Preparation method of traditional Chinese medicine compound enzyme for regulating qi and blood

Abstract

The invention discloses a preparation method of a traditional Chinese medicine compound enzyme for regulating qi and blood, and relates to the crossing field of biological fermentation technology and traditional Chinese medicine application, in particular to the preparation method of the traditional Chinese medicine compound enzyme for regulating qi and blood. The preparation method comprises the following steps: mixing the raw materials in proportion, performing vacuum freeze drying to obtain freeze-dried composite stem and leaf powder, mixing the freeze-dried composite stem and leaf powder with red date particles, adding water for enzymolysis, performing solid-liquid separation, taking residues, inoculating a mixed bacterium solution of candida utilis and lactobacillus plantarum, and performing discontinuous oxygen supply solid-state fermentation and aging to obtain a finished product. And a carbon source is derived from natural sugar and enzymolysis products of the red dates. The water content of the obtained enzyme is about 30-40%, the pH value is 3.8-4.2, and the enzyme has unique fermentation flavor and can be used for regulating qi and blood.

Description

Technical Field

[0001] This invention relates to the intersection of bio-fermentation technology and traditional Chinese medicine application, specifically a method for preparing a compound enzyme of traditional Chinese medicine for regulating qi and blood. Background Technology

[0002] The theory of "Qi and Blood" is one of the core components of traditional Chinese medicine's theoretical system, and the harmony of Qi and Blood is considered a fundamental state for maintaining bodily health. In modern life, irregular work and rest schedules, mental stress, and other factors easily lead to disharmony of Qi and Blood, manifesting as fatigue, weakness, pale complexion, and a series of sub-health conditions. Therefore, seeking safe and effective ways to regulate Qi and Blood daily has received widespread attention.

[0003] Enzymes are proteins produced by living organisms that possess catalytic functions and play a vital role in life activities. In recent years, the preparation of enzyme products through the fermentation of plant raw materials has become an emerging research direction in the health food industry. The principle is to utilize microorganisms or the plant's own enzyme system to decompose and transform plant substrates, generating small-molecule nutrients and various active enzymes that are more easily absorbed and utilized by the human body.

[0004] In the existing technology, there are some reports on the preparation of enzymes using Chinese medicinal materials or fruits and vegetables. For example, some technologies disclose methods that use one or more root and rhizome medicinal materials as the main ingredients, which are chopped and then directly added to sugar sources and water for natural fermentation or fermentation by inoculating a single yeast strain; other technologies focus on using mature fruits for long-term liquid fermentation. However, these existing technologies generally have the following limitations: First, they mostly focus on utilizing soluble sugars or starches in raw materials, resulting in low utilization rates of plant stems and leaves rich in cellulose, hemicellulose, and lignin, leading to a huge waste of the effective components of the raw materials; Second, the fermentation process often relies on a high-sugar environment (such as honey and brown sugar) to inhibit unwanted bacteria and provide nutrients for yeast. This high-sugar condition not only alters the natural properties of the product but may also be unsuitable for specific populations; Third, the fermentation mode is singular, either using only natural fermentation (uncontrollable) or only pure microbial fermentation (single flavor and active ingredients), making it difficult to form a multi-layered and complex metabolic network, resulting in a narrow spectrum of product activity; Fourth, for the traditional Chinese medicine functional claim of "regulating qi and blood," existing technologies mostly remain at the conceptual level, lacking targeted design in terms of raw material compatibility and preparation processes, and failing to establish an intrinsic process link between the characteristics of raw materials and the potential functions of the final product.

[0005] Specifically, existing technologies lack any publicly available methods for combining the stems and leaves of Ligusticum chuanxiong and carrots (two commonly discarded plant stems and leaves) with jujubes, activating endogenous enzyme activity through specific physical cell wall disruption pretreatment, and then employing a stepwise complex enzymatic hydrolysis and discontinuous oxygen supply microbial solid-state fermentation strategy to prepare the target product. Therefore, developing a novel compound enzyme preparation method that can deeply explore the value of specific plant stems and leaves, has controllable processes, and supports its potential function of regulating qi and blood from both a material basis and preparation logic perspective, has significant practical implications and application prospects. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a traditional Chinese medicine compound enzyme that regulates qi and blood. Through specific gradient cell wall breaking, stepwise enzymatic hydrolysis and discontinuous oxygen-sugar fermentation processes, a method is used to prepare a traditional Chinese medicine compound enzyme that can regulate qi and blood without added sugar, thereby improving the utilization rate of active ingredients and product uniqueness, and meeting the needs of natural health conditioning.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood, comprising the following steps: S1. Raw Material Pretreatment: Collect the stems and leaves of Ligusticum chuanxiong that are in their vigorous growth period and are green without yellowing. Select plant parts that are free from mold and insect infestation. Gently rinse the plant parts with running purified water to remove surface dirt and impurities. Spread them evenly on a clean bamboo sieve or breathable mesh rack. Through natural ventilation and moderate turning, allow the stems and leaves to gradually lose water until the moisture content reaches 60%-70%. Then, use a stainless steel knife to cut them into segments 0.5-1.5 cm in length with a flat cross-section, ensuring uniform segment length for subsequent processing. Select freshly picked carrot stems and leaves that are plump and not wilted. After thoroughly washing them with running purified water to remove mud and residual pesticides, immediately place them in a constant temperature water bath. In the equipment or hot air circulation equipment, the temperature is kept constant at 50℃-60℃ for 10-15 minutes. The gentle heat deactivates the oxidases (such as polyphenol oxidase) in the raw materials that are prone to browning and nutrient loss. Then, it is quickly cooled to room temperature and cut into 1-2 cm pieces with slightly fibrous edges to preserve the natural structure of the raw materials. Select fully dried red dates with thick flesh and no insect infestation or mold. Remove the pits manually or mechanically, and use stone mills or stainless steel roller presses for physical crushing. By controlling the pressure, the date flesh is broken into irregularly shaped particles with a diameter of 3-5 mm to avoid over-crushing, which may cause gelatinization or clumping during subsequent extraction.

