Coconut-based metagen with uric acid reducing and oxidation resisting functions and preparation method of coconut-based metagen

Abstract

The invention discloses a coconut-based metagen with uric acid reducing and antioxidant functions and a preparation method thereof, and the coconut-based metagen is prepared by taking coconut water as a fermentation culture medium and adding lactobacillus DO28L8 for fermentation. The coconut water is fermented by applying a specific strain (lactobacillus DO28L8), so that the coconut-based metagen obtained by fermentation can reduce the uric acid level of a hyperuricemia cell model and relieve hyperuricemia, and can be applied to drugs for relieving hyperuricemia; the coconut water is fermented under specific conditions, so that the functional activity of the product can be improved, and the control effect is improved; the old coconut water is used as a raw material, high-value utilization is realized by fully utilizing the old coconut water, and the high additional value of the product is improved; the composition not only can reduce uric acid, but also can effectively scavenge free radicals and improve cell antioxidant enzyme activity, so that the antioxidant effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a coconut-based postbiotic having uric acid-lowering and antioxidant functions and a preparation method thereof. Background Art

[0002] Uric acid is produced in the liver, muscle, and adipose tissue, and is the end product of purine metabolism. It is the main antioxidant in human plasma and can protect cells or neurons from oxidative damage. Under normal circumstances, there is a dynamic balance among the intestinal absorption of purine, the generation of uric acid, and the excretion of uric acid through the kidneys to maintain the normal physiological state of the body.

[0003] Hyperuricemia (HUA) refers to a disease in which purine metabolism in the human body is abnormal, uric acid production increases or excretion decreases, resulting in an increase in blood uric acid level, which can cause various complications such as chronic kidney disease, cardiovascular disease, and gout, threatening human health.

[0004] Existing HUA treatment methods include drug treatment and non-drug treatment, but both have adverse reactions. Among them, the drugs applied clinically are mainly uric acid-excreting drugs, uric acid-production-inhibiting drugs, and alkaline drugs, which relieve hyperuricemia symptoms by increasing uric acid excretion, inhibiting uric acid production, or alkalizing urine. However, the above drugs all have problems such as low tolerance, high drug dependence, and many complication side effects. In terms of diet control, since most foods contain purine due to the presence of animal and plant cells, it is extremely difficult to relieve hyperuricemia simply by restricting purine intake, and the restriction of purine acid intake is likely to cause nutritional imbalance. Based on the defects and deficiencies of the existing treatment methods, there is an urgent need to seek an HUA treatment strategy that is obvious, stable, safe, and convenient.

[0005] In recent years, due to the high safety, non-drug resistance, and the effects on host health of probiotics being well-known, they have been widely used in many fields such as food processing and human health. Currently, there are studies showing that probiotics have functions such as enhancing immunity, improving intestinal function, and relieving metabolic diseases. Therefore, with the continuous in-depth research on probiotics and other microecological preparations, using microecological preparations such as probiotics and postbiotics to regulate the intestinal microecology and improve intestinal immunity can promote the excretion of uric acid from the body through the intestine and relieve hyperuricemia, becoming an effective and safe means of lowering uric acid. Summary of the Invention

[0006] Therefore, the purpose of the present invention is to provide a new, safe, and effective natural biological preparation, using a specific strain to ferment old coconut water to prepare a postbiotic having uric acid-lowering and antioxidant functions, which can reduce uric acid while effectively scavenging free radicals and improving the added value of the product.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] The first aspect of the present invention is to provide coconut-based postbiotics with uric acid-lowering and antioxidant functions, which are fermented with coconut water as the fermentation medium and Lactobacillus DO28L8 added.

[0009] The second aspect of the present invention is to provide a preparation method of coconut-based postbiotics with uric acid-lowering and antioxidant functions, including: filtering the original coconut water through gauze, sterilizing it at 121 °C for 15 min to obtain the coconut water fermentation medium; activating Lactobacillus DO28L8, inoculating the obtained seed liquid into the coconut water fermentation medium at an inoculation amount of 2-5%, and statically fermenting and culturing at a temperature of 33-41 °C for 18-36 h, and inactivating after the culture ends.

