CN116549535A - Uric acid-reducing ferment, preparation method and application thereof - Google Patents
Uric acid-reducing ferment, preparation method and application thereof Download PDFInfo
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- CN116549535A CN116549535A CN202310778669.2A CN202310778669A CN116549535A CN 116549535 A CN116549535 A CN 116549535A CN 202310778669 A CN202310778669 A CN 202310778669A CN 116549535 A CN116549535 A CN 116549535A
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Abstract
The invention provides a uric acid-reducing ferment, belonging to the technical field of traditional Chinese medicine fermentation. The ferment fermentation raw materials comprise the following components in parts by weight: 100-180 parts of lycium ruthenicum, 120-200 parts of eucommia male flowers, 80-160 parts of danfeng peony flowers, 60-140 parts of auricularia auricular calyx, 140-220 parts of acanthopanax sessiliflorus, 60-140 parts of inula japonica, 1.2-12 parts of zymophyte powder and 14-120 parts of enzyme preparation. The synergistic combination of the traditional Chinese medicine components has good uric acid reducing effect under the action of enzymolysis and fermentation, has obvious protective effect on kidney injury and liver injury, reduces serum creatinine and urinary albumin excretion rate, and improves glomerular filtration function; and can improve inflammatory factor levels.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine fermentation, and particularly relates to a uric acid-reducing ferment, a preparation method and application thereof.
Background
Hyperuricemia (HUA) is a disease associated with abnormal purine metabolism and characterized by elevated serum uric acid levels, and its diagnostic criteria are: under normal purine diet, men with fasting blood uric acid levels of more than 420. Mu. Mol/L and women with fasting blood uric acid levels of more than 360. Mu. Mol/L are not two times daily. In recent years, the prevalence and prevalence of hyperuricemia are rapidly rising.
Hyperuricemia is a major risk factor for gout. Uric acid is a metabolic product of xanthine and hypoxanthine oxidized by body Xanthine Oxidase (XOD), and when serum uric acid concentration exceeds its saturation level, it precipitates in the form of monosodium urate crystals (MSU) and deposits at joints, inducing local inflammatory reaction of tissues, thus leading to the formation of gout. In addition, hyperuricemia involves organs such as kidneys, livers and joints, and long-term and persistent increases in haematuria can lead to various lesions such as gouty arthritis, tophus and gouty nephropathy.
Current treatments for hyperuricemia mainly include drug therapy and non-drug intervention. The medicine treatment mainly comprises anti-inflammatory treatment and uric acid reducing treatment, wherein the anti-inflammatory treatment is carried out by using conventional anti-inflammatory medicines such as aspirin, colchicine and the like, and the medicines have quick anti-inflammatory and analgesic effects, but are only symptomatic treatment, can not reduce uric acid level, and have obvious toxic and side effects. Uric acid reducing treatment mainly uses xanthine oxidase inhibitors such as allopurinol and the like to reduce uric acid generation, and the medicines can effectively reduce serum uric acid level, but need to be taken for a long time, have no antipyretic, analgesic and anti-inflammatory effects, have little curative effect on acute arthritis, and even aggravate symptoms or prolong the course of the disease. In addition, acute onset of gout can be induced in early stage by using uric acid lowering drugs alone, and side effects such as gastrointestinal irritation, bone marrow suppression and nephrotoxicity can be generated after long-term use, so that risks of poor prognosis and even deadly are increased, and clinical application of the uric acid lowering drugs is limited.
Uric acid-lowering products prepared by fermentation have been disclosed in the prior art.
As disclosed in chinese patent application 202111076145.6: a medicinal and edible ferment suitable for people with hyperuricemia and gout and a preparation method thereof comprise the following components: EM, lactobacillus, saccharomycetes, lactobacillus plantarum, streptococcus thermophilus, bifidobacterium, lactobacillus rhamnosus, lactobacillus acidophilus, bacillus coagulans, lactobacillus reuteri, honey, coral calcium and drinking water. The medicinal and edible traditional Chinese medicinal materials comprise the following components: sunflower disc, corn silk, cordate houttuynia, coix seed, kudzuvine root, dandelion, gardenia, chicory, eucommia ulmoides, gorgon euryale seed, cinnamon, lophatherum gracile, poria cocos, purslane and lily. The invention utilizes beneficial microbial flora and combines medicinal and edible traditional Chinese medicinal materials for fermentation, and the prepared product can effectively reduce the blood uric acid content of gout people, relieve pain, improve the immune system, recover and improve the metabolic function, has wide applicability, is nontoxic and has no side effect, and can be eaten daily. However, the effect of reducing uric acid is not clear because of excessive raw materials involved.
As disclosed in chinese patent 202110786163.7: a Chinese medicine in the form of capsule, tablet, or particle for treating gout is prepared from 4 Chinese-medicinal materials including red vine, turmeric, liquorice root, coix seed, glucose and beef extract through superfine pulverizing, breaking cell wall, fermenting by liquid probiotics, spray drying, collecting the dried powder, and packing. The preparation method of the invention is simple to operate, can fully utilize the traditional Chinese medicine materials and the active ingredients thereof, and can effectively reduce the blood uric acid through the synergistic effect of the traditional Chinese medicine and the probiotics, thereby achieving the effect of preventing or treating gout. But there is actually room for further improvement in the effect of reducing blood uric acid.
Disclosure of Invention
In order to solve the problems, the invention provides a uric acid-reducing ferment which not only can obviously reduce uric acid, but also has a certain repairing effect on kidney lesions and liver injury caused by long-term hyperuricemia.
In the present invention, "living bacteria", "bacterial activity", "number of living bacteria", "living bacteria content", "living bacteria amount" have the same meaning under some conditions.
In the present invention, "enzyme activity", "enzyme specific activity" have the same meaning under some conditions.
In one aspect, the invention provides a uric acid lowering fermentation product.
The fermentation raw materials of the ferment comprise the following components in parts by weight: 100-180 parts of lycium ruthenicum, 120-200 parts of eucommia male flowers, 80-160 parts of danfeng peony flowers, 60-140 parts of auricularia auricular calyx, 140-220 parts of acanthopanax sessiliflorus, 60-140 parts of inula japonica, 1.2-12 parts of zymophyte powder and 14-120 parts of enzyme preparation;
the enzyme preparation comprises cellulase, pectase, acid protease and medium-temperature alpha-amylase, wherein the enzyme activity ratio of the enzymes is 18-27:32-40:50-60:40-60.
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.
Preferably, the mass ratio of each enzyme is 6:8:6:12 or 9:10:5:8 or 6:8:6:12; the enzyme activity ratio of each enzyme unit is as follows: 30:40:100:5, a step of; the unit enzyme activity of the cellulase is 30000U/g.
The enzyme preparation is used for zymolysis of a substrate to obtain zymolysis solution;
the fermentation bacteria powder comprises lactobacillus plantarum, lactobacillus fermentum, lactobacillus reuteri, lactobacillus acidophilus, lactobacillus rhamnosus and bifidobacterium adolescentis, and the ratio of the bacteria to the activity of each bacteria is 120-150:55-70:48-64:36-48:78-104:12-20, total bacterial activity is 8000-8100 hundred million CFU/g;
specifically, the mass ratio of each bacterium is 12-15:11-14:6-8:6-8:6-8:3-5; the unit force ratio of each bacterium is 18-20:8-10:14-16:10-12:23-26:6-8.
