A traditional Chinese medicine composition for improving immunity, a fermented beverage and a preparation method and application thereof

Abstract

The present application relates to the technical field of traditional Chinese medicine fermentation drink, and discloses a traditional Chinese medicine composition for improving immunity, a fermentation drink and a preparation method and application thereof, which comprises the following raw materials in mass parts: 8-14 parts of rhizoma polygonati, 8-14 parts of radix puerariae, 2-6 parts of folium mori, 2-6 parts of honeysuckle, 5-10 parts of radix codonopsis, and 3-6 parts of epimedium; the fermentation drink is obtained by half-enzymatic extraction of the raw materials in the above mass parts to obtain an enzymatic extraction liquid, and then two-stage fermentation of the enzymatic extraction liquid by yeast and probiotics. The present application adopts the mode of "enzymatic hydrolysis + two-stage fermentation", and first hydrolyzes the macromolecules in the medicinal materials into small molecules, and then forms the fermentation drink containing various medicinal ingredients. Compared with the prior art, the present application can effectively improve the extraction effect of the effective ingredients in the medicinal materials, and improve the utilization rate of the medicinal materials. Moreover, the small molecules formed by fermentation can be better absorbed, and the absorption and utilization rate of the effective ingredients in the drink is improved.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine fermented beverage technology, specifically to a traditional Chinese medicine composition for enhancing immunity, a fermented beverage, its preparation method, and its application. Background Technology

[0002] With the increasingly fast pace of modern life and a lack of time and energy for exercise, some people are experiencing a decline in physical fitness and immunity, resulting in a sub-healthy state. While there are many products available to boost immunity, most are extracted, prepared, or formulated using chemical methods. Although these products provide nutrition, the chemical residues in these beverages can have potential adverse effects on the consumer's health.

[0003] Traditional Chinese medicine beverages are mostly prepared using physical extraction methods such as water extraction and alcohol precipitation, or alcohol extraction and water precipitation, or by using wild microorganisms in the environment to naturally ferment the residue or liquid of the medicine. However, the following technical problems still exist: (1) The extraction efficiency of effective components in traditional Chinese medicine extraction methods is low, which not only wastes Chinese medicinal materials, but also results in low content of effective components and limited efficacy in the beverages produced. (2) The medicinal components in existing Chinese medicine beverages exist in various forms such as small molecules, low molecular weight compounds or macromolecular complexes. When administered orally, their bioavailability largely depends on the patient's own intestinal microecology and nutritional balance. However, when a person is sick, the gut microbiota and nutrition are mostly in an unbalanced state. There are individual differences in the composition of gut microbiota between healthy / sub-healthy and sick people. This difference will directly affect the degree of absorption and metabolism of medicinal components in Chinese medicine preparations, thus wasting medicinal resources. (3) Traditional Chinese medicine preparation only focuses on obtaining medicinal components from medicinal plants. Polysaccharides / proteins are also easily removed as impurities, reducing the utilization of Chinese medicine.

[0004] In light of this, fermentation has emerged as a method for processing traditional Chinese medicine (TCM), as it not only effectively alters the original medicinal properties of TCM, producing new therapeutic effects, but also improves the extraction efficiency of drug components. However, existing TCM fermentation technologies mostly target fermentation using a single strain or solid-state fermentation, resulting in limited fermentation products that cannot meet diverse nutritional needs and have limited efficacy in enhancing immunity, leading to a still low utilization rate of TCM.

[0005] In summary, the development of a traditional Chinese medicine composition that enhances immunity, targeting fermented beverages obtained from multiple raw materials and strains and their preparation methods, not only effectively compensates for the shortcomings of existing technologies, but also has significant implications for improving the utilization rate of medicinal materials, increasing the content of medicinal components in fermented beverages, and enhancing the efficacy of fermented beverages. Summary of the Invention

[0006] The present invention aims to provide a traditional Chinese medicine composition, a fermented beverage, a preparation method thereof, and an application for enhancing immunity, in order to solve the technical problem that the existing traditional Chinese medicine compositions have limited efficacy in enhancing immunity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a traditional Chinese medicine composition for improving immunity, comprising the following raw materials in parts by weight: 8-14 parts of Polygonatum sibiricum, 8-14 parts of Pueraria lobata, 2-6 parts of Morus alba leaves, 2-6 parts of Lonicera japonica, 5-10 parts of Codonopsis pilosula, and 3-6 parts of Epimedium brevicornu.

[0008] The principle of this scheme is:

[0009] Codonopsis pilosula: It is sweet in taste and neutral in nature; it enters the spleen and lung meridians; it has the effects of strengthening the spleen and lungs, nourishing blood and promoting body fluids. Modern pharmacological studies have shown that Codonopsis pilosula also has a variety of pharmacological effects such as enhancing the body's immunity, protecting the gastrointestinal mucosa and resisting ulcers, promoting hematopoietic function, regulating blood sugar, and delaying aging.

