Uric acid-lowering fermentation product and preparation method and use thereof

Abstract

The present invention provides a fermented product for reducing uric acid, belonging to the technical field of Chinese medicine fermentation. The fermented product fermentation raw materials include by weight: 100-180 parts of black wolfberry, 120-200 parts of eucommia male flowers, 80-160 parts of red phoenix peony flowers, 60-140 parts of ear leaf cow hide Xiao, 140-220 parts of short-stemmed acanthopanax, 60-140 parts of venous inula, 1.2-12 parts of fermented bacteria powder and 14-120 parts of enzyme preparation. The synergistic cooperation of each Chinese medicine component has a good effect of reducing uric acid under the action of enzymatic fermentation, and can also have a significant protective effect on kidney damage and liver damage, reduce serum creatinine, urine albumin excretion rate, improve glomerular filtration function; and can improve the level of inflammatory factors.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine fermentation, and particularly relates to a uric acid-lowering fermentation product and a preparation method and use thereof. Background Art

[0002] Hyperuricemia (HUA) is a disease related to abnormal purine metabolism characterized by elevated serum uric acid levels. Its diagnostic criteria are: fasting blood uric acid levels in males and females on two different days are higher than 420 μmol / L and 360 μmol / L, respectively, under a normal purine diet. In recent years, the prevalence and morbidity of hyperuricemia are increasing rapidly.

[0003] Hyperuricemia is the main risk factor for gout. Uric acid is a metabolite of xanthine and hypoxanthine oxidized by xanthine oxidase (XOD). When the serum uric acid concentration exceeds its saturation, it will precipitate in the form of monosodium urate crystals (MSU) and deposit in the joints, inducing local inflammatory reactions in tissues, leading to the formation of gout. In addition, hyperuricemia can also affect organs such as the kidneys, liver, and joints, and long-term and persistent increases in blood uric acid can lead to various damages such as gouty arthritis, tophi, and gouty kidney disease.

[0004] At present, the treatment of hyperuricemia mainly includes drug therapy and non-drug intervention. Drug therapy mainly focuses on anti-inflammatory therapy and uric acid-lowering therapy. Anti-inflammatory therapy is the use of conventional anti-inflammatory drugs such as aspirin and colchicine for treatment. Although these drugs have fast anti-inflammatory and analgesic effects, they are only symptomatic treatments and cannot reduce uric acid levels. Most of them have obvious toxic and side effects. Uric acid-lowering therapy mainly uses xanthine oxidase inhibitors such as allopurinol to reduce uric acid production. Although these drugs can effectively reduce serum uric acid levels, they need to be taken for a long time and do not have antipyretic, analgesic and anti-inflammatory effects. They have little effect on acute arthritis and may even aggravate its symptoms or prolong the course of the disease. In addition, the use of uric acid-lowering drugs alone may induce acute attacks of gout in the early stage, and long-term use will produce side effects such as gastrointestinal irritation, bone marrow suppression and nephrotoxicity, increasing the risk of poor prognosis or even death, which in turn limits their clinical application.

[0005] Products for lowering uric acid prepared by fermentation have been disclosed in the prior art.

[0006] As disclosed in Chinese patent application 202111076145.6: A medicinal and edible enzyme suitable for people with high uric acid and gout and its preparation method, including the following components: EM bacteria, lactic acid bacteria, yeast, Lactobacillus plantarum, Streptococcus thermophilus, Bifidobacterium, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bacillus coagulans, Lactobacillus reuteri, honey, coral calcium, and drinking water. The medicinal and edible Chinese medicinal raw materials include the following components: sunflower disc, corn silk, houttuynia, coix seed, kudzu root, dandelion, gardenia, chicory, eucommia, Euryale ferox, cinnamon, light bamboo leaf, Poria, purslane, and lily. The invention uses beneficial microbial flora and combines medicinal and edible Chinese medicinal raw materials for fermentation. The product can effectively reduce the blood uric acid content of people with gout, relieve pain, improve the immune system, restore and improve metabolic function, has a wide range of applicability, is non-toxic and has no side effects, and can be eaten daily. However, it involves too many raw materials and its uric acid-lowering effect is not clear.

[0007] For example, Chinese patent 202110786163.7 discloses: a fermented Chinese medicine preparation for the treatment of gout and its preparation method, which uses four Chinese medicines, namely, Rhizoma Coptidis, Curcuma Longa, Licorice and Coix Seed, as well as glucose and beef extract. The Chinese medicinal materials are ultrafinely ground, and the cells are broken and then subjected to liquid fermentation by probiotics. The obtained Chinese medicinal fermentation liquid is spray-dried, and the collected dry powder is directly made into powder packaging or made into any one of capsules, tablets, and granules. The preparation method of this invention is simple to operate, can make full use of Chinese medicinal materials and their effective ingredients, and can effectively reduce blood uric acid through the synergistic effect of Chinese medicine and probiotics, thereby achieving the effect of preventing or treating gout. However, there is room for further improvement in its actual effect on reducing blood uric acid. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a uric acid-lowering fermentation product, which can not only significantly reduce uric acid, but also has a certain repair effect on kidney lesions and liver damage caused by long-term hyperuricemia.

[0009] In the present invention, "bacterial activity", "bacterial vitality", "viable bacteria count", "viable bacteria content" and "viable bacteria amount" have the same meaning under some conditions.

[0010] In the present invention, "enzyme activity", "enzyme activity", "enzyme activity" and "enzyme specific activity" have the same meaning under some conditions.

[0011] In one aspect, the present invention provides a uric acid-lowering fermentation product.

[0012] The fermentation raw materials of the fermented product include, by weight: 100-180 parts of black wolfberry, 120-200 parts of eucommia male flowers, 80-160 parts of red peony flowers, 60-140 parts of auricularia acuta, 140-220 parts of Acanthopanax short-stalked, 60-140 parts of Inula japonica, 1.2-12 parts of fermentation bacteria powder and 14-120 parts of enzyme preparation;

[0013] The enzyme preparation comprises cellulase, pectinase, acid protease and medium-temperature alpha-amylase, and the enzyme activity ratio of each enzyme is 18-27:32-40:50-60:40-60.

[0014] Specifically, the mass ratio of each enzyme is 6-9:8-10:5-6:8-12, and the unit enzyme activity ratio is 30-40:40-50:90-100:5-6.

[0015] Preferably, the mass ratio of each enzyme is 6:8:6:12 or 9:10:5:8 or 6:8:6:12; the unit enzyme activity ratio of each enzyme is 30:40:100:5; and the unit enzyme activity of the cellulase is 30,000 U / g.

[0016] The enzyme preparation is used for enzymolysis of substrate to obtain enzymolysis solution;

[0017] The fermented bacteria powder includes plant lactobacillus, fermented mucus lactobacillus, reuteri mucus lactobacillus, acidophilus lactobacillus, rhamnosus lactobacillus and adolescent bifidobacterium, the bacterial activity ratio of each bacteria is 120-150:55-70:48-64:36-48:78-104:12-20, and the total bacterial activity is 800-810 billion CFU / g;

[0018] Specifically, the mass ratio of each bacteria is 12-15:11-14:6-8:6-8:6-8:3-5; the unit activity ratio of each bacteria is 18-20:8-10:14-16:10-12:23-26:6-8.

[0019] Preferably, the mass ratio of each bacteria is 12:14:6:8:8:3 or 15:11:8:6:6:5; the unit activity ratio of each bacteria is 20:10:16:12:26:8; and the unit activity of the plant lactobacillus is 200 billion CFU / g.

