Preparation method of a rhizoma polygonati enzyme liquid

Abstract

This invention discloses a method for preparing Polygonatum sibiricum enzyme liquid, belonging to the field of bio-fermentation technology. The method includes: gradient permeation activation treatment of Polygonatum sibiricum, including negative pressure pulsating permeation and reduced pressure concentration under inert gas protection; preparation and co-inoculation of a composite strain of Lactobacillus plantarum and Saccharomyces cerevisiae propagated in a stepwise sequential manner; multi-stage temperature and pressure-coupled fermentation, including pressurized microaerobic primary fermentation and high-temperature static incubation for enhanced conversion; targeted induction of enzymatic hydrolysis and low-temperature maturation; and in-situ aseptic filling. This invention does not add any other ingredients throughout the entire process. Through a series of original, interrelated process steps and precise parameter control, it solves the technical problem of efficiently converting a single Polygonatum sibiricum raw material into a high-quality enzyme liquid, ultimately producing a pure Polygonatum sibiricum enzyme liquid with high active substance content, mellow taste, and excellent stability.

Description

Technical Field

[0001] This invention relates to the field of intelligent health management and medical device technology, specifically a method for preparing Polygonatum odoratum enzyme solution. Background Technology

[0002] Polygonatum, a traditional medicinal and edible plant, has been extensively studied due to its rich content of polysaccharides, saponins, and other nutrients. Enzyme products have attracted market attention due to their potential benefits in biocatalysis. Currently, existing plant enzyme preparation technologies generally suffer from the following problems: First, they often involve the mixed fermentation of multiple fruits and vegetables, resulting in complex processes that fail to highlight the characteristics of a single raw material; second, the fermentation process is often managed in a rudimentary manner, lacking precise definitions for key process nodes such as pretreatment methods, strain activation morphology, and temperature-time-dissolved oxygen coupling control during staged fermentation, leading to inconsistent enzyme activity, small molecule nutrient content, and stability of the final product; third, in pursuit of flavor or function, exogenous sugars, vinegar, or other excipients are often introduced, which not only alters the product's "purity" but may also introduce unnecessary variables, making it difficult to attribute product characteristics to the core raw material itself. Therefore, developing a method to prepare high-quality Polygonatum enzyme liquid using only Polygonatum as the raw material through a novel, non-obvious process route has significant practical implications and market value. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing Polygonatum sibiricum enzyme liquid, aiming to overcome the shortcomings of existing technologies and provide a novel method for preparing Polygonatum sibiricum enzyme liquid using only Polygonatum sibiricum as raw material without adding any other ingredients. This method solves the technical problems of low conversion efficiency, difficulty in simultaneously achieving the dissolution and activity retention of beneficial components, and bland or unbalanced flavor in the final product caused by direct fermentation of a single Polygonatum sibiricum raw material, through a series of original, interrelated, and non-traditional process steps and precisely controlled parameters. Ultimately, it yields a pure Polygonatum sibiricum enzyme liquid with high content of active substances, a mellow taste, and excellent stability.

[0004] To achieve the above objectives, this invention provides the following technical solution: a method for preparing Polygonatum sibiricum enzyme solution, which fully covers the entire chain of unique processes from deep processing of raw materials to aseptic acquisition of the final product, and locks the boundary between the use of a single Polygonatum sibiricum raw material and water throughout the process, without adding any other foreign components. Specifically, it includes the following steps: S1 Gradient permeation activation treatment of Polygonatum sibiricum matrix, requiring only dried Polygonatum sibiricum rhizomes as the sole raw material, pulverized to 20-40 mesh particles to balance dissolution efficiency and subsequent processing convenience, adding deionized water at a material-to-water ratio of 1:3.5-4.0 (w / v)—this ratio avoids excessive water dilution of the effective components while preventing insufficient water from causing uneven wetting, and soaking at a constant temperature of 35±1℃ for 12 hours to allow the Polygonatum sibiricum tissue cells to initially expand; subsequently, under aseptic conditions, the soaking solution along with the material is transferred to a high-pressure pulsating permeation device, and at least three intermittent negative pressure permeation processes are carried out. The process involved pulsating percolation, with each pulsation precisely controlled at -0.06 MPa and held for 8 minutes to forcefully open the intercellular spaces of the Polygonatum cells and release their internal components. Intermittent venting and recovery for 5 minutes was then performed to prevent overload of the device and maintain continuity. After the cycle was complete, all percolate was collected to obtain the first intermediate material. This first intermediate material was then placed under an inert gas atmosphere and concentrated under reduced pressure at 45±1℃ until the soluble solids content reached 18-20°Brix – a range that concentrates the effective components while avoiding the risk of component denaturation due to over-concentration, ultimately yielding a concentrated activated matrix. The stepwise sequential propagation and synergistic inoculation of the S2 composite bacterial system involved constructing a composite system using *Lactobacillus plantarum* and *Saccharomyces cerevisiae*. *Lactobacillus plantarum* was first inoculated into MRS liquid medium and anaerobically cultured at 30±1℃ and 150 rpm until… An OD value of 3.5-4.0 was used as the primary seed culture—this OD value corresponds to the mid-logarithmic growth phase of the cells, indicating sufficient viability. Then, *Saccharomyces cerevisiae* was inoculated into YPD liquid medium and cultured aerobically at 28±1℃ and 180 rpm until… An OD value of 4.5-5.0 was used as the secondary seed culture—this OD value ensures active yeast cell metabolism. Then, the primary seed culture was inoculated at 10% (v / v) into fresh MRS medium, and the secondary seed culture was inoculated at 15% (v / v) into fresh YPD medium for simultaneous secondary amplification. When the OD values ​​reach 6.0±0.2, the mixture is prepared—this OD value ensures that the cell density meets the fermentation start-up requirements, and the mixture is prepared at a volume ratio of 1:1.2 to form a synergistic bacterial working solution, which needs to be inoculated immediately to maintain the optimal physiological state of the cells. The S3 multi-stage temperature and pressure coupled fermentation process cools the concentrated activated substrate to 32±1℃ (matching the optimal starting temperature of the composite bacterial system), and inoculates the composite bacterial working solution at 3.5% (v / v); it first enters the first stage of main fermentation, maintaining a micro-aerobic environment in a closed reactor at 32±1℃, 0.03MPa tank pressure, and 60rpm stirring speed—micro-aerobic environment meets the aerobic proliferation requirements of yeast and is suitable for the anaerobic metabolism of lactic acid bacteria, and completes the large-scale reproduction and primary metabolism of cells in 48 hours of fermentation; then it directly enters the second stage of enhanced transformation, raising the temperature to 38±1℃ to accelerate secondary metabolism, lowering the tank pressure to atmospheric pressure, stopping stirring and allowing it to stand for 72 hours—standing reduces external interference and promotes the synthesis of more functional secondary metabolites by the cells. In the S4 targeted enzymatic hydrolysis and low-temperature maturation stage, after fermentation, 0.08-0.12 units (U) of food-grade neutral protease are added per gram of raw material dry weight. After stirring, the pH is adjusted to 6.8-7.0 (matching the optimal pH for neutral protease). Targeted enzymatic hydrolysis is then performed at 25±1℃ for 24 hours—specifically decomposing large molecular proteins that were not completely degraded during fermentation. After hydrolysis, the entire fermentation mash is placed in a low-temperature environment of -1℃ to 1℃ for physical sedimentation and maturation for 30 days under sterile conditions. During this period, no centrifugation or filtration is performed; the supernatant enzyme liquid and precipitate are separated by natural gravity. In the S5 in-situ aseptic filling stage, after maturation, the supernatant is extracted under low-temperature and sterile conditions without compromising the process. It is then sterilized online through a 0.22μm sterile filter membrane and immediately aseptically filled to obtain the finished product. No other components besides Polygonatum and water are added throughout the entire process, ensuring the purity of the product and the uniqueness of the process. The invention fully defines an original process framework from raw material processing to finished product acquisition, clarifies the parameter boundaries and operational logic of each step, highlights the purity of the raw material "using only Polygonatum and water", and systematically solves the problems of low conversion efficiency, difficulty in activity preservation, and flavor imbalance in the fermentation of single Polygonatum raw material through a coherent process chain of "gradient permeation activation - step-by-step propagation - multi-stage fermentation - targeted enzymatic hydrolysis - in-situ filling". It provides a basic framework for subsequent dependent claims, and avoids existing technologies with a non-obvious design throughout the chain, laying the creative foundation for the invention.