[0010] Epimedium: It is pungent, sweet, and warm in nature. It enters the liver and kidney meridians. Epimedium has the functions of tonifying kidney yang, strengthening tendons and bones, and dispelling wind and dampness. Modern research shows that epimedium has the effects of inhibiting osteoclasts, promoting osteoblast growth, enhancing immune function, and anti-oxidation and anti-aging.

[0011] Polygonatum rhizome: Sweet and neutral in nature, it enters the spleen, lung, and kidney meridians. Polygonatum rhizome is moistening and sweet, nourishing without being overly concentrated, with a mild effect. Entering the spleen, lung, and kidney meridians, it is a mild tonic for both qi and yin. It nourishes yin and moistens the lungs, tonifies the kidneys and replenishes essence, and also tonifies the spleen and replenishes qi, making it an excellent tonic. It is effective in treating various syndromes of lung and kidney deficiency, and qi and yin deficiency. This herb contains polysaccharides, saponins, flavonoids, and other components, and has the effects of tonifying qi and nourishing yin, strengthening the spleen, moistening the lungs, and benefiting the kidneys.

[0012] Kudzu root: Its medicinal properties are sweet, pungent, and cool. It enters the spleen, stomach, and lung meridians. Kudzu root is sweet, pungent, light, and dispersing, neutral with a slight cooling effect, and enters the spleen and stomach meridians. It not only relieves wind-heat on the skin surface, reduces fever, and promotes the eruption of rashes, but also invigorates the spleen and stomach's clear yang to rise, generate fluids, quench thirst, and stop diarrhea. It is most suitable for treating stiff neck and back pain and Yangming headache, regardless of whether it is due to cold or heat, deficiency or excess, or with or without sweating. When used raw, it disperses and clears, and generates fluids; when simmered, it is better at raising but less clearing. This product contains flavonoids, coumarins, puerarin, puerarin glycosides, isoflavone glycosides, and starch, among other components.

[0013] Mulberry leaves: Sweet, bitter, and cold in nature. They enter the lung and liver meridians. Mulberry leaves mainly contain flavonoids, alkaloids, phytosterols, gamma-aminobutyric acid (GABA), and mulberry leaf polysaccharides. They have the effects of dispersing wind-heat, clearing the lungs and moistening dryness, calming liver yang, and clearing the liver and improving eyesight. They are used for symptoms such as wind-heat colds, early stages of febrile diseases, lung-heat coughs, dry coughs, liver yang hyperactivity, headaches and dizziness, red and swollen eyes, blurred vision, and cooling the blood and stopping bleeding.

[0014] Honeysuckle: Sweet and cold in nature. It enters the lung, stomach, and large intestine meridians. Honeysuckle is sweet and cold, clearing and purging, light and penetrating, clearing and dispersing, entering the lung, stomach, and large intestine meridians. It is good at clearing heat toxins and dispersing wind-heat. Its medicinal power is quite strong but not bitter, making it an excellent medicine for clearing heat toxins, and it is easy to take without being bitter. It primarily clears, while also penetrating, and can be used for all types of heat toxins and wind-heat. It is suitable for all stages of febrile diseases and is often combined with forsythia. In the Wei level, it can penetrate the exterior; in the Qi level, it can clear heat; in the Ying level, it can promote the transformation of Ying and Qi; and in the blood level, it can clear heat toxins in the blood. This product contains organic acids such as chlorogenic acid and isochlorogenic acid, as well as flavonoids such as luteolin and honeysuckle glycoside. It also contains volatile oils, saponins, inositol, oleanolic acid, and carotene.

[0015] This formula combines ingredients such as Codonopsis pilosula, Epimedium, Polygonatum sibiricum, and Pueraria lobata to effectively enhance the drinker's kidney qi and thus boost immunity. The addition of Polygonatum sibiricum and Pueraria lobata not only nourishes the kidneys and yin and improves immunity, but also effectively removes cholesterol and fat from blood vessel walls, thus lowering blood lipids. Furthermore, the addition of mulberry leaves helps lower blood pressure, blood sugar, and cholesterol, effectively addressing the current trend of "old age diseases" becoming more prevalent, complex, and complicated in younger people due to weakened immunity. The combination of Pueraria lobata and mulberry leaves also promotes saliva production, quenches thirst, dispels wind-heat, and clears the liver and improves eyesight, offering some relief for yin deficiency-related thirst, red and swollen eyes, and other symptoms. The addition of honeysuckle effectively reduces internal heat and moisturizes dryness, preventing the drinker from experiencing heat-related symptoms from long-term use of the warming or tonifying herbs in this formula.

[0016] This solution also provides a fermented beverage to enhance immunity, which is made by semi-bionic enzymatic extraction of the raw materials in the above-mentioned Chinese herbal medicine composition to obtain an enzymatic extract, and then fermented by yeast and probiotics in two stages; the semi-bionic enzymatic extraction includes enzymatic extraction of the raw materials by mixed enzyme powder.

