Low-alcohol high-activity astragalus secondary fermentation milk wine and preparation method thereof

Abstract

The application provides a low-alcohol high-activity milk wine with astragalus membranaceus secondary fermentation and a preparation method thereof, and belongs to the field of fermentation and brewing. The application takes traditional Chinese medicine astragalus membranaceus as a core raw material, improves the extraction rate of active ingredients of astragalus membranaceus and generates synergistically functional substances through a unique two-stage solid-state fermentation process, realizes the ternary synchronous symbiotic fermentation of edible fungi, lactic acid bacteria and yeast, and solves the technical problems of low extraction rate of active ingredients of astragalus membranaceus, difficult to balance high activity and low alcohol content, microbial competition inhibition, and poor compatibility of active ingredients and milk base. The astragalus membranaceus secondary fermentation milk wine has low alcohol content and high biological activity, has the functions of enhancing immunity and antioxidant and anti-fatigue, and can be widely applied to the fields of functional food, healthy drinks and new fermentation flavors.

Description

Technical Field

[0001] This invention relates to the field of fermented brewing, specifically to a low-alcohol, high-activity Astragalus secondary fermentation milk wine and its preparation method. Background Technology

[0002] Milk wine is a traditional alcoholic beverage made from whey, cow's milk, or mare's milk through microbial fermentation or distillation. It is rich in amino acids, vitamins, and trace elements. Traditional processes primarily utilize yeast and lactic acid bacteria to ferment lactose, producing alcohol and organic acids, thus achieving a wine-like aroma and yogurt-like flavor. However, in traditional fermented milk wine production, a prolonged fermentation time and significant sugar consumption are often required to obtain sufficient flavor compounds, resulting in a relatively high alcohol content.

[0003] To reduce the alcohol content of milk beer, yeasts with high peptide production and low alcohol production have begun to be used in the fermentation process. CN115820357A discloses a method for preparing bioactive peptide milk beer. This method involves fermenting a milk protein-containing stock solution with *Lactobacillus casei* to obtain a first concentrate, followed by fermentation with low-alcohol yeast to obtain a second concentrate with low alcohol content. The two concentrates are then mixed, additives are added, and after heating and dissolving processes, a low-alcohol milk beer with an alcohol content below 0.1% and a bioactive peptide content of more than 2.5 wt% (molecular weight less than 10 kDa) is obtained. However, the product prepared by this method largely depends on subsequent additives and compound stabilizers in terms of flavor and stability. This indicates that the system formed by separately fermenting lactic acid bacteria and yeast and then mixing them later lacks physical stability and has a relatively simple functional composition, mainly relying on bioactive peptides derived from milk protein.

[0004] Astragalus, a traditional Chinese medicine, is rich in various bioactive components such as polysaccharides, saponins, and isoflavones. By extracting astragalus extract through methods such as decoction or ultrasonic extraction, and then adding it to beverages, the active components, including astragalus polysaccharides and saponins, can be incorporated into the beverage, giving it certain immune-enhancing properties. Simultaneously, astragalus extract can effectively scavenge free radicals in the body, extending the shelf life of the beverage and providing anti-aging and energy-boosting benefits. A paper published in the *Journal of Gansu Agricultural University*, Vol. 60, No. 4 (2025), pp. 325-334, describes the use of microwave-assisted extraction of astragalus polysaccharides and the subsequent blending of astragalus extract with jujube extract to prepare an astragalus-jujube composite beverage. The resulting product exhibits a high DPPH free radical scavenging rate, and the astragalus also contributes antioxidant capacity to the jujube beverage, extending its shelf life. However, this product's system is still an unstable system obtained through physical mixing, requiring stabilizers such as sodium carboxymethyl cellulose to address sedimentation and stratification issues.

[0005] To address the shortcomings of physically mixed beverage systems, targeted microbial fermentation can be used to achieve the biotransformation and deep integration of raw material components. This not only improves the bioavailability of the original components but also creates new flavor substances and active ingredients, breaking through the functional limits of the raw materials themselves. At the same time, through microbial metabolites (such as extracellular polysaccharides) and the fermentation process, an internal, self-stable system can be built for the product, thereby reducing dependence on various chemical stabilizers and pursuing a more natural state for the product. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a low-alcohol, high-activity Astragalus secondary fermentation milk wine and its preparation method. This invention solves the technical problems of low extraction rate of Astragalus active ingredients, difficulty in achieving both high activity and low alcohol content, microbial competitive inhibition, and poor compatibility between active ingredients and milk base by constructing a secondary fermentation system, a ternary symbiotic fermentation mechanism, and a two-stage solid-state fermentation system with seamless transitions between the two stages.

