Preservation method of pseudo-ginseng cell line

Abstract

The invention relates to the technical field of fermentation of medicinal material production, and discloses a preservation method of a pseudo-ginseng cell strain, which comprises the following steps: acquiring a working cell strain; obtaining a liquid culture medium; carrying out mixed culture on the working cell strain and the liquid culture medium, and dehydrating; and soaking the dehydrated biomass in a cryoprotectant, and then carrying out cryopreservation or refrigerated preservation. Through cryopreservation or refrigerated preservation of the mixed solution, the preserved working cell strain can be adopted for fermentation in real time according to the requirements of a user, the richness of the strain can be guaranteed, the preparation period of the seed solution is saved, multi-batch fermentation can be realized, and the yield is stable.

Description

TECHNICAL FIELD

[0001] The present application relates to the fermentation technology field of medicinal material production, and in particular to a preservation method of Panax notoginseng cell lines. BACKGROUND

[0002] The production technology of rare medicinal materials is a key link in the pharmaceutical industry and the health industry. However, the current medicinal material planting technology is facing multiple technical bottlenecks, and the industry upgrading is limited. The following is the main difficulties and specific performance of the medicinal material industry:

[0003] (1) The germplasm resources and breeding technology are backward. The high-quality seed source is scarce: the germplasm of the local medicinal materials is seriously degraded, the hybrid breeding technology is weak, and most of the medicinal materials still use traditional breeding methods. The seed seedling standard is missing.

[0004] (2) The planting mode is extensive, and it is difficult to land the standardization. The traditional experience dominates, and the scientific data is lacking. The implementation rate of GAP (Good Agricultural Practice) is less than 10%.

[0005] (3) The short board of green prevention and control technology of diseases and pests. High dependence on chemical pesticides: high-toxicity organochlorine pesticides are still widely used in ginseng planting. Difficult to popularize biological control technology: the application cost of natural enemy insects (such as predatory mites to control red spider mites) is twice that of chemical pesticides, and farmers have low acceptance. The stability of microbial agents is poor.

[0006] (4) The application of mechanization and intelligentization is insufficient. The special crop adaptation machinery is missing: the damage rate of root and stem medicinal materials (such as salvia miltiorrhiza and angelica dahurica) harvesting machinery is more than 20%. The mechanization rate of mountain medicinal material production areas is less than 15%. The digital technology landing barrier is difficult for small farmers to bear.

[0007] The planting of Panax notoginseng medicinal materials also inevitably encounters the above-mentioned industrial problems, and is particularly serious in germplasm degradation, continuous cropping obstacles, and product pesticide residue exceeding standards. Therefore, a new method should be developed to partially or completely solve the problems of Panax notoginseng planting.

[0008] At present, the acquisition of Panax notoginseng raw materials can use a new method - Panax notoginseng cell fermentation method. This method obtains tissues on the same or different Panax notoginseng plants, induces callus, introduces the callus into a sealed fermentation tank, and performs aerobic fermentation to efficiently obtain high-quality Panax notoginseng cell biomass. When Panax notoginseng cell fermentation is realized, many of the above problems can be solved.

[0009] In the fermentation process of Panax notoginseng plant cells, obtaining and preserving Panax notoginseng cell lines is a crucial step, the starting point of the process, and one of the key technological bottlenecks. Failure to effectively preserve Panax notoginseng cell lines will result in waste, affect the seed liquid preparation time during fermentation, and ultimately impact the yield, output, and product quality. Simultaneously, this method will also address the difficulties of traditional Panax notoginseng germplasm preservation.

[0010] Panax notoginseng seeds are not the conventional type for drying and preservation; they are resilient seeds, also known as "seeds intolerant to dehydration." This is quite different from the "normal seeds" such as rice and wheat, which can be dried and stored at low temperatures for a long time: (1) High water content and vigorous physiological activity. Such a high water content makes the seed's respiration very vigorous, its metabolism active, and its nutrients consumed very quickly. Once separated from the parent plant, life activities proceed rapidly, and its lifespan is very short; (2) Intolerant to dehydration and low temperature: The core difficulty lies in the fact that resilient seeds are extremely sensitive to dehydration and low temperature. If dried and stored at low temperature (0-5℃ in a conventional cold storage) like ordinary seeds, the seeds will quickly lose their vitality and die. This is because dehydration will damage the cell membrane structure, leading to irreversible damage. Therefore, conventional seed bank preservation methods cannot be used; (3) Short lifespan (post-ripening characteristics): Panax notoginseng seeds have physiological post-ripening characteristics. The embryo of freshly harvested seeds is not yet fully developed and needs to go through a period of "dormancy" (actually the process of the embryo continuing to develop) under suitable humidity and temperature for a period of time (40-60 days) before germination. This post-ripening process itself continuously consumes the nutrients of the seeds. The essence of preservation is how to "pause" or "extremely slow down" this consumption process, but it is difficult to do so with current technology; (4) The preservation conditions are harsh and the shelf life is extremely short. The only feasible short-term preservation method at present is sand stratification. Mix the seeds with moist river sand and pile them up, keeping the humidity at 20%-25% and the temperature at around 10-15℃. Even under these conditions, the preservation period is very short. Shelf life: Usually only about 45-60 days. Beyond this period, the germination rate will drop sharply, or even lose all vitality. This strictly limits the sowing to be completed in a very short time after seed collection. (5) Susceptible to pathogens: The high temperature and high humidity sand stratification environment is also conducive to the growth of fungi and bacteria, which can easily cause seed rot and further reduce the germination rate.

[0011] These preservation challenges have had a profound impact on the Panax notoginseng industry. High planting costs: Seeds and seedlings need to be purchased annually, making self-storage impossible. High transportation risks: Propagation materials must be transported long distances in a very short time, placing extremely high demands on logistics. Easy spread of pests and diseases: Seedling transportation is a major route for the long-distance spread of soil-borne diseases such as root rot. Long breeding cycle: The propagation and promotion of superior varieties is slow because rapid propagation through seed banks is not possible.

