A method and device for preparing saussurea involucrata callus powder by combining low-temperature suspension culture with enzymolysis

Abstract

The application discloses a method for preparing saussurea involucrata callus powder by combining low-temperature suspension culture and enzymolysis, and belongs to the technical field of saussurea involucrata preparation, and comprises the following steps: S1: after sterile in-vitro leaves of saussurea involucrata are surface-disinfected, the leaves are inoculated on MS solid induction culture medium to perform primary callus induction culture, the culture temperature is 22-24 DEG C, daily illumination is 10 h, the culture period is 18-22 d, and loose granular callus is obtained through screening. In the application, a low-temperature liquid suspension expansion culture system is used to replace traditional solid static culture, the problems of cell aggregation and uneven nutrition transmission are completely solved, the cell proliferation efficiency is increased by 85%, the proliferation period is shortened by 40%, meanwhile, the low-temperature condition promotes endogenous synthesis of core active substances such as flavones and ECM repair polysaccharides, the total flavone content can be more than 9 times of that of wild saussurea involucrata high-temperature extract, wild saussurea involucrata is not cut in the whole process, the method completely meets the cosmetic raw material compliance mass production requirements, and ton-level continuous industrial production is realized.

Description

Technical Field

[0001] This invention belongs to the field of snow lotus preparation technology, specifically a method and apparatus for preparing snow lotus callus powder by combining low-temperature suspension culture and enzymatic hydrolysis. Background Technology

[0002] As a rare medicinal plant, the extract of Tian Shan snow lotus has good development prospects in the field of skin care and repair. However, wild snow lotus is a national second-class protected plant and its resources are limited. Existing technologies mainly rely on solid agar static callus culture for laboratory-scale preparation, or directly use wild plants for high-temperature organic solvent extraction, centrifugation coarse filtration and high-temperature spray drying to obtain liquid or powder extracts. The above methods constitute the main technical routes for the preparation of active ingredients of snow lotus, which are widely seen in relevant patents and literature reports.

[0003] However, existing technologies still have significant shortcomings: solid static culture suffers from cell aggregation and uneven nutrient delivery, making it impossible to achieve continuous industrial-scale production at the ton level; high-temperature extraction and high-temperature drying processes easily cause significant degradation of heat-sensitive active ingredients such as flavonoids and polysaccharides, with activity loss reaching over 60%, and residual organic solvents are highly irritating to damaged and sensitive skin; single centrifugation or chromatography purification methods cannot balance impurity removal efficiency with equipment investment costs, making it difficult for small and medium-sized enterprises to implement; in addition, the products obtained from existing processes are mostly liquid concentrates or coarse powders with poor water solubility, which are limited in storage and transportation and have poor compatibility in formulation compounding, making it difficult to meet the specific requirements of barrier repair products after minimally invasive medical aesthetic procedures for low-irritant, fully water-soluble, and highly active raw materials, resulting in a significant gap in the supply of high-end repair-specific raw materials. Summary of the Invention

[0004] The purpose of this invention is to provide a method and apparatus for preparing snow lotus callus powder by combining low-temperature suspension culture and enzymatic hydrolysis, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing snow lotus callus powder by combining low-temperature suspension culture and enzymatic hydrolysis, comprising the following steps:

[0006] S1: After surface disinfection, sterile detached leaves of Tian Shan snow lotus were inoculated onto MS solid induction medium for primary callus induction culture. The culture temperature was 22-24℃, the light intensity was 10h per day, and the culture period was 18-22d. Loose granular callus tissue was obtained by screening.

[0007] S2: After cutting the loose granular callus obtained in S1 into small pieces, it was placed in agar-free MS liquid suspension medium for low-temperature liquid suspension culture. The culture temperature was 23-26℃, the shaking speed was 90-120r / min, and the cell suspension was cultured in alternating light and dark for 16-20 days. Half of the culture medium was replaced with fresh culture medium every 7 days to obtain cell suspension.

[0008] S3: Collect the cell suspension obtained in S2, adjust the pH of the system to 4.2-4.8, control the temperature at 40-50℃, add the cellulase and pectinase complex enzyme system, and enzymatically hydrolyze at a constant temperature for 1.5-2.5h to obtain the enzymatic hydrolysis mixture;

[0009] S4: The enzymatic hydrolysis mixture obtained in S3 was subjected to two-stage microfiltration purification: first, it was coarsely filtered through a 5μm microporous membrane to retain large cell wall fragments and insoluble fiber residues, and then filtered through a 0.45μm precision microfiltration membrane to remove tiny cell debris and collect the clear active filtrate.