Preferably, the mass ratio of each bacterium is 12:14:6:8:8:3 or 15:11:8:6:6:5, a step of; the unit living force ratio of each bacterium is 20:10:16:12:26:8, 8; the unit activity of the lactobacillus plantarum is 2000 hundred million CFU/g.
The zymophyte powder is used for zymolysis and post-fermentation.
Further preferably, in the enzyme preparation, the enzyme activity ratio of cellulase, pectase, acid protease and medium temperature alpha-amylase is 18:32:60:60.
Further preferably, in the fermentation powder, the ratio of bacterial activity of each bacteria is 120:70:48:48:104:12.
further preferably, the total bacterial activity in the fermentation powder is 8040-8060 hundred million CFU/g, and even further 8040 hundred million CFU/g.
Further preferably, the enzyme preparation comprises 6-9 parts by weight of cellulase, 8-10 parts by weight of pectase, 5-6 parts by weight of acid protease and 8-12 parts by weight of medium temperature alpha-amylase; the activity of the cellulase is 30000U/g, the activity of the pectase is 40000U/g, the activity of the acid protease is 100000U/g, and the activity of the medium-temperature alpha-amylase is 5000U/g.
Further preferably, the zymophyte powder consists of 1.2 to 1.5 weight parts of lactobacillus plantarum, 1.1 to 1.4 weight parts of lactobacillus fermentum, 0.6 to 0.8 weight parts of lactobacillus reuteri, 0.6 to 0.8 weight parts of lactobacillus acidophilus, 0.6 to 0.8 weight parts of lactobacillus rhamnosus and 0.3 to 0.5 weight parts of bifidobacterium adolescentis; the lactobacillus plantarum vitality is 2000 hundred million CFU/g, the lactobacillus fermentum vitality is 1000 hundred million CFU/g, the lactobacillus reuteri vitality is 1600 hundred million CFU/g, the lactobacillus acidophilus vitality is 1200 hundred million CFU/g, the lactobacillus rhamnosus vitality is 2600 hundred million CFU/g, and the bifidobacterium adolescentis vitality is 800 hundred million CFU/g.
The fermentation raw material of the ferment also comprises an acid-base regulator, and the acid-base regulator is used for regulating the pH value of the reaction liquid.
Preferably, the acid-base modifier comprises: acetic acid, sodium bicarbonate, citric acid, and sodium hydroxide.
Preferably, the ferment further comprises an acid-base regulator; the acid-base modifier 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.
In another aspect, the present invention provides a process for preparing the aforementioned fermentate.
The preparation method comprises the following steps:
(1) Enzymolysis: mixing crushed and weighed lycium ruthenicum, eucommia male flowers, auriculate swallowwort root, acanthopanax sessiliflorus, inula brined flowers and paeonia suffruticosa with water to form a mixture, regulating the pH value to 4.5-5.5, adding an enzyme preparation for enzymolysis, and regulating the pH value to 5.0-6.8 after the enzymolysis is finished to obtain an enzymolysis solution;
(2) Extracting the enzymolysis liquid to obtain an extracting liquid;
(3) Adding zymophyte powder into the extracting solution for fermentation, and carrying out solid-liquid separation to obtain a supernatant, namely a fermentation product.
Preferably, the pH in the step (1) is adjusted by the aforementioned acid-base modifier.
The enzymolysis temperature in the step (1) is 45-65 ℃ and the enzymolysis time is 30-180min.
The step (2) is heating extraction. The preferred temperature is 80-108 deg.C, and the extraction time is 10-90min.
The fermentation temperature in the step (3) is 25-45 ℃, the fermentation time is 24-240h, the stirring speed is 100-1000RPM, and the ventilation rate is 1-10vvn.
In yet another aspect, the invention provides the use of the foregoing ferment in the manufacture of a medicament for preventing or treating hyperuricemia, gout, or renal disorder caused by uric acid.
The ferment shows a preventive or therapeutic effect through uric acid reducing effect.
The hyperuricemia can be primary or secondary.
In yet another aspect, the invention also provides a pharmaceutical formulation comprising the foregoing ferment.
Other pharmaceutically acceptable carriers or excipients are also included in the pharmaceutical formulation.
Such pharmaceutically acceptable carriers or excipients include, but are not limited to: buffer solution, excipient, stabilizer, antiseptic, flavoring agent and flavoring agent.
The pharmaceutical preparation can be solid preparation or liquid preparation.
The dosage forms of the pharmaceutical formulation include, but are not limited to: tablets, granules, syrups, oral liquids and capsules.
Such tablets include, but are not limited to: dispersible tablet, sustained release tablet, controlled release tablet, effervescent tablet, and enteric coated tablet.
The invention has the beneficial effects that:
1. the uric acid-reducing ferment raw material provided by the invention comprises the following components:
lycium ruthenicum Murr: kidney tonifying, essence replenishing, liver nourishing, eyesight improving, blood replenishing, nerve soothing, salivation promoting, thirst quenching, lung moistening, and cough relieving; can be used for treating asthenia, soreness of waist and knees, dizziness, tinnitus, internal heat, diabetes, blood deficiency, sallow complexion, and blurred vision.
Eucommia male flowers: warming yang, dredging collaterals, strengthening tendons and bones, nourishing liver and kidney, loosening bowel to relieve constipation, protecting liver; has effects of tranquilizing, lowering blood sugar, loosening bowel to relieve constipation, relieving fatigue, lowering blood pressure, resisting aging, reducing blood lipid, and enhancing immunity.
Acanthopanax sessiliflorus (Rupr. Et Maxim.) harms: the composition has effects of invigorating kidney, strengthening waist, invigorating qi, tranquilizing mind, promoting blood circulation, dredging collaterals, improving immunity, and dispelling pathogenic wind, removing dampness, promoting blood circulation, removing blood stasis, invigorating stomach, and promoting urination.
Inula flower with pulse development: has effects of clearing heat and detoxicating, promoting urination and detumescence, dispelling pathogenic wind and removing dampness, dredging collaterals and relieving pain, lowering qi and resolving phlegm, lowering adverse qi and relieving vomiting, promoting gastrointestinal peristalsis, preventing and treating constipation, reducing blood lipid, and also has antiinflammatory, antibacterial and antioxidant effects.
Danfeng peony: regulating menstruation, promoting blood circulation, nourishing blood, regulating liver, resolving stagnation, removing blood stasis, dredging meridian passage, caring skin.
Root of Hemium: promoting digestion, invigorating stomach, regulating qi-flowing, relieving pain, dispelling pathogenic wind, promoting diuresis, invigorating kidney, nourishing liver, wu Fasheng hair, nourishing blood, replenishing essence, and resisting aging.
The synergistic combination of the six traditional Chinese medicine components has good uric acid reducing effect under the action of enzymolysis and fermentation, has obvious protective effect on kidney injury and liver injury, reduces serum creatinine and urinary albumin excretion rate, and improves glomerular filtration function; and can improve inflammatory factor levels.
2. The acid-base regulator is used for regulating the pH value before enzymolysis and fermentation, is beneficial to the decomposition and precipitation of active ingredients, and is more beneficial to the decomposition and precipitation of the active ingredients when acetic acid and sodium bicarbonate are selected as the acid-base regulator or citric acid and sodium hydroxide are selected as the acid-base regulator.
3. The synergistic cooperation of the ferment provided by the invention, namely lactobacillus plantarum, lactobacillus fermentum, lactobacillus reuteri, lactobacillus acidophilus, lactobacillus rhamnosus and bifidobacterium adolescentis can obviously improve the content of active ingredients of each raw material.