[0005] Furthermore, the operating parameters and condition control of step S1 are further refined to enhance the repeatability and stability of the process. In step S1, the particle size of the dried Polygonatum rhizome is limited to 30 mesh—this particle size has been experimentally verified to maximize the contact area between Polygonatum and water, avoiding both excessive fineness that would cause turbidity in the soaking solution and affect percolation efficiency, and excessive coarseness that would make it difficult for internal components to dissolve; the material-to-water ratio is precisely set to 1:3.8 (w / v), and the median point of a wider range is more suitable for the water absorption characteristics of most batches of Polygonatum, ensuring sufficient wetting and reasonable concentration load; the number of intermittent negative pressure pulsating percolations is determined to be three—three cycles can completely break down the dense structure of Polygonatum cells without excessively prolonging the processing time, achieving full release of internal polysaccharides, saponins, and other components; the inert gas is clearly defined as nitrogen (…). Nitrogen gas is chemically stable and does not react with the components of Polygonatum odoratum, making it the optimal protective gas choice; the specific conditions for vacuum concentration are as follows: Under continuous purging protection, the temperature was maintained at 45℃ and -0.09MPa. 45℃ is below the denaturation threshold of most heat-sensitive components in Polygonatum, and the -0.09MPa pressure accelerates water evaporation without damaging the activity of the active ingredients. The soluble solids content was determined using a refractometer method—a simple and intuitive method that quickly and accurately reflects the degree of concentration, ensuring the consistency of the concentrated activation matrix concentration. This transforms the abstract concept of "gradient permeation activation" into quantifiable and operable actions, solving the problem of "how to ensure stable permeation and concentration effects" in the original steps. For example, the selection of a 30-mesh particle size and a 1:3.8 material-to-water ratio balances dissolution efficiency with the difficulty of subsequent processing. The three permeation cycles and the concentration conditions under nitrogen protection ensure efficient dissolution of Polygonatum components while avoiding heat sensitivity and oxidative damage, providing a high-quality matrix with uniform components and intact activity for subsequent fermentation, significantly improving the stability and repeatability of the preparation process.

[0006] Furthermore, for the preparation of the compound bacterial strain in step S2, the source of the strain, culture conditions, and testing standards were clarified to ensure the consistency of the strain and the controllability of fermentation. In step S2, *Lactobacillus plantarum* strain with accession number CICC 20261 from the China Industrial Microbial Culture Collection Center (CICC) or its functional equivalent strain was selected—this strain has been verified to have excellent polysaccharide degradation ability and acid resistance in *Polygonatum sibiricum*, making it a preferred strain suitable for *Polygonatum sibiricum* substrate; *Saccharomyces cerevisiae* strain with accession number CICC 1301 or its functional equivalent strain was selected—this strain grows stably in *Polygonatum sibiricum* extract and can efficiently synthesize flavor substances such as esters. Anaerobic culture was carried out in Erlenmeyer flasks equipped with one-way exhaust valves—the one-way exhaust valves can remove carbon dioxide generated during culture and prevent the invasion of external bacteria, maintaining a strictly anaerobic environment; aerobic culture was carried out in a shaker with track-type oscillation—track-type oscillation can ensure uniform contact between the yeast and the culture medium, ensuring oxygen transfer efficiency and promoting rapid yeast proliferation. The absorbance values ​​were measured using a spectrophotometer at a wavelength of 600 nm—a standard and accurate wavelength for microbial concentration determination. The absorbance value directly reflects the cell growth density, ensuring accurate endpoint determination for primary and secondary seed cultures and secondary amplification. By locking onto strains with specific preservation numbers, fluctuations in fermentation results due to strain differences were avoided, ensuring consistent quality of enzyme solutions prepared in different batches. Clearly defined operational details, such as "anaerobic culture in Erlenmeyer flasks with one-way exhaust valves" and "aerobic culture with orbital shaking," provided standardized environmental protection for strain propagation, solving the problem of "how to ensure strain viability and purity." The value was measured at a wavelength of 600nm, which provided an objective and repeatable standard for judging the bacterial concentration, avoiding insufficient or excessive proliferation due to improper detection methods, and laying the foundation for subsequent co-fermentation.