[0010] S5: The clarified active filtrate obtained in S4 is transferred to a low-temperature vacuum concentration tank and concentrated at 40°C until the solid content of the system is 12% to 18%. After sterilization by 0.22μm sterile filtration, it is vacuum freeze-dried, pulverized and sieved to obtain the dry powder product of snow lotus callus tissue.

[0011] As a further preferred embodiment of this technical solution: the formulation of the MS solid induction medium in S1 is: MS basal medium supplemented with 1.0 mg / L 6-BA, 2.0 mg / L NAA and 30 g / L sucrose;

[0012] As a further preferred embodiment of this technical solution: the screening criteria in S1 are: only loose granular callus tissue is retained, and browned, hardened and necrotic cells are removed;

[0013] As a further preferred embodiment of this technical solution: the temperature of the low-temperature liquid suspension expansion culture in S2 is 25℃, the shaking speed is 100r / min, and the culture period is 18d;

[0014] As a further preferred embodiment of this technical solution: the enzymatic hydrolysis temperature in S3 is 45℃, the enzymatic hydrolysis time is 2h, and the pH of the system is 4.5;

[0015] As a further preferred embodiment of this technical solution: the composite enzyme system described in S3 is composed of cellulase and pectinase;

[0016] As a further preferred embodiment of this technical solution: the maximum temperature throughout the entire process in S3 does not exceed 50°C, and no organic solvents are added throughout the entire process;

[0017] As a further preferred embodiment of this technical solution: the solid content of the system is concentrated to 15% in step S5;

[0018] As a further preferred embodiment of this technical solution: the dried snow lotus callus powder described in S5 is a light brownish-yellow powder, 100% water-soluble, and the powder purity is 100%.

[0019] As a further preferred embodiment of this technical solution: the method adopts general basic equipment for bioactive raw materials throughout the process, the equipment including: sterile cell culture reactor, low temperature cold water circulation unit, constant temperature oscillating suspension reaction tank, 5μm / 0.45μm two-stage microfiltration device, low temperature vacuum concentration tank and vacuum freeze dryer.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. In this invention, a low-temperature liquid suspension culture system is used to replace the traditional solid static culture system, which completely solves the problems of cell aggregation and uneven nutrient delivery. The cell proliferation efficiency is increased by 85% and the proliferation cycle is shortened by 40%. At the same time, the low temperature conditions promote the endogenous synthesis of core active substances such as flavonoids and ECM repair polysaccharides. The total flavonoid content can reach more than 9 times that of the high-temperature extract of wild snow lotus. Moreover, wild snow lotus is not harvested throughout the process, which fully meets the requirements for compliant mass production of cosmetic raw materials and realizes continuous industrial production at the ton level.

[0022] 2. In this invention, a compound bio-enzyme at 40-50℃ is used for gentle cell wall disruption. There is no high-temperature oxidation or addition of organic solvents throughout the process. The product has no solvent residue and extremely low irritation, making it safe for use on fragile skin such as wounds after water light therapy and steroid-induced rosacea. Combined with two-stage gradient purification of 5μm coarse filtration and 0.45μm precision microfiltration, insoluble residues are completely removed. The finished product is 100% water-soluble and is stable when combined with various repairing active ingredients such as hyaluronic acid and ceramides. Moreover, the dry powder form has a shelf life that is 24 months longer than that of liquid raw materials, and the storage and transportation costs are reduced by 60%.

[0023] 3. In this invention, the entire process relies on industry-standard basic equipment, eliminating the need for high-pressure extraction, liquid chromatography, or specialized bioreactors. The entire production chain can be completed in a single workshop, reducing the capital threshold for factory construction and mass production by 70%. The resulting snow lotus callus powder can simultaneously repair the epidermal stratum corneum barrier and the dermal ECM extracellular matrix, specializing in high-end medical aesthetic repair such as minimally invasive water-light therapy damage and severe sensitivity caused by hormones, thus building an exclusive cell repair raw material technology barrier that is difficult for brands to replicate. Attached Figure Description

[0024] Figure 1 This is a flowchart of a method and apparatus for preparing snow lotus callus powder using a combination of low-temperature suspension culture and enzymatic hydrolysis according to the present invention. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1:

[0027] Please see Figure 1 As shown, this invention provides a technical solution: a method for preparing snow lotus callus powder using a combination of low-temperature suspension culture and enzymatic hydrolysis, comprising the following steps:

[0028] Step 1: Induction of primary callus tissue using aseptic explants of Tian Shan snow lotus

[0029] Sterile detached leaves of *Saussurea involucrata* were selected, surface-sterilized with 75% ethanol for 30 seconds, sterilized with 0.1% mercuric chloride solution for 8 minutes, rinsed 5 times with sterile water, and then inoculated onto MS solid induction medium. The MS solid induction medium formula was: MS basal medium + 6-BA 1.0 mg / L + NAA 2.0 mg / L + sucrose 30 g / L, agar added at 7 g / L, pH adjusted to 5.8–6.0, and culture conditions were: temperature 23℃, 10 h light per day, 14 h dark culture, culture period 20 days. After culture, the screening criteria were: only pale yellow, loosely structured granular callus tissue was retained, and browned, water-soaked, or hardened necrotic cells were removed to establish a stable cell seed bank.

[0030] Step 2: Low-temperature liquid suspension propagation

[0031] The loose granular callus obtained in step 1 was cut into small pieces with a particle size of about 2-3 mm using sterile surgical scissors. It was then added to agar-free MS liquid suspension medium at an inoculation rate of 10 g / L. The suspension culture conditions were: constant temperature 25℃, shaking speed 100 r / min, alternating light and dark culture for 18 days, and half of the culture medium was replaced with fresh culture medium every 7 days. After the culture was completed, samples were taken for testing.

[0032] Step 3: Low-temperature complex enzyme gentle cell disruption

[0033] Collect all the cell suspension obtained in step 2 and pump it into an enzymatic hydrolysis reactor. Adjust the pH of the system to 4.5 using a citrate-sodium citrate buffer system and control the temperature to 45℃. Add a complex enzyme system of cellulase (activity ≥10000U / g) and pectinase (activity ≥3000U / g), where the amount of cellulase added is 0.8% of the mass of the cell suspension and the amount of pectinase added is 0.4% of the mass of the cell suspension. Enzymatic hydrolysis is carried out at a constant temperature of 45℃ and a stirring speed of 60r / min for 2 hours. During the enzymatic hydrolysis, samples are taken for microscopic examination every 20 minutes until the number of intact cells in the microscopic field of view is less than 5%, at which point the enzymatic hydrolysis is terminated to obtain an enzymatic hydrolysate.

[0034] Step 4: Two-stage gradient microfiltration purification

[0035] The enzymatic hydrolysis mixture obtained in step 3 was sequentially pumped into a two-stage microfiltration device for fractional purification.

[0036] First-stage filtration: Coarse filtration is performed using a 5μm microporous polypropylene filter element at an operating pressure of 0.15–0.20 MPa, which traps large cell wall fragments and insoluble fiber residues.

[0037] Second-stage filtration: The first-stage filtrate was collected and subjected to precision microfiltration using a 0.45μm microporous polyethersulfone filter cartridge at an operating pressure of 0.10–0.15 MPa to remove minute cell debris and insoluble particles. The clear, active filtrate was collected. Testing showed that the turbidity of the filtrate after two-stage filtration decreased from the initial 856 NTU to 4.2 NTU, and the removal rate of insoluble particles (≥10μm) reached 99.6%.

[0038] Step 5: Low-temperature aseptic concentration and vacuum freeze-drying to prepare standardized snow lotus callus powder

[0039] The clarified active filtrate obtained in step 4 was transferred to a low-temperature vacuum concentration tank. The concentration temperature was controlled at 40℃ and the vacuum degree was -0.085 to -0.095 MPa. The concentration was carried out until the solid content of the system was 15%, and the concentrated snow lotus callus tissue was obtained. The concentrated solution was filtered through a 0.22μm polyethersulfone sterile filter for sterilization and then dispensed into freeze-drying trays with a material thickness of 8-10mm. The trays were then transferred to a vacuum freeze dryer for freeze-drying. The freeze-drying process parameters were as follows: pre-freezing temperature -45℃, pre-freezing time 4h; primary drying temperature -25℃→0℃, vacuum degree ≤10Pa, drying time 20h; secondary drying temperature 0℃→30℃, vacuum degree ≤5Pa, drying time 6h. After freeze-drying, the solution was pulverized in a low-humidity environment and passed through a 100-mesh sieve to obtain the finished snow lotus callus tissue dry powder. The finished product is a light brownish-yellow powder with the characteristic odor of the raw material. It is completely water-soluble, with a dry powder purity of 100%. The addition amount in cosmetic residue and rinse products is ≤4%.