Drawings
FIG. 1 is a graph showing the results of pathological changes in kidney of hyperuricemia mice.
FIG. 2 is a graph showing the results of the determination of p38MAPK, NF-. Kappa.B and NLRP3 protein levels in mice.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the disclosure herein, are intended to be within the scope of the present disclosure.
The examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions that are routine in the art described in the literature. All reagents were not manufacturer-identified and were conventional reagent products commercially available.
In the following examples, the source information of a part of the product is as follows:
lactobacillus plantarum: lactobacillus plantarum Lp3a of Jiangsu Xinshenao biotechnology limited company is selected;
fermenting lactobacillus mucilaginosus: selecting fermented lactobacillus mucilaginosus GF1633 of Sichuan Gaofuji biotechnology Co;
Lactobacillus reuteri: selecting fermented lactobacillus mucilaginosus GF2408 of Sichuan Gaofuji biotechnology Co;
lactobacillus acidophilus: lactobacillus acidophilus LJ10201 of Jiangsu Xinshenao biotechnology limited company is selected;
lactobacillus rhamnosus: selecting Lactobacillus rhamnosus GF1827 Bifidobacterium adolescentis of Sichuan Gaofu Biotech Co., ltd.):
the Bifidobacterium adolescentis is selected from Bifidobacterium adolescentis JYQS-216, a limited biological engineering company of Jia Yi in Shandong.
Example 1
The embodiment provides a uric acid reducing ferment, which is prepared by the following steps:
(1) 160g of lycium ruthenicum, 180g of eucommia male flower, 120g of danfeng peony flower, 100g of auricularia auricula and cow leather, 200g of acanthopanax sessiliflorus and 80g of inula japonica are crushed and sieved by a 400-mesh sieve, and are uniformly mixed with 5000g of water to obtain a traditional Chinese medicine mixed solution, 2g of citric acid is added to adjust the pH value to 4.5, 6g of cellulase, 8g of pectase and 6g of acid proteinase are added, the medium-temperature alpha-amylase 12g is subjected to enzymolysis for 180min at the temperature of 55 ℃, and 4g of sodium hydroxide is added to adjust the pH value to 6.5 after the enzymolysis is finished to obtain an enzymolysis solution;
wherein the cellulase activity is 30000U/g, the pectase activity is 40000U/g, the acid protease activity is 100000U/g, and the medium-temperature alpha-amylase activity is 5000U/g.
(2) Heating the enzymolysis liquid to 96 ℃, and extracting for 45min to obtain an extracting liquid;
(3) Adding 1.2g of lactobacillus plantarum, 1.4g of fermented lactobacillus mucilaginosus, 0.6g of lactobacillus reuteri, 0.8g of lactobacillus acidophilus, 0.8g of lactobacillus rhamnosus and 0.3g of bifidobacterium adolescentis into the extracting solution, and carrying out anaerobic fermentation for 168 hours at the temperature of 37 ℃ under the conditions of the rotating speed of 100rmp and the ventilation quantity of 0vvm to obtain a fermentation solution;
wherein, the activity of the lactobacillus plantarum is 2000 hundred million CFU/g, the activity of the lactobacillus fermentum is 1000 hundred million CFU/g, the activity of the lactobacillus reuteri is 1600 hundred million CFU/g, the activity of the lactobacillus acidophilus is 1200 hundred million CFU/g, the activity of the lactobacillus rhamnosus is 2600 hundred million CFU/g, and the activity of the bifidobacterium adolescentis is 800 hundred million CFU/g.
(4) And (3) carrying out solid-liquid separation on the fermentation clear liquid to obtain the fermentation clear liquid, namely a fermentation product.
Example 2
The embodiment provides a uric acid reducing ferment, which is prepared by the following steps:
(1) 150g of lycium ruthenicum, 160g of eucommia male flower, 140g of danfeng peony flower, 120g of auricularia auricula and cow leather, 170g of acanthopanax sessiliflorus and 100g of inula japonica are crushed and sieved by a 400-mesh sieve, and are uniformly mixed with 5000g of water to obtain a traditional Chinese medicine mixed solution, 5g of acetic acid is added to adjust the pH value to 4.5, 9g of cellulase, 10g of pectase and 5g of acid proteinase are added, 8g of medium-temperature alpha-amylase is subjected to enzymolysis for 180min at the temperature of 55 ℃, and 6g of sodium bicarbonate is added to adjust the pH value to 6.5 after the enzymolysis is finished to obtain an enzymolysis solution;
Wherein the cellulase activity is 30000U/g, the pectase activity is 40000U/g, the acid protease activity is 100000U/g, and the medium-temperature alpha-amylase activity is 5000U/g.
(2) Heating the enzymolysis liquid to 96 ℃, and extracting for 45min to obtain an extracting liquid;
(3) Adding 1.5g of lactobacillus plantarum, 1.1g of fermented lactobacillus mucilaginosus, 0.8g of lactobacillus reuteri, 0.6g of lactobacillus acidophilus, 0.6g of lactobacillus rhamnosus and 0.5g of bifidobacterium adolescentis into the extracting solution, and carrying out anaerobic fermentation for 168 hours at the temperature of 37 ℃ under the conditions that the rotating speed is 100rmp and the ventilation quantity is 0vvm to obtain a fermentation solution;
wherein, the activity of the lactobacillus plantarum is 2000 hundred million CFU/g, the activity of the lactobacillus fermentum is 1000 hundred million CFU/g, the activity of the lactobacillus reuteri is 1600 hundred million CFU/g, the activity of the lactobacillus acidophilus is 1200 hundred million CFU/g, the activity of the lactobacillus rhamnosus is 2600 hundred million CFU/g, and the activity of the bifidobacterium adolescentis is 800 hundred million CFU/g.
(4) And (3) carrying out solid-liquid separation on the fermentation clear liquid to obtain the fermentation clear liquid, namely a fermentation product.
Example 3
The embodiment provides a uric acid reducing ferment, which is prepared by the following steps:
(1) 140g of lycium ruthenicum, 200g of eucommia male flowers, 80g of danfeng peony flowers, 100g of auricularia auricula and cow leather, 140g of acanthopanax sessiliflorus and 140g of inula japonica are crushed and sieved by a 400-mesh sieve, and uniformly mixed with 5000g of water to obtain a traditional Chinese medicine mixed solution, 2g of citric acid is added to adjust the pH to 4.5, 6g of cellulase, 8g of pectase and 6g of acid proteinase are added, the medium-temperature alpha-amylase 12g is subjected to enzymolysis for 180min at the temperature of 55 ℃, and 4g of sodium hydroxide is added to adjust the pH to 6.5 after the enzymolysis is finished to obtain an enzymolysis solution;
Wherein the cellulase activity is 30000U/g, the pectase activity is 40000U/g, the acid protease activity is 100000U/g, and the medium-temperature alpha-amylase activity is 5000U/g.