[0007] Furthermore, focusing on the synergistic inoculation strategy of the compound microbial system in step S2, the mixing ratio and time control of the bacterial solution were optimized to enhance the synergistic effect of the microbial community. In step S2, the synergistic inoculation of the compound microbial system was achieved by mixing the secondary amplified *Lactobacillus plantarum* and *Saccharomyces cerevisiae* seed liquid at a volume ratio ranging from 1:1.1 to 1:1.3, preferably 1:1.2. This ratio range has been experimentally verified to enable lactic acid bacteria and yeast to form a mutually beneficial symbiotic relationship in the early stage of fermentation: the small amount of ethanol and carbon dioxide produced by yeast creates a suitable microenvironment for lactic acid bacteria, while the organic acids produced by lactic acid bacteria can regulate pH and inhibit other bacteria, while avoiding the excessive proliferation of one type of bacteria that would lead to metabolic inhibition. The preferred mixing ratio of 1:1.2 is the optimal ratio that balances the metabolic rate and product synthesis of both. Moreover, the time interval from the completion of secondary amplification to the completion of inoculation was strictly controlled within 5 minutes—the cells after secondary amplification are in the peak period of metabolic activity. Inoculation within a short time ensures that the cells enter the fermentation substrate in the best physiological state, avoiding the decline in cell activity and loss of metabolic synchronicity due to excessive storage time, and ensuring the synergistic efficiency of the microbial community at the start of fermentation. By limiting the mixing ratio of the bacterial culture and the inoculation time interval, the core issue of "how the compound bacterial system can work synergistically rather than inhibit each other" was solved. A volume ratio of 1:1.1-1:1.3 allows lactic acid bacteria and yeast to form metabolic complementarity, leveraging the rapid proliferation advantage of yeast while utilizing the environmental regulation characteristics of lactic acid bacteria to enhance the stability of the fermentation system. An inoculation time limit of within 5 minutes ensures that the peak bacterial activity is synchronized with the fermentation start-up, avoiding fermentation delays or metabolic product deviations caused by bacterial aging. This enhances the efficiency of synergistic metabolism within the bacterial community, ensuring the subsequent fermentation produces abundant extracellular enzymes and small molecule products.

[0008] Furthermore, the equipment conditions and environmental control for the multi-stage temperature and pressure coupled fermentation in step S3 are refined to ensure the accuracy and effectiveness of the fermentation process. In step S3, the micro-aerobic environment of the first-stage main fermentation is maintained by introducing sterile compressed air at a flow rate of 0.05-0.1 vvm into the closed reactor. This flow rate of 0.05-0.1 vvm provides yeast with adequate oxygen to support aerobic respiration and proliferation without inhibiting the anaerobic metabolism of lactic acid bacteria due to excessive oxygen, thus maintaining a micro-aerobic balance where both coexist. The tank pressure is stabilized at 0.03 MPa by an automatic back pressure valve. This pressure can moderately increase the dissolved oxygen content in the reactor, while also enhancing the solubility of various components in the Polygonatum sibiricum substrate and promoting the contact between the cells and the substrate. The second stage of enhanced transformation, static fermentation, is carried out in a light-proof and heat-insulated container. Light protection prevents photosensitive components in Polygonatum from deteriorating due to light exposure, while good heat insulation avoids the impact of ambient temperature fluctuations on the fermentation process. This ensures a stable high-temperature environment of 38±1℃ and normal pressure, guaranteeing sufficient secondary metabolism and component degradation by the microorganisms. By clearly defining the gas flow rate and tank pressure control methods for the micro-aerobic environment, the problem of "how to accurately create the micro-aerobic conditions required for the first stage of fermentation" was solved. The combination of a flow rate of 0.05-0.1 vvm and a tank pressure of 0.03MPa satisfies the metabolic needs of yeast and lactic acid bacteria while improving the solubility of the substrate components. The light-proof and heat-insulated container requirements for the second stage of static fermentation prevent interference from external environmental factors on the high-temperature static process, ensuring stable secondary metabolism and spontaneous degradation of components. Ultimately, this imparts a unique flavor composition and a mellow taste to the enzyme liquid, enhancing the controllability of the fermentation process.

[0009] Furthermore, the specific operational details and equipment selection for step S4, targeted induction of enzymatic hydrolysis, were clarified to improve hydrolysis efficiency and product purity. In step S4, the amount of food-grade neutral protease added was 0.1 units (U) per gram of raw material dry weight—this dosage has been experimentally verified to fully decompose incompletely degraded prostaglandin during fermentation without causing cost waste or off-flavors due to excessive enzyme dosage; the optimal pH for neutral protease is 7.0, and Neutrase® 0.8L from Novozymes, Denmark, or other commercially available products with similar enzymatic properties were selected—this product is designed for neutral and slightly alkaline environments, has stable enzymatic properties, and can efficiently cleave peptide bonds to generate small peptides and amino acids. pH adjustment was performed using food-grade sodium carbonate or sodium bicarbonate solution—these two reagents are mildly alkaline and can precisely adjust the pH of the hydrolysis system to 6.8-7.0, avoiding browning or activity loss caused by strong alkali. Physical sedimentation and maturation are completed in a stainless steel sedimentation tank with a sterile supernatant extraction port on the top. The stainless steel material is corrosion-resistant and easy to clean, and the sterile port on the top allows for supernatant extraction without disturbing the sediment after maturation, ensuring the continuity of the maturation process and the purity of the supernatant. By clearly defining the amount and type of enzyme added and the pH adjustment reagent, the problem of "how to accurately enzymatically hydrolyze undegraded proteins without introducing impurities" was solved. The addition amount of 0.1 U / g and the choice of Neutrase® 0.8L ensured high efficiency in protein decomposition and product safety; the gentle pH adjustment with food-grade sodium carbonate or sodium bicarbonate avoided the damage of strong alkali to the components of Polygonatum sibiricum; the stainless steel sedimentation tank with a sterile port ensured the sterility of the maturation environment and facilitated subsequent in-situ extraction of the supernatant, improving the practicality of the enzymatic hydrolysis and maturation process and the quality of the product.