[0008] S2. Gradient cell wall disruption and endogenous enzyme activation: Weigh the Ligusticum chuanxiong stem and leaf segments and the carrot stem and leaf fragments at a mass ratio of 1:(0.8-1.2), mix them evenly, and spread them evenly in a clean tray with a thickness of 2-3 cm to avoid excessive stacking that would affect the drying effect; perform vacuum freeze-drying treatment, first place the material in the pre-freezing chamber of the freeze dryer, control the pre-freezing temperature at -40℃ to -50℃, so that the internal moisture of the material is completely frozen into ice crystals, and the pre-freezing time is 4-6 hours to ensure uniform freezing; then start the vacuum system, and slowly raise the temperature to 25℃-30℃ under a vacuum of 20-30Pa, using the low-temperature vacuum environment to directly sublimate the ice crystals, maintain drying for 12-16 hours until the moisture content of the material is less than 8%, and obtain a freeze-dried composite stem and leaf powder with loose texture, preliminary cell structure destruction, and retention of some endogenous enzyme active ingredients.

[0009] S3. Stepwise compound enzymatic hydrolysis: Weigh and mix the freeze-dried compound stem and leaf powder and the jujube granules at a mass ratio of (2-3):1, add 1.5-2 times the total mass of the materials of purified water, and stir at a low speed for 5-8 minutes to ensure that the solid materials and water are fully in contact to form a uniform slurry. Then, adjust the temperature of the slurry to 45℃ and keep it stable by jacket heating or water bath temperature control. First, add 0.05%-0.1% of the mass of the freeze-dried compound stem and leaf powder of food-grade cellulase (enzyme activity ≥5000U / g), and then... The first stage of enzymatic hydrolysis is carried out at a constant temperature of 45℃ for 90-120 minutes. Cellulase decomposes the cellulose components in the raw materials, destroys the plant cell wall structure, and releases the small molecules encapsulated therein. Then, food-grade pectinase (enzyme activity ≥10000U / g) equivalent to 0.03%-0.06% of the mass of the freeze-dried compound stem and leaf powder is added, and the second stage of enzymatic hydrolysis is carried out at a constant temperature of 45℃ for 60-90 minutes. Pectinase decomposes pectin substances, further reducing the viscosity of the material and improving the dissolution efficiency of soluble components.

[0010] S4. Construction of Complex Microbial Community and Discontinuous Aerobic Solid-State Fermentation: The enzymatic hydrolysis mixture from step S3 is subjected to solid-liquid separation using an 80-100 mesh filter cloth. All solid residue (including incompletely dissolved plant fibers and enzymatic hydrolysis residue) is collected, and the filtrate is collected separately for later use. When the residue temperature naturally cools to 32℃-35℃ (close to the suitable temperature range for microbial growth), 10 microbial cultures are inoculated per gram of residue. 6 -10 7The proportion of CFU mixed bacterial solution is as follows: A pre-prepared activated seed culture of one strain of *Candida utilis* and one strain of *Lactobacillus plantarum* is evenly mixed into the residue. The ratio of viable *Candida utilis* to *Lactobacillus plantarum* in the mixed bacterial solution is 1:(1.5-2.5). During mixing, manual kneading or low-speed stirring equipment is used to ensure full contact and even distribution of the bacterial solution and residue. After mixing, the material is piled into a circular or strip pile approximately 20-30 cm high and 40-50 cm in diameter. Fermentation is carried out in a sealed, anaerobic environment for 48 hours, during which the temperature at the center of the pile is monitored every 6-8 hours by covering. Use breathable burlap sacks or adjust the tightness of the pile to control the core temperature of the pile at 30℃-35℃ to promote the initial colonization and metabolism of anaerobic microorganisms. Then switch to forced convection oxygen supply once a day for 2 hours. During oxygen supply, uncover the covering and turn the surface of the pile appropriately, or use a blower to introduce clean air to the bottom of the pile. After the oxygen supply is finished, cover and seal the pile again to restore the anaerobic environment. Repeat this process until the entire fermentation cycle reaches 15-20 days. At the end of the fermentation, the pH value of the pile is measured by a precision pH meter to be 3.8-4.2. At this time, the accumulation of organic acids is moderate and the flavor substances are initially formed.

[0011] S5. Maturation and Finished Product: After fermentation, the piled material is crushed into coarse particles of 2-5 mm using a pulverizer. The particles are then transferred to a clean container that has undergone high-temperature sterilization and sealed. A small vent is left at the container opening to balance the internal and external air pressure. The material is then left to mature and age for 30-45 days in a cool, dark environment (ambient temperature 15℃-25℃, relative humidity 50%-60%) and free from odor interference. During this period, the container is opened every 7-10 days to observe the material and gently stir it to promote the uniform transformation of internal components. Finally, the traditional Chinese medicine compound enzyme for regulating qi and blood is obtained.

[0012] Furthermore, in step S2, the pre-freezing temperature of the vacuum freeze-drying is -45℃, and the pre-freezing time is 5 hours, to ensure that the internal moisture of the material is fully frozen and the ice crystals are of uniform size; the vacuum degree during the drying stage is controlled at 25Pa, and the temperature is raised to 28℃ and maintained for 14 hours, so that the ice crystals sublimate slowly, avoiding the shrinkage of the material or the destruction of the effective components due to excessive heating. The resulting freeze-dried composite stem and leaf powder is green in color, has good rehydration properties, and the cell wall breakage rate can reach 60%-70%.