[0007] This invention discloses a low-alcohol, high-activity Astragalus secondary fermentation milk wine, the specific technical solution of which is as follows:

[0008] A low-alcohol, high-activity Astragalus secondary fermented milk wine, whose core functional component is derived from Astragalus-whey synergistic active peptides transformed through a time-sequential ternary solid-state symbiotic fermentation system. This system uses functionalized microbial matter obtained from the pre-fermentation of Astragalus by edible fungi as the primary active matrix, which together with whey protein forms a complex fermentation substrate. Subsequently, through the synergistic symbiotic effect of lactic acid bacteria and low-alcohol yeast, the directed enzymatic hydrolysis of proteins and the self-assembly of flavor substances are completed, ultimately forming an active fermentation product that integrates natural antibacterial properties, a self-stabilizing system, and harmonious flavor.

[0009] This invention also discloses a method for preparing a low-alcohol, high-activity Astragalus secondary fermentation milk wine, such as... Figure 1 As shown, the specific technical solution is as follows:

[0010] Step 1: Mix the ultrafine powder of Astragalus membranaceus with the excipients and adjust the moisture content. After sterilization, obtain the solid fermentation substrate of Astragalus membranaceus. Inoculate the substrate with a specific edible fungus strain and carry out pure solid fermentation under controlled temperature and humidity conditions. This allows the mycelium of the edible fungus to grow fully and penetrate the Astragalus membranaceus substrate, completing the primary biotransformation and functional modification of Astragalus membranaceus cells, and obtaining edible fungus-Astragalus membranaceus fermented mycelium rich in primary active ingredients.

[0011] Step 2: The edible fungus-Astragalus fermentation microbial material obtained in Step 1 is combined with whey protein powder and a limited carbon source in a precise ratio to construct a secondary fermentation solid substrate. Then, a specific ratio of lactic acid bacteria and low-alcohol-producing yeast is simultaneously inoculated into the substrate, and a ternary symbiotic fermentation is carried out under specific temperature and humidity conditions. This allows the three types of microorganisms to undergo complex metabolic interactions and synergistic transformations in the solid system, resulting in a solid fermented cake-like product that integrates the target flavor and synergistic active peptides.

[0012] Step 3: The solid fermented cake obtained in Step 2 is extracted with warm water. A clear extract is obtained through solid-liquid separation. The extract is then allowed to stand and mature at low temperature to promote further association of flavor substances and system stabilization, ultimately obtaining a low-alcohol, high-activity Astragalus secondary fermented milk wine with a clear body and harmonious flavor.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] 1. By utilizing the powerful enzymatic hydrolysis ability of edible fungi, the cell walls of Astragalus membranaceus are completely broken down, resulting in the degradation of complex polysaccharides and macromolecular saponins into easily absorbed small molecules, which improves the extraction rate and bioavailability of effective ingredients. At the same time, multi-stage fermentation produces new products, giving the product more comprehensive functional activities.

[0015] 2. A ternary synchronous fermentation mode of edible fungi, lactic acid bacteria and yeast was constructed in a solid substrate, which avoided the problem of mutual inhibition between strains in traditional mixed fermentation. The bioreactor effect of edible fungi mycelium can improve the mass transfer bottleneck of solid fermentation and stably support the symbiotic metabolism of lactic acid bacteria and yeast, thus successfully coordinating the functions of the three types of microorganisms in the same system.

[0016] 3. By designing a low-nutrient substrate and using low-alcohol-producing yeast, the alcohol production is fundamentally reduced to an extremely low level, achieving the goal of low-alcoholization in fermented beverages. At the same time, the synergistic effect of lactic acid bacteria and yeast significantly increases the content of organic acids and esters in the fermentation products, which can mask the bitter taste of Astragalus membranaceus itself and balance the flavor and functionality of the product. Attached Figure Description

[0017] Appendix Figure 1 This is a flowchart illustrating the preparation process of the Astragalus secondary fermentation milk wine of the present invention.

