Preparation method of hippophae rhamnoides fruit compound enzyme

Abstract

The invention relates to the technical field of food biology, in particular to a preparation method of a sea-buckthorn compound enzyme. Aiming at the technical problems of low active ingredient content, single function, poor process stability and the like of a traditional sea-buckthorn enzyme product, efficient extraction and biotransformation of active substances are realized through innovative process design. The method comprises the following steps: increasing the juice yield of the sea-buckthorn by adopting a gradient freezing and crushing technology and retaining heat-sensitive components; an ultrafiltration and secondary fermentation combined process is introduced to optimize the acidity and taste of the product; and finally, the product stability is guaranteed through high-pressure homogenization and an automatic control technology. According to the process, the content of core components such as total flavonoids and SOD activity of the sea-buckthorn is effectively increased, excessive accumulation of ethanol is inhibited, a compound enzyme product with multiple functions of resisting oxidation, regulating intestinal flora and the like is formed, and a green and efficient technical solution is provided for the deep processing industry of the sea-buckthorn.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, and in particular to a method for preparing a sea buckthorn complex enzyme. Background Technology

[0002] Sea buckthorn, a plant used both as food and medicine, is rich in flavonoids, polyphenols, vitamins, and unsaturated fatty acids, possessing physiological functions such as antioxidation, anti-inflammation, and immune regulation. In recent years, the market demand for functional beverages made primarily from sea buckthorn has continued to grow. However, existing sea buckthorn enzyme products generally face three major technical bottlenecks: First, traditional fermentation processes often use single strains or simple bacterial combinations, resulting in single metabolites and insufficient conversion efficiency of active ingredients, making it difficult to achieve efficient bioactivation of flavonoids in sea buckthorn. Second, the complex interactions between components in the preparation of compound plant-based enzymes often lead to degradation or antagonism of active ingredients, especially the synergistic effect of Polygonatum polysaccharides and malt starch enzymatic hydrolysis products is difficult to effectively utilize. Third, existing processes lack a systematic design for preserving rose fragrance components and synergistically extracting antioxidant active substances, resulting in insufficient aroma intensity and significant loss of functional components in the final product.

[0003] In the preparation of sea buckthorn pulp, traditional pressing processes generally yield less than 75% of the juice, and high-speed centrifugation easily leads to the oxidation and browning of heat-sensitive components. For the extraction of Polygonatum sibiricum, conventional water extraction results in low extraction rates and high impurity content, while organic solvent extraction, although increasing the concentration of active ingredients, leaves residual solvents that affect product safety. During malt saccharification, traditional α-amylase and saccharifying enzymes exhibit substrate competition, leading to significant fluctuations in reducing sugar production. Regarding fermentation processes, existing research focuses primarily on the acid-producing or enzyme-producing characteristics of single strains, lacking systematic optimization of multi-strain synergistic metabolic networks. This results in insufficient activity of functional components such as superoxide dismutase (SOD) in the final product, and high levels of residual ethanol. Furthermore, the high-temperature sterilization commonly used in post-processing easily destroys heat-sensitive active substances, while simple filtration is insufficient to effectively remove large molecular impurities, affecting product clarity and stability.

[0004] The existing functional evaluation system for sea buckthorn enzyme products also has shortcomings. Most studies only focus on the content determination of single components, lacking research on the synergistic effects of multiple components. For example, the synergistic antioxidant mechanism of sea buckthorn flavonoids and polygonatum saponins is not yet clear, and the influence of maltose products on the growth and metabolism of probiotics has not been revealed. These technical blind spots lead to the problem that the functional claims of commercially available products generally do not match the actual effects, hindering the upgrading and development of the sea buckthorn compound enzyme industry. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing sea buckthorn compound enzyme.

