A fermenting liquid of a Sparassis crispa with improved immune function, and a preparation method and application thereof

Abstract

The present application relates to the technical field of deep processing of edible fungi, and particularly relates to a fermented liquid of Sparassis crispa for improving immune function and a preparation method and application thereof. The preparation method comprises the following steps: crushing and sieving Sparassis crispa to obtain Sparassis crispa powder, uniformly mixing the Sparassis crispa powder with water, adding cellulase and pectinase for enzymolysis to obtain an enzymolysis liquid; inoculating lactic acid bacteria powder into the enzymolysis liquid for fermentation to obtain a fermentation initial liquid; performing solid-liquid separation on the fermentation initial liquid, and taking the supernatant to obtain the fermented liquid of Sparassis crispa; wherein the lactic acid bacteria powder is at least two or three kinds of combinations of Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02 and Bifidobacterium bifidum BGLP1. Through the synergistic effect of the above-mentioned enzymolysis pretreatment and specific multi-strain combination fermentation, the obtained fermented liquid of Sparassis crispa has significant macrophage activation activity and immune gene expression up-regulation effect, and has excellent polysaccharide content and antioxidant performance, and can be used for the preparation of food, health products or medicines for improving immune function.

Description

Technical Field

[0001] This invention relates to the field of edible fungi deep processing technology, and in particular to a *Hydrangea macrophylla* fermentation broth with immune-enhancing functions, its preparation method, and its application. Background Technology

[0002] Sprassiscrispa, also known as hydrangea or coconut mushroom, is a rare and precious medicinal and edible fungus in my country. Its core characteristics include a high content of highly active β-glucan, fungal polysaccharides, polypeptides, amino acids, and various trace elements. The β-glucan content is significantly higher than that of common edible and medicinal fungi such as Ganoderma lucidum, shiitake mushroom, and Agaricus blazei, making it a recognized natural immune-active substance. Modern pharmacological and immunological studies have shown that the characteristic active components in Sprassiscrispa can effectively activate the activity of immune cells such as macrophages, T lymphocytes, and B lymphocytes, promote cytokine secretion, regulate non-specific and specific immune functions, and improve immunodeficiency. It also possesses antioxidant, anti-inflammatory, and metabolic-regulating effects, making it highly valuable for development and application in functional foods, immune-modulating health products, and biological agents.

[0003] Currently, the industry's development and utilization of *Hydrangea macrophylla* mainly focuses on traditional methods such as wild fruiting body harvesting, artificial solid-state cultivation, and crude extraction and processing of fruiting bodies. However, wild *Hydrangea macrophylla* resources are scarce, have harsh growth environments, and long growth cycles, making them unsuitable for large-scale industrial development. Artificial solid-state cultivation suffers from long cultivation cycles, large land areas, significant susceptibility to environmental temperature and humidity, unstable content of active ingredients in fruiting bodies, and large batch-to-batch variations. Furthermore, cultivated fruiting bodies are mostly used for primary processing such as fresh sales and drying, resulting in extremely low added value. Traditional fruiting body extraction processes require multiple steps including crushing, extraction, and filtration. High-temperature treatment easily causes the degradation and loss of heat-sensitive active polysaccharides and polypeptides in *Hydrangea macrophylla*, leading not only to low extraction and utilization rates and high production costs but also to inconsistent immune activity in the final product, severely restricting the standardized and large-scale production of *Hydrangea macrophylla* immune function products.

[0004] Liquid submerged fermentation technology is currently the mainstream technology for the efficient preparation of active products from edible and medicinal fungi, offering advantages such as short cycle time, high controllability, stable yield, and suitability for industrial-scale production. Existing technologies for *Hydrangea macrophylla* fermentation mostly focus on increasing mycelial biomass, exhibiting significant technical shortcomings: First, current fermentation medium formulations are crude, with unreasonable ratios of carbon, nitrogen, and trace elements, failing to specifically induce *Hydrangea macrophylla* to synthesize highly active immunopolysaccharides and β-glucan, resulting in low immunogenicity of the fermentation products. Second, fermentation process parameters are rigid, failing to optimize key parameters such as dissolved oxygen, temperature, pH, and fermentation time based on the growth and metabolic characteristics of *Hydrangea macrophylla* strains, leading to disordered mycelial growth and metabolism, and low accumulation of active substances. Third, conventional fermentation systems have weak inhibitory capabilities against contaminating microorganisms, making contamination during fermentation easy and resulting in poor batch-to-batch product stability. Fourth, current fermentation products often only collect mycelium, neglecting the utilization of the large amount of soluble immunogenic components in the fermentation broth, causing serious waste of active substances and extremely low raw material utilization.

[0005] Crucially, current technologies lack a fermentation process specifically optimized for enhancing the body's immune function. Conventional fermentation products exhibit poor targeting of active ingredients and weak immunomodulatory effects, failing to produce Fermented Hydrangea syrup products with enriched active ingredients, clearly defined efficacy, and high safety. This makes it difficult to meet current consumer demand for precise immune-regulating natural functional products. Furthermore, fermentation products prepared by existing processes are complex in composition and contain numerous impurities, making subsequent purification difficult and costly, significantly limiting the industrial application and promotion of Fermented Hydrangea syrup in the field of immunomodulation.

[0006] In summary, existing processing and fermentation technologies for *Hydrangea macrophylla* suffer from core technical deficiencies, including low content of active ingredients, unclear immunomodulatory effects, poor product stability, low raw material utilization, and weak industrial adaptability. Therefore, developing a simple, short-cycle, low-cost method for preparing *Hydrangea macrophylla* fermentation broth that can selectively enrich immunomodulatory active ingredients, significantly enhance immune function, and exhibit good batch stability, thereby producing a highly active, safe, and directly applicable *Hydrangea macrophylla* fermentation broth, has become a pressing technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0007] To address the problems of significant loss of active ingredients, insufficient immune-enhancing efficacy of conventional fermentation methods, and lack of synergy between pretreatment and fermentation processes in existing *Hydrangea macrophylla* processing technologies, this invention provides a method for preparing *Hydrangea macrophylla* fermentation broth with improved immune function, comprising the following steps: S1. Pretreatment and enzymatic hydrolysis: The *Hydrangea macrophylla* is crushed and sieved to obtain *Hydrangea macrophylla* powder, which is mixed evenly with water, and cellulase and pectinase are added for enzymatic hydrolysis to obtain enzymatic hydrolysate; S2. Fermentation: Lactic acid bacteria powder is added to the enzymatic hydrolysate for fermentation. After fermentation, the bacteria are inactivated to obtain the initial fermentation liquid. S3. Post-processing: The initial fermentation broth is subjected to solid-liquid separation, and the supernatant is taken to obtain the *Hydrangea macrophylla* fermentation broth. The lactic acid bacteria powder is Lactobacillus plantarum (Lactobacillus plantarum) Lactobacillus plantarum BXM2, Pediococcus pentosaceus ( Pediococcus pentosaceus SUN02 and Bifidobacterium bifidum ( Bifidobacterium bifidum At least two or three combinations of BGLP1; The Lactobacillus plantarum BXM2 has the accession number CGMCC NO.16436, the Pediococcus pentosaceus SUN02 has the accession number CGMCC No.29705, and the Bifidobacterium bifidum BGLP1 has the accession number CGMCC No.35718.