[0010] Optionally, the preparation of the seed liquid includes the following steps:

[0011] (1) First activation

[0012] Inoculate Lactobacillus DO28L8 on the MRS solid medium by streaking, and incubate it upside down at a temperature of 37-40 °C for 24-48 h. Then pick the single colony with a larger calcium dissolution circle on the petri dish and inoculate it into the MRS liquid medium, and statically culture it in a constant temperature incubator at 37-40 °C for 15-24 h for the first activation.

[0013] (2) Second activation

[0014] Transfer the bacterial liquid after the first activation to a new MRS liquid medium at an inoculation amount of 3-5%, and repeat the culture for 15-24 h for the second activation.

[0015] (3) Preparation of the seed liquid

[0016] Centrifuge the bacterial liquid after the second activation at a temperature of 4 °C and a rotation speed of 8000 r / min for 10 min, discard the supernatant, then wash the precipitate with sterile normal saline 2-3 times, and finally resuspend the bacterial cells with sterile normal saline to adjust the bacterial cell concentration to 108-109 CFU / mL to obtain the seed liquid.

[0017] Optionally, the inactivation step includes: after the fermentation culture ends, ultrasonically break the fermentation broth for 5 min, then centrifuge the fermentation broth at a temperature of 4 °C and a rotation speed of 8000 r / min for 15 min to separate the supernatant, and perform pasteurization on the supernatant at a temperature of 70-80 °C for 15-20 min.

[0018] Optionally, the method further comprises freeze-drying and storing after inactivation, wherein the freeze-drying and storing comprises: filtering the inactivated sterile fermentation liquid through a sterile filter membrane, placing the obtained sterile fermentation filtrate in a freeze dryer, first pre-freezing it at a temperature of -40°C for 2 to 3 hours, and then sublimating and drying it at a temperature of -20°C to -10°C and a vacuum degree of 0.1 to 0.2 mbar for 24 to 36 hours, and then desorbing and drying it at a temperature of 20°C to 25°C and a vacuum degree of 0.05 to 0.1 mbar for 4 to 6 hours to obtain a coconut-based postbiotic freeze-dried material, grinding the coconut-based postbiotic freeze-dried material to obtain a coconut-based postbiotic freeze-dried powder, and packaging the coconut-based postbiotic freeze-dried powder and storing it in a -80°C refrigerator.

[0019] The third aspect of the present invention provides the use of coconut-based postbiotics with uric acid-lowering and antioxidant functions in the preparation of uric acid-lowering and / or antioxidant products.

[0020] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0021] 1. The present invention uses a specific strain (Lactobacillus DO28L8) to ferment coconut water, so that the coconut-based postbiotics obtained by fermentation can reduce the uric acid level in the hyperuricemia cell model, relieve hyperuricemia, and can be used in drugs for relieving hyperuricemia.

[0022] 2. The present invention uses a specific strain (Lactobacillus DO28L8) to ferment coconut water under specific conditions, which can enhance the functional activity of the product and improve its prevention and treatment effect.

[0023] 3. The present invention utilizes old coconut water as raw material, fully utilizes old coconut water to achieve high-value utilization, and improves the high added value of the product.

[0024] 4. In addition to lowering uric acid, the postbiotics of the present invention can also effectively scavenge free radicals and enhance the activity of cellular antioxidant enzymes, thereby improving the antioxidant effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a colony morphology diagram of Lactobacillus DO28L8 of the present invention.

[0026] Figure 2 This is the influence curve of the present invention on the XOD (xanthine oxidase) inhibition rate at different fermentation times.

[0027] Figure 3 This is the influence curve of coconut-based postbiotics on XOD (xanthine oxidase) inhibition rate at different fermentation temperatures of the present invention.

[0028] Figure 4The influence curve of coconut-based postbiotics on the inhibition rate of XOD (xanthine oxidase) under different inoculation amounts of the present invention.

[0029] Figure 5 The measurement result of the influence of the embodiment of the present invention on cell viability; abscissa: the concentration of coconut-based postbiotics; ordinate: cell viability.

[0030] Figure 6 The measurement result of the influence of the embodiment of the present invention on the uric acid (UV) level in the hyperuricemia cell model.

[0031] Figure 7 The measurement result of the influence of the embodiment of the present invention on the inflammatory indexes in the hyperuricemia cell model; A: the influence of the level of TNF-α; B: the influence of the level of IL-1β.

[0032] Figure 8 The measurement result of the influence of the embodiment of the present invention on the oxidative stress indexes in the hyperuricemia cell model; A: the content of MDA; B: the activity content of SOD.

