Compound fermented plant beverage with uric acid-lowering and antioxidant activity and fermentation method

Abstract

The application discloses a compound fermented plant beverage with uric acid reducing and antioxidant activities and a fermentation method, and belongs to the technical field of probiotic fermentation of plant raw materials. The beverage takes hawthorn, Chinese wolfberry and celery as main raw materials, degrades cell wall polysaccharides of the raw materials into fermentable substrates through selective enzymolysis technology of raw materials, adopts three-strain stepwise inoculation and three-stage anaerobic fermentation process, and obtains a plant beverage rich in various active ingredients such as oligomeric procyanidins, short-chain inulin, short-chain fatty acids and oleanolic acid and having excellent sensory quality and storage stability. The application solves the technical bottlenecks of low raw material utilization rate, poor functional component conversion efficiency and easy stratification and deterioration of the existing compound plant fermented beverage, and the process is stable and controllable and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of probiotic fermentation technology of plant raw materials, and in particular to a compound fermented plant beverage with uric acid-lowering and antioxidant activities and a fermentation method thereof. Background Technology

[0002] Microbial fermentation technology is a core technology for achieving the biotransformation, enrichment, and synthesis of new functional components in plants. Through the metabolic action of probiotics, active precursors bound to plant cell walls can be converted into free, highly bioavailable components, while simultaneously synthesizing various beneficial secondary metabolites. Compared with traditional physical extraction processes, fermentation technology has significant advantages such as mild reaction conditions, environmental friendliness, the ability to produce new functional components, and improved product flavor, and has become the mainstream technology direction for the development of functional plant foods.

[0003] Most existing plant fermentation technologies employ a pure liquid fermentation model of extraction followed by fermentation: that is, a water-soluble extract of the plant material is first obtained through water extraction or alcohol extraction, and then probiotics are inoculated for liquid fermentation. However, this fermentation method has inherent drawbacks that are difficult to overcome: 1. Extremely low raw material utilization: The extraction process can only recover water-soluble components. A large number of fat-soluble components, glycoside precursors, and polysaccharides bound in plant cell walls are discarded with the residue, and the total substrate utilization rate is usually only 60-70%. For example, about 30% of apigenin in celery exists in a bound state in the cell wall and cannot be obtained by water extraction, which directly leads to insufficient substrate for subsequent biotransformation.

[0004] 2. Low conversion efficiency of functional components: The lack of a solid-phase carrier in the liquid fermentation system makes it difficult for microorganisms to attach and form a stable biofilm, resulting in significantly lower secretion of metabolic enzymes compared to the solid-liquid mixed system. Experimental data show that, under the same conditions, the activity of β-glucosidase in liquid fermentation is only 40-50% of that in solid-liquid mixed fermentation, leading to a conversion rate of apigenin to apigenin that is generally below 60%, and a depolymerization rate of high-polymer proanthocyanidins that is below 50%.

[0005] 3. Low product concentration and poor production efficiency: To ensure uniform mass transfer, liquid fermentation typically uses a low substrate concentration (solid-liquid ratio of 1:5 or higher), resulting in limited product yield per unit volume of fermentation broth. Simultaneously, the extraction process consumes significant amounts of energy and solvents, increasing production costs and environmental pressure.

[0006] For multi-raw material fermentation systems such as celery, hawthorn, and goji berries, existing technologies still face the dual challenges of multi-raw material interaction inhibition and multi-strain synergy difficulties: 1. Mutual inhibition among raw materials: The chemical composition of different plant raw materials is complex and diverse, and antagonistic effects are easily produced when they are mixed. For example, the high concentration of organic acids in hawthorn can significantly inhibit the dissolution and biological activity of wolfberry polysaccharides; flavonoids in celery can inhibit the growth of some probiotics; in untreated compound raw material systems, the initial growth lag phase of strains can be extended by 2 to 4 times.

[0007] 2. Significant differences in the physiological characteristics and metabolic requirements of bacterial strains: Complex fermentation typically requires the simultaneous use of multiple probiotics for synergistic fermentation. Current technologies generally employ a crude approach of one-time mixed inoculation and uniform culture conditions. For example, aerobic lactobacilli consume large amounts of oxygen in the early stages of fermentation, causing a large number of bifidobacteria to die due to premature oxygen exposure. Conversely, if an anaerobic environment is established in advance to protect bifidobacteria, it will inhibit the aerobic proliferation of lactobacilli, resulting in insufficient bacterial concentration and inability to complete subsequent biotransformation tasks.

[0008] 3. Substrate competition and metabolic inhibition among strains: Existing multi-strain formulations are mostly based on experience, lacking clear functional division and optimization of inoculum ratios. Different strains often compete for the same carbon and nitrogen sources, resulting in dominant strains being unable to fully utilize their metabolic functions, while inferior strains struggle to survive; the metabolites of some strains can also inhibit other strains, for example, excessive lactic acid can significantly reduce the metabolic activity of Bifidobacteria.

[0009] Therefore, there is a need for a fermentation method for compound fermented plant beverages that can achieve synergistic transformation and enrichment of multiple plant functional components, clear functional division of strains, and finely controlled fermentation process, in order to solve the technical problems of low raw material utilization, poor synergy among multiple strains, and low product conversion efficiency in existing technologies. Summary of the Invention

[0010] To address the aforementioned technical problems in the prior art, this invention aims to provide a compound fermented plant beverage with uric acid-lowering and antioxidant activities, as well as a fermentation method thereof.

[0011] One objective of this invention is to provide a fermentation method for a compound fermented plant beverage with uric acid-lowering and antioxidant activities. The fermentation method employs a three-stage stepwise fermentation process and includes the following steps: (1) Raw material pretreatment Hawthorn, wolfberry and celery were enzymatically hydrolyzed to obtain hawthorn enzymatic hydrolysate, wolfberry enzymatic hydrolysate and celery enzymatic hydrolysate; (2) Combine and adjust the viscosity The three enzymatic hydrolysates were combined and added to the fermentation tank at a mass ratio of 1:1~2:3~5 of dried hawthorn, wolfberry and celery. The viscosity of the system at 30~40℃ was adjusted to 200~600 mPa·s using maltitol solution and water. Then, the pH was adjusted to 5.6-5.8 using a composite buffer of potassium citrate and dipotassium hydrogen phosphate, and then calcium carbonate microsphere slow-release buffer was added. (3) First stage fermentation Simultaneously, *Lactobacillus reuteri* CGMCC 1.2838 and *Lactobacillus rhamnosus* CGMCC 1.2467 were inoculated into the fermenter. The inoculation amount of *Lactobacillus reuteri* was 0.8 × 10⁻⁶ based on the total dry weight of the raw materials. 8 CFU / g, the inoculum size of Lactobacillus rhamnosus was 0.3 × 10⁻⁶. 8 CFU / g; After inoculation, start timing from 0h, control the fermentation temperature at 37±1℃, and ferment until 12h; (4) Second stage fermentation Within 30 minutes of the completion of the first stage of fermentation, Bifidobacterium bifidum CGMCC 1.5029 was inoculated at a rate of 0.2 × 10⁻⁶. 8 CFU / g; After inoculation, the fermentation temperature was controlled at 37±1℃ and ended at 24h; (5) Third stage fermentation The third stage of fermentation is carried out at a temperature of 34±1℃, and fermentation is terminated at 36 hours or when the conversion rate of apigenin in the fermentation broth is ≥90%. Based on 0h, purified water equal to 5% of the total dry weight of the initial raw materials was added at 24h and 30h.

[0012] Preferably, the first step of inoculation is top spray inoculation. Before inoculation, turn on the agitator to 12 r / min and stir for 2 min. Then, inoculate at a speed of 5~10 L / min under stirring conditions. After inoculation, continue stirring for 5 min and then turn off the agitator.

[0013] Preferably, the second step of vaccination employs bottom-distributed vaccination.

[0014] Preferably, before the enzymatic hydrolysis in step (1), the hawthorn is pulverized to 100 mesh, the wolfberry to 40 mesh, and the celery to 120 mesh.

[0015] Preferably, the chitosan membrane coating calcium carbonate microspheres has a particle size of 10~20μm and a thickness of 0.8~1.2μm.

[0016] Preferably, in the step (2), inulin is added as a dispersant, and the amount of inulin added is 0.2~0.3% of the dry weight of the raw material.

[0017] Preferably, the dry weight ratio of hawthorn, wolfberry and celery is 1:1.5:4.

[0018] Preferably, the fermentation method further includes step (6): after the fermentation is terminated, the mixture is filtered, homogenized and sterilized in sequence to obtain a compound fermented plant beverage.

[0019] Preferably, the homogenization adopts a two-stage homogenization process, with the first-stage pressure controlled at 18~22MPa and the second-stage pressure controlled at 3~7MPa.

[0020] The second objective of this invention is to provide a compound fermented plant beverage with uric acid-lowering and antioxidant activities prepared using the fermentation method described above.

[0021] This invention employs a two-step inoculation and three-stage fermentation process to construct a synergistic metabolic network consisting of liquid-phase proliferation to create the environment, solid-phase colonization to establish the system, and biphase synergistic synthesis. This network enables the synergistic enrichment of five classes of active functional components by *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium bifidum*. In the first stage (0-12h), Lactobacillus and Lactobacillus work synergistically to build the basis of anaerobic metabolism.

[0022] Simultaneously inoculated *Lactobacillus reuteri* and *Lactobacillus rhamnosus* formed a metabolically complementary proliferation system in a liquid-phase microaerobic environment: As a dominant aerobic strain, *Lactobacillus reuteri* rapidly consumes dissolved oxygen in the system through aerobic respiration, reducing the dissolved oxygen concentration to ≤0.3 mg / L, thus creating a strictly anaerobic environment for subsequent inoculation with *Bifidobacterium*. At the same time, it begins to secrete basic extracellular enzymes, initially degrading the plant cell wall structure.