[0006] To achieve the above objectives, this invention provides the following technical solution: a method for preparing sea buckthorn compound enzymes, the core of which lies in constructing a basic preparation process framework for sea buckthorn compound enzymes, achieving efficient extraction and conversion of active ingredients through multi-stage refined processing. The preparation of sea buckthorn pulp employs low-temperature pressing technology, releasing juice by physically breaking down cell walls. Controlling the juice yield to ≥85% maximizes the retention of flavonoids, polyphenols, and vitamins in sea buckthorn. The enzymatic hydrolysis system uses a combination of cellulase and pectinase. Experimental verification shows that the concentration range can destroy plant cell walls while avoiding flavor degradation caused by excessive hydrolysis. An acidic environment of pH 3.8-4.5 simulates the natural fermentation environment of sea buckthorn, activating endogenous enzyme activity. Constant temperature stirring at 50-60℃ for 2-3 hours balances enzymatic hydrolysis efficiency and energy consumption. Vacuum microwave drying of Polygonatum rhizome preserves the fat-soluble properties of its active ingredients. A 60-mesh particle size improves mass transfer efficiency during subsequent ethanol extraction. Reflux extraction with a 75% ethanol solution at 70-80℃ effectively dissolves polar components such as Polygonatum saponins while avoiding damage to heat-sensitive substances at high temperatures. Malt, after being pulverized to 20-40 mesh, allows its starch granules to fully contact the phosphate buffer, achieving gradient saccharification under the synergistic action of α-amylase and saccharifying enzymes. The liquefaction stage at 90-95℃ disrupts the starch crystal structure, while the saccharification stage at 60℃ precisely controls the amount of reducing sugar produced, providing a suitable carbon source for subsequent fermentation. Sea buckthorn pulp, Polygonatum extract, and maltose syrup are mixed in a 4:2:3 ratio. This ensures the presentation of the main flavor of sea buckthorn while enhancing the nutritional value of the fermentation substrate through the synergistic effect of Polygonatum polysaccharides and maltose syrup products. The addition of 0.5-1.0 wt% rose petal homogenate not only imparts a unique aroma to the product but also enhances the system's antioxidant capacity due to its rich vitamin C and flavonoid content. Adjusting the initial sugar content to 18-22°Brix meets the growth requirements of probiotics, and the slightly acidic environment of pH 4.0-4.2 inhibits the growth of miscellaneous bacteria, laying the foundation for subsequent compound fermentation.

[0007] Furthermore, the gradient freeze-thaw process optimizes the disruption of sea buckthorn cells through three-stage temperature control: pre-freezing at -18℃ causes intracellular water to form ice crystals; a gradient heating rate of 5℃ / min avoids mechanical damage to cell structure caused by sudden temperature changes; and after three freeze-thaw cycles, the cell membrane integrity is completely destroyed, significantly increasing the juice yield during pressing. Compared to traditional pressing processes, this technology increases the retention rate of active ingredients in sea buckthorn pulp by more than 15%, especially reducing the heat-sensitive destruction of vitamin C and polyphenols by 40%, while also reducing the impact of heat generated by mechanical pressing on the material, ensuring the efficient execution of subsequent enzymatic hydrolysis reactions.

[0008] Furthermore, after treatment with β-cyclodextrin, the hydrophobic saponin components of the Polygonatum extract are encapsulated within the cyclodextrin cavities, forming stable nanoscale inclusion complexes. The mild conditions of stirring at 40℃ for 2 hours ensure efficient inclusion while preventing cyclodextrin decomposition caused by high temperatures. Drying at 45℃ to ≤5% moisture content prevents the inclusion complex from absorbing moisture and agglomerating. This technology improves the stability of the Polygonatum extract by 3 times, effectively inhibiting the oxidative degradation of active ingredients during storage, while also improving its dispersion uniformity in the fermentation matrix and promoting the utilization efficiency of active substances by probiotics during subsequent fermentation.

[0009] Furthermore, 0.1-0.3 wt% calcium chloride is added to the malt saccharification liquid to stabilize the spatial conformation of α-amylase and saccharifying enzyme through ionic bonding, thereby improving the enzyme's thermostability and catalytic activity. A thermostable α-amylase derived from Aspergillus niger is used, maintaining over 85% enzyme activity during the liquefaction stage at 90-95℃, a 20% improvement over ordinary α-amylase, ensuring efficient starch liquefaction. A specialized saccharifying enzyme is used in the saccharification stage, with its optimal pH and temperature perfectly matched to the malt composition, increasing the reducing sugar production rate by 25% and providing a sufficient carbon source for subsequent co-fermentation by lactic acid bacteria and yeast.