(2) Heating the enzymolysis liquid to 96 ℃, and extracting for 45min to obtain an extracting liquid;
(3) Adding 1.2g of lactobacillus plantarum, 1.4g of fermented lactobacillus mucilaginosus, 0.6g of lactobacillus reuteri, 0.8g of lactobacillus acidophilus, 0.8g of lactobacillus rhamnosus and 0.3g of bifidobacterium adolescentis into the extracting solution, and carrying out anaerobic fermentation for 168 hours at the temperature of 37 ℃ under the conditions of the rotating speed of 100rmp and the ventilation quantity of 0vvm to obtain a fermentation solution;
wherein, the activity of the lactobacillus plantarum is 2000 hundred million CFU/g, the activity of the lactobacillus fermentum is 1000 hundred million CFU/g, the activity of the lactobacillus reuteri is 1600 hundred million CFU/g, the activity of the lactobacillus acidophilus is 1200 hundred million CFU/g, the activity of the lactobacillus rhamnosus is 2600 hundred million CFU/g, and the activity of the bifidobacterium adolescentis is 800 hundred million CFU/g.
(4) And (3) carrying out solid-liquid separation on the fermentation clear liquid to obtain the fermentation clear liquid, namely a fermentation product.
Example 4
The embodiment provides a uric acid reducing ferment, which is prepared by the following steps:
(1) 180g of lycium ruthenicum, 120g of eucommia male flower, 160g of danfeng peony flower, 100g of auricularia auricula and cow leather, 220g of acanthopanax sessiliflorus and 60g of inula japonica are crushed and pass through a 400-mesh sieve, uniformly mixed with 5000g of water to obtain a traditional Chinese medicine mixed solution, 2g of citric acid is added to adjust the pH value to 4.5, 6g of cellulase, 8g of pectase and 6g of acid proteinase are added, the medium-temperature alpha-amylase 12g is subjected to enzymolysis for 180min at the temperature of 55 ℃, and 4g of sodium hydroxide is added to adjust the pH value to 6.5 after the enzymolysis is finished to obtain an enzymolysis solution;
Wherein the cellulase activity is 30000U/g, the pectase activity is 40000U/g, the acid protease activity is 100000U/g, and the medium-temperature alpha-amylase activity is 5000U/g.
(2) Heating the enzymolysis liquid to 96 ℃, and extracting for 45min to obtain an extracting liquid;
(3) Adding 1.2g of lactobacillus plantarum, 1.4g of fermented lactobacillus mucilaginosus, 0.6g of lactobacillus reuteri, 0.8g of lactobacillus acidophilus, 0.8g of lactobacillus rhamnosus and 0.3g of bifidobacterium adolescentis into the extracting solution, and carrying out anaerobic fermentation for 168 hours at the temperature of 37 ℃ under the conditions of the rotating speed of 100rmp and the ventilation quantity of 0vvm to obtain a fermentation solution;
wherein, the activity of the lactobacillus plantarum is 2000 hundred million CFU/g, the activity of the lactobacillus fermentum is 1000 hundred million CFU/g, the activity of the lactobacillus reuteri is 1600 hundred million CFU/g, the activity of the lactobacillus acidophilus is 1200 hundred million CFU/g, the activity of the lactobacillus rhamnosus is 2600 hundred million CFU/g, and the activity of the bifidobacterium adolescentis is 800 hundred million CFU/g.
(4) And (3) carrying out solid-liquid separation on the fermentation clear liquid to obtain the fermentation clear liquid, namely a fermentation product.
Example 5
The embodiment provides a uric acid reducing ferment, which is prepared by the following steps:
(1) 100g of lycium ruthenicum, 200g of eucommia male flower, 150g of danfeng peony flower, 60g of auricularia auricula and cow leather, 200g of acanthopanax sessiliflorus and 130g of inula japonica are crushed and sieved by a 400-mesh sieve, and are uniformly mixed with 5000g of water to obtain a traditional Chinese medicine mixed solution, 2g of citric acid is added to adjust the pH value to 4.5, 6g of cellulase, 8g of pectase and 6g of acid proteinase are added, the medium-temperature alpha-amylase 12g is subjected to enzymolysis for 180min at the temperature of 55 ℃, and 4g of sodium hydroxide is added to adjust the pH value to 6.5 after the enzymolysis is finished to obtain an enzymolysis solution;
Wherein the cellulase activity is 30000U/g, the pectase activity is 40000U/g, the acid protease activity is 100000U/g, and the medium-temperature alpha-amylase activity is 5000U/g;
(2) Heating the enzymolysis liquid to 96 ℃, and extracting for 45min to obtain an extracting liquid;
(3) Adding 1.2g of lactobacillus plantarum, 1.4g of fermented lactobacillus mucilaginosus, 0.6g of lactobacillus reuteri, 0.8g of lactobacillus acidophilus, 0.8g of lactobacillus rhamnosus and 0.3g of bifidobacterium adolescentis into the extracting solution, and carrying out anaerobic fermentation for 168 hours at the temperature of 37 ℃ under the conditions of the rotating speed of 100rmp and the ventilation quantity of 0vvm to obtain a fermentation solution;
wherein, the activity of the lactobacillus plantarum is 2000 hundred million CFU/g, the activity of the lactobacillus fermentum is 1000 hundred million CFU/g, the activity of the lactobacillus reuteri is 1600 hundred million CFU/g, the activity of the lactobacillus acidophilus is 1200 hundred million CFU/g, the activity of the lactobacillus rhamnosus is 2600 hundred million CFU/g, and the activity of the bifidobacterium adolescentis is 800 hundred million CFU/g;
(4) And (3) carrying out solid-liquid separation on the fermentation clear liquid to obtain the fermentation clear liquid, namely a fermentation product.
Example 6
This example provides a fermented powder obtained by concentrating the fermented product obtained in example 1 at 65deg.C under vacuum degree of-0.08 MPa to obtain fermented concentrated extract with density of 1.18g/cm 3 The soluble solids content was 40.6%. And drying the fermented concentrated paste by adopting a spray drying process to obtain fermented powder, wherein the moisture of the fermented powder is 6.3%.
Comparative example 1
This comparative example provides a fermented product made only of 840g of acanthopanax sessiliflorus. The remainder was the same as in example 1.
Comparative example 2
This comparative example provides a ferment consisting of only 840g of Inula venosua. The remainder was the same as in example 1.
Comparative example 3
This comparative example provides a fermented product, the fermentation powder consisting of 5.1g of Lactobacillus reuteri, the remainder being the same as in example 1.
Comparative example 4
The formulation provided in this comparative example was not fermented and was otherwise identical to example 1.
Comparative example 5
Compared with example 1, lactobacillus reuteri is not added, the viable bacteria proportion of other zymophytes is unchanged, and the total bacteria number is unchanged.
Experimental example 1 detection of active ingredient
The purpose of this experiment was to verify the variation of active ingredients of the fermentate/formulation obtained in example 1, and comparative example 3 and comparative example 4.
1) Procyanidine B2 content detection
Detecting procyanidin B2 by reversed phase high performance liquid chromatography (RP-HPLC), and performing gradient elution on a chromatographic column C18 (250 mm×4.6mm,5 μm) with 2% glacial acetic acid solution-acetonitrile as mobile phase; the flow rate is 1mL/min; the detection wavelength was 280nm. Column temperature: 30 ℃; sample injection amount: 20. Mu.L; the elution order is shown in Table 1.
TABLE 1 order of mobile phase elution
Preparation of procyanidine B2 control solution:
1mg of procyanidine B2 reference substance is precisely weighed, and 1mL of methanol is added to prepare a 1mg/mL solution. 1. Mu.L, 2.5. Mu.L, 5. Mu.L, 10. Mu.L, 25. Mu.L and 50. Mu.L of the above solution are respectively taken, methanol is added for constant volume, and 1. Mu.g/mL, 2.5. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 25. Mu.g/mL and 50. Mu.g/mL of the solution are respectively prepared, and the solution is preserved at-18 ℃ for standby.
