A preparation method of ganoderma acid and jujube compound freeze-dried flash release tablets by biological enzymolysis and fermentation

Abstract

This invention discloses a method for preparing Ganoderma lucidum and jujube seed compound freeze-dried flash release tablets by enzymatic hydrolysis and fermentation, which relates to the field of traditional Chinese medicine preparation technology. The method includes the following steps: (1) Raw material pretreatment: Jujube seed is crushed and mixed with citrate buffer to obtain jujube seed slurry; Ganoderma lucidum fruiting body is ultra-finely pulverized and mixed with cut lily petals to obtain mixed raw materials. This invention uses a specific ratio of compound enzymes (cellulase, pectinase, β-glucanase) in an optimized acidic environment for enzymatic hydrolysis, which can efficiently and selectively destroy the plant cell walls of jujube seed, Ganoderma lucidum and lily, and fully release the active ingredients in the cells; the subsequent fermentation of Lactobacillus plantarum can further decompose the substrate and may produce new beneficial metabolites, forming a synergistic biotransformation, which greatly improves the extraction rate of total active ingredients and product value.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine preparation technology, specifically a method for preparing Ganoderma lucidum and Ziziphus jujuba compound freeze-dried flash release tablets using biological enzymatic hydrolysis and fermentation. Background Technology

[0002] Sour jujube seed, Ganoderma lucidum, and lily bulb are all traditional medicinal and edible ingredients, possessing effects such as calming the nerves, aiding sleep, and enhancing immunity. Currently, most related products on the market are made by simply physically pulverizing them into granules, capsules, or traditional tablets, or using conventional extraction processes such as water extraction and alcohol extraction.

[0003] However, the above-mentioned traditional methods have some shortcomings: First, the cell wall structure of plants is dense, and conventional treatment methods are not enough to fully destroy the cell wall, resulting in low dissolution rates of active ingredients (such as jujube seed saponins, Ganoderma lucidum polysaccharides, and lily glycosides) and limited bioavailability; Second, some active ingredients (such as γ-aminobutyric acid) are easily affected by environmental factors (such as degradation under alkaline conditions) during processing or storage and become inactive, resulting in poor stability; Third, traditional solid dosage forms may have problems such as slow disintegration and poor taste, affecting compliance with medication. Summary of the Invention

[0004] This invention aims to overcome the shortcomings of existing technologies and provide a method for preparing Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using enzymatic hydrolysis and fermentation. This method significantly improves the extraction rate and stability of the active ingredients through the synergistic effect of biotransformation technology, and utilizes freeze-drying technology to produce flash-release tablets with rapid disintegration and excellent taste.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using enzymatic hydrolysis and fermentation, comprising the following steps: (1) Raw material pretreatment: After crushing the jujube kernels, they are mixed with citric acid buffer solution to obtain jujube kernel slurry; after ultra-fine pulverization of Ganoderma lucidum fruiting body, it is mixed with cut lily petals to obtain mixed raw materials; (2) Biotransformation: The jujube seed slurry is combined with the mixed raw materials, the pH is adjusted, and a compound enzyme is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, Lactobacillus plantarum seed liquid is inoculated for fermentation to obtain fermentation broth. (3) Formulation: Centrifuge the fermentation broth and collect the supernatant; microencapsulate γ-aminobutyric acid to obtain γ-aminobutyric acid microcapsules; mix the supernatant, the γ-aminobutyric acid microcapsules and pharmaceutically acceptable excipients evenly, fill them into aluminum-plastic blister packs, and then freeze-dry them to obtain freeze-dried flash-release tablets.

[0006] Preferably, in step (1), the jujube kernels are crushed to 60 mesh; the concentration of the citric acid buffer solution is 0.1 mol / L and the pH is 4.5; the ratio of jujube kernels to citric acid buffer solution is 1:12 (g / mL). The Ganoderma lucidum fruiting body is ultra-finely pulverized to a particle size D50 of 15μm; the lily petals are cut into thin slices with a thickness of 2mm; the mass mixing ratio of the Ganoderma lucidum fruiting body and the lily petals is 1:1.