[0008] Further, in step S2, the lactic acid bacteria powder is 0.01% to 0.3% of the water mass.

[0009] Furthermore, the lactic acid bacteria powder is a mixture of two strains among Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02, and Bifidobacterium bifidum BGLP1; the mass ratio of the two different strains is (0.1-10):(0.1-10).

[0010] Further, in step S2, when the lactic acid bacteria powder is a mixture of three strains among Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02 and Bifidobacterium bifidum BGLP1; the mass ratio of Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02 and Bifidobacterium bifidum BGLP1 is (0.1-10):(0.1-10):(0.1-10).

[0011] Further, in step S1, the *Hydrangea macrophylla* powder is pulverized and passed through a sieve with a mesh size of 0.05–0.85 mm; and / or, the mass ratio of the *Hydrangea macrophylla* powder to water is 1:(15–40).

[0012] Further, in step S1, the amount of cellulase added is 0.2% to 0.6% of the water mass, and the amount of pectinase added is 0.1% to 0.3% of the water mass.

[0013] Further, in step S1, the enzymatic hydrolysis temperature is 35–55°C, and the hydrolysis time is 0.5–4.5 h; after enzymatic hydrolysis, enzyme inactivation treatment is performed, followed by cooling to obtain the enzymatic hydrolysate; the enzyme inactivation temperature is 85–90°C, and the enzyme inactivation time is 10–20 min. Further, in step S1, the enzymatic hydrolysis temperature is 50°C, and the hydrolysis time is 3 h.

[0014] Further, in step S2, the fermentation temperature is 35–45°C, and the fermentation time is 10–50 h; after fermentation, inactivation treatment is performed to obtain the initial fermentation liquid; the inactivation temperature is 85–100°C, and the inactivation time is 10–20 min. Further, in step S2, the fermentation temperature is 37°C, and the fermentation time is 15 h.

[0015] The present invention also provides a *Hydrangea macrophylla* fermentation broth, which is prepared by any of the above-described preparation methods.

[0016] The present invention also provides the application of the above-mentioned *Hydrangea macrophylla* fermentation broth in the preparation of functional products that improve immune function.

[0017] Based on the above, the method for preparing the *Hydrangea macrophylla* fermentation broth with improved immune function provided by the present invention, compared with the prior art, effectively breaks down the dense cell wall by pulverizing *Hydrangea macrophylla* and performing targeted enzymatic hydrolysis pretreatment with cellulase and pectinase. This synergistic effect is achieved with the subsequent fermentation step using at least two or three specific strains from *Lactobacillus plantarum* BXM2, *Pediococcus pentosaceus* SUN02, and *Bifidobacterium bifidum* BGLP1. This significantly promotes the release and high-activity transformation of immune-active polysaccharides in *Hydrangea macrophylla*. The resulting fermentation broth has outstanding macrophage activation ability and immune gene expression upregulation activity, while greatly improving the antioxidant properties and polysaccharide content of the product. This effectively solves the problems of weak immune-enhancing efficacy and low utilization rate of active ingredients in the prior art. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments, and the technical features designed in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention provides a *Hydrangea macrophylla* fermentation broth with improved immune function, prepared by the following steps: S1. After crushing the *Hydrangea macrophylla*, pass it through a 0.05-0.85 mm sieve to obtain *Hydrangea macrophylla* powder. Add water at a mass ratio of 1:(15-40), mix evenly, then add 0.2%-0.6% (by water weight) of cellulase and 0.1%-0.3% (by water weight) of pectinase. Enzymatically hydrolyze at 35-55℃ for 0.5-4.5 h, then inactivate the enzyme at 85-90℃ for 10-20 min, cool down, and obtain the enzymatic hydrolysate. S2. Add 0.01% to 0.3% of water by weight of lactic acid bacteria powder to the enzymatic hydrolysate, ferment at 35 to 45°C for 10 to 50 hours, and then inactivate at 85 to 100°C for 10 to 20 minutes to obtain the initial fermentation broth. S3. Separate the initial fermentation broth into solid and liquid components, and take the supernatant to obtain the *Hydrangea macrophylla* fermentation broth.

[0020] The lactic acid bacteria powder contains two or three of the following: Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02, and Bifidobacterium bifidum BGLP1. When the lactic acid bacteria powder is composed of Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02, and Bifidobacterium bifidum BGLP1, the mass ratio of Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02, and Bifidobacterium bifidum BGLP1 is (0.1-10):(0.1-10):(0.1-10).

[0021] Example 1 This embodiment provides a *Hydrangea macrophylla* fermentation broth, which is prepared by the following steps: S1. After crushing the hydrangea, pass it through a 0.85mm sieve to obtain hydrangea powder. Take 50g of hydrangea powder and add it to 1.5kg of water. Mix well, then add 6g of cellulase and 3g of pectinase. Enzymatically hydrolyze at 50℃ for 3h, then inactivate the enzyme at 90℃ for 20min. Allow it to cool naturally to obtain the enzymatic hydrolysate. S2. Inoculate the enzymatic hydrolysate with a mixture of 0.15g of Lactobacillus plantarum BXM2, 0.15g of Pediococcus pentosaceus SUN02 and 0.15g of Bifidobacterium bifidum BGLP1, ferment at 37℃ for 15h, then inactivate at 100℃ for 20min, and cool naturally to obtain the initial fermentation liquid. S3. The initial fermentation broth is subjected to solid-liquid separation to obtain the *Hydrangea macrophylla* fermentation broth.

