A morinda officinalis protein fermentation liquor, a preparation method thereof and application thereof in reducing uric acid

Abstract

The application discloses a milk vetch root protein fermentation liquor, a preparation method thereof and application thereof in reducing uric acid, and relates to the field of milk vetch root protein fermentation liquor. The preparation method comprises the following steps: adding milk vetch root protein, sucrose and probiotic powder into water, mixing uniformly after sealing, and then performing fermentation; and after fermentation is completed, centrifugal collection is performed on supernatant to obtain the milk vetch root protein fermentation liquor; wherein the probiotic is at least one of lactic acid bacteria, bifidobacterium and yeast. It is found through experiments that the milk vetch root protein fermentation liquor can significantly reduce the uric acid content in a liver cell HepG2 high uric acid model, and therefore, the milk vetch root protein fermentation liquor can be used for developing products for preventing and treating hyperuricemia or resisting gout, and further improving the application value of milk vetch root.

Description

Technical Field

[0001] This invention belongs to the field of functional food or health product technology, and specifically relates to a fermentation broth of *Millettia dielsiana* protein, its preparation method, and its application in lowering uric acid. Background Technology

[0002] *Millettia dielsiana*, a legume belonging to the genus *Millettia*, is mainly cultivated in subtropical regions, such as Guangdong, Guangxi, Fujian, Hainan, and Hunan provinces. It has a long history of being used for both food and medicine, belonging to the category of plants with both medicinal and edible uses, possessing high nutritional and health-promoting value. Reports indicate that the root of *Millettia dielsiana* is used medicinally to strengthen muscles and tendons, invigorate the lungs, and replenish deficiencies. It is commonly used as a raw material for making medicinal wine, tea, soups, or medicinal cuisine, possessing effects such as clearing heat and detoxifying, and promoting blood circulation and heat dissipation. Numerous studies have shown that *Millettia dielsiana* contains various active substances such as polysaccharides, flavonoids, esters, and glycosides, thus exhibiting antioxidant, anti-tumor, anti-inflammatory, and immune-enhancing effects. Some studies have also shown that using enzymatic hydrolysis to prepare *Millettia dielsiana* protein into bioactive peptides also possesses antioxidant effects. Currently, there are no reports on the use of *Millettia dielsiana* protein fermentation broth for lowering uric acid. Summary of the Invention

[0003] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing Daphne genkwa protein fermentation broth.

[0004] Another object of the present invention is to provide the fermentation broth of *Mammillaria gracilis* protein prepared by the method.

[0005] Another object of the present invention is to provide the application of the aforementioned *Millettia dielsiana* protein fermentation broth.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for preparing a fermented liquid of *Mammillaria gracilis* protein includes the following steps: adding *Mammillaria gracilis* protein, sucrose, and probiotic powder to water, sealing and mixing, and then fermenting. After fermentation, centrifuging is performed to collect the supernatant to obtain the fermented liquid of *Mammillaria gracilis* protein; wherein the probiotics are at least one of lactic acid bacteria, bifidobacteria, and yeast.

[0008] The *Smilax glabra* protein comprises crude and / or purified protein extracted from the root and / or leaf tissues of *Smilax glabra*; it can be extracted using conventional methods in the art; preferably, it is extracted using the following method:

[0009] (1) Pretreatment

[0010] Fresh leaves and / or root tissues of *Smilax glabra* are washed, cleaned, dried, then crushed and sieved to obtain *Smilax glabra* powder.

[0011] (2) Extraction

[0012] Adding Radix Millettiae Speciosae powder into water, adjusting pH to 11-13, and performing extraction at 35-50°C to obtain a Radix Millettiae Speciosae protein extract;

[0013] (3) Preparation of Radix Millettiae Speciosae protein

[0014] ① Centrifuging the Radix Millettiae Speciosae protein extract obtained in step (2) to remove precipitate, and freeze-drying to obtain crude Radix Millettiae Speciosae protein;

[0015] or

[0016] ② Adjusting pH of the Radix Millettiae Speciosae protein extract obtained in step (2) to 1.5-5.0, standing, centrifuging to remove supernatant, then adding water for reconstitution, adjusting pH to 7.0, dialyzing, and lyophilizing to obtain purified Radix Millettiae Speciosae protein.