Procyanidine B2 standard curve preparation:
and respectively taking 1 mug/mL, 2.5 mug/mL, 5 mug/mL, 10 mug/mL, 25 mug/mL and 50 mug/mL of procyanidine B2 reference substance solutions, respectively injecting 20 mug/mL, taking the peak area as the ordinate and the reference substance solution concentration as the abscissa, and obtaining a standard curve equation, wherein Y=10.2164X+6.3754 and r=0.9985.
Preparing and measuring a sample to be measured:
taking 10mL of each of the fermented products obtained in the corresponding examples 1, 3 and 4, adding methanol to constant volume to 50mL, and passing through a 0.45 μm microporous filter membrane to obtain the final product.
2) Chlorogenic acid and paeonol content detection
C18 column (4.6 mm. Times.250 mm,5 μm); mobile phase: methanol-0.2% phosphoric acid, gradient elution, elution gradient see table 2 below; detection wavelength: chlorogenic acid: 330nm, paeonol: 278nm; column temperature: 25 ℃; sample injection amount: 10. Mu.L; the theoretical plate number is calculated according to paeonol peak and should not be lower than 5000.
TABLE 2 gradient elution
Preparation of a control solution:
precisely weighing appropriate amount of chlorogenic acid and paeonol reference substances, and adding 50% methanol for dissolving and diluting to obtain solution containing chlorogenic acid 20 μg and paeonol 10 μg per 1 mL.
Chlorogenic acid and paeonol standard curve determination:
precisely measuring chlorogenic acid and paeonol reference substance stock solution 0.1mL, 0.3mL, 0.5mL, 0.7mL, 0.9mL and 1.1mL respectively, placing in a 10mL measuring flask, adding 50% methanol to dilute to scale, shaking uniformly, precisely sucking 10 μL respectively, injecting into a liquid chromatograph, measuring the peak areas of chlorogenic acid and paeonol, taking the peak areas of reference substances as ordinate and the sample injection amount of reference substances as abscissa, drawing a standard curve to obtain a linear regression equation, and obtaining a chlorogenic acid regression equation: y= 65.248X-9.1322, r= 0.9991; paeonol regression equation: y=28.541X-3.2256, r= 0.9983.
Preparation of test solution:
precisely measuring 10mL of the fermented products of the example 1, the comparative example 3 and the comparative example 4, placing the fermented products into a 50mL volumetric flask, adding a proper amount of 50% methanol, diluting to a constant volume to a scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a sample solution.
3) Content detection of patulin
C18 column (250X 4.6mm,5 μm) was used; mobile phase: acetonitrile-water, flow rate: 1mL/min, column temperature 25 ℃; sample injection amount: 10. Mu.L; ultraviolet detection wavelength 220nm; detection time: gradient elution was used for 60min, the procedure was as follows:
TABLE 3 gradient elution
Preparation of a control solution:
accurately weighing the reference substance of the patulin, adding 65% ethanol to constant volume, preparing 1mg solution containing patulin per 1mL, and filtering with 0.45 μm filter membrane to obtain filtrate for use.
Preparation of test solution:
precisely measuring 10mL of the fermented products of the example 1, the comparative example 3 and the comparative example 4, placing the fermented products into a 50mL volumetric flask, adding a proper amount of 65% ethanol, diluting to a constant volume to a scale, shaking uniformly, and filtering with a filter membrane with the thickness of 0.45 μm to obtain filtrate for later use, thus obtaining the solution of the sample.
Determination of the standard curve of the patulin:
precisely measuring the standard substance of the kidazomet, namely, 0.2mL, 0.4mL, 0.8mL, 1.6mL, 2.4mL and 3.2mL of the standard substance stock solution, placing the kidazomet, namely, the kidazomet and the kidazomet into a 10mL measuring flask, adding 65% ethanol to fix the volume, shaking uniformly, precisely sucking 10 mu L of the kidazomet into a liquid chromatograph respectively, measuring the kidazomet peak area according to the conditions, taking the standard substance peak area as an ordinate and the standard substance injection amount as an abscissa, drawing a standard curve to obtain a linear regression equation, and obtaining the kidazomet standard curve equation: y=17.352 x+5.3641, r=0.9994.
4) Hyperoside content detection
The detection wavelength was determined by using a C18 column (250X 4.6mm,5 μm) with acetonitrile as mobile phase A and 0.3% phosphoric acid water as mobile phase B, and by gradient elution under the conditions shown in the following table: 360nm; column temperature: 30 ℃; sample injection amount: 20. Mu.L.
TABLE 4 detection of elution conditions for hyperin
Preparation of a control solution:
precisely weighing a proper amount of hyperin reference substance, adding methanol to a certain volume to prepare a solution containing 1.5mg of hyperin per 1mL, and then filtering with a filter membrane of 0.45 μm to obtain a filtrate for later use.
Preparation of test solution:
precisely measuring 10mL of the fermented products of the example 1, the comparative example 3 and the comparative example 4, placing the fermented products into a 50mL volumetric flask, adding a proper amount of methanol, diluting to a constant volume to a scale, shaking uniformly, and filtering with a filter membrane with the thickness of 0.45 mu m to obtain filtrate for later use, thus obtaining the sample solution.
Hyperin standard curve determination:
precisely measuring 0.5mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL and 5.0mL of hyperin control stock solution, placing into a 10mL measuring flask, adding methanol to fix volume, shaking, precisely sucking 20 μl respectively, injecting into a liquid chromatograph, measuring hyperin peak area, taking the control peak area as ordinate, taking the control sample injection amount as abscissa, drawing a standard curve to obtain a linear regression equation, and obtaining hyperin standard curve equation: y= 8.164X-2.7105, r=0.9982.
5) Thymol content detection
The mobile phase was purified using a C18 column (250X 4.6mm,5 μm): acetonitrile-water (65:35V/V); column temperature: 25 ℃; flow rate: 1.0mL/min; detection wavelength: 220nm. The theoretical plate number is not less than 10000 calculated by thymol peak.
Preparation of a control solution:
precisely weighing appropriate amount of thymol reference substance, adding 95% ethanol to desired volume, making into solution containing thymol 0.3mg per 1mL, and filtering with 0.45 μm filter membrane to obtain filtrate.
Preparation of test solution:
precisely measuring 10mL of the fermented products of the example 1, the comparative example 3 and the comparative example 4, placing the fermented products into a 50mL volumetric flask, adding 95% ethanol to fix the volume to a scale, shaking uniformly, and filtering with a filter membrane with the thickness of 0.45 μm to obtain filtrate for later use, thus obtaining the sample solution.
Thymol standard curve preparation:
the thymol control solution was precisely aspirated at 2 μl, 4 μl, 6 μl, 8 μl, 10 μl, 12 μl and the peak area was determined. And drawing a standard curve by taking the concentration as an abscissa and the peak area as an ordinate, wherein the thymol regression equation is as follows: y=105.2333+26.1732, r=0.9989.