[0022] Example 2 This embodiment provides a *Hypericum hygroscopicum* fermentation broth, the preparation steps of which differ from those of Example 1 only in that the bacterial powder in step S2 is replaced with an equal mass of a mixed bacterial powder of *Lactobacillus plantarum* BXM2 and *Pediococcus pentosaceus* SUN02. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0023] Example 3 This embodiment provides a *Hydrangea macrophylla* fermentation broth, the preparation steps of which differ from those of Example 1 only in that the bacterial powder in step S2 is replaced with an equal mass of a mixed bacterial powder of *Lactobacillus plantarum* BXM2 and *Bifidobacterium bifidum* BGLP1. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0024] Example 4 This embodiment provides a *Pediococcus spp.* fermentation broth, the preparation steps of which differ from those of Example 1 only in that the bacterial powder in step S2 is replaced with an equal mass of a mixed bacterial powder of *Pediococcus pentosaceus* SUN02 and *Bifidobacterium bifidum* BGLP1. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0025] Comparative Example 1 This comparative example provides a *Hydrangea macrophylla* fermentation broth, the preparation steps of which differ from those of Example 1 only in that: in step S1, cellulase and pectinase are not added for enzymatic hydrolysis, and instead, extraction is performed at 100°C for 1 hour; and step S2 is omitted, without microbial fermentation. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0026] Comparative Example 2 This comparative example provides a *Hydrangea macrophylla* fermentation broth, the preparation steps of which differ from those of Example 1 only in that: in step S1, cellulase and pectinase are not added for enzymatic hydrolysis, and instead, extraction is performed at 100°C for 1 hour. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0027] Comparative Example 3 This comparative example provides a *Hydrangea macrophylla* fermentation broth, the preparation steps of which differ from those of Example 1 only in that step S2 is omitted, and microbial fermentation is not performed. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0028] Comparative Example 4 This comparative example provides a *Hydrangea macrophylla* fermentation broth, the preparation steps of which differ from those of Example 1 only in that the bacterial powder in step S2 is replaced with a mixed bacterial powder of 0.015g *Lactobacillus plantarum* BXM2, 0.015g *Pediococcus pentosaceus* SUN02, and 0.015g *Bifidobacterium bifidum* BGLP1. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0029] Comparative Example 5 This comparative example provides a *Hydrangea macrophylla* fermentation broth, the preparation steps of which differ from those of Example 1 only in that the bacterial powder in step S2 is replaced with a mixed bacterial powder of 1.5g *Lactobacillus plantarum* BXM2, 1.5g *Pediococcus pentosaceus* SUN02, and 1.5g *Bifidobacterium bifidum* BGLP1. All other components, proportions, operating steps, and process parameters remain consistent with those of Example 1.

[0030] Comparative Example 6 This comparative example provides a *Hydrangea macrophylla* fermentation broth, the preparation steps of which differ from those of Example 1 only in that the bacterial powder in step S2 is replaced with an equal mass of *Lactobacillus plantarum* BXM2. All other components, proportions, operating steps, and process parameters remain consistent with Example 1.

[0031] Comparative Example 7 This comparative example provides a *Pediococcus spp.* fermentation broth, the preparation steps of which differ from those of Example 1 only in that the bacterial powder in step S2 is replaced with an equal mass of *Pediococcus pentosaceus* SUN02. All other components, proportions, operating steps, and process parameters remain consistent with Example 1.

[0032] Comparative Example 8 This comparative example provides a *Hydrangea macrophylla* fermentation broth, the preparation steps of which differ from those of Example 1 only in that the bacterial powder in step S2 is replaced with an equal mass of *Bifidobacterium bifidum* BGLP1. All other components, proportions, operating steps, and process parameters remain consistent with Example 1.

[0033] Comparative Example 9 This comparative example provides a *Hydrangea macrophylla* fermentation broth, the preparation steps of which differ from those of Example 1 only in that the bacterial powder in step S2 is replaced with an equal mass of *Lactobacillus plantarum* YYS-99. All other components, proportions, operating steps, and process parameters remain consistent with Example 1.

[0034] Based on the above embodiments and comparative examples, the present invention also conducted the following performance tests: Test 1: Effect of different preparation processes on the content of glucan and total polysaccharides in *Hydrangea rubra* 1.1 Effect of different preparation processes on glucan content in *Hydrangea macrophylla* The content of glucan in *Hylocereus undatus* prepared by different processes was detected. The determination of glucan in the *Hylocereus undatus* fermentation broth was carried out according to the detection methods specified in the *Detection Methods for Efficacy Components of Health Foods* (2011 edition). The results are shown in Table 1.

[0035] Table 1. Effects of different preparation processes on glucan content in *Hydrangea macrophylla* Group Dextran (mg / 100mL) average value Standard deviation Example 1 134、132.86、135.12 133.99 1.13 Example 2 126.32、125.18、127.45 126.32 1.14 Example 3 118.45、117.23、119.68 118.45 1.23 Example 4 110.23、109.12、111.35 110.23 1.12 Comparative Example 1 25、24.36、25.62 24.99 0.63 Comparative Example 2 65.45、64.23、66.68 65.45 1.23 Comparative Example 3 35.23、34.12、36.35 35.23 1.12 Comparative Example 4 45.45、44.23、46.68 45.45 1.23 Comparative Example 5 55.23、54.12、56.35 55.23 1.12 Comparative Example 6 102.45、101.23、103.68 102.45 1.23 Comparative Example 7 94.23、93.12、95.35 94.23 1.12 Comparative Example 8 86.45、85.23、87.68 86.45 1.23 Comparative Example 9 78.23、77.12、79.35 78.23 1.12 Comparing Examples 1 with Examples 2-4, it can be seen that Example 1, which uses a combination of three microorganisms (BXM2, SUN02, and BGLP1) for fermentation, has a significantly higher glucan content in the *Hydrangea hydrangea* fermentation broth than the groups fermented with any two microorganisms or with a single microorganism (Comparative Examples 6-8). This indicates that there is a significant synergistic effect among the three microorganisms (BXM2, SUN02, and BGLP1). Fermentation with all three can more efficiently decompose the cell wall structure of *Hydrangea hydrangea*, promote the release, transformation, and accumulation of endogenous glucan in *Hydrangea hydrangea*, and the three-microorganism combined fermentation process has significant advantages over the two-microorganism combined and single-microorganism fermentation processes.

[0036] Comparative Examples 1 and 4 and 5 show that the amount of the three strains added in the fermentation system is one of the key parameters affecting the formation and accumulation of glucan. When the amount of strains added is too low, the total number of microorganisms in the system is insufficient, the ability to metabolize and decompose the *Hydrangea spp.* substrate is weak, and the amount of glucan produced is low. When the amount of strains added is too high, the microorganisms proliferate in large quantities and consume the nutrients of the fermentation substrate, which inhibits the synthesis and accumulation of glucan, both of which will cause a significant decrease in glucan content.

[0037] As can be seen from the comparison between Example 1 and Comparative Example 1, the combined process of "pulverization + enzymatic hydrolysis + three-strain compound fermentation" adopted in this invention can significantly increase the glucan content in the *Hydrangea hydrangea* fermentation broth compared to the traditional direct water extraction process without pulverization. The traditional water extraction process does not break down the *Hydrangea hydrangea* raw material; the intact mycelial cell walls form a dense barrier, making it difficult to dissolve intracellular glucan, resulting in extremely low extraction efficiency. In contrast, the combined process of "pulverization + enzymatic hydrolysis + compound fermentation" in this invention can break down the cell wall structure barrier of *Hydrangea hydrangea* layer by layer, greatly increasing the amount of glucan dissolved and the amount generated during fermentation.

