Method for improving uric acid reducing function of safflower enzyme by adopting lactobacillus fermentation

Abstract

The invention discloses a method for improving the uric acid reducing function of safflower enzyme through lactobacillus fermentation. According to the method, Weissella cibaria X1 is inoculated into a safflower culture medium for fermentation, after centrifugation and filtration, the safflower enzyme with the improved uric acid reducing function is obtained, and the preservation number of the Weissella cibaria X1 is CGMCC No.31306. The invention further discloses a preparation method of the safflower enzyme with the improved uric acid reducing function. The X1 fermented safflower enzyme prepared by the method disclosed by the invention is obviously superior to natural fermented safflower enzyme (without inoculation) in uric acid reducing effect, can be used for preparing health food or medicines with the uric acid reducing effect, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of fermented products, and in particular to a lactic acid bacteria fermented safflower product with uric acid lowering function and a preparation method thereof. Background Art

[0002] Hyperuricemia is a metabolic disease caused by abnormal purine metabolism in the body, which leads to excessive uric acid concentration in the blood, and then causes urate precipitation in the joints and kidneys, causing symptoms such as gout. According to reports, the number of patients with hyperuricemia is increasing year by year worldwide. In my country's economically developed cities and coastal areas, the prevalence of hyperuricemia reaches 5%-23.5%, close to the level of developed Western countries. At present, the clinical treatment of hyperuricemia is mainly through drugs that inhibit uric acid synthesis (allopurinol, etc.) and promote uric acid excretion (probenecid, etc.). However, these drugs show symptoms such as skin itching, intestinal discomfort, and allopurinol hypersensitivity syndrome (AHS) while treating hyperuricemia. Therefore, it is necessary to develop natural substances with uric acid-lowering function without side effects.

[0003] Safflower belongs to the Asteraceae family and is an annual herb. It is a commonly used traditional Chinese medicine in China. It is a drug included in the Chinese Pharmacopoeia from 1963 to 2015. There are a large number of flavonoids in safflower. Before the invention of chemical synthetic dyes, safflower was one of the most important sources of dyes in the world. In recent years, studies have shown that flavonoids such as serotonin yellow A (HSYA)11, rutin, and quercetin in safflower silk have antioxidant, anti-inflammatory, blood sugar-lowering, and ischemic stroke treatment effects. In addition to flavonoids, safflower also contains other active ingredients such as fatty acids, amino acids, and polysaccharides, which give safflower physiological activities such as immunomodulation, anti-inflammatory, and blood pressure reduction. However, there are few studies on the ability of safflower to reduce uric acid. Therefore, it is of great significance to further study the biological activity of safflower, improve its bioavailability, and reduce uric acid levels in the human body. Summary of the invention

[0004] In response to the above problems, the present invention provides a method for improving the uric acid-lowering ability of safflower by fermenting safflower with lactic acid bacteria, studies the changes in key metabolites during the fermentation process and the xanthine oxidase inhibitory ability of the fermented product, and preliminarily explores the potential mechanism of the uric acid-lowering function of fermented safflower through the established hyperuricemia zebrafish model.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A method for improving the uric acid lowering function of safflower enzyme by using lactic acid bacteria fermentation. Weissella cibaria X1 is inoculated into safflower culture medium for fermentation. After centrifugation and filtration, safflower enzyme with improved uric acid lowering function is obtained. The preservation number of Weissella cibaria X1 is CGMCC No.31306.

[0007] The safflower culture medium is prepared by taking 3 g of safflower powder (naturally dried safflower silk is dried at 40° C. for 10 min to further remove excess water and then made into powder), 1 g of glucose, adding 100 mL of distilled water, and heating at 65° C. for 20 min for pasteurization.

[0008] The inoculation amount of the inoculation is 3% by volume.

[0009] The fermentation was carried out at 37°C for 24 hours.

[0010] The centrifugation was carried out at 7000×g and 4° C. for 20 min.