[0040] Example 2

[0041] This embodiment provides a method for preparing callus powder by low-temperature suspension enrichment of active substances from snow lotus cells, wherein some process parameters adopt the lower limit of the range, and the remaining steps are the same as in embodiment 1.

[0042] The specific parameters that differ from those in Example 1 include:

[0043] The low-temperature liquid suspension propagation conditions in step 2 are: temperature 23℃, shaker speed 90r / min, and culture period 20d;

[0044] The enzymatic hydrolysis conditions in step 3 are: temperature 40℃, pH 4.2, and hydrolysis time 1.5h;

[0045] In step 5, the mixture is concentrated until the solids content is 12%.

[0046] The remaining operations are exactly the same as in Example 1. The dried snow lotus callus powder obtained in this example is a light brownish-yellow powder, 100% water soluble, with a total flavonoid content of 1.02%, a total polysaccharide content of 16.9%, a moisture content of 2.5%, and is determined to be non-irritating in human skin patch test (same as in Example 1).

[0047] Example 3

[0048] This embodiment provides a method for preparing callus powder by low-temperature suspension enrichment of active substances from snow lotus cells, wherein some process parameters adopt the upper limit of the range, and the remaining steps are the same as in embodiment 1.

[0049] The specific parameters that differ from those in Example 1 include:

[0050] The low-temperature liquid suspension propagation conditions in step 2 are: temperature 26℃, shaker speed 120r / min, and culture period 16d;

[0051] The enzymatic hydrolysis conditions in step 3 are: temperature 50℃, pH 4.8, and hydrolysis time 2.5h;

[0052] In step 5, the mixture is concentrated until the solids content is 18%.

[0053] The remaining operations are exactly the same as in Example 1. The dried snow lotus callus powder obtained in this example is a light brownish-yellow powder, 100% water soluble, with a total flavonoid content of 1.15%, a total polysaccharide content of 17.5%, and a moisture content of 2.1%. The human skin patch test (same as in Example 1) determined it to be non-irritating.

[0054] Comparative Example 1 (Static Solid Culture Control)

[0055] The only difference between this comparative example and Example 1 is that in step 2, traditional solid static agar callus culture is used instead of low-temperature liquid suspension culture.

[0056] Specifically, step 2 of this comparative example is as follows: the loose callus tissue obtained in step 1 is transferred to MS solid agar medium (agar addition amount 7g / L), and statically cultured at 23℃ constant temperature and 10h light per day for 30 days. The remaining steps (primary induction, enzymatic hydrolysis and cell wall disruption, two-stage microfiltration, concentration and freeze drying, etc.) are exactly the same as in Example 1.

[0057] The total flavonoid content of the dried snow lotus callus powder prepared in this comparative example was 0.13%. The results showed that the total flavonoid content was increased by about 9 times compared with the traditional solid static culture. At the same time, due to severe cell aggregation and uneven nutrient transfer, the biomass of the callus obtained by solid static culture was only 25% of that in Example 1.

[0058] Comparative Example 2 (High-Temperature Ethanol Reflux Extraction Control)

[0059] The difference between this comparative example and Example 1 is that steps 2 to 3 of the present invention are replaced by conventional high-temperature ethanol reflux extraction process (i.e., the suspension expansion and enzymatic hydrolysis steps are omitted), and high-temperature ethanol extraction is performed directly from the primary callus tissue obtained in step 1.

[0060] Specifically, the comparative example steps are as follows: the primary callus tissue obtained in step 1 is dried and pulverized, and 70% ethanol is added at a material-to-liquid ratio of 1:10 (g / mL). The mixture is refluxed at 75°C twice for 2 hours each time. The extracts are combined, filtered, and the ethanol is recovered by vacuum concentration. The dry powder is obtained by two-stage microfiltration in step 4 and concentration freeze drying in step 5, which are the same as in Example 1.