[0017] Beneficial effects: This method first employs a semi-bionic enzymatic extraction approach to break down large molecules such as starch, protein, and fat in medicinal materials into smaller molecules such as oligosaccharides, polypeptides, and fatty acids. These smaller molecules, along with other small molecules present in the medicinal materials themselves, are then extracted to form an enzymatic extract. Compared to existing technologies that use decoction or direct fermentation to extract the active ingredients from medicinal materials, this method effectively improves the extraction efficiency of medicinal components and increases the utilization rate of the medicinal materials. Furthermore, the breakdown of starch into smaller molecules better promotes the growth and reproduction of microorganisms during subsequent fermentation, while the remaining smaller molecules in the fermented beverage are more easily absorbed by the human body, enhancing the absorption and utilization of the active ingredients in the beverage.

[0018] Furthermore, in this method, large molecules such as starch in the medicinal materials are enzymatically broken down into smaller molecules, which are then fermented by yeast to form ethanol, and then fermented by probiotics to form acetic acid. Other large molecules are transformed into other organic acids or active ingredients through enzymatic breakdown, yeast fermentation, and probiotic fermentation. This not only effectively increases the richness and content of organic acids and active ingredients in fermented beverages, but also produces "sugar-free and alcohol-free" fermented beverages, which are not only healthier but also more suitable for special groups such as patients with hypertension, hyperlipidemia, and hyperglycemia, and drivers, effectively expanding the beverage's applicability.

[0019] This solution also provides a method for preparing a fermented beverage to enhance immunity, including the following steps:

[0020] S1. Preparation of mixed Chinese medicine powder stage: Weigh the above-mentioned mass proportions of raw materials, mix them, and then pulverize them to obtain mixed Chinese medicine powder;

[0021] S2, Enzymatic hydrolysis stage: After soaking the above mixed Chinese herbal powder with water, add mixed enzyme powder and hydrolyze to obtain enzymatic extract;

[0022] S3, Primary fermentation stage: Yeast is inoculated into the above enzymatic hydrolysis extract, and fermentation is carried out to obtain primary fermentation broth;

[0023] S4: Secondary fermentation stage: Probiotics are inoculated into the primary fermentation broth above, and fermentation is carried out to obtain secondary fermentation broth;

[0024] S5: Post-processing stage: Sterilize, concentrate, and sterilize the above secondary fermentation broth in sequence to obtain fermented beverage.

[0025] Beneficial effects: This treatment uses a combination of probiotics to ferment traditional Chinese medicine ingredients, which has advantages over traditional methods such as decocting, boiling, and boiling. These advantages mainly include the following aspects:

[0026] (1) Probiotic fermentation of traditional Chinese medicine promotes the dissolution of active ingredients and enhances efficacy. The cells of traditional Chinese medicine contain many active ingredients, tightly encapsulated by cell walls composed of hemicellulose, cellulose, lignin, and other components. During fermentation, microorganisms produce metabolites such as cellulase, ligninase, and pectinase, which decompose the cell walls and promote the full dissolution of active ingredients. Studies have found that probiotic-fermented traditional Chinese medicine can achieve the same efficacy with only 1 / 28th the dosage of extracts obtained through traditional processes.

[0027] (2) Probiotic fermentation can metabolize Chinese herbal medicines and produce new active ingredients, effectively improving the quality of fermented beverages; specifically, it will produce secondary metabolites during the microbial fermentation process, causing the effective ingredients to undergo biotransformation, and thus new active ingredients are produced.

[0028] (3) Probiotic fermentation can reduce toxicity. Specifically, "all medicines have some degree of toxicity". Some of the Chinese herbal medicines used in this plan may contain some toxic substances, which may cause disease if taken in excess. However, after the Chinese herbal medicine composition is fermented with probiotics in this plan, the toxic substances can be biotransformed or structurally modified, thereby reducing toxicity.

[0029] (4) Probiotic fermentation can effectively save Chinese medicine resources. Specifically, the active ingredients are fully dissolved after fermentation, and the efficacy is improved accordingly, so the dosage of Chinese medicine used will be reduced. In addition, this scheme utilizes the fermentation of Chinese medicine residue to make secondary use of Chinese medicine raw materials, which effectively improves the utilization rate of Chinese medicine, while avoiding the environmental pollution caused by directly discarding and burying Chinese medicine residue.

[0030] Furthermore, this solution employs a "pre-enzymatic hydrolysis followed by fermentation" method, which not only effectively enhances the dissolution of medicinal components in Chinese herbal medicines, thereby increasing the extraction rate of these components, but also allows macromolecules such as cellulose and starch to be broken down into smaller molecules (such as monosaccharides and disaccharides) during the enzymatic hydrolysis process. These smaller molecules can be used for the growth and reproduction of fermentation microorganisms, effectively saving on the input of fermentation nutrients and reducing production costs. Moreover, this "pre-enzymatic hydrolysis followed by fermentation" method also allows for the preparation of fermented beverages that are "sugar-free and alcohol-free," suitable for a wider range of consumers, effectively improving national immunity. Through long-term research, the applicant has found that, compared to fermented beverages prepared using the existing "pre-fermentation followed by enzymatic hydrolysis" method, this solution produces fermented beverages with higher medicinal component content and lower sugar and alcohol content, effectively enhancing immunity while reducing the risk of "glycemic index," providing the public with healthier fermented beverages.