[0033] Figure 9 The measurement result of the influence of the embodiment of the present invention on the change of mitochondrial membrane potential (MMP) in the hyperuricemia cell model.

[0034] Figure 10 The measurement result of the influence of the embodiment of the present invention on the level of reactive oxygen species (ROS) in the hyperuricemia cell model. Detailed implementation manners

[0035] In order to enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0036] The fermentation strain used in the following experiment is a strain with the potential of reducing uric acid and antioxidant properties screened from the feces of long-lived elderly people in Hainan area. The screened strain is inoculated on the MRS solid medium for growth, and the colony morphology diagram of the strain growth is as Figure 1 shown, from Figure 1As can be seen, the colonies are round, with neat edges, smooth and moist surfaces, milky white in color, and about 1-2 mm in diameter. This figure clearly presents the characteristics of the colony size, shape, color, surface texture, etc. By observing these characteristics, samples were collected, strains were isolated, cultured and amplified in MRS medium, DNA was extracted, specific gene fragments were amplified by PCR and then sequenced. After comparison and identification, the strain was named Lactobacillus DO28L8 (preserved in the Microbiology Laboratory of Hainan University).

[0037] Example 1

[0038] This example provides a preparation method of coconut-based postbiotics with uric acid-lowering and antioxidant functions, including the following steps:

[0039] I. Raw material pretreatment

[0040] Select fresh and mature old coconuts. After breaking the shells, collect the coconut water, filter out impurities through four layers of gauze to obtain clear coconut water stock solution. Sterilize the coconut water stock solution at 121 °C for 15 min to kill the miscellaneous bacteria therein, then add appropriate amounts of nutrients, stir evenly, and adjust the pH to 6.5 to prepare a coconut water fermentation medium. Among them, the nutrients include 2% glucose, 0.1% ammonium sulfate, 0.1% magnesium sulfate, 0.5% peptone, and 0.5% yeast extract powder.

[0041] II. Fermentation culture

[0042] Streak inoculate the preserved Lactobacillus DO28L8 on MRS solid medium and incubate it upside down at 37 °C for 24 h until obvious single colonies grow. Then pick the single colonies and inoculate them into a triangular flask containing 100 mL of MRS liquid medium, and incubate them statically at 37 °C for 24 h for the first activation. Then transfer the activated bacterial liquid after the first activation to fresh MRS liquid medium at an inoculation amount of 3% and repeat the culture for 24 h for the second activation. The activated bacterial liquid after the second activation is centrifuged at 4 °C and 8000 r / min for 10 min, the supernatant is discarded, the precipitate is washed twice with sterile normal saline, and finally the bacterial cells are resuspended with sterile normal saline to adjust the bacterial cell concentration to 10 8 CFU / mL to obtain a seed solution. Inoculate the seed solution into the prepared coconut water fermentation medium at an inoculation amount of 3% and ferment statically at a temperature of 37 °C for 24 h.

[0043] III. Inactivation and freeze-drying

[0044] After fermentation, the fermentation broth was quickly cooled to 4°C. After ultrasonically disrupting the fermentation broth for 5 min, it was centrifuged at 4°C and a rotation speed of 8000 r / min for 15 min to obtain the supernatant. The supernatant was transferred to a sterile container, ultrasonically disrupted for 5 min, working at 220 W for 5 s and stopping for 5 s, and pasteurized at 70°C for 15 min to kill residual microorganisms. Then it was filtered through a 0.22 μm sterile filter membrane to remove impurities and macromolecular substances, obtaining a sterile filtrate. The sterile filtrate was dispensed into petri dishes and placed in a freeze dryer for freeze-drying. The freeze-drying program was as follows: pre-freezing temperature -40°C, pre-freezing time 2 h; sublimation drying temperature -20°C to -10°C, vacuum degree 0.2 mbar, sublimation drying time 24 h; desorption drying temperature 20°C to 25°C, vacuum degree 0.1 mbar, desorption drying time 4 h. After freeze-drying, it was dispensed, the bag mouth was sealed, obtaining freeze-dried coconut-based postbiotics powder, which was stored in a -80°C refrigerator for standby.