[0018] Appendix Figure 2 The diagram shows the alcohol content of the products in the embodiments and comparative examples of this invention.

[0019] Appendix Figure 3 The diagram shows the astragalus polysaccharide content of the embodiments and comparative products of this invention;

[0020] Appendix Figure 4 The quality standard curve of astragaloside A;

[0021] Appendix Figure 5 The graph shows the total saponin content of the products in the embodiments and comparative examples of this invention. Detailed Implementation

[0022] The following embodiments further explain and illustrate the technical solutions of the present invention. It should be specifically noted that each specific embodiment is a concretization and explanation of the technical solution and should not be considered as a limitation on the scope of protection of the present invention. Those skilled in the art still have the right to modify the technical solutions of these embodiments and make equivalent substitutions for some or all of the technical features, and these modifications or substitutions do not change the essence of the corresponding technical solutions, nor do they cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the present invention.

[0023] This invention proposes a low-alcohol, high-activity Astragalus secondary fermentation milk wine. It uses functionalized microbial products obtained from the pre-fermentation of Astragalus by edible fungi and whey protein to form a composite fermentation matrix. Through a time-sequential ternary solid-state symbiotic fermentation system, the final product has the characteristics of promoting the synergistic effect of Astragalus and whey protein active ingredients, constructing a self-stabilizing system that does not rely on exogenous additions, and naturally achieving low-alcohol properties through microbial metabolism.

[0024] This invention also proposes a method for preparing a low-alcohol, high-activity Astragalus secondary fermentation milk wine, such as... Figure 1 As shown, the specific technical solution is as follows:

[0025] 1. Directed solid-state pre-fermentation of Astragalus membranaceus was conducted using macroed edible fungi as the fermentation strain. Under pure culture conditions without the addition of other microorganisms, the pretreated Astragalus membranaceus solid-state fermentation substrate was fermented. The mycelium of the edible fungi grew rapidly in the solid substrate, utilizing its secreted enzyme systems such as cellulase, hemicellulase, pectinase, and protease to comprehensively degrade the fibrous and macromolecular substances in Astragalus membranaceus. Through this process, the previously difficult-to-release active ingredients of Astragalus membranaceus were completely released, increasing the leaching rate of polysaccharides, saponins, and other effective components. Simultaneously, under the metabolic action of the edible fungi, some components of Astragalus membranaceus could be transformed into new functional products as precursors, or secondary metabolites produced by the edible fungi could combine with Astragalus membranaceus components to generate new synergistic active substances. Furthermore, the edible fungi had a certain modifying effect on the original bitter flavor of Astragalus membranaceus, and the enzymatic reactions produced during the fermentation process also partially reduced the herbal odor. The product obtained in the first stage of fermentation was fermented Astragalus membranaceus mycelium, which was rich in Astragalus membranaceus components and mycelial networks that had been initially degraded by the edible fungi.

[0026] 2. After dry mixing the fermented Astragalus mycelium with whey protein powder, a secondary fermentation with multiple components is carried out. In this stage, lactic acid bacteria and yeast are introduced into the solid substrate, forming a ternary microbial symbiotic system together with the remaining edible fungi mycelium. Whey protein powder is used as an excipient and uniformly mixed with the fermented Astragalus mycelium to form a protein-rich and low-sugar solid fermentation substrate. Because this substrate contains a large amount of mycelial network from the first stage, the edible fungi can quickly resume growth and occupy a spatial advantage after mixing. Therefore, the inoculated lactic acid bacteria and low-alcohol-producing yeast colonize and symbiotically ferment in the natural mycelial reactor constructed by the edible fungi. The edible fungi mycelial network helps increase the substrate porosity and improve the efficiency of oxygen and nutrient transfer, which is equivalent to establishing a micro-circulation channel in the solid system, which is beneficial to the subsequent growth and metabolism of bacteria and yeast. Lactic acid bacteria and yeast interact and proliferate synergistically in this network. Lactic acid bacteria decompose and utilize some whey protein to produce lactic acid and other organic acids and various enzymatic hydrolysis products; yeast utilizes fermentable sugars for metabolism, producing a small amount of ethanol and synthesizing various volatile aroma components such as esters and alcohols. Through this series of complex microbial metabolic interactions and biochemical transformations, the dry mixed substrate gradually becomes moist and viscous, eventually forming a solid fermented cake-like substance with a compact structure, uniform texture, and rich in microbial metabolites.

