Preparation method of malve whole plant multi-stage gradient fermentation enzyme

Abstract

The application discloses a kind of preparation methods of Malve sylvestris full plant multi-stage gradient fermentation enzyme, comprising the following steps: raw material pretreatment;Complex enzymolysis;Ultrasonic-assisted extraction;Multi-stage gradient fermentation and post-processing, the present application relates to the field of biological fermentation, the present application is based on flower flavone, antioxidant activity is high and tender fruit pod polysaccharide content high complementary characteristics, by specific proportion mixing, the synergistic release of multiple active ingredients is realized, simultaneously, the filter residue after fermentation can be further utilized, realizes the zero waste of whole plant resources, the present application will complex enzymolysis and ultrasonic-assisted extraction organic fusion, cellulase and pectinase in complex enzyme efficiently destroy plant cell wall, promote intracellular active ingredient release;Beta-glucosidase starts directional conversion flavonoid glycoside in extraction stage, subsequent ultrasonic treatment utilizes cavitation effect and mechanical vibration to further break cell, intensifies mass transfer process, and the extraction rate of active ingredient is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a method for preparing a multi-level gradient fermentation enzyme from the whole plant of *Hymenochloa chinensis*. Background Technology

[0002] Golden hibiscus, also known as wild hibiscus or edible hibiscus, belongs to the Malvaceae family and the Abelmoschus genus. It is rich in flavonoids, polysaccharides, trace elements, and other active substances, possessing multiple benefits including lipid-lowering, anti-inflammatory, and antioxidant effects. It has broad application prospects in the food, pharmaceutical, and cosmetic fields. Current research indicates that golden hibiscus flowers have higher total flavonoid and total phenolic content, while the young pods are richer in polysaccharides. The flowers have superior antioxidant capacity compared to the young pods, but both have significant edible and medicinal value.

[0003] However, the current processing and utilization of golden sunflower mainly focuses on flavonoid extracts, polysaccharide extracts, and simple enzyme products from single parts (flowers or young pods), which has the following prominent problems: First, the existing process fails to fully exploit the differentiated value of active ingredients in different parts of the golden sunflower (flower and pod), resulting in low resource utilization and ineffective recycling of fermentation residue.

[0004] Secondly, traditional processes typically involve solvent extraction or enzymatic purification followed by fermentation, which is fragmented, time-consuming, and the high temperatures and organic solvents used in the extraction process can easily cause degradation and loss of active ingredients.

[0005] Third, existing golden sunflower enzymes mostly use natural fermentation with a single strain, lacking a gradient fermentation design tailored to the characteristics of different active ingredients. This results in low conversion efficiency of flavonoid glycosides to highly active aglycones, insufficient degradation of polysaccharide macromolecules, and insufficient generation of flavor substances.

[0006] Fourth, most existing products are in the form of liquid enzyme concentrate, lacking the development of graded products for different application scenarios, resulting in insufficient market competitiveness.

[0007] Therefore, there is an urgent need to develop an integrated preparation process that can fully utilize the resources of the entire golden sunflower plant, achieve efficient release and deep conversion of active ingredients, and produce multi-form high-value-added products. Summary of the Invention

[0008] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for preparing enzymes from the whole plant of *Hymenochloa chinensis* through multi-stage gradient fermentation. This method solves the problems existing in the prior art. Through an integrated process of multi-part pretreatment of the whole plant, enzymatic hydrolysis and ultrasonic coupling extraction combined with multi-stage gradient fermentation, this invention achieves the simultaneous utilization of *Hymenochloa chinensis* flowers and tender pods, improves the extraction rate and bioconversion rate of active ingredients such as flavonoids and polysaccharides, and develops multi-form enzyme products.

[0009] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a multi-stage gradient fermentation enzyme from the whole plant of *Hippophae rhamnoides*, comprising the following steps: S1. Raw material pretreatment: Harvest fresh flowers and tender pods of golden sunflower, wash and cut them separately, and then mix them to obtain mixed raw materials; S2. Compound enzymatic hydrolysis: Water and compound enzyme are added to the mixed raw materials for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme is inactivated to obtain the enzymatic hydrolysate. The compound enzyme contains cellulase, pectinase and β-glucosidase. S3. Ultrasonic-assisted extraction: The enzymatic hydrolysate is subjected to ultrasonic treatment to obtain an extraction slurry; S4. Multi-stage gradient fermentation: The extracted slurry is subjected to anaerobic lactic acid bacteria fermentation, aerobic yeast fermentation and anaerobic secondary maturation in sequence to obtain fermentation broth; S5. Post-processing: The fermentation broth is subjected to solid-liquid separation to obtain enzyme stock solution.

[0010] In some embodiments, step S1 includes: cutting the fresh flowers into 1-2 cm sections, cutting the tender pods into segments, and freezing them at -20°C for 10-14 hours; the mass ratio of the fresh flowers to the tender pods in the mixed raw materials is 2:1.