[0013] Furthermore, in step S3, during the preparation of the slurry, the ratio of the total mass of the material to the mass of the added purified water is 1:1.5 to 1:1.8, ensuring that the slurry is a thick paste, which facilitates sufficient contact between the enzyme and the substrate and avoids excessive moisture leading to spoilage during subsequent fermentation. The first stage of enzymatic hydrolysis takes 100 minutes, and the second stage takes 75 minutes. This combination of times allows for more thorough decomposition of cellulose and pectin, increasing the soluble solids dissolution rate by 15%-20%. The amount of cellulase added is 0.08% of the mass of the freeze-dried composite stem and leaf powder, and the amount of pectinase added is 0.04% of the mass of the freeze-dried composite stem and leaf powder. This ratio can ensure enzymatic hydrolysis efficiency while reducing the interference of excessive enzyme residue on the fermentation system.

[0014] Further, in step S4, the preparation of the activated seed liquid involves inoculating the corresponding strains (Candida utilis and Lactobacillus plantarum) onto slant culture media. The slant culture media are prepared according to conventional microbial culture methods (e.g., yeast slant culture media contains glucose, peptone, yeast extract, etc., and lactic acid bacteria slant culture media contains MRS agar, etc.). The media are then activated in a constant temperature incubator at 28℃-30℃ for 24 hours until uniform colonies appear on the slant and are plump. Single colonies are then picked and transferred to liquid seed culture media (yeast liquid culture media contains malt extract, peptone, etc., and lactic acid bacteria liquid culture media contains MRS broth, etc.). The media are cultured at 28℃-30℃ with shaking at 150-200 rpm for 18-24 hours, during which the cell growth curve is monitored until the cell concentration reaches 10⁻⁶. 8 A seed culture with a concentration of CFU / mL or higher is obtained, resulting in a highly viable seed culture.

[0015] Further, in step S4, the total inoculation amount of the mixed bacterial solution is 5 × 10⁻⁶ per gram of residue. 6 CFU, this inoculation amount can ensure that microorganisms can quickly colonize in the slag and form a dominant bacterial community, avoiding contamination by other bacteria; the ratio of the viable number of the Candida utilis to the viable number of Lactobacillus plantarum is 1:2. This ratio can enable the extracellular enzyme secretion of yeast and the organic acid metabolism of lactic acid bacteria to work synergistically, promoting the conversion and enrichment of active ingredients such as flavonoids and saponins in the raw materials.

[0016] Furthermore, in step S4, the pile is a cone-shaped pile with a height of about 30-40 cm and a width of about 50-60 cm, with the cone apex angle controlled at 60°-70° to facilitate heat accumulation and gas diffusion balance. A gas guide pipe with an inner diameter of 1-2 cm (made of food-grade stainless steel or ceramic) is inserted in the center of the pile. The bottom end of the gas guide pipe is 5-10 cm from the bottom of the pile, and the top end extends 5 cm above the surface of the pile. This is used to connect with the outside atmosphere during the forced convection oxygen supply stage, guide air to permeate evenly into the interior of the pile, and avoid local oxygen deficiency or over-supply.

[0017] Furthermore, in step S4, the forced convection oxygen supply operation is set to run from 9:00 AM to 11:00 AM daily, when the ambient temperature is relatively stable, which can reduce the impact of external temperature fluctuations on the internal micro-ecology of the reactor. The ambient temperature during oxygen supply is controlled at 25°C-28°C, and is adjusted by air conditioning or temperature control equipment to ensure that the center temperature of the reactor does not exceed 38°C during oxygen supply, so as to prevent high temperature from inhibiting microbial activity or causing decomposition of beneficial components.

[0018] Furthermore, in step S5, the container used for aging is made of ceramic (wall thickness 3-5 mm, inner wall glazed with food grade) or stainless steel (304 or 316L material, inner wall polished). Such containers have moderate air permeability and chemical stability, which can prevent container components from migrating and contaminating the material. The aging time is 40 days. During this period, the organic acids and esters in the material further associate, resulting in a more mellow and harmonious flavor and significantly improved stability of active ingredients.

[0019] Furthermore, the preparation method does not add any form of sugar (including but not limited to sucrose, glucose, fructose, maltose, honey, syrup, etc.) as a fermentation substrate or flavoring agent throughout the entire process. The carbon source required for fermentation comes entirely from the natural sugars contained in the jujube granules (mainly glucose, fructose and a small amount of sucrose) and the polysaccharide degradation products (such as oligosaccharides generated by the hydrolysis of cellulose and hemicellulose) produced by the enzymatic hydrolysis of the raw materials.

[0020] Furthermore, the finished product is a paste or lumpy substance with a water content of approximately 30%-40%, with a soft and delicate texture and a certain degree of viscosity and elasticity, making it difficult to break apart; its pH value is 3.8-4.2, which is weakly acidic, conducive to the preservation of active ingredients and conforms to the adaptability of the human digestive tract environment; and it has a unique fermented aroma (combining plant fragrance with ester and sour aroma produced by microbial metabolism) and flavor (slightly sweet with sourness, and a smooth taste without bitterness) produced by the specific raw materials (Ligusticum chuanxiong stems and leaves, carrot stems and leaves, and dried red dates) and processes (gradient cell wall breaking, stepwise enzymatic hydrolysis, non-continuous oxygen supply fermentation of compound microbial groups, and long-term aging), which is different from existing enzyme products.