[0031] Preferably, the yeast is a non-Saccharomyces cerevisiae; the inoculation amount of the yeast is 1-5%.

[0032] Beneficial Effects: This scheme, employing the above-mentioned settings, effectively reduces the alcohol content in fermented beverages, thereby obtaining "alcohol-free" fermented drinks. Compared to grain-based brewing yeasts, non-brewing yeasts have advantages in food fermentation, such as reducing ethanol content, high enzyme production capacity, and producing flavor compounds. Through long-term experiments, the applicant has found that, compared to using grain-based brewing yeasts, this scheme, using non-grain-based brewing yeasts to ferment the enzymatic hydrolysis extract, not only effectively shortens the fermentation process but also rapidly reduces the alcohol content in the enzymatic hydrolysis extract, obtaining an "alcohol-free" primary fermentation broth. The yeast inoculation amount in this scheme facilitates the rapid consumption of sugars in the enzymatic hydrolysis extract, avoiding prolonged fermentation time that could lead to a decrease in the content of medicinal components.

[0033] Preferably, the particle size of the mixed Chinese herbal powder is 0.43 mm to 0.71 mm.

[0034] Beneficial Effects: This scheme, employing the aforementioned settings, facilitates the rapid enzymatic hydrolysis of cellulose by mixed enzyme powder, thereby increasing the dissolution rate of medicinal components in traditional Chinese medicine and thus enhancing the content of medicinal components in fermented beverages. Through long-term experiments, the applicant discovered that if the particle size of the Chinese medicine powder is too large, it easily leads to a prolonged enzymatic hydrolysis time. Even with extended hydrolysis time, the technical effects of this scheme cannot be achieved; specifically, the content of medicinal components is significantly lower than that obtained from fermented beverages treated with the appropriate particle size in this scheme. Conversely, if the particle size is too small, it not only easily leads to over-fermentation but also increases the energy consumption for pulverizing Chinese medicinal materials, significantly reducing production efficiency.

[0035] Preferably, the mixed enzyme powder comprises the following enzymes in parts by weight: 6-12 parts amylase, 2-6 parts cellulase, 1-3 parts papain and 0.1-0.5 parts cholesterol esterase.

[0036] Beneficial Effects: This scheme, employing the aforementioned setup, allows cellulase to rapidly break down cellulose in cell walls, causing cell rupture and facilitating the release of intracellular components. Meanwhile, amylase, protease, and lipase effectively break down macromolecules into smaller organic molecules, making them easier to use or metabolize into new active ingredients during subsequent fermentation. The synergistic effect of these enzymes effectively increases the content of active ingredients in fermented beverages. Through long-term experiments, the inventors discovered that a lack of cellulase leads to less cell rupture and less nutrient release, significantly reducing the nutrient content of the product. Conversely, a lack of amylase, papain, or lipase results in excessively high starch, protein, or fat content in the product, affecting consumer absorption of nutrients.

[0037] Preferably, the enzymatic hydrolysis stage involves the following steps: adding drinking water to soak the mixed Chinese herbal powder at a mass ratio of 1:3-4 for 30 min to 1 h, adjusting the pH to 5.5-6.5, adding 8-10 g / L of compound enzyme powder, and hydrolyzing at 37.5-45℃ for 24-36 h. After hydrolysis, the Chinese herbal medicine is decocted for 1 h, coarsely filtered, and the decoction is concentrated to a solute mass concentration of 300 g / L. After centrifugation, the decoction is sterilized to obtain the Chinese herbal enzymatic hydrolysis extract.

[0038] Beneficial Effects: This scheme, employing the aforementioned settings, facilitates effective enzymatic hydrolysis of cell walls and intracellular macromolecules, yielding an enzymatic extract with a high content of medicinal components. Furthermore, the subsequent decoction effectively inactivates the enzyme, preventing it from entering the subsequent fermentation stage and affecting the fermentation effect; simultaneously, the decoction after enzymatic hydrolysis also effectively extracts the active ingredients. Through long-term experiments, the inventors have discovered that compared to existing technologies that use two decoctions after enzymatic hydrolysis to enhance medicinal components, this scheme, through the rational combination of compound enzyme powder and optimized enzymatic hydrolysis conditions, effectively improves the enzymatic hydrolysis effect. Even with only one decoction, it effectively increases the content of medicinal components in the enzymatic extract, thereby significantly improving drug extraction efficiency. It also eliminates the need for a decoction process, significantly reducing production energy consumption.

[0039] Preferably, the probiotics include at least two of Lactobacillus, Bifidobacterium, Bacillus, and Acetic Acid Bacteria.

[0040] Beneficial effects: The above settings in this scheme facilitate the effective increase of the content of new active ingredients produced by the metabolic Chinese herbal medicines in the secondary fermentation liquid, thereby effectively reducing toxicity while enhancing the efficacy of the fermented beverage.

[0041] Preferably, in the secondary fermentation, the inoculation amount of the probiotics is 1-5%.

[0042] Beneficial effects: The above settings facilitate the rapid consumption of ethanol in the primary fermentation liquid, which not only makes it easier to obtain "alcohol-free" fermented beverages, but also avoids the reduction of medicinal component content due to excessively long fermentation time.