[0023] Lactobacillus rhamnosus utilizes oligosaccharide substrates for synchronous proliferation, secretes α-rhamnosidase to fill the enzyme gap in Lactobacillus reuteri, and hydrolyzes glycosides containing rhamnosyl groups; the trace organic acids produced by its metabolism can moderately lower the local pH, creating conditions for the activation of subsequent functional enzymes.

[0024] With the synergistic effect of the two strains, the total viable count reached 2.0 × 10⁻⁶ after 12 hours. 9 When CFU / g or higher, the cells simultaneously enter the mid-logarithmic growth phase, and the cell viability and enzyme secretion capacity reach their optimal levels.

[0025] In the second stage (12-24 hours), Bifidobacteria colonize in a solid phase, and the three bacteria initially work together to establish a lineage.

[0026] After inoculation with Bifidobacterium, the three strains initially established a synergistic relationship between Lactobacillus enzyme production, Bifidobacterium colonization, and substrate stepwise degradation: Both lactobacilli strains entered a stationary phase, with their proliferation rate slowing significantly and shifting towards highly efficient secretion of functional enzyme systems. *Lactobacillus reuteri* secreted large amounts of β-glucosidase and proanthocyanidin depolymerase, initiating the initial conversion of apigenin and high-polymer proanthocyanidins; *Lactobacillus rhamnosus* continuously secreted α-rhamnosidase, further expanding its substrate utilization range.

[0027] Bifidobacteria rapidly attach to the surface of raw material particles, forming an anaerobic biofilm. They utilize the solid-phase polysaccharide substrate for metabolism, producing a small amount of short-chain fatty acids. Their metabolic acid production, in synergy with the double buffer system, stabilizes the system pH at 4.8–5.2, providing a suitable environment for the catalytic activity of functional enzymes. The above-described pH synergistic regulation mechanism during fermentation is merely an experimental derivation and does not limit the scope of protection of this invention.

[0028] By 24 hours, the colonization rate of Bifidobacteria was ≥90%, the complex enzyme system was basically mature, and the transformation of functional components officially began.

[0029] In the third stage (24-36h), the two-phase closed-loop synergy allows for the simultaneous enrichment of five functional components.

[0030] Based on the bacterial cell and enzyme system constructed in the first and second stages, the three strains form a closed-loop synergistic metabolic network of substrate hydrolysis, product utilization, and environmental optimization, achieving targeted and efficient synthesis of functional components. As the main functional strain, *Lactobacillus reuteri* secretes β-glucosidase to efficiently hydrolyze apigenin to produce free apigenin, and secretes proanthocyanidin depolymerase to depolymerize high-polymer proanthocyanidins into low-polymer proanthocyanidins with antioxidant activity; at the same time, it releases bound oleanolic acid through plant cell wall disruption.

[0031] Lactobacillus rhamnosus metabolizes inulin precursors in celery, hydrolyzing them into highly active short-chain inulin with a molecular weight of 2000-3000 Da, which has strong uric acid-lowering activity. The organic acids it secretes can further reduce the local pH, promote the diffusion of apigenin from plant cells into the liquid phase, and assist Lactobacillus reuteri in increasing the apigenin dissolution rate.

[0032] Bifidobacterium utilizes the monosaccharides produced by the hydrolysis of the former two to metabolize short-chain fatty acids such as acetic acid, propionic acid, and butyric acid; its continuous acid production can precisely stabilize the pH of the system within the optimal catalytic range of enzymes of 4.5~5.0, which in turn significantly improves the activity of functional enzymes secreted by Lactobacillus reuteri; at the same time, through deep cell wall disruption, it further releases bound oleanolic acid, thereby increasing the total enrichment of oleanolic acid.

[0033] In addition, *Lactobacillus reuteri* continuously consumes trace amounts of oxygen in the system, protecting Bifidobacteria and metabolic enzymes from oxidative damage; the metabolic flows of the three strains are interconnected and mutually promote each other, ultimately achieving the simultaneous and efficient enrichment of five functional components: apigenin, oligomeric proanthocyanidins, short-chain inulin, short-chain fatty acids, and oleanolic acid. Detailed Implementation

[0034] The following description includes certain specific details to provide a comprehensive understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments can be implemented without employing one or more of these specific details, but using other methods, components, materials, etc.

[0035] Unless otherwise required by the present invention, throughout the specification and the following claims, the words “comprising” and “including” shall be interpreted in an open-ended, inclusive sense, meaning “including but not limited to”.

[0036] Throughout this specification, the terms "an embodiment," "an embodiment," "a preferred embodiment," or "some embodiments" refer to including, in at least one embodiment, a specific reference element, structure, or feature associated with that embodiment. Therefore, the phrases "in an embodiment," "in a preferred embodiment," or "in some embodiments" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, specific elements, structures, or features may be combined in one or more embodiments in any suitable manner.

[0037] According to a first aspect of the present invention, a fermentation method for a compound fermented plant beverage having uric acid-lowering and antioxidant activities is provided, the fermentation method employing a three-stage stepwise fermentation, the fermentation method comprising the following steps: (1) Raw material pretreatment Hawthorn, wolfberry and celery were enzymatically hydrolyzed to obtain hawthorn enzymatic hydrolysate, wolfberry enzymatic hydrolysate and celery enzymatic hydrolysate; Specifically, hawthorn is pulverized and then enzymatically hydrolyzed using a compound pectinase. The pH is controlled at 4.5-5.0 during the hydrolysis process to obtain hawthorn hydrolysate. The pH of the hawthorn enzymatic hydrolysis process is controlled at 4.5~5.0, which can efficiently degrade pectin and moderately remove methyl ester groups. This invention uses potassium citrate as a pH adjuster to form a buffer pair with the natural citric acid of hawthorn to maintain pH stability during the enzymatic hydrolysis process.

[0038] Specifically, after crushing the hawthorn, add purified water at a solid-liquid mass ratio of 1:1. First, adjust the pH to 4.5-5.0 with potassium citrate, then add 0.04-0.06% of food-grade compound pectinase by dry weight of the raw material, and incubate at 40-50℃ for 10-20 minutes.

[0039] The food-grade compound pectinase contains protopectinase, polygalacturonase, and pectin methylesterase. The food-grade compound pectinase used in this invention is a commercially available industrial-grade product with the following enzyme activity composition: protopectinase ≥ 500 U / g, polygalacturonase ≥ 10000 U / g, and pectin methylesterase ≥ 2000 U / g. Products from different suppliers, as long as they meet the above enzyme activity composition requirements, can be used in this invention and can achieve essentially the same enzymatic hydrolysis effect.

[0040] After the hawthorn enzymatic hydrolysis is completed, the hawthorn enzymatic hydrolysate can be tested. First, the hawthorn enzymatic hydrolysate is pretreated by ethanol precipitation purification to remove small molecule impurities and acid-base interferences. Then, gel permeation chromatography is used to determine that pectin is degraded into small molecule oligosaccharides with a molecular weight of 5000~10000 Da under the action of compound pectinase; the removal rate of methyl ester groups is determined to be 60~70% by acid-base titration.

[0041] Specifically, after the wolfberries are crushed, they are enzymatically hydrolyzed using β-glucanase and amylase, with the pH controlled at 5-6 during the hydrolysis process, to obtain wolfberry hydrolysate.

[0042] The pH of the enzymatic hydrolysis process of wolfberry is controlled at 5-6, which is the common optimal pH for β-glucanase and amylase, and also the stable pH range for wolfberry polysaccharides. The natural pH of the wolfberry raw material is exactly in this range, so no additional adjustment is required.

[0043] Specifically, after crushing the goji berries, purified water is added at a solid-liquid mass ratio of 1:1.5. Then, 0.03% β-glucanase and 0.02% amylase (based on the dry weight of the goji berry raw material) are added, and the mixture is incubated at 35-45℃ for 10-15 minutes. This process decomposes the starch and β-glucan in the goji berries while preserving the bioactivity of the polysaccharides, reducing the viscosity of the system and preventing particle aggregation and stratification during subsequent fermentation.

[0044] Specifically, after celery is crushed, it is enzymatically hydrolyzed using β-glucanase and cellulase, with the pH controlled at 5.5-6 during the hydrolysis process, to obtain celery hydrolysate.

[0045] The celery enzymatic hydrolysis process is controlled at a pH of 5.5–6.0, which takes into account the activity of the complex enzyme, the stability of apigenin, and the compatibility with subsequent fermentation. This invention uses sodium bicarbonate as a pH adjuster, which on the one hand avoids the non-enzymatic acid hydrolysis of apigenin, and on the other hand, the introduced sodium ions can promote the growth of probiotics.

[0046] Specifically, celery was added to purified water at a solid-liquid mass ratio of 1:1. The pH was first adjusted to 5.5-6.0 with sodium bicarbonate. Then, 0.05% β-glucanase and 0.02% cellulase (by dry weight of celery) were added, and the mixture was incubated at 45-55℃ for 15-20 minutes. This process disrupts the dense structure of the celery fiber cell walls, releasing intracellularly bound apigenin, inulin precursors, and oleanolic acid, providing ample substrate for subsequent biotransformation by probiotics.

[0047] The pH control described above is achieved through initial adjustment. Due to the short enzymatic hydrolysis time and the fact that the raw materials themselves have a certain buffering capacity, the pH fluctuation during the enzymatic hydrolysis process is small, and there is no need to add additional acid or alkali for adjustment.

[0048] (2) Combine and adjust the viscosity The three enzymatic hydrolysates were combined and added to the fermentation tank at a mass ratio of 1:1~2:3~5 for hawthorn, wolfberry and celery powder dry materials. The viscosity of the system at 30~40℃ was adjusted to 200~600 mPa·s using maltitol solution and water.