[0010] Furthermore, the activation treatment of the compound fermentation agent optimized the activity of the strains through staged cultivation: the freeze-dried bacterial powder was activated for 12 hours in MRS medium (for lactic acid bacteria) and YPD medium (for yeast) respectively to restore the metabolic function of the cells. After being transferred to sterilized malt extract medium, it was cultured at 30°C with shaking until the end of the logarithmic growth phase, at which point the cell concentration reached 1×10^8 CFU / mL, and the viable cell ratio exceeded 95%. This activation process enhanced the symbiotic adaptability of the two strains, allowing them to quickly occupy a dominant position in fermentation after inoculation, inhibiting contamination by other microorganisms, and providing a stable microbial system for the subsequent 7-day anaerobic fermentation and 14-day facultative anaerobic fermentation.

[0011] Furthermore, the dynamic oxygen partial pressure control technology optimizes the fermentation environment through a phased nitrogen-filling strategy: for the first 3 days, nitrogen is used to maintain the tank pressure at 0.03-0.05 MPa, creating an absolutely anaerobic environment that encourages *Lactobacillus plantarum* to preferentially utilize glucose to produce lactic acid, while avoiding the inhibitory effect of ethanol accumulation on yeast. From the 4th day onwards, intermittent nitrogen filling is used (10 minutes every 6 hours) to maintain a micro-oxygen environment and activate the ethanol metabolism pathway of *Kluyveromyces martensii*, enabling it to efficiently convert sugars under low-oxygen conditions. Ultimately, the ethanol volume fraction is strictly controlled below 0.5%, achieving a synergistic effect between lactic acid and ethanol, and improving the product's tangy taste and preservative properties.

[0012] Furthermore, after ultrafiltration, acetic acid bacteria are introduced for secondary fermentation. Acetobacter pasteurianus CGMCC1.41 is fermented at 30℃ for 3 days, converting residual sugars into acetic acid and increasing the total acidity to 6.5-7.0 g / L (calculated as malic acid). This process not only enhances the product's acidity and taste but also extends shelf life through the antibacterial effect of acetic acid. Simultaneously, the trace amounts of acetate esters generated undergo esterification with sea buckthorn flavonoids, forming novel antioxidant components that further enhance the product's functionality.

[0013] Furthermore, the preparation of rose petal homogenate utilizes liquid nitrogen flash-freezing technology. Instant freezing at -196℃ weakens the cell walls, achieving a cell wall breakage rate of ≥90%, more than three times higher than traditional grinding methods. During homogenization, 0.05wt% ascorbic acid is added; its reducing properties effectively inhibit polyphenol oxidase activity, preserving the integrity of the rose's red color and aroma components. This technology allows for an 85% retention rate of terpenoids in rose essential oil, giving the product a long-lasting fragrance while avoiding aroma loss due to traditional high-temperature processing.

[0014] Furthermore, high-pressure homogenization, using 30-40 MPa pressure, ensures a particle size distribution (D50) of ≤5 μm in the fermentation broth, while temperature control at 40-50℃ prevents the inactivation of heat-sensitive components. The homogenized system exhibits a 60% reduction in viscosity, resulting in a smoother and more delicate texture, while simultaneously increasing the specific surface area to promote the release and absorption of active ingredients. This process doubles the bioavailability of total flavonoids from sea buckthorn and increases SOD activity retention by 40%, significantly enhancing the product's functional performance.

[0015] Furthermore, the automated control system achieves precise control through real-time monitoring and feedback adjustment of multiple parameters: a temperature sensor monitors fermentation temperature with an accuracy of ±0.5℃, a pH electrode dynamically adjusts pH fluctuations within a range of ≤0.1 units, and a dissolved oxygen probe provides real-time feedback on oxygen concentration changes and automatically adjusts the nitrogen purging frequency. This system strictly controls process parameter fluctuations within ±5% of the set values, ensuring consistency across different batches of products while reducing human error and improving production efficiency and quality control. Data shows that automated control increases the product qualification rate from 85% with traditional processes to 98%, significantly reducing production costs.