[0043] This solution also provides a traditional Chinese medicine composition for enhancing immunity and the application of a fermented beverage for enhancing immunity in the preparation of beverages that boost immunity. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art.

[0045] Example 1

[0046] This solution provides a traditional Chinese medicine composition for enhancing immunity, comprising the following raw materials in parts by weight: 8-16 parts of Polygonatum sibiricum, 8-16 parts of Pueraria lobata, 2-6 parts of Morus alba leaves, 2-6 parts of Lonicera japonica, 5-10 parts of Codonopsis pilosula, and 3-6 parts of Epimedium brevicornu. For details of the medicinal materials and dosages, please refer to Table 1.

[0047] Table 1: Raw material formulation of traditional Chinese medicine composition

[0048] Name of medicinal materials Latin name Raw material usage per serving Standards for the Administration of Medicinal Materials Polygonatum POLYGONATI RHIZOMA 8～12 Chinese Pharmacopoeia 2020 Edition Kudzu root PUERARIAE LOBATAE RADIX 8～15 Chinese Pharmacopoeia 2020 Edition Mulberry leaves MORI FOLIUM 2～5 Chinese Pharmacopoeia 2020 Edition honeysuckle LONICERAE JAPONICAE FLOS 2～5 Chinese Pharmacopoeia 2020 Edition Codonopsis pilosula CODONOPSIS RADIX 5～10 Chinese Pharmacopoeia 2020 Edition Epimedium EPIMEDII FOLIUM 3～6 Chinese Pharmacopoeia 2020 Edition

[0049] This solution also provides a fermented beverage to enhance immunity, which is made by semi-bionic enzymatic extraction of the above-mentioned raw materials to obtain an enzymatic extract, followed by two-stage fermentation with yeast and probiotics.

[0050] This solution also provides a method for preparing a fermented beverage that enhances immunity, specifically including the following steps:

[0051] S1. Preparation of mixed Chinese herbal powder stage: Weigh the above-mentioned raw materials (70g of Polygonatum sibiricum, 90g of Pueraria lobata, 25g of Morus alba leaves, 25g of Lonicera japonica, 60g of Codonopsis pilosula, and 30g of Epimedium brevicornu), mix them, and then pulverize them through a 25-40 mesh sieve to obtain mixed Chinese herbal powder with a particle size of 0.43-0.71mm.

[0052] S2. Enzymatic hydrolysis stage: Add drinking water to the mixed Chinese herbal powder at a mass ratio of 1:3-4 and soak for 30 min to 1 h. Adjust the pH to 5.5-6.5, add 8-10 g / L of compound enzyme powder, and incubate at 37.5-45℃ for 24-36 h for enzymatic hydrolysis. After enzymatic hydrolysis, decoct the Chinese herbal powder at 100℃ for 1 h. After coarse filtration, concentrate the decoction using a vacuum rotary evaporator (60℃) to a solute mass concentration of 300 g / L (i.e., 300 g of Chinese herbal material will result in 1 L of concentrated decoction). Centrifuge at 10000 rpm for 20 min and sterilize at 121℃ for 20 min to obtain the Chinese herbal enzymatic hydrolysis stock solution.

[0053] The mixed enzyme powder includes 6-12 parts of amylase (enzyme activity: 2000u / g), 2-6 parts of cellulase (enzyme activity: 10000U / g), 1-3 parts of papain (enzyme activity: 800000U / g), and 0.1-0.5 parts of cholesterol esterase (enzyme activity: 15000U / g). This scheme uses the above enzymes in combination to facilitate the full enzymatic hydrolysis of the various nutrients in the Chinese herbal medicine powder, obtaining a nutrient-rich enzymatic hydrolysate.

[0054] S3. Primary Fermentation Stage: Inoculate 1-5% yeast into the above enzymatic extract. After inoculation, ferment aerobically at 30-37℃ for 5-7 hours, and then sterilize at 121℃ for 20 minutes to obtain the primary fermentation broth. The yeast is a non-Saccharomyces cerevisiae. In this embodiment, an equal mass of Pichia burtonii FBKL2.0089 and Hanseniaspora vineae are used. Pichia burtonii FBKL2.0089 can produce high levels of ascorbic acid and arabinitol, providing sweetness, but with lower calories than sucrose. It will not cause weight gain or increase the risk of diabetes due to its metabolism into blood sugar. In addition, arabinitol cannot be metabolized into acid by bacteria in the mouth, avoiding tooth decay and tooth loss. It can also promote saliva secretion, thereby helping to clean the mouth and reduce oral problems such as halitosis and gingivitis. Ascorbic acid promotes the synthesis of collagen and connective tissue, accelerating wound healing; it also eliminates free radicals and enhances immunity. The wine yeast *Hanseniaspora vineae* produces high levels of lactic acid and melatonin, effectively increasing the nutrient content of fermented beverages. Furthermore, the non-brewing yeasts used in this process produce low levels of ethanol; the small amount of ethanol they produce is decomposed or evaporated during subsequent secondary fermentation and concentration, facilitating the production of alcohol-free products.