[0045] Example 2

[0046] The preparation method of the coconut-based postbiotics with uric acid lowering and antioxidant functions in this example was the same as that in Example 1 above, except that the inoculation amount of the seed liquid when inoculating the seed liquid into the coconut water fermentation medium in the step "II. Fermentation culture" was different. The inoculation amount of the seed liquid used in this example was 2%.

[0047] Example 3

[0048] The preparation method of the coconut-based postbiotics with uric acid lowering and antioxidant functions in this example was the same as that in Example 1 above, except that the inoculation amount of the seed liquid when inoculating the seed liquid into the coconut water fermentation medium in the step "II. Fermentation culture" was different. The inoculation amount of the seed liquid used in this example was 4%.

[0049] Example 4

[0050] The preparation method of the coconut-based postbiotics with uric acid lowering and antioxidant functions in this example was the same as that in Example 1 above, except that the inoculation amount of the seed liquid when inoculating the seed liquid into the coconut water fermentation medium in the step "II. Fermentation culture" was different. The inoculation amount of the seed liquid used in this example was 5%.

[0051] Example 5

[0052] The preparation method of the coconut-based postbiotics with uric acid lowering and antioxidant functions in this example was the same as that in Example 1 above, except that the temperature of static fermentation in the step "II. Fermentation culture" was different. The fermentation temperature used in this example was 33°C.

[0053] Example 6

[0054] The preparation method of the coconut-based postbiotic with uric acid-lowering and antioxidant functions in this example is the same as that in Example 1 above, except that the temperature for static fermentation in the step of "II. Fermentation culture" is different. The fermentation temperature used in this example is 41°C.

[0055] Example 7

[0056] The preparation method of the coconut-based postbiotic with uric acid-lowering and antioxidant functions in this example is the same as that in Example 1 above, except that the time for static fermentation in the step of "II. Fermentation culture" is different. The fermentation time used in this example is 18 h.

[0057] Example 8

[0058] The preparation method of the coconut-based postbiotic with uric acid-lowering and antioxidant functions in this example is the same as that in Example 1 above, except that the time for static fermentation in the step of "II. Fermentation culture" is different. The fermentation time used in this example is 30 h.

[0059] Example 9

[0060] The preparation method of the coconut-based postbiotic with uric acid-lowering and antioxidant functions in this example is the same as that in Example 1 above, except that the time for static fermentation in the step of "II. Fermentation culture" is different. The fermentation time used in this example is 36 h.

[0061] Comparative Example 1

[0062] The preparation method of the coconut-based postbiotic with uric acid-lowering and antioxidant functions in this comparative example is the same as that in Example 1 above, except that the inoculation amount of the seed liquid when inoculating the seed liquid into the coconut water fermentation medium in the step of "II. Fermentation culture" is different. The inoculation amount of the seed liquid used in this example is 1%.

[0063] Comparative Example 2

[0064] The preparation method of the coconut-based postbiotic with uric acid-lowering and antioxidant functions in this comparative example is the same as that in Example 1 above, except that the temperature for static fermentation in the step of "II. Fermentation culture" is different. The fermentation temperature used in this example is 29°C.

[0065] Comparative Example 3

[0066] The preparation method of the coconut-based postbiotic with uric acid-lowering and antioxidant functions in this comparative example is the same as that in Example 1 above, except that the temperature for static fermentation in the step of "II. Fermentation culture" is different. The fermentation temperature used in this example is 45°C.

[0067] Comparative Example 4

[0068] The preparation method of the coconut-based postbiotic with uric acid lowering and antioxidant functions in this comparative example is the same as that in Example 1 above, except that the standing fermentation time in the step of "II. Fermentation culture" is different. The fermentation time adopted in this example is 12h.

[0069] I. Effects of coconut-based postbiotics on the inhibition rate of XOD (xanthine oxidase) under different fermentation times, fermentation temperatures, and fermentation inoculation amounts

[0070] Examine the effects of different fermentation, fermentation temperature, and seed liquid (strain) inoculation amounts on the inhibition rate of xanthine oxidase during the fermentation of coconut water. The process is as follows:

[0071] Single-factor experiment: The fermentation temperature is 37°C, the fermentation time is 24h, and the strain inoculation amounts are set to 1%, 2%, 3%, 4%, and 5% respectively; the strain inoculation amount is 3%, the fermentation time is 24h, and the fermentation temperatures are set to 29°C, 33°C, 37°C, 41°C, and 45°C respectively; the inoculation amount is 3%, the fermentation temperature is 37°C, and the fermentation times are set to 12h, 18h, 24h, 30h, and 36h respectively. Ferment the coconut water. The fermentation broth is centrifuged at 4°C and 8000 r / min, and then pasteurized at 70°C for 15 min. Using the inhibition rate of xanthine oxidase as the evaluation value, examine the effects of inoculation amount, fermentation temperature, and fermentation time on the function of fermented coconut water.