[0038] Comparing Example 1 and Comparative Example 2, it can be seen that the enzymatic hydrolysis process is an important guarantee for increasing the glucan content. Direct compound fermentation of *Hydrangea hydrangea* after crushing without enzymatic hydrolysis lacks the pre-degradation effect of enzyme preparation on the cell wall, resulting in insufficient decomposition of the raw material matrix and a significant reduction in glucan release. Enzymatic hydrolysis pretreatment on the basis of raw material crushing can pre-disrupt the dense mycelial structure of *Hydrangea hydrangea*, creating favorable conditions for subsequent fermentation and degradation of polysaccharides, and effectively increasing the glucan content in the fermentation broth.

[0039] Comparing Example 1 and Comparative Example 3, it can be seen that the compound fermentation process is the core and key step in the preparation of high-content glucan. Using only "pulverization + enzymatic hydrolysis" without microbial fermentation, the glucan in the *Hydrangea macrophylla* substrate cannot be fully converted and released by enzymatic hydrolysis alone, resulting in extremely low glucan dissolution. By adding a three-strain compound fermentation step on the basis of pulverization and enzymatic hydrolysis pretreatment, the continuous metabolic decomposition of polysaccharide macromolecules by the strains and the release of soluble glucan can significantly increase the glucan content, fully verifying the necessity and core role of the compound fermentation process in the preparation process of this invention.

[0040] 1.2 Effect of different preparation processes on the total polysaccharide content in *Hydrangea macrophylla* The total polysaccharide content in *Hylocereus undatus* fermentation broth prepared using different processes was determined. The determination method for total polysaccharides in the *Hylocereus undatus* fermentation broth followed the 2011 edition of the "Detection Methods for Efficacy Components in Health Foods". The results are shown in Table 2.

[0041] Table 2. Effects of different preparation processes on the total polysaccharide content in *Hydrangea macrophylla*. Group Total polysaccharides (mg / 100mL) average value Standard deviation Example 1 392.2、331.56、311.88 345.21 41.86 Example 2 321.56、318.23、324.78 321.52 3.28 Example 3 296.12、293.45、298.76 296.11 2.66 Example 4 271.56、268.32、274.78 271.55 3.23 Comparative Example 1 39.4、16.06、27.73 27.73 11.67 Comparative Example 2 141.12、138.45、143.76 141.11 2.66 Comparative Example 3 66.56、63.32、69.78 66.55 3.23 Comparative Example 4 91.12、88.45、93.76 91.11 2.66 Comparative Example 5 116.56、113.32、119.78 116.55 3.23 Comparative Example 6 246.12、243.45、248.76 246.11 2.66 Comparative Example 7 221.56、218.32、224.78 221.55 3.23 Comparative Example 8 196.12、193.45、198.76 196.11 2.66 Comparative Example 9 171.56、168.32、174.78 171.55 3.23 Comparing Examples 1 with Examples 2-4, it is evident that Example 1, which uses a three-strain co-fermentation method (BXM2, SUN02, and BGLP1), exhibits a significantly higher total polysaccharide content in its fermentation broth compared to the groups using any two strains or the single-strain fermentation groups (Comparative Examples 6-8). This indicates a significant synergistic effect among the three strains (BXM2, SUN02, and BGLP1). The three-strain co-fermentation method can effectively degrade the dense mycelial cell walls of *Hydrangea hydrangea*, efficiently promoting the dissolution, transformation, and enrichment of *Hydrangea hydrangea* polysaccharides. The three-strain co-fermentation process demonstrates significant advantages over the two-strain co-fermentation and single-strain fermentation processes.

[0042] Comparative Examples 1 and 4 and 5 show that the amount of microbial strains added in the fermentation system is one of the key parameters affecting the total polysaccharide enrichment. When the amount of microbial strains added is too low, the total number of microorganisms in the system is insufficient, the ability to decompose the *Hydrangea spp.* substrate is weak, and the polysaccharide release is low. When the amount of microbial strains added is too high, the microorganisms will consume a large amount of nutrients from the fermentation substrate, inhibiting the dissolution and accumulation of polysaccharides. Both of these will lead to a significant decrease in the total polysaccharide content. In this invention, the optimal inoculation ratio is 0.01% for BXM2, SUN02, and BGLP1. Under this condition, the metabolic activity of the microorganisms is balanced, which can maximize the total polysaccharide content of the fermentation broth.

[0043] As can be seen from the comparison between Example 1 and Comparative Example 1, the combined process of "pulverization + enzymatic hydrolysis + three-strain compound fermentation" adopted in this invention can significantly increase the total polysaccharide content of *Hydrangea hydrangea* fermentation broth compared to the traditional direct water extraction process without pulverization. The traditional water extraction process does not break down the *Hydrangea hydrangea* raw material; the intact mycelial cell walls form a structural barrier, making it difficult for intracellular polysaccharides to dissolve, resulting in extremely low extraction efficiency. In contrast, the combined process of this invention can break down the mycelial cell wall barriers layer by layer, greatly increasing the amount of polysaccharides dissolved and the fermentation conversion efficiency.

[0044] Comparing Example 1 and Comparative Example 2, it can be seen that the enzymatic hydrolysis process is an important guarantee for increasing the total polysaccharide content. Direct compound fermentation after crushing the raw materials without enzymatic hydrolysis pretreatment lacks the pre-degradation effect of enzyme preparations on the cell wall, resulting in insufficient decomposition of the *Hydrangea spp.* matrix and a significant reduction in polysaccharide release. Enzymatic hydrolysis in addition to the crushing process can pre-destroy the dense structure of the mycelium, creating favorable conditions for subsequent microbial fermentation and degradation of polysaccharides, and effectively increasing the total polysaccharide content of the fermentation broth.

[0045] Comparing Example 1 and Comparative Example 3, it can be seen that the compound fermentation process is the core and key step in obtaining a high total polysaccharide content. Using only "pulverization + enzymatic hydrolysis" without microbial fermentation, the polysaccharides in the *Hydrangea macrophylla* matrix cannot be fully degraded and released by enzymatic hydrolysis alone, resulting in extremely low total polysaccharide dissolution. By adding a three-strain compound fermentation step on the basis of pulverization and enzymatic hydrolysis pretreatment, the total polysaccharide content can be significantly increased by relying on the continuous metabolic decomposition of macromolecular polysaccharides and the release of soluble polysaccharides by the strains. This fully verifies the necessity and core role of the compound fermentation process in the preparation process of this invention.

[0046] Test 2: In vitro antioxidant activity of *Hydrangea sylvestris* fermentation broth prepared by different processes The in vitro antioxidant activity of *Hydrangea macrophylla* fermentation broth prepared by different processes was detected. The methods for detecting hydroxyl radical, superoxide anion, and DPPH radical scavenging rates were all performed according to the standard in vitro antioxidant activity testing procedures and protocols. The results are shown in Tables 3-5.