[0016] This invention provides a method for preparing sea buckthorn compound enzyme, which has the following beneficial effects: 1. This invention significantly improves the overall quality and functional properties of sea buckthorn compound enzymes by constructing a multi-dimensional synergistically optimized preparation process system. Firstly, by employing gradient freeze-crushing combined with enzymatic hydrolysis technology, the juice yield of sea buckthorn pulp reaches over 85%, an improvement of 12%-15% compared to traditional processes. Simultaneously, by controlling the pH value and enzymatic hydrolysis temperature, efficient degradation of macromolecules such as pectin and cellulose is achieved, significantly increasing the dissolution rate of active ingredients. This process innovatively solves the problem of easy oxidation of heat-sensitive components in traditional pressing processes, retaining over 90% of the vitamin C and polyphenols in sea buckthorn fruit.

[0017] Regarding the synergistic processing of Polygonatum and malt, this invention employs a gradient-temperature microwave drying combined with ethanol fractional extraction technology, increasing the extraction rate of Polygonatum saponins to 18.7 mg / g, more than three times higher than conventional water extraction. By introducing a phosphate buffer system and a high-temperature resistant saccharifying enzyme, the reducing sugar concentration in the malt saccharification liquid can reach over 150 g / L, and the saccharification efficiency is increased by 25% compared to traditional processes. Most importantly, the polysaccharide components in the Polygonatum extract form a synergistic network with the malt saccharification products, significantly promoting the generation of short-chain fatty acids during probiotic fermentation, resulting in acetic acid and butyric acid content in the final product reaching 2.3 times that of traditional processes.

[0018] This invention's innovative compound fermentation system achieves targeted enrichment of functional components through precise regulation of multi-strain symbiotic metabolism. The synergistic effect of *Lactobacillus plantarum* and *Kluyveromyces martensii* increases SOD activity to over 300 U / mL, a 60% improvement compared to single-strain fermentation. Dynamic oxygen partial pressure regulation technology effectively inhibits excessive ethanol accumulation, strictly controlling the ethanol volume fraction in the finished product below 0.5%, overcoming the technical bottleneck of traditional enzyme products' susceptibility to rancidity. The combined ultrafiltration and secondary fermentation process not only improves product clarity but also increases the total acidity to 6.5-7.0 g / L through the secondary metabolism of acetic acid bacteria, giving the product a unique tangy taste and a longer shelf life.

[0019] In terms of sensory quality optimization, liquid nitrogen quick-freezing and crushing technology achieves a cell wall breakage rate of over 90% for rose petals. Combined with an ascorbic acid color-protecting system, this effectively preserves terpenoid compounds in rose essential oil, increasing the aroma intensity of the finished product by 40%. High-pressure homogenization further refines the particle size of the materials; the microstructure with D50≤5μm significantly improves the smoothness and stability of the taste. The full-process automated control system ensures that fluctuations in key process parameters are controlled within ±5%, guaranteeing a high degree of consistency in product quality between batches.

[0020] This invention integrates multiple functions, including anti-oxidation, gut microbiota regulation, and immune enhancement, through an innovative design of a synergistic effect mechanism among its components. Experimental data show that in a mouse model that took this product continuously for 8 weeks, serum MDA levels decreased by 32%, IL-6 inflammatory factor expression decreased by 45%, and the abundance of beneficial gut bacteria increased by 2.1 times, demonstrating significant in vivo bioactivity. These technological breakthroughs enable the product of this invention to reach international leading levels in functionality, safety, and sensory quality, providing a brand-new technical solution for the sea buckthorn deep processing industry. Detailed Implementation

[0021] How to use In the raw material pretreatment stage, fresh sea buckthorn berries are selected and processed using a gradient freeze-thaw technique. Three cycles of freeze-thaw cycles are used to fully loosen the pulp tissue, followed by low-temperature pressing to obtain a high-concentration pulp. During this process, the juice yield is strictly controlled to be no less than 85%, and a compound enzyme preparation is added to perform targeted enzymatic hydrolysis of the pulp. The enzymatic hydrolysis system is a scientifically formulated combination of cellulase and pectinase, which, under specific pH and temperature conditions for a certain period of time, effectively decomposes the plant cell wall structure and improves the dissolution efficiency of active substances.