[0012] Therefore, plant cell fermentation technology has not only changed the landscape of Panax notoginseng cultivation technology, but its cell line preservation method has also improved the preservation and transmission of Panax notoginseng germplasm, enabling long-term preservation of rich germplasm in a closed, controlled, and clean environment. This technology can be used for Panax notoginseng cell fermentation, and it can also facilitate the regeneration of plants from cell lines preserved using this technology in the traditional Panax notoginseng cultivation industry, making it a technology of great practical significance.

[0013] This invention relates to a method for preserving Panax notoginseng cell lines. Summary of the Invention

[0014] This invention proposes a method for preserving Panax notoginseng cell lines to solve the technical problem that it is difficult to preserve Panax notoginseng cell lines in the fermentation methods of existing technologies.

[0015] To address the aforementioned technical problems, this invention proposes a method for preserving Panax notoginseng cell lines, comprising:

[0016] Obtain working cell lines;

[0017] Obtain liquid culture medium;

[0018] After culturing the working cell line in the liquid culture medium for a period of time, it is dehydrated to obtain Panax notoginseng cell clusters.

[0019] The dehydrated Panax notoginseng cell clusters are immersed in a cryoprotectant for cryopreservation or refrigerated storage.

[0020] Optionally, after immersing the dehydrated Panax notoginseng cell clusters in a cryoprotectant for freezing or refrigeration, the method further includes:

[0021] Upon revival, the mixture, which has been frozen or refrigerated, is dehydrated to obtain the working cell line.

[0022] Optionally, the revived, cryopreserved, or refrigerated mixture used to obtain the working cell line comprises:

[0023] The two cryogenic tubes containing the mixture are placed in a water bath at 40°C to 45°C to liquefy the mixture and obtain liquefied contents.

[0024] Pour the liquefied contents into a test tube containing sterile rinsing solution;

[0025] The cryoprotectant is rinsed away using the sterile rinsing solution to obtain biomass;

[0026] The biomass is left to stand for a second preset time to obtain a supernatant and a resuspended biomass.

[0027] The supernatant and the resuspended biomass were separated by vacuum filtration.

[0028] The filter membrane containing resuspended biomass was cultured on the first solidified culture medium for 1-2 hours;

[0029] The filter membrane was transferred to a second curing medium and cultured for 22-28 hours in a dark environment at 23-27°C to obtain the working cell line.

[0030] Optionally, the first solidified culture medium comprises 0.4-0.6M sorbitol and 0.03-0.05% calcium chloride dihydrate.

[0031] Optionally, the second solidified culture medium comprises 0.08-0.12M sorbitol and 0.03-0.05% calcium chloride dihydrate.

[0032] Optionally, the cryoprotectant is liquid nitrogen.

[0033] Optionally, the liquid culture medium includes a high-sugar culture medium or a high-sugar alcohol culture medium.

[0034] Optionally, the cryoprotectant includes a cryoprotectant and a hypertonic solution.

[0035] Optionally, the cryoprotectant includes sorbitol and ethylene glycol.

[0036] Optionally, the dehydration of the mixture of the working cell line and the liquid culture medium includes:

[0037] The working cell line was pre-cultured in a mixture of the working cell line and MS20 growth medium supplemented with sorbitol;

[0038] The mixture is incubated in a mixed solution containing a preset concentration of sugar and ethylene glycol for a first preset time to dehydrate the mixture.

[0039] Compared with the prior art, the method for preserving Panax notoginseng cell lines in this invention preserves the richness of the cell lines by immersing the mixture in a cryoprotectant solution for freezing or refrigeration. It can use working cell lines for fermentation in real time according to user needs, quickly obtain sufficient quality seed liquid, ensure stable quality of multiple batches of fermentation and product quality, and save various costs of cell line breeding starting from explants. It has advantages over traditional planting in terms of germplasm stability, process controllability, component stability and quality safety. Attached Figure Description

[0040] Figure 1 This is a flowchart of the method for constructing a Panax notoginseng cell line and the liquid fermentation method in one embodiment of the present invention;

[0041] Figure 2 This is a graph showing experimental data for saponin testing in one embodiment of the present invention;

[0042] Figure 3 This is a schematic flow diagram of a 300L reactor process in one embodiment of the present invention;

[0043] Figure 4 This is a process data diagram of cultivating pan097 in a 16L bioreactor according to one embodiment of the present invention;

[0044] Figure 5 This is a graph showing the changes of pan097 during cultivation in a 16L bioreactor in one embodiment of the present invention;

[0045] Figure 6 This is data on the changes over time during the cultivation of PanO23 in a 300L reactor according to one embodiment of the present invention;

[0046] Figure 7 This is a diagram showing the changes in the culture medium in one embodiment of the present invention;

[0047] Figure 8 This is a schematic diagram of the biomass of pan023 encapsulated in one embodiment of the present invention. Detailed Implementation

[0048] A method for preserving a Panax notoginseng cell line, comprising:

[0049] Obtain working cell lines;

[0050] Obtain liquid culture medium;

[0051] Dehydrate the mixture of the working cell line and the liquid culture medium;

[0052] The dehydrated mixture is then immersed in a cryoprotectant for freezing or refrigeration.

[0053] In the method for preserving Panax notoginseng cell lines of the present invention, the mixture is frozen or refrigerated by immersing it in a cryoprotectant. This allows for real-time fermentation using working cell lines according to user needs, enabling semi-continuous fermentation in multiple batches with stable yield.

[0054] In one embodiment, after immersing the dehydrated mixture in a cryoprotectant for freezing or refrigeration, the method further includes:

[0055] The mixture, which has been frozen or refrigerated, is revived to obtain the working cell line.

[0056] In one embodiment, the revived cryopreserved or refrigerated mixture for obtaining the working cell line comprises:

[0057] The two cryogenic tubes containing the mixture are placed in a water bath at 40°C to 45°C to liquefy the mixture and obtain liquefied contents.

[0058] Pour the liquefied contents into a test tube containing sterile rinsing solution;

[0059] The cryoprotectant is rinsed away using the sterile rinsing solution to obtain biomass;

[0060] The biomass is left to stand for a second preset time to obtain a supernatant and a resuspended biomass.

[0061] The supernatant and the resuspended biomass were separated by vacuum filtration.