[0049] In this invention, the mass ratio of hawthorn, goji berries, and celery is 1:1~2:3~5. This ratio is beneficial for the synergistic effect of the functional components of the three raw materials. Celery is the main source of apigenin, inulin precursor, and dietary fiber in this invention, and is the core ingredient for the product's uric acid-lowering function. Hawthorn provides proanthocyanidins, pectin, and organic acids, which are the basis for the product's antioxidant function and fermentation pH regulation. Goji berries provide goji polysaccharides and oleanolic acid, which are the core of the product's intestinal health and immune regulation functions. Secondly, this ratio helps meet the metabolic balance requirements of the bacterial strains. The inulin precursor provided by celery is the only substrate for *Lactobacillus rhamnosus* to synthesize short-chain inulin, and the polysaccharides provided by hawthorn and goji berries are the main carbon source for *Bifidobacterium* to synthesize short-chain fatty acids. The 1:1~2:3~5 ratio ensures that the concentration ratio of the three substrates matches the inoculum ratio of the three bacterial strains, avoiding substrate competition and waste. Thirdly, it helps to balance the taste and flavor of the product. Too much celery will result in an overly grassy taste, too much hawthorn will result in an overly acidic taste, and too much goji berries will result in an overly sweet taste. A ratio of 1:1 to 2:3 to 5 makes the product taste sweet and sour, with a fresh plant aroma and no obvious off-flavors.

[0050] In this invention, food-grade maltitol solution and purified water are used to adjust the viscosity of the system to 200-600 mPa·s. During fermentation, this viscosity range ensures stable suspension of solid particles while maintaining good mass transfer efficiency, and provides suitable attachment sites for microorganisms, promoting biofilm formation. Below 200 mPa·s, the particle settling rate increases sharply, forming dense sediment at the bottom, which prevents effective microbial attachment. Above 600 mPa·s, mass transfer efficiency decreases significantly, the system becomes too viscous, and oxygen and nutrients cannot diffuse effectively. Increasing the stirring speed to improve mass transfer leads to increased oxygen intake, which not only disrupts the anaerobic microenvironment of the bottom solid layer, increasing the mortality rate of Bifidobacteria, but also increases the degradation rate of easily oxidized functional components such as oligomeric proanthocyanidins and apigenin. Simultaneously, excessive shear force can damage the existing biofilm structure, causing microbial detachment and a decrease in metabolic enzyme secretion. If metabolites cannot diffuse in time and the local concentration is too high, a significant product inhibition effect will occur, reducing the activity of key functional enzymes such as β-glucosidase and proanthocyanidin depolymerase, and decreasing the conversion rate of apigenin. At the same time, excessively high local organic acid concentrations will cause a sudden drop in pH, further inhibiting the metabolic activity of the cells, and may even trigger cell autolysis, ultimately leading to fermentation failure.

[0051] The maltitol solution is a maltitol solution with a solids content of 75%. Maltitol solution is chosen as a thickening agent because it is not utilized by probiotics, does not interfere with the fermentation process, and can improve the taste and flavor of the product, while having the functional advantage of a low glycemic index. The amount of maltitol solution used is 2 to 2.5 times the total dry weight of the raw materials.

[0052] Then, a composite buffer of potassium citrate and dipotassium hydrogen phosphate was used to adjust the pH to 5.6-5.8, and then calcium carbonate microsphere slow-release buffer was added to construct a dual-buffer pH control system with liquid-phase immediate buffer and solid-phase slow-release buffer.

[0053] Specifically, the mass ratio of potassium citrate to dipotassium hydrogen phosphate is 3:1. The amount of calcium carbonate microsphere slow-release buffer used is 0.02~0.03% of the total dry weight of the raw materials, where the total dry weight of the raw materials refers to the total weight of dried hawthorn, wolfberry, and celery.

[0054] A composite buffer composed of potassium citrate and dipotassium hydrogen phosphate in a 3:1 mass ratio serves as an immediate liquid-phase buffer system. It plays a role in the early and middle stages of fermentation, neutralizing the large amount of lactic acid produced by the rapid proliferation of lactobacilli and maintaining the pH stable within the optimal growth range of 5.6-5.8 for lactobacilli.

[0055] The calcium carbonate microspheres are chitosan-coated calcium carbonate microspheres. As a solid-phase slow-release buffer system, the chitosan coating material begins to slowly degrade when the pH drops below 5.0, releasing calcium carbonate to neutralize the organic acids produced in the later stage of fermentation and stabilizing the pH at 4.5~5.0.

[0056] The above explanation of the mechanism of action of the liquid-phase instantaneous buffer and the solid-phase slow-release buffer forming a dual-buffer pH regulation system is only a reasonable explanation for the experimental phenomena of this invention, and is only used to help understand this technical solution. The scope of protection of this invention is not limited by this theoretical speculation.

[0057] (3) First stage fermentation Simultaneously, *Limosilactobacillus reuteri* CGMCC 1.2838 and *Lactobacillus rhamnosus* CGMCC 1.2467 were inoculated into the fermenter. The inoculum size of *Limosilactobacillus reuteri* was 0.8 × 10⁻⁶ based on the total dry weight of the raw materials. 8 CFU / g, the inoculum size of Lactobacillus rhamnosus was 0.3 × 10⁻⁶. 8 CFU / g; After inoculation, start timing from 0h and ferment for 12 hours.

[0058] Specifically, before inoculation, high-purity nitrogen gas is introduced into the fermenter for 1.5–2 minutes at a flow rate of 0.5–1.0 m³ / min. 3 / h, control the dissolved oxygen in the liquid phase at 0.8~1.2 mg / L, and seal the tank.

[0059] This invention employs a method of simultaneous co-inoculation of *Lactobacillus reuteri* and *Lactobacillus rhamnosus*, based on the optimized metabolic synergy and dissolved oxygen utilization efficiency of the two strains. Simultaneous inoculation allows both strains to enter the logarithmic growth phase concurrently, forming a complementary metabolic network and precisely controlling the rate of dissolved oxygen decay. At 12 hours, the viable cell count and enzyme activity of both strains reach their peak simultaneously, laying the foundation for subsequent inoculation with *Bifidobacterium* and the three-stage synergistic fermentation.

[0060] If a sequential inoculation method is used, it may lead to substrate competition and growth inhibition, causing the dissolved oxygen decay curve to deviate from the design value, and ultimately resulting in a significant decrease in the amount of functional components synthesized.

[0061] After inoculation, time is taken as 0h. The fermentation period from 0 to 12h is the liquid-phase microaerobic proliferation phase. At the end of the first stage of fermentation, the dissolved oxygen concentration drops to ≤0.3mg / L, and the OD... 600 =0.8~1.0, and pH=5.5~5.8.

[0062] The OD 600The optical density value of the fermentation broth at a wavelength of 600 nm is used to characterize the cell concentrations of *Lactobacillus reuteri* and *Lactobacillus rhamnosus* in the system. When the OD... 600 When the value is 0.8~1.0, it indicates that the two Lactobacillus strains have entered the mid-logarithmic growth phase, and the cell viability and enzyme secretion capacity have reached the optimal level, meeting the inoculation conditions for Bifidobacterium.

[0063] In the first stage from 0 to 12 hours, the simultaneous proliferation of Lactobacillus reuteri and Lactobacillus rhamnosus was the core, accompanied by synergistic changes in dissolved oxygen, pH, enzyme system, substrate and system state, ultimately constructing a strictly anaerobic fermentation environment suitable for Bifidobacterium inoculation, with sufficient cell activity and system stability.

[0064] During this stage, both *Lactobacillus reuteri* and *Lactobacillus rhamnosus* simultaneously transitioned from the recovery phase to the logarithmic growth phase, reaching a total viable count of 2.0 × 10⁻⁶ at 12 hours. 9 With CFU / g or higher, the bacteria enter the mid-logarithmic growth phase, exhibiting peak activity. Dissolved oxygen concentration decreases from the initial 0.8–1.2 mg / L to ≤0.3 mg / L, establishing a strictly anaerobic preconditioning environment. pH is maintained at 5.5–5.8 with minimal fluctuations thanks to the presence of potassium citrate and dipotassium hydrogen phosphate liquid buffers. Lactobacillus begins secreting extracellular enzymes (β-glucosidase, pectinase, cellobiase), with enzyme activity gradually increasing, preparing for the third-stage transformation. Monosaccharides and small-molecule sugars in the liquid phase are rapidly utilized; celery inulin precursor, hawthorn pectin, and wolfberry polysaccharides undergo mild enzymatic hydrolysis, releasing readily available carbon sources. Intermittent stirring maintains the raw material particles in suspension within the fermentation system. *Lactobacillus reuteri* and *Lactobacillus rhamnosus* produce small amounts of lactic acid and CO2, which accumulates within the fermenter, further enhancing the anaerobic environment. Apigenin, proanthocyanidins, and oleanolic acid are released in small amounts but not extensively transformed, remaining in their precursor state.

[0065] Preferably, after inoculation, the fermentation temperature is controlled at 37±1℃, and an intermittent stirring mode of 11~13r / min for 8min and stopping for 22min is adopted to ferment until the 12th hour.

[0066] At the system viscosity of 200-600 mPa·s in this invention, a stirring speed of 11-13 r / min is more conducive to forming a gentle axial circulation throughout the tank, and more conducive to balancing the concentration difference between the upper and lower substrates and the bacterial cells, thus meeting the mass transfer requirements. Simultaneously, there are no obvious vortices on the liquid surface, allowing for controllable oxygen intake that matches the oxygen consumption rate of Lactobacillus. Furthermore, at this speed, the shear force is gentle, preventing damage to the integrity of the raw material particles and preserving the porous structure of the coarse wolfberry particles, which is more conducive to providing a carrier for the colonization of Bifidobacterium in the second stage. The preferred stirring speed is 12 r / min.

[0067] An 8-minute stirring time ensures complete homogenization of the entire tank, guaranteeing system uniformity; a 22-minute settling time provides a stable microaerobic metabolic environment for lactobacilli, allowing dissolved oxygen to steadily decrease, enabling continuous cell proliferation and secretion of extracellular enzymes, while avoiding shear damage and oxygen introduction caused by continuous stirring.