[0022] In the co-processing stage of Polygonatum and malt, the Polygonatum tubers are vacuum microwave-dried, then pulverized and sieved. Multi-stage reflux extraction with ethanol solution is then performed, and the resulting extract is incorporated to form a stable complex. The malt raw material is finely pulverized and combined with a phosphate buffer system, undergoing a gradient saccharification reaction under the action of specific enzymes to generate sugars suitable for fermentation. The processed sea buckthorn pulp, Polygonatum extract, and malt saccharification liquid are mixed in a specific ratio, and rose petal homogenate is added for flavor blending to form a balanced fermentation substrate.

[0023] The fermentation process employs a co-fermentation system of multiple microbial strains, achieving multi-stage fermentation through precise control of parameters such as temperature, humidity, and oxygen partial pressure. Initially, a strictly anaerobic environment promotes lactic acid bacteria-dominated metabolism; in the middle stage, a facultative anaerobic environment enhances yeast activity; and in the later stage, dynamic oxygen partial pressure management achieves the synergistic accumulation of flavor compounds and functional components. Various physicochemical indicators are regularly monitored during fermentation to ensure a stable and controllable fermentation process.

[0024] The post-processing stage employs a combination of multi-stage filtration and ultrafiltration technologies to remove large molecular impurities while retaining active ingredients. The ultrafiltration permeate undergoes a secondary fermentation process to further optimize acidity and taste, and finally, high-pressure homogenization ensures a uniform and delicate product texture. An automated control system monitors and adjusts production parameters in real time throughout the entire process, ensuring precise execution of process indicators at each stage.

[0025] This method achieves highly efficient preparation of sea buckthorn compound enzymes through innovative process design. The finished product significantly enhances the content and bioactivity of active ingredients while retaining the natural properties of the raw materials. The entire process emphasizes green environmental protection and energy conservation, aligning with the development trend of modern food processing technology.

[0026] Example 1: Preparation and Enzymatic Hydrolysis Optimization of Seabuckthorn Pulp Fresh sea buckthorn berries are selected and processed using a gradient freeze-thaw technique. Three cycles of freeze-thaw cycles thoroughly loosen the pulp tissue, followed by low-temperature pressing to obtain a high-concentration pulp. During this process, the juice yield is strictly controlled to be no less than 85%, and a compound enzyme preparation is added for targeted enzymatic hydrolysis of the pulp. The enzymatic hydrolysis system is a scientifically formulated combination of cellulase and pectinase, which, under specific pH and temperature conditions for a certain period of time, effectively decomposes the plant cell wall structure and enhances the dissolution efficiency of active substances. After enzymatic hydrolysis, the residue is removed by centrifugation, retaining the clear and transparent pulp for later use.

[0027] Example 2: Extraction and Inclusion Processing of Active Components from Polygonatum After vacuum microwave drying, the rhizomes of Polygonatum sibiricum were pulverized and sieved. They were then extracted using a multi-stage reflux extraction with 75% ethanol solution at a specific temperature. The resulting extract was then treated with β-cyclodextrin to form a stable complex. During the inclusion process, the binding degree between cyclodextrin and Polygonatum sibiricum saponins was controlled by adjusting the temperature and stirring rate, resulting in uniformly dispersed nanoscale inclusion complexes. This treatment effectively improved the stability of the Polygonatum sibiricum extract, inhibited the oxidative degradation of active ingredients during storage, and improved its dispersion uniformity in the fermentation matrix.

[0028] Example 3: Optimization of Malt Gradient Saccharification Process After being pulverized and refined, malt raw materials are mixed with phosphate buffer in a specific ratio, and a gradient saccharification reaction is completed under the synergistic action of α-amylase and saccharifying enzyme. The liquefaction stage uses high-temperature treatment to break down the starch crystal structure, while the saccharification stage achieves efficient production of reducing sugars through precise temperature control. An appropriate amount of calcium chloride is added to the saccharification solution to stabilize enzyme activity, ultimately producing a saccharified solution rich in fermentable sugars such as maltose and glucose, providing a sufficient carbon source for subsequent probiotic fermentation.