[0062] The filter membrane containing resuspended biomass was cultured on the first solidified culture medium for 1-2 hours;

[0063] The filter membrane was transferred to a second curing medium and cultured for 20-28 hours in a dark environment at 23-27°C to obtain the working cell line.

[0064] In one embodiment, the first solidified culture medium comprises 0.4-0.6M sorbitol and 0.03-0.05% calcium chloride dihydrate.

[0065] In one embodiment, the second solidified culture medium comprises 0.09-0.11M sorbitol and 0.04% calcium chloride dihydrate.

[0066] In one embodiment, the cryoprotectant is liquid nitrogen.

[0067] In one embodiment, the liquid culture medium includes a high-sugar culture medium or a high-sugar alcohol culture medium.

[0068] In one embodiment, the cryoprotectant includes a cryoprotectant and a hypertonic solution.

[0069] In one embodiment, the cryoprotectant includes sorbitol and ethylene glycol.

[0070] In one embodiment, dehydrating the mixture of the working cell line and the liquid culture medium includes:

[0071] The working cell line was pre-cultured in a mixture of the working cell line and MS20 growth medium supplemented with sorbitol;

[0072] The mixture is incubated in a mixed solution containing a preset concentration of sugar and ethylene glycol for a first preset time to dehydrate the mixture.

[0073] For ease of explanation, the method for preserving Panax notoginseng cell lines proposed in this invention is presented as follows: Figure 1 The construction and fermentation methods of the Panax notoginseng cell lines shown will be explained.

[0074] Specifically, the methods for constructing Panax notoginseng cell lines include:

[0075] A1. Obtain the target organ from the Panax notoginseng plant;

[0076] A2. Callus tissue was induced from the target organ using a solid culture medium.

[0077] A3. The callus tissue is cultured in a shaking fermentation system to obtain working cell lines and liquid culture medium.

[0078] Plant cell suspension culture or fermentation technology is a synthetic biology technique that utilizes the natural instincts of plant cells, such as their "dryness" and "totipotency," combined with fermentation engineering, cell engineering, enzyme engineering, and genetic engineering. Plant cell fermentation technology can improve the pain points of traditional medicinal herb cultivation from multiple dimensions.

[0079] First, plant cell fermentation technology can optimize germplasm resources and standardize seeds, thereby ensuring stable fermentation yield, product quality, and product profiles for each batch, overcoming the dependence of authentic medicinal materials on comprehensive geographical resources such as latitude, longitude, hydrology, climate, and weather.

[0080] Secondly, plant cell fermentation technology can solve the problems of extensive planting methods and difficulty in standardization in traditional Chinese medicine cultivation. Plant cell fermentation technology uses stable seed preservation techniques to ensure stable metabolic synthesis of cell lines, a closed fermentation environment, and stable and controllable fermentation conditions, thus ensuring stable and controllable separation, extraction, and purification conditions. Therefore, it is highly conducive to achieving standardized production.

[0081] Third, plant cell fermentation technology can solve the pollution problems caused by pesticides and fertilizers in the traditional Chinese medicine planting process. The plant cell fermentation process is a closed, sterile, pure culture process that naturally avoids contact with pesticide and fertilizer residues, resulting in pollution-free production, and the product is naturally free of bacterial endotoxins.

[0082] Fourth, plant cell fermentation technology inherently possesses the potential to integrate with and continuously iterate upon mechanized and intelligent technologies. By combining mechanized and intelligent technologies, plant cell fermentation technology leverages the genetic potential, reaction rate, and product quality of cell lines, achieving yields that surpass those of traditional Chinese medicine production by 3 to 5 geometric progressions. Furthermore, this technology significantly reduces the need for manual labor, thus representing intelligent manufacturing.

[0083] In terms of germplasm resource assurance, this technology has obvious advantages: (1) Germplasm stability. Since it is asexual reproduction, once the cell line is determined, the stability of the variety and the high efficiency of rejuvenation can be guaranteed under long-term operation. Traditional Panax notoginseng seeds have a short lifespan, surviving only for 1 week at room temperature and less than 1 year at low temperature. However, plant cell fermentation technology uses liquid nitrogen low-temperature refrigeration, which can guarantee preservation for several years or even 10 years; (2) Germplasm resource diversity: This technology can quickly and economically form cell lines, including cell lines formed from wild Panax notoginseng from different habitats such as Yunnan and Guangxi, and can also establish cell lines from different organs of different Panax notoginseng plants, such as roots, stems, leaves, flowers, fruits, and seeds, to establish a library, which can not only ensure germplasm stability, but also have rich diversity to meet the raw material needs of a wider range of health products; (3) Breeding efficiency. The rejuvenation of Panax notoginseng cell lines can be completed in 30-45 days (basically within 2-3 fermentation cycles), while the breeding of cultivated Panax notoginseng varieties requires 5-10 years. This is because Panax notoginseng takes 3-5 years to flower and bear fruit; (4) Quality standardization. Because the definition and evaluation standards of traditional Chinese medicine (TCM) systems are difficult to standardize within the industry, it is challenging to achieve uniformity in molecular identity, geographical environment, and medicinal component fingerprinting. Plant cell fermentation, however, can control the basic characteristics based on a few indicators such as active component profiles and fermentation performance characteristics, thus possessing standardized evaluation criteria and component stability.

[0084] In terms of production efficiency, the annual yield per mu (a Chinese unit of area, approximately 0.165 acres) of plant cell fermentation technology surpasses that of Panax notoginseng by several geometric progressions. Based on the data from this experiment, a 100-mu (a Chinese unit of area, approximately 6.65 acres) plant cell fermentation plant can produce 259.2 tons of Panax notoginseng cell dry matter annually (=12 tanks * 60 tons * 24 fermentations * 15 kg / ton), while planting 100 mu of Panax notoginseng can only produce 3 tons annually (100 mu * 1 / 5 * 150 kg / mu), resulting in a yield per mu ratio of 86.4 and saving approximately 99.2% of cultivated land.

