Exocarpium citri grandis compound fermentation liquor for assisting in reducing uric acid, preparation method thereof and application of exocarpium citri grandis compound fermentation liquor in beer

Abstract

The invention relates to the technical field of biological medicine, and discloses exocarpium citri grandis compound fermentation liquor for assisting in reducing uric acid, a preparation method of the exocarpium citri grandis compound fermentation liquor and application of the exocarpium citri grandis compound fermentation liquor in beer. Comprising the following steps: mixing exocarpium citri grandis, perilla frutescens, folium mori, poria cocos, coix seeds, chicory roots, licorice roots, radix puerariae, fructus gardeniae and lily bulbs to obtain a raw material mixture; mixing the raw material mixture with water, and decocting to obtain a composite extracting solution; performing enzymolysis on the composite extracting solution to obtain enzymatic hydrolysate; inoculating probiotic strains into the enzymatic hydrolysate, and culturing to obtain exocarpium citri grandis compound fermentation liquor for assisting in reducing uric acid; the exocarpium compound fermentation liquor for assisting in reducing uric acid has the effects of reducing uric acid and treating gout; furthermore, the exocarpium citri grandis is applied to beer preparation, the exocarpium citri grandis beer with the gout treatment effect is obtained, the problem that gout patients cannot drink wine is solved through development of the exocarpium citri grandis beer, and the exocarpium citri grandis beer has wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a compound fermentation liquid of Citrus reticulata extract for assisting in lowering uric acid, its preparation method, and its application in beer. Background Technology

[0002] Gout is a disease caused by reduced uric acid excretion or abnormal purine metabolism. It is associated with hyperuricemia and the deposition of monosodium urate crystals and is a type of metabolic rheumatic disease. Gout can lead to kidney disease and, in severe cases, is often accompanied by diabetes, arteriosclerosis, coronary heart disease, hyperlipidemia, hypertension, and other diseases, potentially causing joint damage and kidney dysfunction. In daily life, gout patients need to reduce their intake of high-purine foods, such as animal organs, seafood, and meat broth, as these foods increase uric acid production and worsen the condition. In addition, gout patients should avoid alcohol, especially beer.

[0003] Western medicine treatment for gout mainly focuses on two aspects: controlling hyperuricemia and preventing acute gouty arthritis. The main approaches are to inhibit uric acid production, promote uric acid excretion, and provide anti-inflammatory and analgesic effects. Commonly used drugs include febuxostat, benzbromarone, and allopurinol. While these drugs are highly effective in rapidly lowering uric acid levels, controlling inflammation, and relieving pain, they also have numerous side effects, low safety profiles, and can easily damage liver and kidney function. Furthermore, they can cause gastrointestinal reactions such as nausea and vomiting, as well as allergic reactions such as rashes and asthma. Long-term use is difficult for patients. Therefore, finding a safe and effective treatment method is an urgent problem to be solved. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a method for preparing a compound fermentation broth containing Citrus reticulata peel that helps lower uric acid, comprising the following steps: Step 1: Weigh out the following ingredients in proportion: Citrus reticulata peel, Perilla frutescens, Mulberry leaf, Poria cocos, Coix seed, Chicory root, Licorice, Kudzu root, Gardenia, and Lily bulb. Mix them together, pulverize them, and sieve them to obtain the raw material mixture. Step 2: Mix the raw material mixture with water, decoct, collect the filtrate, add water to the residue and continue to decoct, repeat three times, combine the three filtrates to obtain a compound extract; Cellulase and pectinase were added to the compound extract, and the mixture was sterilized to obtain the enzymatic hydrolysate. Step 3: Inoculate the plant lactobacillus strain and the Bacillus subtilis strain into the enzymatic hydrolysate and ferment to obtain the Huajuhong compound fermentation broth that helps lower uric acid.

