A method for preparing a three-stage herbal fermentation-assisted alcoholism-relieving probiotic product

Abstract

This invention discloses a three-stage herbal fermentation method for preparing probiotic products with auxiliary effects in relieving hangovers and protecting against chemically induced liver damage. The method is based on a compound water extract of kudzu root, Japanese raisin tree fruit, turmeric, poria cocos, and licorice root, combined with black tea extract, along with sugar sources, fruit juice, and functional microorganisms. It employs a three-stage fermentation process: anaerobic-aerobic-low-temperature live bacteria fermentation. The first stage involves anaerobic yeast fermentation to generate flavor precursors; the second stage involves aerobic fermentation with acetic acid bacteria to regulate acidity and pasteurize and inactivate the precursor bacteria; the third stage involves low-temperature inoculation with probiotics for post-fermentation. The product can be further processed into a liquid beverage or freeze-dried powder. The resulting product has a harmonious flavor, high live bacteria content, and a live bacteria count ≥1×10⁻⁶ at 4℃. 8 It has the effects of assisting in sobering up, relieving discomfort after drinking, assisting in protecting against chemical liver damage, protecting the stomach and regulating the intestines.

Description

Technical Field

[0001] This invention relates to a three-stage herbal fermentation method for preparing probiotic products that aid in hangover relief, belonging to the field of food biotechnology and functional fermented beverages. Background Technology

[0002] Excessive alcohol consumption leads to the accumulation of ethanol and acetaldehyde in the body, causing discomfort such as dizziness, nausea, and vomiting. It can also cause oxidative stress in liver cells, manifested as significantly elevated serum ALT and AST levels, decreased SOD and GSH-Px activity, and increased MDA content. To address the discomfort and related liver damage risks associated with alcohol consumption, various hangover remedies have emerged on the market. However, most are single plant extracts or simple compound formulations, and generally suffer from low utilization rates of functional ingredients, bitter taste, and poor stability of live bacteria.

[0003] In recent years, multi-stage fermentation has been applied in products such as kombucha and fruit vinegar, but there are no reports of using a three-stage fermentation process—yeast → acetic acid bacteria → probiotics—to create a hangover remedy. In particular, there is a lack of technical solutions for mild pasteurization to inactivate the preceding microorganisms after the second stage of fermentation, followed by low-temperature post-fermentation with probiotics. This makes it difficult to simultaneously achieve flavor depth, functional component stability, and live bacteria activity. The main challenges include antagonistic effects among multiple microbial strains within the same system, rapid probiotic deterioration during storage, insufficient live bacteria counts, inability to segmentally and directionally regulate the generation of organic acids and functional products during fermentation, and limited product functionality—only relieving hangover symptoms and failing to simultaneously leverage the gut-liver axis for intestinal regulation and adjuvant protection against chemically induced liver damage.

[0004] Therefore, there is an urgent need for a preparation method that can systematically integrate the efficacy of herbal medicine, black tea polyphenols, fruit juice nutrition and the metabolic advantages of multiple microbial strains, and achieve synergistic enhancement of product function and sensory appeal through precise control of the fermentation stage. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a three-stage fermentation method for preparing a probiotic herbal liquid beverage with the functions of assisting in sobering up and protecting against chemically induced liver damage. By introducing different functional microorganisms in stages, flavor construction, acidity adjustment and live bacteria colonization are completed in sequence, and finally a functional beverage with a harmonious flavor, rich in active probiotics and with the potential to assist in sobering up, relieving post-drinking discomfort and protecting against chemically induced liver damage is obtained.

[0006] To address the aforementioned technical problems, this invention provides a method for preparing a herbal probiotic fermentation product, comprising the following steps: (1) The herbal raw materials are soaked in water and separated to obtain herbal extract; (2) Extract the black tea raw material in hot water and quickly cool it to obtain black tea extract; (3) Mix the extracts obtained in steps (1) and (2) and use white sugar as the sugar source as the fermentation base liquid; (4) Inoculate the fermentation liquid with the yeast strain CICC30089 and carry out the first stage of anaerobic fermentation. (5) Inoculate the fermentation base liquid with the preservation number CICC7010 of Acetobacter pasteurellium to carry out the second stage of aerobic fermentation. After the fermentation is completed, sterilize the fermentation liquid. (6) Inoculate the fermentation base liquid with Lactobacillus plantarum (CCICC25283), Lactobacillus acidophilus (CCICC6081), and Bifidobacterium longum (DSM23233) for the third stage of deep fermentation to obtain the herbal probiotic fermentation product.

[0007] This invention combines herbal formulation, extraction, microbial fermentation, and probiotics. Extraction effectively extracts various nutrients and active substances from the raw materials, providing essential material support for subsequent steps. Microbial fermentation produces unique aromas and acidic substances, enriching the product's flavor. Simultaneously, microbial fermentation produces cellulase and protease, which break down large molecules in the raw materials, such as proteins and polysaccharides, into smaller molecules that are more easily absorbed by the human body, thereby improving the product's nutritional value and its protective effect against chemically induced liver damage.

[0008] Acetic acid bacteria are used to oxidize the ethanol produced by yeast into acetic acid under aerobic conditions, thereby adjusting the total acidity of the product, creating a refreshing and slightly acidic taste, inhibiting contamination by other microorganisms, and extending shelf life.

