Preparation method of a gyromitrin enzyme and application thereof

Abstract

The application provides a preparation method and application of an agaricus blazei murill enzyme, and the preparation method comprises the following steps: preparing agaricus blazei murill enzyme powder by using mashed agaricus blazei murill fresh fruiting bodies; preparing mixed enzyme powder by using sugar beets, iron stick yam, mulberries and pineapples; mixing the agaricus blazei murill enzyme powder and the mixed enzyme powder in a mass ratio of 8-10:1 to obtain the agaricus blazei murill enzyme. The agaricus blazei murill enzyme prepared by the application has high antioxidant effect and SOD enzyme activity. Meanwhile, the agaricus blazei murill enzyme powder of the application has good stability, can be stored for a long time in the shade after being opened, and can still maintain the stability of product quality, improves the use value of the product, and has more market competitiveness.

Description

Technical Field

[0001] This invention belongs to the field of enzyme preparation technology, specifically relating to a method for preparing *Hydrangea macrophylla* enzyme and its application. Background Technology

[0002] Sprassis Crispa enzymes refer to enzyme preparations or fermentation products produced through the fermentation process of Sprassis Crispa. Sprassis Crispa is an edible fungus with numerous health benefits, containing abundant beta-glucan, protein, polysaccharides, vitamins, minerals, and antioxidants. When Sprassis Crispa is used in the fermentation process, it can produce a series of enzymes, such as proteases, amylases, and cellulases. These enzymes help break down large molecules in food, making them easier to absorb. They may also produce beneficial metabolites, such as antioxidants, which help eliminate free radicals in the body, protect cell structure and function from damage, slow down the aging process, and enhance the body's immune function.

[0003] Currently, many commercially available *Hydrangea macrophylla* enzyme products advertise antioxidant capabilities, making them suitable for use in dietary supplements, health products, or functional foods. However, their actual antioxidant effects are not ideal, and their SOD enzyme activity is also low. Furthermore, with long-term storage, enzyme activity gradually decreases, and antioxidant components degrade, leading to a decline in the product's effectiveness and reducing consumer purchasing desire.

[0004] To maintain the antioxidant effects of *Hydrangea macrophylla* enzyme products, current practices primarily involve storing the product in a cool, dry place, avoiding direct sunlight and high temperatures. Using airtight containers reduces contact with air and moisture, extending the product's shelf life. Alternatively, refrigeration can further inhibit enzyme activity and microbial growth. Adding natural or synthetic antioxidants, such as vitamin C, tocopherol, and hydroxyanisole, to the product formulation can help slow down the oxidation of other components. However, some natural antioxidants may be unstable and easily degrade during processing or storage, thus losing their antioxidant capacity. The safety of synthetic antioxidants is questionable; long-term intake may have adverse effects on human health, as some synthetic antioxidants are suspected of having carcinogenic or endocrine-disrupting effects.

[0005] Therefore, there is an urgent need for a method to prepare *Hydrangea macrophylla* enzyme with good antioxidant effect, high SOD enzyme activity and good stability. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing *Hydrangea macrophylla* enzyme and its application.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing a *Hydrangea macrophylla* enzyme, the method comprising the following steps:

[0009] (1) Mix 100 parts by weight of crushed fresh fruiting bodies of *Hydrangea macrophylla*, 30-40 parts by weight of granulated sugar and 300-350 parts by weight of sterile water, add *Lactobacillus brevis*, and anaerobic ferment at 30-35℃ for 80-90 days. Then add *Lactobacillus plantarum* and anaerobic ferment at 30-35℃ for 60-70 days. Finally add *Lactobacillus helveticus* and anaerobic ferment at 30-35℃ for 30-40 days to obtain fermentation liquid A.

[0010] (2) The fermentation liquid A is concentrated and evaporated under reduced pressure until the solid content is ≥55wt% to obtain concentrated liquid A. The concentrated liquid A is freeze-dried until the moisture content is ≤3wt%, subjected to airflow micro-grinding, and sieved to obtain Hydrangea spp. enzyme powder.