[0005] Preferably, in step one, the components in the raw material mixture, by weight, are: 2-4 parts of Citrus reticulata peel, 1.5-2 parts of Perilla frutescens, 3.5-4 parts of Mulberry leaf, 2-3 parts of Poria cocos, 3.5-4 parts of Coix lacryma-jobi, 4-6 parts of Chicory root, 1-1.5 parts of Glycyrrhiza uralensis, 3.5-4 parts of Pueraria lobata, 2-3 parts of Gardenia jasminoides, and 2-3 parts of Lilium brownii. The effects of each component in the above process are as follows: Citrus reticulata peel is pungent, bitter and warm in nature. It is pungent and aromatic, bitter and warm, and enters the lung, spleen and stomach meridians. It is good at regulating qi and dispelling cold, drying dampness and resolving phlegm, and also helps with digestion. Its functions are to regulate qi and relieve chest tightness, dry dampness and resolve phlegm, and help with digestion. It is mainly used to treat cough due to wind-cold, itchy throat and excessive phlegm, and is most suitable for those with food stagnation. Perilla leaves are pungent and warm in nature, and enter the lung and spleen meridians. They have the effects of opening up and dispersing lung qi, releasing exterior cold, and regulating qi and relieving chest tightness. They are suitable for symptoms such as colds due to wind-cold, aversion to cold and fever, cough, asthma, and chest and abdominal distension. Mulberry leaves are sweet and bitter in taste, cold in nature, and enter the lung and liver meridians. They are mainly used for wind-heat colds, dry coughs due to lung heat, or dizziness, headaches, red and blurred vision, and watery eyes caused by liver heat. They also help prevent and treat wind-heat colds, dry coughs due to lung heat, sore throats, headaches due to blood deficiency, eye fatigue, habitual constipation, difficulty urinating, and rheumatic joint pain. They also help lower blood pressure, blood sugar, and blood lipids, prevent cerebral thrombosis, lose weight, and maintain health. Poria cocos is sweet, bland, and neutral in nature. It enters the heart, lung, spleen, and kidney meridians. It promotes diuresis and eliminates dampness, strengthens the spleen and resolves phlegm, calms the mind and soothes the nerves, detoxifies and fights cancer. Its medicinal properties are mild. It promotes diuresis without harming the body's vital energy. It can treat symptoms such as difficulty urinating, edema and abdominal distension, phlegm and cough, vomiting, morning sickness, diarrhea, seminal emission, urinary tract infection, palpitations, and forgetfulness. Job's tears are sweet, bland, and cool in nature, and enter the spleen, stomach, and lung meridians. They have the effects of strengthening the spleen and removing dampness, relieving numbness and stopping diarrhea, clearing heat and draining pus. They can treat edema, beriberi, difficulty urinating, dampness-induced numbness and contracture, spleen deficiency diarrhea, lung pain, intestinal abscess, and flat warts. Chicory root is the dried root of the plant *Chicorydalis yanhusuo* or *Chicorydalis tangutica* in the Asteraceae family. It has a slightly bitter, salty, and cool taste and enters the liver, gallbladder, and stomach meridians. It clears the liver and gallbladder, strengthens the stomach and aids digestion, promotes urination and reduces swelling. It is mainly used for damp-heat jaundice, stomach pain with poor appetite, and edema with scanty urine. Licorice is sweet and neutral in nature. It enters the heart, lung, spleen, and stomach meridians. It has the effects of tonifying the spleen and replenishing qi, clearing heat and detoxifying, eliminating phlegm and relieving cough, relieving spasms and pain, harmonizing various medicines, and reducing the toxicity and harshness of drugs. It can treat spleen and stomach weakness, fatigue, palpitations and shortness of breath, cough with phlegm, abdominal and limb spasms and pain, carbuncles and boils, etc. Kudzu root, sweet, pungent and cool in taste, enters the spleen and stomach meridians. Its functions and indications are: relieving muscle tension and reducing fever, promoting body fluid production, promoting rash eruption, raising yang and stopping diarrhea. It is used for exogenous fever and headache, stiff neck and back pain, thirst, diabetes, incomplete measles eruption, dysentery, diarrhea, and hypertension with stiff neck and back pain. Gardenia, bitter and cold in nature, non-toxic, enters the heart, lung, stomach, and triple burner meridians. Its functions and indications include: purging fire and relieving irritability, clearing heat and promoting diuresis, cooling blood and detoxifying; used for feverish irritability, depression, restlessness, damp-heat stagnation, jaundice, scanty dark urine, hematemesis, epistaxis, hematuria, carbuncles, red and swollen eyes, boils and carbuncles caused by heat toxins; externally used for sprains and contusions; charred gardenia cools blood and stops bleeding, used for hematemesis, epistaxis, hematuria, and metrorrhagia due to blood heat. Lily bulb is sweet, slightly bitter, and slightly cold in nature. It enters the heart and lung meridians. Its main functions are to nourish yin and moisten the lungs, clear the heart and calm the mind. It is used for chronic cough due to yin deficiency, hemoptysis, late stage of febrile disease, lingering heat, or restlessness, palpitations, insomnia, dreaminess, mental confusion, carbuncles, and damp sores caused by emotional distress.

[0006] Preferably, in step two, during the preparation of the compound extract, the mass ratio of the raw material mixture to water is 1:10, the mass ratio of the filter residue to water is 1:10, and the decoction time is 30 minutes each time.

[0007] Preferably, in step two, the cellulase has an enzyme activity of 20,000 U / g and is used in an amount of 4-6 wt% of the raw material mixture, and the pectinase has an enzyme activity of 20,000 U / g and is used in an amount of 2-3 wt% of the raw material mixture.

[0008] Preferably, in step two, the enzymatic hydrolysis conditions are: a temperature of 55 ℃ and an enzymatic hydrolysis time of 5 h.

[0009] Preferably, in step three, the concentration of the Bacillus subtilis strain is 1.0 × 10⁻⁶. 8 -1.5×10 8 The inoculum concentration was 6-8% (cfu / mL); the concentration of the *Lactobacillus plantarum* strain was 1.0 × 10⁻⁶. 8 -1.5×10 8 cfu / mL, inoculation amount is 3-5%.

[0010] Preferably, in step three, the fermentation culture conditions are: fermentation culture for 3 days at a temperature of 37 ℃ and a stirring speed of 160 rpm.

[0011] The uric acid-lowering compound fermentation broth was prepared using the aforementioned method.

[0012] The compound fermentation liquid of Huajuhong, which helps lower uric acid, is used to prepare Huajuhong beer.

[0013] Furthermore, the preparation method of the orange-red beer includes the following steps: Step S1: Mix malt and rice at a mass ratio of 7.5:2.5 to obtain brewing raw materials; mix the brewing raw materials with water at a material-to-water ratio of 1:4, add the raw materials at 35 ℃, raise the temperature to 53 ℃ after 40 min and hold for 30 min, then slowly raise the temperature to 62 ℃, raise the temperature to 68 ℃ after 60 min and hold for 15 min, until saccharification is complete as detected by iodine solution, and obtain wort; Step S2: Take the wort obtained in Step 1 and boil it for 90 minutes. During the boiling process, add 0.01 wt% bitter hops, 0.03 wt% bitter hops, and 0.03 wt% aroma hops at the 15th, 45th, and 75th minutes of the total wort volume, respectively. After boiling, remove the hops and heat-coagulated material, then steam sterilize at 115 °C for 20 minutes and cool to obtain the brewing fermentation stock solution. Step S3: Mix the brewing fermentation liquid and the Huajuhong compound fermentation liquid at a mass ratio of 100:(16-24), cool to 7 ℃, inoculate with activated dry yeast at an inoculation amount of 1 g / L, and aerate the mixture to achieve an oxygenation level of 8 mg / L. Set the fermentation temperature to 25 ℃. After fermentation for 24 h, when the sugar content drops below 5 degrees, control the fermentation pressure to 0.15 MPa. Set the temperature to 0 ℃ on day 11 of fermentation, and set the temperature to -1 ℃ on day 13. Brewing is completed on day 14, and the lees are discharged on day 15 to obtain Huajuhong beer.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention discloses a compound fermented liquid of Citrus reticulata peel for assisting in lowering uric acid, also known as "gout fermented liquid". It is prepared by enzymatic hydrolysis and fermentation using Citrus reticulata peel, Perilla frutescens, Mulberry leaf, Poria cocos, Coix seed, Chicory root, Licorice, Kudzu root, Gardenia jasminoides, and Lilium brownii as raw materials. The components of the gout fermented liquid of this invention work together to effectively reduce the levels of uric acid, creatinine, blood urea nitrogen, plasma pro-inflammatory factors TNF-α, TNF-1β, and IL-6 in plasma, inhibit xanthine oxidase levels, increase CAT content in plasma, and reduce PO+HX-induced hyperuricemic liver and kidney damage. Therefore, the compound fermented liquid of Citrus reticulata peel for assisting in lowering uric acid of this invention provides a safe and efficient treatment for gout. 2. This invention also discloses a citrus-infused beer, namely red beer. The citrus-infused beer of this invention incorporates a citrus-infused compound fermentation liquid that helps lower uric acid during its preparation. The citrus-infused beer of this invention is superior to the citrus-infused compound fermentation liquid that helps lower uric acid in terms of lowering uric acid and inhibiting xanthine oxidase levels. Therefore, the citrus-infused beer of this invention also possesses excellent gout treatment effects. Furthermore, the development of the citrus-infused beer of this invention solves the problem of gout patients being unable to drink alcohol, improves the quality of life of gout patients, and has broad application prospects. Attached Figure Description

