the ratio of plastids to 1:9. Set up blank wells, standard wells and test sample wells respectively. Accurately add 50 μL of standard sample to the standard well, add 40 μL of sample diluent to the test sample well first, and then add 10 μL of the test sample (the final dilution of the sample is 5 times). Add the sample to the bottom of the ELISA plate well, avoid touching the well wall, and mix gently. Seal the plate with a sealing film and incubate at 37°C for 30 minutes. Dilute the 30-fold concentrated washing solution 30 times with distilled water for later use. Remove the sealing film, discard the liquid, fill each well with washing solution, let it stand for 30 seconds and discard it, repeat 5 times, and pat dry. Add 50 μL of enzyme labeling reagent to each well (except blank wells), seal the plate with a sealing film, and incubate at 37°C for 30 minutes. Wash again 5 times and pat dry. Add 50μL of color developer A and 50μL of color developer B to each well, mix well, and color at 37℃ in the dark for 10 minutes. Add 50μL of stop solution to stop the reaction (blue turns to yellow). Take the blank well as the zero point and measure the absorbance (OD value) of each well at a wavelength of 450nm. The measurement should be completed within 15 minutes after adding the stop solution.

[0061] 4.6. Examination of plasma creatinine (Cr) content;

[0062] Take mouse plasma samples and preheat the matrix solution at 37°C. Add 20 μL of matrix solution and 5 μL of the sample to be tested to the sample well and mix well; add 20 μL of matrix solution to the control well. Incubate all wells at 37°C for 30 minutes. Then add creatinine amidohydrolase and continue incubation for 15 minutes. Then add creatine amidohydrolase and sarcosine oxidase and incubate for another 15 minutes. Subsequently, add 2,4-(6-triiodo-3-hydroxybenzoic acid) and 4-aminoantipyrine, add peroxidase, mix well and react at room temperature for 10 minutes. Finally, use a spectrophotometer with a wavelength of 546 nm to measure the absorbance (OD value) of the purple-red compound to calculate the creatinine content in plasma.

[0063] 4.7. Plasma urea nitrogen (BUN) content examination;

[0064] Take mouse plasma samples and preheat the matrix solution at 37°C. Add 20 μL of matrix solution and 5 μL of the sample to be tested to the sample well and mix well; add 20 μL of matrix solution to the control well. Then incubate at 37°C for 30 minutes. Then add urease and continue incubation for 15 minutes. After that, add 20 μL of phenol color developer and alkaline sodium hypochlorite to each well and leave it at room temperature for 10 minutes to develop color. Finally, use a spectrophotometer with a wavelength of 640 nm to measure the absorbance (OD value) of each well.

[0065] Experimental results and analysis:

[0066] Figure 2 The invention discloses the effects of Lactobacillus paracasei Lpc-C66 on plasma uric acid (UA) and xanthine oxidase (XOD) activity in hyperuricemia mice. Figure 2 The results showed that compared with the CON group, the levels of uric acid (UA) and xanthine oxidase (XOD) in the serum of mice in the MOD group were significantly increased, both more than twice the normal levels (p<0.05). After 14 days of oral gavage treatment, the results showed that the levels of UA and XOD in the serum of mice decreased significantly after administration of L. paracasei Lpc-C66 (p<0.05). The levels of UA and XOD in the Lpc-C66 group were similar to those in the Allopurinol group and significantly lower than those in the MOD group (p<0.05). These results indicate that L. paracasei Lpc-C66 has a potential therapeutic effect and can exert its effect by reducing the serum biochemical indicators of HUA model mice.

[0067] Figure 3 The invention relates to the effect of Lactobacillus paracasei Lpc-C66 on the activities of adenosine deaminase (ADA) and xanthine oxidase (XOD) in the liver of hyperuricemia mice; Figure 3The results showed that the levels of adenosine deaminase (ADA) and xanthine oxidase (XOD) in the liver of the MOD group were significantly increased compared with the CON group (p<0.05). In contrast, the ADA and XOD enzyme activities in the Lpc-C66 group were inhibited, and their levels were similar to those in the Allopurinol group. This suggests that L. paracasei Lpc-C66 may reduce uric acid levels by inhibiting the activity of hepatic urate synthase.