[0029] Example 4: Construction and Dynamic Control of a Compound Fermentation System Sea buckthorn pulp, Polygonatum extract, and maltose syrup were mixed in a specific ratio and then blended with rose petal homogenate to create a balanced fermentation substrate. A compound starter culture consisting of Lactobacillus plantarum and Kluyveromyces martensii in a specific ratio was inoculated, and fermentation was carried out in stages under anaerobic and facultative anaerobic conditions. During fermentation, the microbial metabolic environment was optimized using dynamic oxygen partial pressure regulation technology. In the early stage, an absolutely anaerobic environment was created to promote lactic acid bacteria-dominated metabolism; in the middle stage, a microaerobic environment was established to activate yeast activity; and in the later stage, nitrogen purging was used to maintain a stable fermentation environment, achieving the synergistic accumulation of lactic acid, ethanol, and functional components.

[0030] Example 5: Post-processing and quality improvement technology After fermentation, a diatomaceous earth filter is used for coarse filtration, followed by fine filtration through an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa to remove large molecular impurities while retaining active ingredients. The ultrafiltration permeate undergoes secondary fermentation with acetic acid bacteria, converting residual sugars into acetic acid, thus increasing the product's acidity and preservative properties. The final product undergoes high-pressure homogenization to refine particle size, and an automated control system is used to regulate temperature, pH, and dissolved oxygen concentration in real time, ensuring that fluctuations in each process parameter are controlled within ±5% of the set values, resulting in a uniformly textured and smooth-tasting sea buckthorn compound enzyme product.

[0031] Example 6: Synergistic Enhancement Process of Composite Enzymatic Hydrolysis System In the enzymatic hydrolysis of sea buckthorn pulp, a combined system of cellulase and pectinase is used. By adjusting the pH to 3.8-4.5 and the temperature to 50-60℃, efficient cell wall degradation is achieved. The enzyme ratio in the hydrolysis system is optimized, with the ratio of cellulase to pectinase activity units controlled within a specific range to ensure efficient decomposition of macromolecules such as pectin and cellulose, while avoiding flavor deterioration caused by excessive hydrolysis. After enzyme inactivation treatment, the hydrolysate is mixed with other treatment solutions to form a pre-fermentation liquid rich in active ingredients.

[0032] Example 7: Preparation and Color Protection Process of Rose Petal Homogenization Fresh rose petals are processed using liquid nitrogen flash-freezing technology, significantly improving cell wall disruption. Combined with an ascorbic acid color-protecting system, this effectively inhibits polyphenol oxidase activity. A trace amount of ascorbic acid is added during homogenization to maintain the integrity of the rose's red color and aroma components. This technology ensures that over 85% of the terpenoids in rose essential oil are retained, giving the product a long-lasting fragrance while avoiding aroma loss due to traditional high-temperature processing.

[0033] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a sea buckthorn complex enzyme, characterized in that, Includes the following steps: (a) Fresh sea buckthorn berries are pressed at low temperature to obtain sea buckthorn pulp, with a juice yield of ≥85%, and purified water is added at a mass ratio of 1:3~5 for enzymatic hydrolysis. The enzymatic hydrolysis system contains 0.3-0.8 U / g of cellulase and 0.5-1.2 U / g of pectinase, and the pH is adjusted to 3.8-4.

5. The mixture is stirred at a constant temperature of 50-60℃ for 2-3 hours. (b) After taking the rhizome of Polygonatum odoratum, slice it and dry it in vacuum microwave until the moisture content is ≤8%. Then, pulverize it through a 60-mesh sieve, add 75% ethanol solution at a material-to-liquid ratio of 1:10, and reflux extract it twice at 70-80℃ for 1.5 hours each time. Combine the filtrates and concentrate them under reduced pressure to obtain an extract with a relative density of 1.20-1.30 (60℃). (c) Grind the malt to 20-40 mesh, add phosphate buffer at pH 5.0 at a solid-liquid ratio of 1:8, add α-amylase 0.6-1.0 U / g raw material, liquefy at 90-95℃ for 30 minutes, then cool to 60℃ and add saccharifying enzyme 0.8-1.5 U / g raw material, saccharify for 4 hours; (d) Mix the above three treatment solutions in a ratio of sea buckthorn pulp: polygonatum extract: maltose syrup = 4:2:3, add 0.5-1.0wt% rose petal homogenate, adjust the initial sugar content to 18-22°Brix, and adjust the pH to 4.0-4.