[0020] The fermentation bacteria powder is used for fermentation after enzymolysis.

[0021] Further preferably, in the enzyme preparation, the enzyme activity ratio of cellulase, pectinase, acid protease and mesophilic α-amylase is 18:32:60:60.

[0022] Further preferably, in the fermented bacterial powder, the bacterial activity ratio of each bacteria is 120:70:48:48:104:12.

[0023] Further preferably, the total bacterial activity of the fermented bacteria powder is 804-806 billion CFU / g, and further is 804 billion CFU / g.

[0024] Further preferably, the enzyme preparation comprises 6-9 parts by weight of cellulase, 8-10 parts by weight of pectinase, 5-6 parts by weight of acid protease and 8-12 parts by weight of medium-temperature α-amylase; the activity of the cellulase is 30,000 U / g, the activity of the pectinase is 40,000 U / g, the activity of the acid protease is 100,000 U / g, and the activity of the medium-temperature α-amylase is 5,000 U / g.

[0025] Further preferably, the fermented bacteria powder is composed of 1.2-1.5 weight parts of Lactobacillus plantarum, 1.1-1.4 weight parts of fermented mucus Lactobacillus, 0.6-0.8 weight parts of Lactobacillus reuteri, 0.6-0.8 weight parts of Lactobacillus acidophilus, 0.6-0.8 weight parts of Lactobacillus rhamnosus, and 0.3-0.5 weight parts of Bifidobacterium adolescentis; the activity of Lactobacillus plantarum is 200 billion CFU / g, the activity of fermented mucus Lactobacillus is 100 billion CFU / g, the activity of Lactobacillus reuteri is 160 billion CFU / g, the activity of Lactobacillus acidophilus is 120 billion CFU / g, the activity of Lactobacillus rhamnosus is 260 billion CFU / g, and the activity of Bifidobacterium adolescentis is 80 billion CFU / g.

[0026] The fermentation raw materials of the fermented product also include an acid-base regulator, and the acid-base regulator is used to adjust the pH of the reaction solution.

[0027] Preferably, the acid-base regulator includes: acetic acid, sodium bicarbonate, citric acid and sodium hydroxide.

[0028] Preferably, the fermented product further comprises an acid-base regulator; the acid-base regulator comprises 2-6 parts by weight of acetic acid and 2-8 parts by weight of sodium bicarbonate or consists of 1-4 parts by weight of citric acid and 2-6 parts by weight of sodium hydroxide.

[0029] In another aspect, the present invention provides a method for preparing the aforementioned fermentation product.

[0030] The preparation method comprises:

[0031] (1) Enzymatic hydrolysis: crush and weigh the black wolfberry, eucommia male flower, oxalis aurantii, Acanthopanax short-stalked, Inula venosus, and Paeonia suffruticosa flowers, and mix with water to form a mixture, adjust the pH to 4.5-5.5, then add an enzyme preparation for enzymatic hydrolysis, and adjust the pH to 5.0-6.8 after the enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0032] (2) extracting the enzymatic hydrolyzate to obtain an extract;

[0033] (3) Adding fermentation bacteria powder to the extract to carry out fermentation, and separating the solid and liquid to obtain the supernatant is the fermentation product.

[0034] Preferably, the pH in step (1) is adjusted by the aforementioned acid-base regulator.

[0035] The enzymatic hydrolysis temperature in step (1) is 45°C-65°C, and the enzymatic hydrolysis time is 30-180 min.

[0036] The extraction in step (2) is a temperature-raising extraction, preferably at a temperature of 80-108° C. and an extraction time of 10-90 min.

[0037] In step (3), the fermentation temperature is 25-45° C., the fermentation time is 24-240 h, the stirring speed is 100-1000 RPM, and the ventilation volume is 1-10 vvn.

[0038] Beneficial effects of the present invention:

[0039] 1. In the fermentation raw material for lowering uric acid provided by the present invention:

[0040] Black wolfberry: nourishes the kidney and replenishes essence, nourishes the liver and improves eyesight, replenishes blood and calms the mind, promotes fluid production and quenches thirst, moistens the lungs and relieves cough; can be used for deficiency of essence, soreness of waist and knees, dizziness and tinnitus, internal heat and thirst, blood deficiency and sallow complexion, and blurred vision.

[0041] Eucommia male flowers: warm the yang and dredge the meridians, strengthen the tendons and bones, nourish the liver and kidneys, moisten the intestines and promote bowel movements, and protect the liver; it has the effects of calming the nerves, lowering blood sugar, moistening the intestines and promoting bowel movements, resisting fatigue, lowering blood pressure, anti-aging, lowering blood lipids, and enhancing immunity.

[0042] Acanthopanax short-stemmed: It can nourish the kidney and strengthen the waist, replenish qi and calm the mind, promote blood circulation and dredge collaterals, can improve human immunity, is rich in cardiac glycosides, and has multiple functions such as dispelling wind and dampness, promoting blood circulation and removing blood stasis, and strengthening the stomach and promoting diuresis.

[0043] Xianmai Xuanfuhua: clears away heat and detoxifies, promotes diuresis and reduces swelling, dispels wind and dampness, dredges meridians and relieves pain, descends Qi and resolves phlegm, reduces adverse reactions and stops vomiting, promotes gastrointestinal motility, prevents and treats constipation, lowers blood lipids, and also has anti-inflammatory, antibacterial and antioxidant effects.

[0044] Danfeng Peony Flower: regulates menstruation and activates blood circulation, nourishes blood and liver, relieves depression and removes blood stasis, promotes blood circulation and activates collaterals, and beautifies the skin.

[0045] Ear leaf cowhide: helps digestion and strengthens the stomach, regulates qi and relieves pain, dispels wind and promotes diuresis. It also has the functions of nourishing the kidney and liver, blackening hair, nourishing blood and improving essence, and anti-aging.

[0046] The components in the above fermentation raw materials work synergistically under the action of enzymatic fermentation to have a good effect of lowering uric acid, and can also have a significant protective effect on kidney damage and liver damage, reduce serum creatinine, urinary albumin excretion rate, improve glomerular filtration function; and can improve the level of inflammatory factors.

[0047] 2. The fermented product provided by the present invention has an acid-base regulator used for adjusting the pH before enzymatic hydrolysis and fermentation, which is beneficial to the decomposition and precipitation of effective ingredients. When acetic acid and sodium bicarbonate or citric acid and sodium hydroxide are selected as the acid-base regulator, it is more conducive to the decomposition and precipitation of effective ingredients.

[0048] 3. The fermented product provided by the present invention, the synergistic combination of Lactobacillus plantarum, Lactobacillus mucilaginosus fermentation, Lactobacillus mucilaginosus reuteri, Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium adolescentis can significantly increase the content of effective ingredients of each raw material. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This figure shows the pathological changes in the kidneys of hyperuricemia mice.

[0050] Figure 2 The graph shows the results of measuring the p38MAPK, NF-κB and NLRP3 protein levels in mice. DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following embodiments are generally conventional conditions unless otherwise specified, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified. Based on the embodiments disclosed in the present invention, all other embodiments obtained by ordinary technicians in the art without creative work are within the scope of protection of the present disclosure.

[0052] If no specific experimental steps or conditions are specified in the examples, the experiments can be carried out according to the conventional steps or conditions described in the literature in the field. All reagents without manufacturer specified are conventional reagent products that can be obtained through commercial purchase.