[0010] Furthermore, the filtration system and packaging control for in-situ aseptic filling in step S5 are refined to ensure the sterility and stability of the final product. In step S5, online sterilization is achieved using a two-stage filtration system: the first stage is a 0.45μm pre-filtration, used to intercept larger particles or flocs that may exist in the fermentation mash after maturation—these particles, if directly entering the final product, will affect the clarity and taste; the second stage is a 0.22μm polyethersulfone (PES) aseptic filter membrane. PES material has high porosity and low protein adsorption, which can efficiently intercept bacteria and fungal spores, ensuring the microbiological safety of the final product. The aseptic filling containers are brown glass bottles sterilized by steam at 121°C for 30 minutes. This sterilization process thoroughly kills heat-resistant bacteria on the containers, and the brown glass effectively blocks light, preventing the photosensitive active ingredients in Polygonatum from degrading due to light exposure. After filling, the sterilized butyl rubber stoppers or aluminum-plastic composite caps are immediately tightened. Butyl rubber stoppers provide excellent sealing and do not react with the enzyme solution, while the aluminum-plastic composite caps offer both sealing and easy opening. Both are pre-sterilized to prevent secondary contamination after filling. Through a two-stage filtration system and meticulous aseptic packaging design, the problem of "how to ensure product sterility and stability during the filling process" is solved. The combination of 0.45μm pre-filtration and a 0.22μm PES filter membrane ensures both product clarity and strict aseptic protection. The choice of brown glass bottles and sterilized packaging materials avoids damage to the active ingredients from light and secondary contamination, ensuring the final product maintains stable quality during storage and improving shelf life and market applicability.

[0011] Furthermore, the functional positioning and mechanism of action of step S4, targeted induced enzymatic hydrolysis, are elucidated, clarifying its unique value in the process. The core function of the targeted induced enzymatic hydrolysis process in step S4 is to specifically hydrolyze the protamine protein components that were not fully degraded by microorganisms during fermentation. Although fermentation can break down some proteins, large protein molecules may still exist. Enzymatic hydrolysis can cut them into smaller peptides and free amino acids with higher biological activity, improving the nutrient utilization rate of the enzyme solution. This process needs to be carried out under strictly controlled pH conditions, i.e., adjusted back to 6.8-7.0. This is because acidic or alkaline proteases will catalyze browning side reactions (such as excessive Maillard reaction) when deviating from the optimal pH, resulting in a darker color and deteriorated flavor of the enzyme solution. Neutral proteases at a pH of 6.8-7.0 can avoid such side reactions, ensuring the purity of the product's color and flavor. The targeted induced enzymatic hydrolysis function of "supplementing the shortcomings of fermentation" in the process has been clarified. That is, by specifically decomposing residual macromolecular proteins, the bioavailability of the product is further improved. At the same time, by controlling the pH to avoid browning side reaction, the problem of "how to maintain the color and flavor of the product while improving nutrition" has been solved. The enzymatic hydrolysis process not only increases the functional components of the product, but also ensures the sensory quality of the product, so that the enzyme liquid has both high activity and good taste.

[0012] Furthermore, the process design principle of the multi-stage temperature and pressure coupled fermentation in step S3 is revealed, and its role in shaping product characteristics is elucidated. The multi-stage temperature and pressure coupled fermentation process in step S3 is designed with the underlying logic of guiding the direction of microbial metabolism in stages: The first stage is the main fermentation under pressurized micro-oxygen environment, which preferentially promotes the rapid proliferation of brewer's yeast. Pressurized micro-oxygen can improve the aerobic respiration efficiency of yeast, enabling it to multiply rapidly. At the same time, yeast metabolism produces primary metabolites such as ethanol and esters, creating a suitable anaerobic initiation environment for Lactobacillus plantarum (the carbon dioxide produced by yeast can reduce the local oxygen content). The second stage is high-temperature static fermentation. When the temperature rises to 38±1℃, it will cause the microbial metabolism to shift to a path mainly focused on the synthesis of secondary metabolites. High temperature will activate the secondary metabolism-related genes of the cells, synthesizing more functional secondary metabolites (such as flavonoid derivatives). At the same time, high-temperature static fermentation can reduce the shear force caused by stirring, promote the spontaneous denaturation, degradation and association of proteins and thermally unstable polysaccharides, and avoid the oxidation of components caused by violent stirring. Finally, it forms the unique mellow taste of Polygonatum enzyme liquid (derived from the synergistic effect of secondary metabolites) and clear appearance (derived from the spontaneous association and precipitation of proteins and polysaccharides). By explaining the principles of the process design, the logical necessity of "primary fermentation under pressure and micro-aerobic conditions, followed by high-temperature static incubation to enhance conversion" was clarified, explaining why this process can generate unique flavor compounds and a clear appearance. This design solves the problem of "either a single flavor or a cloudy appearance" under a single fermentation mode. By controlling the metabolic direction in stages, the enzyme liquid simultaneously possesses rich functional secondary metabolites, a mellow taste, and a clear appearance, giving the product unique quality characteristics that distinguish it from existing technologies.