[0047] Table 3 OH removal rate (%) Group OH removal rate % average value Standard deviation Example 1 20.81、22.22、20.55 21.19 0.9 Example 2 20.12、20.35、20.28 20.25 0.12 Example 3 19.65、19.82、19.76 19.74 0.09 Example 4 19.22、19.45、19.38 19.35 0.12 Comparative Example 1 13.42、14.93、10.22 12.86 2.41 Comparative Example 2 16.85、17.02、16.96 16.94 0.09 Comparative Example 3 15.45、15.62、15.56 15.54 0.09 Comparative Example 4 15.92、16.15、16.08 16.05 0.12 Comparative Example 5 16.38、16.55、16.46 16.46 0.09 Comparative Example 6 18.72、18.95、18.86 18.84 0.12 Comparative Example 7 18.25、18.42、18.36 18.34 0.09 Comparative Example 8 17.78、17.95、17.86 17.86 0.09 Comparative Example 9 17.32、17.55、17.48 17.45 0.12 Table 4 Superoxide anion scavenging rate (%) Group Superoxide anion scavenging rate % average value Standard deviation Example 1 11.92、15.31、17.38 14.87 2.76 Example 2 11.62、11.75、11.68 11.68 0.07 Example 3 11.32、11.45、11.38 11.38 0.07 Example 4 10.98、11.12、11.05 11.05 0.07 Comparative Example 1 5.89、6.85、7.99 6.91 1.05 Comparative Example 2 9.32、9.45、9.38 9.38 0.07 Comparative Example 3 8.32、8.45、8.38 8.38 0.07 Comparative Example 4 8.65、8.78、8.72 8.72 0.07 Comparative Example 5 8.98、9.12、9.05 9.05 0.07 Comparative Example 6 10.65、10.78、10.72 10.72 0.07 Comparative Example 7 10.32、10.45、10.38 10.38 0.07 Comparative Example 8 9.98、10.12、10.05 10.05 0.07 Comparative Example 9 9.65、9.78、9.72 9.72 0.07 Table 5 DPPH removal rate (%) Group DPPH removal rate % average value Standard deviation Example 1 74.54、74.00、73.69 74.08 0.43 Example 2 72.12、72.85、72.48 72.48 0.37 Example 3 70.32、71.05、70.68 70.68 0.37 Example 4 68.52、69.25、68.88 68.88 0.37 Comparative Example 1 52.01、51.14、50.58 51.24 0.72 Comparative Example 2 59.52、60.25、59.88 59.88 0.37 Comparative Example 3 54.12、54.85、54.48 54.48 0.37 Comparative Example 4 55.92、56.65、56.28 56.28 0.37 Comparative Example 5 57.72、58.45、58.08 58.08 0.37 Comparative Example 6 66.72、67.45、67.08 67.08 0.37 Comparative Example 7 64.92、65.65、65.28 65.28 0.37 Comparative Example 8 63.12、63.85、63.48 63.48 0.37 Comparative Example 9 61.32、62.05、61.68 61.68 0.37 As can be seen from Tables 3-5: Comparing Examples 1 with Examples 2-4 and Comparative Examples 6-9, it can be seen that Example 1, which uses a three-strain co-fermentation of BXM2, SUN02, and BGLP1, exhibits significantly higher hydroxyl radical scavenging rates, superoxide anion scavenging rates, and DPPH radical scavenging rates than the fermentation groups using any two strains or the single-strain fermentation groups. This indicates a significant synergistic effect among the three strains (BXM2, SUN02, and BGLP1). The co-fermentation of these three strains can effectively degrade the mycelial structure of *Hydrangea hydrangea*, promote the release and transformation of antioxidant active substances such as polyphenols and polysaccharides, and simultaneously enhance the triple free radical scavenging capacity. The three-strain co-fermentation process has significant advantages over the two-strain co-fermentation and single-strain fermentation processes.

[0048] Comparing Example 1 with Comparative Examples 4 and 5, it can be seen that the amount of the three functional microbial strains added in the fermentation system is the key parameter determining the antioxidant activity of the *Hydrangea macrophylla* fermentation broth. When the amount of strains added is too low, the total number of microorganisms in the system is insufficient, resulting in low degradation and transformation efficiency of the *Hydrangea macrophylla* substrate, low generation of antioxidant active substances, and weak free radical scavenging ability. When the amount of strains added is too high, it will cause a large proliferation of microorganisms, consuming the nutrient substrate of the fermentation system, which will inhibit the enrichment of antioxidant active ingredients, and the scavenging rate of the three free radicals will decrease significantly. In this invention, the optimal inoculation ratio is 0.01% for each of BXM2, SUN02, and BGLP1. Under this condition, the metabolic degradation ability of the strains is balanced and sufficient, and the comprehensive antioxidant activity of the fermentation broth is the strongest.

[0049] Comparing Example 1 and Comparative Example 1, it can be seen that the combined process of "pulverization + enzymatic hydrolysis + three-strain compound fermentation" adopted in this application can significantly improve the ability of *Hydrangea macrophylla* products to scavenge three types of free radicals compared to the traditional water extraction process that does not involve pulverization and direct extraction. The traditional water extraction process does not pulverize or break down the *Hydrangea macrophylla* raw material, resulting in a dense barrier formed by the intact hyphal cell walls, making it difficult for intracellular polysaccharides and other substances to dissolve, and thus having a weak in vitro antioxidant effect. In contrast, the multi-stage combined process of this invention can break down the hyphal structural barriers layer by layer, releasing and transforming a large amount of highly active antioxidant components, significantly improving the efficiency of triple free radical scavenging, and demonstrating significant process advantages.

[0050] Comparing Example 1 and Comparative Example 2, it can be seen that the enzymatic hydrolysis pretreatment process is an important guarantee for improving the antioxidant activity of *Hydrangea macrophylla* fermentation broth. Simply conducting three-strain compound fermentation directly after raw material crushing, omitting the enzymatic hydrolysis step, lacks the pre-degradation effect of enzymes on the mycelial cell walls, resulting in insufficient decomposition of the *Hydrangea macrophylla* matrix, limited release of antioxidant active substances, and significantly lower scavenging rates of hydroxyl radicals, superoxide anions, and DPPH radicals compared to Example 1. Enzymatic hydrolysis, combined with the crushing process, can pre-destroy the dense mycelial structure, creating favorable conditions for subsequent microbial fermentation and transformation of antioxidant active substances, and significantly improving the overall antioxidant level of the fermentation broth.