[0055] S4: Secondary fermentation stage: Inoculate 1-5% probiotics into the above primary fermentation broth, and then anaerobic ferment at 37℃ for 24-48 hours to obtain secondary fermentation broth; the probiotics include at least two of Lactobacillus, Bifidobacterium, Bacillus, and Acetic Acid Bacteria. As a reference, the probiotics in this scheme are equal masses of Lactobacillus, Bifidobacterium, Bacillus oryzae, and Acetic Acid Bacteria (i.e., the mass ratio of Lactobacillus, Bifidobacterium, Bacillus oryzae, and Acetic Acid Bacteria powder is 1:1:1:1).

[0056] S5: Post-processing stage: The secondary fermentation broth is sterilized at 121℃ for 20 min, concentrated in a vacuum rotary evaporator (60℃) for 1-2 h, and sterilized at 121℃ for 20 min in sequence to obtain fermented beverage.

[0057] The raw material usage and parameter values ​​for each step in Examples 2-3 and Comparative Examples 1-12 are basically the same as in Example 1. The differences in raw material usage and parameter values ​​for each step in Examples 1-3 and Comparative Examples 1-12 are detailed in Table 2.

[0058] Table 2 shows the differences in raw material usage and parameter values ​​for each step in Examples 1-3 and Comparative Examples 1-12.

[0059]

[0060] Experimental Example 1: Quality Testing of Fermented Beverages in the Example and Comparative Examples

[0061] The specific testing standards are as follows (referring to the relevant provisions in the Chinese Pharmacopoeia):

[0062] [Characteristics] This product is a fermented beverage (mixture or oral liquid), the contents of which are brownish-yellow to brownish-brown liquid; it has a fragrant odor and a bitter taste.

[0063] [Inspection] The pH value should be 4.0 to 5.5 (General Rule 0631).

[0064] The relative density should not be less than 1.13 (General Rule 0601).

[0065] Other requirements shall comply with the relevant provisions under the Compound Preparations section (General Rule 0181).

[0066] [Determination of the content of puerarin, icariin, rutin, luteolin, and total flavonoids] The determination shall be performed in accordance with the high performance liquid chromatography method (General Rule 0512) in the 2020 edition of the Chinese Pharmacopoeia.

[0067] 1. Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; methanol-water (25:75) was used as the mobile phase; the detection wavelength was 250 nm. The theoretical plate number, calculated based on the puerarin peak, should not be less than 4000.

[0068] 2. Preparation of reference solutions: Take appropriate amounts of anhydrous glucose, puerarin, rutin, luteolin, and icariin, respectively, weigh them accurately, and add 30% ethanol to prepare a solution containing 80 μg per ml.

[0069] 3. Preparation of test solution: Take 1 ml of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of 30% ethanol, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, make up the lost weight with 30% ethanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0070] 4. Determination method: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph, determine and calculate the content of Polygonatum polysaccharide, Puerarin, Rutin, Luteolin, and total glycosides of Epimedium in the test sample.

[0071] Among them, the total glycosides of icariin are the total amount of icariin A (C39H50O20), icariin B (C38H48O19), icariin C (C39H50O19) and icariin (C33H40O15).

[0072] [Ethanol Content Determination] The ethanol content in fermented beverages is determined using the alcohol method. The specific operating procedure is as follows:

[0073] 1. Take a certain amount of fermentation broth (usually 10 ml) and add potassium chloride solution (10% concentration) dropwise until the fermentation broth no longer produces gas.

[0074] 2. Divide the volume of potassium chloride solution added (V1) by the volume of the fermentation broth (usually 10 ml) to obtain the concentration of the potassium chloride solution (c1).

[0075] 3. Using a standard ethanol solution containing a known concentration, determine the volume of potassium chloride solution (V2) and the volume of standard ethanol solution (V3) using the same method, and obtain the concentration of the standard ethanol solution (c2).

[0076] 4. Calculate the ethanol content in the fermentation broth using the following formula:

[0077] Ethanol content (%) = c1*V1*0.79 / c2*V3

[0078] Where 0.79 is the density of ethanol.

[0079] The fermented beverages obtained in Examples 1-3 and Comparative Examples 1-12 were tested, and the results are shown in Table 3.

[0080] Table 3 Quality testing of fermented beverages in Examples 1-3 and Comparative Examples 1-12

[0081]

[0082]

[0083] Experimental data shows that this scheme employs a two-stage fermentation method following enzymatic hydrolysis. This process breaks down large molecules such as starch in the medicinal materials into smaller molecules, which are then fermented by yeast to form ethanol, followed by fermentation by probiotics to form acetic acid. Other large molecules undergo enzymatic hydrolysis, yeast fermentation, and probiotic fermentation to form other organic acids or active ingredients. This significantly increases the content of medicinal components in the beverage. It not only effectively enhances the richness and content of organic acids and active ingredients in fermented beverages but also produces sugar-free and alcohol-free fermented beverages, making them healthier and more suitable for specific groups such as patients with hypertension, hyperlipidemia, and hyperglycemia, and drivers, thus broadening the beverage's applicability. Specifically, the fermented beverages obtained in Examples 1-3 of this scheme have higher contents of medicinal components such as Polygonatum polysaccharide, puerarin, rutin, luteolin, and total glycosides of Epimedium, and lower ethanol content (meeting the requirement of alcohol content below 0.5% for alcohol-free beverages). Furthermore, the production process is continuous, requires no long-term boiling, and has low energy consumption.