[0027] 3. The solid-state fermented cake-like material is extracted using warm water extraction technology to target and dissolve the active ingredients. Warm water efficiently dissolves the astragalus active ingredients (such as fermented and modified polysaccharides and saponins), whey protein source synergistic active peptides, and flavor precursors produced by yeast, while avoiding damage to heat-sensitive components. After extraction, solid-liquid separation is performed by centrifugation or filtration to remove undissolved mycelia and solid residues, yielding the initial extract. This extract is then allowed to mature under low-temperature conditions to obtain a low-alcohol functional milk wine with excellent taste and flavor, possessing both astragalus bioactivity and protein peptide nutrition. During this process, various molecules in the solution undergo slow and complex physicochemical reactions, enhancing the product's flavor. Simultaneously, any small suspended particles or colloidal substances present in the solution gradually settle or aggregate during this stage, greatly improving the product's physical stability.

[0028] The following are some specific embodiments of the present invention, and Table 1 shows the raw material information used in the embodiments.

[0029] Table 1 Raw Material Information Table

[0030]

[0031] Example 1

[0032] S1: After pulverizing Astragalus membranaceus, pass it through an 80-mesh sieve. Weigh 150g of Astragalus membranaceus powder and 50g of wheat bran into a sterile fermentation dish, mix thoroughly, add 200g of deionized water and stir well. Seal the fermentation dish with heat-resistant plastic wrap and autoclave at 121℃ for 30 minutes. After sterilization, transfer to a sterile workbench and cool to room temperature. Then, under sterile conditions, evenly sprinkle 1.0g of Hericium erinaceus mycelium powder on the surface of the sterilized substrate. Seal the fermentation dish again and place it in a constant temperature incubator. Ferment at 26℃ and 80% humidity in the dark for 96 hours until the substrate is completely covered by white mycelium and forms clumps, obtaining Hericium erinaceus-Astragalus membranaceus fermented mycelium. Store it in a sealed container at 4℃ for later use.

[0033] S2: 1.0g of *Lactobacillus casei* powder was inoculated into 100mL of sterilized MRS broth medium and incubated at 37℃ for 18h to obtain activated bacterial solution 1; 1.0g of *Kluyveromyces martensii* powder was inoculated into 100mL of sterilized YPD broth medium and incubated at 28℃ with shaking at 150rpm for 24h to obtain activated bacterial solution 2. In a sterile operating table, the *Hericium erinaceus*-*Astragalus membranaceus* fermentation mycelium obtained in S1 was crushed, and 100g of whey protein powder and 5g of glucose were added and mixed evenly. Activated bacterial solution 1 and activated bacterial solution 2 were simultaneously added to this mixed matrix. After the bacterial solutions were evenly absorbed by the matrix, the inoculated mixture was compacted under sterile conditions, sealed in a fermentation container, and then placed in a constant temperature incubator at 22℃ for static fermentation for 144h to obtain a dense, fermented solid fermented cake-like substance with a fermented flavor.

[0034] S3: Transfer the solid fermented cake obtained in S2 to a clean container, add sterile deionized water at 45℃ to make the solid-liquid ratio 1:5, and slowly stir and extract for 2 hours. Then filter using eight layers of sterile gauze and collect the filtrate. Centrifuge the filtrate at 8000 rpm for 15 minutes at 4℃ and collect the supernatant. Refrigerate the supernatant at 4℃ and allow it to mature for 168 hours. Then, aliquot the product into sterile sample bottles and store at -20℃. This sample is the low-alcohol, high-activity Astragalus secondary fermented milk wine.

[0035] Example 2

[0036] The preparation method is the same as in Example 1, except that:

[0037] S1: Weigh 100g of Astragalus powder, replace Hericium erinaceus powder with Shiitake mushroom powder, and ferment the fermentation tray in a constant temperature incubator at a temperature of 24℃, a humidity of 75%, and a fermentation time of 72h.