[0011] The filtration was performed using a 0.22 μm filter (water system).

[0012] The present invention also provides a safflower enzyme, which is prepared according to the above method.

[0013] The present invention also provides the use of safflower enzyme in preparing products with uric acid lowering function.

[0014] The products include health foods or medicines.

[0015] The present invention also provides the use of safflower enzyme in preparing a pharmaceutical composition for treating diseases related to hyperuricemia.

[0016] The diseases associated with hyperuric acid include hyperuricemia and gout.

[0017] The present invention also provides a product with uric acid lowering function, comprising safflower enzyme as an active ingredient.

[0018] The present invention has the following beneficial effects:

[0019] 1. The present invention found that the safflower enzyme obtained by inoculating Weissella cibaria X1 into a culture medium containing safflower powder is superior to the naturally fermented safflower enzyme (uninoculated) in reducing the levels of uric acid (p < 0.05), creatinine (p < 0.05), XO, and urea nitrogen.

[0020] 2. Compared with the naturally fermented safflower enzyme (uninoculated), the safflower enzyme of the present invention significantly increased the content of total acid, and the lactic acid bacteria count in the fermentation liquid of X1 fermented safflower reached 10.94±0.54LogCFU / mL.

[0021] 3. Compared with naturally fermented safflower enzyme (uninoculated), the safflower enzyme of the present invention significantly improves its ability to inhibit xanthine oxidase.

[0022] 4. The safflower enzyme of the present invention significantly increases the content of total flavonoids compared with the naturally fermented safflower enzyme (not inoculated with bacteria).

[0023] 5. The preparation method of the safflower enzyme of the present invention is simple and easy to operate, with a short fermentation time, and significantly improves the ability of safflower to reduce uric acid.

[0024] Weissella cibaria X1, the strain was deposited on July 15, 2024 at the General Microbiology Center of China Microorganism Culture Collection (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Accession Number: CGMCC No.31306. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are the physical and chemical properties of fermented safflower, including (A) pH; (B) lactic acid content.

[0026] Figure 2 It is the content of active substances in fermented safflower, including (A) reducing sugar content; (B) total flavonoids content; (C) total phenol content.

[0027] Figure 3 The XO inhibitory capacity of fermented safflower in vitro.

[0028] Figure 4 The effect of fermented safflower on zebrafish with high uric acid, including (A) morphology of zebrafish; (B) uric acid content; (C) XO activity; (D) creatinine content; (E) urea nitrogen content. DETAILED DESCRIPTION

[0029] It will be appreciated by those skilled in the art that the disclosed techniques in the following examples represent techniques that the inventors have found to work well in the practice of the present invention. However, many changes may be made in the disclosed specific embodiments, and still the same or similar results may be obtained without departing from the spirit and scope of the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0030] The lactic acid bacteria used in this study are as follows, which were isolated and preserved in the laboratory of the inventor and disclosed in the patent application with application number 2024111596229:

[0031] Weissella cibaria X1, the strain was deposited on July 15, 2024 at the General Microbiology Center of China Microorganism Culture Collection (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Accession Number: CGMCC No.31306.

[0032] Example 1X1 Preparation of fermented safflower enzyme and naturally fermented safflower enzyme

[0033] The safflower silk sample was dried at 40°C for 10 minutes to constant weight, ground into powder with a mortar and passed through a 200-mesh sieve. Take 3g of safflower powder, add 1g of glucose to 100mL of distilled water, heat at 65°C for 20 minutes for pasteurization, and obtain safflower culture medium. Weissella cibaria X1 was inoculated into the above safflower culture medium at an inoculum rate of 3% (v / v), fermented at 37°C for 24 hours, centrifuged at 7000×g, 4°C for 20 minutes, and the supernatant was filtered with a 0.22μm filter (water system) to obtain a cell-free supernatant for subsequent experiments. The safflower fermented with X1 was recorded as the X1 group, and the safflower fermentation medium without inoculation of X1 was the natural fermentation group, recorded as the Natural group (N group).