[0053] In the following examples, the source information of some products is as follows:

[0054] Lactobacillus plantarum: Lactobacillus plantarum Lp3a from Jiangsu Xinshenao Biotechnology Co., Ltd.

[0055] Fermented mucus lactobacillus: Fermented mucus lactobacillus GF1633 from Sichuan Gaofuji Biotechnology Co., Ltd.

[0056] Lactobacillus reuteri: fermented Lactobacillus reuteri GF2408 from Sichuan Gaofuji Biotechnology Co., Ltd.

[0057] Lactobacillus acidophilus: Lactobacillus acidophilus LJ10201 from Jiangsu Xinshenao Biotechnology Co., Ltd.

[0058] Lactobacillus rhamnosus: Use Lactobacillus rhamnosus GF1827 Bifidobacterium adolescentis from Sichuan Gaofuji Biotechnology Co., Ltd.:

[0059] The Bifidobacterium adolescentis selected was Bifidobacterium adolescentis JYQS-216 produced by Shandong Zhongke Jiayi Bioengineering Co., Ltd.

[0060] Example 1

[0061] This embodiment provides a uric acid-lowering fermented product, the preparation method of which is as follows:

[0062] (1) 160 g of black wolfberry, 180 g of Eucommia ulmoides male flower, 120 g of Danfeng peony flower, 100 g of Auricularia auricula, 200 g of Acanthopanax short-stalked and 80 g of Inula obtusifolia were crushed and passed through a 400-mesh sieve, and mixed evenly with 5000 g of water to obtain a mixed solution, 2 g of citric acid was added to adjust the pH to 4.5, 6 g of cellulase, 8 g of pectinase, 6 g of acid protease and 12 g of medium-temperature α-amylase were added, and enzymolysis was performed at a temperature of 55° C. for 180 min. After the enzymolysis was completed, 4 g of sodium hydroxide was added to adjust the pH to 6.5 to obtain an enzymolysis solution;

[0063] Among them, the cellulase activity is 30000U / g, the pectinase activity is 40000U / g, the acid protease activity is 100000U / g, and the medium-temperature α-amylase activity is 5000U / g.

[0064] (2) heating the above enzymatic hydrolyzate to 96° C. and extracting for 45 minutes to obtain an extract;

[0065] (3) Add 1.2 g of Lactobacillus plantarum, 1.4 g of Lactobacillus mucilaginosus fermentation, 0.6 g of Lactobacillus mucilaginosus reuteri, 0.8 g of Lactobacillus acidophilus, 0.8 g of Lactobacillus rhamnosus, and 0.3 g of Bifidobacterium adolescentis to the above extract, and perform anaerobically fermentation for 168 h at a temperature of 37° C., a rotation speed of 100 rpm, and a ventilation volume of 0 vvm to obtain a fermentation liquid;

[0066] Among them, the activity of Lactobacillus plantarum is 200 billion CFU / g, the activity of Lactobacillus fermentum is 100 billion CFU / g, the activity of Lactobacillus reuteri is 160 billion CFU / g, the activity of Lactobacillus acidophilus is 120 billion CFU / g, the activity of Lactobacillus rhamnosus is 260 billion CFU / g, and the activity of Bifidobacterium adolescentis is 80 billion CFU / g.

[0067] (4) The fermentation supernatant is subjected to solid-liquid separation to obtain the fermentation supernatant, i.e., the fermentation product.

[0068] Example 2

[0069] This embodiment provides a uric acid-lowering fermented product, the preparation method of which is as follows:

[0070] (1) 150 g of black wolfberry, 160 g of male flowers of Eucommia ulmoides, 140 g of red peony flowers, 120 g of radix scutellariae, 170 g of Acanthopanax short-stalked and 100 g of Inula obtusifolia were crushed and passed through a 400-mesh sieve, and mixed evenly with 5000 g of water to obtain a mixed solution, 5 g of acetic acid was added to adjust the pH to 4.5, and then 9 g of cellulase, 10 g of pectinase, 5 g of acid protease and 8 g of medium-temperature α-amylase were added to perform enzymolysis at 55° C. for 180 min. After the enzymolysis was completed, 6 g of sodium bicarbonate was added to adjust the pH to 6.5 to obtain an enzymolysis solution;

[0071] Among them, the cellulase activity is 30000U / g, the pectinase activity is 40000U / g, the acid protease activity is 100000U / g, and the medium-temperature α-amylase activity is 5000U / g.

[0072] (2) heating the above enzymatic hydrolyzate to 96° C. and extracting for 45 minutes to obtain an extract;

[0073] (3) adding 1.5 g of Lactobacillus plantarum, 1.1 g of Lactobacillus mucilaginosus fermentation, 0.8 g of Lactobacillus mucilaginosus reuteri, 0.6 g of Lactobacillus acidophilus, 0.6 g of Lactobacillus rhamnosus, and 0.5 g of Bifidobacterium adolescentis to the above extract, and performing anaerobically fermentation for 168 h at a temperature of 37° C., a rotation speed of 100 rpm, and a ventilation volume of 0 vvm to obtain a fermentation liquid;

[0074] Among them, the activity of Lactobacillus plantarum is 200 billion CFU / g, the activity of Lactobacillus fermentum is 100 billion CFU / g, the activity of Lactobacillus reuteri is 160 billion CFU / g, the activity of Lactobacillus acidophilus is 120 billion CFU / g, the activity of Lactobacillus rhamnosus is 260 billion CFU / g, and the activity of Bifidobacterium adolescentis is 80 billion CFU / g.

[0075] (4) The fermentation supernatant is subjected to solid-liquid separation to obtain the fermentation supernatant, i.e., the fermentation product.

[0076] Example 3

[0077] This embodiment provides a uric acid-lowering fermented product, the preparation method of which is as follows:

[0078] (1) 140 g of black wolfberry, 200 g of male flowers of Eucommia ulmoides, 80 g of red peony flowers, 100 g of Auricularia auricula, 140 g of Acanthopanax short-stalked and 140 g of Inula obtusifolia were crushed and passed through a 400-mesh sieve, and mixed evenly with 5000 g of water to obtain a mixed solution, 2 g of citric acid was added to adjust the pH to 4.5, 6 g of cellulase, 8 g of pectinase, 6 g of acid protease and 12 g of medium-temperature α-amylase were added, and enzymolysis was performed at a temperature of 55° C. for 180 min. After the enzymolysis was completed, 4 g of sodium hydroxide was added to adjust the pH to 6.5 to obtain an enzymolysis solution;

[0079] Among them, the cellulase activity is 30000U / g, the pectinase activity is 40000U / g, the acid protease activity is 100000U / g, and the medium-temperature α-amylase activity is 5000U / g.

[0080] (2) heating the above enzymatic hydrolyzate to 96° C. and extracting for 45 minutes to obtain an extract;

[0081] (3) Add 1.2 g of Lactobacillus plantarum, 1.4 g of Lactobacillus mucilaginosus fermentation, 0.6 g of Lactobacillus mucilaginosus reuteri, 0.8 g of Lactobacillus acidophilus, 0.8 g of Lactobacillus rhamnosus, and 0.3 g of Bifidobacterium adolescentis to the above extract, and perform anaerobically fermentation for 168 h at a temperature of 37° C., a rotation speed of 100 rpm, and a ventilation volume of 0 vvm to obtain a fermentation liquid;

[0082] Among them, the activity of Lactobacillus plantarum is 200 billion CFU / g, the activity of Lactobacillus fermentum is 100 billion CFU / g, the activity of Lactobacillus reuteri is 160 billion CFU / g, the activity of Lactobacillus acidophilus is 120 billion CFU / g, the activity of Lactobacillus rhamnosus is 260 billion CFU / g, and the activity of Bifidobacterium adolescentis is 80 billion CFU / g.