[0015] Figure 1 The changes in body weight of mice in the blank group, model group, red beer group, gout fermentation liquid group, tangerine peel fermentation liquid group and allopurinol group; Figure 2 The results show the plasma uric acid levels in mice from the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Figure 3 The results show the detection of xanthine oxidase levels in the plasma of mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Figure 4 Optical images of kidney tissue from mice in the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group; Figure 5 The results show the kidney index of mice in the blank group, model group, red beer group, gout fermentation liquid group, tangerine peel fermentation liquid group and allopurinol group. Figure 6 The results show the kidney injury scores of mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Figure 7 The results of H&E staining of kidney tissue from mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group and allopurinol group are as follows: Figure 8 The results show the plasma creatinine levels in mice from the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Figure 9 The results show the blood urea nitrogen levels in the plasma of mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Figure 10 The results show the plasma levels of TNF-α (A), IL-1β (B), IL-6 (C), and CAT (D) in mice from the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Figure 11Optical images of liver tissue from mice in the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group; Figure 12 The results show the liver index of mice in the blank group, model group, red beer group, gout fermentation liquid group, tangerine peel fermentation liquid group and allopurinol group. Figure 13 The results show the liver injury scores of mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group and allopurinol group. Figure 14 The results are H&E staining of liver tissue from mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0017] Example 1

[0018] This embodiment discloses a method for preparing a compound fermentation broth of Citrus reticulata peel that helps lower uric acid, including the following steps: Step 1: Take 2 parts of Citrus reticulata peel, 1.5 parts of Perilla frutescens, 3.5 parts of Mulberry leaf, 2 parts of Poria cocos, 3.5 parts of Coix seed, 4 parts of Chicory root, 1 part of Licorice root, 3.5 parts of Kudzu root, 2 parts of Gardenia jasminoides, and 2 parts of Lilium brownii by weight, mix them, pulverize them, and pass them through an 80-mesh sieve to obtain the raw material mixture. Step 2: Mix the raw material mixture with water at a mass ratio of 1:10, decoct for 30 minutes, collect the filtrate, add water to the residue and continue decoction, with the mass ratio of residue to water being 1:10, repeat three times, and combine the three filtrates to obtain a composite extract; add cellulase and pectinase to the composite extract, enzymatically hydrolyze at 55 ℃ for 5 h, sterilize, and obtain an enzymatic hydrolysate; wherein the cellulase has an enzyme activity of 20000 U / g, and its dosage accounts for 4wt% of the mass of the raw material mixture, and the pectinase has an enzyme activity of 20000 U / g, and its dosage accounts for 2wt% of the mass of the raw material mixture; Step 3: Inoculate the cryopreserved *Lactobacillus plantarum* and *Bacillus subtilis* strains into the enzymatic hydrolysate. Ferment and culture at 37 ℃ and 160 rpm for 3 days. After the culture is complete, centrifuge the resulting fermentation mixture at 4000 rpm for 10 min, collect the supernatant, and sterilize at 121 ℃ for 15 min to obtain the *Citrus reticulata* compound fermentation broth for uric acid reduction. Store it in a dark, dry place for later use. The concentration of the *Bacillus subtilis* strain is 1.0 × 10⁻⁶. 8 The concentration of *Lactobacillus plantarum* was 1.0 × 10⁻⁶ cfu / mL, and the inoculum size was 6%. 8 cfu / mL, inoculum size 3%.

[0019] Example 2

[0020] This embodiment discloses a method for preparing a compound fermentation broth of Citrus reticulata peel that helps lower uric acid, including the following steps: Step 1: Take 4 parts of Citrus reticulata peel, 2 parts of Perilla frutescens, 4 parts of Mulberry leaf, 3 parts of Poria cocos, 4 parts of Coix lacryma-jobi, 6 parts of Chicory root, 1.5 parts of Glycyrrhiza uralensis, 4 parts of Pueraria lobata, 3 parts of Gardenia jasminoides, and 3 parts of Lilium brownii by weight, mix them together, pulverize them, and pass them through an 80-mesh sieve to obtain the raw material mixture. Step 2: Mix the raw material mixture with water at a mass ratio of 1:10, decoct for 30 minutes, collect the filtrate, add water to the residue and continue decoction, with the mass ratio of residue to water being 1:10, repeat three times, and combine the three filtrates to obtain a composite extract; add cellulase and pectinase to the composite extract, enzymatically hydrolyze at 55 ℃ for 5 h, sterilize, and obtain an enzymatic hydrolysate; wherein the cellulase has an enzyme activity of 20000 U / g, and its dosage accounts for 6wt% of the mass of the raw material mixture, and the pectinase has an enzyme activity of 20000 U / g, and its dosage accounts for 3wt% of the mass of the raw material mixture; Step 3: Inoculate the cryopreserved *Lactobacillus plantarum* and *Bacillus subtilis* strains into the enzymatic hydrolysate. Ferment and culture at 37 ℃ and 160 rpm for 3 days. After the culture is complete, centrifuge the resulting fermentation mixture at 4000 rpm for 10 min, collect the supernatant, and sterilize at 121 ℃ for 15 min to obtain the *Citrus reticulata* compound fermentation broth for uric acid reduction. Store it in the dark and dry for later use. The concentration of the *Bacillus subtilis* strain is 1.5 × 10⁻⁶. 8 The concentration of *Lactobacillus plantarum* was 1.5 × 10⁻⁶ CFU / mL, and the inoculum size was 8%. 8 cfu / mL, inoculum size 5%.