[0051] Comparing Example 1 and Comparative Example 3, it can be seen that the compound microbial fermentation process is the core and key step in obtaining the highly antioxidant active *Hydrangea macrophylla* product. Using only pulverization and enzymatic hydrolysis without microbial fermentation, enzymatic hydrolysis alone is insufficient to fully transform and modify the antioxidant components in the raw materials, resulting in very limited scavenging effects on the three types of free radicals. Combining pulverization and enzymatic hydrolysis pretreatment with compound fermentation of the three microorganisms, relying on the continuous metabolic generation and modification of polysaccharides and polyphenolic antioxidants by microorganisms, can simultaneously enhance the scavenging capacity of hydroxyl radicals, superoxide anions, and DPPH radicals, fully verifying the necessity and core role of the compound fermentation process in the preparation process of this invention.

[0052] In summary, the *Hydrangea spp.* fermentation broth prepared by this application using a combined process of "pulverization + enzymatic hydrolysis with specific parameters + compound fermentation of specific three strains" exhibits excellent scavenging ability against hydroxyl radicals, superoxide anions, and DPPH radicals. Its comprehensive in vitro antioxidant activity is significantly superior to other process schemes such as dual-strain compounding, single-strain fermentation, traditional water extraction, omitting enzymatic hydrolysis, omitting fermentation, and excessive or insufficient strain addition.

[0053] Test 3: Enhanced immune activity of *Hydrangea macrophylla* fermentation broth prepared by different processes The effects of different fermentation broths of *Hylocereus undatus* prepared using different processes on nitric oxide (NO) release and the expression levels of immune-related genes TNF-α and iNOS in RAW264.7 cells were investigated. The cell culture method, NO detection (Griess method), and PCR detection methods for the aforementioned gene expression levels were all performed according to standard cell biology testing procedures and gene expression PCR detection protocols. The results are shown in Tables 6–8.

[0054] Table 6. NO release (μM) of *Hydrangea macrophylla* fermentation broth prepared by different processes. Group NO release amount (μM) average value Standard deviation Blank group 18.42、19.11、18.87 18.8 0.35 Positive control group (lipopolysaccharide) 125.76、125.78、129.64 127.06 2.23 Example 1 49.68、49.83、49.68 49.73 0.09 Example 2 47.25、47.31、47.22 47.26 0.05 Example 3 45.18、45.22、45.15 45.18 0.04 Example 4 43.05、43.12、43.02 43.06 0.05 Comparative Example 1 34.10、34.29、34.25 34.21 0.1 Comparative Example 2 35.02、35.05、35.04 35.04 0.02 Comparative Example 3 34.45、34.42、34.48 34.45 0.03 Comparative Example 4 34.65、34.62、34.68 34.65 0.03 Comparative Example 5 34.85、34.82、34.88 34.85 0.03 Comparative Example 6 41.20、41.15、41.23 41.19 0.04 Comparative Example 7 39.08、39.12、39.05 39.08 0.04 Comparative Example 8 37.15、37.20、37.12 37.16 0.04 Comparative Example 9 35.62、35.58、35.65 35.62 0.04 Table 7. Relative expression levels of TNF-α in *Hylocereus undatus* fermentation broth prepared by different processes. Group TNF-α relative expression level average value Standard deviation Blank group 1、1、1 1 0 Positive control group (lipopolysaccharide) 5.58、5.38、5.53 5.5 0.1 Example 1 4.00、3.92、3.88 3.93 0.06 Example 2 3.75、3.68、3.62 3.68 0.07 Example 3 3.52、3.46、3.40 3.46 0.06 Example 4 3.30、3.25、3.18 3.24 0.06 Comparative Example 1 2.09、2.14、2.23 2.15 0.07 Comparative Example 2 2.28、2.23、2.26 2.26 0.03 Comparative Example 3 2.11、2.17、2.22 2.17 0.06 Comparative Example 4 2.15、2.18、2.26 2.2 0.06 Comparative Example 5 2.20、2.26、2.28 2.25 0.04 Comparative Example 6 3.08、3.02、2.96 3.02 0.06 Comparative Example 7 2.86、2.81、2.75 2.81 0.06 Comparative Example 8 2.65、2.60、2.54 2.6 0.06 Comparative Example 9 2.43、2.38、2.32 2.38 0.06 Table 8. Relative expression levels of iNOS in *Hylocereus undatus* fermentation broth prepared by different processes. Group iNOS relative expression level average value Standard deviation Blank group 1、1、1 1 0 Positive control group (lipopolysaccharide) 4.18、4.27、4.00 4.15 0.14 Example 1 3.88、3.77、2.72 3.46 0.64 Example 2 3.65、3.52、2.58 3.25 0.58 Example 3 3.42、3.30、2.45 3.06 0.53 Example 4 3.18、3.06、2.31 2.85 0.47 Comparative Example 1 1.00、1.59、1.86 1.48 0.44 Comparative Example 2 2.10、1.99、1.68 1.92 0.22 Comparative Example 3 1.80、1.69、1.42 1.64 0.2 Comparative Example 4 1.89、1.78、1.51 1.73 0.2 Comparative Example 5 2.01、1.90、1.60 1.84 0.21 Comparative Example 6 2.95、2.83、2.18 2.65 0.41 Comparative Example 7 2.72、2.60、2.05 2.46 0.36 Comparative Example 8 2.48、2.37、1.92 2.26 0.3 Comparative Example 9 2.25、2.14、1.79 2.06 0.24 As can be seen from Tables 6-8: Comparison of the blank group, the lipopolysaccharide positive control group, and Examples 1-4 and Comparative Examples 1-9 shows that the blank group had extremely low NO release levels and the lowest basal expression levels of TNF-α and iNOS genes. The lipopolysaccharide positive control group significantly stimulated macrophages to release large amounts of NO and upregulated the expression of TNF-α and iNOS genes, proving that this cell model can be effectively used for evaluating immune activation activity. After treatment of the samples in each example and comparative example, the amount of NO release, TNF-α, and iNOS gene expression in macrophages were significantly higher than those in the blank group, indicating that the *Hydrangea hydrangea* samples prepared by each process in this application all have the effect of activating macrophages and regulating innate immunity, but there are significant differences in the immune activation effects of samples prepared by different processes.

[0055] Comparing Examples 1 with Examples 2-4 and Comparative Examples 6-9, it is evident that Example 1, employing a three-strain fermentation method (BXM2, SUN02, and BGLP1), exhibited the best immune activation effect. Its induction of NO release from RAW264.7 cells and the extent of upregulation of TNF-α and iNOS immune gene expression were significantly higher than those of the two-strain fermentation groups and the single-strain fermentation groups. This demonstrates a significant synergistic effect among BXM2, SUN02, and BGLP1. The three-strain fermentation method can fully transform the polysaccharide-rich immunomodulatory substances in the *Hylocereus undatus* raw material, more effectively activating the macrophage innate immune pathway. The three-strain fermentation scheme demonstrates significant advantages over two-strain and single-strain fermentation methods.