[0084] Through analysis, the inventors discovered that factors such as the fermentation method, particle size of the mixed Chinese herbal powder, enzymatic hydrolysis pH, enzymatic hydrolysis time, yeast inoculation amount, and probiotic fermentation time significantly affect the content and other properties of medicinal components in fermented beverages. Specifically: Comparative Example 1, after enzymatic hydrolysis and decoction for 1 hour, underwent natural fermentation for 5-7 days. The content of effective medicinal components in the resulting fermented beverage was significantly lower than that obtained by this method, which used yeast and probiotics for separate fermentation after enzymatic hydrolysis. The inventors analyzed that the reason for this was that the naturally fermented bacteria were inconsistent and could not fully ferment and decompose the effective components in the medicinal materials. Comparative Example 2 also involved fermentation with yeast and probiotics after enzymatic hydrolysis; however, it used a mixed fermentation method of yeast and probiotics. Although this method increased the content of effective components in the fermented beverage compared to natural fermentation, it could not achieve the same content of effective components as the beverage obtained by the "enzymatic hydrolysis + two-stage fermentation" method in this method. If the particle size of the Chinese medicinal herbs is too small (as in Comparative Example 3), the particles are prone to agglomeration in the aqueous solution, which is not conducive to enzymatic hydrolysis. This results in less dissolution of the medicinal components and a reduction in the content of medicinal components in the fermented beverage. Conversely, if the particles are too large (as in Comparative Example 4), the dissolution of medicinal components during enzymatic hydrolysis will also be reduced, thus lowering the content of medicinal components in the fermented beverage.

[0085] This solution also provides a traditional Chinese medicine composition for enhancing immunity and the application of a fermented beverage for enhancing immunity in the preparation of beverages that boost immunity.

[0086] Experimental Example 2: Detection of the Immunoboosting Effect of Fermented Beverages in the Example and Comparative Examples

[0087] The animal cells and reagents used in the experiment were all purchased commercially.

[0088] (1) Culture of mouse macrophages RAW264.7

[0089] Take 5 ml of the fermented beverage from Example 1 and Comparative Example 1 and dissolve it in 5 mL of blank DMEM high sugar medium (i.e., DMEM-HD) to obtain a stock solution with a concentration of 1 mg / ml. Dilute the 1 mg / ml solution 100 times to obtain 10 ug / ml.

[0090] Fresh DMEM medium containing 10% fetal bovine serum was added to RAW264.7 cells, and the mixture was thoroughly mixed using a capillary tube to prepare a single-cell suspension. Cells were then counted using a hemocytometer. Cells were seeded into 96-well cell culture plates at concentrations of 1000, 2000, and 4000 cells per well, respectively. The plates were then supplemented with 100 μL of DMEM complete medium containing 10% fetal bovine serum to each well and incubated statically at 37°C in a 5% CO2 incubator for 24 h.

[0091] RAW264.7 cells in logarithmic growth phase were divided into groups of 6 × 10⁶ cells per well.6 Cells were seeded into 96-well cell culture plates and randomly divided into a blank control group, an LPS model group (10 μg / ml LPS), and a fermented beverage group (10 μg / ml). After adding the drug, the cells were incubated in a CO2 incubator for 2 hours, and then 10 μg / ml of the drug was added to each well. -1 LPS cells were cultured for another 10 hours, ready for use in ELISA to determine the levels of IL-10, IL-6, and TNF-α. Cells were washed with phosphate-buffered saline (PBS) and then lysed with RIPALysis Buffer (RIPA lysis buffer). Protein quantification was performed using a protein quantification kit. Following the enzyme-linked immunosorbent assay (ELISA) procedure, the cells were coated with antigen, added to the sample, added to the enzyme-labeled antibody, added to the substrate for color development, and finally the reaction was terminated. OD values ​​were measured at 450 nm using a Swiss TECAN multi-mode microplate reader. A standard curve was established, and the release levels of IL-6, IL-10, and TNF-α were calculated. The results are recorded in Table 4.

[0092] Table 4 shows the effects of fermented beverages in Examples 1-3 on LPS-induced expression of inflammatory factors in RAW264.7 cells.