[0068] Figure 4 The invention discloses the influence of Lactobacillus paracasei Lpc-C66 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in the plasma of hyperuricemia mice. Figure 4 The results showed that compared with the CON group, the ALT and AST levels in the liver of the MOD group were significantly increased (p<0.05). Compared with the MOD group, the ALT and AST levels in the Lpc-C66 group and the Allopurinol group were significantly decreased (p<0.05). At the same time, the plasma ALT and AST levels in the Lpc-C66 group were significantly lower than those in the Allopurinol group, indicating that L. paracasei Lpc-C66 has a better protective effect on the liver.

[0069] Figure 5 The invention discloses the influence of Lactobacillus paracasei Lpc-C66 on blood urea nitrogen (BUN) and creatinine (Cr) in the plasma of hyperuricemia mice. Figure 5 The results showed that the levels of BUN and Cr in the liver of the MOD group were significantly higher than those of the CON group (p<0.05). Compared with the MOD group, the levels of BUN and Cr in the Lpc-C66 group and the Allopurinol group were significantly lower (p<0.05). In addition, the plasma BUN and Cr levels in the Lpc-C66 group were significantly lower than those in the Allopurinol group, indicating that L. paracasei Lpc-C66 has a better protective effect on the kidneys.

[0070] Embodiment 3, the preparation of inactivation Lactobacillus paracasei Lpc-C66 bacterial liquid:

[0071] 1 g of Lpc-C66 bacterial powder was dissolved in 500 mL of LB liquid culture medium, and after culturing at 37°C for 24 h, the bacterial suspension was inactivated at 121°C for 30 min under high temperature and high pressure, and centrifuged at 3000 rpm for 15 min at room temperature. After the centrifugation, the supernatant was removed, and an appropriate amount of PBS was added to wash away the residual culture medium, and the mixture was centrifuged again to remove the supernatant to obtain the inactivated animal Bifidobacterium lactis subsp. Lpc-C66 bacterial cells. 1 mL of physiological saline was added to every 50 mg of the bacterial cells to dissolve the inactivated Lactobacillus paracasei Lpc-C66 bacterial liquid.

[0072] Example 4: Inactivation of Lactobacillus paracasei Lpc-C66 to relieve hyperuricemia

[0073] 1. Experimental materials:

[0074] Thirty-two C57BL / 6 male mice were purchased from Shanghai Slake Experimental Animal Center (company license number: SCXK(Shanghai)2013-0016) and were kept in the SPF-level animal breeding room of the Experimental Animal Center of Zhejiang Chinese Medical University.

[0075] 2. Reagents: ALT kit (C009-2 Nanjing Jiancheng Bioengineering Institute), AST kit (C010-2 Nanjing Jiancheng Bioengineering Institute), TG kit (C110-1-1 Nanjing Jiancheng Bioengineering Institute), TC kit (C111-1-1 Nanjing Jiancheng Bioengineering Institute), urea nitrogen kit (C013-2-1 Nanjing Jiancheng Bioengineering Institute), creatinine kit (C011-2-1 Nanjing Jiancheng Bioengineering Institute), mouse xanthine oxidase (XOD) ELISA kit (C10361-Muco Abbio), mouse adenosine deaminase (ADA) ELISA kit (C10711-Muco Abbio)

[0076] 3. Rearing environment: room temperature 20-25℃, light and dark alternation every 12 hours, relative humidity about 50%, feed, drinking water, etc. are uniformly matched, keep the indoor environment well ventilated, clean every day, and maintain a good hygienic environment in the cage. All experiments comply with the Animal Ethics Code and the relevant regulations of the Laboratory Animal Ethics Committee, and are specifically carried out in accordance with IAC and other relevant standard operating procedures (SOP). After the experiment, blood is collected from the abdominal aorta under anesthesia, and the bodies are uniformly handled harmlessly.