[0072] Response surface experiment: Based on the results of the single-factor experiment, according to the response surface experimental design principle, using the inhibition rate of xanthine oxidase as the response value, design a response surface analysis optimization experiment and conduct a regression analysis. The results are as shown in Figure 2 、 Figure 3 and Figure 4 shown.

[0073] As can be seen from Figure 2 , when the fermentation time is 12h, the inhibition rate is about 45%. When it rises from 12h to 24h, the inhibition rate rises rapidly and reaches the highest at about 73% at 24h. After that, when it rises from 24h to 36h, the inhibition rate begins to decline. At 36h, the inhibition rate is about 61%.

[0074] As can be seen from Figure 3 , when the temperature is 29°C, the inhibition rate is about 54%. When it rises from 29°C to 37°C, the inhibition rate rises and reaches the highest at 37°C, about 68.5%. After that, when it rises from 37°C to 45°C, the inhibition rate begins to decline. When it rises to 45°C, the inhibition rate drops to about 46%.

[0075] As can be seen from Figure 4As can be seen, when the inoculation amount is 1%, the inhibition rate is about 56%. When the inoculation amount increases from 1% to 3%, the inhibition rate increases significantly and reaches the highest at 3%, about 69.5%. When it continues to increase to 5%, it decreases, and the inhibition rate is about 64%.

[0076] To sum up, when the present invention ferments coconut water, the coconut-based postbiotics obtained under the fermentation conditions of an inoculation amount of 3%, a temperature of 37 °C, and a time of 24 h have the best inhibitory effect on xanthine oxidase.

[0077] II. Cell experiments

[0078] (I) Cultivation of cells BRL-3A

[0079] 1. Cell resuscitation and cultivation

[0080] After taking out the cryopreservation tube from liquid nitrogen or a -80 °C refrigerator, immediately immerse it in a 37 °C water bath and quickly shake it for 1 min until it is completely dissolved. Place the dissolved cells in a centrifuge tube, centrifuge once at a speed of 1200 r / min for 5 min, discard the supernatant, resuspend the cells with 2 mL of complete medium, then inject the cell suspension into a T25 flask, add another 5 mL of complete medium, and place it in a 37 °C, 5% concentration CO 2 incubator for cultivation.

[0081] 2. Cell passage

[0082] When the cells are in good growth condition and the confluence reaches 80% - 90%, start passage. Take cells in good condition and operate in a laminar flow hood. Aspirate the medium in the T25 culture flask, wash the cells with 1 mL of PBS, shake well, aspirate the PBS, add 1 mL of trypsin for digestion, place it in the incubator for 2 min, add 3 mL of medium to terminate digestion, centrifuge at 1200 r / min for 3 min, discard the supernatant, add 1 mL of complete medium, pipette well and then aspirate it into a T25 flask, add 3 mL of medium, shake well and culture in the incubator.

[0083] 3. Cell cryopreservation

[0084] Take out the cells to be cryopreserved from the incubator (the remaining cells that do not need to be cryopreserved are used as cell materials for subsequent experiments), remove the medium, add 1 mL of PBS for washing, add 1 mL of trypsin, place it in the incubator for 2 min, then add 2 mL of medium to terminate digestion, pipette well, add it to a centrifuge tube, centrifuge at 1200 r / min for 5 min, discard the supernatant, add cryopreservation solution (add as many mL of cryopreservation solution as the number of tubes to be cryopreserved), pipette well and then add it to a cryopreservation tube, place it in a -80 °C refrigerator, and transfer it to liquid nitrogen after 24 h.

[0085] (II) Cell cytotoxicity test

[0086] The standard CCK-8 method was used to verify the cytotoxicity of coconut-based postbiotics and screen out the appropriate concentration of coconut-based postbiotics. The specific steps are as follows:

[0087] (1) Inoculate the BRL-3A (rat hepatocytes) cells obtained from the above subculture into a 96-well cell culture plate, culture each well with 100 μL of culture medium, and place it in an incubator (37 °C, 5% CO 2 ), and discard the culture medium when the cells reach 80% - 90%.