[0017] The drying temperature in step (1) is 30-60°C; preferably 30°C.

[0018] The sieving in step (1) is preferably passing through a 100-mesh sieve.

[0019] The material-to-liquid ratio of the Radix Millettiae Speciosae powder to water in step (2) is 1:10-50 (g / ml); preferably 1:40 (g / ml).

[0020] The pH adjustment in step (2) is preferably adjusting pH to 12.

[0021] The pH adjustment in step (2) is performed with NaOH solution; preferably with 1-6 mol / L NaOH solution; more preferably with 6 mol / L NaOH solution.

[0022] The extraction temperature in step (2) is preferably 50°C.

[0023] The extraction time in step (2) is 2-3 h; preferably 2 h.

[0024] The water in steps (2) and (3) is preferably sterile water, deionized water or ultrapure water.

[0025] The centrifugation conditions in steps (3) ① and ② are: centrifuging at 4000-10000 rpm / min for 15-40 min; preferably: centrifuging at 4000 rpm / min for 15 min.

[0026] The pH adjustment in step (3) ② is performed with HCl solution; preferably with 1-6 mol / L HCl solution; more preferably with 6 mol / L HCl solution.

[0027] The pH value adjustment to 7.0 in step (3) ② is performed using NaOH solution; preferably, a NaOH solution of 1 to 6 mol / L is used; more preferably, a NaOH solution of 1 mol / L is used.

[0028] The pH adjustment mentioned in step (3) ② is preferably adjusted to pH 3.0.

[0029] The settling time mentioned in step (3) ② is at least 1 hour.

[0030] The molecular weight cutoff of the dialysis bag used in step (3) ② is less than or equal to 1000 Da.

[0031] The amount of the *Smilax glabra* protein is calculated based on its mass percentage in the fermentation system of 0.6-4%; preferably, it is calculated based on its mass percentage in the fermentation system of 0.6-1.2%.

[0032] The amount of sucrose used is calculated based on its mass percentage in the fermentation system of 1.6 to 4%; preferably, it is calculated based on its mass percentage in the fermentation system of 0.6 to 1.2%.

[0033] The number of live cells at the time of inoculation of the probiotics is 10. 6 ~10 8 CFU / mL; preferably 10 7 CFU / mL.

[0034] The amount of probiotics used is calculated based on 1-5% of their volume percentage in the fermentation system; preferably, it is calculated based on 1% of their volume percentage in the fermentation system.

[0035] The probiotics are preferably at least one of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus gasseri, and Bifidobacterium; more preferably Lactobacillus plantarum; and even more preferably Lactobacillus plantarum FS4722.

[0036] The fermentation process is preferably dynamic fermentation, i.e., fermentation is carried out on a shaker.

[0037] The fermentation conditions are: 200-500 rpm, 25-37 ℃ for 24-48 h; preferably: 300 rpm, room temperature or 37 ℃ for 24-48 h.

[0038] The centrifugation conditions after fermentation are: 8000-15000 rpm / min for 5-20 min; preferably: 8000 rpm / min for 20 min.

[0039] A fermentation broth containing *Mammillaria gracilis* protein is prepared by any of the methods described above.

[0040] The application of the aforementioned *Millettia dielsiana* protein fermentation broth in the preparation of products for the prevention and treatment of hyperuricemia and / or anti-gout.

[0041] The products mentioned include pharmaceuticals, etc.

[0042] The glycine and lactic acid components in the fermented broth of *Millettia dielsiana* protein have the effect of reducing uric acid content; the glycine and acetic acid components in the fermented broth of *Millettia dielsiana* protein can reduce the enzyme activity of XOD, a key enzyme in the uric acid synthesis pathway; the fermented broth of *Millettia dielsiana* protein can reduce the uric acid level in liver cells, thereby achieving the purpose of lowering uric acid and preventing gout.