[0085] In terms of product quality, Panax notoginseng cell fermentation technology has two typical advantages over cultivated Panax notoginseng: (1) Advantage in quality. Panax notoginseng cell fermentation technology can be implemented in GMP-level workshops, with no chemical fertilizers or pesticides used throughout the process, naturally eliminating the residue of pollutants. In contrast, the cultivation process of Panax notoginseng faces the challenges of chemical fertilizer application and pest control. In particular, to resist the typical continuous cropping obstacles of Panax notoginseng, the land must either be left fallow for 5-10 years or high-input agricultural processes such as high-concentration soil disinfection and process pest control must be implemented. Therefore, the quality of Panax notoginseng products often faces the problem of pollutant residues; (2) Advantage in function. Based on the components required by the human body, this technology can use controllable culture medium components, stimulant types and their concentrations, fermentation strategies, etc., to achieve the goal of the active components of the product meeting the needs of the human body. Moreover, the rich cell lines have a wide range of medicinal expertise, which can be applied to the treatment or conditioning needs of different indications or sub-health conditions.

[0086] In terms of its advantages in integrating with future technologies for innovation, plant cell technology, being a product of interdisciplinary collaboration, naturally absorbs and innovates upon the outstanding achievements of advanced manufacturing fields such as synthetic biology, fermentation engineering, and smart science. For example, Panax notoginseng cell fermentation technology can incorporate single-cell sequencing and gene editing from synthetic biology to form cell lines with extremely high yields; it can also incorporate the latest achievements of smart science in fermentation engineering, combining "dry" prediction with "wet" detection to continuously unlock yield potential.

[0087] Therefore, developing Panax notoginseng cell fermentation technology can solve the inefficiency problems in the Panax notoginseng planting industry, such as the "joint liability barrier" and competition for land with grain crops. It can also address the pollution risks associated with fertilizers and pesticides during the planting process, and ensure the richness of product efficacy and quality safety. This provides an optimal solution for critical areas such as land health, environmental safety, and human food and health, possessing significant practical and economic value. Furthermore, as a common plant cell fermentation technology, Panax notoginseng cell fermentation technology can also provide valuable insights for the development of cell fermentation technologies for other rare medicinal plants, exhibiting extremely high academic value.

[0088] Liquid fermentation methods for Panax notoginseng cell lines include:

[0089] Step 1 – Callus induction process: This mainly involves randomly selecting tissues from the six major organs of healthy Panax notoginseng plants, and after rapid and slow sterilization, cutting tissue blocks of 1-10 mm, placing them in solid culture media such as MS20, MS2, NTM52, B5.6, B5.7, B5.7h&h, B5.12, and IND30, and culturing them at 23-27℃ for 10-25 days to form callus tissue.

[0090] Step 2 – The construction of cell lines mainly involves cutting the callus tissue obtained in Step 1 into 4-8 portions, and then performing 1-4 cycles as in Step 1 to obtain pure cell lines. These cell lines are cultured on plates until they reach a fresh weight of 1-5g, then inoculated into MS20 series liquid medium containing specific phytostimulants, and cultured at 60-120 rpm and 23-27℃ for 7-21 days. Biomass density and total saponin titer are measured every 7 days.

[0091] Step 3 – The cryopreservation process of cell lines mainly involves pre-culturing, pre-treatment, and freezing. Pre-culturing involves initial dehydration in a high-sugar / high-sugar-alcohol medium; pre-treatment involves immersing the culture in a cryoprotectant solution; and freezing involves rapid freezing (vitrification) or gradual freezing (slow freezing) in a cryoprotectant and hypertonic solution.

[0092] Step 4 – Resurrection: This mainly involves post-thawing processing, removing toxic cryoprotectants, readjusting the osmotic pressure to a physiological state, restoring the tissue on a plate, and allowing callus tissue to regrow from the successfully restored cells.

[0093] Step 5 – Preparation of seed culture: The selected cell line obtained in Step 2 is inoculated into the liquid culture medium obtained in Step 2, with an inoculation amount of 60-120 g / L of fresh cell weight after inoculation. The cells are cultured at 80-150 rpm and 23-27℃ for 7-21 days. The fermentation is stopped when the total saponin titer is greater than 1.0 g / L.

[0094] It is important to note that steps 3 and 4 are not necessary for every fermentation. These steps are only required to rejuvenate the seeds when seed yield has declined, in order to obtain satisfactory seeds.

[0095] Step 6 (Step B4) – Liquid submerged fermentation: Inoculate the seed culture obtained in Step 5 into the fermenter at an inoculum size of 60-120 g / L of cell fresh weight after inoculation. Culture at 80-150 rpm and 23-27℃ for 14-21 days. Use B5.7 medium for the first 7 days and MS20 medium for the following 7-14 days. Fermentation is stopped when the total saponin titer reaches a value greater than 1.0 g / L in cell fresh weight.

[0096] Step 7 - Dehydration: Remove water from the fermentation broth obtained in Step 6 by centrifugation or pressure filtration, adjusting the operating parameters to ensure that the damage to cell biomass is less than 3%.

[0097] Step 8 - Dry product preparation: The concentrated slurry obtained in step 7 is dried to remove moisture. The principle of adjusting the operating parameters is to reduce the total saponin content to less than 3%, and the specific drying methods are not limited to fluidized bed drying.

[0098] Step 9 - Fresh Product Preparation: The concentrated slurry obtained in Step 7 is mixed with food preservatives such as tea polyphenols, potassium sorbate, and e-polylysine, so that the material can be stored in a sealed container away from light at 8-15 degrees Celsius for more than 1 year.

[0099] The screening of Panax notoginseng cell lines and the evaluation of shake-flask yields include the following aspects:

[0100] 1.1 Materials

[0101] Panax notoginseng seedlings were randomly purchased by Langoufei Aiteng Biomedical Co., Ltd. in Wenshan, Yunnan Province, planted in nutrient soil, and used as explant material after two weeks of growth.

[0102] 1.2 Culture medium

[0103] MS20 was used as the basal medium. Meanwhile, to create diverse cell lines and compare fermentation yields, NTM52, IND30, and B5.6 were also used to evaluate the fermentation yields of different cell lines. Their formulations are shown in Table 1.

[0104]

[0105] 1.3 Disinfection of explants

[0106] First, rinse the Panax notoginseng seedlings with running water and cut them into appropriate sizes. Then, place them in a clean bench, add 75% ethanol (by volume) and soak for 30 seconds. After that, discard the ethanol and rinse three times with sterile water. Next, pour in 0.1% mercuric chloride solution for 5 minutes to disinfect. After that, discard the mercuric chloride solution and rinse five times with sterile water. Finally, use sterile filter paper to absorb the moisture.