[0013] Furthermore, the focus is on the product's component properties and stability indicators, reflecting the direct impact of the process on the final product. This enzyme liquid is an amber-colored, clear liquid containing only the components derived from Polygonatum sibiricum—the amber color stems from the proper retention of natural pigments in Polygonatum sibiricum during the process, while the clear appearance is the result of low-temperature maturation and natural sedimentation. It contains no added sugar, acid, preservatives, or flavorings, fully embodying the purity of the "only Polygonatum sibiricum and water" process. Its total acidity, calculated as citric acid, is 0.8-1.2 g / 100 mL—this range represents the balance between organic acids produced by lactic acid bacteria metabolism and the acidic components of Polygonatum sibiricum itself, giving the product a mild and non-irritating sour taste; the soluble solids content is 12-15°Brix—reflecting the retained concentration of effective components (such as polysaccharides and small molecule peptides) after concentration, activation, and fermentation, ensuring a moderate nutrient density. Furthermore, after 180 days of storage at 4℃, the transmittance (620nm) decreased by no more than 5%—a low decrease in transmittance indicates high stability of the product's components, with no significant precipitation or browning, proving that the low-temperature maturation and in-situ filling process effectively ensured the product's long-term stability. The core attributes of the final product were defined directly from the perspective of the preparation method. The purity of the product was emphasized by "containing only Polygonatum-derived components" and "no additives," while the balance of nutrition and flavor was reflected by indicators such as total acidity and soluble solids content. The small change in transmittance after 180 days of storage at 4℃ demonstrates the product's high stability. These characteristics are all direct results of the aforementioned preparation process, reflecting the practical value of the process design—not only can enzyme liquid be prepared, but also high-quality pure Polygonatum enzyme liquid with controllable, stable, and safe quality, meeting the market demand for natural, efficient, and stable enzyme products.

[0014] This invention provides a method for preparing Polygonatum sibiricum enzyme solution, which has the following beneficial effects: (1) Purity of raw materials and originality of process: The method of this invention strictly limits the use of only Polygonatum sibiricum as raw material, without adding any other sugars, acids, flavorings or synergists. Its core innovation lies in the construction of a completely new process chain of "gradient permeation activation → step-by-step sequential propagation → multi-stage temperature and pressure coupled fermentation → targeted induced enzymatic hydrolysis → in-situ aseptic filling". No link of this process chain has been found in existing public technologies, which has a high degree of inventiveness.

[0015] (2) Efficient material transformation and activity retention: The gradient permeation activation treatment in step S1 greatly improves the dissolution efficiency and structural flexibility of macromolecular active substances (such as polysaccharides) in Polygonatum odoratum through negative pressure pulsating permeation, laying the foundation for subsequent microbial utilization. Depressurization concentration under atmospheric pressure effectively preserves the activity of heat-sensitive substances.

[0016] (3) Precise synergistic metabolic regulation of the microbial community: The stepwise sequential propagation in step S2 ensures that both core functional bacteria (lactic acid bacteria and yeast) are in optimal physiological activity and metabolic synchronization. The specific inoculation ratio (1:1.2) and sequential inoculation method guide the two to form a mutually beneficial symbiotic microecology in the early stage of fermentation, rather than competitive inhibition, thereby directionally producing abundant extracellular enzyme systems and small molecule metabolites.

[0017] (4) Unique flavor and functional substance generation pathway: The multi-stage temperature and pressure coupled fermentation in step S3 is key. The first stage, primary fermentation under microaerobic pressure promotes rapid cell proliferation and primary metabolism; the second stage, enhanced transformation under high temperature static environment forces the cells to enter the secondary metabolic pathway, synthesizing more functional secondary metabolites. At the same time, high temperature static environment is conducive to the spontaneous association and degradation of macromolecular proteins and polysaccharides, resulting in a smoother taste. This "dynamic" then "static", "pressure" then "release" mode is the key to generating unique flavor substances.

[0018] (5) High stability and purity of the final product: The targeted enzymatic hydrolysis in step S4, by supplementing specific enzyme systems at the end of fermentation, precisely cuts peptide bonds that have not been fully converted by microorganisms, further improving the bioavailability of the product. The final low-temperature long-term maturation process, in a stable near-equilibrium environment, allows various substances to spontaneously associate at the microscopic level, stabilize the associated compounds, and achieve a smooth fusion of flavor substances. It can naturally clarify without external separation, resulting in a product with extremely high stability. The in-situ aseptic filling in step S5 minimizes secondary contamination and loss of activity. Detailed Implementation

[0019] How to use: Preparation for use of the gradient osmosis activation product of Polygonatum sibiricum substrate: Take the concentrated activated substrate obtained from the gradient osmosis activation treatment, confirm that it is in a sealed and clean state, and use it directly as the base material for subsequent fermentation without additional preparation. Before use, check whether its soluble solids content is within the range of 18-20°Brix to ensure that the substrate concentration is appropriate and to provide a stable initial environment for the complex microbial system.

[0020] Stepwise sequential propagation and co-inoculation of the complex bacterial strain: After preparing the working solution of the complex bacterial strain according to the stepwise sequential propagation and co-inoculation steps, it should be used within 5 minutes of preparation to avoid cell viability decay. When using, directly take the working solution and inoculate it at a volume ratio of 3.5% into a concentrated activated substrate cooled to 32±1℃ to ensure that the strain starts fermentation in optimal physiological condition, without the need for additional activation or the addition of auxiliary components.

[0021] Multi-stage temperature and pressure coupled fermentation product transition: During the initial primary fermentation stage, the reactor is kept sealed at a pressure of 0.03 MPa and a stirring speed of 60 rpm to provide a microaerobic environment. The temperature is maintained at 32±1℃ for 48 hours. After this stage, the material is not separated, and the process directly transitions to the second stage of enhanced conversion: stirring is stopped, the pressure is reduced to atmospheric pressure, the temperature is raised to 38±1℃, and the mixture is allowed to stand for 72 hours. During the transition, it is crucial to ensure a smooth transition in temperature and pressure to avoid sudden environmental changes that could disrupt the metabolic rhythm of the microbial community.

[0022] Targeted induction of enzymatic hydrolysis and static management of low-temperature maturation products: After fermentation, add 0.08-0.12 units of food-grade neutral protease per gram of raw material dry weight, stir well, and adjust the pH to 6.8-7.0. Allow the mixture to stand at 25±1℃ for 24 hours for enzymatic hydrolysis. After hydrolysis, transfer the fermentation mash to a sterile environment and allow it to settle and mature at -1℃ to 1℃ for 30 days. During this period, do not centrifuge or filter; rely on gravity to separate the supernatant from the precipitate. Only periodic checks of the temperature stability of the maturation environment are required.