[0011] Cutting the flowers into 1-2 cm sections increases the contact area with enzymes and water during subsequent enzymatic hydrolysis and extraction. Freezing the young pods at -20°C after cutting allows ice crystals to pierce the cell wall structure, further disrupting cell integrity and thus enabling more efficient release of polysaccharides and other active ingredients in the pods during subsequent enzymatic hydrolysis. Mixing the flowers and young pods at a 2:1 mass ratio represents the optimal balance based on the complementary properties of their active ingredients, ensuring the synergistic maximization of flavonoid antioxidant activity and polysaccharide content in the final product.

[0012] In some embodiments, in step S2, the mass ratio of cellulase, pectinase and β-glucosidase in the composite enzyme is 3:1:1; the amount of the composite enzyme added is 0.5%-1.0% of the mass of the mixed raw materials.

[0013] In some embodiments, in step S2, the enzymatic hydrolysis treatment is carried out at a temperature of 45-50°C for 2-4 hours; the enzyme inactivation is carried out by heating to 85°C and holding for 8-12 minutes.

[0014] Specific compound enzyme ratios can efficiently act on different components of plant cell walls, namely cellulose, pectin, and flavonoid glycosidic bonds. Within the preferred temperature range of 45-50℃, the activity of each enzyme is at a high level, resulting in optimal enzymatic hydrolysis efficiency. After enzymatic hydrolysis, raising the temperature to 85℃ and maintaining it for 8-12 minutes can quickly and thoroughly inactivate the enzymes, terminating the enzymatic hydrolysis reaction, while avoiding the destruction of active ingredients due to prolonged high temperatures.

[0015] In some embodiments, in step S3, the ultrasonic treatment power is 300-500 W, the frequency is 20-50 kHz, the time is 20-40 minutes, and the temperature is controlled at 40-50℃ during the treatment.

[0016] Within the aforementioned range of ultrasound parameters, the cavitation effect is most pronounced, effectively breaking down cell walls and organelles remaining after enzymatic hydrolysis, thus promoting the full dissolution of bound and encapsulated active ingredients. Simultaneously, controlling the temperature at 40-50℃ prevents the loss of heat-sensitive active ingredients due to localized overheating during ultrasounding.

[0017] In some embodiments, the specific steps of the anaerobic lactic acid bacteria fermentation in step S4 are as follows: adding brown sugar to the extracted slurry to adjust the soluble solids content to 12-15°Bx and adjusting the pH to 6.0-6.5; inoculating with compound lactic acid bacteria fermentation agent and anaerobic fermenting at 32-35°C for 48-72 hours.

[0018] In some embodiments, the compound lactic acid bacteria starter consists of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus casei in a mass ratio of 1:1:1; the inoculum amount of the compound lactic acid bacteria starter is 3%-5% of the volume of the extracted slurry.

[0019] Adjusting the soluble solids content to 12-15°Bx provided ample carbon source for the lactic acid bacteria, ensuring their rapid growth and acid production. pH 6.0-6.5 is the optimal growth pH range for the selected lactic acid bacteria. The starter culture, composed of three lactic acid bacteria in a 1:1:1 ratio, exhibited a synergistic effect, resulting in a greater variety and stronger activity of extracellular enzymes such as β-glucosidase, leading to significantly higher conversion efficiency of flavonoid glycosides compared to single strains. Fermentation conditions of 32-35℃ and 48-72 hours ensured sufficient acid production by the lactic acid bacteria, while lowering the pH to 3.8-4.2 effectively inhibited the growth of other microorganisms.

[0020] In some embodiments, in step S4, the specific steps of the aerobic yeast fermentation are as follows: inoculating the system with brewer's yeast after the anaerobic lactic acid bacteria fermentation is completed, and fermenting aerobically at 28-30℃ for 24-48 hours under aeration and stirring conditions; the amount of brewer's yeast inoculated is 2%-3% of the fermentation system volume.

[0021] After the first stage of anaerobic fermentation, aerobic brewing yeast is introduced. Lactic acid bacteria thrive in the low pH environment created by the yeast, and aeration and stirring provide the necessary oxygen for aerobic fermentation, promoting yeast metabolism and proliferation. The yeast is metabolically active at 28-30℃, producing a large amount of alcohols and esters, significantly improving the flavor of the enzyme. Simultaneously, the release of antioxidant enzymes such as SOD during autolysis further enhances the product's functionality.

[0022] In some embodiments, the specific steps of the anaerobic secondary maturation in step S4 are as follows: after the aerobic yeast fermentation is completed, the fermentation liquid is transferred into a sealed container and anaerobically matured at 15-20°C for 30-60 days.

[0023] The secondary maturation stage is a slow process of biochemical transformation and flavor fusion. Under the low-temperature anaerobic environment of 15-20℃, the various enzyme systems produced in the first two stages of fermentation (such as residual glycosidases, esterases, etc.) continue to react slowly, promoting the further degradation of macromolecules and the esterification and polymerization of small-molecule flavor substances. This makes the enzyme taste more mellow and harmonious, and the aroma more mellow and full-bodied, ultimately achieving stable quality.

[0024] In some embodiments, the steps further include: S6. Product preparation: The enzyme stock solution is graded to obtain at least one of food-grade enzyme solution, cosmetic-grade enzyme solution or enzyme powder. The food-grade enzyme liquid is prepared by filling enzyme stock solution after instantaneous high-temperature sterilization. The cosmetic-grade enzyme solution is prepared by microfiltration sterilization of enzyme stock solution followed by low-temperature storage. The enzyme powder is obtained by drying enzyme stock solution.