[0021] Example 3

[0022] This embodiment discloses a method for preparing a compound fermentation broth of Citrus reticulata peel that helps lower uric acid, including the following steps: Step 1: By weight, take 3 parts of Citrus reticulata peel, 1.75 parts of Perilla frutescens, 3.75 parts of Mulberry leaf, 2.5 parts of Poria cocos, 3.75 parts of Coix seed, 5 parts of Chicory root, 1.25 parts of Licorice root, 3.75 parts of Kudzu root, 2.5 parts of Gardenia jasminoides, and 2.5 parts of Lilium brownii. Mix them together, pulverize them, and pass them through an 80-mesh sieve to obtain the raw material mixture. Step 2: Mix the raw material mixture with water at a mass ratio of 1:10, decoct for 30 minutes, collect the filtrate, add water to the residue and continue decoction, with the mass ratio of residue to water being 1:10, repeat three times, and combine the three filtrates to obtain a composite extract; add cellulase and pectinase to the composite extract, enzymatically hydrolyze at 55 ℃ for 5 h, sterilize, and obtain an enzymatic hydrolysate; wherein the cellulase has an enzyme activity of 20000 U / g and its dosage accounts for 5wt% of the mass of the raw material mixture, and the pectinase has an enzyme activity of 20000 U / g and its dosage accounts for 2.5wt% of the mass of the raw material mixture; Step 3: Inoculate the cryopreserved *Lactobacillus plantarum* and *Bacillus subtilis* strains into the enzymatic hydrolysate. Ferment and culture at 37 ℃ and 160 rpm for 3 days. After the culture is complete, centrifuge the resulting fermentation mixture at 4000 rpm for 10 min, collect the supernatant, and sterilize at 121 ℃ for 15 min to obtain the *Citrus reticulata* compound fermentation broth for uric acid reduction. Store it in a dark, dry place for later use. The concentration of the *Bacillus subtilis* strain is 1.25 × 10⁻⁶. 8 The concentration of *Lactobacillus plantarum* was 1.25 × 10⁻⁶ CFU / mL, and the inoculum size was 7%. 8 cfu / mL, inoculum size 4%.

[0023] Example 4

[0024] This embodiment discloses a method for preparing Citrus aurantium beer, including the following steps: Step S1: Mix malt and rice at a mass ratio of 7.5:2.5 to obtain brewing raw materials; mix the brewing raw materials with water at a material-to-water ratio of 1:4, add the raw materials at 35 ℃, raise the temperature to 53 ℃ after 40 min and hold for 30 min, then slowly raise the temperature to 62 ℃, raise the temperature to 68 ℃ after 60 min and hold for 15 min, until saccharification is complete as detected by iodine solution, and obtain wort; Step S2: Take the wort obtained in Step 1 and boil it for 90 minutes. During the boiling process, add 0.01 wt% bitter hops, 0.03 wt% bitter hops, and 0.03 wt% aroma hops at the 15th, 45th, and 75th minutes of the total wort volume, respectively. After boiling, remove the hops and heat-coagulated material, then steam sterilize at 115 °C for 20 minutes and cool to obtain the brewing fermentation stock solution. Step S3: Mix the brewing fermentation liquid with the uric acid-lowering compound fermentation liquid prepared in Example 3 at a mass ratio of 100:20, cool to 7 ℃, inoculate with activated dry yeast at an inoculation amount of 1 g / L, and aerate the mixture to achieve an oxygenation level of 8 mg / L. Set the fermentation temperature to 25 ℃. After fermentation for 24 h, when the sugar content drops below 5 degrees, control the fermentation pressure to 0.15 MPa. Set the temperature to 0 ℃ on day 11 of fermentation, and set the temperature to -1 ℃ on day 13. Brewing is completed on day 14, and the lees are discharged on day 15 to obtain citrus beer, i.e., red beer.

[0025] Comparative Example 1 This comparative example discloses a method for preparing Citrus reticulata fermentation broth, including the following steps: Step 1: Take pulverized Citrus reticulata peel, pass it through an 80-mesh sieve to obtain Citrus reticulata peel powder; mix the Citrus reticulata peel powder with water at a mass ratio of 1:10, decoct for 30 minutes, collect the filtrate, add water to the residue and continue decoction, the mass ratio of residue to water is 1:10, repeat three times, combine the three filtrates to obtain a composite extract; add cellulase and pectinase to the composite extract, enzymatically hydrolyze at 55 ℃ for 5 hours, sterilize to obtain an enzymatic hydrolysate; wherein, the cellulase has an enzyme activity of 20000 U / g, and its amount accounts for 5wt% of the mass of the raw material mixture, and the pectinase has an enzyme activity of 20000 U / g, and its amount accounts for 2.5wt% of the mass of the raw material mixture; Step 3: Inoculate the cryopreserved *Lactobacillus plantarum* and *Bacillus subtilis* strains into the enzymatic hydrolysate and culture for 3 days at 37 ℃ and 160 rpm. After culturing, centrifuge the resulting fermentation mixture at 4000 rpm for 10 min, collect the supernatant, and sterilize it at 121 ℃ for 15 min to obtain the *Citrus reticulata* fermentation broth. Store it in the dark and dry for later use. The concentration of the *Bacillus subtilis* strain is 1.25 × 10⁻⁶. 8 The concentration of *Lactobacillus plantarum* was 1.25 × 10⁻⁶ CFU / mL, and the inoculum size was 7%. 8 cfu / mL, inoculum size 4%.