[0043] The present invention has the following advantages and effects compared with the prior art:

[0044] 1. The *Millettia dielsiana* protein fermentation broth of this invention is prepared by fermentation of *Millettia dielsiana* protein powder (root protein and leaf protein), probiotic powder, sucrose, and sterile water. Experiments have shown that the *Millettia dielsiana* protein fermentation broth can significantly reduce uric acid content in a HepG2 hepatocyte hyperuricemia model. The components glycine and lactic acid in the *Millettia dielsiana* protein fermentation broth have a significant effect on reducing uric acid content. The components glycine and acetic acid in the *Millettia dielsiana* protein fermentation broth can significantly inhibit the enzyme activity of XOD, a key enzyme in the uric acid synthesis pathway. Therefore, it can be used to develop drugs with the effects of preventing and treating hyperuricemia (lowering uric acid) and anti-gout.

[0045] 2. This invention utilizes probiotics to prepare fermented protein broth (product) from the roots and leaves of *Smilax glabra* via bio-fermentation, evaluates its uric acid-lowering effect, broadens the biological efficacy of *Smilax glabra*-related products, and further enhances the application value of *Smilax glabra*. Attached Figure Description

[0046] Figure 1 These are different types of oxalis protein fermentation broth. 1 ¹H NMR spectra; where A is the pure protein fermentation broth from the root of *Achyranthes bidentata*; and B is the pure protein fermentation broth from the leaf tissue of *Achyranthes bidentata*.

[0047] Figure 2 This is a statistical graph showing the intracellular uric acid content of HepG2 liver cells after treatment with different Daphne genkwa protein fermentation broths (in the graph, different letters between samples indicate significant differences, while the opposite indicates no significant differences).

[0048] Figure 3This is a statistical graph showing the intracellular uric acid content and XOD enzyme activity of HepG2 hepatocytes after different component treatments (in the graph, different letters between samples indicate significant differences, and vice versa); where A represents uric acid content and B represents XOD enzyme activity. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0050] Example 1: Extraction of Daphne odora protein

[0051] (1) Extraction of crude protein from *Smilax glabra*: Fresh leaves and roots of *Smilax glabra* (Guangdong Mailinco Biotechnology Co., Ltd.) were classified, cleaned and cleaned to remove impurities, and placed in a 30℃ oven until the moisture was dried. Then, they were crushed separately and sieved through a 100-mesh sieve to obtain *Smilax glabra* root powder and *Smilax glabra* leaf powder. The *Smilax glabra* root powder and *Smilax glabra* leaf powder were then suspended in deionized water at a material-to-liquid ratio of 1:40 (g / ml). The pH was adjusted to 12 with 6 mol / L NaOH solution, and the extracts were soaked in a 50℃ constant temperature water bath for 2 hours. After centrifugation at 4000 rpm / min for 15 minutes, the precipitate was removed to obtain the protein extracts of *Smilax glabra* root and leaves. A portion of the extracts was freeze-dried to obtain crude protein from *Smilax glabra* leaves and crude protein from *Smilax glabra* roots.

[0052] (2) Extraction of pure protein from *Smilax glabra* root and leaves: The protein extracts from *Smilax glabra* root and leaves before freeze-drying in step (1) were adjusted to pH 3.0 with 6 mol / L HCl solution, allowed to stand for 1 h, and then centrifuged at 4000 rpm / min for 15 min to remove the supernatant. The protein precipitates were redissolved with ultrapure water, and the pH was adjusted to 7.0 with 1 mol / L NaOH solution. The precipitates were then dialyzed with a dialysis bag with a molecular weight cutoff of 1000 Da to remove impurities. The *Smilax glabra* protein solution in the dialysis bag was then freeze-dried to obtain pure protein from *Smilax glabra* root and leaves.