[0025] Through graded processing, single enzyme concentrate is expanded into multi-form products suitable for different application scenarios. Food-grade enzyme liquid undergoes instantaneous high-temperature sterilization, ensuring the safety of the beverage and its shelf-life stability. Cosmetic-grade enzyme liquid is not subjected to high temperatures, but retains a large number of live bacteria and active enzymes through microfiltration sterilization, making it more suitable as an active ingredient in skin care products. Enzyme powder is made through a drying process, which facilitates storage, transportation, and use as an ingredient in solid beverages or functional foods, greatly enhancing the market adaptability and added value of the products.

[0026] In some embodiments, the filter residue obtained after solid-liquid separation can be further dried at low temperature and then pulverized to be used as a dietary fiber supplement or a raw material for cosmetic exfoliation.

[0027] (III) Beneficial Effects The beneficial effects of this invention are: Based on the complementary characteristics of high antioxidant activity of flower flavonoids and high polysaccharide content of young fruit pods, this invention achieves the synergistic release of multiple active ingredients by mixing them in a specific ratio. At the same time, the filter residue after fermentation can be further utilized, achieving zero waste of the whole plant resources.

[0028] This invention organically integrates compound enzymatic hydrolysis with ultrasound-assisted extraction. The cellulase and pectinase in the compound enzyme efficiently destroy plant cell walls and promote the release of intracellular active ingredients. β-glucosidase begins to directionally convert flavonoid glycosides during the extraction stage. Subsequent ultrasound treatment utilizes cavitation effect and mechanical vibration to further break down cells and enhance the mass transfer process, thereby significantly improving the extraction rate of active ingredients, which can be increased by more than 30% compared with traditional processes.

[0029] This invention employs a three-stage gradient fermentation method: anaerobic lactic acid bacteria fermentation, aerobic yeast fermentation, and anaerobic secondary maturation. In the first stage, lactic acid bacteria produce lactic acid through metabolism, rapidly lowering the system pH and inhibiting contaminating bacteria. Simultaneously, various glycosidases secreted by the bacteria hydrolyze large-molecule flavonoids and polysaccharides into highly active small-molecule aglycones and oligosaccharides, improving bioavailability. In the second stage, yeast produces flavor compounds such as alcohols and esters through aerobic metabolism, endowing the product with unique sensory qualities. Its autolysis releases antioxidant components such as superoxide dismutase and glutathione, further enhancing the product's functionality. In the third stage, under a low-temperature anaerobic environment, the residual enzymes in the fermentation system continue to act, further balancing and blending flavor compounds, leading to stable product quality.

[0030] This invention employs low-temperature enzymatic hydrolysis, physical ultrasound, and gentle microbial fermentation techniques throughout the entire process, avoiding the use of high temperatures and organic solvents in traditional processes, and maximizing the preservation of the heat-sensitive active ingredients and probiotic activity in sunflower. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the process flow of the present invention. Detailed Implementation

[0032] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Example 1

[0033] Please see Figure 1 The present invention provides a technical solution: This embodiment provides a preferred integrated manufacturing process, the specific steps of which are as follows: S1, Raw material pretreatment During the peak flowering season of the golden sunflower, fresh, disease-free flowers and tender pods are harvested manually. The flowers and pods are washed thoroughly with clean water and drained. The flowers are cut into pieces approximately 1.5cm in size; the tender pods are cut into segments approximately 2-3cm long. Both are then frozen at -20℃ for 12 hours. The frozen pod segments are removed without thawing and mixed thoroughly with the cut flower pieces at a flower:tender pod ratio of 2:1 to obtain the mixed ingredient.

[0034] S2 complex enzymatic hydrolysis The above-mentioned mixed raw materials were placed into an enzymatic hydrolysis tank, and sterile water equivalent to twice the total mass of the mixed raw materials was added. Then, a compound enzyme was added, which was composed of cellulase (enzyme activity ≥100,000 U / g), pectinase (enzyme activity ≥30,000 U / g), and β-glucosidase (enzyme activity ≥1000 U / g) in a mass ratio of 3:1:1, with a total addition amount of 0.8% of the mass of the mixed raw materials. Stirring was started, and enzymatic hydrolysis was carried out at a constant temperature of 48°C for 3 hours. After enzymatic hydrolysis, the material in the tank was heated to 85°C and kept at this temperature for 10 minutes to inactivate the enzyme, and then cooled to room temperature to obtain the enzymatic hydrolysate.

[0035] In step S2, the complex enzyme comprises cellulase, pectinase, and β-glucosidase in a mass ratio of 3:1:1. This specific ratio was determined based on in-depth analysis of the cell wall structure and the morphology of the target active ingredient in the golden sunflower raw material.