[0034] Example 2X1 Physicochemical Properties of Fermented Safflower Enzyme and Naturally Fermented Safflower Enzyme

[0035] (1) pH and total acid content

[0036] The pH of fermented safflower was measured by a pH meter (PHS-25, INESA, ShangHai, China), and each sample was repeated three times. The total acid content was determined according to the determination method of total acid in food in GB 12456-2021. Take 30mL of cell-free fermentation supernatant, add 2-3 drops of phenolphthalein indicator, titrate with 0.1mol / L NaOH solution, record the volume of NaOH consumed, and the total acid content is expressed as lactic acid per liter, calculated according to the following formula.

[0037]

[0038] X is the lactic acid content per liter of fermentation sample (g / L); c is the concentration of NaOH standard solution (mol / L); K is the acid conversion coefficient, and this study uses the lactic acid conversion coefficient (0.090g / mmoL); F is the dilution factor of the sample; 30 is the sampling volume of the sample (mL).

[0039] The results showed that the pH of safflower fermented by X1 was significantly lower than that of safflower fermented by natural fermentation ( Figure 1 A), and the pH of safflower fermented with X1 after 24h of fermentation was significantly lower than that after 0h of fermentation. Figure 1 B) (expressed in lactic acid content in the figure) is significantly higher than that of naturally fermented safflower. The total acid content of X1 fermented safflower after 24h of fermentation is significantly higher than that of fermentation 0h. This may be because lactic acid bacteria produce lactic acid during the fermentation process, resulting in a decrease in pH and an increase in total acid content.

[0040] (2) Lactic acid bacteria colony count

[0041] The plate count method was used to count the live lactic acid bacteria in the sample. The specific method was as follows: take 1 mL of sample solution, dilute it with sterile physiological saline (0.85%, w / v), select the appropriate dilution gradient and spread it on MRS agar plates, and after culturing at 37°C for 48 hours, count the plates with colony counts between 30 and 300, which were recorded as log CFU / mL. The results showed that the lactic acid bacteria colony count of X1 fermented safflower was 6.31±0.37logCFU / mL at 0 hours of fermentation, and the lactic acid bacteria colony count increased significantly to 10.94±0.54logCFU / mL at 24 hours of fermentation, while no lactic acid bacteria growth was observed in the naturally fermented safflower. The above results show that the safflower culture medium used in this study is suitable for the growth of X1.

[0042] Example 3X1 Active substance content of fermented safflower enzyme and naturally fermented safflower enzyme

[0043] (1) Determination of reducing sugar content

[0044] The content of reducing sugar in fermented safflower was determined by dinitrosalicylic acid (DNS) method. Take 1 mL of sample and add 2 mL of DNS reagent, mix thoroughly and heat in a boiling water bath for 5 min, cool and dilute to 20 mL, and measure the absorbance at 550 nm. Draw a standard curve by the absorbance of the glucose standard solution (0.1 mg / mL-1.0 mg / mL), substitute the above measured absorbance into the standard curve, and the result is expressed as the corresponding glucose concentration (mg / mL).

[0045] (2) Determination of total flavonoid content

[0046] Take 4mL of sample to be tested, add 2mL of distilled water and 1mL of NaNO2 solution (5%, w / v), shake up and leave standstill for 6min, add 1mL of Al(NO)3 solution (10%, w / v), shake up and leave standstill for 6min, then add 10mL of NaOH solution (5%, w / v), make up to 25mL with distilled water, shake up and leave standstill for 15min, use corresponding reagent as blank to measure absorbance at 500nm. Draw standard curve by the absorbance of rutin (0.02mg / mL-0.2mg / mL), substitute the above-mentioned absorbance measured into standard curve, the result is represented by corresponding rutin concentration (mg / mL).