[0009] Acetic acid bacteria and yeast ferment together, secreting extracellular enzymes such as proteases and cellulases, which promote the release of active ingredients such as puerarin, flavonoids and polyphenols in herbal raw materials and improve the utilization rate of functional substances.

[0010] In some embodiments, the herbal ingredients include kudzu root, Japanese raisin tree fruit, and one or more of turmeric, poria cocos, and licorice.

[0011] Furthermore, the raw material components and their mass parts are 10-15 parts of kudzu root, 10-15 parts of Japanese raisin tree fruit, 3-5 parts of turmeric, 6-10 parts of poria cocos, and 3-5 parts of licorice, and the mass of purified water added is 5 times the total mass of all herbal raw materials.

[0012] The raw materials for the black tea include 10-20g of black tea per 100mL of water and 50-100g of white sugar per L.

[0013] The preferred raw material components and their weight parts are 10 parts kudzu root, 10 parts Japanese raisin tree fruit, 3 parts turmeric, 8 parts poria cocos, and 4 parts licorice. The black tea raw material includes 10g of black tea per 100mL of water and 50-100g of white sugar per L.

[0014] The raw materials are selected from medicinal and edible materials to avoid the metabolic burden on the body. At the same time, the effects of kudzu root, Japanese raisin tree fruit, turmeric, poria cocos, and licorice work synergistically, and the components work together to make the final product have good effects in helping to relieve hangovers, alleviate discomfort after drinking, and help protect against chemical liver damage.

[0015] The core functions of the raw material, kudzu root, include relieving muscle tension and fever, promoting the upward movement of clear yang energy, detoxifying alcohol, and protecting the stomach. Kudzu root specifically targets the spleen and stomach, enabling it to promote the upward movement of clear yang energy and dissipate dampness from alcohol, thus alleviating headaches, thirst, and nausea after drinking. It is rich in puerarin, which can increase the activity of liver ADH (alcohol dehydrogenase) and ALDH (acetaldehyde dehydrogenase).

[0016] The core functions of the raw material, Hovenia dulcis, are to quench thirst and relieve irritability, clear damp heat, detoxify alcohol, and promote urination. It specifically clears dampness and detoxifies alcohol, expelling toxins from the body through urination. It also promotes acetaldehyde metabolism, reduces liver damage, and alleviates post-drinking dizziness, irritability, and dry mouth.

[0017] The core function of the raw material turmeric is to promote blood circulation, regulate qi, relieve pain, and address the issue that alcohol is considered a damp-heat evil that, with prolonged consumption, damages the liver and causes qi and blood stagnation. Turmeric can soothe the liver, promote bile secretion, and invigorate blood circulation. It also has an auxiliary protective effect against chemically induced liver damage, preventing alcohol-induced liver injury and compensating for the deficiency of kudzu root and Japanese raisin tree fruit in that they "only relieve alcohol but do not protect the liver."

[0018] The core functions of the raw material Poria cocos are to strengthen the spleen and eliminate dampness, calm the mind and soothe the nerves, and promote diuresis and reduce swelling. Alcohol-induced dampness is most harmful to the spleen; spleen deficiency leads to dampness, dizziness, fatigue, and nausea. Poria cocos strengthens the spleen and eliminates dampness, thus removing alcohol-induced dampness from the body.

[0019] The core functions of licorice, the main ingredient, are to invigorate qi and replenish the middle jiao, clear heat and detoxify, harmonize the effects of other herbs, moderate their properties, prevent excessive dispersion of kudzu root and turmeric, and protect stomach qi. It also has a certain liver-protective effect and can alleviate stomach irritation after drinking alcohol.

[0020] Further, weigh out kudzu root, Japanese raisin tree fruit, turmeric, poria cocos, and licorice root, mix and grind them according to the specified ratio, add 5 times the amount of purified water, continue heating and extracting for 1 hour, and then use after coarse filtration through a filter cloth. Furthermore, the black tea extract is prepared by steeping black tea in hot water at 90-95°C for 10-15 minutes and then cooling before use.

[0021] Furthermore, the black tea extract was rapidly separated into tea liquor, and the cooling condition was to reduce the temperature to below 10°C within 10 minutes.

[0022] Furthermore, after mixing the medicinal and edible extract and the black tea extract, sugar is added for further preparation. The sugar includes, but is not limited to, sucrose, and the amount of sugar added is 5-10% of the total mass of the fermentation liquid.

[0023] In some embodiments, in step (3), the volume ratio of the herbal extract is 20% and the volume ratio of the black tea extract is 10%.

[0024] In some embodiments, in step (4), the brewing yeast is inoculated using a liquid seed culture obtained through stepwise expansion, and the viable cell concentration of the seed culture is ≥1×10⁻⁶. 8 CFU / mL, the seed liquid is added at a volume of 5% to 10% of the total volume of the main fermentation broth, and fermented in a sealed environment at a temperature of 18 to 22°C for 4 to 7 days.

[0025] Furthermore, the yeast is *Saccharomyces cerevisiae*.