[0011] (3) Add 120-150 parts of sterile water to 100 parts of mixed fruits and vegetables and pulverize. Add 2-3 parts of white sugar and stir well. Add Lactobacillus bulgaricus and ferment at 30-35℃ for 40-50 days. Continue to add Lactobacillus casei and ferment at 30-35℃ for 20-30 days. Add Bifidobacterium longum and ferment at 30-35℃ for 5-10 days to obtain fermentation liquid B.

[0012] (4) The fermentation liquid B is concentrated and evaporated under reduced pressure until the solid content is ≥55wt% to obtain concentrated liquid B. The concentrated liquid B is freeze-dried until the moisture content is ≤3wt%, subjected to airflow micro-grinding, and sieved to obtain mixed enzyme powder.

[0013] (5) Mix the hydrangea enzyme powder and mixed enzyme powder at a mass ratio of 8-10:1 to obtain hydrangea enzyme.

[0014] Furthermore, in step (1), the mass of Lactobacillus brevis accounts for 0.3-0.5% of the total mass of the crushed fresh fruiting bodies of *Hylocereus undatus*, granulated sugar, and sterile water.

[0015] Furthermore, in step (1), the mass of *Lactobacillus plantarum* accounts for 0.1-0.3% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar, and sterile water.

[0016] Furthermore, in step (1), the mass of Lactobacillus helveticus accounts for 0.1-0.3% of the total mass of the crushed fresh fruiting bodies of Hydrangea macrophylla, granulated sugar, and sterile water.

[0017] Existing technologies typically use a single strain to ferment fresh fruiting bodies of *Hydrangea macrophylla*, but the resulting *Hydrangea macrophylla* enzyme has low antioxidant effects and SOD enzyme activity, failing to meet market demands. The inventors attempted to use a compound strain for fermentation, which improved the antioxidant effect, but the SOD enzyme activity remained unsatisfactory. This invention sequentially adds specific enzymes for gradient fermentation, simultaneously improving both antioxidant effects and SOD enzyme activity. Analysis shows that the enzymes selected in this invention facilitate the release or activation of substrates or precursors for subsequent enzyme action. Furthermore, adding enzymes in stages reduces the impact of inhibitors on enzyme activity, allowing the enzymes to exert maximum efficiency with less inhibition, thereby producing more active ingredients. This simultaneously improves antioxidant effects and SOD enzyme activity, enhancing the product's value and market competitiveness. However, the resulting *Hydrangea macrophylla* enzyme powder exhibits unsatisfactory stability; after prolonged storage in a cool, dark place after opening, tests show varying degrees of decline in both antioxidant effects and SOD enzyme activity.

[0018] Furthermore, in step (3), the mixed fruits and vegetables are a mixture of beets, Chinese yam, mulberries and pineapples in a mass ratio of 10:1-4:6-8:3-5.

[0019] Furthermore, in step (3), the mass of Lactobacillus bulgaricus accounts for 2-3% of the total mass of the mixed fruits and vegetables, sterile water and white sugar.

[0020] Furthermore, in step (3), the mass of Lactobacillus casei accounts for 1-1.5% of the total mass of the mixed fruits and vegetables, sterile water and white sugar.

[0021] Furthermore, in step (3), the mass of Bifidobacterium longum accounts for 1-1.5% of the total mass of the mixed fruits and vegetables, sterile water and white sugar.

[0022] During the experiment, this invention used Vitamin C as an antioxidant added to the *Hydrangea macrophylla* enzyme powder. However, this weakened the overall antioxidant effect of the system. This may be because Vitamin C reacts chemically with other components in the enzyme powder, such as binding to the active sites of certain enzymes or reacting with other antioxidants like phenolic compounds, leading to a decrease in the system's antioxidant capacity. This invention attempted to use mixed fruit fermentation products mixed with *Hydrangea macrophylla* enzyme powder, but the unsuitable pH environment of both fruit enzyme powder and *Hydrangea macrophylla* enzyme powder may affect enzyme stability, resulting in unstable SOD enzyme activity. This invention improves the storage stability of *Hydrangea macrophylla* enzyme by using a mixed enzyme powder prepared from a mixture of vegetables and fruits as raw materials and mixing it with *Hydrangea macrophylla* enzyme powder. The mixed enzyme powder and *Hydrangea macrophylla* enzyme powder have a suitable pH. Furthermore, the fermented products of beets, Chinese yam, mulberries, and pineapples are rich in different antioxidant components and active enzymes. The antioxidant components have a synergistic effect, and the enzymes in different raw materials complement each other functionally, improving the stability of the system.