[0007] Preferably, in step (2), the pH is adjusted to 4.5; the amount of the compound enzyme added is 2% of the total mass of the mixture, and the compound enzyme is composed of cellulase, pectinase and β-glucanase in a mass ratio of 3:2:1; the process conditions for enzymatic hydrolysis are: temperature 50℃, reaction for 2 hours under shaking conditions at 120 rpm.

[0008] Preferably, in step (2), the inoculation amount of the Lactobacillus plantarum seed liquid is 5% of the total volume of the enzymatically hydrolyzed mixture; the fermentation process conditions are: temperature 28℃, static fermentation for 48 hours.

[0009] Preferably, in step (3), the centrifugation process conditions are: 8000 rpm and 15 minutes.

[0010] Preferably, in step (3), the microencapsulation process involves encapsulating γ-aminobutyric acid with chitosan and sodium alginate to form microcapsules.

[0011] Preferably, in step (3), the mixing and filling are performed by sterilization filtration filling using a microporous filter membrane with a pore size of 0.22 μm.

[0012] Preferably, in step (3), the freeze-drying process is a three-stage freeze-drying process, specifically including: First stage of pre-freezing: Place the filled aluminum-plastic blister packs at -45℃ for 2 hours to pre-freeze; Second stage of sublimation drying: raise the temperature to -25℃ and maintain it for 16 hours to carry out sublimation drying; Third-stage desorption drying: Raise the temperature to 35℃ and maintain it for 6 hours to carry out desorption drying.

[0013] Preferably, the freeze-drying process results in a three-dimensional porous network structure in the final product with rapid disintegration characteristics. Beneficial effects

[0014] This invention provides a method for preparing Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using enzymatic hydrolysis and fermentation. The invention employs a specific ratio of compound enzymes (cellulase, pectinase, and β-glucanase) in an optimized acidic environment for enzymatic hydrolysis, which efficiently and selectively destroys the cell walls of jujube seed, Ganoderma lucidum, and lily, fully releasing intracellular active ingredients. Subsequent fermentation with Lactobacillus plantarum further decomposes the substrate and may generate new beneficial metabolites, forming a synergistic biotransformation that significantly improves the extraction rate of total active ingredients and the product value.

[0015] To address the instability of γ-aminobutyric acid (GABA), a novel chitosan / sodium alginate microencapsulation technology was employed for encapsulation. This effectively isolates the GABA from adverse external environments (especially alkaline conditions), preventing its decarboxylation and degradation. This significantly improves the stability of this key active ingredient during processing and storage, as well as the bioavailability of the final product.

[0016] Using a three-stage freeze-drying technology, the liquid medicine is directly formed into a freeze-dried tablet with a high porosity three-dimensional network structure inside the aluminum-plastic blister pack. This structure allows the product to be quickly absorbed by saliva and disintegrated and dispersed after entering the mouth, achieving "flash release". It does not require water to take, has a delicate taste, and greatly improves the user experience. It is especially suitable for people who have difficulty swallowing or do not like taking medicine.

[0017] By organically integrating modern technologies such as enzymatic hydrolysis, fermentation, microencapsulation, aseptic filling, and freeze drying, the process flow is clear and controllable. The microporous filling and freeze drying processes are completed within the final packaging, reducing the risk of contamination in intermediate links. The product has a uniform appearance and moisture content, and stable quality.

[0018] The final product is packaged in aluminum blister packs, combined with freeze-dried solid form, which has excellent moisture-proof, light-proof and sealing properties, which is conducive to the long-term stable storage of active ingredients. Detailed Implementation

[0019] The present invention will be further described in detail below through specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0020] Example 1: A method for preparing Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using enzymatic hydrolysis and fermentation, comprising the following steps: (1) Raw material pretreatment: a. Weigh 1.0 kg of dry, mold-free jujube kernel raw material, crush it using a universal pulverizer (e.g., DFY-500 type), and pass it through a 60-mesh standard sieve to obtain uniform jujube kernel coarse powder.