[0038] S2: Replace Lactobacillus casei with Lactobacillus acidophilus, replace Kluyveromyces martensii with Candida albicans, add 37.5g of whey protein powder, replace glucose with fructose, ferment at 20℃, and ferment for 96 hours;

[0039] S3: The temperature of the sterile deionized water during extraction is 40℃, the extraction time is 1 hour, and the post-ripening time is 72 hours. All other steps are the same.

[0040] Example 3

[0041] The preparation method is the same as in Example 1, except that:

[0042] S1: The amount of Astragalus powder is 250g. The Hericium erinaceus powder is replaced with Poria cocos powder. The fermentation temperature in the fermentation tray in the constant temperature incubator is 28℃, the fermentation humidity is 90%, and the fermentation time is 120h.

[0043] S2: Replace Lactobacillus casei with Lactobacillus plantarum, replace Kluyveromyces martensii with Pichia pastoris, add 450g of whey protein powder, replace glucose with sucrose, ferment at 25℃, and ferment for 168h.

[0044] S3: The temperature of the sterile deionized water during extraction is 55℃, the extraction time is 3 hours, and the post-ripening time is 240 hours. All other steps are the same.

[0045] Example 4

[0046] The preparation method is the same as in Example 1, except that:

[0047] S1: The amount of Astragalus powder weighed is 200g. The fermentation temperature in the fermentation tray in the constant temperature incubator is 25℃, the fermentation humidity is 85%, and the fermentation time is 108h.

[0048] S2: 250g of whey protein powder was added, the fermentation temperature was 24℃, and the fermentation time was 136h;

[0049] S3: The temperature of the sterile deionized water during extraction is 50℃, the extraction time is 1.5h, and the post-ripening time is 120h. All other steps are the same.

[0050] Comparative Example 1

[0051] The preparation method is the same as in Example 1, except that:

[0052] S1: After grinding Astragalus membranaceus into powder, pass it through an 80-mesh sieve. Weigh out 150g of Astragalus membranaceus powder, 50g of wheat bran, 100g of whey protein powder, and 5.0g of glucose, mix them evenly, and use them as a mixing matrix.

[0053] S2: Simultaneously inoculate activated bacterial solution 1 and activated bacterial solution 2 into the mixed substrate obtained in S1. After mixing evenly, place the mixture in a constant temperature incubator at 22℃ and let it ferment for 144 hours. The remaining steps are the same.

[0054] This comparative preparation omits the first pre-fermentation and directly uses raw Astragalus powder that has not been treated with edible fungi in the second fermentation to prepare Astragalus milk wine.

[0055] Comparative Example 2

[0056] The preparation method is the same as in Example 1, except that:

[0057] S2: Inoculate activated bacterial solution 1 into the mixed substrate and ferment at 22°C for 48 hours. Then inoculate activated bacterial solution 2 into the fermented substrate and ferment at 22°C for 96 hours. The remaining steps are the same.

[0058] This comparative preparation altered the inoculation sequence, first inoculating lactic acid bacteria to establish dominance, and then inoculating yeast to create a competitive environment, rather than simultaneously inoculating to construct a ternary synchronous symbiotic fermentation system to prepare Astragalus secondary fermented milk wine.

[0059] Comparative Example 3

[0060] The preparation method is the same as in Example 1, except that:

[0061] S1: After crushing Astragalus membranaceus into powder, pass it through an 80-mesh sieve. Weigh out 150g of Astragalus membranaceus powder, 50g of wheat bran, 100g of whey protein powder, and 5.0g of glucose. Mix them evenly and add 305g of deionized water while stirring continuously. Then, put the mixture into a fermentation pan and seal it. Transfer the fermentation pan to an autoclave and sterilize it at 121℃ for 30 minutes as the mixing substrate.

[0062] S2: Simultaneously inoculate Hericium erinaceus powder, activated bacterial solution 1, and activated bacterial solution 2 into the mixed substrate obtained in S1, and ferment continuously at 26℃ for 240 hours. All other steps are the same.

[0063] This comparative preparation of three strains of bacteria competed with each other, resulting in insufficient conversion of Astragalus membranaceus and thus Astragalus membranaceus milk wine, which was not obtained through sequential secondary fermentation.