[0083] (4) The fermentation supernatant is subjected to solid-liquid separation to obtain the fermentation supernatant, i.e., the fermentation product.

[0084] Example 4

[0085] This embodiment provides a uric acid-lowering fermented product, the preparation method of which is as follows:

[0086] (1) 180 g of black wolfberry, 120 g of male flowers of Eucommia ulmoides, 160 g of red peony flowers, 100 g of Auricularia auricula, 220 g of Acanthopanax short-stalked and 60 g of Inula obtusifolia were crushed and passed through a 400-mesh sieve, and mixed evenly with 5000 g of water to obtain a mixed solution, 2 g of citric acid was added to adjust the pH to 4.5, 6 g of cellulase, 8 g of pectinase, 6 g of acid protease and 12 g of medium-temperature α-amylase were added, and enzymolysis was performed at a temperature of 55° C. for 180 min. After the enzymolysis was completed, 4 g of sodium hydroxide was added to adjust the pH to 6.5 to obtain an enzymolysis solution;

[0087] Among them, the cellulase activity is 30000U / g, the pectinase activity is 40000U / g, the acid protease activity is 100000U / g, and the medium-temperature α-amylase activity is 5000U / g.

[0088] (2) heating the above enzymatic hydrolyzate to 96° C. and extracting for 45 minutes to obtain an extract;

[0089] (3) Add 1.2 g of Lactobacillus plantarum, 1.4 g of Lactobacillus mucilaginosus fermentation, 0.6 g of Lactobacillus mucilaginosus reuteri, 0.8 g of Lactobacillus acidophilus, 0.8 g of Lactobacillus rhamnosus, and 0.3 g of Bifidobacterium adolescentis to the above extract, and perform anaerobically fermentation for 168 h at a temperature of 37° C., a rotation speed of 100 rpm, and a ventilation volume of 0 vvm to obtain a fermentation liquid;

[0090] Among them, the activity of Lactobacillus plantarum is 200 billion CFU / g, the activity of Lactobacillus fermentum is 100 billion CFU / g, the activity of Lactobacillus reuteri is 160 billion CFU / g, the activity of Lactobacillus acidophilus is 120 billion CFU / g, the activity of Lactobacillus rhamnosus is 260 billion CFU / g, and the activity of Bifidobacterium adolescentis is 80 billion CFU / g.

[0091] (4) The fermentation supernatant is subjected to solid-liquid separation to obtain the fermentation supernatant, i.e., the fermentation product.

[0092] Example 5

[0093] This embodiment provides a uric acid-lowering fermented product, the preparation method of which is as follows:

[0094] (1) 100 g of black wolfberry, 200 g of Eucommia ulmoides male flower, 150 g of Danfeng peony flower, 60 g of Auricularia auricula, 200 g of Acanthopanax short-stalked and 130 g of Inula obtusifolia were crushed and passed through a 400-mesh sieve, and mixed evenly with 5000 g of water to obtain a mixed solution, 2 g of citric acid was added to adjust the pH to 4.5, 6 g of cellulase, 8 g of pectinase, 6 g of acid protease and 12 g of medium-temperature α-amylase were added, and enzymolysis was performed at a temperature of 55° C. for 180 min. After the enzymolysis was completed, 4 g of sodium hydroxide was added to adjust the pH to 6.5 to obtain an enzymolysis solution;

[0095] Among them, the cellulase activity is 30000U / g, the pectinase activity is 40000U / g, the acid protease activity is 100000U / g, and the medium-temperature α-amylase activity is 5000U / g;

[0096] (2) heating the above enzymatic hydrolyzate to 96° C. and extracting for 45 minutes to obtain an extract;

[0097] (3) Add 1.2 g of Lactobacillus plantarum, 1.4 g of Lactobacillus mucilaginosus fermentation, 0.6 g of Lactobacillus mucilaginosus reuteri, 0.8 g of Lactobacillus acidophilus, 0.8 g of Lactobacillus rhamnosus, and 0.3 g of Bifidobacterium adolescentis to the above extract, and perform anaerobically fermentation for 168 h at a temperature of 37° C., a rotation speed of 100 rpm, and a ventilation volume of 0 vvm to obtain a fermentation liquid;

[0098] Among them, the activity of Lactobacillus plantarum is 200 billion CFU / g, the activity of Lactobacillus fermentum is 100 billion CFU / g, the activity of Lactobacillus reuteri is 160 billion CFU / g, the activity of Lactobacillus acidophilus is 120 billion CFU / g, the activity of Lactobacillus rhamnosus is 260 billion CFU / g, and the activity of Bifidobacterium adolescentis is 80 billion CFU / g;

[0099] (4) The fermentation supernatant is subjected to solid-liquid separation to obtain the fermentation supernatant, i.e., the fermentation product.

[0100] Example 6

[0101] This embodiment provides a fermentation powder, which is prepared by concentrating the fermented product prepared in Example 1, the concentration temperature is 65°C, the vacuum degree is -0.08MPa to prepare a fermentation concentrated paste, the density is 1.18g / cm 3 The soluble solid content is 40.6%. The fermentation concentrate is dried into powder by spray drying to obtain baking powder, and the moisture content of the baking powder is 6.3%.

[0102] Comparative Example 1

[0103] This comparative example provides a fermented product, which is made from only 840 g of Acanthopanax brevis. The rest is the same as in Example 1.

[0104] Comparative Example 2

[0105] This comparative example provides a fermented product, which is composed of only 840 g of Inula venosa. The rest is the same as in Example 1.

[0106] Comparative Example 3

[0107] This comparative example provides a fermentation product, wherein the fermentation bacterial powder consists of 5.1 g of Lactobacillus reuteri, and the rest is the same as that of Example 1.

[0108] Comparative Example 4

[0109] The preparation provided in this comparative example is not fermented, and the rest is the same as Example 1.

[0110] Comparative Example 5

[0111] Compared with Example 1, Lactobacillus reuteri was not added, the proportion of live bacteria of other fermentation bacteria remained unchanged, and the total bacterial count remained unchanged.

[0112] Experimental Example 1 Active ingredient detection

[0113] The purpose of this experiment is to verify the changes in the active ingredients of the fermented products / preparations obtained in Example 1, Comparative Examples 3 and 4.

[0114] 1) Proanthocyanidin B2 content detection

[0115] Reverse phase high performance liquid chromatography (RP-HPLC) was used to detect proanthocyanidin B2, with a chromatographic column C18 (250 mm × 4.6 mm, 5 μm), a mobile phase of 2% glacial acetic acid solution-acetonitrile, gradient elution, a flow rate of 1 mL / min, and a detection wavelength of 280 nm. Column temperature: 30 °C; injection volume: 20 μL; elution order is shown in Table 1.

[0116] Table 1 Mobile phase elution order

[0117]

[0118] Preparation of proanthocyanidin B2 reference solution:

[0119] Accurately weigh 1 mg of proanthocyanidin B2 reference substance, add 1 mL of methanol to prepare a 1 mg / mL solution. Take 1 μL, 2.5 μL, 5 μL, 10 μL, 25 μL, and 50 μL of the above solution, add methanol to the volume, and prepare 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL solutions, respectively, and store at -18°C for later use.