[0026] In the first stage of anaerobic fermentation, yeast can utilize the sugar source in the fermentation base liquid to produce ethanol and flavor precursors such as esters and alcohols. At the same time, it secretes a variety of hydrolytic enzymes to promote the release of active ingredients from the herbs, improve the utilization rate of functional substances, and provide substrates for subsequent acetic acid bacteria fermentation.

[0027] In some embodiments, step (5) involves aseptically inoculating acetic acid bacteria into the fermentation broth. The acetic acid bacteria are prepared into a liquid seed culture using a stepwise aerobic expansion method, with continuous aeration throughout to ensure dissolved oxygen. The viable bacterial concentration in the mature seed culture is ≥1×10⁻⁶. 8 CFU / mL, the seed liquid is added at 5% to 10% of the total volume of the main fermentation broth, and fermented at 28 to 32°C, 100 to 200 rpm, with oxygenation for 3 to 5 days.

[0028] Furthermore, the acetic acid bacillus is Pasteurella multocida.

[0029] In the second stage of aerobic fermentation, acetic acid bacteria oxidize ethanol to acetic acid, adjusting the product's acidity, improving flavor, and inhibiting unwanted microorganisms, while further promoting the dissolution and stability of functional components. The step-by-step fermentation by yeast and acetic acid bacteria achieves a synergistic effect in flavor development, functional enhancement, and quality stability.

[0030] The second stage of acetic acid bacteria aerobic fermentation is a strictly aerobic process. During fermentation, the dissolved oxygen concentration is controlled at 15% to 30% saturation (DO), preferably 20% ± 5%. The dissolved oxygen is maintained by adjusting the aeration rate to 0.05 to 0.20 vvm and the stirring speed to 180 to 220 rpm. The temperature is controlled at 28 to 32℃. Fermentation continues until the pH reaches 4.5, after which pasteurization is carried out.

[0031] Furthermore, after the second stage of aerobic fermentation is completed, pasteurization is carried out under the following conditions: temperature 65-70℃, time 20-30min. After pasteurization, the temperature is rapidly cooled to 20-25℃ for later use.

[0032] In some embodiments, in step (6), *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Bifidobacterium longum* are prepared into seed cultures through anaerobic expansion, and the viable bacterial concentration of each strain in the seed culture is ≥1×10⁻⁶. 8The inoculum concentration was 5%–10% CFU / mL, the fermentation temperature was 20–25℃, and the fermentation time was 2–3 days.

[0033] In some implementations, step (6) also includes fruit juice.

[0034] Further, aseptic fruit juice is added to the cooled aseptic liquid at a rate of 5%–10% of the final product volume, and the mixture is stirred and mixed. Then, probiotic seed liquid is inoculated at a rate of 5%–10% (v / v), and the mixture is subjected to low-temperature anaerobic fermentation at 20–25°C for 48–72 hours to allow the probiotics to colonize and proliferate. After fermentation, the mixture is aseptically bottled to obtain a probiotic herbal liquid beverage with the effects of assisting in the relief of hangovers and assisting in the protection against chemically induced liver damage.

[0035] Furthermore, the juice may include one or more of apple juice, kiwi juice, pear juice, and mango juice.

[0036] The fruit juice can replenish nutrients and energy. It is rich in various vitamins, organic acids, minerals and natural glycogen, which can quickly replenish the body's energy, relieve discomfort such as fatigue, hypoglycemia and dizziness after drinking, and synergistically enhance the body's recovery effect.

[0037] The juice can adjust the taste and flavor, neutralize the bitterness of the herbs and the sourness of fermentation, give the product a natural fruity aroma, make the overall taste harmonious in sweet and sour, refreshing and palatable, and significantly improve the drinking experience and acceptance.

[0038] The juice can optimize the fermentation environment, provide natural carbon sources and trace elements needed for the growth of probiotics, promote the colonization and proliferation of probiotics, and increase the number of live bacteria and product stability.

[0039] Furthermore, after fermentation, the fermentation liquid is cooled to 4–10°C to maintain the activity of probiotics and functional components.

[0040] Furthermore, the probiotics are Lactobacillus plantarum, Lactobacillus acidophilus, and Bifidobacterium longum.

[0041] During the third stage of fermentation, the probiotics colonize and proliferate, forming a highly active live bacteria system with a live bacteria count ≥1×10⁻⁶ at 4℃. 8 CFU / mL, once it enters the human body, can regulate the balance of intestinal flora, help maintain the intestinal barrier, and improve intestinal digestive and absorptive functions.

[0042] The probiotics metabolize to produce prebiotics, including short-chain fatty acids (acetic acid, propionic acid, butyric acid), extracellular polysaccharides, soluble peptides, etc., which can provide nutritional substrates for beneficial bacteria in the intestines and enhance the body's antioxidant and immune regulation capabilities.

[0043] During the third stage of fermentation, the probiotics continue to transform the functional components of the herbs. Through the hydrolytic enzyme system such as β-glucosidase and esterase secreted by the bacteria, they biotransform the flavonoid glycosides and flavonoid precursors in kudzu root, Japanese raisin tree fruit, and turmeric, generating more easily absorbed and more active flavonoid aglycones and secondary metabolites. This further enhances the effects of assisting in hangover relief, assisting in protecting against chemical liver damage, and anti-oxidation, making the product's functional effects more significant and its effects longer-lasting.