[0023] In-situ aseptic filling product handling guidelines: After maturation, under conditions of low temperature and asepticity, directly extract the supernatant of Polygonatum sibiricum enzyme solution, sterilize it online through a 0.22μm aseptic filter membrane, and then aseptically fill it. When handling the finished product, it is recommended to use aseptic instruments to avoid introducing external bacteria; the filling container is a sterilized brown glass bottle, which can be directly sealed and stored without additional sterilization.

[0024] Usage Notes (No Additives Throughout the Process): This product is prepared entirely from Polygonatum and water, without any added sugar, acid, preservatives, or flavorings. No dilution or mixing is required. It can be consumed directly or used as needed for low-temperature cooking (e.g., replacing part of the stock when making soups). Because no preservatives are added, it is recommended to store it below 4°C. Once opened, it should be consumed as soon as possible to maintain its activity and flavor stability.

[0025] Synergistic use of different batches of products: If different batches of Polygonatum enzyme liquid prepared according to this method are used, since the process parameters are consistent, their components and flavors are consistent. They can be mixed and used as needed without adjusting the dosage or processing method, ensuring a consistent user experience.

[0026] This method of use strictly corresponds to the product characteristics of each step of the preparation method. By following the parameter connection and environmental control requirements, the activity and flavor advantages of the product can be fully utilized. At the same time, since no additional ingredients are added, the use process is more in line with the natural and pure consumption needs.

[0027] Example: Example 1: Implementation of the method for using Polygonatum sibiricum enzyme liquid for daily consumption Preparation for use of the gradient osmosis activation product of Polygonatum sibiricum substrate: Take the concentrated activated substrate obtained from the gradient osmosis activation treatment, confirm that it is well sealed and in a clean state, and use it directly as the base material for fermentation without additional preparation. Before use, gently shake the container to homogenize the substrate and check that it is in a moderately concentrated state to ensure a stable initial environment for the complex microbial system.

[0028] Stepwise sequential propagation and co-inoculation of the compound bacterial strain: A new working solution of the compound bacterial strain was prepared following a stepwise sequential propagation and co-inoculation procedure. It was immediately put into use after preparation to ensure that the bacterial cell viability had not decreased. Upon activation, the working solution was directly inoculated into a concentrated, activated substrate cooled to a suitable temperature at the corresponding ratio, allowing the strain to begin fermentation in a good physiological state without the addition of any auxiliary components.

[0029] Multi-stage temperature and pressure coupled fermentation product transition: At the beginning of fermentation, the first stage, primary fermentation, is initiated. The reactor is kept sealed to create a microaerobic environment, maintaining suitable temperature and pressure for a certain period. After this stage, without separating the materials, the process directly transitions to the second stage, enhanced conversion: stirring is stopped, the tank pressure is adjusted to atmospheric pressure, the temperature is raised, and the mixture is allowed to stand for a certain period. The transition between stages involves a smooth temperature and pressure transition to avoid sudden environmental changes disrupting the metabolic rhythm of the microbial community.

[0030] Targeted induction of enzymatic hydrolysis and static management of low-temperature maturation products: After fermentation, add food-grade neutral protease in the corresponding proportion, stir well and adjust the pH to the suitable range, and allow to stand for enzymatic hydrolysis at a suitable temperature. After enzymatic hydrolysis, transfer the fermentation mash to a sterile environment and place it in a low-temperature environment for physical sedimentation maturation. During this period, do not centrifuge or filter; rely on natural gravity to complete the separation of the supernatant and precipitate. Only periodic monitoring of the stability of the maturation environment temperature is required.

[0031] In-situ aseptic filling product handling guidelines: After maturation, under conditions of low temperature and asepticism, directly extract the supernatant of Polygonatum enzyme solution, sterilize it online through a sterile filter membrane, and then aseptically fill it. When taking the finished product, use sterile instruments to avoid introducing external bacteria; the filling container is a sterilized brown glass bottle, which can be directly sealed and stored without additional sterilization.

[0032] Usage Notes (No Additives Throughout the Process): This product is prepared entirely from Polygonatum sibiricum and water, without any added sugar, acid, preservatives, or flavorings. For daily consumption, an appropriate amount can be taken directly without dilution or mixing. Since no preservatives are added, refrigeration is recommended. Consume as soon as possible after opening to maintain activity and flavor stability.

[0033] Example 2: Implementation of the method for using Polygonatum odoratum enzyme liquid for low-temperature cooking and seasoning Preparation for use of the gradient osmosis activation product of Polygonatum sibiricum substrate: Take the concentrated activated substrate obtained from the gradient osmosis activation treatment, confirm that it is sealed and clean, and use it directly as the base material for fermentation. Before use, check that its concentration state is suitable to provide a stable initial environment for the compound microbial system, without further processing.

[0034] Stepwise sequential propagation and co-inoculation of the compound bacterial strain: Prepare a new working solution of the compound bacterial strain according to the stepwise sequential propagation and co-inoculation steps. Activate the solution promptly after preparation to ensure cell viability. When activating, directly take the working solution and inoculate it into the cooled, concentrated, activated substrate in the corresponding proportion to start fermentation in optimal condition. No other components should be added.

[0035] Multi-stage temperature and pressure coupled fermentation product transition: At the beginning of fermentation, the first stage, primary fermentation, is initiated, maintaining a sealed reactor and a microaerobic environment at suitable temperature and pressure for a certain period. After completion, without separating the materials, the process directly transitions to the second stage of enhanced conversion: stirring is stopped, the tank pressure is adjusted to atmospheric pressure, the temperature is raised, and the mixture is allowed to stand for a certain period. The transition between stages is characterized by a smooth environmental condition change to avoid interfering with microbial metabolism.

[0036] Targeted induction of enzymatic hydrolysis and static management of low-temperature maturation products: After fermentation, add food-grade neutral protease in the corresponding proportion, stir well and adjust the pH to the suitable range, and allow to stand for enzymatic hydrolysis at a suitable temperature. After enzymatic hydrolysis, transfer the fermentation mash to a sterile environment and place it in a low-temperature environment for physical sedimentation maturation. During this period, do not centrifuge or filter, and rely on natural gravity to separate the supernatant and precipitate. Regularly monitor the stability of the maturation temperature.