[0023] The combined action of these enzymes can improve the bioavailability of enzyme powder, making its active ingredients easier for the body to absorb and utilize. Their synergistic effect can enhance the system's biological activity.

[0024] Furthermore, steps (2) and (4) are both passed through a 100-300 mesh sieve.

[0025] This invention also provides the application of the above preparation method in the preparation of *Hydrangea macrophylla* enzyme.

[0026] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0027] 1. The *Hydrangea macrophylla* enzyme prepared by this invention has high antioxidant effect and SOD enzyme activity. At the same time, the *Hydrangea macrophylla* enzyme powder of this invention has good stability. After opening the can, it can still maintain the stability of product quality even after long-term storage in a cool place, which improves the use value of the product and makes it more competitive in the market.

[0028] 2. This invention sequentially adds specific enzymes for gradient fermentation, which simultaneously improves the antioxidant effect and SOD enzyme activity, thereby increasing the product's usability and making it more competitive in the market.

[0029] 3. This invention improves the storage stability of *Hydrangea macrophylla* enzyme by mixing a mixture of vegetables and fruits as raw materials with *Hydrangea macrophylla* enzyme powder. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] The bacteria used in this invention are all commercially available products, and specific information is as follows:

[0032] Lactobacillus brevis, accession number: SHBCC D14346. Deposited at the Shanghai Center for Microbiological Collections.

[0033] Lactobacillus plantarum, accession number: CCTCC AB 2010210, is deposited at the China Center for Type Culture Collection.

[0034] Lactobacillus casei, accession number: SHBCC D24737, deposited at the Shanghai Center for Microbiological Collection.

[0035] Bifidobacterium longum, accession number: CCTCC HB 20082718, is deposited at the China Center for Type Culture Collection.

[0036] Lactobacillus bulgaricus, accession number: CCTCC CB 20082295, is deposited at the China Center for Type Culture Collection.

[0037] Lactobacillus helveticus, accession number: CCTCC AB 2010205, is deposited at the China Center for Type Culture Collection.

[0038] Example 1

[0039] This embodiment provides a method for preparing *Hydrangea macrophylla* enzyme, the preparation method comprising the following steps:

[0040] (1) Mix 100 parts by weight of crushed fresh fruiting bodies of *Hydrangea macrophylla*, 35 parts by weight of granulated sugar and 320 parts by weight of sterile water, add *Lactobacillus brevis*, the mass of which accounts for 0.4% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 85 days. Then add *Lactobacillus plantarum*, the mass of which accounts for 0.2% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 65 days. Then add *Lactobacillus helveticus*, the mass of which accounts for 0.2% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 35 days to obtain fermentation liquid A;

[0041] (2) The fermentation liquid A was concentrated and evaporated under reduced pressure until the solid content was 56 wt% to obtain concentrated liquid A. The concentrated liquid A was freeze-dried until the moisture content was 2.5 wt%, and then subjected to air-flow micro-grinding and passed through a 200-mesh sieve to obtain Hydrangea spp. enzyme powder.

[0042] (3) The mixed fruits and vegetables are a mixture of beets, Chinese yam, mulberries and pineapples in a mass ratio of 10:3:7:4. Add 130 parts of sterile water to 100 parts of the mixed fruits and vegetables and blend. Add 2.5 parts of white sugar and stir well. Add Lactobacillus bulgaricus. The mass of Lactobacillus bulgaricus accounts for 2.4% of the total mass of the mixed fruits and vegetables, sterile water and white sugar. Ferment at 32℃ for 46 days. Continue to add Lactobacillus casei. The mass of Lactobacillus casei accounts for 1.2% of the total mass of the mixed fruits and vegetables, sterile water and white sugar. Ferment at 32℃ for 25 days. Add Bifidobacterium longum. The mass of Bifidobacterium longum accounts for 1.3% of the total mass of the mixed fruits and vegetables, sterile water and white sugar. Ferment at 32℃ for 7 days to obtain fermentation liquid B.