[0077] 4. Methods:

[0078] Laboratory animal husbandry;

[0079] Rearing environment: room temperature 20-25℃, light and dark alternation every 12 hours, relative humidity about 50%, feed, drinking water, etc. are uniformly matched, keep the indoor environment well ventilated, clean every day, and maintain a good hygienic environment in the cage. All experiments comply with the Animal Ethics Code and the relevant regulations of the Experimental Animal Ethics Committee, and are specifically carried out in accordance with IAC and other relevant standard operating procedures (SOP). After the experiment, blood was collected from the abdominal aorta under anesthesia, and the bodies were uniformly handled harmlessly.

[0080] After one week of adaptation feeding in an SPF animal laboratory, C57BL / 6 mice were randomly divided into a normal control group (CON group) (n=10); a hyperuric acid model group (MOD group) (n=10); an Lpc-C66+ model group (Lpc-C66 group) (n=10); and an inactivated Lpc-C66+ model group (Lpc-C66heat-killed group) (n=10). The CON group was fed a normal diet and water, while the other groups were fed a high-purine diet. To induce hyperuricemia (HUA), mice were intraperitoneally injected with 200 mg / kg of potassium oxonate dissolved in 0.5% CMC-Na solution for 2 weeks. After the model was successfully established, oral administration was started. The mice in the Lactobacillus paracasei group were given Lactobacillus paracasei Lpc-C66 oral administration (4×10^9 CFU / mL, 0.25mL / day / mouse), and the mice in the inactivated Lactobacillus paracasei group were given an equal volume of inactivated Lactobacillus paracasei Lpc-C66 bacterial solution. The intervention lasted for 2 weeks, and all mice were free to eat and drink, and their body weight was measured every week. The feed consumed by the mice was replaced with new feed every 5 days, and the weight of the mice was recorded every week to detect changes. After feeding, the mice were anesthetized with 1% pentobarbital intraperitoneal injection, and blood was taken from the inferior vena cava to measure biochemical indicators such as ALT and AST.

[0081] Index determination:

[0082] 4.1. Detection of plasma uric acid (UA) level;

[0083] Serum or plasma samples can be measured directly. If there are suspended solids in the sample, they can be removed by centrifugation. Depending on the expected uric acid concentration in the sample, the sample may need to be appropriately diluted. The diluent is usually physiological saline (0.9% NaCl) or PBS (0.01M, pH7.4). In a 96-well microplate, add 25 μL of different concentrations of standard to each standard tube, and add 25 μL of the sample to be tested to each assay tube. Add 250 μL of reagent 2 to each well. Vortex mix for 3 seconds, let stand at room temperature for 5 minutes, then centrifuge at 2000×g for 5 minutes and take the supernatant. Transfer 160 μL of supernatant to a new microplate well. Add 50 μL of reagent 3 and reagent 4 to each well. Use an enzyme reader to vibrate the plate for 10 seconds and let stand at room temperature for 15 minutes. Use an enzyme reader to measure the absorbance (OD) value of each well at a wavelength of 690 nm. Calculate the uric acid content in the sample using the standard curve. The standard curve is drawn by measuring the OD values ​​of a series of standards with known concentrations.

[0084] 4.2. Detection of plasma and liver xanthine (XOD) enzyme activity;

[0085] Dilute the standard according to the instructions. Then set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same), standard wells, and test sample wells. Accurately add 50μL of the standard on the enzyme-labeled plate, add 40μL of sample diluent to the test sample wells, and then add 10μl of the test sample (the final dilution of the sample is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix. Seal the plate with a sealing film and incubate at 37℃ for 30min. Dilute the 30-fold concentrated washing solution with distilled water 30 times for use. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30s and discard it, repeat this 5 times, and pat dry. Add 50μL of enzyme-labeled reagent to each well, except for the blank well. Once again, seal the plate with a sealing film and incubate at 37°C for 30 minutes. Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard it. Repeat this 5 times and pat dry. First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color at 37°C in the dark for 10 minutes. Add 50μL of stop solution to each well to stop the reaction (the blue color immediately turns yellow). Use the blank air conditioner as zero and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0086] 4.3. Plasma ALT detection;

[0087] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding ALT / GPT activity unit.