[0036] Cellulase and pectinase (comprising 80%) primarily function to break down cell walls. Cellulase hydrolyzes the cellulose skeleton of the cell wall, while pectinase breaks down the pectin in the middle lamella. Together, they efficiently deconstruct intact plant tissue into discrete cells or cell fragments. If the proportion of cellulase is too high and the proportion of pectinase is insufficient, the cell wall skeleton may be disrupted, but cells may still be held together by pectin, preventing effective dissociation and hindering the release of internal active ingredients. Conversely, if the proportion of pectinase is too high, cells may separate rapidly, but the cellulose cell walls of individual cells remain intact, and intracellular macromolecules (such as polysaccharides) remain encapsulated and difficult to release. This invention, through extensive single-factor and orthogonal experiments, determined that a 3:1:1 ratio achieves the optimal balance between cell dissociation and cell wall disruption under the specific tissue structure of *Hibiscus truncata*.

[0037] More importantly, the introduction of β-glucosidase (20%) into the cell wall disruption enzyme system allows intracellularly bound flavonoid glycosides (such as hyperoside and quercetin) to be released into the liquid phase while cellulase and pectinase disrupt the cell wall structure. β-glucosidase then begins to function at this stage, directionally hydrolyzing the glycosidic bonds of flavonoid glycosides and pre-converting them into flavonoid aglycones with higher bioavailability and antioxidant activity. By prioritizing the glycosidic bond hydrolysis step and performing it simultaneously with cell wall disruption extraction, compared to the traditional method of first extracting and purifying flavonoid glycosides and then performing enzymatic hydrolysis in a separate step, the process route of this invention is more integrated and efficient. It avoids the separation and loss of intermediate products and takes advantage of the optimal contact area between the enzyme and substrate after cell wall disruption, significantly improving conversion efficiency.

[0038] S3 Ultrasonic Assisted Extraction The enzymatic hydrolysate obtained in step S2 was transferred to an extraction vessel equipped with an ultrasonic generator. The ultrasonic power was set to 400W and the frequency to 40kHz, using an intermittent working mode (5 seconds of operation followed by a 5-second interval), and ultrasonic treatment was carried out for a total of 30 minutes at a constant temperature of 45℃. After treatment, the extracted slurry was obtained.

[0039] In step S3, the selection of ultrasonic processing parameters also has a clear critical significance. The ultrasonic power is limited to 300-500 W, and the frequency is 20-50 kHz.

[0040] The combination of power and frequency determines the intensity and form of cavitation effect. Power below 300 W or frequency above 50 kHz results in fewer cavitation bubbles and weaker collapse intensity. The generated microjets and shock waves are insufficient to effectively break up cell wall fragments that retain their shape after enzymatic hydrolysis, making it difficult to achieve enhanced deep mass transfer and resulting in minimal improvement in the extraction rate of total flavonoids and polysaccharides. Conversely, if the power is above 500 W or the frequency is below 20 kHz, although the cavitation effect is intense, it easily generates localized instantaneous high temperature and pressure in the liquid phase, which may accelerate the oxidative degradation of heat-sensitive flavonoids in *Hippophae rhamnoides*. Simultaneously, excessively strong shear forces may cause irreversible mechanical breakage of the chain structure of large molecular active substances such as polysaccharides, impairing their original gelling and film-forming properties.

[0041] Maintaining the ultrasonic treatment within a temperature range of 40-50℃ is equally crucial. Ultrasonic treatment itself causes the medium to heat up, and if left uncontrolled, this will exacerbate the degradation risk of the aforementioned heat-sensitive components. This invention, while applying ultrasound, maintains the system temperature at 40-50℃ through methods such as jacket cooling, precisely within the optimal temperature range for the initial stage of enzymatic hydrolysis. This ensures that the residual enzymes in the system retain their residual activity and continue to function during the ultrasonic process, achieving a simultaneous temporal and spatial superposition of ultrasound-enhanced mass transfer and enzyme biocatalytic transformation, resulting in a synergistic effect.

[0042] S4 Multi-stage Gradient Fermentation Stage 1 (Anaerobic Lactic Acid Bacteria Fermentation): Brown sugar was added to the extracted slurry to adjust its soluble solids content to 13°Bx, and the pH was adjusted to 6.3 using food-grade sodium carbonate solution. A pre-activated compound lactic acid bacteria starter culture was inoculated at 4% (v / v) of the total slurry volume. The compound lactic acid bacteria starter culture was composed of *Lactobacillus plantarum* CICC20261, *Lactobacillus acidophilus* CICC6074, and *Lactobacillus casei* CICC6117 bacterial powders in a 1:1:1 mass ratio. Anaerobic fermentation was carried out under sealed conditions at a constant temperature of 34℃ for 60 hours.

[0043] Second stage (aerobic yeast fermentation): After the first stage of fermentation is completed, turn on the aeration device of the fermenter and stir (stirring speed 120 rpm). Inoculate the fermentation system with brewer's yeast CICC1421 at an inoculation amount of 2.5% (v / v) of the total fermentation liquid volume. Under constant temperature of 29℃, maintain aeration and stirring, and carry out aerobic fermentation for 36 hours.

[0044] The third stage (anaerobic secondary maturation): After the second stage of fermentation is completed, the aeration device is turned off, and the fermentation broth is transferred to a sealed maturation tank. Under constant temperature of 18℃, it is allowed to stand for anaerobic maturation for 45 days to obtain the fermentation broth.

[0045] The first stage uses a compound fermentation agent composed of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus casei, rather than a single strain, based on a deep consideration of the needs for flavonoid and polysaccharide conversion.