[0044] Fruit juice can replenish vitamins, glycogen and other healthy components, and quickly relieve fatigue after drinking; at the same time, it can adjust the sweet and sour taste, mask the bitterness of herbs, and provide nutrients for the proliferation of probiotics, thus achieving a dual improvement in function and sensory experience.

[0045] Furthermore, step (6) includes a filtration and impurity removal step after fermentation.

[0046] Furthermore, after the third stage of low-temperature probiotic fermentation is completed, the fermentation liquid is subjected to low-temperature freeze-drying to prepare a probiotic powder that helps with hangover relief and protects against chemically induced liver damage.

[0047] Freeze-drying removes moisture under low-temperature, vacuum, and anhydrous conditions, maximizing the preservation of probiotic cell activity and avoiding damage to live bacteria caused by high-temperature drying, allowing the product to be stored stably at room temperature for extended periods. Simultaneously, freeze-drying completely preserves the flavonoids, short-chain fatty acids, extracellular polysaccharides, and other functional substances in the fermentation system, preventing degradation and loss of these components. The resulting powder has a high live bacteria content, good stability, and is easy to carry, and can be further processed into various dosage forms such as solid beverages, compressed candies, and capsules, expanding its application scenarios.

[0048] The second technical solution provided by the present invention is a herbal probiotic fermented product prepared using the method described in the first technical solution.

[0049] The resulting fermented product has a viable cell count ≥1×10⁻⁶ at 4℃. 8 CFU / mL, shelf life ≥14 days.

[0050] In some embodiments, the fermented product is a beverage, powder, or tablet. The effects corresponding to the above-mentioned technical advantages cannot be achieved through simple raw material compounding or single-stage fermentation, resulting in unique and unexpected technical effects from the combined process of segmented + pasteurization isolation of this invention.

[0051] The third technical solution provided by the present invention is the application of the fermented product described in the second technical solution in the preparation of hangover remedies.

[0052] The fourth technical solution provided by the present invention is the application of the fermented product described in the second technical solution in the preparation of drugs or health products that alleviate and / or reduce chemical liver damage.

[0053] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The three-stage process synergistically enhances efficiency, overcoming the technical obstacle of mutual inhibition among multiple bacterial species.

[0054] The first stage involves yeast fermentation to produce ethanol and flavor precursors such as pyrazines and esters, and promotes the release of functional components. The second stage involves acetic acid bacteria oxidizing ethanol to acetic acid, which regulates acidity, improves taste, inhibits unwanted bacteria, reduces irritation, and enhances freshness. The third stage involves low-temperature inoculation of probiotics to avoid high-temperature inactivation and ensure that the final product contains highly active probiotics. This achieves three-stage fermentation with each strain working in succession without interference and with synergistic effects.

[0055] 2. Biotransformation of functional components yields unexpected synergistic effects.

[0056] The hydrolytic enzymes produced by multi-strain fermentation convert flavonoid glycosides into aglycones that are more easily absorbed and have stronger activity. The effects of assisting in the detoxification of alcohol and protecting against chemical liver damage are significantly better than those of the same formula in physical mixtures. This is a technical effect that cannot be expected with a single raw material or simple compounding.

[0057] 3. Overcome the technical obstacles of herbal beverages being bitter, unpleasant to drink, and difficult to industrialize. By using yeast to produce aroma, acetic acid to adjust acidity, and fruit juice to correct flavor, the bitterness of the herbs is significantly neutralized, resulting in a sweet and refreshing taste with a harmonious blend of fruit and herbal aromas. This achieves simultaneous improvement in efficacy and taste, addressing the industry pain point of "effective but unpalatable, palatable but ineffective." It also achieves synergistic regulation of the liver, intestines, and stomach, with unexpectedly comprehensive functional benefits.

[0058] The product not only accelerates ethanol metabolism and relieves post-drinking discomfort, but also regulates gut microbiota, helps maintain the intestinal barrier, and helps protect against chemical liver damage, forming a synergistic protection of the liver-gut axis. Its comprehensive functions are significantly superior to existing single hangover remedies.

[0059] 5. High viable bacteria retention rate solves the industry problem of low viable bacteria survival rate in fermented beverages. Through staged fermentation and sterilization isolation, the viable bacterial count of the product is kept stable at ≥1×10⁻⁶ at 4℃. 8 With a concentration of CFU / mL, probiotics can colonize efficiently, possessing both functional activity and food shelf stability, and have good prospects for industrialization. Attached Figure Description

[0060] Figure 1 This is a process flow diagram of the method described in this invention. Detailed Implementation

[0061] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0062] Raw materials used in the examples: In all the following examples, kudzu root, Japanese raisin tree fruit, turmeric, poria cocos, and licorice were purchased from the JD.com Guoyao Medicinal Herbs Health JD.com self-operated flagship store. The quality of the medicinal materials met the relevant standards for food and medicine homology, the batches were consistent, and the performance fluctuations were controllable.

[0063] In all the following examples, the black tea used is food-grade Qimen black tea, free from mold and aging.

[0064] In all the examples below, the concentrated fruit juice is UHT ultra-high temperature instantaneous sterilized concentrated juice, which does not contain artificial flavors, colorings, or preservatives, and the soluble solids are fixed at 70 Brix.