[0064] Experimental Example 1

[0065] Measure 100 mL of the products prepared in Examples 1-4 and Comparative Examples 1-3 respectively, and place each into a 500 mL distillation flask. Rinse the graduated cylinder three times with 50 mL of deionized water, and add the washings to the distillation flask. Add a small amount of boiling chips to prevent bumping. Set up the distillation apparatus, insert the outlet of the condenser into a 100 mL volumetric flask, turn on the cooling water, and start heating the system simultaneously. Control the heating temperature so that the distillate flow rate is about 3-4 mL / min. When the volume of the distillate reaches about 95 mL, stop heating, rinse the inner wall of the condenser with a small amount of water, add the washings to the volumetric flask, and finally add water to the 100 mL mark. Mix well. This distillate is the test solution. Wash and dry the density flask and weigh it, recording the mass as m0. Fill the density flask with freshly boiled and cooled distilled water to 15-20°C, insert a thermometer, and place the density flask in a constant temperature water bath at 20°C. Immerse the bottle for at least 30 minutes, ensuring the temperature inside and outside is uniform. Quickly absorb the water overflowing from the capillary side hole with filter paper. Remove the density bottle, wipe the outer wall dry, immediately cap the side hole, and weigh it. Record the mass as m_water. Empty and dry the density bottle, rinse it 2-3 times with distillate, then fill it with distillate. Insert a thermometer and place the density bottle in a constant temperature water bath at 20°C. Immerse the bottle for at least 30 minutes, ensuring the temperature inside and outside is uniform. Quickly absorb the water overflowing from the capillary side hole with filter paper. Remove the density bottle, wipe the outer wall dry, immediately cap the side hole, and weigh it. Record the mass as m_sample. Calculate the relative density d of the alcohol in the effluent at 20°C. The calculation method is as follows:

[0066] d=

[0067] Based on the alcohol relative density and mass fraction comparison table, the alcohol content of each product at 20℃ was calculated and expressed as %vol. The test results are shown in Table 2 and [Table 3]. Figure 2 As shown.

[0068] Table 2. Alcohol content in the products of the examples and comparative examples

[0069]

[0070] From Table 2 and Figure 2As can be seen, the alcohol content of the example samples was lower than that of the comparative samples, and the alcohol content of the example samples did not exceed 1.5% vol. According to the requirements of GB 2758-2012 National Food Safety Standard - Fermented Wines and Their Blended Wines, the products meet the low-alcohol standard. In Comparative Example 1, because the fermentable sugars in the astragalus powder were not pre-consumed by the edible fungi, more substrate was provided for the yeast, leading to increased alcohol production, exceeding the low-alcohol level specified in the standard, and therefore it does not belong to the low-alcohol beverage category. In Comparative Example 2, the lactic acid bacteria fermented and produced acid first, consuming some nutrients and severely inhibiting the growth and metabolism of the subsequently added yeast, resulting in a significant decrease in the yeast's ability to produce alcohol and esters, thus resulting in a lower alcohol content. However, this also led to a slightly acidic taste, affecting the drinkability. In Comparative Example 3, all strains and raw materials coexisted initially, and the yeast had the richest sugar source from astragalus, wheat bran, and glucose, resulting in the highest alcohol production, which also did not meet the low-alcohol requirement for the product.

[0071] Experiment Example 2

[0072] Take 1.0 mL of each of the milk wine products prepared in Examples 1-4 and Comparative Examples 1-3, add them to a centrifuge tube, add 24.0 mL of deionized water, mix well, and then transfer 2.0 mL to another centrifuge tube. Add 2.0 mL of 30% trichloroacetic acid solution, mix well, and let stand for 10 min. Take 1.0 mL of the supernatant in a test tube, then add 0.5 mL of 5% phenol aqueous solution to the test tube, mix immediately, then add 2.5 mL of concentrated sulfuric acid, vortex immediately, and place in a 25°C water bath for 20 min. At the same time, take a clean test tube, add 1.0 mL of deionized water, and then add phenol aqueous solution and concentrated sulfuric acid to react, as a blank control group. After the reaction, remove the test tubes and cool them to room temperature. Set the wavelength of the ultraviolet spectrophotometer to 490 nm, zero the absorbance with the absorbance of the blank control group, and measure the absorbance of the solution in each sample tube. Calculate the glucose mass based on the absorbance, and use the formula to calculate the astragalus polysaccharide content in the milk wine product:

[0073] Astragalus polysaccharide content (mg / 100mL) = (glucose mass × 25 × 0.9) × 100

[0074] The test results are shown in Table 3 and Figure 3 As shown.