[0021] This invention provides a method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood, which has the following beneficial effects: 1. This method uses the stems and leaves of Ligusticum chuanxiong (which invigorates blood and promotes qi circulation) during its vigorous growth period and fresh carrot stems and leaves (rich in carotenoids and dietary fiber) as core medicinal and edible raw materials, supplemented with pitted red dates (which tonify the middle energizer and replenish qi, and provide natural sugar). Differential pretreatment maximizes efficacy: Ligusticum chuanxiong stems and leaves are spread out to control moisture (60%-70%) before being cut into sections, preserving volatile active ingredients while preventing the loss of effective substances; carrot stems and leaves are subjected to constant temperature inactivation at 50℃-60℃ to inactivate and deactivate enzymes, preventing oxidative browning while retaining enzyme precursors; red dates are physically crushed into 3-5 mm particles to increase the contact area with the enzymatic hydrolysis system. Gradient cell wall disruption (vacuum freeze-drying to moisture content <8%) further breaks down the plant cell wall structure, allowing for the full release of insoluble components such as ferulic acid from Ligusticum chuanxiong and β-carotene from carrots, providing a high-concentration active matrix for subsequent enzymatic hydrolysis and fermentation, ensuring the material basis for "regulating qi and blood" from the source.

[0022] Employing a stepwise enzymatic hydrolysis system of "cellulase + pectinase" (staged action at 45℃), this method specifically decomposes the lignocellulose in the stems and leaves of Ligusticum chuanxiong and the pectin in the stems and leaves of carrots, converting macromolecular polysaccharides and fibers into fermentable monosaccharides and small peptides. This reduces residue viscosity, improves mass transfer efficiency, and provides a high-quality carbon and nitrogen source for microbial metabolism. An innovative non-continuous aerobic solid-state fermentation mode of "48 hours of closed-loop anoxic environment + 2 hours of forced convection aerobic supply daily" is designed. The initial anoxic environment promotes anaerobic microorganisms (such as Candida utilis) to preferentially utilize monosaccharides to synthesize amino acids, B vitamins, and other key factors for blood and qi regulation. Later, periodic aerobic supply activates the metabolism of aerobic bacteria (such as Lactobacillus plantarum), producing organic acids (lactic acid, acetic acid) to regulate the intestinal microecology, while simultaneously promoting the accumulation of secondary metabolites such as flavonoids and saponins. The synergistic effect of the two microorganisms (live bacteria ratio 1:1.5-2.5) further enhances the variety and content of active substances through metabolic complementarity, resulting in a more comprehensive "blood and qi regulation" effect compared to single-strain fermentation products.

[0023] This method strictly limits the addition of any sugars (sucrose, glucose, etc.) as fermentation substrates or flavorings throughout the entire process. The carbon source relies entirely on the natural fructose of jujubes and the oligosaccharides and monosaccharides produced by the enzymatic hydrolysis of the raw materials. This avoids the problem of high osmotic pressure introduced by exogenous sugars inhibiting microbial activity, ensuring that the fermentation process is dominated by the components of the raw materials themselves, reducing the risk of contamination by other microorganisms; at the same time, it avoids the potential for blood sugar fluctuations caused by additional sugars, making it particularly suitable for people who need to control their blood sugar. In the targeted fermentation system, Candida utilis synthesizes glutathione (antioxidant, liver-protecting) from amino acids, and Lactobacillus plantarum metabolizes to produce short-chain fatty acids (regulating spleen and stomach function). The two work together to transform the traditional "qi and blood replenishing" effects of the raw materials into quantifiable bioactivity indicators (such as a 30% increase in reducing sugar content and a 25% increase in free amino acids), making the product's efficacy purer and more in line with modern health needs.

[0024] From raw material pretreatment to fermentation, meticulous temperature control is implemented throughout the entire process: the inactivation temperature of Sichuan lovage / carrot stems and leaves (50℃-60℃) is precisely matched to the critical value for enzyme inactivation, avoiding excessive heating that could damage heat-sensitive components; during the freeze-drying stage, pre-freezing (-40℃ to -50℃) is combined with vacuum drying (25-30℃) to maximize the preservation of the raw material's color and volatile aroma; enzymatic hydrolysis is carried out at a constant temperature of 45℃ to stabilize enzyme activity; in the early stage of fermentation, the temperature at the center of the pile is controlled at 30℃-35℃ (suitable for the logarithmic growth phase of yeast), and in the later oxygen supply stage, the ambient temperature is 25℃-28℃ (to balance the metabolic rate of aerobic bacteria). In addition, the conical pile (30-40cm high and 50-60cm wide) combined with the gas duct design achieves uniform oxygen distribution through forced convection oxygen supply (9-11 am daily), avoiding localized excessive anaerobic conditions that could lead to rancidity or excessive aerobic conditions that could cause inactivation. Multi-dimensional environmental regulation ensures a stable fermentation process, with the product pH maintained at 3.8-4.2 (inhibiting unwanted bacteria and facilitating the preservation of activity), ultimately resulting in an enzyme product with uniform color and harmonious flavor.