[0053] Protein content was determined using the spectrophotometric method according to GB 5009.5-2016. Based on pure protein, the protein content in the leaves and roots of *Smilax glabra* was 15.4% and 14.6%, respectively. The protein extraction rates from the leaves and roots were obtained in the above experiments, ranging from 70% to 88%; the molecular weights were both in the range of 12 to 19 kDa.

[0054] Example 2: Preparation of *Millettia dielsiana* protein fermentation broth

[0055] (1) Probiotic activation:

[0056] The probiotic selected in this embodiment is Lactobacillus plantarum FS4722 (accession number CGMCC No. 22750), which has been disclosed in a patent (patent number 202210167880.6, entitled "A strain of Lactobacillus plantarum and its application in lowering uric acid, weight loss, anti-inflammation, etc.").

[0057] Add 1 g of the above probiotic powder to 100 mL of sterilized MRS broth (MRS broth medium purchased from Guangdong Huankai Microbial Technology Co., Ltd.; its formula is as follows: per liter, it contains 10 g of casein digest, 10 g of beef extract powder, 4 g of yeast extract powder, 2 g of triammonium citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate tetrahydrate, 2 g of dipotassium hydrogen phosphate, 20 g of glucose, 1.08 g of Tween-80, pH 5.7 ± 0.2. Usage method: take 54 g, add 1 L of deionized water, dissolve completely, and autoclave at 121 ℃ for 15 min) and seal the container. Incubate overnight at 37 ℃ to obtain a probiotic solution (approximately 10 mL). 7 (CFU / mL).

[0058] (2) Fermentation of *Smilax glabra* protein: The crude root protein, pure root protein, crude leaf protein, and pure leaf protein extracted in Example 1 were fermented. 1% (v / v) probiotic solution was added to a sterile fermenter containing 0.6–4 wt% (the amounts added in this experiment were 0.6 wt% and 1.2 wt%, respectively; the fermentation supernatant obtained at 1.2 wt% was used for component detection and uric acid reduction assessment) of *Smilax glabra* protein (crude root protein, pure root protein, crude leaf protein, and pure leaf protein) and 1.6–4 wt% (the amounts added in this experiment were 0.6 wt% and 1.2 wt%, respectively; the fermentation supernatant obtained at 1.2 wt% was used for component detection and uric acid reduction assessment). After sealing and shaking, the mixture was cultured at 37 ℃ and 300 rpm for 24–48 h. At this time, the pH of the system was 3.5–5.4. After fermentation, the supernatant was collected after centrifugation at 8000 rpm for 20 min to obtain crude protein fermentation broth from *Lactobacillus plantarum* root, pure protein fermentation broth from root, crude protein fermentation broth from leaf, and pure protein fermentation broth from leaf (Note: *Lactobacillus plantarum* FS4722 can lower blood uric acid through the degradation of nucleosides by its cellular contents (3 intracellular enzymes) and the combined effect of the intact strain in regulating the intestinal flora structure. Therefore, in order to eliminate experimental interference from *Lactobacillus plantarum* FS4722 and its cellular contents, this invention uses the supernatant obtained by centrifugation after fermentation of *Lactobacillus plantarum* protein with *Lactobacillus plantarum* FS4722 for uric acid reduction evaluation).

[0059] Example 3: Detection of components in the fermentation broth of *Smilax glabra* protein

[0060] The supernatant obtained in Example 2 (i.e., the pure protein fermentation broth from the root and leaf of *Smilax glabra*) was mixed with the extraction solvent (100 mM potassium phosphate buffer and D2O mixed at a volume ratio of 1:9) at a volume ratio of 1:7. The mixture was vortexed for 5 min, and 60 μL was collected in an NMR tube. Detection was performed using a 600 M superconducting nuclear magnetic resonance spectrometer (Bruker AVANCE III HD 600). The full spectrum was acquired using the NOESYPR1D sequence (recycledelay-90-t1-90-tm-90-freeinductiondecay (FID)). Water peaks were suppressed using a pre-saturation method. The sampling points were 32K, the spectral width was 20 ppm, the mixing time was 0.1 s, the relaxation delay time was 2 s, and the number of accumulations was 128. The collected spectral peaks were baseline corrected, and metabolite structures were analyzed using a database. All of the above processing was performed in MestReNova (Mnova 9.0) and determined according to the reference (Cai Yi, Hao Fuhua, Wang Yulan. NMR analysis of common body fluids and tissue metabolomics in Kunming mice [J]. Journal of Spectroscopy, 2013 (1): 14. DOI: 10.3969 / j.issn.1000-4556.2013.01.006.). 1 Metabolite attribution from H NMR spectra.