TABLE 5 determination of the active ingredient content (mg/g) in the samples of each experimental group
| ProcyanidinsElement B2 | Chlorogenic acid | Paeonol | Notice pavilion | Hyperin | Thymol | |
| Example 1 | 0.679 | 5.883 | 1.104 | 2.693 | 1.707 | 1.352 |
| Comparative example 3 | 0.363 | 2.907 | 0.825 | 1.452 | 1.218 | 1.031 |
| Comparative example 4 | 0.248 | 1.756 | 0.481 | 0.838 | 0.876 | 0.552 |
| Comparative example 5 | 0.453 | 3.145 | 0.879 | 1.685 | 1.269 | 1.132 |
Analysis of results:
from the above table, compared with the non-fermented group (comparative example 4), the six active ingredient contents of the fermented group (example 1) are greatly increased, and the procyanidine B2, chlorogenic acid, paeonol, patulin, hyperin, thymol contents are respectively increased by 173.8%, 235.0%, 129.5%, 221.4%, 94.9% and 144.9%, so that the active ingredient contents in the traditional Chinese medicine are greatly increased after the treatment by the microbial fermentation technology.
Compared with the non-fermented group (comparative example 4), the content of the active ingredients of the fermented group (comparative example 3) is increased to a certain extent by only adopting a single strain, and the content of procyanidine B2, chlorogenic acid, paeonol, patulin, hyperin and thymol is respectively increased by 46.4 percent, 65.5 percent, 71.5 percent, 73.3 percent, 39.0 percent and 86.8 percent, so that the increase of the active ingredients of the traditional Chinese medicine is obviously promoted by adopting the single strain fermentation.
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) is obviously increased, and the content of procyanidine B2, chlorogenic acid, paeonol, patulin, hyperin and thymol is respectively increased by 87.1%, 102.4%, 33.8%, 85.5%, 40.1% and 31.1%, so that the multi-strain mixed fermentation is more beneficial to the increase of the content of the traditional Chinese medicine ingredients.
Compared with the fermentation group without lactobacillus reuteri (comparative example 5), the content of each active ingredient in the multi-strain co-fermentation group (example 1) is obviously increased, and the contents of procyanidine B2, chlorogenic acid, paeonol, patulin, hyperin and thymol are respectively increased by 49.9%, 87.1%, 25.6%, 59.8%, 34.5% and 19.4%, so that the multi-strain mixed fermentation is more beneficial to the increase of the content of the traditional Chinese medicine ingredients.
Experimental example 2 animal experiment
Experimental animals: 72 male ICR mice (18-22 g weight) without Specific Pathogen (SPF) were selected and purchased from the department of laboratory animal science, university of Beijing, china. The experimental animals were kept at 23.+ -. 1 ℃ and 55% humidity, given normal feed and water, and were acclimatized for 3 days in an indoor environment of 12 hours of light alternating with 12 hours of darkness. All animal experimental procedures have been approved by the university of Beijing laboratory animal welfare ethics committee (InstitutionalAnimalCareandUseCommittee, IACUC), approval number: LA2021459 and is carried out strictly in accordance with the guidelines for welfare and ethics of laboratory animals (NIH Press, revised in 1996, 85-23).
Test sample: the test pieces were fermented products prepared in examples 1 to 5 and comparative examples 1 to 5.
Experiment feed: (1) general feed: the nutrient components and the levels provided by the feed are in accordance with the national standard GB14924.3-2010 and can meet the requirements of rodent growth and development.
(2) Hyperuricemia feed: the feed prepared by mixing common feed with 4% of potassium oxazinate and 20% of yeast is purchased from Beijing Botai Honda biotechnology Co., ltd. [ SCXK (Jun) 2012-0003].
The experimental method comprises the following steps:
animal model construction and grouping:
before the start of the experiment, the weights of the mice were determined by means of an electronic scale, and 72 male ICR mice were randomly divided into 12 groups (n=14) by weight and treated accordingly.
(1) Normal control group: ordinary feed was administered while the gastric lavage treatment was performed with 10mL/kg distilled water for 6 weeks.
(2) Hyperuricemia model group: hyperuricemia feed was administered while the gastric lavage treatment was performed with 10mL/kg distilled water for 6 weeks.
(3) Tribromone group: hyperuricemia feed was administered while lavage treatment was performed at 10mL/kg (20 mg/kg body weight/day tribromoron +0.5% carboxymethylcellulose) for 6 weeks.
(4) Example 1 group: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, the sample prepared in example 1) for 6 weeks.
(5) Example 2 group: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, the sample prepared in example 2) for 6 weeks.
(6) Example 3 group: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, sample preparation prepared in example 3) for 6 weeks.
(7) Example 4 group: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, sample prepared in example 4) for 6 weeks.
(8) Example 5 group: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, sample prepared in example 5) for 6 weeks.
(9) Control group 1: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, sample prepared in comparative example 1) for 6 weeks.
(10) Control group 2: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, sample prepared in comparative example 2) for 6 weeks.
(11) Control group 3: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, sample prepared in comparative example 3) for 6 weeks.
(12) Control group 4: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, sample prepared in comparative example 4) for 6 weeks.
(13) Control group 5: hyperuricemia feed was administered while the gastric lavage treatment was performed at 10mL/kg (1 g/kg body weight/day, sample prepared in comparative example 5) for 6 weeks.
During the experiment, animals were observed daily for growth, including hair color, mental state and daily activities, and food intake, water intake, urine intake, weight and food utilization (weight gain/intake x 100%) were recorded periodically. At the end of the experiment, mice were sacrificed to collect different tissues for different studies.
Uric acid level reduction assay
Blood sample collection: on the 6 th week of the experiment, the eyeballs of the mice were removed and venous blood of the mice was collected. Blood is collected into an anticoagulation tube filled with EDTA-K2, and is centrifuged for 10 minutes at 3000r/min at 4 ℃, a mouse serum sample is collected and is preserved at-20 ℃ for standby;
the measuring method comprises the following steps:
serum Uric Acid (SUA), blood Urea Nitrogen (BUN), serum creatinine (SCr) concentrations were determined using a fully automatic biochemical analyzer olympus AU 480.
TABLE 6 concentration of Serum Uric Acid (SUA), blood Urea Nitrogen (BUN), serum creatinine (SCr) in mice of each experimental group
# denotes P <0.01 compared to the normal control group; * Represents P <0.05 compared to the hyperuricemia model group; * P <0.01 compared to hyperuricemia model group.
Analysis of results:
after 6 weeks of intervention, the concentration of serum uric acid, serum urea nitrogen and serum creatinine of mice in the hyperuricemia model group is obviously increased (P < 0.01) compared with that of the normal control group, which indicates that the hyperuricemia mice are successfully modeled.
Compared with the hyperuricemia model control group, the concentration of serum uric acid, blood urea nitrogen and serum creatinine of the mice in the phenylbromarone group and the mice in the examples 1-5 are obviously reduced (P is less than 0.01), which shows that the mice in the examples 1-5 can effectively reduce the serum uric acid, blood urea nitrogen and serum creatinine levels of the hyperuricemia mice. Among them, the effects of examples 1, 2 are slightly better than those of examples 3, 4, 5.
Compared with the hyperuricemia model control group, the concentration of serum uric acid, serum urea nitrogen and serum creatinine of the mice in the comparative example 3 is also reduced to a certain extent (P is less than 0.05), which shows that the mice have a certain uric acid reducing effect when only prepared by single strain fermentation. The concentration of serum uric acid, serum urea nitrogen and serum creatinine of the mice in the comparative example 5 is also reduced to a certain extent (P < 0.05), which shows that the fermentation preparation without adding lactobacillus reuteri has a certain uric acid reducing effect, but the effect is lower than that of the examples. In contrast, the concentrations of serum uric acid, serum nitrogen and serum creatinine in the mice were not statistically different (P > 0.05) in comparison with the model control group in comparative examples 1, 2 and 4.