[0025] After fermentation, a 30-45 day (preferably 40 days) sealed maturation period is set in a ceramic / stainless steel container. Utilizing a micro-aerobic environment and slow enzymatic reactions (such as residual pectinase continuing to break down fibers), Maillard reactions (generating flavor compounds), and microbial autolysis (releasing intracellular active substances), the process promotes further polymerization or transformation of small molecules. This process not only naturally forms the enzyme into a paste / clump with 30%-40% water content (easy to consume and delays spoilage), but also significantly enhances the synergistic effect of active ingredients: for example, the volatile oil of Ligusticum chuanxiong forms an inclusion complex with jujube polysaccharides, reducing irritation and prolonging the duration of action; the metabolites of Lactobacillus plantarum combine with flavonoids to enhance intestinal absorption. The final product possesses both a "unique fermented aroma" (a complex aroma derived from the original flavor of the raw materials and fermentation byproducts) and a "deeply regulating qi and blood" function (improving microcirculation and enhancing the activity of enzymes related to hemoglobin synthesis), distinguishing it from the bland flavor and singular efficacy of commercially available short-fermented enzymes, and better aligning with the traditional Chinese medicine concept of "slowly regulating qi and blood." Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] How to use: I. Applicable Scenarios and Core Functions This product is designed for people who need gentle conditioning of Qi and blood. It is suitable for symptoms such as fatigue, pale complexion, and cold hands and feet caused by daily Qi and blood deficiency. Through the synergistic effect of food-grade medicinal ingredients and fermentation products, it helps to improve the circulation of Qi and blood.

[0028] II. Basic Usage It is recommended to take it on an empty stomach or 30 minutes before meals to facilitate the absorption of the active ingredients. The amount should be about the size of your thumb or palm (about 5-10 grams), and can be taken in divided doses. If you find the texture too thick, you can add a small amount of room temperature purified water (about 10-20 ml) to make a thin paste. If you prefer the original flavor, you can take it directly or with warm water to avoid high temperatures destroying the active ingredients.

[0029] III. Frequency and Period Initial adaptation period (first 2 weeks): Once daily, observe the body's reaction (such as gastrointestinal comfort and mental state); after adaptation, the frequency can be adjusted to 1-2 times daily. One month of continuous use constitutes one basic conditioning cycle. Because the fermented products contain natural organic acids and enzymes, it is recommended to pause for one day every 3-5 days to avoid long-term continuous irritation to the gastrointestinal tract.

[0030] IV. Precautions Body type suitability: This product contains Ligusticum chuanxiong (pungent and warm). Those with yin deficiency and excessive internal heat, or those prone to getting overheated, are advised to halve the dosage for first-time use, or consult a professional. Pregnant women, breastfeeding women, and patients with serious chronic diseases should follow their doctor's advice before use.

[0031] Storage instructions: As no preservatives are added, it must be sealed and stored in a cool, dark place (such as a cupboard). It should be consumed within 30-45 days after aging. Once opened, it is recommended to use it within 1 week to prevent contamination by other bacteria that may affect the quality.

[0032] Flavor Tolerance: The unique fermented aroma may take some getting used to. If you are bothered by the smell, you can mix it with a small amount of warm honey water (not necessary, as this product itself is not sweet), but please note that the water temperature should not exceed 40℃.

[0033] V. Collaboration Recommendations Regulating Qi and blood requires regular sleep and moderate exercise (such as Baduanjin or walking). This product is an auxiliary method and cannot replace medication or a balanced diet. If mild abdominal bloating occurs during use, it is mostly a normal reaction to the metabolism of fermentation products. Reducing the dosage or stopping use for 1-2 days will relieve the symptoms.

[0034] Example: Example 1: Typical Application of Basic Processes This embodiment adopts the basic process of "a method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood", focusing on the synergy of raw material compatibility and key process parameters.

[0035] Raw material pretreatment: Collect the stems and leaves of Ligusticum chuanxiong during its vigorous growth period, rinse them with purified water, spread them out to dry to a suitable moisture content, and cut them into short sections; select fresh carrot stems and leaves, wash them, and inactivate the enzymes at a moderate temperature, then cool them and cut them into small pieces; remove the pits from dried red dates and crush them into irregular particles by physical crushing.

[0036] Gradient cell wall disruption and endogenous enzyme activation: The stem and leaf segments of Ligusticum chuanxiong and the stem and leaf fragments of carrot are mixed in proportion, spread out and then subjected to vacuum freeze drying. First, the moisture is fully frozen at a low pre-freezing temperature. After several hours of pre-freezing, the temperature is gradually increased to a mild temperature under a suitable vacuum and maintained to obtain low-moisture freeze-dried composite stem and leaf powder, which not only retains the active ingredients of the raw materials, but also improves the efficiency of subsequent enzymatic hydrolysis through cell wall disruption.

[0037] Stepwise compound enzymatic hydrolysis: Freeze-dried compound stem and leaf powder is mixed with jujube granules in a certain proportion, and an appropriate amount of purified water is added to stir into a slurry and adjusted to the enzymatic hydrolysis temperature. First, a small amount of food-grade cellulase is added and kept at a constant temperature for a period of time to decompose the fiber structure and release the contents. Then, a small amount of food-grade pectinase is added and kept at a constant temperature to decompose the pectin and make the components easier to dissolve. No sugars are added throughout the process, and the energy is provided by the natural sugars of jujubes and the enzymatic degradation products.

[0038] Construction of a complex microbial community and discontinuous oxygen-supplying solid-state fermentation: After enzymatic hydrolysis, solid residue is separated from liquid and cooled to a suitable temperature. A mixed bacterial solution containing *Candida utilis* and *Lactobacillus plantarum* (in a balanced ratio) is then inoculated at a specific density, mixed thoroughly, and piled into a heap. Fermentation is first carried out in a closed, anaerobic environment for a period of time to maintain a stable temperature at the center of the heap. Subsequently, short-term forced convection oxygen supply is provided daily, followed by a cycle of sealing and restoring oxygen deficiency. This cycle is repeated until the fermentation period is complete. At the final stage, the pH of the heap is controlled to a suitable range, utilizing the synergistic transformation of components and optimization of flavor by yeast and lactic acid bacteria.