[0021] b. Prepare 12 L of 0.1 mol / L citrate-sodium citrate buffer solution and calibrate the pH to 4.5 ± 0.1 using a pH meter.

[0022] c. Mix the coarse jujube seed powder with the above buffer solution at a ratio of 1:12 (g / mL), place the mixture in a mixing tank equipped with a stirrer, and stir at 50 rpm for 15 minutes at room temperature to ensure thorough saturation, thereby obtaining a jujube seed slurry. The slurry is in a uniform suspension.

[0023] d. Take another 0.5 kg of dried Ganoderma lucidum fruiting body and pulverize it using an airflow ultrafine pulverizer (e.g., QLM-80K type). Monitor the pulverization endpoint with a laser particle size analyzer and control the particle size distribution D50 = 15 ± 2 μm to obtain Ganoderma lucidum ultrafine powder.

[0024] e. Weigh 0.5 kg of dried lily petals and slice them into uniform thin slices with a thickness of 2.0 ± 0.5 mm using a slicer (e.g., GQ-200).

[0025] f. Add the Ganoderma lucidum ultrafine powder and lily slices into a three-dimensional motion mixer and mix for 30 minutes to ensure that the two are mixed evenly to obtain the mixed raw materials.

[0026] (2) Biotransformation: a. Transfer the jujube seed slurry and mixed raw materials together to a 50L bioreactor with jacketed heating and stirring functions, and start low-speed stirring (30rpm).

[0027] b. Using online pH monitoring, a small amount of 10% (v / v) citric acid solution was added dropwise to precisely adjust and maintain the pH of the entire mixture at 4.5±0.1.

[0028] c. Enzymatic hydrolysis: Weigh 40g of the compound enzyme (pre-mixed evenly according to the mass ratio of cellulase:pectinase:β-glucanase = 3:2:1, with enzyme activity ≥100,000 U / g). Under continuous stirring, slowly add the compound enzyme to the reactor until the final concentration is 2% of the total mass of the mixture. Raise the reactor temperature to 50.0±0.5℃ using a jacketed water bath and increase the stirring speed to 120±5 rpm. Under these conditions, perform enzymatic hydrolysis with shaking for 2 hours. During the enzymatic hydrolysis process, the system gradually becomes viscous and darkens in color.

[0029] d. Fermentation: After enzymatic hydrolysis, the material temperature is lowered to 28.0±0.5℃ using jacketed circulating water. *Lactobacillus plantarum*, activated to the logarithmic growth phase, is inoculated at 5% (v / v) of the total current material volume. Stirring is stopped, and the material is allowed to stand in the reactor for anaerobic fermentation at a constant temperature of 28℃ for 48 hours. After fermentation, a brownish-red fermentation broth with a rich, sour aroma and uniform texture is obtained.

[0030] (3) Formulation: a. Centrifugation clarification: Divide the fermentation broth into centrifuge cups of a high-speed centrifuge (e.g., TGL-20M model) and centrifuge at 4°C and 8000 rpm for 15 minutes. Carefully aspirate the supernatant and filter it sequentially through a 200-mesh stainless steel sieve and a 0.45 μm microporous membrane to obtain a clear, reddish-brown extract. Record the volume for later use.

[0031] b. Preparation of γ-aminobutyric acid (GABA) microcapsules: Microcapsules were prepared using a complex coagulation method. 100g of GABA raw material was weighed. A 2% (w / v) sodium alginate solution was mixed with a uniformly dispersed GABA powder at 40℃. While continuously stirring, a 1.5% (w / v) chitosan acetate solution was added dropwise to adjust the pH to approximately 5.0, inducing a complex coagulation reaction. After the reaction was complete, the microcapsules were filtered, washed, and freeze-dried to obtain white powdery GABA microcapsules.