[0088] 4.4. Plasma AST detection;

[0089] Mouse plasma samples were directly sampled for determination. The matrix solution was preheated at 37°C in advance. 20 μL matrix solution and 5 μL sample to be tested were added to the sample wells and mixed. 20 μL matrix solution was added to the control wells and incubated at 37°C for 30 min. 20 μL of 2,4-dinitrophenylhydrazine solution was added to the determination wells and control wells, and 5 μL sample to be tested was added to the control wells, mixed, and incubated at 37°C for 20 min. 200 μL of 0.4 mol / L sodium hydroxide solution was added to each well, mixed, and placed at room temperature for 15 min. The OD value of each well was measured by an enzyme marker at a wavelength of 510 nm, and the standard curve was checked to obtain the corresponding AST / GPT activity unit.

[0090] 4.5. Detection of liver ADA content;

[0091] Take 20 mg of mouse liver and grind the liver with PBS as solvent according to the ratio of plastids to 1:9. Set up blank wells, standard wells and test sample wells respectively. Accurately add 50 μL of standard sample to the standard well, add 40 μL of sample diluent to the test sample well first, and then add 10 μL of the test sample (the final dilution of the sample is 5 times). Add the sample to the bottom of the ELISA plate well, avoid touching the well wall, and mix gently. Seal the plate with a sealing film and incubate at 37°C for 30 minutes. Dilute the 30-fold concentrated washing solution 30 times with distilled water for later use. Remove the sealing film, discard the liquid, fill each well with washing solution, let it stand for 30 seconds and discard it, repeat 5 times, and pat dry. Add 50 μL of enzyme labeling reagent to each well (except blank wells), seal the plate with a sealing film, and incubate at 37°C for 30 minutes. Wash again 5 times and pat dry. Add 50μL of color developer A and 50μL of color developer B to each well, mix well, and color at 37℃ in the dark for 10 minutes. Add 50μL of stop solution to stop the reaction (blue turns to yellow). Take the blank well as the zero point and measure the absorbance (OD value) of each well at a wavelength of 450nm. The measurement should be completed within 15 minutes after adding the stop solution.

[0092] 4.6. Examination of plasma creatinine (Cr) content;

[0093] Take mouse plasma samples and preheat the matrix solution at 37°C. Add 20 μL of matrix solution and 5 μL of the sample to be tested to the sample well and mix well; add 20 μL of matrix solution to the control well. Incubate all wells at 37°C for 30 minutes. Then add creatinine amidohydrolase and continue incubation for 15 minutes. Then add creatine amidohydrolase and sarcosine oxidase and incubate for another 15 minutes. Subsequently, add 2,4-(6-triiodo-3-hydroxybenzoic acid) and 4-aminoantipyrine, add peroxidase, mix well and react at room temperature for 10 minutes. Finally, use a spectrophotometer with a wavelength of 546 nm to measure the absorbance (OD value) of the purple-red compound to calculate the creatinine content in plasma.

[0094] 4.7. Plasma urea nitrogen (BUN) content examination;

[0095] Take mouse plasma samples and preheat the matrix solution at 37°C. Add 20 μL of matrix solution and 5 μL of the sample to be tested to the sample well and mix well; add 20 μL of matrix solution to the control well. Then incubate at 37°C for 30 minutes. Then add urease and continue incubation for 15 minutes. After that, add 20 μL of phenol color developer and alkaline sodium hypochlorite to each well and leave it at room temperature for 10 minutes to develop color. Finally, use a spectrophotometer with a wavelength of 640 nm to measure the absorbance (OD value) of each well.

[0096] Experimental results and analysis

[0097] Figure 6 The invention discloses the effect of the inactivated Lactobacillus paracasei Lpc-C66 on the plasma uric acid (UA) and xanthine oxidase (XOD) activity in hyperuricemia mice; Figure 6 The results showed that compared with the CON group, the levels of uric acid (UA) and xanthine oxidase (XOD) in the serum of mice in the MOD group were significantly increased (p<0.05). After 14 days of oral gavage treatment, the levels of UA and XOD in the serum of mice in the Lpc-C66 group and the Lpc-C66 inactivated group were significantly lower than those in the MOD model group (p<0.05). These results indicate that the inactivated L. paracasei Lpc-C66 also has a therapeutic effect and helps to reduce hyperuricemia-related indicators.