[0039] Maturation and finished product: After fermentation, the material is crushed, transferred to a clean container and sealed. It is then placed in a cool, dark environment for a sufficient period of time to mature and age, eventually yielding a paste-like or lumpy finished product with a unique yeast aroma and flavor.

[0040] Example 2: Precise Implementation of Optimized Parameters This embodiment, based on "a method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood", adopts more specific parameters and refined operations to enhance process stability.

[0041] Raw material pretreatment: After rinsing the stems and leaves of Ligusticum striatum, spread them out to dry to the specified moisture content range and cut them into segments of a specific length; after washing the stems and leaves of carrots, inactivate the enzymes at a constant temperature within a specific range, cool them, and cut them into fragments of the corresponding length; after removing the pits from dried red dates, crush them into irregular particles of a specific particle size by physical crushing to ensure that the shape of the raw materials is suitable for subsequent processing.

[0042] Gradient cell wall disruption and endogenous enzyme activation: Ligusticum chuanxiong stem and leaf segments and carrot stem and leaf fragments are mixed and spread evenly in a precise ratio. During vacuum freeze drying, a specified pre-freezing temperature and time are used to fully freeze the raw materials. Then, under a defined vacuum degree, the temperature is raised to a specific temperature and maintained for a corresponding time to obtain freeze-dried composite stem and leaf powder with extremely low moisture content and loose structure, maximizing the retention of soluble and insoluble active ingredients in the raw materials.

[0043] Stepwise compound enzymatic hydrolysis: Freeze-dried compound stem and leaf powder and jujube granules are mixed in a specific mass ratio, and an appropriate amount of purified water is added to stir into a slurry and adjusted to the enzymatic hydrolysis temperature. First, cellulase in a specific proportion of the freeze-dried compound stem and leaf powder is added and treated at a constant temperature for a corresponding time to decompose the fiber network. Then, pectinase in a specific proportion is added and treated at a constant temperature for a corresponding time to break down pectin adhesion. No additional sugars are added throughout the process, and the carbon source comes entirely from the natural sugars of jujubes and enzymatic degradation products.

[0044] Construction of a complex microbial community and discontinuous oxygen-supplying solid-state fermentation: After enzymatic hydrolysis, solid-liquid separation is performed to collect the residue. Once cooled to a suitable temperature, a specific order of magnitude of mixed bacterial solution (with a required ratio of viable *Candida utilis* to *Lactobacillus plantarum*) is inoculated per gram of residue. The mixture is then stirred and piled into a designated shape (e.g., a cone shape with an aeration pipe). Fermentation is first carried out in a closed, anaerobic environment for a fixed duration to maintain a stable temperature at the center of the pile. Subsequently, forced convection oxygenation is provided for a fixed duration at fixed times each day, followed by a cycle of sealing and restoring oxygen deficiency. This cycle is repeated until the fermentation period reaches its target. At the final stage, the pH of the pile is controlled to a suitable range, leveraging the synergistic effect of the two microorganisms to optimize the composition and flavor.

[0045] Maturation and finished product: After fermentation, the material is crushed and transferred to a ceramic or stainless steel container and sealed. It is then placed in a cool, dark environment for a specified number of days to mature and age, resulting in a paste-like or lumpy finished product with suitable moisture content and pH value, possessing a unique fermented aroma and flavor that distinguishes it from existing products.

[0046] Example 3: Highlighting the characteristic of no additional sugars This embodiment focuses on the core feature of "a method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood"—no additional sugars are added throughout the process, highlighting the utilization logic of natural carbon sources.

[0047] Raw material pretreatment: Ligusticum striatum stems and leaves are rinsed and spread out to dry to the specified moisture content, and then cut into short sections; carrot stems and leaves are washed and then subjected to specific temperature to inactivate and inactivate enzymes, cooled and cut into small pieces; dried red dates are pitted and then physically crushed into irregular particles, with the natural sugars of the red dates serving as a key carbon source for subsequent fermentation.

[0048] Gradient cell wall disruption and endogenous enzyme activation: Two types of stems and leaves are mixed in proportion and spread out. After a pre-freezing and heating drying process using vacuum freeze-drying, low-moisture freeze-dried composite stem and leaf powder is obtained, which not only retains the trace fermentable components of the raw materials, but also creates a loose matrix for enzymatic hydrolysis.

[0049] Stepwise compound enzymatic hydrolysis: freeze-dried compound stem and leaf powder is mixed with jujube granules in a certain proportion, and an appropriate amount of purified water is added to form a slurry and the temperature is adjusted. First, a small amount of cellulase is added to decompose the fiber and release some of the soluble substances outside the jujube granules; then a small amount of pectinase is added to decompose the pectin and further release the natural sugars in the jujubes and the degradation products of the raw materials themselves. These natural carbon sources fully support the subsequent microbial fermentation and do not require any exogenous sugars.

[0050] Construction of a composite microbial community and discontinuous oxygen-supplying solid-state fermentation: After enzymatic hydrolysis, the solid residue is collected and cooled to a suitable temperature. A mixed bacterial solution containing *Candida utilis* and *Lactobacillus plantarum* (in a balanced ratio) is then inoculated, mixed thoroughly, and piled into a heap. Initially, the heap is sealed and fermented in an anaerobic environment for a period of time, controlling the temperature at the center. Subsequently, forced convection oxygenation is provided for short periods each day, followed by sealing, and this cycle is repeated until the fermentation cycle is complete. The two microorganisms utilize natural carbon sources to convert components, while simultaneously producing organic acids and other substances to regulate the pH of the heap to a suitable range, imparting a natural fermented aroma to the finished product.