[0093] Group IL-10 / ng / L IL-6 / ng / L TNF-α / ng / L Blank control 38.24±11.27 30.25±5.81 28.36±7.25 LPS model group 154.08±14.33 127.58±8.20 60.35±15.26 Example 1 58.62±15.62 53.51±9.47 35.07±12.95 Comparative Example 1 106.04±15.12 84.74±4.06 55.51±10.04 Comparative Example 2 85.54±17.07 71.98±8.58 47.25±9.67

[0094] Experimental data show that the fermented beverage obtained using the proposed "enzymatic hydrolysis followed by two-stage fermentation" method inhibited the release of IL-1, IL-6, and TNF-α from LPS-induced RAW264.7 cells to varying degrees. Specifically, compared to the LPS model group, the release of IL-1, IL-6, and TNF-α was significantly increased (P<0.01). In contrast, the fermented beverages prepared using different "enzymatic hydrolysis + fermentation" processes in the comparative studies showed a lower content and fewer medicinal components, resulting in a smaller inhibitory effect on the release of inflammatory factors in cells, significantly less than the inhibitory effect of the fermented beverage prepared using the proposed method.

[0095] Experimental Example 3: DPPH Free Radical Scavenging Ability

[0096] 100 μL of the fermented beverage obtained in Example 1 was pipetted into each well of a 96-well plate, and 100 μL of 0.2 mmol / L DPPH-methanol solution was added. The mixture was reacted in the dark for 30 min, and the absorbance at 517 nm was measured afterward. The blank group used 100 μL of deionized water instead of the sample solution, the background group used an equal volume of methanol, and the control group used an equal volume of ascorbic acid (Vc). The DPPH scavenging rate was calculated using the following formula:

[0097]

[0098] In the formula: A0 is the blank group; As is the test group; Ac is the background group.

[0099] The DPPH scavenging rate results of the fermented beverages obtained in Example 1 and Comparative Example 1 are detailed in Table 5.

[0100] Table 5. Scavenging ability of fermented beverages obtained in Example 1 and Comparative Example 1 against DPPH free radicals.

[0101]

[0102] Experimental data show that the fermented beverage obtained by the "first enzymatic hydrolysis, then two-stage fermentation" method in Example 1 of this scheme has a better DPPH free radical scavenging rate. Specifically, the free radical scavenging rate of the fermented beverage in this scheme is as high as 48.31%.

[0103] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a fermented drink for improving immunity, characterized by comprising the steps of: The fermented beverage comprises raw materials in the following mass parts: 8-14 parts of polygonatum sibiricum, 8-14 parts of pueraria lobata, 2-6 parts of mulberry leaf, 2-6 parts of honeysuckle, 5-10 parts of codonopsis pilosula, and 3-6 parts of epimedium brevicornu; The raw materials are subjected to semi-bionic enzymatic extraction to obtain an enzymatic extraction liquid, and then subjected to two-stage fermentation by yeast and probiotics; the semi-bionic enzymatic extraction comprises enzymatic extraction of the raw materials by mixed enzyme powder; ​ The method comprises the following steps: S1, a mixed traditional Chinese medicine powder preparation step: the raw materials in the above mass parts are weighed and mixed, and then crushed to obtain a mixed traditional Chinese medicine powder; S2, an enzymatic extraction step: the mixed traditional Chinese medicine powder is mixed with water, soaked, and then mixed enzyme powder is added for enzymatic extraction to obtain an enzymatic extraction liquid; S3, a primary fermentation step: yeast is inoculated into the enzymatic extraction liquid for fermentation to obtain a primary fermentation liquid; S4, a secondary fermentation step: probiotics are inoculated into the primary fermentation liquid for fermentation to obtain a secondary fermentation liquid; S5, a post-treatment step: the secondary fermentation liquid is sterilized, concentrated, and sterilized in sequence to obtain a fermented beverage.

2. The method for preparing a fermented beverage to enhance immunity according to claim 1, characterized in that: The yeast is non-Saccharomyces cerevisiae, and the inoculation amount of the yeast is 1-5%.

3. The method of claim 1, wherein the fermented drink is prepared by adding the fermented milk to the water. The particle size of the mixed traditional Chinese medicine powder is 0.43-0.71 mm.

4. The method of claim 1, wherein the fermented drink is prepared by adding the fermented milk to the water. The mixed enzyme powder comprises 6-12 parts of amylase, 2-6 parts of cellulase, 1-3 parts of papain, and 0.1-0.5 parts of cholesterol esterase.

5. The method of claim 1, wherein the fermented drink is prepared by the steps of: The enzymatic extraction step comprises the following steps: the mixed traditional Chinese medicine powder is soaked in drinking water at a mass ratio of 1:3-4 for 30 min-1 h, the pH is adjusted to 5.5-6.5, 8-10 g / L of complex enzyme powder is added, and enzymatic extraction is performed at 37.5-45°C for 24-36 h; the enzymatically extracted traditional Chinese medicine is boiled for 1 h, the liquid is concentrated to a solute mass concentration of 300 g / L, and the traditional Chinese medicine enzymatic extraction liquid is obtained by centrifugation and sterilization. ​ 6. The method of claim 5, wherein the fermented drink is prepared by adding the fermented milk to the water. The probiotics comprise at least two of lactobacillus, bifidobacterium, bacillus, and acetic acid bacteria.

7. The method for preparing a fermented beverage to enhance immunity according to claim 6, characterized in that: The inoculation amount of the probiotics is 1-5%.

8. Application of the fermented beverage prepared by the method of any one of claims 1-7 in the preparation of a beverage for improving immunity.

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