[0068] The stirring mode in the first stage of fermentation is more conducive to the uniform and synchronous proliferation of cells and the control of the rate of dissolved oxygen decay.

[0069] (4) Second stage fermentation Within 30 minutes of the completion of the first stage of fermentation, Bifidobacterium bifidum CGMCC 1.5029 was inoculated at an inoculation rate of 0.2 × 10⁸ CFU / g. The fermentation temperature was controlled at 37 ± 1℃, and fermentation was terminated at 24 h.

[0070] In this invention, the second stage of fermentation takes place from 12 to 24 hours. The second stage of fermentation is the solid-phase anaerobic colonization period. During this stage, the solid-phase anaerobic colonization of Bifidobacterium and the formation of biofilm are the core. The pH of the system is regulated by a double-buffered pH regulation system. The three strains initially establish a synergistic metabolic relationship, and the transformation of functional components is officially initiated.

[0071] In this stage, the main focus is on the solid-phase anaerobic colonization and biofilm formation of Bifidobacterium. By 24 hours, the colonization rate of Bifidobacterium on the surface of the raw material particles reaches over 90%, while Lactobacillus enters the stationary phase and begins enzyme production. During this stage, dissolved oxygen is consistently maintained at ≤0.3 mg / L, reaching ≤0.1 mg / L at the bottom layer. The pH slowly decreases to 4.8–5.2, with the liquid buffer continuing to act. Simultaneously, the degradation of chitosan-coated calcium carbonate microspheres begins at pH ≤5.0, further stabilizing the system pH. The formation of a dense anaerobic biofilm by Bifidobacterium on the surface of the raw material particles lays the foundation for efficient synthesis in the third stage.

[0072] Lactobacillus enzyme production increased significantly, and Bifidobacterium began to produce auxiliary enzymes; forming a complex enzyme system of β-glucosidase, proanthocyanidin depolymerase, and α-rhamnosidase.

[0073] Solid-phase polysaccharides (hawthorn pectin, wolfberry polysaccharide, and celery fiber) undergo extensive enzymatic hydrolysis, releasing aglycone precursors and monosaccharides to supply the metabolism of the three strains.

[0074] Bifidobacteria begin to metabolize polysaccharides to produce short-chain fatty acids (acetic acid, propionic acid); the system maintains a slight positive pressure (0.02~0.05MPa), and CO2 is retained inside the tank.

[0075] Apigenin and proanthocyanidins initiate the conversion, oleanolic acid begins to accumulate, and inulin precursors are initially synthesized into inulin.

[0076] Preferably, after the second stage of fermentation inoculation is completed, the fermentation tank is sealed, the fermentation temperature is controlled at 37±1℃, and an intermittent stirring mode of 10r / min for 5min and stopping for 25min is adopted. The pressure inside the tank is controlled at 0.02~0.05MPa slightly positive pressure, pH=4.8~5.2, and fermentation continues until 24h with 0h as the baseline.

[0077] In this invention, a stirring speed of 10 r / min is used, resulting in gentle shear force that does not interfere with the biofilm assembly process of Bifidobacteria. Simultaneously, it only forms a slow axial circulation throughout the tank with extremely low oxygen content. This is just enough to keep the raw material particles in suspension, preventing sedimentation and clumping, ensuring continuous nutrient transfer to the solid phase surface, and supporting the steady metabolic proliferation of Bifidobacteria. If the stirring speed is too high, exceeding 12 r / min, the liquid phase shear force will increase significantly, and the Bifidobacteria newly attached to the surface of the goji berry particles will easily be washed away by the water flow, hindering the secretion of extracellular polysaccharides to form a stable biofilm, and reducing the 24-hour colonization rate. If the speed is too low, below 8 r / min, it is not conducive to forming an effective axial circulation throughout the tank. Monosaccharides and water-soluble nutrients in the liquid phase are difficult to effectively transport to the surface of the bottom solid phase particles, and Bifidobacteria may experience metabolic stagnation due to insufficient substrate. At the same time, the bottom particles will gradually settle and clump, significantly reducing the effective colonization area, and the clumped area is prone to localized rancidity and the growth of other bacteria.

[0078] A 5-minute stirring cycle completes a full nutrient refresh, evenly transporting monosaccharides and functional enzymes secreted by lactobacilli from the liquid phase to the solid surface at the bottom of the tank. This meets the metabolic needs of Bifidobacteria for one cycle, while gentle agitation prevents particle aggregation. A 25-minute settling period provides Bifidobacteria with a sufficient stable anaerobic window, allowing them to complete the entire process of attachment, extracellular polysaccharide secretion, and biofilm formation, thus improving colonization rate. The overall rhythm matches the colonization cycle of Bifidobacteria, ensuring basic mass transfer efficiency while minimizing interference with cell attachment and significantly reducing the total amount of oxygen introduced.

[0079] Maintaining a slight positive pressure of 0.02~0.05MPa inside the tank, combined with a double-buffered pH system and intermittent stirring, is more conducive to ensuring a strict anaerobic environment in the second stage.

[0080] The stirring mode in the second stage of fermentation can take into account both mass transfer and biofilm formation, and assist in the solid-phase colonization of Bifidobacterium.

[0081] (5) Third stage fermentation The fermentation temperature was controlled at 34±1℃, and fermentation was terminated when the fermentation time reached 36 hours or the conversion rate of apigenin in the fermentation broth was ≥90%. Based on 0h, purified water equal to 5% of the total dry weight of the initial raw materials was added at 24h and 30h.

[0082] Specifically, the third stage of fermentation corresponds to the interval from 24 to 36 hours.

[0083] This stage is the core stage of the targeted synthesis of functional components. The core objective is to optimize the synergistic metabolic efficiency of the three strains and achieve the simultaneous and efficient enrichment of five functional components: apigenin, oligomeric proanthocyanidins, inulin, short-chain fatty acids, and oleanolic acid.

[0084] The fermentation temperature was controlled at 34±1℃, which takes into account the thermal stability of metabolic enzymes, the direction of cell metabolism, the stability of functional components, and the synergy of the dual buffer system.

[0085] There is a significant difference between the optimal growth temperature of probiotics and the optimal catalysis temperature of metabolic enzymes: the core objectives of the first and second stages are bacterial proliferation and colonization, so the optimal growth temperature of Lactobacillus and Bifidobacterium is 37℃; in the third stage, the bacteria have completed sufficient proliferation, and the core objective shifts to the targeted synthesis of functional components. At this time, lowering the temperature can shift the metabolic flow of bacteria from bacterial growth to product synthesis.

[0086] At 34℃, the relative activities of β-glucosidase, proanthocyanidin depolymerase, and α-rhamnosidase are very high, and the enzyme half-life is 16 hours, which perfectly covers the entire 12-hour fermentation cycle of the third stage, eliminating the need for additional enzyme supplementation. If the temperature is increased to 37℃, the enzyme half-life will shorten to 5 hours, and at 36 hours, the enzyme activity will be less than 20% of the initial value, leading to a significant decrease in conversion efficiency.

[0087] At 34°C, the cell growth rate slows down significantly, with most of the carbon source flowing to the product synthesis pathway, which is more conducive to improving product conversion rate and avoiding excessive cell proliferation that consumes substrate.

[0088] 34℃ can significantly reduce the 12h degradation rate of apigenin, oligomeric proanthocyanidins and oleanolic acid, thereby significantly improving the retention rate of the final product.

[0089] At 34℃, the degradation rate of chitosan membrane is moderate, and the release rate of calcium carbonate is more conducive to synergistic with the acid production rate of bacteria, so that the pH is kept stable in the optimal range of enzymes from 4.5 to 5.0 throughout the later stage of fermentation.

[0090] At 24 and 30 hours of fermentation, 5% of the total dry weight of the raw materials was added with sterile purified water. Each addition was performed using a closed, constant-pressure drip method, completed uniformly within 10 minutes. The purified water was preheated to 34±1℃. The addition process maintained a slight positive pressure within the tank and the original intermittent stirring mode. This is because short-chain fatty acids and aglycones produced during fermentation inhibit the activity of metabolic enzymes; when their mass concentration exceeds 1%, enzyme activity decreases significantly. Two equal-volume gradient additions dilute the product concentration, keeping it below the inhibition threshold, while avoiding the introduction of new nutrients, thus preventing contamination and excessive cell proliferation.

[0091] Preferably, the third stage of fermentation adopts an intermittent stirring mode of stirring at 8 r / min for 3 min and stopping for 27 min.

[0092] In this invention, the shear force is extremely low at a stirring speed of 8 r / min, which does not damage the spatial structure of mature biofilms and functional enzymes, thus stably maintaining the catalytic activity of the enzymes. Furthermore, the oxygen content is minimal, which helps reduce the oxidative loss of functional components. Simultaneously, it maintains a uniform suspension of particles throughout the container, preventing sedimentation and clumping, and ensuring basic contact efficiency between the substrate and enzyme. A 3-minute stirring cycle completes a gentle circulation of the entire container, diluting the high concentration of product on the particle surface, relieving product inhibition, and simultaneously replenishing fresh substrate to the enzyme reaction interface. Combined with the water replenishment operations at 24h and 30h, the added purified water can be quickly mixed evenly, achieving uniform concentration throughout the system without impacting the biofilm structure. A 27-minute settling period provides a stable window for the enzymatic reaction, allowing the enzyme and substrate to form a stable complex for continuous and efficient catalytic conversion. During the settling period, almost no oxygen is introduced, minimizing the oxidative degradation of functional components, and the Bifidobacterium biofilm remains undisturbed, enabling continuous metabolic acid production and synergistic pH stabilization of the system.

[0093] In a preferred embodiment of the present invention, the first step of inoculation is top spray inoculation. Before inoculation, the agitator is turned on to 12 r / min and stirred for 2 min. Then, under stirring conditions, the inoculation is carried out at a speed of 5~10 L / min. After the inoculation is completed, the agitator is turned off after stirring for 5 min.