[0120] Preparation of proanthocyanidin B2 standard curve:

[0121] Take 1μg / mL, 2.5μg / mL, 5μg / mL, 10μg / mL, 25μg / mL, and 50μg / mL proanthocyanidin B2 reference solution, and inject 20μL of each solution. Use the peak area as the ordinate and the concentration of the reference solution as the abscissa to obtain the standard curve equation, Y=10.2164X+6.3754, r=0.9985.

[0122] Preparation and determination of samples to be tested:

[0123] Take 10 mL of the fermented products obtained in Example 1, Comparative Example 3 and Comparative Example 4, add methanol to make the volume 50 mL, and filter through a 0.45 μm microporous filter membrane for use.

[0124] 2) Detection of chlorogenic acid and paeonol content

[0125] A C18 chromatographic column (4.6 mm × 250 mm, 5 μm) was used; mobile phase: methanol-0.2% phosphoric acid, gradient elution, the elution gradient is shown in Table 2 below; detection wavelength: chlorogenic acid: 330 nm, paeonol: 278 nm; column temperature: 25 °C; injection volume: 10 μL; the theoretical plate number was calculated based on the paeonol peak and should not be less than 5000.

[0126] Table 2 Gradient elution

[0127]

[0128] Preparation of reference solution:

[0129] Accurately weigh an appropriate amount of chlorogenic acid and paeonol reference substances, add 50% methanol to dissolve and dilute to make a solution containing 20 μg of chlorogenic acid and 10 μg of paeonol per 1 mL.

[0130] Chlorogenic acid and paeonol standard curve determination:

[0131] Accurately measure 0.1mL, 0.3mL, 0.5mL, 0.7mL, 0.9mL and 1.1mL of chlorogenic acid and paeonol reference stock solutions respectively and place them in 10mL volumetric flasks, add 50% methanol to dilute to the scale, shake well, accurately pipette 10μL of each and inject into liquid chromatograph to determine the peak areas of chlorogenic acid and paeonol, draw standard curves with the peak area of ​​reference substance as the ordinate and the injection volume of reference substance as the abscissa, and obtain the linear regression equation: chlorogenic acid regression equation: Y=65.248X-9.1322, r=0.9991; paeonol regression equation: Y=28.541X-3.2256, r=0.9983.

[0132] Preparation of test solution:

[0133] Accurately measure 10 mL of the fermentation products of Example 1, Comparative Example 3 and Comparative Example 4, place in a 50 mL volumetric flask, add an appropriate amount of 50% methanol to dilute to the scale, shake well, filter, and take the filtrate to obtain the test solution.

[0134] 3) Detection of the content of cadaverine

[0135] A C18 chromatographic column (250×4.6mm, 5μm) was used; mobile phase: acetonitrile-water, flow rate: 1mL / min, column temperature 25°C; injection volume: 10μL; UV detection wavelength 220nm; detection time: 60min, gradient elution was used, and the procedure was as follows:

[0136] Table 3 Gradient elution

[0137]

[0138] Preparation of reference solution:

[0139] Accurately weigh an appropriate amount of the caldatine reference substance, add 65% ethanol to make up to volume, to prepare a solution containing 1 mg of caldatine per 1 mL, and then filter with a 0.45 μm filter membrane to obtain the filtrate for use.

[0140] Preparation of test solution:

[0141] Accurately measure 10 mL of the fermentation products of Example 1, Comparative Example 3 and Comparative Example 4, place in a 50 mL volumetric flask, add an appropriate amount of 65% ethanol to dilute to the scale, shake well, filter with a 0.45 μm filter membrane, and obtain the filtrate for use to obtain the test solution.

[0142] Determination of the standard curve of cadaverine:

[0143] Accurately measure 0.2mL, 0.4mL, 0.8mL, 1.6mL, 2.4mL, and 3.2mL of the gabutin reference stock solution and place them in a 10mL volumetric flask, add 65% ethanol to make up to volume, shake well, and accurately pipette 10μL of each solution and inject it into the liquid chromatograph. Determine the gabutin peak area according to the above conditions, plot the standard curve with the reference peak area as the ordinate and the reference injection volume as the abscissa, and obtain the linear regression equation. The gabutin standard curve equation is: Y=17.352X+5.3641, r=0.9994.

[0144] 4) Hyperoside content detection

[0145] A C18 chromatographic column (250×4.6mm, 5μm) was used, with acetonitrile as mobile phase A and 0.3% phosphoric acid water as mobile phase B, gradient elution, elution conditions as shown in the table below, detection wavelength: 360nm; column temperature: 30℃; injection volume: 20μL.

[0146] Table 4 Elution conditions for hyperoside detection

[0147]

[0148] Preparation of reference solution:

[0149] Accurately weigh an appropriate amount of hyperoside reference substance, add methanol to make up to volume, to prepare a solution containing 1.5 mg of hyperoside per 1 mL, then filter with a 0.45 μm filter membrane to obtain the filtrate for use.

[0150] Preparation of test solution:

[0151] Accurately measure 10 mL of the fermentation products of Example 1, Comparative Example 3 and Comparative Example 4, place in a 50 mL volumetric flask, add an appropriate amount of methanol to dilute to the scale, shake well, filter with a 0.45 μm filter membrane, and obtain the filtrate for use to obtain the test solution.

[0152] Hyperoside standard curve determination:

[0153] Accurately measure 0.5mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, and 5.0mL of the hyperoside reference stock solution and place it in a 10mL volumetric flask, add methanol to make up to volume, shake well, and accurately pipette 20μL of each into a liquid chromatograph to determine the peak area of ​​hyperoside. Use the peak area of ​​the reference substance as the ordinate and the injection volume of the reference substance as the abscissa to draw a standard curve and obtain the linear regression equation. The equation of the hyperoside standard curve is: Y=8.164X-2.7105, r=0.9982.

[0154] 5) Thymol content detection

[0155] A C18 chromatographic column (250×4.6mm, 5μm) was used, with the mobile phase being acetonitrile-water (65:35V / V); the column temperature being 25°C; the flow rate being 1.0mL / min; and the detection wavelength being 220nm. The theoretical plate number should be no less than 10,000 based on the thymol peak.

[0156] Preparation of reference solution:

[0157] Accurately weigh an appropriate amount of thymol reference substance, add 95% ethanol to make up to volume to prepare a solution containing 0.3 mg of thymol per 1 mL, then filter with a 0.45 μm filter membrane to obtain the filtrate for use.

[0158] Preparation of test solution:

[0159] Accurately measure 10 mL of the fermentation products of Example 1, Comparative Example 3 and Comparative Example 4, place in a 50 mL volumetric flask, add 95% ethanol to the mark, shake well, filter with a 0.45 μm filter membrane, and obtain the filtrate for use to obtain the test solution.

[0160] Preparation of thymol standard curve:

[0161] Accurately pipette 2μL, 4μL, 6μL, 8μL, 10μL, and 12μL of thymol reference solution and measure its peak area. Draw a standard curve with concentration as the horizontal axis and peak area as the vertical axis. The regression equation of thymol is: Y=105.233X+26.1732, r=0.9989.