[0043] (4) The fermentation liquid B was concentrated and evaporated under reduced pressure to a solid content of 57wt% to obtain concentrated liquid B. The concentrated liquid B was freeze-dried to a moisture content of 3wt%, subjected to airflow micro-grinding, and passed through a 200-mesh sieve to obtain mixed enzyme powder.

[0044] (5) Mix the hydrangea enzyme powder and the mixed enzyme powder at a mass ratio of 9:1 to obtain hydrangea enzyme.

[0045] Example 2

[0046] This embodiment provides a method for preparing *Hydrangea macrophylla* enzyme, the preparation method comprising the following steps:

[0047] (1) Mix 100 parts by weight of crushed fresh fruiting bodies of *Hydrangea macrophylla*, 30 parts by weight of granulated sugar and 350 parts by weight of sterile water, add *Lactobacillus brevis*, the mass of which accounts for 0.3% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 35°C for 90 days. Then add *Lactobacillus plantarum*, the mass of which accounts for 0.1% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 35°C for 60 days. Then add *Lactobacillus helveticus*, the mass of which accounts for 0.3% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 30°C for 40 days to obtain fermentation liquid A;

[0048] (2) The fermentation liquid A was concentrated and evaporated under reduced pressure to a solid content of 55wt% to obtain concentrated liquid A. The concentrated liquid A was freeze-dried to a moisture content of 3wt%, subjected to airflow micro-grinding, and passed through a 100-mesh sieve to obtain Hydrangea spp. enzyme powder.

[0049] (3) The mixed fruits and vegetables are a mixture of beets, Chinese yam, mulberries and pineapples in a mass ratio of 10:1:8:3. Add 150 parts of sterile water to 100 parts of the mixed fruits and vegetables and blend. Add 2 parts of white sugar and stir well. Add Lactobacillus bulgaricus, with the mass of Lactobacillus bulgaricus accounting for 3% of the total mass of the mixed fruits and vegetables, sterile water and white sugar. Ferment at 35°C for 50 days. Continue to add Lactobacillus casei, with the mass of Lactobacillus casei accounting for 1% of the total mass of the mixed fruits and vegetables, sterile water and white sugar. Ferment at 35°C for 20 days. Add Bifidobacterium longum, with the mass of Bifidobacterium longum accounting for 1.5% of the total mass of the mixed fruits and vegetables, sterile water and white sugar. Ferment at 35°C for 10 days to obtain fermentation liquid B.

[0050] (4) The fermentation liquid B is concentrated and evaporated under reduced pressure to a solid content of 55wt% to obtain concentrated liquid B. The concentrated liquid B is freeze-dried to a moisture content of 3wt%, subjected to airflow micro-grinding, and passed through a 300-mesh sieve to obtain mixed enzyme powder.

[0051] (5) Mix the hydrangea enzyme powder and the mixed enzyme powder at a mass ratio of 10:1 to obtain hydrangea enzyme.

[0052] Comparative Example 1

[0053] The difference between this comparative example and Example 1 is that no mixed enzyme powder was added.

[0054] This comparative example provides a method for preparing *Hydrangea macrophylla* enzyme, the preparation method comprising the following steps:

[0055] (1) Mix 100 parts by weight of crushed fresh fruiting bodies of *Hydrangea macrophylla*, 35 parts by weight of granulated sugar and 320 parts by weight of sterile water, add *Lactobacillus brevis*, the mass of which accounts for 0.4% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 85 days. Then add *Lactobacillus plantarum*, the mass of which accounts for 0.2% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 65 days. Then add *Lactobacillus helveticus*, the mass of which accounts for 0.2% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 35 days to obtain fermentation liquid A;

[0056] (2) The fermentation liquid A was concentrated and evaporated under reduced pressure until the solid content was 56 wt% to obtain concentrated liquid A. The concentrated liquid A was freeze-dried until the moisture content was 2.5 wt%, and then subjected to air-flow micro-grinding and passed through a 200-mesh sieve to obtain physalis enzyme.