[0094] Inoculation is performed via spraying from the main inoculation port at the top center of the fermenter. The inoculated bacterial solution at the top will naturally settle and disperse evenly throughout the liquid phase from top to bottom. The inoculation rate is 5-10 L / min. Too fast a rate will lead to excessively high local bacterial concentrations and abnormal local metabolism, while too slow a rate will prolong the inoculation time and increase the risk of contamination by other microorganisms. For example, if the inoculation volume for a 1000L fermenter is 50L, the inoculation time should be controlled at 5-10 minutes.

[0095] The agitator must be turned on first (12 rpm) and stirred for 2 minutes before inoculation. After inoculation, continue stirring for 5 minutes, then turn off the agitator. Turning on the agitator first creates a slight vortex on the liquid surface, which helps the bacterial solution disperse quickly. After inoculation, continue stirring for 5 minutes to ensure that the bacterial solution is completely and evenly dispersed throughout the liquid phase.

[0096] In a preferred embodiment of the present invention, the second inoculation step employs bottom-distributed inoculation.

[0097] Because of the significant vertical gradient of dissolved oxygen within the fermenter—highest at the top (approximately 0.3–0.5 mg / L) and lowest at the bottom solid layer (≤0.1 mg / L)—Bifidobacteria inoculated at the bottom can directly enter the strictly anaerobic zone without passing through the hyperaerobic liquid phase, which is more conducive to their colonization. Experimental tests showed that the survival rate of Bifidobacteria inoculated at the bottom was 91.2%, while the survival rate of those inoculated at the top was only 28.7%.

[0098] Specifically, the second step of inoculation is carried out simultaneously through 3 to 4 dedicated inoculation ports evenly distributed at the bottom of the fermenter. The inoculation port pipes extend upward to 5 to 10 cm above the bottom of the tank. Stirring is turned off 10 minutes before the second step of inoculation. The inoculation pipes are purged with high-purity nitrogen for 5 minutes before inoculation. During the inoculation process, high-purity nitrogen is introduced through each inoculation port to form a nitrogen barrier. After inoculation, the tank is left to stand for 30 minutes.

[0099] The pipe at each bottom inoculation port extends upwards to 5-10cm above the bottom of the tank and is directly inserted into the solid phase material layer.

[0100] Inoculate at multiple points simultaneously through 3-4 bottom inoculation ports to ensure that Bifidobacteria are evenly distributed throughout the solid material layer at the bottom of the tank, avoiding excessively high local concentrations.

[0101] Before inoculation, purge the inoculation pipeline with high-purity nitrogen for 5 minutes to completely replace the air in the pipeline.

[0102] During the inoculation process, high-purity nitrogen gas is slowly introduced into each inoculation port at a flow rate of 0.1 m³ / s. 3 / h, forming a nitrogen barrier.

[0103] Ensure the mixing system is off 10 minutes before vaccination, and do not turn it on during vaccination or for 30 minutes after vaccination. After vaccination, let it stand for 30 minutes before starting the second stage of mixing (intermittent mixing mode with a mixing speed of 10 r / min, mixing for 5 minutes, and stopping for 25 minutes).

[0104] When dissolved oxygen ≤ 0.3 mg / L, OD 600 Once the three indicators (pH=0.8~1.0, pH=5.5~5.8) are simultaneously met, the inoculation should be completed within 30 minutes.

[0105] In a preferred embodiment of the present invention, before the enzymatic hydrolysis in step (1), hawthorn is pulverized to 100 mesh, wolfberry is pulverized to 40 mesh, and celery is pulverized to 120 mesh.

[0106] In this invention, pulverizing hawthorn to 100 mesh is more conducive to balancing pectin hydrolysis efficiency and system viscosity. Hawthorn contains up to 15% pectin, and pulverizing to 100 mesh allows pectinase to fully contact the substrate, resulting in higher pectin degradation and methyl ester removal rates. Pulverizing to 120 mesh can easily lead to a large amount of pectin dissolving, causing a sharp increase in system viscosity, preventing uniform enzyme diffusion, reducing hydrolysis efficiency, and potentially encapsulating the cells and inhibiting their metabolic growth. Pulverizing to 80 mesh results in a lower pectin release rate, leaving a large amount of pectin residue after hydrolysis, which can easily cause the system viscosity to rise above 800 mPa·s in the later stages of fermentation, affecting mass transfer and stirring. 100-mesh hawthorn particles are mainly distributed in the lower part of the liquid phase, forming a transition layer connecting the solid and liquid phases, promoting bidirectional diffusion of substrates and metabolites, and improving overall mass transfer efficiency. The organic acids and pectinates contained in hawthorn can act as auxiliary buffering substances, working synergistically with the double buffer system to further reduce pH fluctuations in the early stages of fermentation.

[0107] Grinding goji berries to 40 mesh facilitates the colonization of Bifidobacteria. 40-mesh goji berry particles have high density and a suitable particle size. Within 30 minutes of stopping stirring, over 92% of the particles settle to the bottom of the container, forming a stable solid layer 5-8 cm thick. This area has dissolved oxygen ≤0.1 mg / L, constituting a strictly anaerobic zone in the system. After bottom-distributed inoculation, most Bifidobacteria colonize the surface of the goji berry particles at the bottom. The rough, porous surface of 40-mesh goji berry particles, with a suitable specific surface area and rich in neutral goji berry polysaccharides, serves as a biofilm framework component, promoting the secretion of extracellular polysaccharides by Bifidobacteria and forming a dense and stable anaerobic biofilm. Simultaneously, 40-mesh grinding moderately disrupts the goji berry cell walls, allowing for a slow and continuous release of goji berry polysaccharides, perfectly meeting the nutritional needs of Bifidobacteria during their 24-hour colonization period and preventing contamination from other microorganisms due to rapid nutrient release.

[0108] If goji berries are ground to 60 mesh, about 40% of the goji berry particles will be suspended in the liquid phase, which is not conducive to the colonization of Bifidobacterium bifidum. If the goji berry particles are ground to 20 mesh, the particles are too large, and the internal mass transfer is hindered. Since Bifidobacterium bifidum can only attach to the surface, the effective area of ​​the biofilm is reduced, and the production of short-chain fatty acids decreases.

[0109] Celery pulverized to 120 mesh facilitates the release of functional components and improves the utilization efficiency of lactobacilli. Celery is high in fiber and has a dense cell wall structure; over 90% of functional components such as celery glycosides, inulin precursors, and oleanolic acid exist in a bound state within the cell wall. Ultrafine pulverization is necessary to thoroughly disrupt the cell wall and release these functional precursors, providing substrates for subsequent biotransformation.

[0110] Celery particles of 120 mesh exhibit a sedimentation rate of no more than 30% over 24 hours at a system viscosity of 200–600 mPa·s, which is more conducive to maintaining uniform suspension. This is especially beneficial as it highly overlaps with the spatial distribution of lactobacilli during the first stage of fermentation, facilitating the lactobacilli's contact with and utilization of the released substrate, leading to rapid proliferation and secretion of functional enzymes. If celery is pulverized to 150 mesh, the celery fiber breaks into tiny particles, significantly increasing the system viscosity and causing the adsorption of some enzymes and bacterial cells, thus reducing metabolic efficiency.

[0111] Three different particle sizes—hawthorn, goji berry, and celery—were used to construct a gradient particle size distribution, forming a three-dimensional structure consisting of a bottom solid-phase colonization zone, a middle transitional mass transfer zone, and an upper liquid-phase synthesis zone. This provided exclusive attachment sites and growth microenvironments for the three strains, enabling zoned metabolism among the three strains. The colonization rate of Bifidobacterium reached over 90%. A gradient substrate release rhythm was also established, synchronized with the metabolic needs of the three stages, avoiding substrate inhibition and improving raw material utilization.

[0112] In a preferred embodiment of the present invention, the chitosan membrane coating calcium carbonate microspheres have a particle size of 10-20 μm and a thickness of 0.8-1.2 μm.

[0113] In this invention, the chitosan membrane-coated calcium carbonate microspheres have a particle size of 10-20 μm and a membrane thickness of 0.8-1.2 μm. This is the result of comprehensive optimization based on the spatial distribution of the fermentation system, pH trigger response rate, double buffer relay aging, and complete degradation. This particle size can uniformly fill the gaps between raw material particles, matching the system viscosity to achieve stable suspension throughout the entire cycle, without disrupting the anaerobic microenvironment of Bifidobacteria at the bottom. This membrane thickness is beneficial for controlling the trigger pH at 4.9-5.1, synergistically with the liquid buffer, allowing calcium carbonate to be released at a rate of 0.02-0.03 mmol / L / h, which is conducive to synchronizing with the acid production rate of the third-stage bacteria. Furthermore, it can be completely degraded into food-grade safe substances with no residue upon completion of fermentation. This parameter combination is also deeply synergistic with gradient particle size design and the three-stage fermentation process, ultimately achieving a pH fluctuation of ≤±0.15 throughout the entire fermentation cycle, providing a core guarantee for maintaining the activity of functional enzymes and the efficient synthesis of functional components.

[0114] The chitosan membrane-coated calcium carbonate microspheres used in this invention can be obtained commercially or prepared using food-grade processes. The preparation method involves adding food-grade lightweight calcium carbonate microspheres to a 2% chitosan-acetic acid solution, stirring and adsorbing for 30 minutes, then spray-drying, washing with anhydrous ethanol, and vacuum drying. Both commercially available and homemade products achieve the same pH-controlled release effect, ensuring stable fermentation.

[0115] In a preferred embodiment of the present invention, inulin is added as a dispersant in step (2), and the amount of inulin added is 0.2 to 0.3% of the dry weight of the raw material.