[0061] The dry powder product obtained in this comparative example is a dark brown powder with a total flavonoid content of only 0.14%. Furthermore, the detection of residual organic solvents during the extraction process showed that the ethanol residue was 0.8%. In the skin patch test, 4 out of 30 subjects showed moderate erythema reactions. The results indicate that the high-temperature alcohol extraction process not only results in a serious loss of active ingredients, but also causes significant skin irritation due to residual organic solvents. Therefore, it is not suitable for repair applications on sensitive skin after procedures such as mesotherapy.

[0062] The comparative results of the above embodiments show that the complete technical solution of the present invention, which adopts low-temperature liquid suspension expansion culture, low-temperature compound enzyme cell disruption, two-stage microfiltration purification and low-temperature concentration freeze drying, achieves synergistic effects among the various processes in terms of active ingredient enrichment (total flavonoid content 1.02%~1.28%), dry powder water solubility (100% water solubility) and product safety (no skin irritation). In particular, the optimal parameter combination of Example 1 has achieved the best technical effect.

[0063] The comparative experimental data are shown in the table below:

[0064]

[0065] 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 snow lotus callus powder using a combination of low-temperature suspension culture and enzymatic hydrolysis, characterized in that, Includes the following steps: S1: After surface disinfection, sterile detached leaves of Tian Shan snow lotus were inoculated onto MS solid induction medium for primary callus induction culture. The culture temperature was 22-24℃, the light intensity was 10h per day, and the culture period was 18-22d. Loose granular callus tissue was obtained by screening. S2: After cutting the loose granular callus obtained in S1 into small pieces, it was placed in agar-free MS liquid suspension medium for low-temperature liquid suspension culture. The culture temperature was 23-26℃, the shaking speed was 90-120r / min, and the cell suspension was cultured in alternating light and dark for 16-20 days. Half of the culture medium was replaced with fresh culture medium every 7 days to obtain cell suspension. S3: Collect the cell suspension obtained in S2, adjust the pH of the system to 4.2-4.8, control the temperature at 40-50℃, add the cellulase and pectinase complex enzyme system, and enzymatically hydrolyze at a constant temperature for 1.5-2.5h to obtain the enzymatic hydrolysis mixture; S4: The enzymatic hydrolysis mixture obtained in S3 was subjected to two-stage microfiltration purification: first, it was coarsely filtered through a 5μm microporous membrane to retain large cell wall fragments and insoluble fiber residues, and then filtered through a 0.45μm precision microfiltration membrane to remove tiny cell debris and collect the clear active filtrate. S5: The clarified active filtrate obtained in S4 is transferred to a low-temperature vacuum concentration tank and concentrated at 40°C until the solid content of the system is 12% to 18%. After sterilization by 0.22μm sterile filtration, it is vacuum freeze-dried, pulverized and sieved to obtain the dry powder product of snow lotus callus tissue.

2. The method according to claim 1, characterized in that: The MS solid induction medium formulation described in S1 is: MS basal medium supplemented with 1.0 mg / L 6-BA, 2.0 mg / L NAA and 30 g / L sucrose.

3. The method according to claim 1, characterized in that: The screening criteria described in S1 are: retain only loose, granular callus tissue and remove browned, hardened, and necrotic cells.

4. The method according to claim 1, characterized in that: The low-temperature liquid suspension expansion culture described in S2 is at a temperature of 25℃, a shaking speed of 100r / min, and a culture period of 18d.

5. The method according to claim 1, characterized in that: The enzymatic hydrolysis temperature described in S3 is 45℃, the hydrolysis time is 2h, and the pH of the system is 4.

5.

6. The method according to claim 1, characterized in that: The complex enzyme system described in S3 consists of cellulase and pectinase.

7. The method according to claim 1, characterized in that: The maximum temperature throughout the process described in S3 does not exceed 50°C, and no organic solvents are added throughout the process.

8. The method according to claim 1, characterized in that: S5 describes a concentration to a system solids content of 15%.

9. The method according to claim 1, characterized in that: The snow lotus callus powder described in S5 is a light brownish-yellow powder that is 100% water-soluble and has a purity of 100%.

10. The method according to claim 1, characterized in that: The method uses general basic equipment for bioactive raw materials throughout the process. The equipment includes: a sterile cell culture reactor, a low-temperature cold water circulation unit, a constant temperature oscillating suspension reaction tank, a 5μm / 0.45μm two-stage microfiltration device, a low-temperature vacuum concentration tank, and a vacuum freeze dryer.

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