[0057] Comparative Example 2

[0058] The difference between this comparative example and Example 1 is that in step (1), Lactobacillus brevis is replaced with Lactobacillus bulgaricus; Lactobacillus plantarum is replaced with Lactobacillus casei; and Lactobacillus helveticus is replaced with Bifidobacterium longum.

[0059] Comparative Example 3

[0060] The difference between this comparative example and Example 1 is that the order of adding the bacterial strains in step (1) is different.

[0061] (1) Mix 100 parts by weight of crushed fresh fruiting bodies of *Hydrangea macrophylla*, 35 parts by weight of granulated sugar and 320 parts by weight of sterile water, add *Lactobacillus helveticus*, the mass of *Lactobacillus helveticus* being 0.4% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 85 days. Then add *Lactobacillus plantarum*, the mass of *Lactobacillus plantarum* being 0.2% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 65 days. Then add *Lactobacillus brevis*, the mass of *Lactobacillus brevis* being 0.2% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 35 days to obtain fermentation broth A.

[0062] Comparative Example 4

[0063] The difference between this comparative example and Example 1 is that in step (1), the mass of *Lactobacillus brevis* accounts for 0.1% of the total mass of the crushed fresh fruiting body of *Hylocereus undatus*, granulated sugar, and sterile water; the mass of *Lactobacillus plantarum* accounts for 0.35% of the total mass of the crushed fresh fruiting body of *Hylocereus undatus*, granulated sugar, and sterile water; and the mass of *Lactobacillus plantarum* accounts for 0.35% of the total mass of the crushed fresh fruiting body of *Hylocereus undatus*, granulated sugar, and sterile water.

[0064] Comparative Example 5

[0065] The difference between this comparative example and Example 1 is that the mixed fruits and vegetables in step (3) are a mixture of beets, Chinese yam, mulberries and pineapples in a mass ratio of 1:1:1:1.

[0066] Comparative Example 6

[0067] The difference between this comparative example and Example 1 is that the mixed fruits and vegetables are replaced with a mixed fruit consisting of lemon, grape, apple and blueberry in a mass ratio of 10:3:7:4.

[0068] Comparative Example 7

[0069] The difference between this comparative example and Example 1 is that in step (3), Lactobacillus bulgaricus is replaced with Lactobacillus brevis; Lactobacillus casei is replaced with Lactobacillus plantarum; and Bifidobacterium longum is replaced with Lactobacillus helveticus.

[0070] Comparative Example 8

[0071] The difference between this comparative example and Example 1 is that the order in which the bacterial strains were added is different.

[0072] (3) The mixed fruits and vegetables are a mixture of beets, Chinese yam, mulberries and pineapples in a mass ratio of 10:3:7:4. Add 130 parts of sterile water to 100 parts of the mixed fruits and vegetables and blend. Add 2.5 parts of white sugar and stir well. Add Bifidobacterium longum, with the mass of Bifidobacterium longum accounting for 2.4% of the total mass of the mixed fruits and vegetables, sterile water and white sugar. Ferment at 32℃ for 46 days. Continue to add Lactobacillus casei, with the mass of Lactobacillus casei accounting for 1.2% of the total mass of the mixed fruits and vegetables, sterile water and white sugar. Ferment at 32℃ for 25 days. Add Lactobacillus bulgaricus, with the mass of Lactobacillus bulgaricus accounting for 1.3% of the total mass of the mixed fruits and vegetables, sterile water and white sugar. Ferment at 32℃ for 7 days to obtain fermentation liquid B.

[0073] Comparative Example 9

[0074] The difference between this comparative example and Example 1 is that in step (1), fermentation broth A is prepared using a compound bacteria.