[0116] Inulin is a water-soluble polysaccharide that forms a low-viscosity colloidal solution in water. It adsorbs onto the surface of fine particles such as celery powder and chitosan-coated calcium carbonate microspheres, effectively preventing particle aggregation through a combination of electrostatic repulsion and steric hindrance. Furthermore, it can systematically regulate the system viscosity, making it more stable.

[0117] Inulin, as a natural prebiotic, is not rapidly utilized by lactobacilli when added at a low concentration of 0.25%. Instead, it acts as a mild growth promoter, which can shorten the recovery time of lactobacilli by 15% without altering the metabolic rhythm of the three-stage fermentation process.

[0118] Specifically, inulin is dissolved in purified water at 10 times its weight, stirred at 35-40℃ and 50 rpm until completely dissolved, with a dissolution time of 5-8 minutes. The dissolved solution is colorless and transparent, with no visible lumps. The pretreated hawthorn, wolfberry, and celery enzymatic hydrolysates are added to the fermentation tank in proportion, and stirred at 30 rpm until homogeneous. The inulin solution is then slowly added to the fermentation tank over 2-3 minutes. After the inulin solution is added, the stirring speed is increased to 80 rpm and stirred continuously for 10 minutes to ensure that the inulin molecules are fully adsorbed onto the surface of the celery particles and chitosan-coated calcium carbonate microspheres, forming a stable dispersion layer. Then, the viscosity and pH are adjusted.

[0119] In a preferred embodiment of the present invention, the dry weight ratio of hawthorn, wolfberry and celery is 1:1.5:4.

[0120] In this invention, the dry weight ratio of hawthorn, goji berries, and celery is 1:1.5:4, corresponding to a dry weight percentage of 15.4% for hawthorn, 23.1% for goji berries, and 61.5% for celery. Firstly, this ratio, combined with the gradient particle size distribution, is more conducive to constructing a precise three-dimensional metabolic structure. The coarse goji berry particles form a stable solid-phase colonization layer 7-8 cm thick at the bottom of the fermenter, providing ample attachment sites for Bifidobacteria and ensuring smooth mass transfer within the layer. The medium-sized hawthorn particles form a continuous transitional mass transfer layer, preventing oxygen diffusion downwards and enabling efficient bidirectional transfer of substrates and metabolites. The fine celery particles are uniformly suspended in the upper part of the liquid phase, constructing a liquid-phase synthesis zone that matches the spatial distribution of Lactobacillus. Secondly, this ratio is more conducive to matching the nutritional needs and metabolic characteristics of the three strains at different fermentation stages. The specific polysaccharides continuously released by goji berries can meet the nutritional needs of Bifidobacteria during colonization; the rapid release of hawthorn pectin provides a fast-acting carbon source for Lactobacillus in the early stages of inoculation; and the continuous release of nutrients from celery supports the full-cycle proliferation and enzyme production of Lactobacillus. Meanwhile, under this ratio, the amount of monosaccharides produced by Lactobacillus hydrolysis matches the consumption by Bifidobacterium, and the total carbon source utilization rate reaches over 85%. Thirdly, under this ratio, the initial viscosity after enzymatic hydrolysis is 210 mPa·s, and it remains stable at 380~450 mPa·s during fermentation, which is more conducive to balancing the suspension requirements of raw material particles and mass transfer resistance, stabilizing fermentation fluctuations. Fourthly, under this ratio, the product has a moderate sweet-sour ratio, a refreshing and harmonious taste, and no obvious astringency or bitterness; the natural fruit aroma produced after fermentation blends with the sweetness of maltitol, eliminating the need for additional flavorings.

[0121] In a preferred embodiment of the present invention, the fermentation method further includes step (6): after the fermentation is terminated, the mixture is filtered, homogenized and sterilized in sequence to obtain a compound fermented plant beverage.

[0122] Specifically, a 200-mesh filter is used, and a plate and frame filter press is employed for atmospheric pressure filtration. The filtration temperature is controlled at 30~35℃ to avoid degradation of functional components due to high temperatures. The 200-mesh filter removes large particle residues first, which can prevent them from clogging the homogenization valve during the homogenization process and improve homogenization efficiency.

[0123] The sterilization method is pasteurization at 85℃ for 15 minutes, followed by immediate cooling to below 25℃ to reduce the time spent at high temperatures.

[0124] The homogenization temperature is controlled at 25~30℃, and the homogenization is performed twice. The homogenization process is kept in a sealed environment to prevent oxygen from entering.

[0125] In a preferred embodiment of the present invention, the homogenization adopts a two-stage homogenization process, with the first-stage pressure controlled at 18~22MPa and the second-stage pressure controlled at 3~7MPa.

[0126] Specifically, the homogenization process employs a continuous two-stage homogenization process, involving two homogenization passes. Each homogenization pass sequentially through a primary homogenizing valve and a secondary homogenizing valve. The pressure of the primary homogenizing valve is 18–22 MPa, and the pressure of the secondary homogenizing valve is 3–7 MPa. The feed flow rate is controlled to be 80–90% of the homogenizer's rated capacity, ensuring a total residence time of the material within the homogenizing valves of 0.15–0.2 s. Simultaneously, the valve chamber cooling temperature is controlled to be ≤35℃, and the dissolved oxygen in the system is controlled to be ≤0.1 mg / L.

[0127] The primary homogenizer undertakes the main task of particle breaking, using high-pressure shearing and cavitation effects to break down the tiny fibrous particles and aggregates remaining after filtration. If the pressure is below 18 MPa, particle breaking is insufficient; if the pressure is above 22 MPa, it will cause the glycosidic bonds of short-chain inulin and oligomeric proanthocyanidins to break, increasing the degradation rate.

[0128] Secondary homogenization further disperses and homogenizes the particles after primary homogenization under low pressure, eliminating particle agglomeration and resulting in a more uniform particle size distribution. It also breaks down fat globules and protein aggregates, forming a stable emulsion system. If the pressure is below 3 MPa, the dispersion effect is insufficient, and the product is prone to stratification. If the pressure is above 7 MPa, energy consumption increases without a significant improvement in homogenization, and the introduction of more oxygen may lead to the oxidation of functional components.

[0129] According to a second aspect of the present invention, a compound fermented plant beverage having uric acid-lowering and antioxidant activities is provided, which is prepared using the fermentation method described above.

[0130] The compound fermented plant beverage prepared by this invention is rich in free apigenin and short-chain inulin, which work synergistically to regulate uric acid metabolism: apigenin competitively inhibits the catalytic activity of xanthine oxidase and xanthine dehydrogenase, blocking the conversion of hypoxanthine and xanthine into uric acid and reducing the production of endogenous uric acid in the human body; short-chain inulin, as a prebiotic, can reshape the intestinal flora structure, increase the abundance of uric acid-degrading bacteria, and promote uric acid excretion through the intestine, thus achieving uric acid metabolism regulation through a dual pathway. The apigenin and oligomeric proanthocyanidins enriched in this invention are both natural polyphenolic flavonoids that can scavenge free radicals such as DPPH, reduce oxidative damage to the system, and endow the product with excellent antioxidant capacity.

[0131] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0132] In the following embodiments, unless otherwise specified, all raw material components are commercially available products.

[0133] Example 1 Raw material formula, based on a 1000L fermentation tank: Hawthorn: Grind into 100 mesh, 15kg; Goji berries: Grind into 40 mesh, 22.5 kg; Celery: Crush to 120 mesh, 60 kg; Maltitol solution with 75% solids content: 200 kg; Inulin: 0.24 kg, which is 0.25% of the total dry weight of the raw materials; Compound pectinase: 0.06 kg, which is 0.04% of the dry weight of hawthorn; β-glucanase: 0.03675 kg, of which 0.00675 kg (0.03% of dry weight of wolfberry) is used for enzymatic hydrolysis of wolfberry and 0.003 kg (0.05% of dry weight of celery) is used for enzymatic hydrolysis of celery. Amylase: 0.0045 kg, which is 0.02% of the dry weight of wolfberries; Cellulase: 0.012 kg, which is 0.02% of the dry weight of celery; Potassium citrate: 0.6 kg; Dipotassium hydrogen phosphate: 0.2 kg; Chitosan membrane-coated calcium carbonate microspheres: particle size 10~20μm, membrane thickness 0.8~1.2μm, 0.024kg; Lactobacillus reuteri CGMCC 1.2838 bacterial culture: 8L, concentration 1×10⁻⁶ 9 CFU / mL; Lactobacillus rhamnosus CGMCC 1.2467 bacterial culture: 3L, concentration 1×10⁻⁶ 9 CFU / mL; Bifidobacterium bifidum CGMCC 1.5029 bacterial culture: 2L, concentration 1×10 9 CFU / mL.

[0134] Fermentation process steps: (1) Raw material pretreatment Hawthorn enzymatic hydrolysis: Hawthorn powder was added to purified water at a solid-liquid mass ratio of 1:1, the pH was adjusted to 4.8 with potassium citrate, compound pectinase was added, and the mixture was kept at 45℃ for 15 min to obtain hawthorn enzymatic hydrolysate (pectin molecular weight 5000~10000 Da, methyl ester group removal rate is about 65%).

[0135] Goji berry enzymatic hydrolysis: Goji berry powder was added to purified water at a solid-liquid mass ratio of 1:1.5. After stirring evenly, the pH was measured to be 5.4, which did not require adjustment. β-glucanase and amylase were added, and the mixture was incubated at 40℃ for 12 min to obtain the goji berry enzymatic hydrolysate.

[0136] Celery enzymatic hydrolysis: Celery was added to purified water at a solid-liquid mass ratio of 1:1, the pH was adjusted to 5.8 with sodium bicarbonate, β-glucanase and cellulase were added, and the mixture was incubated at 50℃ for 18 min to obtain celery enzymatic hydrolysate.