[0088] (2) Divide the cells into a blank group, a postbiotic group, and a control group.

[0089] (3) The blank group has no cells and only adds 100 μL of complete medium; the postbiotic group adds 100 μL of postbiotics with concentrations of 50, 100, 150, 200, 250, and 300 μg / mL respectively; the control group adds complete medium and cultures in a cell incubator for 24 h.

[0090] (4) After the induction ends, process according to the steps required by the cell viability detection kit. After 24 h, remove the culture medium containing postbiotics, wash 3 times with PBS, and add 10 μL of cell viability detection solution to each well. Incubate the culture plate in the incubator for 5 - 6 h. Set the excitation wavelength at 530 nm and the emission wavelength at 590 nm with an enzyme-linked immunosorbent assay (ELISA) reader to detect the fluorescence value of each well, calculate the cell viability, and the results are as Figure 5 shown. The cell viability test is calculated by the following formula:

[0091]

[0092] In the formula: A 后生元 represents the absorbance of the well with cells, cell viability detection solution, and postbiotics; A 空白 represents the absorbance of the well with culture medium and cell viability detection solution but without cells; A 对照 represents the absorbance of the well with cells and cell viability detection solution but without postbiotics.

[0093] The results show that as the concentration of postbiotics increases, the cells all have a certain viability, and when the concentration is 200 μg / mL, the cell viability is close to 140%, indicating that postbiotics have no obvious toxic effect on the cells.

[0094] As can be seen from the figure, when the concentration of postbiotics is 100 μg / mL, 150 μg / mL, and 200 μg / mL, the cell viability is good. Therefore, in the following determination experiments, the concentrations of postbiotics selected are 100 μg / mL, 150 μg / mL, and 200 μg / mL for determination.

[0095] (3) Determination of uric acid lowering effect

[0096] First, 1 mM xanthine was selected as an inducer to induce BRL-3A cells to establish a hyperuricemia cell model. Then, coconut-based postbiotics were added and co-cultured with the hyperuricemia cells. After culturing for a certain period of time, the uric acid in the cells was measured. The specific steps are as follows:

[0097] Inoculate BRL-3A cells into 6 six-well plates. After culturing in a conventional medium for 24 h until they adhered, the 6 six-well plates were grouped and then continued to be cultured. The specific grouping is as follows: 3 six-well plates were respectively the blank group (NC), the model group (MD), and the positive control group (Allopurine), and the other 3 six-well plates were experimental groups with different concentrations (100 μg / mL, 150 μg / mL, 200 μg / mL). When culturing after grouping, the components of the culture medium for each group were as follows:

[0098] Blank group: Conventional medium;

[0099] Model group: Conventional medium + 1% 1 mM xanthine;

[0100] Experimental groups: Conventional medium + 100 μg / mL postbiotics + 1% 1 mM xanthine; Conventional medium + 150 μg / mL postbiotics + 1% 1 mM xanthine; Conventional medium + 200 μg / mL postbiotics + 1% 1 mM xanthine;

[0101] Positive control group: Conventional medium + 50 μg / mL allopurinol.

[0102] After culturing each group in the corresponding culture medium for 48 h, the cell supernatants of each group were collected respectively. An uric acid detection kit (Nanjing Jiancheng uric acid detection kit) was used, and the operation was carried out according to the kit instructions to measure the uric acid content in the cell supernatants, and the uric acid inhibition rate was calculated. The results are as Figure 6 shown.

[0103] The results showed that: The uric acid level of the cells in the model group was significantly higher than that in the blank group, rising from nearly 25 μmol / L to nearly 250 μmol / L, indicating that the hyperuricemia cell model was successfully established. After treating the hyperuricemia cells with coconut-based postbiotics at different concentrations, the uric acid level in the experimental groups was significantly lower than that in the model group and was close to the level of the positive control group, about 150 μmol / L. The results indicated that: Coconut-based postbiotics have the effect of lowering uric acid, and the inhibition rates among different concentrations do not differ much, and all have an inhibitory effect on uric acid.