[0047] (3) Determination of total phenol content

[0048] 1 mL of sample was added to 5 mL of Folin-Ciocalteu reagent, reacted at room temperature for 3 min, then 4 mL of NaCO3 solution (7.5%, w / v) was added, vortexed for 1 min to fully mix, reacted at room temperature in the dark for 60 min, and the absorbance was measured at 760 nm. A standard curve was obtained by measuring the absorbance of gallic acid (0-400 mg / L), and the absorbance measured above was substituted into the standard curve, and the result was expressed as the corresponding gallic acid concentration (g / L).

[0049] The results showed that the reducing sugar content in safflower fermented by X1 after 24h of fermentation was significantly lower than that in safflower fermented by natural fermentation. This was because X1 consumed reducing sugars during the fermentation of safflower ( Figure 2 A). At 0h of fermentation, the total flavonoids content in naturally fermented safflower was significantly higher than that in X1 fermented safflower, and after 24h of fermentation, the total flavonoids content in X1 fermented safflower was significantly higher than that in naturally fermented safflower ( Figure 2 B). At 0h of fermentation, the total phenol content in the naturally fermented safflower was significantly higher than that in the X1 fermented safflower, but at 24h of fermentation, there was no significant difference in the total phenol content between the naturally fermented safflower and the X1 fermented safflower ( Figure 2 C). In summary, X1 fermentation significantly increased the flavonoid content of safflower at 24 h of fermentation.

[0050] Example 4 XO Inhibition Capacity of X1 Fermented Safflower Enzyme and Naturally Fermented Safflower Enzyme

[0051] Xanthine oxidase (XO) is a key enzyme in uric acid synthesis and an important target for the treatment of hyperuricemia. Drugs such as allopurinol and febuxostat treat hyperuricemia by inhibiting the activity of xanthine oxidase. Therefore, the ability of fermented safflower to inhibit XO was determined to determine its ability to lower uric acid. Take 0.1mL of sample and add 0.6mL PBS buffer (pH7.5), 0.2mL xanthine (0.15mM) and 0.1mL xanthine oxidase (0.2U / mL). After reacting at 37°C for 15min, add 1mL HCl (1N) to terminate the reaction. The absorbance measured at 290nm is recorded as A. The sample blank group is replaced by PBS instead of the enzyme solution, and the absorbance measured is recorded as A0; the sample is replaced by PBS as the control group, and the absorbance measured is recorded as A CK The XO inhibition rate was calculated according to the following formula.

[0052]

[0053] The results show that ( Figure 3 ), after 24h of fermentation, the inhibition rate of XO of safflower fermented with X1 was 68.05±1.69%, indicating that safflower fermented with X1 has a high ability to reduce uric acid. Compared with safflower fermented with X1 at 0h, the ability of safflower fermented with X1 at 24h to inhibit XO was significantly improved, and the ability of safflower fermented with X1 to inhibit XO was significantly higher than that of naturally fermented safflower. In summary, safflower fermented with X1 will significantly improve its ability to inhibit XO.

[0054] Example 5X1 Therapeutic effects of fermented safflower enzyme and naturally fermented safflower enzyme in hyperuricemia zebrafish

[0055] To construct the HUA zebrafish model, 40 zebrafish at 5 dpf (fifth day after fertilization) were randomly selected and placed in a 6-well plate, with 3 parallels in each group (120 in each group). The model was constructed according to Table 1, where the concentration of potassium oxonate (PO) was 200 μmol / L, the concentration of xanthine sodium salt (XSS) was 10 μmol / L, the concentration of allopurinol (APL) was 1 mg / mL, and the fermented safflower supernatant was diluted 10 3 After culturing at 28.5℃ for 24h, the contents of various substances were determined using uric acid kit, XO kit, creatinine kit and urea nitrogen kit.

[0056] Table 1 Construction of hyperuricemia zebrafish model

[0057]

[0058] Note: Except for the CK group, PO and XSS were added to each group first, and their solutions were added 1 hour later.