[0061] Mass spectra of pure protein fermentation broth from the roots and leaves of *Smilax glabra* are shown below. Figure 1 As shown: After metabolite classification, both fermentation broths were found to contain glycine, lactic acid and acetic acid components.

[0062] Example 4: Establishment of a HepG2 hepatocyte hyperuricemia model

[0063] HepG2 cells (Chinese Academy of Sciences Cell Bank, No. TCHu72) were stored at a density of 10-1 5 Cells were seeded in 96-well plates and diluted to 200 μL / well with DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin / streptomycin. The plates were incubated at 37°C and 5% CO2 for 12 h, followed by pouring out the culture medium and washing three times with sterile PBS buffer. 100 μL of a modeling agent (composed of inosine and guanosine, both at 1.3 mM) was added, and the plates were incubated for 24 h to complete modeling. The same volume of PBS buffer was used instead of the modeling agent, and the plates were incubated for the same time; this was designated as the control group. The culture medium and cells were collected, and after sonication to disrupt the cells, the uric acid levels in both the control and model groups were measured using a uric acid assay kit (Nanjing Jiancheng, No. C01221). The experiment was performed in triplicate. The results showed that the uric acid content in the model group was significantly higher than that in the control group (see...). Figure 2 The presence of a control group and a model group indicates successful modeling.

[0064] Example 5: Effect of *Millettia dielsiana* protein fermentation broth on uric acid synthesis in a HepG2 hepatocyte hyperuricemia model.

[0065] (1) Take the fermentation broth of *Smilax glabra* protein from Example 2 (fermentation broth of crude protein from *Smilax glabra* root, pure protein from root, crude protein from leaf, and pure protein from leaf), dilute it 100 times with sterile PBS buffer, filter it through a sterile filter membrane, and add 100 μL to the HepG2 hepatocyte hyperuricemia model from Example 4. After culturing for 24 h, lyse the cells and detect the uric acid content using a uric acid kit. The experiment was repeated three times.

[0066] The results are as follows Figure 2 As shown, the results indicate that all four types of *Millettia dielsiana* protein fermentation broths significantly reduced uric acid levels in the model group. Furthermore, based on the fermentation broth components measured in Example 3, the potential uric acid-lowering substances in the fermentation broth may be glycine, emulsion, and acetic acid.

[0067] (2) The successfully constructed model group in Example 4 was further treated with glycine (final concentration 1 mg / mL) and lactic acid and acetic acid (final concentration 1 μM), respectively, to divide the experiment into 5 groups: ① The successfully constructed model group cells in Example 4 were not treated in any way and were named the model group; ② The model group cells were treated with PBS buffer as a control and were named the control group; ③ The model group cells were treated with 1 mg / mL glycine and were named the glycine group; ④ The model group cells were treated with 1 μM lactic acid and were named the lactic acid group; ⑤ The model group cells were treated with 1 μM acetic acid and were named the acetic acid group. After culturing for 24 h, the cells were lysed, and the uric acid content was detected using a uric acid kit, and the XOD enzyme activity was detected using an XOD enzyme activity detection kit (Nanjing Jiancheng, No. A00211). The experiment was set up in triplicate.