[0137] (2) Combine and adjust the viscosity All three enzymatic hydrolysates were added to the fermenter, along with pre-dissolved inulin solution (0.24 kg inulin dissolved in 2.4 kg purified water at 38℃), and stirred at 30 rpm for 10 min. Maltitol solution and purified water were added to bring the volume to 950 L, and the viscosity of the system at 35℃ was adjusted to 420 mPa·s (measured using a rotational viscometer, rotor No. 3, rotation speed 60 rpm, 35℃). Potassium citrate and dipotassium hydrogen phosphate were added to adjust the pH to 5.7. Finally, chitosan membrane-coated calcium carbonate microspheres were added and stirred for 5 min to mix thoroughly.

[0138] (3) First stage fermentation (0~12h) High-purity nitrogen gas was introduced into the fermenter for 2 minutes at a flow rate of 0.8 m³ / min. 3 At 12 h, the dissolved oxygen in the liquid phase was controlled to be 1.0 mg / L, and the tank was immediately sealed. *Lactobacillus reuteri* and *Lactobacillus rhamnosus* were inoculated using a top spray method. Before inoculation, the mixture was stirred at 12 rpm for 2 min, and inoculated at a rate of 8 L / min. After inoculation, stirring was continued for 5 min, then the stirring was stopped. This was marked as 0 h of fermentation. The fermentation temperature was 37 ± 1℃, and an intermittent stirring mode of 12 rpm for 8 min followed by a 22 min stop was used. When fermentation reached 12 h, the dissolved oxygen was measured to be ≤0.3 mg / L and the OD value was ≤0.5 mg / L. 600 =0.9, pH=5.6, meeting the inoculation conditions for Bifidobacterium bifidum.

[0139] (4) Second stage fermentation (12~24h) Ensure the agitator is off for 10 minutes before inoculating with Bifidobacterium. Inoculate Bifidobacterium simultaneously through the three inoculation ports at the bottom of the fermenter, controlling the inoculation rate at 2-3 L / min. Purge the pipeline with high-purity nitrogen for 5 minutes before inoculation, controlling the nitrogen flow rate at 0.1 m³ / min. 3 / h, completely replace the residual air in the pipeline; after purging, maintain a slight nitrogen flow, close all inoculation port valves and nitrogen valve after inoculation, let stand for 30 minutes, seal the fermentation tank, and maintain the fermentation temperature at 37±1℃. After standing, use an intermittent stirring mode of 10r / min for 5 minutes and stop for 25 minutes, control the gauge pressure inside the tank at 0.03MPa, maintain pH=4.8~5.2, and ferment for 24 hours.

[0140] (5) Third stage fermentation (24~36h) The fermentation temperature was lowered to 34±1℃, and an intermittent stirring mode of 8r / min for 3min and stopping for 27min was adopted. At 24h and 30h, 5L of sterile purified water preheated to 34℃ (5% of the total dry weight of the initial raw materials) was added respectively. After 36h of fermentation, the conversion rate of apigenin was determined by high performance liquid chromatography to be 92.3%, and the fermentation was terminated.

[0141] The apigenin content and conversion rate were determined by high-performance liquid chromatography (HPLC). Chromatographic conditions included a C18 column, column temperature of 30℃, detection wavelength of 335 nm, mobile phase of methanol-0.1% phosphoric acid aqueous solution (volume ratio 55:45), and flow rate of 1.0 mL / min. The total apigenin content before fermentation was determined after hydrolysis with hydrochloric acid, while the free apigenin content after fermentation was directly determined. The apigenin conversion rate was calculated as follows: Apigenin conversion rate (%) = (Free apigenin content after fermentation ÷ Total apigenin content before fermentation) × 100% (6) Post-processing The fermentation broth was filtered through a 200-mesh plate and frame filter (32℃). The filtrate was homogenized twice using a two-stage homogenization process (first stage pressure 20MPa, second stage pressure 5MPa, feed temperature 28℃). After homogenization, it was sterilized in a tubular sterilizer at 85℃ for 15 minutes and then immediately cooled to 25℃ before being bottled to obtain the finished product.

[0142] Example 2 Raw material formula (based on a 1000L fermenter): Inulin: 0.192 kg, which is 0.2% of the total dry weight of the raw materials; the amounts of the other raw materials are the same as in Example 1.

[0143] Fermentation process steps: (2) Combine and adjust the viscosity All three enzymatic hydrolysates were added to the fermenter, followed by pre-dissolved inulin solution (0.192 kg inulin was dissolved in 1.92 kg purified water at 38°C, stirred at 35°C and 50 rpm for 6 min until completely dissolved), and stirred at 30 rpm for 10 min. Maltitol solution and purified water were added to bring the volume to 950 L, and the viscosity of the system at 35°C was adjusted to 410 mPa·s (rotational viscometer method). 0.56 kg potassium citrate and 0.187 kg dipotassium hydrogen phosphate were added to adjust the pH to 5.7. Finally, chitosan membrane-coated calcium carbonate microspheres were added and stirred for 5 min to mix thoroughly.

[0144] The remaining steps are the same as in Example 1, and fermentation is terminated after 36 hours.

[0145] Example 3 The raw material formula is the same as in Example 1, except that hawthorn, wolfberry and celery are all pulverized to 100 mesh.

[0146] The fermentation process steps are the same as in Example 1.

[0147] Example 4 The raw material formula is the same as in Example 1. The fermentation process steps are the same as in Example 1, except that the post-treatment homogenization parameters are adjusted to a first-stage pressure of 22 MPa and a second-stage pressure of 7 MPa.

[0148] Example 5 The raw material formula is the same as in Example 1.

[0149] In the fermentation process, the stirring mode is the same for the first, second, and third stages of fermentation: an intermittent stirring mode of 12 r / min for 8 min followed by a 22 min pause. The remaining steps are the same as in Example 1.

[0150] Comparative Example 1 Raw material formula Same as Example 1 Fermentation process steps After combining and adjusting the viscosity, three strains of probiotics were inoculated simultaneously and fermented at a constant temperature of 37°C for 36 hours. The remaining processes were the same as in Example 1.

[0151] Comparative Example 2 Raw material formula Same as Example 1, but without the addition of calcium carbonate microsphere slow-release buffer.

[0152] The fermentation process steps are the same as in Example 1.

[0153] Performance testing 1. Content of active ingredients Apigenin and oleanolic acid were determined by high-performance liquid chromatography (HPLC); oligomeric proanthocyanidins were determined by vanillin-hydrochloric acid spectrophotometry; short-chain fatty acids were determined by gas chromatography (GC); and short-chain inulin was determined by a combination of phenol-sulfuric acid and gel permeation chromatography (GPC). The results of the active ingredient content tests are shown in Table 1. Table 1 Results of Active Ingredient Content Test

[0154] Examples 1-4 employed a three-stage gradient decreasing intermittent stirring, step-by-step inoculation, and liquid-solid double buffering process, resulting in high levels of five active substances: apigenin, oligomeric proanthocyanidins, short-chain inulin, short-chain fatty acids, and oleanolic acid. Example 5 maintained a uniform high stirring intensity throughout, without gradient weakening shear design, and continuously introduced oxygen to disrupt the Bifidobacterium biofilm and reduce functional enzyme activity, resulting in significantly lower levels of various active ingredients compared to Examples 1-4. Comparative Example 1 simultaneously inoculated three strains, resulting in severe strain competition inhibition, a significant decline in substrate conversion efficiency, and the lowest content of active substances. Comparative Example 2 lacked a calcium carbonate solid-phase slow-release buffer, and pH loss during the later stages of fermentation inhibited enzyme activity, resulting in lower levels of active ingredients than in Example 5. This demonstrates that gradient stirring, double buffering, and step-by-step inoculation are key technical features for enhancing the enrichment of functional components.

[0155] 2. Total carbon source utilization rate The phenol-sulfuric acid method was used. The initial total sugar content C0 was measured before fermentation, and the final total sugar content C was measured after fermentation was terminated. t Total carbon utilization rate = (1-C) t / C0)×100%.

[0156] 3. Total particle settling rate over 24 hours The total particle settling rate over 24 hours was determined using the graduated cylinder settling method, with the settling rates of the fermentation broth and the finished product measured separately. Fermentation broth sedimentation rate: Measured for the original fermentation broth that has just finished fermentation and been cooled to 25°C.

[0157] Settling rate of finished product: This is measured for the finished product obtained after all steps (6) are completed.

[0158] 4. Accelerated stability test Experimental conditions: 37℃±2℃, relative humidity 75±5%, stored away from light, and the retention rate of effective ingredients was measured after 3 months.

[0159] The results of the total carbon utilization rate, 24-hour total particle settling rate, and accelerated stability test are shown in Table 2. Table 2. Results of accelerated stability tests on total carbon utilization rate and 24-hour total particle settling rate.

[0160] In Examples 1-4, the carbon source utilization rate was over 85% due to the full hydrolysis and utilization of the raw polysaccharide. In Example 5, the stirring shear was consistently high, the bacterial biofilm was easily detached, and the polysaccharide conversion was incomplete, resulting in a utilization rate of 81.5%. In Comparative Example 1, multiple strains competed for carbon sources simultaneously, leading to serious substrate waste and a utilization rate of only 67.2%. In Comparative Example 2, there was no solid-phase buffer, and the enzyme activity decreased in the later stages, resulting in a carbon utilization rate of 74.8%.

[0161] The sedimentation rate of the fermentation broth after 24 hours was determined by gradient stirring, which gradually reduced shear and particle breakage and agglomeration. Combined with the inulin dispersion system, the sedimentation rates of the fermentation broth and finished products in Examples 1-4 were extremely low. In Example 5, high stirring throughout the process led to an increase in fine particles, which easily settled upon standing. The two comparative groups had poor system stability and significantly higher sedimentation values ​​due to the lack of gradient oxygen control and long-term pH buffer.

[0162] Accelerated at 37℃ for 3 months, the retention rate of active ingredients in Examples 1-4 was 88.6%-92.1%, with no stratification throughout the process; Example 5 showed only trace precipitation, with a retention rate of 83.6%; Comparative Example 1 showed severe oxidative degradation and obvious stratification, while Comparative Example 2 showed slight stratification, verifying that gradient stirring can reduce the oxidation of active substances and maintain the uniformity and stability of the system for a long time.