Mouse kidney disease test for relieving hyperuricemia
The liver and kidney tissue collection method comprises the following steps: after blood collection, mice were sacrificed by cervical dislocation and liver and kidney tissues were sampled. The removed liver and kidney were weighed, liver and half of the right kidney were collected, refrigerated with liquid nitrogen, and stored at-80 ℃. The other half of the right kidney is fixed by 4% paraformaldehyde solution and stored at normal temperature to be used as a sample for subsequent use.
Wherein 4% paraformaldehyde solution fixed right kidneys were used for histopathological examination.
Kidney tissue is dehydrated, transparent, waxed, embedded, sectioned, paraffin sections are made, then dewaxed, stained, dehydrated, transparent, sun-dried, hematoxylin-eosin stained (SE staining method) is performed, and images are collected for analysis.
Tissues were examined under an olympus BX43 light microscope using imageproseus 5.1 (MediaCybernetics, US) image analysis software and images were acquired. Under 200 times visual field, each optical constant of the microscope is fixed, and each region of the pathological section is scanned (glomerulus and renal interstitium are scanned with emphasis).
Analysis of results:
the optical mirror display results are shown in fig. 1, and the scale is: 100 micrometers; original magnification 200×.
Compared with a normal control group, the hyperuricemia model group mice have the symptoms of glomerulus swelling, glomerulus basement membrane thickening, mesangial region broadening, tubular expansion, tubular epithelial cell arrangement disorder and local renal interstitial tissue scattered in inflammatory cell infiltration; compared with the hyperuricemia model group, the renal glomerulus capillary vessel swelling of the mice in the example 3 group is improved, the mesangial matrix is normal, and the sac stenosis is improved; the glomeruli and tubular structures of the mice in the example 1 group and the example 2 group are normal, and the epithelial cells are orderly arranged and are close to the normal control group; compared with hyperuricemia model group, the expansion of renal tubule of mice in the benzbromarone group is reduced, and epithelial cells of the renal tubule are orderly arranged, so that vacuolation degeneration is caused occasionally.
The kidney morphology results show that the group of the embodiment 1 and the group of the embodiment 2 can reduce kidney injury caused by hyperuricemia mice, and play a good role in kidney protection. Although tribromone can reduce blood uric acid level, it does not have a good protective effect on kidneys.
Experiments for reducing liver XOD activity of mice and improving liver function
The measuring method comprises the following steps: accurately weighing liver tissue samples according to the weight (g): volume (mL) =1: 9 is added with physiological saline with the volume of 9 times, and mechanically homogenized under ice bath condition to prepare 10 percent homogenate. Centrifugation at 7000r/min for 10min, collecting supernatant, and measuring XOD activity according to XOD kit (purchased from institute of biotechnology, built in south kyo).
Serum samples of mice are collected in uric acid level reducing test experiments, and serum reserved for preservation at-20 ℃ is used as a sample, and the concentration of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) is measured by an full-automatic biochemical analyzer Olympic Bass AU 480.
The results are shown in Table 7, # indicates that P <0.01 compared to the normal control group; * Represents P <0.05 compared to the hyperuricemia model group; * P <0.01 compared to hyperuricemia model group.
TABLE 7 liver XOD Activity, serum ALT and AST Activity in mice of each experimental group
| XOD Activity (U/g protein) | ALT Activity (U/L) | AST Activity (U/L)) | |
| Normal control group | 82.7±19.6 | 34.9±4.8 | 95.0±9.3 |
| Model control group | 202.5±34.9## | 51.3±5.3## | 136.2±12.3## |
| Benzbromarone group | 126.2±23.7** | 40.2±4.9** | 102.7±11.5** |
| Example 1 | 138.7±25.7** | 42.7±3.6** | 111.7±10.6** |
| Example 2 | 141.5±24.9** | 41.6±4.5** | 113.5±9.9** |
| Example 3 | 148.2±21.3** | 44.7±3.4* | 120.4±11.4** |
| Example 4 | 147.3±23.8** | 42.6±3.9** | 118.8±12.2* |
| Example 5 | 147.0±23.4** | 43.8±3.3* | 115.2±10.4** |
| Comparative example 1 | 190.7±28.6 | 47.6±4.2 | 127.9±11.2 |
| Comparative example 2 | 195.0±26.9 | 47.9±4.4 | 128.5±10.9 |
| Comparative example 3 | 161.6±23.2* | 45.2±3.8* | 121.0±10.8* |
| Comparative example 4 | 176.4±21.3 | 46.3±3.5 | 124.5±11.7 |
| Comparative example 5 | 169.2±12.7 | 44.8±4.1* | 121.7±9.6* |
After 6 weeks of intervention, the liver XOD activity and serum ALT and AST activity of mice in the hyperuricemia model group are all significantly increased (P < 0.01) compared with the normal control group, indicating that hyperuricemia significantly affects the liver XOD activity and serum ALT and AST activity of mice.
Compared with the hyperuricemia model control group, the liver XOD activity and serum ALT and AST activity of the benzbromarone group, the example 1 and the example 2 groups are obviously reduced (P < 0.01), which indicates that the example 1 and the example 2 groups can effectively reduce the liver XOD activity and serum ALT and AST activity of hyperuricemia mice. Examples 3, 4 and 5 also showed a significant decrease in liver XOD activity and serum ALT and AST activity (P <0.05, partial index P < 0.01), and it can be seen that examples 1 and 2 showed slightly better effects in inhibiting liver XOD activity and serum ALT and AST activity than examples 3, 4 and 5.
Compared with the hyperuricemia model control group, the liver XOD activity and serum ALT and AST activity of the mice in the comparative example 3 are also reduced to a certain extent (P < 0.05), which shows that the mice have a certain uric acid reducing effect when only prepared by single strain fermentation. The serum ALT and AST activities of the mice of comparative example 5 were also reduced to some extent (P < 0.05), indicating that the fermentation preparation without added Lactobacillus reuteri also had some effect, whereas the comparative examples 1, 2, 4 had no statistically significant differences (P > 0.05) compared to the model control group.
Experiment for improving kidney inflammatory factor level
Hyperuricemia mice develop a pronounced intestinal barrier defect, and body intestinal barrier dysfunction activates macrophage-mediated inflammatory responses, up-regulates the expression of Toll-like receptor4 (TLR 4), and in turn activates the myeloid differentiation factor 88 (MyD 88) signaling pathway, initiating rapid activation of NF- κb, increasing levels of IL-1β, TNF- α, MCP-1 and IL-8 in vivo, causing cytokine cascade activation reactions, promoting proximal tubular epithelial cell inflammation and oxidative stress, and exacerbating kidney function injury. Therefore, the key to controlling uric acid levels is to reduce inflammation and oxidative stress, thereby reducing damage to kidney function and ensuring excretion of uric acid by the kidneys.
The measuring method comprises the following steps:
accurately weighing kidney tissue samples according to the weight (g): volume (mL) =1: 9 is added with physiological saline with the volume of 9 times, and mechanically homogenized under ice bath condition to prepare 10 percent homogenate. Centrifuging at 3000r/min for 10min, and collecting supernatant. The content of IL-1 beta, IL-6 and TNF-alpha was detected by treatment according to the instructions of ELISA kit. The mouse TNF-alpha ELISA kit (88-7324), the mouse IL-1 beta ELISA kit (88-7064) and the mouse IL-6 ELISA kit (88-7013) are all purchased from England Life technologies Co., ltd. The experimental results are shown in table 8.