[0104] (4) Determination of the levels of inflammatory factors (TNF-α, IL-1β) in cells

[0105] The experiment was designed according to the steps of the above-mentioned “(3) Determination of the effect of coconut-based postbiotics on reducing uric acid”. After culturing for 48 h, the cell supernatants of each group were collected, and an inflammatory factor detection kit (Nanjing Jiancheng Bioengineering Co., Ltd.) was used to measure the inflammatory indexes, including TNF-α and IL-1β. The results are as Figure 7 shown. In Figure 7 , Figure A shows the concentration of the inflammatory factor TNF-α; Figure B shows the concentration of the inflammatory factor IL-1β.

[0106] The results showed that: compared with the blank group, the levels of inflammatory factors (TNF-α and IL-1β) in the model group cells were significantly increased. After treatment with coconut-based postbiotics, the levels of inflammatory factors in the experimental group were significantly decreased and were close to the levels of the blank group and the positive control group. The results indicated that coconut-based postbiotics could inhibit the inflammatory response in the hyperuricemia cell model.

[0107] (5) Cell antioxidant experiment

[0108] 1. Determination of horizontal antioxidant stress indexes (MDA, SOD)

[0109] The experiment was designed according to the steps of the above-mentioned “(3) Determination of the effect of coconut-based postbiotics on reducing uric acid”. After culturing for 48 h, the cells of each group were collected, and the levels of oxidative stress indexes (MDA, SOD) in the cells of each group were detected respectively. The detection processes are as follows;

[0110] (1) Determination of MDA content

[0111] The thiobarbituric acid method was used to determine the MDA content in cells. The cells were collected, an appropriate amount of lysis buffer was added, the cells were ultrasonically broken on ice bath, and the supernatant was taken by centrifugation. According to the MDA detection kit instruction manual, after adding the reagents and reacting, the absorbance was measured at 532 nm, and the MDA content was calculated according to the standard curve.

[0112] (2) Determination of SOD activity

[0113] The xanthine oxidase method was used to determine the SOD activity in cells. The cells were collected, and the treatment method was the same as that in the above “(1) Determination of MDA content”. According to the SOD detection kit instruction manual, after adding the reagents and reacting, the absorbance was measured at 450 nm, and the SOD activity was calculated according to the formula provided by the kit.

[0114] (3) Determination of NO content: It was detected using the Beyotime kit.

[0115] The results are as Figure 8 shown. In Figure 8Among them, in Figure A is the MDA content; in Figure B is the SOD activity content. It can be seen from Figure A that for the MDA content, the model group was significantly higher than the blank group, while after the postbiotic intervention, its MDA content decreased significantly and was lower than the blank group level. It can be seen from Figure B that for the SOD activity content, the model group was lower than the blank group, and after the postbiotic intervention, its activity content increased significantly and exceeded the blank group.

[0116] The results show that coconut-based postbiotics have a certain improvement effect on the cellular redox status and have good antioxidant functions.

[0117] 2. Determination of mitochondrial membrane potential (MMP)

[0118] The experiment was designed according to the steps of the above-mentioned “(III) Determination of the uric acid-lowering effect of coconut-based postbiotics”. After culturing for 48 h, a JC-1 probe mitochondrial membrane potential assay kit was used for determination. Prepare the staining working solution, dilute JC-1 according to the ratio and add it to the staining buffer. After aspirating the culture medium in each well of the six-well plate, add fresh culture medium, and then add the staining working solution, and incubate in a cell culture incubator at 37 °C for 20 minutes. During the incubation, prepare 1×JC-1 staining buffer (ice bath). After the incubation, aspirate the supernatant, wash twice with 1×JC-1 staining buffer, add an appropriate amount of culture medium, and observe under a fluorescence inverted microscope. The change in mitochondrial membrane potential was represented by the ratio of red to green fluorescence. The results are as Figure 9 shown.

[0119] The results showed that the ratio of red to green fluorescence of the cells in the model group decreased compared with the blank group, indicating a decrease in mitochondrial membrane potential; while after the postbiotic intervention, the ratio of red to green fluorescence increased significantly, indicating that the postbiotic has a protective effect on mitochondrial function.