[0065] In all the examples below, the white sugar is commercially available Grade 1 white sugar with a sucrose purity of ≥99.5% and no additional excipients are added.

[0066] The strains in the following examples Example 1 Herbal extract: 10g kudzu root, 10g Japanese raisin tree fruit, 3g turmeric, 6g poria cocos, and 3g licorice root are mixed and pulverized, then 160mL of purified water is added and extracted at 95℃ for 1.5h, followed by filtration.

[0067] Black tea extract: Add 10g of black tea to 100mL of 95℃ hot water, steep for 12 minutes, quickly cool to 8℃, and filter.

[0068] Fermentation base liquid: 200mL herbal extract + 100mL black tea extract + 50g white sugar, bring the volume to 1000mL, sterilize at 100℃ for 15min, and cool to room temperature.

[0069] The first stage of anaerobic fermentation: inoculate with liquid seed culture of brewer's yeast, adding 7% of the total volume of the main fermentation liquid, and anaerobic ferment at 20℃ for 120 hours.

[0070] The second stage is aerobic fermentation: inoculate with liquid seed culture of Acetobacter pasteurization at a ratio of 10% (v / v), ferment at 30°C, DO 20%, aeration 0.1 vvm, stirring at 200 rpm, for 72 h until pH 4.5; pasteurize at 68°C for 20 min, and rapidly cool to 22°C.

[0071] Third stage post-fermentation: Add 10% (v / v) sterile apple juice, and inoculate with 5% (v / v) liquid seed liquid of Lactobacillus plantarum + Lactobacillus acidophilus + Bifidobacterium longum, and anaerobic ferment at 22℃ for 48h; aseptically fill and store at 4℃.

[0072] Product Specifications: Live bacteria count at 4℃ 1×10⁻⁶ 8 With a CFU / mL concentration, it has a refreshing sweet and sour taste and is effective in helping to relieve hangovers and protect against chemically induced liver damage.

[0073] Example 2 The three-stage fermentation was completed according to the steps in Example 1. The fermentation broth was pre-frozen at -40°C for 12 hours, then transferred to a vacuum freeze dryer with a vacuum degree of 15 Pa and a cold trap temperature of -45°C for 36 hours to obtain a light yellow loose powder.

[0074] Product Specifications: Live Bacteria Count ≥ 5 × 10⁻⁵ 8 CFU / g, viable bacteria survival rate ≥85% after 6 months of sealed storage at room temperature.

[0075] Experimental Example 1: Determination of Total Flavonoid Content in a Three-Stage Fermentation Process Experimental methods: Samples of herbal extract (unfermented), after the first stage of yeast fermentation, after the second stage of acetic acid bacteria fermentation, and after the third stage of probiotic fermentation were taken respectively. The total flavonoid content was determined by sodium nitrite-aluminum nitrate spectrophotometry, with rutin as the reference standard. The detection wavelength was 510 nm. The results are expressed as mg / mL.

[0076] The changes in total flavonoid content during the three-stage fermentation process are shown in the table below: Table 1. Changes in total flavonoid content during fermentation.

[0077] Compared with unfermented herbal extracts, the total flavonoid content increased significantly after three-stage fermentation, with an increase of 234%.

[0078] The first stage of yeast fermentation involves the secretion of hydrolytic enzymes, which initially break down plant cell walls, resulting in a significant increase in the release of flavonoids.

[0079] The second stage of acetic acid bacteria fermentation further promotes the dissociation of bound flavonoids, resulting in a continuous increase in total flavonoids.

[0080] The third stage of probiotic fermentation is the most significant, where the β-glucosidase and esterase secreted by probiotics efficiently convert flavonoid glycosides into more easily absorbed and more active flavonoid aglycones, achieving a simultaneous increase in total flavonoid content and bioactivity.

[0081] The three-stage fermentation process of this invention can significantly improve the dissolution and conversion efficiency of flavonoid active ingredients, resulting in a substantial increase in the total flavonoid content. This synergistic effect cannot be achieved by a single component or simple mixing, providing a clear material basis for the product's ability to assist in detoxification and protect against chemical liver damage.

[0082] Experimental Example 2: Determination of Total Polyphenol Content During Three-Stage Fermentation Experimental methods: Samples were taken separately: the original liquid (mixed extract of herbal medicine and black tea, unfermented), the liquid from the first stage of anaerobic fermentation with yeast, the liquid from the second stage of aerobic fermentation with acetic acid bacteria, and the liquid from the third stage of low-temperature fermentation with probiotics. The total polyphenol content was determined by the Folin-Ciocalteu method and standardized with gallic acid. The unit is mg / mL.

[0083] The changes in total polyphenol content during the three-stage fermentation process are shown in the table below: Table 2. Changes in total polyphenol content during fermentation.

[0084] Compared with the initial extract, the total polyphenol content of the product increased by 181.0% after the complete three-stage fermentation. Extracellular enzymes in yeast disrupt the dense cell wall structure of plants, promoting the release of polyphenols, resulting in a significant increase in polyphenols during the first stage of fermentation. Acetic acid bacteria metabolize and produce acid, optimizing the system environment and further promoting the dissolution of bound polyphenols. In the third stage, probiotics secrete esterases and glycosidases, hydrolyzing large-molecule bound polyphenols into small-molecule active polyphenols, which is the core reason for the highest increase in polyphenols in this stage.