2. (e) Inoculate with a compound fermentation agent, which is composed of Lactobacillus plantarum ATCC 8014 and Kluyveromyces marxianus CICC 33027 at a live cell ratio of 3:1, with a total inoculation amount of 5-8%. First, anaerobic fermentation is carried out at 32±1℃ for 7 days, and then transferred to facultative anaerobic fermentation at 28±1℃ for 14 days. (f) After fermentation, the solution is coarsely filtered through a diatomaceous earth filter and then finely filtered through an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa. The permeate is then collected. (g) The final product contains ≥50mg / 100mL of total flavonoids from sea buckthorn, ≥300U / mL of SOD activity, 4.5-6.0g / L of total acidity (calculated as malic acid), and ≤0.5% of ethanol by volume.

2. The method for preparing a sea buckthorn compound enzyme according to claim 1, characterized in that: The seabuckthorn pulp is prepared using a gradient freezing and crushing process, specifically by pre-freezing at -18℃ for 2 hours, then heating to -5℃ at a rate of 5℃ / min and maintaining the temperature for 1 hour, repeating this cycle 3 times before pressing.

3. The method for preparing a sea buckthorn compound enzyme according to claim 1, characterized in that: The extract of Polygonatum sibiricum was concentrated and then subjected to β-cyclodextrin inclusion treatment. The inclusion conditions were β-CD: Polygonatum sibiricum alcohol extract = 1:4 (m / m), stirred at 40°C for 2 hours, and dried at 45°C until the moisture content was ≤5%.

4. The method for preparing a sea buckthorn compound enzyme according to claim 1, characterized in that: The malt saccharification solution contains 0.1-0.3 wt% calcium chloride, and the saccharifying enzyme is a thermostable α-amylase derived from Aspergillus niger.

5. The method for preparing a sea buckthorn compound enzyme according to claim 1, characterized in that: The compound fermentation agent is activated before use. Specifically, the freeze-dried bacterial powder is inoculated into MRS medium and YPD medium, and after being activated by standing at 37°C for 12 hours, it is transferred to sterilized malt extract medium at an inoculation rate of 1% and cultured at 30°C with shaking until the end of the logarithmic growth phase.

6. The method for preparing a sea buckthorn compound enzyme according to claim 1, characterized in that: The fermentation process is subject to dynamic oxygen partial pressure control. Specifically, nitrogen is used to maintain the tank pressure at 0.03-0.05 MPa for the first 3 days of fermentation, and then intermittent nitrogen purging is used from the 4th day onwards (nitrogen is purged for 10 minutes every 6 hours).

7. The method for preparing a sea buckthorn compound enzyme according to claim 1, characterized in that: After ultrafiltration, a secondary fermentation is carried out. Acetobacter pasteurianus CGMCC1.41 is inoculated into the filtrate at a ratio of 0.2-0.4wt%, and fermented at 30℃ for 3 days until the final acidity reaches 6.5-7.0g / L.

8. The method for preparing a sea buckthorn compound enzyme according to claim 1, characterized in that: The rose petal homogenate was prepared using liquid nitrogen quick-freezing and disruption technology, with a cell wall breakage rate of ≥90%. 0.05wt% ascorbic acid was added during homogenization for color protection.

9. The method for preparing a sea buckthorn compound enzyme according to claim 1, characterized in that: The final product undergoes high-pressure homogenization treatment at 30-40 MPa and 40-50 °C for two cycles to achieve a particle size distribution D50 ≤ 5 μm.

10. The method for preparing a sea buckthorn compound enzyme according to claim 1, characterized in that: The method employs an automated control system throughout the entire process, which monitors and adjusts the fermentation temperature, pH value, and dissolved oxygen concentration in real time, with the fluctuation range of each process parameter controlled within ±5% of the set value.