[0120] 3. Determination of reactive oxygen species (ROS)

[0121] The experiment was designed according to the steps of the above-mentioned “(III) Determination of the uric acid-lowering effect of coconut-based postbiotics”. After culturing for 48 h, the cells of each group were collected, washed with phosphate buffered saline and then placed in a culture medium containing 10 μmol·L -1 DCFH-DA fluorescent probe, incubated in the dark for 30 min, washed with PBS and then observed under a fluorescence microscope. The fluorescence intensity of the cells in each group was analyzed using Image J software, and the relative level of ROS in the cells of each group was calculated according to the formula. The results are as Figure 10 shown.

[0122] Relative level of ROS = fluorescence intensity of the experimental group / fluorescence intensity of the control group

[0123] The results showed that the oxidative stress state of the cells in the model group was significantly higher than that in the blank group. After the intervention of postbiotics, the level of reactive oxygen species (ROS) decreased and approached that in the blank group, indicating that postbiotics can regulate the intracellular ROS level in hyperuricemic cells and have an antioxidant stress effect.

[0124] Although the present invention has been described by using the above preferred embodiments, it is not intended to limit the protection scope of the present invention. Any person skilled in the art can still make various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention, and these still belong to the protection scope of the present invention.

Claims

1. A coconut-based postbiotic with uric acid lowering and antioxidant functions, characterized in that: It is made by using coconut water as the fermentation medium and adding Lactobacillus DO28L8 for fermentation.

2. A method for preparing coconut-based postbiotics with uric acid lowering and antioxidant functions, characterized in that: include: The coconut water stock solution was filtered through gauze and sterilized at 121°C for 15 min to serve as the coconut water fermentation medium; Lactobacillus DO28L8 is activated, and the seed liquid obtained after activation is inoculated into coconut water fermentation medium at an inoculation rate of 2-5%, and is statically fermented and cultured at a temperature of 33-41° C. for 18-36 hours, and then inactivated after the culture is completed.

3. The method for preparing the coconut-based postbiotics with uric acid lowering and antioxidant functions according to claim 2, characterized in that: The preparation of the seed solution comprises the following steps: (1) First activation The lactobacillus DO28L8 was streaked onto an MRS solid medium, incubated upside down at 37-40°C for 24-48 hours, and then a single colony with a larger calcium-dissolving circle on the plate was selected and inoculated into an MRS liquid medium, and then statically incubated in a constant temperature incubator at 37-40°C for 15-24 hours for the first activation; (2) Second activation The bacterial liquid after the first activation was transferred to a new MRS liquid culture medium at an inoculation rate of 3-5%, and the culture was repeated for 15-24 hours for the second activation. (3) Preparation of seed solution The second activated bacterial solution was centrifuged at 4°C and 8000 r / min for 10 min, the supernatant was discarded, and the precipitate was washed 2 to 3 times with sterile saline. Finally, the bacterial cells were resuspended with sterile saline to adjust the bacterial concentration to 10 8 ~10 9 CFU / mL, and obtain the seed solution.

4. The method for preparing coconut-based postbiotics with uric acid lowering and antioxidant functions according to claim 2, characterized in that: The inactivation step comprises: after the fermentation culture is completed, the fermentation liquid is ultrasonically crushed for 5 minutes, and then the fermentation liquid is centrifuged for 15 minutes at a temperature of 4° C. and a rotation speed of 8000 r / min to separate the supernatant, and the supernatant is pasteurized at a temperature of 70-80° C. for 15-20 minutes.

5. The method for preparing coconut-based postbiotics with uric acid lowering and antioxidant functions according to claim 2, characterized in that: It also includes freeze-drying and storage after inactivation, and the freeze-drying storage includes: The inactivated sterile fermentation liquid is filtered through a sterile filter membrane, and the obtained sterile fermentation filtrate is placed in a freeze dryer, first pre-frozen at a temperature of -40°C for 2 to 3 hours, and then sublimated and dried at a temperature of -20°C to -10°C and a vacuum degree of 0.1 to 0.2 mbar for 24 to 36 hours, and then analytically dried at a temperature of 20°C to 25°C and a vacuum degree of 0.05 to 0.1 mbar for 4 to 6 hours to obtain a coconut-based postbiotic freeze-dried material, and the coconut-based postbiotic freeze-dried material is ground to obtain a coconut-based postbiotic freeze-dried powder, and the coconut-based postbiotic freeze-dried powder is packaged and stored in a -80°C refrigerator.

6. Use of the coconut-based postbiotics with uric acid-lowering and antioxidant functions as claimed in claim 1 in the preparation of uric acid-lowering and / or antioxidant products.

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