[0026] Experimental Example I. Animal Experiment Protocol All experiments were conducted in accordance with the "Regulations on the Management of Laboratory Animals of the State Science and Technology Commission of the People's Republic of China". The mouse program was reviewed and approved by the Animal Research Ethics Committee of Dalian University of Technology. Male KM mice (18-22 g) aged 4-5 weeks were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (Changchun, China). All mice were housed in a clean room free of specific pathogens, with the clean room maintained at 21-25 °C and 50-55% relative humidity. The dark / light cycle was 12 hours. Mice had free access to sterilized feed pellets and double-distilled water. All animal experiments strictly followed the relevant principles and guidelines formulated by the China Animal Protection Committee and complied with international animal research ethics standards, including the "ARRIVE Guidelines". While ensuring the reliability of experimental results, this study minimized the number of animals used and made every effort to reduce animal suffering.

[0027] After acclimatizing to a standard feeding environment for 3 days, all mice were randomly divided into 6 groups (n=5): blank control group, model group, red beer group, gout fermentation broth group, Huajuhong fermentation broth group, and allopurinol group. The mice in each group underwent pretreatment (as shown in Table 1): Blank control group: 30 mg / kg CMC-Na (sodium carboxymethyl cellulose) injection + 30 mg / kg CMC-Na oral administration; Model group: 30 mg / kg PO (potassium oxonate) injection + 30 mg / kg HX (hypoxanthine) oral administration; Red beer group: 30 mg / kg Huajuhong beer (i.e., red beer) prepared in Example 4 orally + 30 mg / kg PO injection + 30 mg / kg HX oral administration; Huajuhong fermentation broth group: 30 mg / kg Huajuhong fermentation broth prepared in Comparative Example 1 orally + 30 mg / kg PO injection + 30 mg / kg HX oral administration; Gout fermentation broth group: 30 mg / kg CMC-Na (sodium carboxymethyl cellulose) injection + 30 mg / kg CMC-Na oral administration; Model group: 30 mg / kg PO (potassium oxonate) injection + 30 mg / kg HX oral administration; Allopurinol group: 30 mg / kg CMC-Na (sodium carboxymethyl cellulose ... The uric acid-lowering compound fermented liquid of Citrus reticulata (i.e., gout fermented liquid) prepared in Example 3 was administered orally + 30 mg / kg PO injection + 30 mg / kg HX orally; Allopurinol group: 5 mg / kg allopurinol orally + 30 mg / kg PO injection + 30 mg / kg HX orally. The experiment lasted for 6 days. Each group of mice was pretreated every morning. On the 7th day, after fasting for 12 hours, the mice were euthanized. The weight of the mice was recorded throughout the experiment. Table 1. Animal experimental protocol for gout models

[0028] (Note: Table 1 uses the fermented broth of Citrus reticulata as an example. The sample of Citrus reticulata fermented broth (30 mg / kg) represents the Citrus reticulata fermented broth containing 30 mg of Citrus reticulata per kilogram of mouse body weight when administered by gavage. The dosage of 0.1 mL / 20 g is the ratio of the volume of Citrus reticulata fermented broth to the body weight of the mouse. The concentration of Citrus reticulata fermented broth used for gavage administration to the mice is 6 mg / mL.) II. Animal Experiment Content (1) Sample collection After the mice were euthanized, blood was collected into anticoagulant tubes containing EDTA K2, kept in an ice box for 30 min, and then centrifuged at 3500 r / min for 15 min. The mouse plasma was then aspirated into a new EP tube and stored at -80 ℃ for biochemical index analysis. After the mice were dissected, liver and kidney tissues were collected and weighed for organ index calculation to measure the degree of damage. The organ index was calculated as organ mass / mouse weight before euthanasia. In addition, some kidney and liver tissues were taken, fixed with 4% paraformaldehyde, and used for H&E staining of pathological sections; another part of the tissue was stored at -80 ℃ for later use. (2) Measurement of physiological and biochemical indicators of mouse plasma After blood was drawn from the eyeball into an anticoagulant tube, it was centrifuged at 3500 r / min for 10 min, and the supernatant was collected. The changes in the levels of uric acid (UA), xanthine oxidase (XOD), creatinine (CRE), blood urea nitrogen (BUN), inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and catalase (CAT) in the plasma were detected according to the kit instructions. (3) Macroscopic images of liver and kidney tissues and their damage scores The liver and kidney tissues were laid flat on filter paper and photographed for recording. The scoring criteria for liver and kidney damage are shown in Tables 2 and 3 (the higher the score, the more severe the damage).

[0029] (4) Histopathological analysis of liver and kidney tissues Liver and kidney tissues were fixed with 4% paraformaldehyde for 24 h and then stained with hematoxylin and eosin (H&E Stain). (7) Data processing All data are expressed as mean ± standard deviation (Mean ± SD). The significance analysis between groups was performed using SPSS 18.0 data processing software. Experimental data were compared between groups using one-way ANOVA, and P < 0.05 was used as the criterion for statistical significance.