[0075] (1) Mix 100 parts by weight of crushed fresh fruiting bodies of *Hydrangea macrophylla*, 35 parts by weight of granulated sugar and 320 parts by weight of sterile water, add compound bacteria, the mass of the compound bacteria being 0.8% of the total mass of the crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and anaerobic ferment at 32℃ for 185 days to obtain fermentation liquid A; the compound bacteria is a mixture of *Lactobacillus brevis*, *Lactobacillus plantarum* and *Lactobacillus helveticus* in a mass ratio of 2:1:1.

[0076] Comparative Example 10

[0077] The difference between this comparative example and Example 1 is that fermentation broth B was prepared using a compound bacteria.

[0078] Specifically, the mixed fruit and vegetable mixture consisted of beets, Chinese yam, mulberries, and pineapples in a mass ratio of 10:3:7:4. 130 parts by mass of sterile water were added to 100 parts by mass of the mixed fruit and vegetable mixture and blended. 2.5 parts by mass of white sugar were added and stirred until well mixed. A compound microbial culture was then added, with the compound microbial culture accounting for 4.9% of the total mass of the mixed fruit and vegetable mixture, sterile water, and white sugar. Fermentation was carried out at 32℃ for 46 days to obtain fermentation broth B. The compound microbial culture consisted of *Lactobacillus bulgaricus*, *Lactobacillus casei*, and *Bifidobacterium longum* in a mass ratio of 2.4:1.2:1.3. Performance testing was then conducted.

[0079] The *Hydrangea macrophylla* enzymes prepared in Examples 1-2 and Comparative Examples 1-10 were used as samples for performance testing:

[0080] 1. Method for determining DPPH free radical scavenging rate:

[0081] 1) Prepare a 0.2 mM DPPH solution with anhydrous ethanol and store it protected from light;

[0082] 2) Transfer 2 mL of DPPH and 2 mL of sample to mix, shake well, and let stand in the dark at room temperature for 30 min. Then, zero the sample with an equal volume of anhydrous ethanol and measure the absorbance value (Ai) at a wavelength of 517 nm.

[0083] 3) Measure the absorbance (Aj) of 2 mL of sample mixed with 2 mL of anhydrous ethanol;

[0084] 4) Measure the absorbance (A0) of a mixture of 2 mL DPPH and 2 mL anhydrous ethanol.

[0085] The calculation formula is as follows: DPPH free radical scavenging rate (%) = [A0 – (Ai – Aj)] × 100 / A0.

[0086] 2. SOD enzyme activity was determined using the superoxide dismutase SOD kit (NBT method) produced by Suzhou Keming Biotechnology Co., Ltd.

[0087] Table 1 Results of DPPH free radical scavenging rate

[0088] DPPH free radical scavenging rate % SOD enzyme activity (U / mg) Example 1 96.34 259 Example 2 96.25 250 Comparative Example 1 94.78 228 Comparative Example 2 93.52 217 Comparative Example 3 94.13 220 Comparative Example 4 94.54 225 Comparative Example 5 95.04 231 Comparative Example 6 93.18 214 Comparative Example 7 94.34 222 Comparative Example 8 93.68 230 Comparative Example 9 91.54 192 Comparative Example 10 91.76 198

[0089] 2. The products prepared in Examples 1-2 and Comparative Examples 1-10 were filled using conventional methods. The inner sealing film was opened, and the outer plastic cap was placed on top. The products were stored at 25°C and 45% relative humidity for one month. The DPPH free radical scavenging rate and SOD enzyme activity were measured using the same method. The results are shown in Table 2.

[0090] Table 2 Storage stability test results

[0091] DPPH free radical scavenging rate % SOD enzyme activity (U / mg) Example 1 95.43 250 Example 2 95.12 242 Comparative Example 1 90.65 171 Comparative Example 2 91.54 202 Comparative Example 3 92.09 205 Comparative Example 4 92.13 207 Comparative Example 5 93.00 210 Comparative Example 6 91.02 198 Comparative Example 7 91.87 208 Comparative Example 8 91.25 213 Comparative Example 9 89.32 168 Comparative Example 10 89.53 165

[0092] The performance test results above show that the *Hydrangea macrophylla* enzymes in Examples 1-2 have good antioxidant effects and high SOD enzyme activity. In particular, the comprehensive performance of Example 1 is the most outstanding. This is mainly because the two enzymes are prepared through a specific fermentation method and produce a synergistic effect.