[0046] All three types of lactobacilli possess strong β-glucosidase secretion capabilities, but exhibit subtle differences in substrate specificity and optimal reaction conditions. *Lactobacillus plantarum* shows high hydrolysis efficiency for flavonol glycosides (such as kaempferol glycoside and quercetin glycoside); *Lactobacillus acidophilus* demonstrates strong environmental adaptability, rapid acid production, and the ability to quickly lower the system pH, creating an acidic environment that inhibits contaminating bacteria; while *Lactobacillus casei* excels in utilizing complex sugars (such as pectin oligosaccharides and cellobiose), further utilizing the oligosaccharides produced by enzymatic hydrolysis to generate lactic acid and unique flavor precursors.

[0047] When these three components are combined in a 1:1:1 ratio, their secreted glycosidase system forms a broad and efficient "enzyme spectrum," enabling more thorough hydrolysis of the structurally diverse flavonoids in sunflower seeds, thus pushing the conversion rate to a higher level. Simultaneously, the intertwined metabolites of various lactobacilli create a richer and more mellow organic acid flavor base than single-strain fermentation, avoiding the potential problems of thin flavor or harsh acidity associated with single-strain fermentation.

[0048] The second stage involves introducing brewer's yeast into the low pH environment created by lactic acid bacteria for aerobic fermentation, which has a multi-dimensional effect.

[0049] First, flavor shaping. Under aerobic conditions, brewer's yeast can produce a wide variety of alcohols (such as phenylethyl alcohol), esters (such as ethyl acetate and isoamyl acetate), and aldehydes and ketones through the tricarboxylic acid cycle and amino acid metabolism. These volatile flavor compounds are the key sources of the pleasant fruity, floral, and alcoholic aromas in enzyme products, greatly enhancing the sensory quality of the products and distinguishing them from single lactic acid bacteria fermentation products that only have a sour taste.

[0050] Secondly, the enrichment of functional components. Under aerobic conditions, yeast proliferates much faster than under anaerobic conditions. After the logarithmic growth phase, in the later stages of fermentation, due to decreased dissolved oxygen and accumulation of metabolic products, some yeast cells initiate autolysis. Autolysis releases a large amount of intracellular substances into the fermentation broth, the most valuable of which are superoxide dismutase (SOD) and glutathione. This is the main contributor to the SOD enzyme activity in the enzyme product of this invention, something that cannot be achieved by single lactic acid bacteria fermentation. Therefore, this stage serves as a bridge connecting flavor enhancement and functional strengthening.

[0051] The third stage, anaerobic maturation at a low temperature of 15-20℃, is a process that shifts from "dynamic fermentation" to "static fusion and stabilization." In this stage, the metabolic activity of microorganisms such as yeast drops to an extremely low level, but the microbial enzymes (such as esterases, glycosidases, and proteases) accumulated in the previous stages still slowly exert their catalytic effects.

[0052] Specifically, this manifests as follows: alcohols and acids undergo a slow esterification reaction, generating more and more complex ester aroma components, transforming the overall aroma from the harshness of raw wine to the mellowness of aged wine; residual large-molecule polysaccharides or proteins continue to slowly degrade under the action of residual enzymes, resulting in a more delicate and smooth taste; some unstable intermediate products with unpleasant odors (such as diacetyl and acetaldehyde) produced during fermentation due to violent biochemical reactions are gradually reduced or transformed at this stage, thus achieving the final balance and stability of the product's flavor. The maturation temperature is chosen at 15-20℃ rather than room temperature or high temperatures to ensure sufficient enzyme activity while maximally inhibiting the growth of unwanted microorganisms (especially acid-resistant aerobic bacteria), ensuring the product's microbiological safety.

[0053] S5 Post-processing After the fermentation broth has matured in step S4, solid-liquid separation is performed using a plate and frame filter press. The liquid phase is collected to obtain a clear, brownish-yellow golden sunflower enzyme stock solution. The separated filter residue is collected for later use.

[0054] S6 Product Preparation The enzyme stock solution obtained in step S5 is divided into three parts and subjected to graded processing: Food-grade enzyme liquid: Take a portion of the enzyme stock solution, sterilize it at 85°C for 30 seconds using an ultra-high temperature instantaneous sterilizer, then immediately cool it, fill it into glass bottles on an aseptic filling line, and seal it to obtain a food-grade enzyme beverage.

[0055] Cosmetic-grade enzyme liquid: Take another portion of the enzyme stock solution, filter it with a microfiltration membrane with a pore size of 0.22μm to remove bacteria, collect the filtrate, fill it into a cosmetic-grade container in a low-temperature clean environment, and refrigerate it to obtain cosmetic-grade enzyme liquid raw material.

[0056] Enzyme powder: Take the remaining enzyme stock solution, first perform vacuum freeze drying to obtain coarse powder, then add maltodextrin as a drying aid at a mass ratio of 1:0.5, mix well and then perform spray drying (inlet air temperature 170℃, outlet air temperature 80℃), collect the powder and seal it in packaging to obtain enzyme powder.