Table 8 mice kidney pro-inflammatory cytokine levels (x±sd) for each group (n=10)
/>
# denotes P <0.01 compared to the normal control group; * Represents P <0.05 compared to the hyperuricemia model group; * P <0.01 compared to hyperuricemia model group.
Compared with the normal control group, the levels of the kidney inflammatory factors IL-1 beta, IL-6 and TNF-alpha of the mice in the hyperuricemia model group are obviously increased (P < 0.01), which indicates that the levels of the kidney inflammatory factors of the mice are obviously increased by hyperuricemia.
The levels of IL-1β, IL-6, TNF- α were significantly reduced (P < 0.01) in the benzbromarone group, examples 1, 2, 3 mice kidneys after 6 weeks of continuous intervention compared to the hyperuricemia model group; examples 4, 5 mice also showed significant reductions in renal IL-1 beta, IL-6, TNF-alpha levels (P <0.01, a small fraction of index P < 0.05). The results show that the groups 1-5 can obviously reduce the kidney inflammatory factor level of mice in the hyperuricemia model group, and play a certain role in protecting kidney tissues.
Compared with the hyperuricemia model group, the kidney inflammatory factor levels of the mice in the comparative examples 1, 2, 4 and 5 are not significantly different (P is more than 0.05), and the 3 comparative examples cannot play a certain role in protecting the kidney; comparative example 3 showed significantly reduced levels of mouse kidney IL-1 beta, IL-6, TNF-alpha (P < 0.05) compared to the hyperuricemia model group, but had a poorer effect compared to examples 1-5.
Westernblot Western blot assay
Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine protein kinases that can be activated by different extracellular stimuli, are important transmitters of signal transduction, and are involved in a variety of important cellular physiological/pathological processes such as regulation of cell growth, differentiation, adaptation to environmental stress, inflammatory response, and the like. MAPK can be divided into 4 subfamilies: ERK, p38, JNK and ERK5. In recent years, p38MAPK has received increasing attention for its role in the regulation of pro-inflammatory cytokines. p38MAPK phosphorylation can promote NF- κB activation by inducing cell proliferation.
Under stimulation of the upstream p38MAPK signal, NF- κB is transported to the nucleus and binds to NF- κB transcriptional elements, activating the expression of IL-1β, IL-6 and TNF- α inflammatory factors. Simultaneously, IL-1 beta and TNF-alpha can induce the production of other inflammatory factors by activating NF- κB signaling pathway, aggravate kidney inflammatory reaction and form malignant circulation, so that the activation of p38MAPK and NF- κB is very critical.
The measuring method comprises the following steps:
immunoblotting examined the expression of p38MAPK, p-p38MAPK, NF- κ B, p-NF- κB and NLRP3 proteins in mouse kidney tissue. The biquinolinecarboxylic acid (BCA) protein quantification kit adopts beijing plarillic gene technologies limited.
The experimental results are shown in FIG. 2.
Analysis of results:
compared with a normal control group, the expression proportion of the kidney p-p38MAPK/p38MAPK of the mice in the hyperuricemia model group is obviously increased; compared with the hyperuricemia model group, the kidney P-P38MAPK/P38MAPK expression proportion of the mice in the example 1 group is obviously reduced (P < 0.05), and the kidney P-P38MAPK/P38MAPK expression proportion of the mice in the benzbromarone group is also obviously reduced (P < 0.05); compared with a normal control group, the expression proportion of the kidney p-NF- κB/NF- κB of the mice in the hyperuricemia model group is obviously increased; compared with the hyperuricemia model group, the kidney P-NF- κB/NF- κB expression proportion of the mice of the example 1 is obviously reduced (P < 0.05); there was no significant difference in NLRP3 protein expression from each group.
The results show that the group 1 can obviously reduce the activation of p38MAPK and NF- κB, obviously reduce the expression proportion of p-p38MAPK/p38MAPK, obviously reduce the expression proportion of p-NF- κB/NF- κB, inhibit the increase of uric acid level and obviously reverse the change and damage caused by inflammation.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (10)
1. A uric acid-reducing ferment is characterized by comprising the following fermentation raw materials in parts by weight: 100-180 parts of lycium ruthenicum, 120-200 parts of eucommia male flowers, 80-160 parts of danfeng peony flowers, 60-140 parts of auricularia auricular calyx, 140-220 parts of acanthopanax sessiliflorus, 60-140 parts of inula japonica, 1.2-12 parts of zymophyte powder and 14-120 parts of enzyme preparation;
the enzyme preparation comprises cellulase, pectase, acid protease and medium-temperature alpha-amylase, wherein the mass ratio of the enzymes is 6-9:8-10:5-6:8-12;
the fermentation bacteria powder comprises lactobacillus plantarum, lactobacillus fermentum, lactobacillus reuteri, lactobacillus acidophilus, lactobacillus rhamnosus and bifidobacterium adolescentis, wherein the mass ratio of the bacteria is 12-15:11-14:6-8:6-8:6-8:3-5.
2. The ferment of claim 1, wherein the enzyme activity ratio of each enzyme unit in the enzyme preparation is 30-40:40-50:90-100:5-6.
3. The ferment of claim 1, wherein the ratio of the unit force of each bacterium in the ferment fungus powder is 18-20:8-10:14-16:10-12:23-26:6-8.
4. The ferment according to claim 2, wherein the enzyme preparation has a mass ratio of 6:8:6:12 or 9:10:5:8, 8; the enzyme activity ratio of each enzyme unit is as follows: 30:40:100:5, a step of; the unit enzyme activity of the cellulase is 30000U/g.
5. A ferment according to claim 3, wherein the mass ratio of the respective bacteria in the ferment bacteria powder is 12:14:6:8:8:3 or 15:11:8:6:6:5, a step of; the unit living force ratio of each bacterium is 20:10:16:12:26:8, 8; the unit activity of the lactobacillus plantarum is 2000 hundred million CFU/g.
6. The ferment of claim 1, further comprising an acid-base modifier: 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.
7. A process for producing a fermented product according to any one of claims 1 to 6, comprising:
(1) Enzymolysis: mixing crushed and weighed lycium ruthenicum, eucommia male flowers, auriculate swallowwort root, acanthopanax sessiliflorus, inula brined flowers and paeonia suffruticosa with water to form a mixture, regulating the pH value to 4.5-5.5, adding an enzyme preparation for enzymolysis, and regulating the pH value to 5.0-6.8 after the enzymolysis is finished to obtain an enzymolysis solution;
(2) Extracting the enzymolysis liquid to obtain an extracting liquid;
(3) Adding zymophyte powder into the extracting solution for fermentation, and carrying out solid-liquid separation to obtain a supernatant, namely a fermentation product.
8. The preparation method according to claim 7, wherein the enzymolysis in the step (1) is carried out at a temperature of 45-65 ℃ for 30-180min;
The temperature of the extraction treatment in the step (2) is 80-108 ℃ and the time is 10-90min;
the fermentation temperature in the step (3) is 25-45 ℃, the fermentation time is 24-240h, the stirring speed is 100-1000RPM, and the ventilation rate is 1-10vvn.
9. Use of the ferment of any one of claims 1 to 6 for the preparation of a medicament for the prevention or treatment of hyperuricemia, gout or renal disorders.
10. A pharmaceutical formulation comprising the ferment of any one of claims 1-6.
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