[0032] c. Mixing: Transfer the clarified extract to a sterile mixing tank, add 50g of GABA microcapsules (10g based on GABA content) and 50g of mannitol as a lyophilization excipient. Under sterile conditions, stir at 40 rpm for 30 minutes to ensure all components are thoroughly mixed.

[0033] d. Filling: The mixed drug solution is terminally sterilized by passing it through a 0.22μm pore size polyethersulfone (PES) sterile filter membrane. Using a fully automated liquid filling machine (e.g., GZP-10 model), the filling volume per blister is set to 1.00±0.05mL, and the drug solution is precisely filled into pre-sterilized aluminum-plastic blister packs (rigid PVC base and pharmaceutical aluminum foil top cover).

[0034] e. Freeze-drying: Transfer the filled blister packs to a vacuum freeze dryer (e.g., LGJ-18C) for programmed freeze-drying.

[0035] The freeze-drying process parameters are shown in the table below. f. Capping and Packaging: After the freeze-drying process is completed, the top cap is immediately heat-sealed with aluminum foil in a dry, inert gas environment (such as nitrogen). Then, the outer packaging is performed to obtain the finished product: Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets. The product is a porous, honeycomb-shaped solid tablet attached to the bottom of the blister pack, with a crisp texture. Example

[0036] The steps are basically the same as in Example 1, except that the biotransformation conditions are optimized. The specific parameters are changed as follows: (1) Raw material pretreatment: The particle size of jujube kernels was adjusted to 80 mesh in order to obtain a larger specific surface area.

[0037] (2) Biotransformation: a. Enzymatic hydrolysis stage: The amount of compound enzyme added is adjusted to 1.8% of the total mass of the mixture. The hydrolysis temperature is adjusted to 48℃, and the hydrolysis time is extended to 2.5 hours, aiming to process the materials more gently.

[0038] b. Fermentation stage: The fermentation temperature of Lactobacillus plantarum was adjusted to 30℃, and the fermentation time was shortened to 36 hours.

[0039] (3) Formulation: In the freeze-drying process, the temperature of the partition plate for the second-stage sublimation drying was adjusted to -20℃ and the time was extended to 18 hours to explore the effect of different drying rates on the product structure.

[0040] The resulting product has a light brown appearance, uniform pores, and a disintegration time that is slightly longer than that of Example 1 (about 5-15 seconds), but still meets the "flash release" requirement. Example

[0041] The steps are basically the same as in Example 1, with the main adjustment being the formulation. The specific changes are as follows: (1) Raw material ratio: The mixing ratio of Ganoderma lucidum fruiting body and lily petals was adjusted to 2:1 (mass ratio) to enhance the contribution of Ganoderma lucidum related components.

[0042] (2) Formulation: a. The amount of GABA microcapsules added was increased to 75g (15g based on GABA content).

[0043] b. The freeze-drying excipient was changed to a mixture of sorbitol and microcrystalline cellulose (mass ratio 1:1), with a total addition of 60g.

[0044] The resulting product has slightly increased structural strength, disintegration time within 10 seconds, and a slightly sweet taste.

[0045] Effect comparison experiment To verify the advantages of the method of the present invention, a concentrated solution prepared by the traditional water extraction and alcohol precipitation method was set as a control (CK) and compared with the lyophilized flash-release tablet extract prepared in Example 1 of the present invention (sampled before lyophilization) for key indicators. The results are shown in the table below: Table 2: Comparison of the effects of the present invention's process and the traditional process As shown in Table 2, the bio-enzymatic hydrolysis and fermentation synergistic process adopted in this invention can significantly improve the extraction efficiency of the main active ingredients; through microencapsulation technology and optimized freeze-drying process, the storage stability of unstable ingredients and the product's compliance with consumption are greatly improved.