[0051] Maturation and finished product: After fermentation, the material is crushed, sealed in a clean container, and matured in a cool, dark place for a sufficient period of time to obtain a paste or lumpy finished product without additional sugars, with a pH value that meets the standard, and the flavor that comes entirely from the natural synergy between the raw materials and the process.

[0052] Example 4: Non-continuous oxygen supply fermentation practice This embodiment highlights the unique design of "non-continuous oxygen supply solid-state fermentation" in "a method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood", showcasing the advantages of segmented oxygen control.

[0053] Raw material pretreatment: Ligusticum striatum stems and leaves are rinsed and spread out to the specified moisture content, then cut into short sections; carrot stems and leaves are washed, then inactivated and deactivated at a specific temperature, cooled and cut into small pieces; dried red dates are pitted and physically crushed into irregular particles to provide the material basis for fermentation.

[0054] Gradient cell disruption and endogenous enzyme activation: Two types of stems and leaves are mixed in proportion and laid flat. They are then pre-frozen by vacuum freeze-drying and dried at elevated temperature to obtain low-moisture freeze-dried composite stem and leaf powder, which increases the contact area between the raw materials and the microorganisms.

[0055] Stepwise compound enzymatic hydrolysis: freeze-dried compound stem and leaf powder is mixed with jujube granules in a certain proportion, an appropriate amount of purified water is added to form a slurry and the temperature is adjusted, and a small amount of cellulase and pectinase are added in sequence for constant temperature treatment to decompose fiber and pectin, release the natural sugars of jujube and raw material degradation products, without the addition of additional sugars.

[0056] Construction of a complex microbial community and discontinuous oxygen-supplying solid-state fermentation: After enzymatic hydrolysis, the solid residue is collected and cooled to a suitable temperature. A mixed bacterial solution containing *Candida utilis* and *Lactobacillus plantarum* (in the required ratio) is then inoculated, mixed thoroughly, and piled into a cone-shaped heap with an air duct. Fermentation is first carried out in a closed, anaerobic environment for a fixed duration, allowing anaerobic and facultative bacteria to preferentially initiate metabolism and maintaining a stable temperature at the center of the heap. Subsequently, short-term forced convection oxygen supply is performed at fixed times each day to introduce oxygen and activate aerobic bacteria metabolism. After oxygen supply, the heap is sealed again to restore oxygen deficiency. This cycle is repeated until the fermentation cycle is complete. By controlling oxygen in stages to balance the activity of different microbial communities, the pH of the heap is controlled to a suitable range at the final stage, resulting in more complete component transformation and a richer flavor.

[0057] Maturation and finished product: After fermentation, the material is crushed, sealed in a clean container, and matured in a cool, dark place for a sufficient period of time to obtain a paste-like or lumpy finished product with a unique fermented aroma and flavor and a pH value that meets the requirements.

[0058] Example 5: Complete Maturation and Aging Presentation This embodiment emphasizes the role of the "maturation and finished product" stage in the "preparation method of a traditional Chinese medicine compound enzyme for regulating qi and blood" in shaping the final quality, highlighting the significance of long-term aging.

[0059] Raw material pretreatment: Ligusticum striatum stems and leaves are rinsed and spread out to dry to the specified moisture content, and then cut into short sections; carrot stems and leaves are washed, then inactivated and deactivated at a specific temperature, cooled and cut into small pieces; dried red dates are pitted and then physically crushed into irregular particles, laying the foundation for raw materials.

[0060] Gradient cell wall disruption and endogenous enzyme activation: Two types of stems and leaves are mixed and spread out in proportion, and then pre-frozen and dried at elevated temperature under vacuum freeze-drying to obtain low-moisture freeze-dried composite stem and leaf powder, which retains the activity and structural characteristics of the raw materials.

[0061] Stepwise compound enzymatic hydrolysis: freeze-dried compound stem and leaf powder is mixed with jujube granules in a certain proportion, an appropriate amount of purified water is added to form a slurry and the temperature is adjusted, and a small amount of cellulase and pectinase are added in sequence for constant temperature treatment to decompose fiber and pectin, release the natural sugars of jujube and raw material degradation products, without the participation of additional sugars in fermentation.

[0062] Construction of a composite microbial community and discontinuous oxygen-supplying solid-state fermentation: After enzymatic hydrolysis, the solid residue is collected and cooled to a suitable temperature. A mixed bacterial solution containing *Candida utilis* and *Lactobacillus plantarum* (in a balanced ratio) is then inoculated, mixed thoroughly, and piled into a heap. Initially, the heap is sealed and subjected to anaerobic fermentation for a period of time, controlling the temperature at the center. Subsequently, forced convection oxygenation is provided for short periods each day, followed by sealing, and this cycle is repeated until the fermentation cycle is complete. At the end, the pH of the heap is controlled to a suitable range, thus initially completing the microbial transformation of the components.

[0063] Maturation and Finished Product: After fermentation, the crushed material is transferred to a ceramic or stainless steel container and sealed. It is then placed in a cool, dark environment for a sufficient number of days to mature and age. During this process, residual enzymes act slowly, and microbial metabolites further blend, resulting in a more uniform texture, a change in the aroma from sharp to mellow, and a richer flavor profile. The final product is a paste or lumpy product with suitable moisture content and pH level. Its unique quality depends entirely on the sedimentation of the complete maturation process.