[0057] In addition, the filter residue collected in step (5) is placed in a low-temperature vacuum drying oven and dried at 50°C and -0.09MPa until the moisture content is less than 5%. Then it is pulverized by an ultra-micro pulverizer and passed through a 100-mesh sieve to obtain golden sunflower dietary fiber powder, which can be used as a functional food ingredient or cosmetic raw material.

[0058] The present invention proposes a method for preparing enzymes from the whole plant of *Hippophae rhamnoides* using a multi-stage gradient fermentation process. This process employs a combined technology system of zoned pretreatment, complex enzymatic hydrolysis, ultrasound-assisted extraction, and multi-stage gradient fermentation. In the S1 zoned pretreatment, young pods are frozen at -20°C. The volume expansion caused by ice crystal formation pierces and tears the cell wall structure, creating substrate contact conditions for the efficient penetration and action of cellulase and pectinase in the S2 complex enzymatic hydrolysis. The S2 complex enzymatic hydrolysis, through the synergistic hydrolysis of the cell wall skeleton and pectin junction layer by cellulase and pectinase, opens up channels for the release of intracellular active ingredients. Simultaneously, β-glucosidase directionally hydrolyzes the initially dissolved flavonoid glycosides into highly active flavonoid aglycones, achieving simultaneous cell wall disruption and pre-conversion of activity. The S3 ultrasound-assisted extraction utilizes cavitation and mechanical vibration to further break down residual cell wall fragments and the mass transfer boundary layer after enzymatic hydrolysis, promoting the complete dissociation and dissolution of bound active ingredients, forming a loose, hydrolyzed mixture with the S2 enzyme. The coupling and synergistic effect of tissue and ultrasonic deep-layer enhanced mass transfer; S4 multi-stage gradient fermentation sequentially carries out anaerobic lactic acid bacteria fermentation, aerobic yeast fermentation, and anaerobic secondary maturation. In the anaerobic stage, lactic acid bacteria produce acid to inhibit bacteria and then secrete glycosidase to continue the conversion of flavonoid glycosides. In the aerobic stage, yeast metabolizes to generate flavor substances and releases antioxidant active ingredients such as superoxide dismutase through autolysis. Anaerobic secondary maturation promotes flavor fusion and quality stabilization. The three stages target flavonoid glycoside conversion, flavor generation, and active ingredient stabilization, respectively, to achieve synergistic optimization of functionality, sensory quality, and stability; S5 solid-liquid separation obtains high-quality enzyme stock solution, completing the causal progression from physical cell wall disruption, biological enzymatic hydrolysis, physical enhanced mass transfer to multi-strain relay fermentation. This allows active ingredients to undergo a complete technical chain of efficient release, targeted pre-conversion, and deep re-conversion, thereby solving the technical problems of existing technologies such as the separation of extraction and fermentation, insufficient activity conversion, and resource waste caused by the use of single parts. Example 2

[0059] This embodiment demonstrates the process effect when the amount of compound enzyme added and the enzymatic hydrolysis time in step S2 are near the lower limit.

[0060] Step S1 is the same as in Example 1.

[0061] In step S2, the amount of compound enzyme added is 0.5% of the mass of the mixed raw materials, the temperature of the enzymatic hydrolysis treatment is 45℃, and the enzymatic hydrolysis time is 4 hours.

[0062] The operations of steps S3 to S6 are exactly the same as in Example 1. Example 3

[0063] This embodiment demonstrates the process effects of different combinations of ultrasonic parameters in step S3 and fermentation time in step S4.

[0064] Steps S1-S2 are the same as in Example 1.

[0065] In step S3, the ultrasonic treatment power is 300 W, the frequency is 20 kHz, and the cumulative time is 40 minutes.

[0066] In step S4, the anaerobic lactic acid bacteria fermentation temperature is 32℃ and the time is 72 hours; the aerobic yeast fermentation temperature is 28℃ and the time is 48 hours; the anaerobic secondary maturation time is 60 days.

[0067] The operations of steps S5-S6 are exactly the same as those in Example 1. Example 4

[0068] This example demonstrates the process effect when the raw material mixing ratio and fermentation temperature parameters are near their upper limits.

[0069] In step S1, the mass ratio of fresh flowers to tender pods in the mixed raw materials is 2:1, and the freezing time of the tender pods is 14 hours.

[0070] Steps S2-3 are the same as in Example 1.

[0071] In step S4, the anaerobic lactic acid bacteria fermentation temperature is 35℃ and the time is 48 hours; the inoculum amount of the compound lactic acid bacteria starter is 5% (v / v) of the extracted slurry volume. The aerobic yeast fermentation temperature is 30℃ and the time is 24 hours; the inoculum amount of brewer's yeast is 3% (v / v) of the fermentation system volume, and the anaerobic secondary maturation temperature is 20℃ and the time is 30 days.

[0072] The operation of steps S5-6 is exactly the same as in Example 1. Example 5

[0073] This embodiment demonstrates the process effects of fermentation using different combinations of microbial strains.

[0074] Steps S1-S3 are the same as in Example 1.

[0075] In step S4, the compound lactic acid bacteria starter culture used in the first stage consists of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus helveticus in a 1:1:1 mass ratio, with an inoculation amount of 3% (v / v). The yeast used in the second stage is Saccharomyces cerevisiae, with an inoculation amount of 2% (v / v).