[0098] Figure 7 The invention discloses the effect of the inactivated Lactobacillus paracasei Lpc-C66 on the activities of adenosine deaminase (ADA) and xanthine oxidase (XOD) in the liver of hyperuricemia mice; Figure 7 The results showed that compared with the CON group, the levels of adenosine deaminase (ADA) and xanthine oxidase (XOD) in the liver of mice in the MOD group were significantly increased (p<0.05). After 14 days of oral gavage treatment, the levels of ADA and XOD in the serum of mice in the Lpc-C66 group and the Lpc-C66 inactivated group were significantly lower than those in the MOD model group (p<0.05). It is worth noting that the liver XOD level in the Lpc-C66 inactivated group was lower than that in the Lpc-C66 live bacteria group (p<0.01). These results indicate that the inactivated Lactobacillus paracasei Lpc-C66 also has a good therapeutic effect and helps to reduce hyperuricemia-related indicators.

[0099] Figure 8 The invention discloses the effect of the inactivated Lactobacillus paracasei Lpc-C66 on alanine aminotransferase and aspartate aminotransferase (ALT and AST) in the plasma of hyperuricemia mice; Figure 8 The results showed that compared with the CON group, the ALT and AST levels in the liver of the MOD group mice were significantly increased (p<0.05). Compared with the MOD group, the ALT and AST levels in the Lpc-C66 live bacteria group and the inactivated group were significantly decreased (p<0.05). This indicates that the inactivated L. paracasei Lpc-C66 has a protective effect on the liver.

[0100] Fig. 9 The invention discloses the influence of the inactivated Lactobacillus paracasei Lpc-C66 on blood urea nitrogen (BUN) and creatinine (Cr) in the plasma of hyperuricemia mice. Fig. 9The results showed that the BUN and Cr levels in the liver of the MOD group were significantly higher than those of the CON group (p<0.05). Compared with the MOD group, the BUN and Cr levels in the live and inactivated Lpc-C66 groups were significantly lower (p<0.05). In addition, the plasma Cr content in the inactivated Lpc-C66 group was significantly lower than that in the live group, indicating that the Lpc-C66 inactivated by Lactobacillus paracasei has a better protective effect on the kidneys.

[0101] Example 5: Lactobacillus paracasei Lpc-C66 was used as the research object to simulate the gastrointestinal tolerance experiment, and Lactobacillus paracasei K56 was used as the comparative example.

[0102] Tolerance experiment in gastrointestinal fluid: simulated human gastrointestinal fluid was prepared, and Lactobacillus paracasei Lpc-C66 and Lactobacillus paracasei K56 were treated in the simulated gastrointestinal fluid for different time periods; the plate count method was used to determine the number of viable bacteria of Lactobacillus paracasei Lpc-C66 and Lactobacillus paracasei K56 after treatment in the simulated gastrointestinal fluid.

[0103] Preparation of simulated gastric juice: Use PBS at pH 2.5 to prepare a pepsin solution with a concentration of 3 mg / mL, i.e. artificial gastric juice, which is filtered and sterilized through a 0.22 μm microporous filter membrane for later use.

[0104] Preparation of simulated intestinal fluid: Prepare a trypsin solution with a concentration of 1 mg / mL using PBS at pH 8.0, add ox bile salt to make the final concentration of 1.8%, the obtained solution is the artificial intestinal fluid, and filter it through a 0.22 μm microporous filter membrane for sterilization before use.

[0105] The powder of Lactobacillus paracasei Lpc-C66 and Lactobacillus paracasei K56 was added to sterile PBS (pH 7.3), shaken to resuspend, and 500 μL of the resuspended solution was added to 4.5 mL of artificial gastric juice, incubated in a 37°C incubator for 3 hours, and then counted by pouring plate method. Then, 500 μL of the bacterial suspension incubated with artificial gastric juice for 3 hours was added to 4.5 mL of artificial intestinal juice, and incubated in a 37°C incubator for 8 hours, and then counted by pouring plate method. The survival rate was calculated according to the following formula.