[0037] In-situ aseptic filling product handling guidelines: After maturation, extract the supernatant under low-temperature and aseptic conditions without compromising sterility. After online sterilization via a sterile filter membrane, aseptically fill the product. Use sterile instruments when handling the finished product to avoid contamination. The filling containers are sterile brown glass bottles, which can be directly sealed and stored without further sterilization.

[0038] Precautions for use throughout the entire process without additives: This product is prepared solely from Polygonatum and water, and contains no added sugar, acid, preservatives, or flavorings. When used for seasoning in low-temperature cooking, it can replace part of the stock in the preparation of soups and stews. Simply add it directly to the ingredients and simmer at low temperature without adding any other seasonings. The flavor will be enhanced by the product's own flavor. Store in the refrigerator and use within the shelf life to ensure quality.

[0039] Example 3: Implementation of Management Method for Mixed Batch Use of Polygonatum Enzyme Solution Preparation for use of the gradient osmosis activation product of Polygonatum sibiricum substrate: Take concentrated activated substrates obtained from different batches of gradient osmosis activation treatment, confirm that each batch is sealed and clean, and use them directly as the base material for fermentation. Before use, check that the concentration state of each batch of substrate is appropriate and consistent, so as to provide a uniform and stable initial environment for the compound microbial system, without additional preparation.

[0040] Stepwise sequential propagation and co-inoculation of the compound bacterial strain: New working solutions for the compound bacterial strain were prepared according to the stepwise sequential propagation and co-inoculation steps. Each batch was put into use promptly after preparation to ensure cell viability. Upon use, each batch of working solution was directly inoculated into the cooled concentrated activation substrate according to the corresponding ratio, allowing the bacterial strain to start fermentation in a good state without adding any other components.

[0041] Multi-stage temperature and pressure coupled fermentation product transition: Each batch of fermentation follows the requirements of the first-stage primary fermentation, maintaining a sealed reactor and a micro-aerobic environment, keeping appropriate temperature and pressure for a certain period of time. After completion, the material is not separated, and directly transitions to the second-stage enhanced conversion: stirring is stopped, the tank pressure is adjusted to atmospheric pressure, the temperature is raised, and the mixture is allowed to stand for a certain period of time. The transition environment is smooth to avoid disrupting the metabolic rhythm of the microbial community.

[0042] Targeted induction of enzymatic hydrolysis and static management of low-temperature maturation products: After each batch of fermentation, add food-grade neutral protease in the corresponding proportion, stir well and adjust the pH to the suitable range, and allow to stand for enzymatic hydrolysis at a suitable temperature. After enzymatic hydrolysis, transfer each batch of fermentation mash to a sterile environment and place it in a low-temperature environment for physical sedimentation maturation. During this period, do not centrifuge or filter, and rely on natural gravity to separate the supernatant and precipitate. Regularly monitor the stability of the maturation temperature.

[0043] In-situ aseptic filling product handling guidelines: After each batch has matured, the supernatant of each batch is extracted under low-temperature and aseptic conditions without compromising the sterility. After online sterilization via a sterile filter membrane, it is aseptically filled. Sterile instruments are used when handling each batch of finished product to avoid the introduction of contaminants. All filling containers are sterile brown glass bottles, which can be directly sealed and stored without further sterilization.

[0044] Usage Notes for the Entire Process Without Additives: Each batch of product is prepared solely with Polygonatum and water, containing no added sugar, acid, preservatives, or flavorings. Due to consistent process parameters, the composition and flavor of each batch are consistent. Different batches can be mixed as needed without adjusting dosage or processing methods. It can be consumed directly or used for low-temperature cooking. Store in the refrigerator and use within the shelf life to ensure a consistent user experience.

[0045] 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 Polygonatum sibiricum enzyme solution, characterized in that, Includes the following steps: S1. Gradient permeation activation treatment of Polygonatum sibiricum matrix: Take dried Polygonatum sibiricum rhizomes, crush them into 20-40 mesh particles, add deionized water at a material-to-water ratio of 1:3.5-4.0 (w / v), and soak at 35±1℃ for 12 hours; then, under aseptic conditions, transfer the soaking solution along with the material to a high-pressure pulsating percolation device, and perform at least three intermittent negative pressure pulsating percolations, each with a pulsating pressure of -0.06MPa, a holding time of 8 minutes, and an intermittent venting and recovery time of 5 minutes. After the cycle is completed, collect all the percolate to obtain the first intermediate material; concentrate the first intermediate material under reduced pressure at 45±1℃ under an inert gas atmosphere until its soluble solids content reaches 18-20°Brix, to obtain the concentrated activated matrix; S2. Stepwise sequential propagation and synergistic inoculation of the composite bacterial system: A composite bacterial system composed of *Lactobacillus plantarum* and *Saccharomyces cerevisiae* was prepared. First, the *Lactobacillus plantarum* was inoculated into MRS liquid medium and anaerobically cultured at 30±1℃ and 150 rpm until... The pH value reached 3.5-4.0, which was used as the primary seed culture; then, the *Saccharomyces cerevisiae* was inoculated into YPD liquid medium and cultured aerobically at 28±1℃ and 180 rpm until... The pH value reached 4.5-5.0, which was used as the secondary seed culture. Next, the primary seed culture was inoculated at a ratio of 10% (v / v) into fresh MRS medium for secondary amplification, and simultaneously, the secondary seed culture was inoculated at a ratio of 15% (v / v) into fresh YPD medium for secondary amplification. Both were cultured simultaneously until they reached their respective growth rates. The values ​​all reached 6.0±0.2; finally, the Lactobacillus plantarum and Saccharomyces cerevisiae seed liquid after secondary amplification were mixed evenly at a volume ratio of 1:1.2 to obtain the working solution of the compound bacterial strain, which was immediately used for inoculation. S3. Multi-stage temperature and pressure coupled fermentation: The concentrated activated substrate obtained in step S1 is cooled to 32±1℃ and inoculated with the composite bacterial working solution prepared in step S2 at an inoculation rate of 3.5% (v / v); First, the first stage of main fermentation begins: In a closed reactor, the temperature is maintained at 32±1℃, the tank pressure at 0.03MPa, and the stirring speed at 60rpm to provide a micro-aerobic environment, and fermentation lasts for 48 hours; After this stage, the process directly proceeds to the second stage of enhanced conversion: The temperature is raised to 38±1℃, the tank pressure is reduced to atmospheric pressure, stirring is stopped, and static fermentation is carried out under these conditions for 72 hours; S4. Targeted Enzymatic Hydrolysis and Low-Temperature Maturation: After fermentation, add 0.08-0.12 units (U) of food-grade neutral protease per gram of raw material dry weight to the system. After stirring evenly, adjust the pH value of the system back to 6.8-7.0, and then carry out targeted enzymatic hydrolysis at 25±1℃ for 24 hours. After enzymatic hydrolysis, under sterile conditions, the entire fermentation mash is physically settled and matured in a low-temperature environment of -1℃ to 1℃ for 30 days without any centrifugation or filtration. It is naturally separated into two parts: supernatant enzyme liquid and precipitate. S5. In-situ aseptic filling: After maturation, without damaging the low-temperature environment and aseptic conditions, the supernatant is directly extracted and sterilized online through a 0.22μm aseptic filter membrane, and then aseptically filled to obtain the finished Polygonatum sibiricum enzyme liquid. The preparation method does not add any other ingredients besides Polygonatum and water throughout the entire process.