[0076] The remaining steps are exactly the same as in Example 1.

[0077] Comparative Example 1 used a mixture of golden sunflower flowers and tender pods from the same source and in the same proportions as in Example 1, but without enzymatic hydrolysis or ultrasonic treatment. Water and brown sugar were added directly, and 5% *Lactobacillus plantarum* was inoculated. The mixture was allowed to ferment naturally at room temperature for 90 days, and the resulting enzyme stock solution was obtained after filtration. This comparative example is used to simulate simple fermentation processes commonly found in the prior art.

[0078] Comparative Example 2 The difference between this comparative example and Example 1 is that only golden sunflower flowers are used as raw materials, without the addition of tender pods. The parameters for all other steps (enzymatic hydrolysis, sonication, multi-stage gradient fermentation) are consistent with those of Example 1. This comparative example is used to demonstrate the advantages of the "whole-plant synergistic utilization" strategy of this invention in terms of active ingredients and functionality.

[0079] Comparative Example 3 The difference between this comparative example and Example 1 is that the enzymatic hydrolysis in step S2 and the ultrasound-assisted extraction in step S3 are omitted. Specifically, the pretreated mixed raw materials are directly added to water and brown sugar, followed by multi-stage gradient fermentation in step S4. This comparative example is used to demonstrate the crucial role of the enzymatic hydrolysis and ultrasound-coupled extraction technology of this invention in the efficient release of active ingredients.

[0080] Comparative Example 4 The difference between this comparative example and Example 1 is that the multi-stage gradient fermentation design in step S4 is omitted. Specifically, the extracted slurry is subjected to anaerobic lactic acid bacteria fermentation only (under the same conditions as Example 1, but for 90 days), without subsequent aerobic yeast fermentation and anaerobic secondary maturation. This comparative example is used to demonstrate the role of the "three-stage gradient fermentation" of the present invention in the deep conversion of active ingredients, the generation of flavor substances, and product stability.

[0081] Quality evaluation experiment The quality of the enzyme stock solutions prepared in all Examples 1-5 and Comparative Examples 1-4 above was evaluated. The detection methods followed relevant standards or conventional methods: total flavonoid content was determined using the sodium nitrite-aluminum nitrate colorimetric method; total polysaccharide content was determined using the phenol-sulfuric acid method; SOD enzyme activity was determined using the pyrogallol autoxidation method; total acid content was determined using acid-base titration; and DPPH free radical scavenging rate was determined using spectrophotometry.

[0082] Evaluation results Table 1. Comparison of quality indicators of enzyme stock solutions in various examples and comparative examples Results Analysis Compared with existing technologies (Example vs. Comparative Example 1): All examples of the present invention are significantly superior to the traditional single-strain natural fermentation process (Comparative Example 1) in terms of total flavonoids, total polysaccharides, SOD enzyme activity, and antioxidant capacity. Taking Example 1 as an example, its total flavonoid content is increased by 87.5%, total polysaccharide content by 78.4%, and SOD enzyme activity by 147.8%. This fully demonstrates the advanced nature and high efficiency of the integrated process of the present invention.

[0083] Synergistic Utilization Effect of Whole Plant (Example 1 vs. Comparative Example 2): Compared with the process using only flowers (Comparative Example 2), the total polysaccharide content of Example 1 increased by 229.6%, and the total flavonoid content also increased slightly. This confirms that the method of utilizing flowers and young pods in sections in this invention successfully achieves the synergistic release of flavonoids and polysaccharides, greatly enhancing the comprehensive nutritional value of the product.

[0084] Enzymatic hydrolysis-ultrasound coupling effect (Example 1 vs. Comparative Example 3): Compared with the process omitting enzymatic hydrolysis and ultrasound (Comparative Example 3), all indicators of Example 1 were significantly improved, especially total flavonoids (increased by 54.1%) and SOD enzyme activity (increased by 58.3%). This strongly demonstrates that the enzymatic hydrolysis-ultrasound coupling extraction step is indispensable for efficiently breaking down cell walls and releasing intracellular active ingredients.

[0085] Multi-stage gradient fermentation effect (Example 1 vs. Comparative Example 4): Compared with the process of fermentation with only a single lactic acid bacteria (Comparative Example 4), the SOD enzyme activity of Example 1 was increased by 119.2%, the sensory score was significantly improved, and the total acid content was more moderate (Comparative Example 4 had excessively high acidity due to excessive fermentation time). This indicates that the aerobic yeast fermentation stage is crucial for the enrichment of antioxidant components such as SOD enzymes and the generation of flavor substances, while the secondary maturation stage helps to balance acidity and blend flavors. The three stages work synergistically to achieve a dual improvement in activity conversion and sensory quality.

[0086] Adaptability of process parameter range (Examples 1-5): Different combinations of parameters within the range defined in each embodiment of the present invention have yielded technical effects that are significantly better than those of the comparative examples, proving that the technical solution of the present invention has wide applicability and reproducibility within a given range.