[0085] After stepwise biotransformation, black tea polyphenols and herbal polyphenols synergistically enhance each other's antioxidant capacity, thereby strengthening the product's role in protecting against chemically induced liver damage.

[0086] The gradual and continuous increase in total polyphenols is a process gain effect that cannot be achieved by simple physical compounding of herbal raw materials, and is the technical effect produced by the synergistic effect of the three-stage fermentation of this invention.

[0087] Experimental Example 3: Detection of Total Acidity and Organic Acid Content during Three-Stage Fermentation Experimental methods: Samples were collected in the following stages: unfermented stock solution, first-stage yeast fermentation broth, second-stage acetic acid bacteria fermentation broth, and third-stage finished product solution.

[0088] The total acidity (as acetic acid, %) was determined by potentiometric titration. The specific operation should refer to GB12456-2021 "National Food Safety Standard - Determination of Total Acidity in Food".

[0089] Lactic acid and citric acid content (mg / 100mL) were determined by high-performance liquid chromatography (HPLC). Chromatographic column: C18 column (250 mm × 4.6 mm, 5 µm) or equivalent column; mobile phase: methanol:potassium dihydrogen phosphate buffer = 5:95; flow rate: 1.0 mL / min; column temperature: 35℃; wavelength: 210 nm; injection volume: 20 µL. The sample was filtered through a 0.45 μm aqueous microporous membrane for later use, and quantification was performed using the external standard calibration curve method.

[0090] Acetic acid content (mg / 100mL) was determined by gas chromatography (FID). Capillary column: CP-Wax 57CB (CP-97723A) capillary column (50m length, 0.25mm inner diameter, 0.2µm coating); Carrier gas (high-purity nitrogen): flow rate 1.0mL / min, split ratio: 50:1, tail purge 20mL / min, injection volume: 1μL; Hydrogen: flow rate 40mL / min; Air: flow rate 300mL / min; Detector temperature: 270℃; Injector temperature: 220℃; Column temperature: initial temperature 60℃, constant temperature for 3 min, then programmed to increase to 180℃ at 3.5℃ / min, and constant temperature for another 10 min. Amyl acetate solution (2% v / v) was used as the internal standard.

[0091] Table 3 Total acidity and organic acid content of the three segments

[0092] The first stage of yeast fermentation produces a small amount of ethanol and trace amounts of organic acids, resulting in a slight increase in total acidity. The second stage of Pasteurella acetic acid bacteria aerobically oxidizes ethanol to produce a large amount of acetic acid, precisely increasing the total acidity to the suitable range for the product, thus solving the drawbacks of the original herbal liquid's acid-astringency imbalance and bland taste. The third stage involves the low-temperature metabolism of probiotics to enrich lactic acid, which moderately adjusts the acidity. Combined with the natural citric acid in the fruit juice, this creates a flavor system with a harmonious balance of sweet and sour. The total acidity of the finished product remains stable in the range of 0.5% to 0.6%, which can inhibit the growth of unwanted bacteria and extend shelf life without being too acidic and irritating to the stomach and intestines.

[0093] Test Example 4: Sensory Quality Evaluation Test of Products Twenty tasters with no olfactory or gustatory impairments were selected, with a balanced gender ratio. A blind taste test was conducted, with a maximum score of 100 points, weighted by four dimensions: color 25 points, aroma 25 points, taste 35 points, and palatability 15 points, as shown in the table below.

[0094]

[0095] Sample preparation: Place at room temperature in the dark for 30 minutes, then dispense equal amounts into transparent blind sample cups and number them randomly. A control group was set up: a physical mixture of herbs, black tea, and fruit juice in equal proportions, without fermentation. The experimental group consisted of the three-stage fermented product of this invention.

[0096] Table 4: Sensory rating results

[0097] The unfermented control group had a distinct herbal bitterness and strong medicinal taste, resulting in poor palatability. After three stages of fermentation, the yeast produced alcohols and esters, acetic acid neutralized the spiciness and astringency, and the fruit juice masked the medicinal bitterness, forming a complex aroma of fruit, herbal, and fermented sourness.

[0098] The optimized fermentation system produces a clear and uniform color, free from turbidity and sediment, with a balanced sweet and sour ratio, addressing the industrialization shortcomings of traditional herbal beverages that are bitter and difficult to drink.

[0099] Simply relying on compound raw materials cannot improve the inherent bitterness of herbal beverages. This invention relies on three-stage step-by-step fermentation to synergistically optimize flavor, achieving simultaneous improvement in efficacy and palatability, and overcoming the industry's technical barriers to the commercialization of herbal beverages due to their unpleasant taste.

[0100] Experimental Example 5: Behavioral Indicators for Auxiliary Alcohol Detoxification Eighty SPF-grade male Kunming mice, aged 6-8 weeks and with an initial weight of 18-20g, were selected and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were acclimatized for 3 days and fasted for 12 hours before the experiment but allowed free access to water.