[0106] Survival rate = treated colony count (CFU / mL) / initial colony count (CFU / mL) × 100%. The gastrointestinal tolerance of Lactobacillus paracasei Lpc-C66 and Lactobacillus paracasei K56 is shown in Table 1:

[0107]

[0108] Table 1

[0109] It can be seen from the table that after 3 hours in gastric juice, the viable bacterial survival rate of Lactobacillus paracasei Lpc-C66 can still be maintained at 45.17%, while after continuing to be cultured in intestinal juice for 8 hours, the viable bacterial survival rate is reduced to 83.58% of the viable bacterial count after gastric juice treatment. This shows that Lactobacillus paracasei Lpc-C66 has a higher gastrointestinal tolerance and has a higher tolerance to simulated gastrointestinal fluid than Lactobacillus paracasei K56.

[0110] In summary, the Lactobacillus paracasei Lpc-C66 of the present invention can alleviate hyperuricemia by reducing plasma uric acid, reducing plasma xanthine enzyme activity and reducing liver xanthine enzyme activity, can effectively reduce blood uric acid levels, and has good safety and tolerance. Experiments have shown that the live bacteria liquid and the inactivated bacteria liquid of the Lactobacillus paracasei Lpc-C66 of the present invention both have good abilities to improve hyperuricemia. The present invention not only provides a new probiotic strain, but also provides a scientific basis for its application in functional foods and pharmaceutical preparations.

[0111] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with the technical features with similar functions disclosed in the embodiments of the present disclosure (but not limited to) and the technical solutions formed.

Claims

1. A Lactobacillus paracasei Lpc-C66 with the function of improving hyperuricemia, characterized in that: The Lactobacillus paracasei Lpc-C66 was deposited in the China Center for Type Culture Collection on December 7, 2023, with the deposit number: CCTCC NO: M20232496.

2. application of Lactobacillus paracasei Lpc-C66 according to claim 1 in preparing a product for improving hyperuricemia.

3. The use according to claim 2, characterized in that: The product is a bacterial agent, food and / or medicine.

4. The use according to claim 3, characterized in that: The food is one or more of fermented milk, cheese, milk-containing beverage, solid beverage and milk powder; the food contains live bacterial liquid and / or inactivated bacterial liquid of Lactobacillus paracasei Lpc-C66.

5. The use according to claim 3, characterized in that: The medicine comprises live bacterial liquid and / or inactivated bacterial liquid of Lactobacillus paracasei Lpc-C66, and / or other medicines compatible with the live bacterial liquid and / or inactivated bacterial liquid of Lactobacillus paracasei Lpc-C66, as well as pharmaceutically acceptable carriers and / or excipients.

6. The use according to claim 2, characterized in that: The Lactobacillus paracasei Lpc-C66 is used for preparing bacterial agents, foods and / or medicines for reducing plasma uric acid and / or plasma xanthine enzyme activity.

7. The use according to claim 2, characterized in that: The Lactobacillus paracasei Lpc-C66 is used for preparing bacterial agents, foods and / or medicines for reducing the activities of liver adenosine deaminase and / or liver xanthine enzyme.

8. The use according to claim 2, characterized in that: The Lactobacillus paracasei Lpc-C66 is used for preparing a bacterial agent, food and / or medicine for reducing the level of alanine aminotransferase and / or aspartate aminotransferase in plasma.

9. The use according to claim 2, characterized in that: The Lactobacillus paracasei Lpc-C66 is used for preparing a bacterial agent, food and / or medicine for reducing the levels of urea nitrogen and creatinine in the liver.

10. The use according to claim 4, characterized in that: The preparation of the inactivated bacteria liquid of the Lactobacillus paracasei Lpc-C66 is as follows: 1 g of Lpc-C66 bacterial powder was dissolved in 500 mL of LB liquid culture medium, and after culturing at 37°C for 24 h, the bacterial suspension was inactivated at 121°C for 30 min under high temperature and high pressure, and centrifuged at 3000 rpm for 15 min at room temperature. After the centrifugation, the supernatant was removed, and an appropriate amount of PBS was added to wash away the residual culture medium, and the mixture was centrifuged again to remove the supernatant to obtain the inactivated animal Bifidobacterium lactis subsp. Lpc-C66 bacterial cells. 1 mL of physiological saline was added to every 50 mg of the bacterial cells to dissolve the inactivated Lactobacillus paracasei Lpc-C66 bacterial liquid.

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