[0107] 1.4 Explant inoculation and callus induction

[0108] Place the sterilized taproots, lateral roots, stem segments, leaves, flower buds, fruits, and seeds into petri dishes. Use sterile scissors and a scalpel to cut each organ into small pieces of (1-5) mm × (1-5) mm. After inoculation, place the dishes in an incubator for cultivation, controlling the temperature at 23-27℃ in complete darkness, and regularly observe the changes in the explants and the growth of callus tissue. If contamination occurs, promptly remove the contaminated petri dishes.

[0109] Each callus formed on the plate is then divided into 9 equal parts and placed on an MS20 plate to form new callus. This process is repeated 2-3 times until the resulting callus can be cultured in liquid.

[0110] 1.5 Fermentation Yield Evaluation

[0111] After 2-3 divisions and subcultures of the callus tissue, select vigorous and loosely structured callus tissues. Inoculate individual callus tissue clusters at a rate of 60-120 g / L (fresh weight) into 300 mL Erlenmeyer flasks containing 100 mL of sterile liquid culture medium. Incubate on a shaker at 25°C and 100 rpm. Add 50 µM methyl jasmonate and 100 µM silver thiosulfate on days 0, 7, and 14. Measure relevant parameters every 7 days. Details are as follows.

[0112] Determination of five specific saponins: Cell clusters were obtained by centrifuging 10-20 mL of cell fermentation broth. The cell clusters were extracted twice with 40% ethanol solvent at a weight ratio of 1:10, and the contents of the five saponins in the combined solution were determined. HPLC was used for detection. The five saponins are ginsenoside Rd (CAS 52705-93-8), Rb1 (CAS 41753-43-9), Rg1 (CAS 22427-39-0), Re (CAS 52286-59-6), and notoginsenoside R1 (Not R1, CAS 80418-24-2).

[0113] 1.6 Experimental Results

[0114] from Figure 2 It can be seen that after callus induction and callus liquid fermentation, the ginsenoside composition (proportion of 5 saponins) and ginsenoside concentration (proportion of fresh weight) of tissues from the same plant are very different.

[0115] like Figure 2 The figures show the yield of ginsenosides in suspension cultures generated from primary callus. The concentration and composition of total ginsenosides are also shown. The first bar graph for each cell line shows the ginsenoside yield under uninduced conditions. Subsequent bar graphs show the ginsenoside yield after induction with 50 µM MJS and 100 µM SLTS for 7, 14, and 21 days. The origin of the cell lines is labeled R (root), S (stem), L (leaf), and P (petiole). MJS stands for methyl jasmonate, and SLTS stands for silver thiosulfate.

[0116] Therefore, it can be concluded that cells from different tissues of the same plant, under selective and optimized culture conditions, can form cell lines with different yields and compositions. These cell lines with different yields can produce different medicinal properties and different economic values. In these dimensions, it has greater germplasm richness and product application diversity than traditionally cultivated Panax notoginseng.

[0117] Based on the above-described methods for preserving Panax notoginseng cell lines, the fermentation performance of these cell lines at different scales was analyzed as follows:

[0118] 2.1 Materials and Culture Media

[0119] The experiments yielded a large number of cell lines and suitable culture media. To fully demonstrate the advanced nature of the technology, this experiment selected only one preferred cell line and optimized culture medium to study the fermentation performance at different fermentation scales.

[0120] This experiment used the pan097 cell line and selected B5.7 and MS20 culture media as fermentation medium.

[0121] 2.2 Experimental Methods

[0122] 2.2.1 Fermentation method for 16L scale

[0123] Fermentation performance was tested using pan097 as described in Table 2. In a laboratory bioreactor, pan097 was initially cultured for one cycle (14 days), followed by induction at the start of the second cycle. The first culture cycle lasted 14 days, after which the culture medium in the bioreactor was almost entirely replaced with fresh medium. One day after medium replacement, initial induction was performed by adding MJS (final concentration 50 µM) and SLTS (final concentration 100 µM) to the suspension culture. MJS was added again at a final concentration of 50 µM on days 23, 29, and 39. From day 12, glucose was added to the culture at a feeding rate of 5 g / L / d. Osmolarity indicated an adequate glucose supply, so feeding was stopped on day 33. After inoculation with cells at a fresh weight concentration of 37 g / L, the cell lines showed faster growth compared to other cell lines, reaching 104 g / L after 14 days. In the second culture cycle, induction inhibited growth, but the cells reached a final fresh weight concentration of 299 g / L within 35 days.

[0124]

[0125] 2.2.2 Semi-continuous fermentation experiment with a capacity of 300L

[0126] Methods: B5.7 and cell line pan023 were used as culture medium and experimental materials. The cells were cultured in 300L tanks for more than 13 culture cycles. At the beginning of each cycle, the culture medium was changed, and a portion of the biomass was transferred to a 1500L tank. Three partial harvests were performed before complete harvest (total culture time was 176 days, see...). Figure 3 (As shown).

[0127] like Figure 3 As shown, white arrows indicate culture medium B5.7; light blue arrows indicate B5.7h&h; red arrows indicate harvesting or transfer; yellow lightning bolts indicate the addition of MJS; and black lightning bolts indicate the addition of SLTS.

[0128] 2.3 Experimental Results

[0129] 2.3.1 Fermentation performance of pan097 in a 16L laboratory bioreactor

[0130] In the first cycle, osmolarity decreased from 175 to 63 mOsmol / kg, and in the second cycle, it decreased from 204 to 151 mOsmol / kg. The addition of glucose supplementation was associated with an increase in osmolarity around day 32. Conductivity decreased from 5.22 to 3.66 mS / cm in the first cycle, and further decreased to 1.25 mS / cm in the second cycle due to nutrient absorption. pH values ​​ranged from 5.1 to 6.6.

[0131] like Figure 4 As shown, (a) fresh weight concentration [g / L], (b) osmotic pressure [mOsmol / kg], (c) conductivity [mS / cm] and (d) pH value change over time, with hormones changing on day 14 and MJS (yellow arrow) and SLTS (black arrow) added.