[0056] Comparing Example 1 with Comparative Examples 4 and 5, it can be seen that the amount of the three functional strains added in the fermentation system is the key parameter determining the immune-activating activity of the *Hydrangea macrophylla* fermentation broth. When the amount of strains added is too low, the metabolic transformation capacity of the microorganisms is insufficient, the amount of active polysaccharides produced is low, and the immune activation effect is weak. When the amount of strains added is too high, it will cause excessive proliferation of microorganisms and consumption of nutrient substrates, which will reduce the enrichment level of active ingredients, and significantly reduce the release of NO from macrophages and the upregulation of immune genes. In this invention, the optimal ratio of BXM2, SUN02, and BGLP1 inoculation is 0.01%. Under this condition, the metabolic transformation efficiency of the strains is the highest, and the in vitro immune-enhancing activity of the fermentation broth is the strongest.

[0057] Comparing Example 1 and Comparative Example 1, it can be seen that the combined process of "pulverization + enzymatic hydrolysis + three-strain compound fermentation" in this application can significantly enhance the ability of *Hydrangea hydrangea* products to activate macrophages compared to the traditional water extraction process without pulverization. The traditional water extraction process cannot fully break down the cell walls of *Hydrangea hydrangea* hyphae, resulting in low levels of intracellular immunomodulatory polysaccharides and difficulty in effectively stimulating macrophages to secrete NO and upregulate immune-related genes, leading to weak in vitro immune enhancement effects. The multi-stage combined process of this invention can break down the hyphal structural barrier layer by layer, releasing and converting large amounts of highly active polysaccharides, significantly improving the immune activation effect and demonstrating significant process advantages.

[0058] Comparing Example 1 and Comparative Example 2, it can be seen that the enzymatic hydrolysis pretreatment process is an important guarantee for improving the immune activity of *Hydrangea macrophylla* fermentation broth. Simply pulverizing the raw materials and directly carrying out the three-strain compound fermentation without the enzymatic hydrolysis step lacks the pre-degradation effect of enzymes on the mycelial cell walls, resulting in insufficient decomposition of the raw materials, limited release of active substances, and significantly lower levels of macrophage NO release, TNF-α, and iNOS gene expression compared to Example 1. Enzymatic hydrolysis, combined with the pulverization process, can pre-disrupt the dense mycelial structure, creating favorable conditions for subsequent microbial fermentation and transformation, and significantly improving the immunomodulatory activity of the fermentation broth.

[0059] Comparing Example 1 and Comparative Example 3, it can be seen that the compound microbial fermentation process is the core and key step in obtaining the highly immunogenic *Hydrangea rubra* product. Using only pulverization and enzymatic hydrolysis without fermentation, the enzymatic hydrolysis alone is insufficient to fully convert the polysaccharides into highly active immunogenic polysaccharides, resulting in a very limited activation effect on macrophages. However, combining pulverization and enzymatic hydrolysis with three-strain compound fermentation, relying on the continuous metabolic modification of polysaccharide structure by microorganisms and the enhancement of polysaccharide immunogenicity, can significantly increase NO release and immune gene expression levels, fully demonstrating that the compound fermentation process is indispensable in the preparation process of this invention.

[0060] In summary, the *Hydrangea hydrangea* fermentation broth prepared using a combined process of "pulverization + enzymatic hydrolysis with specific parameters + specific three-strain compound fermentation" significantly induces the release of the immune signaling molecule NO from RAW264.7 macrophages, while simultaneously significantly upregulating the expression of key innate immune genes TNF-α and iNOS. Its in vitro immune-enhancing activity is significantly superior to other processes such as two-strain compounding, single-strain fermentation, traditional water extraction, omitting enzymatic hydrolysis, omitting fermentation, or excessive / inadequate strain addition. The various process steps and parameters in this application synergistically maximize the release and transformation of immunomodulatory polysaccharides in *Hydrangea hydrangea*, resulting in a fermentation broth with excellent in vitro immune-enhancing activity, making it a functional raw material for enhancing the body's immunity.

[0061] The design concept and beneficial effects of this application can be summarized as follows: Design Concept The design concept of this application is to construct a multi-stage synergistic process system involving "physical cell wall disruption, targeted enzymatic hydrolysis, and directional fermentation" to solve the core technical problems of insufficient release of immune-active components and unclear immune-enhancing effects in existing *Hydrangea macrophylla* processing technologies. The specific concept is as follows: Synergistic pretreatment of physical cell wall disruption and targeted enzymatic hydrolysis: By pulverizing *Hydrangea macrophylla* to a specific particle size, the specific surface area is increased, exposing more cell wall action sites; then, cellulase and pectinase are specifically selected for enzymatic hydrolysis. Cellulase degrades the cellulose skeleton in the cell wall, and pectinase degrades the pectin in the intercellular layer. The synergistic effect of the two can efficiently and thoroughly disrupt the dense hyphal cell wall structure of *Hydrangea macrophylla*, allowing a large amount of intracellular bound immunomodulatory polysaccharides to dissolve and be exposed in the reaction system, providing abundant metabolizable substrates for subsequent microbial fermentation.

[0062] Targeted screening and synergistic combination of specific functional strains: A specific combination of *Lactobacillus plantarum* BXM2, *Pediococcus pentosaceus* SUN02, and *Bifidobacterium bifidum* BGLP1 was precisely screened from numerous probiotics. Utilizing the complementary enzyme systems of these three strains during metabolism—each secreting different types of glycosidases, hydrolases, and antioxidant enzymes—the combination deeply degrades, structurally modifies, and revitalizes the enzymatically released macromolecular polysaccharides of *Pediococcus pyogenes*, thereby achieving targeted enhancement of immune activity. The "enzymatic hydrolysis followed by fermentation" sequential synergistic design places the enzymatic hydrolysis step strictly before the fermentation step, forming a sequential cascade of "pretreatment - enzymatic release - microbial transformation". Enzymatic pretreatment clears cell wall barriers in advance, providing the fermentation strain with a large amount of easily uptakeable and transformable polysaccharide precursors; the continuous metabolism generated by the strain during fermentation further modifies and enhances the function of the active substances released by enzymatic hydrolysis. The two steps are sequentially synergistic and functionally complementary, maximizing the immunomodulatory activity of the final product through a dual mechanism of "pre-digestion + deep transformation".

[0063] Precise control of key process parameters: The key parameters such as enzymatic hydrolysis temperature, enzymatic hydrolysis time, fermentation temperature, fermentation time, and inoculation amount and ratio of compound microbial powder were systematically optimized, and the optimal process window for each step was determined to ensure that the above synergistic effect mechanism can play a stable and efficient role, thereby ensuring the quality stability and reliability of immune enhancement efficacy between product batches.