[0068] The results are as follows Figure 3 As shown: Experiments have revealed that glycine and lactic acid have a significant effect on reducing uric acid levels. Figure 3 A), glycine and acetic acid significantly reduce the enzyme activity of XOD, a key enzyme in the uric acid synthesis pathway. Figure 3 B) indicates that the components in the fermentation broth can reduce uric acid content and decrease the enzyme activity of XOD, a key enzyme in the uric acid synthesis pathway, thereby achieving the purpose of lowering uric acid and preventing gout.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a fermentation broth containing *Mammillaria gracilis* protein, characterized in that, comprising the following steps: adding Millettia speciosa Champ. protein, sucrose and probiotic powder into water, sealing and mixing uniformly, then performing fermentation, after fermentation is completed, centrifuging and collecting a supernatant to obtain a Millettia speciosa Champ. protein fermentation broth; wherein, the probiotic is *Lactobacillus plantarum* FS4722, with a preservation number of CGMCC No.22750; the Millettia speciosa Champ. protein is extracted by the following method: (1) Pretreatment washing and impurity removing fresh Millettia speciosa Champ. leaves and / or root tissues, drying, then pulverizing and sieving to obtain Millettia speciosa Champ. powder; (2) Extraction adding the Millettia speciosa Champ. powder into water, adjusting pH to 11-13, extracting at 35-50° C to obtain a Millettia speciosa Champ. protein extracting solution; (3) Preparation of Millettia speciosa Champ. protein ① centrifuging the Millettia speciosa Champ. protein extracting solution obtained in step (2) to remove precipitates, and freeze-drying to obtain crude Millettia speciosa Champ. protein; or ② adjusting pH of the Millettia speciosa Champ. protein extracting solution obtained in step (2) to 1.5-5.0, standing still, centrifuging to remove a supernatant, then adding water for re-dissolution, adjusting pH to 7.0, dialyzing and freeze-drying to obtain purified Millettia speciosa Champ. protein; the fermentation condition is culturing at 200-500 rpm and 25-37° C for 24-48 h.

2. The preparation method of the Millettia speciosa Champ. protein fermentation broth according to claim 1, characterized in that: the dosage of the Millettia speciosa Champ. protein is calculated by adding 0.6% - 4% by mass percentage in the fermentation system; the dosage of the sucrose is calculated by adding 1.6% - 4% by mass percentage in the fermentation system; the probiotic powder is added in the following mode: inoculating probiotic powder into a culture medium for culture to obtain probiotic liquid, and then adding the probiotic liquid into the fermentation system; wherein, The number of live cells at the time of inoculation of the probiotic solution is 10. 6 ~10 8 CFU / mL; the dosage of the probiotic liquid is calculated by adding 1% - 5% by volume percentage in the fermentation system.

3. The preparation method of the Millettia speciosa Champ. protein fermentation broth according to claim 1, characterized in that: the material-to-liquid ratio of the Millettia speciosa Champ. powder to water in step (2) is 1 g: 10-50 ml; the pH adjustment in step (2) is adjusting pH to 12; the pH adjustment in step (3) ② is adjusting pH to 3.0; the molecular weight cutoff of a dialysis bag used for dialysis in step (3) ② is less than or equal to 1000 Da.

4. The preparation method of the Millettia speciosa Champ. protein fermentation broth according to claim 1, characterized in that: the drying temperature in step (1) is 30-60° C; the sieving in step (1) is sieving through a 100-mesh sieve; the extraction time in step (2) is 2-3 h; the centrifugation condition in step (3) ① and ② is centrifuging at 4000-10000 rpm / min for 15-40 min; the standing time in step (3) ② is at least 1 h.

5. The preparation method of the Millettia speciosa Champ. protein fermentation broth according to claim 1, characterized in that: the centrifugation condition after fermentation is centrifuging at 8000-15000 rpm / min for 5-20 min.

6. A fermentation broth containing *Mammillaria gracilis* protein, characterized in that: It is prepared by the method described in any one of claims 1 to 5.

7. The use of the *Millettia dielsiana* protein fermentation broth according to claim 6 in the preparation of drugs for the prevention and treatment of hyperuricemia and / or anti-gout.

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