[0030] III. Animal Experiment Results 1. The results of the detection of body weight, uric acid, and xanthine oxidase levels in each group of mice are as follows: Figure 1-3 As shown: Figure 1 This describes the changes in body weight of mice in the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group, such as... Figure 1 As shown, the body weight of mice in each group increased with increasing drug administration time, but there was no statistically significant difference in body weight between the groups (P>0.05). Figure 2 These are the plasma uric acid levels in mice from the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Uric acid is an important indicator of hyperuricemia; it is produced by the liver and excreted by the kidneys. Insufficient uric acid excretion can cause kidney damage. Figure 2 As shown, the serum uric acid level of the model group mice (38.67 μmol / L) was significantly higher than that of the blank group mice (21.75 μmol / L), proving that the hyperuricemia model was successfully established. Compared with the model group mice, the plasma uric acid levels of mice in the red beer group, Huajuhong fermented liquid group and gout fermented liquid group were significantly reduced to 27.67 μmol / L, 33.60 μmol / L and 30.21 μmol / L, respectively. Among them, the plasma uric acid levels after intervention with Huajuhong beer and allopurinol (25.14 μmol / L) were similar, indicating that Huajuhong beer had a better uric acid-lowering effect. Figure 3 These are the results of plasma xanthine oxidase levels in mice from the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Xanthine oxidase (XOD) is a key enzyme in the conversion of xanthine and hypoxanthine into uric acid; high XOD activity leads to excessive uric acid synthesis. Figure 3As shown, compared with the blank group mice, the plasma XOD activity (65.16 U / L) of the model mice was significantly increased by 50.31%, indicating that the catalytic efficiency of uric acid metabolism enzymes in the model group mice was enhanced. Compared with the model group, Huajuhong beer, Huajuhong fermented liquid, and gout fermented liquid (i.e., Huajuhong compound fermented liquid that helps lower uric acid) all inhibited plasma XOD activity. Among them, the reduction in XOD level was most significant in the red beer group and the gout fermented liquid group, which was 51.71 U / L (P<0.05), similar to the allopurinol group. This suggests that the uric acid-lowering effect of Huajuhong beer and gout fermented liquid (i.e., Huajuhong compound fermented liquid that helps lower uric acid) may be due to the inhibitory effect on XOD level.

[0031] 2. The results of kidney injury detection in each group of mice are as follows: Figure 4-9 As shown: Figure 4-6 The images show optical images of kidney tissue, kidney index, and kidney injury scores of mice in different treatment groups. Figure 4 Optical images of kidney tissue from mice in the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group; Figure 5 The results show the kidney index of mice in the blank group, model group, red beer group, gout fermentation liquid group, tangerine peel fermentation liquid group and allopurinol group. Figure 6 The results show the kidney injury scores of mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. like Figure 4 As shown, the kidneys of the control group mice were generally dark red and shiny, while the kidneys of the model group mice were yellow and accompanied by obvious edema. In addition, compared with the control group mice, the kidney index of the model group mice was significantly higher after inducing hyperuricemia. Figure 5 ) and kidney injury score ( Figure 6The renal index (P>0.05) of hyperuricemia mice was significantly increased (P>0.05); however, after intervention with Huajuhong beer, Huajuhong fermented liquid, and gout fermented liquid (i.e., Huajuhong compound fermented liquid for assisting in lowering uric acid), the degree of kidney color change and edema in mice were reduced; in addition, all doses of Huajuhong beer, Huajuhong fermented liquid, and gout fermented liquid (i.e., Huajuhong compound fermented liquid for assisting in lowering uric acid) significantly reduced the renal index (P<0.05) and renal injury score (P>0.05) in hyperuricemic mice, especially gout fermented liquid and Huajuhong beer. These results indicate that oral administration of Huajuhong beer, Huajuhong fermented liquid, and gout fermented liquid (i.e., Huajuhong compound fermented liquid for assisting in lowering uric acid) significantly reduced the renal index (P<0.05) and renal injury score (P>0.05) in hyperuricemic mice. Allopurinol (a fermented tangerine peel compound) can reduce the kidney index and alleviate kidney damage in mice with hyperglycemia. It is worth noting that after intervention with allopurinol, the kidney tissue of mice turned white, and the kidney index and damage score increased. This indicates that allopurinol treatment cannot effectively alleviate kidney damage. On the contrary, it aggravates PO+HX-induced hyperuricemic kidney damage in mice. This is because during a gout attack, allopurinol prevents xanthine from being converted into uric acid in the body, thereby reducing the production of endogenous uric acid. However, the intermediate product xanthine, which is insoluble in urine, increases and is not easily excreted. Even a slight accumulation may lead to xanthine stones or xanthine nephropathy, thereby aggravating kidney damage. Figure 7 These are the H&E staining results of kidney tissue from mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group, such as... Figure 7 As shown, the kidneys of mice in the blank control group had normal structure, with clear and intact renal tubules and glomeruli, and no abnormalities were observed in the interstitium. In the model group, the renal tubular epithelial cells showed moderate vacuolar degeneration, with some tubular epithelial cells sloughing off, cell debris visible in the lumen, and extensive inflammatory cell infiltration in the renal interstitium. The boundaries between adjacent renal tubular cells were indistinct, with cell swelling and proximal tubular necrosis. In the red beer group, the renal tubular epithelial cells showed moderate vacuolar degeneration, and a small amount of inflammatory cell infiltration in the renal interstitium. In the Citrus reticulata fermentation broth group, the renal tubular epithelial cells... In the gout fermentation broth group, the renal tubular epithelial cells showed moderate vacuolar degeneration, with exposed renal tubular basement membranes and moderate inflammatory cell infiltration in the renal interstitium. In the gout fermentation broth group, the renal tubular epithelial cells showed moderate vacuolar degeneration, and the renal interstitium showed a small amount of inflammatory cell infiltration. The boundary between adjacent renal tubular cells was not obvious. The above results indicate that the gout fermentation broth (i.e., the Huajuhong compound fermentation broth that helps lower uric acid) and Huajuhong beer had the best effect in alleviating kidney damage in hyperuricemic mice. In the allopurinol group, the renal tubular epithelial cells showed severe vacuolar degeneration, some renal tubular epithelial cells sloughed off, and there was more inflammatory cell infiltration in the renal interstitium. Figure 8 These are the results of plasma creatinine levels in mice from the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Figure 9This data represents the plasma urea nitrogen levels in mice from the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Creatinine and urea nitrogen are key indicators for assessing renal function, as impairment is usually accompanied by elevated levels of these two indicators in plasma. Figure 8-9 As shown, compared with the blank group, the creatinine and urea nitrogen levels in the model group mice were significantly increased to 75.57 μmol / L and 10.87 mmol / L, respectively. Compared with the model group mice, after intervention with Huajuhong beer, Huajuhong fermentation broth, and gout fermentation broth, the plasma creatinine and urea nitrogen levels in mice were significantly reduced to 60.10 μmol / L, 71.89 μmol / L, 67.49 μmol / L and 8.90 μmol / L, 8.71 μmol / L, 8.64 mmol / L, respectively. These results indicate that Huajuhong beer has the most significant effect in reducing creatinine and urea nitrogen levels, while after allopurinol intervention, the plasma creatinine and urea nitrogen levels in mice increased instead of decreasing. In fact, allopurinol is mainly excreted through the kidneys. When a large dose of allopurinol is used, the drug concentration in the renal tubules increases to a toxic concentration, which can directly damage the renal tubular epithelial cells, thereby leading to renal function damage and increased creatinine and urea nitrogen levels.