[0132] from Figure 5 It is clear from the data that pan097 requires induction. During the first culture cycle before induction with MJS and SLTS, the total ginsenoside concentration remained stable at approximately 100 mg / kg. In contrast, the initial measurements after MJS and SLTS treatment showed an increase in ginsenoside content. This increase continued until day 43, reaching a maximum ginsenoside concentration of 2.3 g / kg. Figure 4 a) reflects that the cell line grew 23-fold after induction in a bioreactor environment. The titer showed a similar trend, reaching 433 mg / L on day 43 (a). Figure 4 b). Ten days after the last addition of MJS (day 49), the total ginsenoside concentration and titer decreased again to 855 mg / kg and 256 mg / L, respectively. The addition of MJS and SLTS also affected this combination. Before the addition of the inducer, only Rg1 and Re could be measured. Quantifiable Rd, Rb1, and NotR1 contents were discovered through induction. The dominant ginsenoside after induction was Rg1 (up to 71%). Re was approximately 50% before induction, subsequently decreasing to 17%. The highest measured Rb1 proportion was 13%. The maximum proportion of Rd among ginsenosides was 8%. In the later stages of cultivation (day 35 and beyond), Figure 4 c) NotR1 was measured and found to account for 1-2% of the ginsenoside composition. The highest titer reached was 433 mg / L by induction of pan097, exceeding the titer obtained in shake flasks, attributed to excellent growth performance in the bioreactor. In conclusion, the induction of MJS and SLTS can be applied in a bioreactor environment.

[0133] Figure 5During the cultivation of pan097 in a 16L bioreactor, the following are the changes over time: (a) ginsenoside concentration [mg / kg], (b) ginsenoside titer [mg / L], and (c) ginsenoside combination. Gray line - culture medium replacement, red line - addition of MJS (yellow arrow) and SLTS (black arrow).

[0134] Therefore, this experiment leads to the conclusion that Panax notoginseng cell lines can be cultured for extended periods in fermenters with excellent fermentation performance, producing the rare Panax notoginseng saponin NotR1, a component scarce in traditionally cultivated Panax notoginseng. Panax notoginseng plant cell fermentation technology has demonstrated advantages in yield and product quality at a scale of 16L.

[0135] 2.3.2 Performance of Pan023 in semi-continuous fermentation using a 300L bioreactor

[0136] In the initial stage of cultivation in a 300L tank, the biomass fresh weight concentration was 15 g / L. The culture medium was changed after only one week. The subsequent cycle length was 14 days ± 2 days, similar to the growth performance of small-scale cultivation. The biomass fresh weight doubled on average every 14 days. The average osmotic pressure at the beginning of the cycle was 124 mOsmol / kg, and the conductivity was 3.71 mS / cm. Due to the consumption of sugars and nutrients by the cells, the osmotic pressure and conductivity decreased to 3.28 mOsmol / kg and 107 mS / cm, respectively, with the pH value between 5.6 and 6.1 (e.g., ...). Figure 6 (As shown).

[0137] like Figure 6 The data shown are as follows: (a) Fresh weight concentration [g / L], (b) Osmotic pressure [mOsmol / kg], (c) Conductivity [mS / cm], and (d) pH value; Light blue area - B7.5h & h medium; White area - medium B5.7; Gray line - medium replacement; Red line - harvest and medium replacement.

[0138] The key to the first five cycles (scale-up process) was the propagation of biomass. A portion of the biomass was transferred to a 1,500L tank. After the transfer, the remaining biomass was grown for another cycle in B5.7. Subsequent cycles used a different medium, changing the carbon source from 20 g / L sucrose to 10 g / L sucrose and 10 g / L glucose. The total carbon concentration remained constant, but the types of carbon changed. All subsequent cycles used B5.7 h&h. Changing the medium did not affect growth performance, conductivity, or pH. Due to the different sugar types used, the osmotic concentration at the start of the new culture cycle was slightly higher than that of B5.7. With the change to B5.7 h&h, the concentration of saponins in the cells increased sharply. Figure 7Prior to this, the saponin content of the biomass fresh weight was only 300 mg / kg. After changing the culture medium to B7.5 h&h, 28 days later, the fresh weight saponin content exceeded 7 g / kg. With a biomass fresh weight >130 g / L, the titer at the first harvest was close to 1 g / L, achieving the expected target (day 104). In the subsequent three culture cycles, the saponin concentration ranged from 6 to 11 g / kg. The saponin titer varied with fresh weight, and the titers at the subsequent two harvests were 0.9–1.6 g / L.

[0139] like Figure 7 As shown: (a) Ginsenoside concentration [mg / kg], (b) Ginsenoside titer [mg / L] and (c) Distribution of ginsenoside combinations over time, light blue area - B7.5h&h medium, white area - medium B5.7, gray line - medium replacement, red line - harvest and medium replacement.

[0140] Therefore, based on the information analysis of the two figures above, the Panax notoginseng cell fermentation technology can achieve semi-continuous fermentation in multiple batches with stable yields. This indicates that plant cell technology that enables semi-continuous fermentation and harvesting can use half of the cells from the previous cycle as seeds each time, saving the fixed investment, operation and maintenance costs, water and electricity costs of multi-stage seed tanks. Compared to the seedling period (3 years) of cultivated ginseng, the semi-continuous fermentation technology of Panax notoginseng cells saves the previous n-1-stage breeding period. For example, a 75-ton scale involves 6 stages of fermentation (40L-200L-1000L-5000L-20ton-75ton), and semi-continuous fermentation can save the operation and maintenance costs of 5 (6-1=5) stages of seed tanks in multiple cycles, reducing operation and maintenance costs by 60-80%. This is a huge technological and economic advantage.

[0141] To further illustrate the effectiveness of the Panax notoginseng cell line preservation method, the low-temperature preservation experiment of Panax notoginseng cell lines is as follows:

[0142] 3.1 Materials

[0143] Panax notoginseng cell lines are complex and diverse. However, according to cryopreservation methods, they can be classified into two types, covering the cryopreservation applications of all cell lines: -196℃ freezing and 6-12℃ refrigeration. The appropriate method should be chosen based on the specific cell line's tolerance. For ease of explanation, this experiment focuses on comparing and discussing strains that conform to these two preservation methods.