[0162] Table 5 Determination of the content of active ingredients in samples of each experimental group (mg / g)

[0163]

[0164] Result analysis:

[0165] As can be seen from the above table, compared with the non-fermented group (Comparative Example 4), the contents of the six major active ingredients in the fermentation group (Example 1) increased significantly, and the contents of proanthocyanidin B2, chlorogenic acid, paeonol, gadolinium, hyperoside, and thymol increased by 173.8%, 235.0%, 129.5%, 221.4%, 94.9%, and 144.9%, respectively. It can be seen that after treatment with microbial fermentation technology, the content of active ingredients in each raw material increased significantly.

[0166] Compared with the non-fermentation group (Comparative Example 4), the content of effective ingredients in the fermentation group using only a single strain of bacteria (Comparative Example 3) also increased to a certain extent. The contents of proanthocyanidin B2, chlorogenic acid, paeonol, calcitonin, hyperoside, and thymol increased by 46.4%, 65.5%, 71.5%, 73.3%, 39.0%, and 86.8%, respectively. It can be seen that the use of a single strain of bacteria for fermentation also has a significant promoting effect on the increase of effective ingredients in the raw materials.

[0167] Compared with the single strain fermentation group (Comparative Example 3), the content of each active ingredient in the multi-strain co-fermentation group (Example 1) also increased significantly. The contents of proanthocyanidin B2, chlorogenic acid, paeonol, calcitriol, hyperoside, and thymol increased by 87.1%, 102.4%, 33.8%, 85.5%, 40.1%, and 31.1%, respectively. It can be seen that multi-strain mixed fermentation is more conducive to the increase in the content of active ingredients.

[0168] Compared with the fermentation group without adding Lactobacillus reuteri (Comparative Example 5), the content of each effective ingredient in the multi-strain co-fermentation group (Example 1) also increased significantly, and the contents of proanthocyanidin B2, chlorogenic acid, paeonol, calcitriol, hyperoside, and thymol increased by 49.9%, 87.1%, 25.6%, 59.8%, 34.5%, and 19.4%, respectively. It can be seen that multi-strain mixed fermentation is more conducive to the increase of the content of each component.

[0169] Experimental Example 2 Animal Experiment

[0170] Experimental animals: 72 specific pathogen-free (SPF) male ICR mice (weight 18-22 g) were purchased from the Department of Laboratory Animal Science, Peking University Health Science Center (Beijing, China). The experimental animals were housed at 23±1°C and 55% humidity, given normal feed and water, and adapted for 3 days in an indoor environment with 12 hours of light and 12 hours of darkness. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University, approval number: LA2021459, and were strictly carried out in accordance with the Ethical Guidelines for Laboratory Animal Welfare (NIH Press, No. 85-23, revised in 1996).

[0171] Test samples: The test substances were the fermented products prepared in Examples 1-5 and Comparative Examples 1-5.

[0172] Experimental feed: (1) Ordinary feed: purchased from Beijing Keao Xieli Feed Co., Ltd. The nutritional composition and level provided by it conform to the national standard GB14924.3-2010 and can meet the needs of rodent growth and development.

[0173] (2) Hyperuricemia feed: A feed made by mixing ordinary feed with 4% potassium oxonate and 20% yeast, purchased from Beijing Botai Hongda Biotechnology Co., Ltd. [SCXK (Jun) 2012-0003].

[0174] Experimental methods:

[0175] Animal model construction and grouping:

[0176] Before the experiment, the weight of mice was measured with an electronic scale, and 72 male ICR mice were randomly divided into 12 groups (n=14) according to their weight and treated accordingly.

[0177] (1) Normal control group: The mice were given normal feed and 10 mL / kg distilled water by gavage for 6 consecutive weeks.

[0178] (2) Hyperuricemia model group: The mice were given a hyperuricemia diet and 10 mL / kg distilled water by gavage for 6 consecutive weeks.

[0179] (3) Benzbromarone group: The mice were given a hyperuricemia diet and gavage with 10 mL / kg (20 mg / kg body weight / day benzbromarone + 0.5% carboxymethyl cellulose) for 6 consecutive weeks.

[0180] (4) Group 1: The mice were given a hyperuricemia feed and gavage at 10 mL / kg (1 g / kg body weight / day, the test sample prepared in Example 1) for 6 consecutive weeks.

[0181] (5) Group 2 of Example 2: The mice were given a hyperuricemia feed and gavage at 10 mL / kg (1 g / kg body weight / day, the test sample prepared in Example 2) for 6 consecutive weeks.

[0182] (6) Group 3: The mice were given a hyperuricemia feed and gavage at 10 mL / kg (1 g / kg body weight / day, the test sample prepared in Example 3) for 6 consecutive weeks.

[0183] (7) Group 4: The mice were given a hyperuricemia diet and gavage at 10 mL / kg (1 g / kg body weight / day, sample prepared in Example 4) for 6 consecutive weeks.

[0184] (8) Group 5: The mice were given a hyperuricemia diet and gavage at 10 mL / kg (1 g / kg body weight / day, sample prepared in Example 5) for 6 consecutive weeks.

[0185] (9) Control group 1: The mice were given a hyperuricemia diet and gavage at 10 mL / kg (1 g / kg body weight / day, sample prepared in comparative example 1) for 6 consecutive weeks.

[0186] (10) Control group 2: The mice were given a hyperuricemia diet and gavage at 10 mL / kg (1 g / kg body weight / day, sample prepared in comparative example 2) for 6 consecutive weeks.

[0187] (11) Control group 3: The mice were given a hyperuricemia diet and gavage at 10 mL / kg (1 g / kg body weight / day, sample prepared in comparative example 3) for 6 consecutive weeks.

[0188] (12) Control group 4: The mice were given a hyperuricemia diet and gavage at 10 mL / kg (1 g / kg body weight / day, sample prepared in comparative example 4) for 6 consecutive weeks.

[0189] (13) Control group 5: The mice were given a hyperuricemia diet and gavage at 10 mL / kg (1 g / kg body weight / day, sample prepared in comparative example 5) for 6 consecutive weeks.

[0190] During the experiment, the growth of the animals, including coat color, mental state, and daily activities, was observed every day, and the animals' food intake, water intake, urine output, body weight, and food utilization rate (weight gain / food intake × 100%) were recorded regularly. At the end of the experiment, the mice were killed to collect different tissues for different studies.

[0191] Uric acid lowering test

[0192] Blood sample collection: At the end of the sixth week of the experiment, the mouse eyeballs were removed and venous blood was collected from the mice. The blood was collected into an anticoagulant tube containing EDTA-K2 and centrifuged at 3000r / min for 10 minutes at 4°C to collect the mouse serum samples, which were stored at -20°C for later use;

[0193] Determination method:

[0194] The concentrations of serum uric acid (SUA), blood urea nitrogen (BUN), and serum creatinine (SCr) were measured using an automatic biochemical analyzer Olympus AU480.

[0195] Table 6 Concentrations of serum uric acid (SUA), blood urea nitrogen (BUN), and serum creatinine (SCr) in mice in each experimental group

[0196]

[0197] ## indicates P<0.01 compared with the normal control group; * indicates P<0.05 compared with the hyperuricemia model group; ** indicates P<0.01 compared with the hyperuricemia model group.

[0198] Result analysis:

[0199] After 6 weeks of intervention, compared with the normal control group, the concentrations of serum uric acid, blood urea nitrogen, and serum creatinine in the hyperuricemia model group were significantly increased (P<0.01), indicating that the hyperuricemia mouse model was successfully established.