[0032] 3. Results of oxidative stress levels in each group of mice are as follows: Figure 10 As shown: Figure 10 The results show the plasma levels of TNF-α (A), IL-1β (B), IL-6 (C), and CAT (D) in mice from the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group. Figure 10 AC represents the plasma levels of tumor necrosis factor TNF-α, interleukin IL-1β, and interleukin IL-6 in mice under different treatment groups. Hyperuricemia is considered an important factor closely related to kidney and liver failure. Notably, PO+HX treatment not only induced hyperuricemia but also triggered oxidative stress and related organ damage. The plasma levels of pro-inflammatory cytokines tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) are important indicators reflecting the degree of inflammation in lung tissue. like Figure 10As shown in AC, compared with the blank group, the plasma levels of TNF-α, TNF-1β, and IL-6 in the model group mice were significantly increased (P<0.05); compared with the model group mice, the plasma levels of TNF-α, TNF-1β, and IL-6 in the red beer group, Huajuhong fermented liquid group, and gout fermented liquid group were significantly decreased (P<0.05). Specifically, the plasma levels of TNF-α, TNF-1β, and IL-6 in the red beer group mice decreased by 12.86%, 27.13%, and 23.72%, respectively; the plasma levels of TNF-α, TNF-1β, and IL-6 in the Huajuhong fermented liquid group mice decreased by 20.54%, 14.96%, and 6.25%, respectively; and the gout group mice showed significantly decreased plasma levels of TNF-α, TNF-1β, and IL-6. In the fermentation broth group, the plasma levels of TNF-α, TNF-1β, and IL-6 in mice decreased by 21.95%, 13.82%, and 11.52%, respectively. These results indicate that Citrus reticulata beer, Citrus reticulata fermentation broth, and gout fermentation broth (i.e., Citrus reticulata compound fermentation broth for assisting in lowering uric acid) can significantly reduce the plasma levels of pro-inflammatory factors TNF-α, TNF-1β, and IL-6, suggesting that they can improve hyperuricemia in mice by regulating the inflammatory response. However, after allopurinol treatment, the inflammatory factors in the plasma of mice increased. In fact, during a gout attack, although the uric acid-lowering drug allopurinol can rapidly lower uric acid, it also promotes the dissolution of the surface of tophi in the joint, forming insoluble crystals and aggravating the inflammatory response. Figure 10 D represents the plasma catalase (CAT) levels in mice from different treatment groups. Excessive uric acid induces oxidative stress and causes oxidative damage. Catalase (CAT) is an antioxidant enzyme universally present in almost all organisms. Figure 10 As shown in Figure D, compared with the blank group mice, the plasma CAT level in the model group mice was significantly decreased (P<0.05); compared with the model group mice, the red beer, Huajuhong fermented liquid and gout fermented liquid groups all increased the plasma CAT content (P>0.05), indicating that the intervention of Huajuhong beer, Huajuhong fermented liquid and gout fermented liquid (i.e. Huajuhong compound fermented liquid that helps lower uric acid) has the potential to improve the body's oxidative stress homeostasis.

[0033] 4. Liver injury detection results for each group of mice are as follows: Figure 11-14 As shown: Figure 11-13 The images show optical images of liver tissue, liver index, and liver injury scores of mice in different treatment groups. Figure 11 Optical images of liver tissue from mice in the blank control group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group; Figure 12 The results show the liver index of mice in the blank group, model group, red beer group, gout fermentation liquid group, tangerine peel fermentation liquid group and allopurinol group. Figure 13The results show the liver injury scores of mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group and allopurinol group. like Figure 11 As shown, the livers of mice in the control group were bright red, smooth, and had sharp edges, with no obvious pathological changes; while the livers of mice in the model group were dark gray, with blunted edges and white punctate necrotic foci, and increased volume; in addition, compared with mice in the control group, after inducing hyperuricemia, the liver index of mice in the model group ( Figure 12 (P>0.05) and liver injury score ( Figure 13 The liver color of mice increased (P<0.05). However, although treatment with Huajuhong beer and Huajuhong fermented liquid slightly improved liver color, the traces of tissue necrosis and the degree of edema were still obvious. The area of ​​liver necrosis in the mice in the gout fermented liquid group was significantly reduced and the color was brighter than that in the model group. At the same time, all doses of Huajuhong beer, Huajuhong fermented liquid and gout fermented liquid (i.e., Huajuhong compound fermented liquid that helps lower uric acid) significantly reduced the liver index (P>0.05) and liver injury score (P<0.05) in hyperuricemic mice, especially the gout fermented liquid (i.e., the compound fermented liquid that helps lower uric acid). The results showed that oral administration of Huajuhong compound fermented liquid and Huajuhong beer could reduce the liver index and alleviate liver damage in mice with hyperglycemia. Notably, after intervention with allopurinol, the liver index and liver damage score of mice increased, indicating that allopurinol treatment did not effectively alleviate but aggravated PO+HX-induced hyperuricemic liver damage in mice. This is because during a gout attack, the intake of allopurinol increased the metabolic burden on the liver, thus causing drug damage to the liver. Figure 14 These are the H&E staining results of liver tissue from mice in the blank group, model group, red beer group, gout fermentation broth group, tangerine peel fermentation broth group, and allopurinol group, such as... Figure 14As shown, the livers of mice in the blank control group exhibited typical healthy liver lobule structures, with regularly round hepatocyte nuclei around the central vein and clearly discernible intercellular spaces. In contrast, the livers of mice in the model group showed significant pathological changes: hepatocyte swelling and necrosis, vacuolation, and cell boundary fusion. Although the red beer group mice showed some relief from liver damage, cell swelling and necrosis, as well as cell boundary fusion, persisted. Mice in the Huajuhong fermented liquid group still exhibited hepatocyte swelling, mild vacuolation, and cell boundary fusion, notably accompanied by a small amount of inflammatory factor infiltration. In contrast, the hepatocytes in the gout fermented liquid group gradually became more orderly, and cell boundary fusion was significantly reduced. These results indicate that the gout fermented liquid (i.e., the Huajuhong compound fermented liquid that helps lower uric acid) showed better liver protection in mice with high gout. The allopurinol intervention group also showed obvious cell boundary fusion, cell swelling and necrosis in liver tissue, which may be liver damage caused by drug metabolism in the liver.