[0144] 3.2 Methods

[0145] All cryopreservation protocols for plant cell lines include the following common steps:

[0146] (1) Pre-culture, followed by initial dehydration in a high-sugar / high-sugar alcohol medium.

[0147] (2) Add cryoprotectant

[0148] (3) Rapid freezing (vitrification) or gradual freezing (slow freezing) in a freezing solution containing cryoprotectants and hypertonic solutions.

[0149] (4) Post-thawing treatment: remove toxic cryoprotectants and readjust osmotic pressure to physiological state.

[0150] (5) Reconstruction on a plate, with callus tissue regrowth from successfully reconstructed cells.

[0151] (I) Taking cell line pan097 as an example, the low-temperature freezing method of Panax notoginseng cell line is explained.

[0152] Biomass obtained from conventional suspension culture was pre-cultured for 7 days in MS20 growth medium supplemented with sorbitol. At the end of the pre-culture, cells were harvested and incubated for 2 hours in a loading solution containing high concentrations of sugar and ethylene glycol. Subsequently, aliquots of the biomass were transferred to cryovials and incubated in a freezing solution containing sorbitol and ethylene glycol before rapid freezing. The incubation process was performed at 4°C to minimize damage to the cells from the high osmotic pressure and cryoprotectants. After thawing in a water bath, the cryoprotectants were removed, and the osmotic pressure was gradually adjusted to physiological conditions. Details are as follows:

[0153] Material

[0154] • Safety cabinet / laminar flow hood

[0155] • Panax notoginseng suspension culture

[0156] • MS20 sterile liquid culture medium at room temperature

[0157] • Sterile solid culture medium MS2 at room temperature (containing 4.6 g / L gellan gum)

[0158] • Refrigerated aseptic loading solution (N-LS6)

[0159] • Refrigerated sterile cryosol (V2N)

[0160] • Sterile washing solution at room temperature (solution 7)

[0161] • Sterile disposable scraper

[0162] • Aseptic transfer spoon

[0163] • Sterile Erlenmeyer flasks (500 ml and 250 ml)

[0164] • Balance

[0165] • Timer

[0166] • Sterile Buchner funnel

[0167] • Sterile Buchner flask

[0168] • Vacuum pump

[0169] • Sterile Miracloth filter paper

[0170] • Sterile filter discs approximately 3 cm in diameter, such as Whatman®

[0171] • Incubator with rotary shaker

[0172] • 4 ml cryotube

[0173] • Sorbitol

[0174] • Trehalose

[0175] • Ethylene glycol

[0176] • Crushed ice for ice bath

[0177] Liquid nitrogen

[0178] • 4°C refrigerator

[0179] • Calcium chloride dihydrate

[0180] plant materials

[0181] For cryopreservation, mature (day 7) Panax notoginseng suspension cultures can be used. To avoid contamination of plant cell materials, all operations involving cell materials must be performed aseptically using sterile materials in a biosafety cabinet or laminar flow hood.

[0182] The culture medium and solution formulations are as follows:

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] Pre-culture start-up procedure, bottling, and cryopreservation (10 bottles)

[0189] 1. Pre-culture

[0190] 1.1 Transfer 6 g of vacuum-filtered fresh Panax notoginseng biomass from 7-day-old suspension culture to 100 mL of MS20 medium containing an additional 5% sorbitol. Pre-culture for 7 days at 25°C in the dark on a rotary shaker. It is recommended to use a 500 mL Erlenmeyer flask for initiating the pre-culture.

[0191] 1.2 After 7 days of cultivation, the biomass of Panax notoginseng was harvested by vacuum filtration.

[0192] 2. Bottling

[0193] 2.1 Transfer all harvested biomass to a 250 mL Erlenmeyer flask containing 50 mL of bottling solution (N-LS6).

[0194] 2.2 Incubate the bottled culture in the refrigerator for 2 hours, gently shaking it every 15 minutes.

[0195] 3. Vitrification

[0196] 3.1 Place 10 cryovials on a rack, remove the caps (but leave them on), and use a small scraper to fill them with 0.47 to 0.57 grams of the biomass of the bottled culture from left to right.

[0197] 3.2 After filling the last bottle, remove all bottle caps and accurately add 3 ml of cold freezing solution V2N to each bottle from left to right using a pipette; start the timer during the filling of the first vial and incubate for 3 minutes.

[0198] 3.3 Starting from the left, tighten the cap of the first vial and shake it to mix the contents until the biomass is completely suspended; repeat the same operation for vials 2 to 10.

[0199] 3.4 Place the vials in the refrigerator (or an ice bath can be used instead), and adjust the remaining 3 minutes as described above; after 2 minutes and 50 seconds, begin to resuspend the contents of the first vial; then use tweezers to immerse the first vial below the surface of the liquid nitrogen; repeat the same operation for vials 2 to 10.

[0200] 3.5. Register the vial and transfer it to a long-term storage container (-180~196℃).

[0201] Revival of cryopreserved Panax notoginseng cells:

[0202] 1) Place two cryotubes of the same origin in a water bath at 40 to 45 degrees Celsius and immerse them for 90 to 120 seconds with stirring. Once the contents of the tubes begin to liquefy, remove them immediately.

[0203] 2) Pour the liquefied contents of each vial into a separate 15 mL Falcon tube containing 10 mL of sterile rinsing solution (see Solution 7).

[0204] 3) Gently shake the two Falcon tubes to flush out the cryoprotectant.

[0205] 4) Let the biomass stand at room temperature for 10 minutes.

[0206] 5) Discard about half of the supernatant in each Falcon tube, combine the contents of the two Falcon tubes into one tube, and then carefully resuspend the biomass.

[0207] 6) Use vacuum filtration to separate the cells from the washing solution.

[0208] 7) Place the filter membrane containing Panax notoginseng cells on a solidification medium containing 0.5M sorbitol and 0.04% calcium chloride dihydrate (see osmo05) for 1-2 hours, then transfer the filter membrane to a solidification medium containing 0.1M sorbitol and 0.04% calcium chloride dihydrate (see osmo01) and incubate for 24 hours in the dark at 25°C.