[0101] The animals were randomly divided into four groups of 20 each: control group 1 and 2, and example group 1 and 2.

[0102] The control group was given an equal dose of pure water by gavage, followed by 42% vol baijiu (0.25 mL / 10 g) by gavage 30 minutes later.

[0103] The control group was given an equal dose of the fermentation base liquid prepared in step three by gavage, and 30 minutes later, 42% vol baijiu (0.25 mL / 10 g) was administered by gavage.

[0104] In Examples 1 and 2, the probiotic products of the corresponding examples were administered by gavage at the same dose, followed by gavage administration of 42% vol baijiu (0.25 mL / 10 g) 30 minutes later.

[0105] Observe mouse behavioral indicators The time of intoxication (time from alcohol administration to the disappearance of the righting reflex) and the time of recovery (time from the disappearance of the righting reflex to the recovery of the righting reflex) of each group of mice were recorded. The results are shown in Table 5.

[0106] Table 5. Effects of the product on behavioral indicators of intoxicated mice.

[0107] As shown in Table 5, the experimental group that took the product of this embodiment had a longer intoxication latency period and a shorter intoxication time than the control group, indicating a significant auxiliary effect in relieving hangovers.

[0108] Experimental Example 6: Effects of Blood Ethanol Metabolism Eighty SPF-grade male Kunming mice, aged 6-8 weeks and with an initial weight of 18-20g, were selected and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were acclimatized for 3 days and fasted for 12 hours before the experiment but allowed free access to water.

[0109] The animals were randomly divided into four groups of 20 each: control group 1 and 2, and example group 1 and 2.

[0110] The control group was given an equal dose of pure water by gavage, followed by 42% vol baijiu (0.25 mL / 10 g) by gavage 30 minutes later.

[0111] The control group was given an equal dose of the fermentation base liquid prepared in step three by gavage, and 30 minutes later, 42% vol baijiu (0.25 mL / 10 g) was administered by gavage.

[0112] In Examples 1 and 2, the probiotic products of the corresponding examples were administered by gavage at the same dose, followed by gavage administration of 42% vol baijiu (0.25 mL / 10 g) 30 minutes later.

[0113] Blood samples were collected at four time points: 1.0, 2.0, 3.0, and 4.0 hours after alcohol infusion. The blood ethanol content was detected by gas chromatography (headspace method).

[0114] Table 6. Blood ethanol content in mice (mg / ml)

[0115] As shown in Table 6, the blood alcohol content of the experimental group that took the product of this embodiment was lower than that of the control group, and the difference was significant, which can accelerate the metabolism of ethanol in the body.

[0116] Experimental Example 7: Adjunctive Protective Effect Against Chemical-Induced Liver Injury Eighty SPF-grade male Kunming mice, aged 6-8 weeks and with an initial weight of 18-20g, were selected and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were acclimatized for 3 days and fasted for 12 hours before the experiment but allowed free access to water.

[0117] The animals were randomly divided into 5 groups, with 20 animals in each group: blank group, control group 1 and 2, and example group 1 and 2.

[0118] The normal group was given an equal dose of pure water by gavage; The control group was given an equal dose of pure water by gavage, followed by 42% vol baijiu (0.25 mL / 10 g) by gavage 30 minutes later.

[0119] The control group was given an equal dose of the fermentation base liquid prepared in step three by gavage, and 30 minutes later, 42% vol baijiu (0.25 mL / 10 g) was administered by gavage.

[0120] In Examples 1 and 2, the probiotic products of the corresponding examples were administered by gavage at the same dose, followed by gavage administration of 42% vol baijiu (0.25 mL / 10 g) 30 minutes later.

[0121] After the last gavage on day 10, the mice were fasted but allowed to drink water for 12 hours. Blood was collected from the eyeballs of the mice, allowed to stand at room temperature for 30 minutes, and then centrifuged at 3000 r / min for 15 minutes to separate the supernatant serum.

[0122] The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected by visible spectrophotometry. The reagent kit was manufactured by Beijing Solarbio Science & Technology Co., Ltd.

[0123] AST and ALT are mainly found in the cytoplasm and mitochondria of hepatocytes and are the most critical biochemical indicators reflecting the integrity and degree of damage to hepatocytes. When alcohol causes hepatocytes to swell, rupture, and mitochondria to be damaged, cell membrane permeability increases significantly, and large amounts of AST and ALT are released into the bloodstream, causing a sharp increase in serum enzyme activity. Therefore, the lower the serum AST and ALT levels, the milder the hepatocyte damage and the better the liver protection effect. The measurement results are shown in Table 7.

[0124] Table 7 Test Results

[0125] As shown in Table 7, compared with the normal group, the serum AST and ALT levels of mice in the model group were significantly increased, indicating that alcohol had caused obvious acute liver damage, and the model was successfully constructed.

[0126] Compared with the model group, the serum AST and ALT levels of mice in Example 1 and Example 2 groups were significantly reduced and close to the levels of the normal group, indicating that the product of the present invention can significantly reduce the effects of alcohol on hepatocytes and has an auxiliary protective effect against chemical liver damage.