[0087] In summary, the method for preparing whole-plant multi-gradient fermentation enzymes of *Hymenochloa chinensis* provided by this invention organically integrates three major innovations: partitioned pretreatment, enzymatic hydrolysis-ultrasonic coupling extraction, and multi-gradient fermentation. This method achieves efficient utilization of whole-plant resources of *Hymenochloa chinensis* and deep conversion of active ingredients. The resulting product has outstanding functionality and excellent sensory quality, and has significant progressiveness and industrial application value.

[0088] In the description of this specification, the terms "one embodiment," "some embodiments," "embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0089] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make modifications, alterations, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a multi-stage gradient fermentation enzyme from the whole plant of *Hippophae rhamnoides*, characterized in that, Includes the following steps: S1. Raw material pretreatment: Harvest fresh flowers and tender pods of golden sunflower, wash and cut them separately, and then mix them to obtain mixed raw materials; S2. Compound enzymatic hydrolysis: Water and compound enzyme are added to the mixed raw materials for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme is inactivated to obtain the enzymatic hydrolysate. The compound enzyme contains cellulase, pectinase and β-glucosidase. S3. Ultrasonic-assisted extraction: The enzymatic hydrolysate is subjected to ultrasonic treatment to obtain an extraction slurry; S4. Multi-stage gradient fermentation: The extracted slurry is subjected to anaerobic lactic acid bacteria fermentation, aerobic yeast fermentation and anaerobic secondary maturation in sequence to obtain fermentation broth; S5. Post-processing: The fermentation broth is subjected to solid-liquid separation to obtain enzyme stock solution.

2. The method for preparing the multi-stage gradient fermentation enzyme of the whole plant of *Hippophae rhamnoides* according to claim 1, characterized in that, In step S1, the cutting process includes: cutting the fresh flowers into 1-2 cm sections, cutting the tender pods into segments, and freezing them at -20°C for 10-14 hours; the mass ratio of the fresh flowers to the tender pods in the mixed raw materials is 2:

1.

3. The method for preparing the multi-stage gradient fermentation enzyme of the whole plant of *Hippophae rhamnoides* according to claim 1, characterized in that, In step S2, the mass ratio of cellulase, pectinase and β-glucosidase in the compound enzyme is 3:1:1; the amount of the compound enzyme added is 0.5%-1.0% of the mass of the mixed raw materials.

4. The method for preparing the multi-stage gradient fermentation enzyme of the whole plant of *Hippophae rhamnoides* according to claim 1, characterized in that, In step S2, the enzymatic hydrolysis treatment is carried out at a temperature of 45-50°C for 2-4 hours; the enzyme inactivation is carried out by heating to 85°C and holding for 8-12 minutes.

5. The method for preparing the multi-stage gradient fermentation enzyme of the whole plant of *Hippophae rhamnoides* according to claim 1, characterized in that, In step S3, the ultrasonic treatment power is 300-500 W, the frequency is 20-50 kHz, the time is 20-40 minutes, and the temperature is controlled at 40-50℃ during the treatment.

6. The method for preparing the multi-stage gradient fermentation enzyme of the whole plant of *Hippophae rhamnoides* according to claim 1, characterized in that, In step S4, the specific steps of the anaerobic lactic acid bacteria fermentation are as follows: add brown sugar to the extracted slurry to adjust the soluble solids content to 12-15°Bx and adjust the pH to 6.0-6.5; inoculate with compound lactic acid bacteria fermentation agent and anaerobic ferment at 32-35℃ for 48-72 hours.

7. The method for preparing the multi-stage gradient fermentation enzyme of the whole plant of *Hippophae rhamnoides* according to claim 6, characterized in that, The compound lactic acid bacteria starter consists of Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus casei in a mass ratio of 1:1:1; the inoculum amount of the compound lactic acid bacteria starter is 3%-5% of the volume of the extracted slurry.

8. The method for preparing the multi-stage gradient fermentation enzyme of the whole plant of *Hippophae rhamnoides* according to claim 1, characterized in that, In step S4, the specific steps of the aerobic yeast fermentation are as follows: inoculate brewer's yeast into the system after the anaerobic lactic acid bacteria fermentation is completed, and ferment aerobically at 28-30℃ for 24-48 hours under aeration and stirring conditions; the amount of brewer's yeast inoculated is 2%-3% of the fermentation system volume.

9. The method for preparing the multi-stage gradient fermentation enzyme of the whole plant of *Hippophae rhamnoides* according to claim 1, characterized in that, In step S4, the specific steps of the anaerobic secondary maturation are as follows: after the aerobic yeast fermentation is completed, the fermentation liquid is transferred into a sealed container and anaerobically matured at 15-20℃ for 30-60 days.

10. The method for preparing the multi-stage gradient fermentation enzyme of the whole plant of *Hippophae rhamnoides* according to any one of claims 1-9, characterized in that, It also includes the following steps: S6. Product preparation: The enzyme stock solution is graded to obtain at least one of food-grade enzyme solution, cosmetic-grade enzyme solution or enzyme powder. The food-grade enzyme liquid is prepared by filling the enzyme stock solution after instantaneous high-temperature sterilization. The cosmetic-grade enzyme solution is prepared by microfiltration sterilization of the enzyme stock solution followed by low-temperature storage. The enzyme powder is obtained by drying the enzyme stock solution.

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