[0075] Table 3. Astragalus polysaccharide content in the products of the examples and comparative examples

[0076]

[0077] From Table 3 and Figure 3As can be seen, the samples in the examples all have high astragalus polysaccharide content, indicating that the products in the examples have a stronger effect on enhancing immunity. Comparative Example 1 did not undergo pre-fermentation and lacked the cell wall-breaking process of edible fungi on astragalus. The water-soluble polysaccharides were physically encapsulated by the tough cell walls, lacking cellulase, pectinase, etc., secreted by the edible fungi. The cell walls remained intact, resulting in extremely low polysaccharide dissolution rate during warm water extraction. In Comparative Example 2, lactic acid bacteria preferentially occupied the ecological niche, and the polysaccharides were released in the first fermentation, but their subsequent transformation pathways were altered. After yeast was inhibited, its metabolic activities lost their further modification or degradation effect on polysaccharides. At the same time, the metabolic direction of the system was completely biased towards acid production, affecting the stability of polysaccharides, resulting in a slightly lower content than in the complete symbiotic system. In Comparative Example 3, the slow-growing edible fungi were strongly inhibited by the rapidly growing lactic acid bacteria and yeast, and their core functions could not be performed. They could not effectively establish a hyphal network and secrete cell wall-breaking enzyme systems. Therefore, the astragalus cell walls remained intact, resulting in the ineffective release of polysaccharides.

[0078] Experimental Example 3

[0079] Weigh 5 mg of astragaloside A standard, dissolve it in methanol, and dilute to 50 mL to obtain a 0.1 mg / mL stock solution. Pipette 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of the stock solution into stoppered test tubes. Evaporate the methanol in a warm water bath. Add 0.2 mL of 5% vanillin-glacial acetic acid solution to the residue in each test tube, followed by 0.8 mL of perchloric acid. Vortex to mix, seal the test tubes tightly, and heat in a 60°C water bath for 15 min. Immediately remove and cool to room temperature in an ice-water bath. Add 5 mL of glacial acetic acid to each tube, vortex to mix, and measure the absorbance (A) at 550 nm, using the tube with 0% as a blank. Plot a standard curve with the mass of astragaloside A as the x-axis and absorbance (A) as the y-axis to obtain the regression equation. Take 5 mL of the milk wine products prepared in Examples 1-4 and Comparative Examples 1-3 into an evaporating dish, evaporate to dryness in a water bath, dissolve the residue in a small amount of methanol and transfer it to a stoppered test tube, heat in a water bath to evaporate the methanol, add 0.2 mL of 5% vanillin-glacial acetic acid solution to the residue in the test tube, then add 0.8 mL of perchloric acid, vortex to mix, seal the test tube tightly, heat in a 60°C water bath for 15 min, immediately remove and cool to room temperature in an ice-water bath, add 5 mL of glacial acetic acid to each tube, vortex to mix, measure the absorbance at a wavelength of 550 nm, calculate the mass of astragaloside A based on the absorbance, and calculate the total saponin content using the formula:

[0080] Total saponin content (mg / 100mL) = [Astragaloside A mass (μg) / 5] × 0.1

[0081] The test results are shown in Table 4 and Figure 4 , Figure 5 As shown.