[0127] Experiment 8: Stability of viable bacteria in product under refrigeration at 4°C The finished product from Example 1 was filled and sealed, and stored at a constant temperature of 4°C in the dark. Samples were taken on days 0, 7, 14, and 21, and the total number of viable probiotics (CFU / mL) was determined by the MRS plate count method.

[0128] Table 8. Measurement of viable bacteria count in products under refrigeration at 4℃

[0129] The product remained at a viable count of 1×10⁶ after being stored at 4℃ for 21 days. 8 With CFU / mL or higher, the viable bacteria retention rate was 76.7% after 21 days of storage, and the viable bacteria decline was gradual. The second stage of pasteurization thoroughly eliminates interference from yeast, acetic acid bacteria, and other miscellaneous bacteria. Combined with a suitable organic acid environment and the natural probiotic nutrients of fruit juice, it creates a stable living environment for probiotics and avoids the rapid death of live bacteria caused by bacterial antagonism.

[0130] In summary, this invention employs a three-stage herbal fermentation process, organically combining the extraction of medicinal and edible raw materials, multi-strain synergistic fermentation, low-temperature colonization of probiotics, and biotransformation of functional components. It is not a simple superposition of existing technologies, but rather overcomes multiple technical obstacles such as multi-strain inhibition, poor taste, low absorption, and limited functionality, achieving unexpected synergistic effects. The prepared probiotic herbal beverage has a harmonious flavor, rich taste, and high live bacteria content. It can significantly prolong the latency period of intoxication, shorten the sobering-up time, accelerate ethanol metabolism, and effectively reduce indicators related to alcohol-induced liver damage. It also possesses multiple functions including assisting in sobering up, assisting in protecting against chemically induced liver damage, and regulating intestinal flora. The product has high safety, wide adaptability, and good industrialization prospects and application value.

[0131] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a herbal probiotic fermented product, characterized in that, Includes the following steps: (1) The herbal raw materials are soaked in water and separated to obtain herbal extract; (2) Add the black tea raw material to hot water for extraction to obtain black tea extract; (3) Mix the extracts obtained in steps (1) and (2), add white sugar as a sugar source, and obtain the fermentation base liquid; (4) Inoculate the fermentation base liquid with the yeast strain with preservation number CICC30089 to carry out the first stage of anaerobic fermentation; (5) After the first stage of anaerobic fermentation, pasteurized acetic acid bacteria with preservation number CICC7010 were inoculated for the second stage of aerobic fermentation. After the fermentation was completed, the fermentation liquid was sterilized. (6) Inoculate the fermentation broth sterilized in step (5) with Lactobacillus plantarum (accession number CICC25283), Lactobacillus acidophilus (accession number CICC6081), and Bifidobacterium longum (accession number DSM23233) for the third stage of deep fermentation to obtain the herbal probiotic fermentation product.

2. The method according to claim 1, characterized in that, In step (1), the herbal raw materials, by weight, include 10-15 parts of kudzu root, 10-15 parts of Japanese raisin tree fruit, 3-5 parts of turmeric, 6-10 parts of poria cocos, and 3-5 parts of licorice root, and the mass of purified water added is 5 times the total mass of all herbal raw materials. In step (2), the raw material of black tea includes 10-20g of black tea per 100mL of water; the extraction conditions are: soaking in hot water at 90-95℃ for 10-15 minutes.

3. The method according to claim 1, characterized in that, In step (3), the amount of white sugar added is 50-100g / L, the volume ratio of the herbal extract added is 20%, and the volume ratio of the black tea extract added is 10%.

4. The method according to claim 1, characterized in that, In step (4), the brewing yeast is inoculated using a liquid seed culture obtained through stepwise expansion, with a viable cell concentration of ≥1×10⁻⁶. 8 CFU / mL, the seed liquid is added at a volume of 5% to 10% of the total volume of the main fermentation broth, and fermented in a sealed environment at a temperature of 18 to 22°C for 4 to 7 days.

5. The method according to claim 1, characterized in that, In step (5), the acetic acid bacteria are prepared into liquid seed culture using a stepwise aerobic expansion culture, with continuous aeration throughout the process to ensure dissolved oxygen. The viable bacterial concentration of the mature seed culture is ≥1×10⁻⁶. 8 The seed culture volume is 5%–10% of the total volume of the main fermentation broth. Fermentation is carried out at 28–32°C, with a rotation speed of 100–200 rpm and dissolved oxygen concentration controlled at 15%–30% saturation for 3–5 days.

6. The method according to claim 1, characterized in that, In step (6), *Lactobacillus plantarum*, *Lactobacillus acidophilus*, and *Bifidobacterium longum* were prepared into seed cultures through anaerobic expansion, and the viable bacterial concentration of each strain in the seed culture was ≥1×10⁻⁶. 8 The inoculum concentration was 5%–10% CFU / mL, the fermentation temperature was 20–25℃, and the fermentation time was 2–3 days.

7. The method according to claim 1, characterized in that, In step (6), fruit juice is also included, with an addition amount of 5% to 10% of the final product volume.

8. Herbal probiotic fermented products prepared by the method according to any one of claims 1 to 7.

9. The use of the fermented product according to claim 8 in the preparation of hangover remedies.

10. The use of the fermented product according to claim 8 in the preparation of pharmaceuticals or health products for relieving and / or reducing chemically induced liver injury.

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