[0046] It should be noted that in this paper, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

Claims

1. A method for preparing Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using enzymatic hydrolysis and fermentation, characterized in that, Includes the following steps: (1) Raw material pretreatment: After crushing the jujube kernels, they are mixed with citric acid buffer to obtain jujube kernel slurry; after ultra-fine pulverizing the Ganoderma lucidum fruiting body, it is mixed with the cut lily petals to obtain mixed raw materials; (2) Biotransformation: The jujube seed slurry is combined with the mixed raw materials, the pH is adjusted, and a compound enzyme is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, Lactobacillus plantarum seed liquid is inoculated for fermentation to obtain fermentation broth. (3) Formulation: Centrifuge the fermentation broth and collect the supernatant; microencapsulate γ-aminobutyric acid to obtain γ-aminobutyric acid microcapsules; mix the supernatant, the γ-aminobutyric acid microcapsules and pharmaceutically acceptable excipients evenly, fill them into aluminum-plastic blister packs, and then freeze-dry them to obtain freeze-dried flash-release tablets.

2. The preparation method of Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using biological enzymatic hydrolysis and fermentation according to claim 1, characterized in that, In step (1), the jujube kernels are crushed to 60 mesh; the concentration of the citric acid buffer solution is 0.1 mol / L and the pH is 4.5; the ratio of jujube kernels to citric acid buffer solution is 1:12 (g / mL). The Ganoderma lucidum fruiting body is ultra-finely pulverized to a particle size D50 of 15μm; the lily petals are cut into thin slices with a thickness of 2mm; the mass mixing ratio of the Ganoderma lucidum fruiting body and the lily petals is 1:

1.

3. The preparation method of Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using biological enzymatic hydrolysis and fermentation according to claim 1, characterized in that, In step (2), the pH is adjusted to 4.5; the amount of the compound enzyme added is 2% of the total mass of the mixture, and the compound enzyme is composed of cellulase, pectinase and β-glucanase in a mass ratio of 3:2:1; the process conditions for enzymatic hydrolysis are: temperature 50℃, reaction for 2 hours under shaking conditions at 120 rpm.

4. The preparation method of Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using biological enzymatic hydrolysis and fermentation according to claim 1, characterized in that, In step (2), the inoculation amount of the Lactobacillus plantarum seed liquid is 5% of the total volume of the enzymatically hydrolyzed mixture; the fermentation process conditions are: temperature 28℃, static fermentation for 48 hours.

5. The preparation method of Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using biological enzymatic hydrolysis and fermentation according to claim 1, characterized in that, In step (3), the centrifugation process conditions are: rotation speed 8000 rpm, time 15 minutes.

6. The method for preparing Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using enzymatic hydrolysis and fermentation according to claim 1, characterized in that, In step (3), the microencapsulation process involves encapsulating γ-aminobutyric acid with chitosan and sodium alginate to form microcapsules.

7. The preparation method of Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using biological enzymatic hydrolysis and fermentation according to claim 1, characterized in that, In step (3), the mixing and filling are performed by sterilization filtration filling using a microporous filter membrane with a pore size of 0.22 μm.

8. The method for preparing Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets by bio-enzymatic hydrolysis and fermentation according to claim 1, characterized in that, In step (3), the freeze-drying process is a three-stage freeze-drying process, specifically including: First stage of pre-freezing: Place the filled aluminum-plastic blister packs at -45℃ for 2 hours to pre-freeze; Second stage of sublimation drying: raise the temperature to -25℃ and maintain it for 16 hours to carry out sublimation drying; Third-stage desorption drying: Raise the temperature to 35℃ and maintain it for 6 hours to carry out desorption drying.

9. A method for preparing Ganoderma lucidum and jujube seed compound freeze-dried flash-release tablets using biological enzymatic hydrolysis and fermentation according to any one of claims 1-8, characterized in that, The freeze-drying process results in a three-dimensional porous network structure in the final product with rapid disintegration characteristics.