[0082] Table 4. Total saponin content in the products of the examples and comparative examples

[0083]

[0084] From Table 4 and Figure 4 , Figure 5 As can be seen, the products in the examples all have high total saponin content, indicating that they all possess good antioxidant and anti-fatigue bioactivity, reflecting their high activity characteristics. However, in Comparative Example 1, the lack of edible fungi to break down the cell walls of Astragalus membranaceus resulted in the physical blockage of saponins by the cell walls. Furthermore, since saponins often exist in glycosylated form, the enzyme systems of edible fungi, such as glycosidases, can biotransform them. The absence of this step meant that saponins were not only difficult to release but also not effectively activated. In Comparative Example 2, the deep transformation of saponins relies on a complex metabolic network involving multiple microorganisms. In this product, the inhibition of yeast disrupted this network. The single action of lactic acid bacteria cannot completely replace the synergistic effect of the ternary system, leading to insufficient biotransformation of saponins and failing to maximize their final yield and active form. In Comparative Example 3, due to the inactivation of the edible fungi, neither of the two key functions of cell wall breaking and release nor enzymatic transformation of saponins could be initiated. The entire system was equivalent to a complex fermentation of raw Astragalus membranaceus, resulting in extremely low saponin dissolution and conversion rates.

Claims

1. A method for preparing a low-alcohol, high-activity Astragalus secondary fermentation milk wine, characterized in that, It is prepared through the following steps: S1. Astragalus powder is mixed with excipients and the moisture content is adjusted. After sterilization, an astragalus solid-state fermentation substrate is obtained. Large edible fungi are inoculated and pure-culture solid-state fermentation is carried out under temperature and humidity controlled conditions to allow the mycelium of the edible fungi to grow fully and penetrate the astragalus substrate, completing the primary biotransformation and functional modification of the astragalus cells, and obtaining edible fungi-astragalus fermented mycelium rich in primary active ingredients; the large edible fungi are one or more of Hericium erinaceus mycelium, Lentinus edodes mycelium, and Poria cocos mycelium. S2. The edible fungus-Astragalus fermentation inoculum obtained in S1 is combined with whey protein powder and a limited carbon source in a certain proportion to construct a secondary fermentation solid substrate. Then, lactic acid bacteria and low-alcohol-producing yeast are simultaneously inoculated into the substrate for ternary symbiotic fermentation, so that the three types of microorganisms undergo complex metabolic interactions and synergistic transformations in the solid system to obtain a solid fermented cake-like product integrating the target flavor and synergistic active peptides. The lactic acid bacteria are one or more of Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus plantarum, and the low-alcohol-producing yeast is one or more of Kluyveromyces martensii, Candida albicans, and Pichia pastoris. S3. The solid fermented cake obtained in S2 is extracted with warm water, and a clear extract is obtained by solid-liquid separation. The extract is then allowed to stand and mature at low temperature to promote further association of flavor substances and system stabilization, and finally, Astragalus secondary fermented milk wine is obtained.

2. The method for preparing a low-alcohol, high-activity Astragalus secondary fermentation milk wine according to claim 1, characterized in that: The mass ratio of Astragalus powder to excipients in the Astragalus solid fermentation substrate of S1 is (2:1) to (5:1), the fermentation temperature of the pure culture solid fermentation is 24 to 28°C, the fermentation humidity is 75% to 90%, and the fermentation time is 72 to 120 hours.

3. The method for preparing a low-alcohol, high-activity Astragalus secondary fermentation milk wine according to claim 1, characterized in that: The primary active ingredients described in S1 are soluble small-molecule polysaccharides, saponins, and flavonoid active components released by enzymatic hydrolysis of the Astragalus cell wall.

4. The method for preparing a low-alcohol, high-activity Astragalus secondary fermentation milk wine according to claim 1, characterized in that: The carbon source mentioned in S2 is one or more of glucose, fructose, and sucrose.

5. The method for preparing a low-alcohol, high-activity Astragalus secondary fermentation milk wine according to claim 1, characterized in that: The mass ratio of edible fungi-Astragalus fermentation microbiota to whey protein powder in S2 is (1:0.25)~(1:1.5), and the fermentation temperature of the ternary symbiotic fermentation is 20~25℃, and the fermentation time is 96~168h.

6. The method for preparing a low-alcohol, high-activity Astragalus secondary fermentation milk wine according to claim 1, characterized in that: The extraction temperature described in S3 is 40~55℃, the extraction time is 1~3h, and the post-ripening time is 72~240h.

7. The milk wine prepared by the method for preparing low-alcohol, high-activity Astragalus secondary fermentation milk wine according to any one of claims 1 to 6, characterized in that: The milk wine has an alcohol content of ≤1.5%, an astragalus polysaccharide content of ≥8.5mg / 100mL, and a total saponin content of ≥2.5mg / 100mL.

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