[0093] The comparative examples, lacking the necessary technical solutions, showed significantly inferior performance compared to the exemplary examples. In Comparative Example 1, no mixed fruit and vegetable enzymes were added; while initial antioxidant effects and SOD enzyme activity were relatively ideal, stability decreased significantly after prolonged storage. In Comparative Examples 2-4, the preparation methods of the *Hydrangea macrophylla* enzyme powder differed, resulting in decreased initial antioxidant effects and SOD enzyme activity, as well as poor storage stability. In Comparative Examples 5-8, the preparation methods of the mixed enzyme powder differed, leading to decreased initial antioxidant effects and SOD enzyme activity, and poor storage stability. In Comparative Examples 9-10, enzymatic hydrolysis of the two enzyme powders using a compound enzyme resulted in poor antioxidant effects and SOD enzyme activity. These experimental results further demonstrate the importance of the technical solutions defined in this invention for its technical effectiveness.

[0094] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a *Hydrangea macrophylla* enzyme, characterized in that, The preparation method includes the following steps: (1) Mix 100 parts by weight of crushed fresh fruiting bodies of *Hydrangea macrophylla*, 30-40 parts by weight of granulated sugar and 300-350 parts by weight of sterile water, add *Lactobacillus brevis*, and anaerobic ferment at 30-35℃ for 80-90 days. Then add *Lactobacillus plantarum* and anaerobic ferment at 30-35℃ for 60-70 days. Finally add *Lactobacillus helveticus* and anaerobic ferment at 30-35℃ for 30-40 days to obtain fermentation broth A. The mass of *Lactobacillus brevis* accounts for 0.3-0.5% of the total mass of crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, the mass of *Lactobacillus plantarum* accounts for 0.1-0.3% of the total mass of crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water, and the mass of *Lactobacillus helveticus* accounts for 0.1-0.3% of the total mass of crushed fresh fruiting bodies of *Hydrangea macrophylla*, granulated sugar and sterile water. (2) The fermentation liquid A is concentrated and evaporated under reduced pressure until the solid content is ≥55wt% to obtain concentrated liquid A. The concentrated liquid A is freeze-dried until the moisture content is ≤3wt%, subjected to airflow micro-grinding, and sieved to obtain Hydrangea spp. enzyme powder. (3) Add 120-150 parts by weight of sterile water to 100 parts by weight of mixed fruits and vegetables and pulverize. Add 2-3 parts by weight of white sugar and stir evenly. Add Lactobacillus bulgaricus and ferment at 30-35℃ for 40-50 days. Continue to add Lactobacillus casei and ferment at 30-35℃ for 20-30 days. Add Bifidobacterium longum and ferment at 30-35℃ for 5-10 days to obtain fermentation liquid B. The mixed fruits and vegetables are a mixture of beets, Chinese yam, mulberries and pineapples with a mass ratio of 10:1-4:6-8:3-5. The mass of Lactobacillus bulgaricus accounts for 2-3% of the total mass of mixed fruits and vegetables, sterile water and white sugar. The mass of Lactobacillus casei accounts for 1-1.5% of the total mass of mixed fruits and vegetables, sterile water and white sugar. The mass of Bifidobacterium longum accounts for 1-1.5% of the total mass of mixed fruits and vegetables, sterile water and white sugar. (4) The fermentation liquid B is concentrated and evaporated under reduced pressure until the solid content is ≥55wt% to obtain concentrated liquid B. The concentrated liquid B is freeze-dried until the moisture content is ≤3wt%, subjected to airflow micro-grinding, and sieved to obtain mixed enzyme powder. (5) Mix the hydrangea enzyme powder and mixed enzyme powder at a mass ratio of 8-10:1 to obtain hydrangea enzyme.

2. The method for preparing *Hydrangea macrophylla* enzyme according to claim 1, characterized in that, Both steps (2) and (4) are passed through a 100-300 mesh sieve.

3. The application of the preparation method according to any one of claims 1-2 in the preparation of *Hydrangea macrophylla* enzyme.