[0209] 8) After 24 hours, transfer the filter tray with biomass to a solidified growth medium, such as MS20.

[0210] (II) Taking cell line pan023 as an example, the low-temperature refrigeration method for Panax notoginseng cell lines is explained.

[0211] 1. Materials

[0212] pan023 cell line

[0213] 2. Method

[0214] Both pan097 and pan023 were maintained in MS20 medium. The main differences between them were cell cluster size and ginsenoside content. The cell clusters of pan097 were less than 1 mm, while the cell clusters of pan023 could reach 4 mm, depending on the growth conditions. The following outlines the modifications made to the preservation protocol of pan0097 after testing for cryopreservation of pan023.

[0215] Encapsulation technology is commonly used for the cryopreservation of large cell clusters or plant organs. We have developed a method for encapsulating plant cell material in alginate. Alginate is a natural polymer extracted from brown algae that dissolves in water and forms a gel upon contact with certain salts.

[0216] The pan023 suspension was treated using the cryopreservation method described above (except for step 3.5). The resulting treated solution was suspended in an alginate solution, and then CaCl2 solution was added dropwise to form gel beads of approximately 4 mm in size. Figure 8 Dry the beads in a clean bench or biosafety cabinet to reduce the moisture content of the beads and biomass. Transfer the beads to sterile cryovials or plastic test tubes and store them in a refrigerator at 8-15°C.

[0217] During cell line recovery, the test tubes were removed from the refrigerator and allowed to slowly recover for 1–2 hours under a clean bench or biosafety cabinet. After thawing, the beads were placed on a plate containing activated carbon to adsorb any potentially toxic protective compounds. Recovery tests were conducted in Erlenmeyer flasks containing both solid and liquid culture media. Successful preservation was defined as the continuous proliferation of the cell beads.

[0218] like Figure 8 As shown, (a) preparation of alginate beads. (b) alginate beads in a shake flask. The image was taken from the bottom of the flask. (c) alginate beads on a plate containing activated carbon.

[0219] 3. Results

[0220] 3.1 The resurrection effect of low-temperature freezing

[0221] Cryopreservation methods for plants are not universally applicable and need to be developed for each species, and sometimes even for each cell line. As shown in Table 3, pan097 can be preserved using cryopreservation because it can be revived on plates.

[0222]

[0223] 3.2 The revival effect of low-temperature refrigeration

[0224] Table 4 shows that pan023 can be preserved using cryopreservation because it can be revived on plates. Moreover, this method is generally applicable to most cell lines. This indicates that the integration of cryoprotectants and sodium alginate beads is generally effective for Panax notoginseng cell lines.

[0225]

[0226] A comprehensive comparison of cryogenic freezing and cryogenic preservation reveals that each method has its advantages. Table 5 below illustrates these advantages to help in the appropriate selection of the technology.

[0227]

[0228] In summary, low-temperature freezing and low-temperature refrigeration constitute a complete germplasm preservation method for cell lines, which is beneficial to supporting fermentation technology and its downstream engineering.

[0229] In summary, the method for preserving Panax notoginseng cell lines can effectively construct cell lines rich in medicinal properties, thus enabling a wide range of applications. Furthermore, the fermentation technology is highly efficient, produces high-quality products, maintains stable cell performance and yield, and is environmentally friendly, energy-saving, water-saving, and land-saving. It is a technology worthy of key support and should be protected by patents.

Claims

1. A method for preserving a Panax notoginseng cell line, characterized in that, include: Obtain working cell lines; Obtain liquid culture medium; The working cell line and the liquid culture medium were mixed and cultured, and then dehydrated. The dehydrated mixture is immersed in a cryoprotectant and then frozen or refrigerated.

2. The method for preserving Panax notoginseng cell lines according to claim 1, characterized in that, After immersing the dehydrated mixture in a cryoprotectant and then freezing or refrigerating it, the process further includes: The mixture, which has been frozen or refrigerated, is revived to obtain the working cell line.

3. The method for preserving Panax notoginseng cell lines according to claim 2, characterized in that, The revived cryopreserved or refrigerated mixture, used to obtain the working cell line, comprises: The two cryogenic tubes containing the mixture are placed in a water bath at 40°C to 45°C to liquefy the mixture and obtain liquefied contents. Pour the liquefied contents into a test tube containing sterile rinsing solution; The cryoprotectant is rinsed away using the sterile rinsing solution to obtain biomass; The biomass is left to stand for a second preset time to obtain a supernatant and a resuspended biomass. The supernatant and the resuspended biomass were separated by vacuum filtration. The filter membrane containing resuspended biomass was cultured on the first solidified culture medium for 1-2 hours; The filter membrane was transferred to a second curing medium and cultured for 20-28 hours in a dark environment at 23-27°C to obtain the working cell line.

4. The method for preserving Panax notoginseng cell lines according to claim 3, characterized in that, The first solidified culture medium comprises 0.4-0.6M sorbitol and 0.03-0.05% calcium chloride dihydrate.

5. The method for preserving Panax notoginseng cell lines according to claim 3, characterized in that, The second solidification culture medium comprises 0.08-0.12M sorbitol and 0.03-0.05% calcium chloride dihydrate.

6. The method for preserving Panax notoginseng cell lines according to claim 1, characterized in that, The cryoprotectant is liquid nitrogen.

7. The method for preserving Panax notoginseng cell lines according to claim 1, characterized in that, The liquid culture medium includes a high-sugar culture medium or a high-sugar alcohol culture medium.

8. The method for preserving Panax notoginseng cell lines according to claim 1, characterized in that, The cryoprotectant includes a cryoprotectant and a hypertonic solution.

9. The method for preserving Panax notoginseng cell lines according to claim 8, characterized in that, The cryoprotectant includes sorbitol and ethylene glycol.

10. The method for preserving Panax notoginseng cell lines according to claim 1, characterized in that, The step of dehydrating the mixture of the working cell line and the liquid culture medium includes: The working cell line was pre-cultured in a mixture of the working cell line and MS20 growth medium supplemented with sorbitol; The mixture is incubated in a mixed solution containing a preset concentration of sugar and ethylene glycol for a first preset time to dehydrate the mixture.

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