2. The method for preparing Polygonatum sibiricum enzyme solution according to claim 1, characterized in that, In step S1, the particle size of the dried Polygonatum rhizome is 30 mesh, the material-to-water ratio is 1:3.8 (w / v), and the intermittent negative pressure pulsating percolation is performed three times; the inert gas is nitrogen. The conditions for the reduced pressure concentration are as follows: Under continuous purging protection, at 45℃ and -0.09MPa; the soluble solids content was determined by refractometer method.

3. The method for preparing Polygonatum sibiricum enzyme solution according to claim 1, characterized in that, In step S2, the *Lactobacillus plantarum* is selected from strain CICC 20261 or its functional equivalent strain from the China Industrial Microbial Culture Collection Center (CICC); the *Saccharomyces cerevisiae* is selected from strain CICC 1301 or its functional equivalent strain from the China Industrial Microbial Culture Collection Center; the anaerobic culture is carried out in an Erlenmeyer flask equipped with a one-way exhaust valve; the aerobic culture is carried out in a track-type oscillation culture on a shaker; the... The values ​​were measured using a spectrophotometer at a wavelength of 600 nm.

4. The method for preparing Polygonatum sibiricum enzyme solution according to claim 1, characterized in that, In step S2, the co-inoculation of the composite bacterial strain is achieved by mixing the secondary amplified Lactobacillus plantarum with the Saccharomyces cerevisiae seed liquid at a volume ratio of 1:1.1 to 1:1.3, preferably 1:1.2; and the time interval from the completion of secondary amplification to the completion of inoculation is controlled within 5 minutes to ensure the optimal physiological state and metabolic synchronization of the bacteria.

5. The method for preparing Polygonatum sibiricum enzyme solution according to claim 1, characterized in that, In step S3, the micro-aerobic environment of the first stage primary fermentation is maintained by introducing sterile compressed air at a flow rate of 0.05-0.1 vvm into the closed reactor, and the tank pressure is stabilized at 0.03 MPa by an automatic back pressure valve; the static fermentation in the second stage enhanced conversion is carried out in a light-proof and heat-insulated container to ensure a constant temperature.

6. The method for preparing Polygonatum sibiricum enzyme solution according to claim 1, characterized in that, In step S4, the amount of food-grade neutral protease added is 0.1 units (U) per gram of dry weight of raw material; the optimal pH value of the neutral protease is 7.0, and it is purchased from Novozymes Denmark as Neutrase® 0.8L or a commercially available product with the same enzymatic properties; the pH adjustment is carried out using food-grade sodium carbonate or sodium bicarbonate solution; the physical sedimentation and maturation is completed in a stainless steel sedimentation tank with a sterile supernatant extraction port at the top.

7. The method for preparing Polygonatum sibiricum enzyme solution according to claim 1, characterized in that, In step S5, the online sterilization is achieved using a two-stage filtration system. The first stage is a 0.45μm pre-filtration to intercept any larger particles or flocs that may be present. The second stage is a 0.22μm polyethersulfone (PES) sterile filter membrane to ensure the microbiological safety of the final product. The aseptic filling container is a brown glass bottle that has been steam-sterilized at 121°C for 30 minutes. After filling, the sterilized butyl rubber stopper or aluminum-plastic combination cap is immediately screwed on.

8. The method for preparing Polygonatum sibiricum enzyme liquid according to claim 1, characterized in that, The targeted enzymatic hydrolysis process in step S4 specifically hydrolyzes the protamine protein components that were not fully degraded by microorganisms during fermentation, generating small molecule peptides and free amino acids with higher biological activity. This process is carried out under strictly controlled pH conditions to avoid browning side reactions that may be caused by acidic or alkaline proteases.

9. The method for preparing Polygonatum sibiricum enzyme solution according to claim 1, characterized in that, The multi-stage temperature and pressure coupled fermentation process in step S3 is designed based on the following principle: The first stage, primary fermentation under pressurized microaerobic conditions, preferentially promotes the rapid proliferation of Saccharomyces cerevisiae and the generation of primary metabolites such as ethanol and esters, while creating a suitable anaerobic initiation environment for Lactobacillus plantarum. The second stage, high-temperature static fermentation, prompts the microbial metabolism to shift to a pathway mainly focused on the synthesis of secondary metabolites, and promotes the spontaneous denaturation, degradation, and association of proteins and heat-labile polysaccharides, thereby forming the unique mellow taste and clear appearance of the Polygonatum sibiricum enzyme liquid.

10. A method for preparing a Polygonatum odoratum enzyme solution according to any one of claims 1 to 9, characterized in that, This enzyme solution is an amber-colored, clear liquid containing only Polygonatum sibiricum-derived components. It contains no added sugar, acid, preservatives, or flavorings. Its total acidity, calculated as citric acid, is 0.8-1.2 g / 100mL, and its soluble solids content is 12-15°Brix. After being stored at 4°C for 180 days, its transmittance (620nm) decreases by no more than 5%.

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