[0200] Compared with the hyperuricemia model control group, the concentrations of serum uric acid, blood urea nitrogen, and serum creatinine in the benzbromarone group and Example 1-5 groups of mice were significantly reduced (P<0.01), indicating that Example 1-5 groups can effectively reduce the levels of serum uric acid, blood urea nitrogen, and serum creatinine in hyperuricemia mice. Among them, the effects of Examples 1 and 2 are slightly better than those of Examples 3, 4, and 5.

[0201] Compared with the hyperuricemia model control group, the concentrations of serum uric acid, blood urea nitrogen, and serum creatinine in mice in Comparative Example 3 were also reduced to a certain extent (P<0.05), indicating that the fermentation preparation using only a single strain also has a certain effect on lowering uric acid. The concentrations of serum uric acid, blood urea nitrogen, and serum creatinine in mice in Comparative Example 5 were also reduced to a certain extent (P<0.05), indicating that the fermentation preparation without adding Lactobacillus reuteri also has a certain effect on lowering uric acid, but the effect is lower than that in the embodiment. In Comparative Examples 1, 2, and 4, there was no statistical difference in the concentrations of serum uric acid, blood urea nitrogen, and serum creatinine in mice compared with the model control group (P>0.05).

[0202] Experiment on reducing renal lesions in mice with hyperuricemia

[0203] Method for collecting liver and kidney tissues: After blood was collected, mice were killed by cervical dislocation, and liver and kidney tissues were sampled. The removed liver and kidney were weighed, and the liver and half of the right kidney were collected, refrigerated with liquid nitrogen, and stored at -80°C. The other half of the right kidney was fixed with 4% paraformaldehyde solution and stored at room temperature for subsequent use as a sample.

[0204] The right kidney fixed with 4% paraformaldehyde solution was used for histopathological examination.

[0205] The kidney tissue was dehydrated, transparent, wax-impregnated, embedded, and sliced ​​to make paraffin sections, which were then dewaxed, stained, dehydrated, transparent, and dried. Hematoxylin-eosin staining (SE staining) was performed, and images were collected for analysis.

[0206] The tissues were examined and images were collected using ImageProPlus5.1 (MediaCybernetics, US) image analysis software under an Olympus BX43 optical microscope. At a magnification of 200, the optical constants of the microscope were fixed and each area of ​​the pathological section was scanned (focusing on the glomerulus and renal interstitium).

[0207] Result analysis:

[0208] The light microscopy results are as follows Figure 1 Shown, scale bar: 100 μm; original magnification 200×.

[0209] Compared with the normal control group, the mice in the hyperuricemia model group had swollen glomeruli, thickened glomerular basement membranes, widened mesangial areas, dilated renal tubules, disordered arrangement of tubular epithelial cells, and scattered inflammatory cell infiltration in local renal interstitial tissues; compared with the hyperuricemia model group, the glomerular capillary swelling of the mice in Example 3 group was improved, the mesangial matrix was normal, and the cystic cavity stenosis was improved; the glomeruli and tubular structures of the mice in Example 1 and Example 2 groups were normal, and the epithelial cells were neatly arranged, which was close to that of the normal control group; compared with the hyperuricemia model group, the renal tubular dilatation of the mice in the benzbromarone group was reduced, the tubular epithelial cells were neatly arranged, and occasional vacuolar degeneration was observed.

[0210] The above kidney morphology results show that Example 1 and Example 2 can reduce the renal damage caused by hyperuricemia mice and play a good role in protecting the kidneys. Although benzbromarone can reduce blood uric acid levels, it does not show a good protective effect on the kidneys.

[0211] Reduce the activity of XOD in mouse liver and improve liver function experiment

[0212] Determination method: Accurately weigh liver tissue samples, add 9 times the volume of physiological saline at a weight (g): volume (mL) ratio of 1:9, and mechanically homogenize under ice bath conditions to prepare a 10% homogenate. Centrifuge at 7000r / min for 10min, collect the supernatant, and detect XOD activity according to the instructions of the XOD kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0213] In the uric acid lowering test experiment, serum samples from mice were collected and stored at -20°C for later use as samples. The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the fully automatic biochemical analyzer Olympus AU480.

[0214] The results are shown in Table 7, ## indicates P<0.01 compared with the normal control group; * indicates P<0.05 compared with the hyperuricemia model group; ** indicates P<0.01 compared with the hyperuricemia model group.

[0215] Table 7 Liver XOD activity, serum ALT and AST activities of mice in each experimental group

[0216]

[0217] After 6 weeks of intervention, compared with the normal control group, the liver XOD activity, serum ALT and AST activities of mice in the hyperuricemia model group were significantly increased (P<0.01), indicating that hyperuricemia significantly affected the liver XOD activity, serum ALT and AST activities of mice.

[0218] Compared with the hyperuricemia model control group, the benzbromarone group, Example 1, and Example 2 groups had significantly reduced liver XOD activity, serum ALT, and AST activities in mice (P<0.01), indicating that Example 1 and Example 2 groups can effectively reduce liver XOD activity, serum ALT, and AST activities in hyperuricemia mice. The liver XOD activity, serum ALT, and AST activities in mice of Examples 3, 4, and 5 were also greatly reduced (P<0.05, and some indicators were P<0.01). It can be seen that Examples 1 and 2 are slightly better than Examples 3, 4, and 5 in terms of inhibiting the liver XOD activity, serum ALT, and AST activities in mice.

[0219] Compared with the hyperuricemia model control group, the liver XOD activity, serum ALT and AST activities of mice in comparison example 3 were also reduced to a certain extent (P<0.05), indicating that the fermentation preparation using only a single strain also has a certain effect on lowering uric acid. The serum ALT and AST activities of mice in comparison example 5 were also reduced to a certain extent (P<0.05), indicating that the fermentation preparation without adding Lactobacillus reuteri also has a certain effect, while comparison examples 1, 2, and 4 had no statistically significant differences compared with the model control group (P>0.05).

[0220] Experiment on improving the level of renal inflammatory factors

[0221] Hyperuricemia mice will have obvious intestinal barrier defects. Intestinal barrier dysfunction will activate macrophage-mediated inflammatory response, upregulate the expression of Toll-like receptor 4 (TLR4), and then activate the myeloid differentiation factor 88 (MyD88) signaling pathway, initiate the rapid activation of NF-κB, increase the levels of IL-1β, TNF-α, MCP-1 and IL-8 in the body, cause cytokine cascade activation reaction, promote proximal tubular epithelial cell inflammation and oxidative stress, and then aggravate kidney function damage. Therefore, the key to controlling uric acid levels is to reduce inflammation and oxidative stress, thereby reducing damage to kidney function and ensuring kidney excretion of uric acid.

[0222] Determination method:

[0223] Accurately weigh the kidney tissue sample, add 9 times the volume of normal saline at a ratio of weight (g): volume (mL) = 1:9, and prepare a 10% homogenate by mechanical homogenization under ice bath conditions. Centrifuge at 3000r / min for 10min and collect the supernatant. Process according to the instructions of the ELISA kit to detect the content of IL-1β, IL-6 and TNF-α respectively. Mouse TNF-α enzyme-linked immunosorbent assay kit (88-7324), mouse IL-1β enzyme-linked immunosorbent assay kit (88-7064), and mouse IL-6 enzyme-linked immunosorbent assay kit (88-7013) were all purchased from Invitrogen Life Sciences, Inc., USA. The experimental results are shown in Table 8.

[0224] Table 8 Pro-inflammatory cytokine levels in the kidneys of mice in each group (n=10) (X±SD)

[0225]

[0226] ## indicates P<0.01 compared with the normal control group; * indicates P<0.05 compared with the hyperuricemia model group; ** indicates P<0.01 compared with the hyperuricemia model group.