[0163] 5. Taste Evaluation The taste evaluation employed a professional sensory evaluation method, conducted by a panel of 20 professionally trained sensory evaluators (10 men and 10 women). The dimensions and weights of the taste evaluation are shown in Table 3. Table 3. Taste Evaluation Dimensions and Weights

[0164] Test method: Take 20mL of sample into a disposable tasting cup, and the evaluators score independently under natural light. The highest and lowest scores are removed, and the average score is taken.

[0165] The taste evaluation results are shown in Table 4: Table 4 Taste Evaluation Results

[0166] Examples 1-4, using gradient oxygen-controlled fermentation, resulted in minimal oxidation of polyphenols and flavonoids, producing a uniform and bright color, intact fruit aroma, a smooth and balanced sweet and sour taste, and a uniform texture free of impurities, achieving a comprehensive score of 92-94. Example 5, with prolonged high-intensity stirring introducing oxygen, resulted in slight oxidation of the raw materials, leading to a dull color, a weaker fruit aroma, and a slight grainy texture, achieving a comprehensive score of 85. Comparative Example 1, with its imbalanced microbial community resulting from a single inoculation, produced a rancid odor, a prominent astringent taste, and turbidity and sedimentation, scoring only 71. Comparative Example 2, lacking solid-phase buffering to regulate acidity, exhibited an abrupt sour taste and a weak aroma, with sensory quality inferior to Example 5. This demonstrates that the gradient fermentation process of this invention can simultaneously optimize the product's appearance, aroma, taste, and uniformity.

[0167] 6. Antioxidant activity assay Antioxidant efficacy was determined using the DPPH free radical scavenging method: Take the finished products obtained from each example and comparative example, filter them through a 0.45 μm microporous membrane, and dilute them with anhydrous ethanol to a total solids concentration of 1.0 mg / mL for later use; prepare a 0.1 mmol / L DPPH anhydrous ethanol solution, and use it immediately in the dark. Add samples according to the following system: Sample group: 2 mL sample solution + 2 mL DPPH solution, mix thoroughly, react at 25℃ in the dark for 30 min, and measure the absorbance at 517 nm, which is recorded as sample A; Sample blank group: 2 mL sample solution + 2 mL anhydrous ethanol, react under the same conditions and measure the absorbance, which is recorded as sample blank A; Control group: 2 mL anhydrous ethanol + 2 mL DPPH solution, reacted under the same conditions and the absorbance was measured, which was recorded as control A; The DPPH free radical scavenging rate was calculated by performing three parallel measurements and taking the average value as follows: Clearance rate (%) = [1 - (Sample A - Blank Sample A) / Control Sample A] × 100%.

[0168] The results of the antioxidant activity test are shown in Table 5.

[0169] Table 5 Results of antioxidant activity test

[0170] Antioxidant activity was positively correlated with the content of free flavonoids and polyphenols in the system. Examples 1-4 showed that gradient fermentation fully converted apigenin and polymeric anthocyanins into highly active free antioxidants, with DPPH scavenging rates of 83.7%-86.2%. In Example 5, continuous high-oxygen stirring led to polyphenol oxidation and loss, reducing the scavenging rate to 74.5%. In Comparative Example 1, the strain failed to synergistically, resulting in insufficient conversion of active ingredients and a scavenging rate of only 42.8%. Comparative Example 2 lacked a solid-phase pH buffer, limiting enzyme activity and resulting in a low accumulation of antioxidants. These data further validated that three-stage gradient stirring can effectively retain and convert antioxidant active ingredients.

[0171] 7. Uric acid-lowering activity assay The uric acid-lowering efficacy was determined using the xanthine oxidase inhibition method. The finished products obtained from each example and comparative example were filtered through a 0.45 μm microporous membrane and then diluted with pH 7.5 phosphate buffer to a total solids concentration of 2.0 mg / mL for later use. Prepare a 0.5 mmol / L xanthine substrate solution and preheat it to 37°C; prepare a 0.1 U / mL xanthine oxidase solution and store it on ice. Add samples according to the following systems (all preheated to 37°C): Control group: 2.5 mL xanthine substrate solution + 0.5 mL buffer solution, mix well, add 0.5 mL xanthine oxidase solution, react at 37℃ for 10 min, immediately add 1 mL 1 mol / L HCl to terminate the reaction, and measure the absorbance at 290 nm, which is recorded as control A. Sample group: 2.5 mL xanthine substrate solution + 0.5 mL sample solution, mix well, add 0.5 mL xanthine oxidase solution, react under the same conditions, and measure absorbance after termination. This is recorded as sample A. Blank group: 2.5 mL xanthine substrate solution + 0.5 mL buffer + 1 mL HCl, then add 0.5 mL enzyme solution, and measure the absorbance as the baseline blank; The xanthine oxidase inhibition rate was calculated by performing three parallel measurements and taking the average value as follows: Inhibition rate (%) = [(ΔA control - ΔA sample) / ΔA control] × 100% Where ΔA is the absorbance of the reaction group minus the absorbance of the corresponding blank baseline.

[0172] The results of the uric acid-lowering activity assay are shown in Table 6.

[0173] Table 6 Results of uric acid-lowering activity assay

[0174] The inhibition capacity of the samples depends on the enrichment of free apigenin and short-chain inulin. Examples 1-4, using a gradient fermentation process, fully released the two core active substances, achieving enzyme inhibition rates of 73.5%-76.8%. Example 5, with its high-shear, high-oxygen environment, disrupted the biofilm, leading to a decrease in functional component conversion and an inhibition rate of only 62.5%. Comparative Example 1, with simultaneous inoculation of antagonistic microbial communities, showed extremely poor conversion of active substrates and the weakest inhibition effect. Comparative Example 2, lacking a solid-phase sustained-release buffer, suffered from pH mismatch with the enzymatic reaction in the later stages, resulting in significantly lower uric acid-lowering activity compared to the other examples. These findings fully demonstrate that the entire process of this invention can synergistically enrich and regulate the effective components for uric acid metabolism.

[0175] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, nor does it mean that the present invention must rely on the above process steps for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A fermentation method for a compound fermented plant beverage with uric acid-lowering and antioxidant activities, characterized in that, The fermentation method employs a three-stage, stepwise fermentation process, comprising the following steps: (1) Raw material pretreatment Hawthorn, wolfberry and celery were enzymatically hydrolyzed to obtain hawthorn enzymatic hydrolysate, wolfberry enzymatic hydrolysate and celery enzymatic hydrolysate; (2) Combine and adjust the viscosity The three enzymatic hydrolysates were combined and added to the fermentation tank at a mass ratio of 1:1~2:3~5 of dried hawthorn, wolfberry and celery. The viscosity of the system at 30~40℃ was adjusted to 200~600 mPa·s using maltitol solution and water. Then, the pH was adjusted to 5.6-5.8 using a composite buffer of potassium citrate and dipotassium hydrogen phosphate, and then calcium carbonate microsphere slow-release buffer was added. (3) First stage fermentation Simultaneously, *Lactobacillus reuteri* CGMCC 1.2838 and *Lactobacillus rhamnosus* CGMCC 1.2467 were inoculated into the fermenter. The inoculation amount of *Lactobacillus reuteri* was 0.8 × 10⁻⁶ based on the total dry weight of the raw materials. 8 CFU / g, the inoculum size of Lactobacillus rhamnosus was 0.3 × 10⁻⁶. 8 CFU / g; After inoculation, start timing from 0h, control the fermentation temperature at 37±1℃, and ferment until 12h; (4) Second stage fermentation Within 30 minutes of the completion of the first stage of fermentation, Bifidobacterium bifidum CGMCC 1.5029 was inoculated at a rate of 0.2 × 10⁻⁶. 8 CFU / g; After inoculation, the fermentation temperature was controlled at 37±1℃ and ended at 24h; (5) Third stage fermentation The third stage of fermentation is carried out at a temperature of 34±1℃, and fermentation is terminated at 36 hours or when the conversion rate of apigenin in the fermentation broth is ≥90%. Based on 0h, purified water equal to 5% of the total dry weight of the initial raw materials was added at 24h and 30h.

2. The fermentation method as described in claim 1, characterized in that, The first step of inoculation is top spray inoculation. Before inoculation, turn on the agitator to 12 r / min and stir for 2 minutes. Then, inoculate at a speed of 5~10 L / min while stirring. After inoculation, continue stirring for 5 minutes and then turn off the agitator.

3. The fermentation method as described in claim 1, characterized in that, The second step of vaccination uses a bottom-distributed vaccination method.

4. The fermentation method as described in claim 3, characterized in that, Before the enzymatic hydrolysis in step (1), hawthorn is pulverized to 100 mesh, wolfberry to 40 mesh, and celery to 120 mesh.

5. The fermentation method as described in claim 1, characterized in that, The chitosan membrane-coated calcium carbonate microspheres have a particle size of 10~20μm and a thickness of 0.8~1.2μm.

6. The fermentation method according to claim 1, characterized in that, In step (2), inulin is added as a dispersant, and the amount of inulin added is 0.2 to 0.3% of the dry weight of the raw material.

7. The fermentation method according to claim 1, characterized in that, The dry weight ratio of hawthorn, wolfberry and celery is 1:1.5:

4.

8. The fermentation method according to any one of claims 1 to 7, characterized in that, The fermentation method further includes step (6): after the fermentation is terminated, the mixture is filtered, homogenized and sterilized in sequence to obtain a compound fermented plant beverage.

9. The fermentation method as described in claim 8, characterized in that, The homogenization process employs a two-stage homogenization process, with the first-stage pressure controlled at 18~22MPa and the second-stage pressure controlled at 3~7MPa.

10. A compound fermented plant beverage with uric acid-lowering and antioxidant activities prepared by the fermentation method according to any one of claims 1 to 9.

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