[0064] Beneficial effects Based on the above design concept, the preparation method provided in this application has the following advantages compared with the prior art: Significantly enhanced immune-enhancing effects: Through the synergistic effect of enzymatic release and targeted transformation of specific three strains, the resulting *Hydrangea hydrangea* fermentation broth can significantly induce RAW264.7 macrophages to release the immune signaling molecule NO, while significantly upregulating the expression of key innate immune genes TNF-α and iNOS. The in vitro immune-activating activity far exceeds that of single-strain fermentation, dual-strain fermentation, and process schemes that omit enzymatic hydrolysis or fermentation.

[0065] Significantly increased content of active ingredients: The combined process of this application can efficiently break down the structural barrier of the cell wall of *Hydrangea hydrangea*, promote the full release and transformation of intracellular active polysaccharides, and significantly increase the content of β-glucan and total polysaccharides in the resulting fermentation broth, thus realizing the efficient utilization of high-value immune active ingredients in *Hydrangea hydrangea* raw materials.

[0066] Synergistic enhancement of antioxidant activity: During fermentation, the three specific strains can not only transform into highly active immunopolysaccharides, but also simultaneously promote the release and generation of antioxidant active substances such as polyphenols and organic acids. This results in a synergistic enhancement of the scavenging ability of the fermentation broth against hydroxyl radicals, superoxide anion radicals and DPPH radicals, demonstrating excellent comprehensive antioxidant performance.

[0067] Good process synergy and stability: The process steps of "pulverization + enzymatic hydrolysis + fermentation" in this application are closely linked and complementary. The precise control of key parameters ensures the stable performance of the synergistic effect mechanism. The process is simple to operate and the conditions are mild, making it suitable for large-scale industrial production. It provides a reliable technical solution for the industrial application of *Hydrangea rubra* in precision immune-regulating functional products.

[0068] It should be noted that: (1) Definition: In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0069] (2) Raw materials used in implementation: All enzymes used in this article are commercially available enzymes that can be purchased and obtained by those skilled in the art.

[0070] (3) Information on strain preservation All the bacterial strains used in this application are publicly available strains, preserved and viable in biological collections. Technicians can obtain them from the collections or from the applicant based on their accession numbers. Therefore, this application does not involve bacterial strain preservation and does not require proof of strain viability.

[0071] Patent application CN118421739A discloses the aforementioned *Lactobacillus plantarum* ( Lactiplantibacillus plantarum BXM2 was deposited on September 6, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNO.16436.

[0072] Patent application CN118240700A discloses the aforementioned Pediococcus pentosaceus ( Pediococcus pentosaceus SUN02 was deposited on January 22, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29705.

[0073] Patent application CN121086957A discloses the aforementioned Bifidobacterium bifidum ( Bifidobacterium bifidum BGLP1 was deposited on August 22, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35718.

[0074] The cellulase used in the examples and comparative examples was Nanning Pangbo cellulase with an enzyme activity of 20,000 Units / g; the pectinase used was Novozymes pectinase with an enzyme activity of 11,010 Units / g.

[0075] In summary, the specific parameters or some commonly used reagents or raw materials in the above embodiments are specific or preferred embodiments under the concept of this application, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of this application.

[0076] In addition, unless otherwise specified, the raw materials used may be commercially available products in the field or prepared by conventional methods in the field; that is, the reagents and instruments used in this embodiment do not specify the manufacturer or other information, and are all conventional products that can be purchased from the market.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0078] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing a *Hydrangea macrophylla* fermentation broth with improved immune function, characterized in that, Includes the following steps: S1. Pretreatment and enzymatic hydrolysis: The *Hydrangea macrophylla* is crushed and sieved to obtain *Hydrangea macrophylla* powder, which is mixed evenly with water, and cellulase and pectinase are added for enzymatic hydrolysis to obtain enzymatic hydrolysate; S2. Fermentation: Lactic acid bacteria powder is added to the enzymatic hydrolysate for fermentation. After fermentation, the bacteria are inactivated to obtain the initial fermentation liquid. S3. Post-processing: The initial fermentation broth is subjected to solid-liquid separation, and the supernatant is taken to obtain the *Hydrangea macrophylla* fermentation broth. The lactic acid bacteria powder is Lactobacillus plantarum (Lactobacillus plantarum) Lactobacillus plantarum BXM2, Pediococcus pentosaceus ( Pediococcus pentosaceus SUN02 and Bifidobacterium bifidum ( Bifidobacterium bifidum At least two or three combinations of BGLP1; The Lactobacillus plantarum BXM2 has the accession number CGMCC NO.16436, the Pediococcus pentosaceus SUN02 has the accession number CGMCC No.29705, and the Bifidobacterium bifidum BGLP1 has the accession number CGMCC No.35718.

2. The preparation method according to claim 1, characterized in that, In step S2, the lactic acid bacteria powder is 0.01% to 0.3% of the water mass.

3. The preparation method according to claim 1, characterized in that, When the lactic acid bacteria powder is a mixture of two strains among Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02 and Bifidobacterium bifidum BGLP1; the mass ratio of the two different strains is (0.1-10):(0.1-10).

4. The preparation method according to claim 1, characterized in that, In step S2, the lactic acid bacteria powder is a mixture of three strains: Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02, and Bifidobacterium bifidum BGLP1. The mass ratio of Lactobacillus plantarum BXM2, Pediococcus pentosaceus SUN02 and Bifidobacterium bifidum BGLP1 was (0.1-10):(0.1-10):(0.1-10).

5. The preparation method according to claim 1, characterized in that, In step S1, the *Hydrangea macrophylla* is pulverized and passed through a sieve with a mesh size of 0.05–0.85 mm; and / or, the mass ratio of the *Hydrangea macrophylla* powder to water is 1:(15–40).

6. The preparation method according to claim 1, characterized in that, In step S1, the amount of cellulase added is 0.2% to 0.6% of the water mass, and the amount of pectinase added is 0.1% to 0.3% of the water mass.

7. The preparation method according to claim 1, characterized in that, In step S1, the enzymatic hydrolysis temperature is 35–55°C, and the enzymatic hydrolysis time is 0.5–4.5 h. The enzyme hydrolysate is then subjected to enzyme inactivation treatment and cooled to obtain an enzyme hydrolysate; the enzyme inactivation temperature is 85-90℃ and the enzyme inactivation time is 10-20 min.

8. The preparation method according to claim 1, characterized in that, In step S2, the fermentation temperature is 35-45℃ and the fermentation time is 10-50 hours. The fermentation process involves inactivation treatment to obtain the initial fermentation broth; the inactivation temperature is 85–100℃, and the inactivation time is 10–20 min.

9. A fermentation broth for *Hydrangea macrophylla*, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The use of the *Hydrangea macrophylla* fermentation broth according to claim 9 in the preparation of functional products that improve immune function.