[0034] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a compound fermentation broth of Citrus reticulata peel that helps lower uric acid, characterized in that, Includes the following steps: Step 1: Weigh out the following ingredients in proportion: Citrus reticulata peel, Perilla frutescens, Mulberry leaf, Poria cocos, Coix seed, Chicory root, Licorice, Kudzu root, Gardenia, and Lily bulb. Mix them together, pulverize them, and sieve them to obtain the raw material mixture. Step 2: Mix the raw material mixture with water, decoct, collect the filtrate, add water to the residue and continue to decoct, repeat three times, combine the three filtrates to obtain a compound extract; Cellulase and pectinase were added to the compound extract, and the mixture was sterilized to obtain the enzymatic hydrolysate. Step 3: Inoculate the plant lactobacillus strain and the Bacillus subtilis strain into the enzymatic hydrolysate and ferment to obtain the Huajuhong compound fermentation broth that helps lower uric acid.

2. The preparation method of the citrus reticulata compound fermentation broth for assisting in lowering uric acid according to claim 1, characterized in that, In step one, the components in the raw material mixture, by weight, are as follows: 2-4 parts of Citrus reticulata peel, 1.5-2 parts of Perilla frutescens, 3.5-4 parts of Mulberry leaf, 2-3 parts of Poria cocos, 3.5-4 parts of Coix lacryma-jobi, 4-6 parts of Chicory root, 1-1.5 parts of Glycyrrhiza uralensis, 3.5-4 parts of Pueraria lobata, 2-3 parts of Gardenia jasminoides, and 2-3 parts of Lilium brownii.

3. The preparation method of the citrus reticulata compound fermentation broth for assisting in lowering uric acid according to claim 1, characterized in that, In step two, during the preparation of the compound extract, the mass ratio of the raw material mixture to water is 1:10, the mass ratio of the filter residue to water is 1:10, and the decoction time is 30 minutes each time.

4. The preparation method of the citrus reticulata compound fermentation broth for assisting in lowering uric acid according to claim 1, characterized in that, In step two, the cellulase has an enzyme activity of 20,000 U / g and is used at a rate of 4-6 wt% of the raw material mixture. The pectinase has an enzyme activity of 20,000 U / g and is used at a rate of 2-3 wt% of the raw material mixture.

5. The preparation method of the citrus reticulata compound fermentation broth for assisting in lowering uric acid according to claim 1, characterized in that, In step two, the enzymatic hydrolysis conditions are: temperature 55 ℃ and hydrolysis time 5 h.

6. The preparation method of the citrus reticulata compound fermentation broth for assisting in lowering uric acid according to claim 1, characterized in that, In step three, the concentration of the Bacillus subtilis strain is 1.0 × 10⁻⁶. 8 -1.5×10 8 The inoculum concentration was 6-8% (cfu / mL); the concentration of the *Lactobacillus plantarum* strain was 1.0 × 10⁻⁶. 8 -1.5×10 8 cfu / mL, inoculation amount is 3-5%.

7. The preparation method of the citrus reticulata compound fermentation broth for assisting in lowering uric acid according to claim 1, characterized in that, In step three, the fermentation conditions are as follows: fermentation for 3 days at a temperature of 37 ℃ and a stirring speed of 160 rpm.

8. A compound fermentation broth for assisting in lowering uric acid, prepared by the method described in any one of claims 1-7.

9. The citrus reticulata-based compound fermentation broth for assisting in lowering uric acid according to claim 8, characterized in that, The compound fermentation liquid of Huajuhong, which helps lower uric acid, is used to prepare Huajuhong beer.

10. The application according to claim 9, characterized in that, The preparation method of the orange-red beer includes the following steps: Step S1: Mix malt and rice at a mass ratio of 7.5:2.5 to obtain brewing raw materials; mix the brewing raw materials with water at a material-to-water ratio of 1:4, add the raw materials at 35 ℃, raise the temperature to 53 ℃ after 40 min and hold for 30 min, then slowly raise the temperature to 62 ℃, raise the temperature to 68 ℃ after 60 min and hold for 15 min, until saccharification is complete as detected by iodine solution, and obtain wort; Step S2: Take the wort obtained in Step 1 and boil it for 90 minutes. During the boiling process, add 0.01 wt% bitter hops, 0.03 wt% bitter hops, and 0.03 wt% aroma hops at the 15th, 45th, and 75th minutes of the total wort volume, respectively. After boiling, remove the hops and heat-coagulated material, then steam sterilize at 115 °C for 20 minutes and cool to obtain the brewing fermentation stock solution. Step S3: Mix the brewing fermentation liquid and the Huajuhong compound fermentation liquid at a mass ratio of 100:(16-24), cool to 7°C, inoculate with activated dry yeast at a rate of 1 g / L, and aerate the mixture to achieve an oxygen content of 8 mg / L. Set the fermentation temperature to 25°C. After fermentation for 24 hours, when the sugar content drops below 5 degrees, control the fermentation pressure to 0.15 MPa. Set the temperature to 0°C on day 11 and -1°C on day 13. Brewing is completed on day 14. On day 15, remove the lees to obtain Huajuhong beer.

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