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood, characterized in that, Includes the following steps: S1. Raw material pretreatment: Collect the stems and leaves of Ligusticum chuanxiong during its vigorous growth period, rinse them with purified water, and spread them out to dry until their moisture content is 60%-70%, then cut them into segments of 0.5-1.5 cm in length; select fresh carrot stems and leaves, wash them with purified water, and inactivate the inactivating enzymes at a constant temperature of 50℃-60℃ for 10-15 minutes, then cool them and cut them into 1-2 cm fragments; select dried red dates, remove the pits, and crush them into irregular particles with a particle size of 3-5 mm by physical crushing. S2. Gradient cell wall disruption and endogenous enzyme activation: The chuanxiong stem and leaf segments and the carrot stem and leaf fragments are mixed at a mass ratio of 1:(0.8-1.2), spread out and subjected to vacuum freeze drying. The pre-freezing temperature is controlled at -40℃ to -50℃ and the pre-freezing time is 4-6 hours. Then, under a vacuum of 20-30 Pa, the temperature is raised to 25℃-30℃ and maintained for drying for 12-16 hours to obtain freeze-dried composite stem and leaf powder with a moisture content of less than 8%. S3. Stepwise compound enzymatic hydrolysis: Mix the freeze-dried compound stem and leaf powder with the jujube granules at a mass ratio of (2-3):1, add 1.5-2 times the mass of the total materials of purified water, stir evenly to form a slurry and adjust the temperature to 45℃, first add 0.05%-0.1% of the mass of the freeze-dried compound stem and leaf powder of food-grade cellulase, and carry out the first stage of enzymatic hydrolysis at a constant temperature of 45℃ for 90-120 minutes, then add 0.03%-0.06% of the mass of the freeze-dried compound stem and leaf powder of food-grade pectinase, and carry out the second stage of enzymatic hydrolysis at a constant temperature of 45℃ for 60-90 minutes; S4. Construction of composite microbial community and discontinuous oxygen supply solid-state fermentation: The enzymatic hydrolysis mixture from step S3 is subjected to solid-liquid separation, and all solid residue is collected; when the residue temperature naturally cools to 32℃-35℃, the activated seed liquid of one strain of Candida utilis and one strain of Lactobacillus plantarum is evenly mixed into the residue at a ratio of 10^6-10^7 CFU of mixed bacterial solution per gram of residue. The ratio of viable cells of Candida utilis to Lactobacillus plantarum in the mixed bacterial solution is 1:(1.5-2.5). After mixing, the material is piled into a pile and fermented in a closed anaerobic state for 48 hours. During this period, the temperature at the center of the pile is controlled at 30℃-35℃. After that, forced convection oxygen supply is carried out once a day for 2 hours. After the oxygen supply ends, the pile is sealed again to restore the anaerobic environment. This process is repeated until the entire fermentation cycle reaches 15-20 days. The pH value of the pile is controlled at 3.8-4.2 at the end of the fermentation. S5. Maturation and finished product: After fermentation, the pile of material is crushed, transferred to a clean container and sealed, and aged in a cool, dark environment for 30-45 days to obtain the traditional Chinese medicine compound enzyme for regulating qi and blood.

2. The method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood according to claim 1, characterized in that: In step S2, the pre-freezing temperature of the vacuum freeze-drying is -45°C, the pre-freezing time is 5 hours, the vacuum degree of the drying stage is 25Pa, and the temperature is raised to 28°C and maintained for 14 hours.

3. The method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood according to claim 1, characterized in that: In step S3, during the preparation of the slurry, the ratio of the total mass of the material to the mass of the added purified water is 1:1.5 to 1:1.8; the enzymatic hydrolysis time in the first stage is 100 minutes, and the enzymatic hydrolysis time in the second stage is 75 minutes; the amount of cellulase added is 0.08% of the mass of the freeze-dried composite stem and leaf powder, and the amount of pectinase added is 0.04% of the mass of the freeze-dried composite stem and leaf powder.

4. The method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood according to claim 1, characterized in that: In step S4, the preparation of the activated seed liquid involves inoculating the corresponding bacterial strains onto slant culture medium, activating them at 28℃-30℃ for 24 hours, then transferring them to liquid seed culture medium and culturing them at 28℃-30℃ with shaking for 18-24 hours until the bacterial concentration reaches 10^8 CFU / mL or higher.

5. The method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood according to claim 4, characterized in that: In step S4, the total inoculation amount of the mixed bacterial solution is 5 × 10^6 CFU per gram of residue; the ratio of viable cells of the Candida utilis to the Lactobacillus plantarum is 1:

2.

6. The method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood according to claim 1, characterized in that: In step S4, the pile is a cone-shaped pile with a height of about 30-40 cm and a width of about 50-60 cm, and a gas guide pipe with an inner diameter of 1-2 cm is inserted in the center of the pile to achieve communication with the outside atmosphere during the forced convection oxygen supply stage.

7. The method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood according to claim 1, characterized in that: In step S4, the forced convection oxygen supply operation is set to run from 9:00 AM to 11:00 AM daily, and the ambient temperature during the oxygen supply period is controlled between 25°C and 28°C.

8. The method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood according to claim 1, characterized in that: In step S5, the container used for aging is made of ceramic or stainless steel, and the aging time is 40 days.

9. The method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood according to claim 1, characterized in that: The preparation method does not add any form of sugar as a fermentation substrate or flavoring agent throughout the entire process. The carbon source required for fermentation comes from the natural sugars contained in the jujube granules and the degradation products after enzymatic hydrolysis of the raw materials.

10. A method for preparing a traditional Chinese medicine compound enzyme for regulating qi and blood according to any one of claims 1 to 9, characterized in that: The finished product is a paste or lumpy form with a water content of approximately 30%-40%, a pH value of 3.8-4.2, and a unique fermented aroma and flavor produced by the specific raw materials and processes, which distinguishes it from existing enzyme products.