[0059] The morphology of zebrafish in each treatment group is shown in Figure 4A. The results showed that the uric acid content and XO activity of the CK group were 2.59±0.34 mg / L and 121.20±64.13 nmol / min / g, respectively, and the uric acid content and XO activity of the HUA group were 22.95±2.58 mg / L and 14972.24±518.38 nmol / min / g, respectively, which were significantly higher than those of the CK group, indicating that the hyperuricemia zebrafish model was successfully established. After adding the supernatant of X1 fermented safflower, the uric acid level was significantly reduced to 4.29±2.68 mg / L compared with the HUA group, which was not significantly different from the positive control group (APL group) and the CK group, and the XO activity was significantly reduced to 145.44±24.24 nmol / min / g, which was not significantly different from the CK group and was significantly lower than the APL group. The uric acid level and XO activity of zebrafish supplemented with natural fermented safflower supernatant were 7.68±0.75 mg / L and 331.28±37.02 nmol / min / g, respectively, which were also significantly lower than those of the HUA group, but significantly higher than those of zebrafish supplemented with X1 fermented safflower supernatant ( Figure 4 B, 4C). This indicates that both X1-fermented safflower and naturally fermented safflower can reduce the uric acid level and XO activity in zebrafish with high uric acid levels, and the uric acid-lowering level of X1-fermented safflower is significantly higher than that of naturally fermented safflower.

[0060] Creatinine and urea nitrogen (BUN) are the end products of creatine phosphate metabolism in muscle and protein metabolism in the body, respectively, and they are usually used to evaluate renal dysfunction. Long-term hyperuricemia can lead to further aggravation of renal damage, which is manifested as a further increase in creatinine and urea nitrogen. Therefore, the levels of creatinine and urea nitrogen (BUN) in zebrafish were measured. The creatinine and urea nitrogen levels in the HUA group were 44.20±1.40μmol / L and 3.70±0.25mmol / L, respectively. After adding X1 fermented safflower supernatant, the creatinine content was significantly reduced to 32.23±7.57μmol / L, and the urea nitrogen content was reduced to 3.37±0.30mmol / L; after adding naturally fermented safflower supernatant, the creatinine content increased slightly to 44.90±4.46μmol / L, and the urea nitrogen content was significantly reduced to 3.17±0.18mmol / L ( Figure 4 D, 4E). This indicates that X1 fermented safflower can significantly reduce the creatinine content in zebrafish with high uric acid, while naturally fermented safflower can significantly reduce the urea nitrogen content in zebrafish with high uric acid.

[0061] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for improving the uric acid-lowering function of safflower enzyme by lactic acid bacteria fermentation, characterized in that: Weissella cibaria X1 is inoculated into safflower culture medium for fermentation, and after centrifugation and filtration, safflower enzyme with enhanced uric acid lowering function is obtained. The preservation number of Weissella cibaria X1 is CGMCC No.31306.

2. The method according to claim 1, characterized in that The safflower culture medium is prepared by taking 3 g of safflower powder and 1 g of glucose, adding 100 mL of distilled water, and heating at 65° C. for 20 minutes for pasteurization.

3. The method according to claim 1, characterized in that The inoculation amount of the inoculation is 3% by volume.

4. The method according to claim 1, characterized in that The fermentation was carried out at 37°C for 24 hours.

5. A safflower enzyme, characterized in that: Prepared according to the method of claim 1.

6. Use of the safflower enzyme according to claim 5 in the preparation of a product having the function of lowering uric acid.

7. The use according to claim 6, characterized in that: The products include health foods or medicines.

8. Use of the safflower enzyme described in claim 5 in the preparation of a pharmaceutical composition for treating diseases related to hyperuricemia.

9. The use according to claim 8, characterized in that The diseases associated with hyperuric acid include hyperuricemia and gout.

10. A product with uric acid lowering function, characterized in that: Contains the safflower enzyme described in claim 5 as an active ingredient.

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