[0227] Compared with the normal control group, the levels of renal inflammatory factors IL-1β, IL-6 and TNF-α in mice in the hyperuricemia model group were significantly increased (P<0.01), indicating that hyperuricemia significantly increased the levels of renal inflammatory factors in mice.

[0228] Compared with the hyperuricemia model group, after 6 weeks of continuous intervention, the levels of IL-1β, IL-6, and TNF-α in the kidneys of the mice in the benzbromarone group, Examples 1, 2, and 3 were significantly reduced (P<0.01); the levels of IL-1β, IL-6, and TNF-α in the kidneys of the mice in Examples 4 and 5 were also significantly reduced (P<0.01, and P<0.05 for a few indicators). The results showed that the Examples 1-5 groups could significantly reduce the levels of inflammatory factors in the kidneys of the mice in the hyperuricemia model group, and played a certain protective role on the kidney tissue.

[0229] Compared with the hyperuricemia model group, there was no significant difference in the levels of renal inflammatory factors in mice in comparison groups 1, 2, 4, and 5 (P>0.05), which also showed that the three comparison groups could not play a certain protective role on the kidneys; compared with the hyperuricemia model group, the levels of IL-1β, IL-6, and TNF-α in the mouse kidneys in comparison group 3 were significantly reduced (P<0.05), but the effect was poorer than that in Examples 1-5.

[0230] Western blot protein blotting assay

[0231] Mitogen-activated protein kinase (MAPK) is a group of serine-threonine protein kinases that can be activated by different extracellular stimuli. It is an important transmitter of signal transduction and participates in regulating many important cellular physiological / pathological processes such as cell growth, differentiation, adaptation to environmental stress, and inflammatory response. MAPK can be divided into four subfamilies: ERK, p38, JNK, and ERK5. In recent years, the role of p38MAPK in the regulation of proinflammatory cytokines has received increasing attention. Phosphorylation of p38MAPK can promote the activation of NF-κB by inducing cell proliferation.

[0232] Under the stimulation of upstream p38MAPK signal, NF-κB is transported to the nucleus, binds to NF-κB transcription elements, and activates the expression of IL-1β, IL-6, and TNF-α inflammatory factors. At the same time, IL-1β and TNF-α can induce the production of other inflammatory factors by activating the NF-κB signaling pathway, aggravating the renal inflammatory response and forming a vicious cycle. Therefore, it is very important to reduce the activation of p38MAPK and NF-κB.

[0233] Determination method:

[0234] The expression of p38MAPK, p-p38MAPK, NF-κB, p-NF-κB and NLRP3 proteins in mouse kidney tissue was detected by immunoblotting. The bicinchoninic acid (BCA) protein quantification kit was purchased from Beijing Pulilai Gene Technology Co., Ltd.

[0235] The experimental results are as follows Figure 2 shown.

[0236] Result analysis:

[0237] Compared with the normal control group, the p-p38MAPK / p38MAPK expression ratio in the kidney of mice in the hyperuricemia model group was significantly increased; compared with the hyperuricemia model group, the p-p38MAPK / p38MAPK expression ratio in the kidney of mice in Example 1 was significantly decreased (P<0.05), and the p-p38MAPK / p38MAPK expression ratio in the kidney of mice in the benzbromarone group was also significantly decreased (P<0.05); compared with the normal control group, the p-NF-κB / NF-κB expression ratio in the kidney of mice in the hyperuricemia model group was significantly increased; compared with the hyperuricemia model group, the p-NF-κB / NF-κB expression ratio in the kidney of mice in Example 1 was significantly decreased (P<0.05); there was no significant difference in the expression of NLRP3 protein among the groups.

[0238] The above results show that Example 1 group can significantly reduce the activation of p38MAPK and NF-κB, the p-p38MAPK / p38MAPK expression ratio is significantly reduced, the p-NF-κB / NF-κB expression ratio is significantly reduced, the increase of uric acid levels is inhibited, and the changes and damage caused by inflammation can be significantly reversed.

[0239] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A fermented product for lowering uric acid, characterized in that: The fermentation raw materials are calculated by weight as follows: 100-180 parts of black wolfberry, 120-200 parts of eucommia male flowers, 80-160 parts of red peony flowers, 60-140 parts of auricularia acuta, 140-220 parts of Acanthopanax short-stalked, 60-140 parts of Inula japonica, 1.2-12 parts of fermentation bacteria powder and 14-120 parts of enzyme preparation; The enzyme preparation is composed of cellulase, pectinase, acid protease and medium-temperature α-amylase, and the mass ratio of each enzyme is 6-9:8-10:5-6:8-12; The fermented bacterial powder consists of plant lactobacillus, fermented mucin lactobacillus, reuteri mucin lactobacillus, acidophilus lactobacillus, rhamnosus lactobacillus and adolescent bifidobacterium, and the mass ratio of each bacteria is 12-15:11-14:6-8:6-8:6-8:3-5.

2. The fermentation product according to claim 1, characterized in that The enzyme activity ratio of each enzyme unit in the enzyme preparation is 30-40:40-50:90-100:5-6.

3. The fermentation product according to claim 1, characterized in that The activity ratio of each bacterial unit in the fermented bacterial powder is 18-20: 8-10: 14-16: 10-12: 23-26: 6-8.

4. The fermentation product according to claim 2, characterized in that In the enzyme preparation, the mass ratio of each enzyme is 6:8:6:12 or 9:10:5:8; the unit enzyme activity ratio of each enzyme is 30:40:100:5; and the unit enzyme activity of the cellulase is 30000U / g.

5. The fermentation product according to claim 3, characterized in that In the fermented bacteria powder, the mass ratio of each bacteria is 12:14:6:8:8:3 or 15:11:8:6:6:5; the unit activity ratio of each bacteria is 20:10:16:12:26:8; and the unit activity of the plant lactobacillus is 200 billion CFU / g.

6. The fermentation product according to claim 1, characterized in that The invention also comprises an acid-base regulator: the acid-base regulator is composed of 2-6 parts by weight of acetic acid and 2-8 parts by weight of sodium bicarbonate, or the acid-base regulator is composed of 1-4 parts by weight of citric acid and 2-6 parts by weight of sodium hydroxide.

7. The method for preparing a fermented product according to any one of claims 1 to 6, characterized in that: include: (1) Enzymatic hydrolysis: crush and weigh the black wolfberry, eucommia male flower, oxalis aurantii, Acanthopanax short-stalked, Inula venosus, and Paeonia suffruticosa flowers, and mix with water to form a mixture, adjust the pH to 4.5-5.5, then add an enzyme preparation for enzymatic hydrolysis, and adjust the pH to 5.0-6.8 after the enzymatic hydrolysis to obtain an enzymatic hydrolyzate; (2) extracting the enzymatic hydrolysate to obtain an extract; the extraction is performed by heating extraction at a temperature of 80-108° C. and an extraction time of 10-90 min; (3) Adding fermentation bacteria powder to the extract to carry out fermentation, and separating the solid and liquid to obtain the supernatant is the fermentation product.

8. The preparation method according to claim 7, characterized in that: The enzymatic hydrolysis temperature in step (1) is 45°C-65°C, and the enzymatic hydrolysis time is 30-180 min; In step (3), the fermentation temperature is 25-45° C., the fermentation time is 24-240 h, the stirring speed is 100-1000 RPM, and the ventilation volume is 1-10 vvn.

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