Medicinal lotus cell suspension factory system and method for directionally producing high-activity components based on healthy target spots

Abstract

The invention discloses a medicine lotus cell suspension factory system and method for directionally producing high-activity components based on a healthy target spot, and the system is characterized in that a health-oriented cell line design module, an intelligent scale culture module, a precise metabolism induction module and a targeted separation and product development module are in closed-loop linkage; and an innovative mode of reversely designing a production process from health requirements is realized. According to the method, aiming at specific targets of epidemic resistance, depression resistance, reproductive health and the like, a high-yield cell line is created through multi-omics analysis screening or gene editing, and large-scale culture is performed in an optimally controlled bioreactor; and the elicitor is utilized to accurately stimulate a metabolic pathway to directionally enrich high-value components such as liensinine, nuciferine, epigallocatechin gallate and the like. And finally, preparing a standardized extract by adopting a green extraction technology (such as ultrasonic extraction and supercritical CO2 extraction), wherein the standardized extract is used for functional foods, dietary supplements or medical products.

Description

Technical Field

[0001] This invention relates to the field of lotus cell suspension factory technology, specifically a lotus cell suspension factory system and method for targeted production of highly active ingredients based on health targets. Background Technology

[0002] Nelumbo nucifera Gaertn., an important medicinal plant in my country, is rich in various bioactive components in different parts. Studies have shown that lotus seed alkaloids have hypotensive, antiarrhythmic, and autophagy-inhibiting effects; lotus polyphenols (including proanthocyanidins and flavonoids) possess significant antioxidant, anti-radiation, anti-tumor, and liver-protective activities; while components such as lotus leaf alkaloids show outstanding potential in antidepressant and lipid-regulating effects. These properties make Nelumbo nucifera a promising candidate for development in the fields of epidemic prevention, antidepressant treatment, and reproductive health.

[0003] However, traditional methods of cultivating and processing lotus roots face many insurmountable bottlenecks:

[0004] 1. High dependence and inconsistent quality: Traditional cultivation depends on land, climate and season, resulting in huge fluctuations in the content of active ingredients. For example, the content of the core active ingredient lotusine in different batches of lotus seeds from the same place can vary by as much as 30% to 50%, making it impossible to standardize the quality of medicinal materials.

[0005] 2. Immense resource and ecological pressures: Large-scale planting occupies a large amount of high-quality arable land and may compete with food production for resources. In addition, the use of pesticides in field management brings the risk of residues, and heavy metals in the soil may be accumulated by plants, introducing safety hazards.

[0006] 3. Low production efficiency and high cost: Plants have a long growth cycle, typically requiring one to two years to harvest. Furthermore, the content of the target active ingredient in the whole plant is extremely low; for example, lotusine typically accounts for less than one percent of the content in dried lotus seeds. This results in extremely low extraction efficiency and requires processing large quantities of plant material, leading to high costs.

[0007] 4. Complete lack of targeting: Traditional breeding has a long cycle and it is difficult to target and enhance the specific component spectrum required for specific health functions, thus failing to achieve the precision agriculture goal of "production on demand".

[0008] While existing plant cell suspension culture technologies have achieved success in species such as yew, they largely focus on simple cell expansion and the acquisition of single components. Current technologies lack a holistic solution that uses clearly defined human health needs as systemic input, employs multi-module closed-loop linkages, and reverse-engineers to maximize the yield of specific target components. This is particularly true for precious species like lotus, with its complex composition and diverse functions; how to construct a direct pathway from "health targets" to "standardized products" remains an unresolved technological gap.

[0009] Plant cell suspension culture technology is considered an effective way to solve the above problems. Currently, this technology has been successfully applied to medicinal plants such as yew and honeysuckle. For example, yew stem cell suspension culture in a 1000L reactor can produce 0.1-0.5 kg of paclitaxel in 4 months, with a direct cost of less than 10,000 yuan. However, existing technologies mostly focus on simple cell culture and expansion, and have not yet formed a comprehensive solution that closely integrates the culture system with clearly defined health targets, and uses systems engineering methods to directionally design and maximize the yield of target components. Especially for lotus, a species with multiple health values, how to construct a closed-loop system that designs the "production process" from "health needs" remains a technological gap. Therefore, those skilled in the art provide a lotus cell suspension factory system and method for the targeted production of highly active components based on health targets to solve the problems mentioned in the background. Summary of the Invention

[0010] The purpose of this invention is to provide a system and method for producing highly active ingredients from lotus cells based on health targets, in order to solve the problems mentioned in the background art.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] A lotus cell suspension factory system for the targeted production of highly active ingredients based on health targets includes four functional modules connected in sequence:

[0013] 1. Health-oriented cell line design module

[0014] Germplasm Resource Bank: Collects and preserves medicinal lotus materials of different genotypes (including lotus seeds, hypocotyls, lotus roots, etc.), covering characteristic germplasm with high content of lotus seed alkaloids, lotus leaf alkaloids, etc.

[0015] Multi-omics analysis unit: Integrates transcriptomic, metabolomic, and proteomic data to identify key genes and enzymes (such as key enzymes in the phenylpropane pathway, such as PAL, C4H, and 4CL) that regulate the synthesis of target active ingredients (such as lotusine and flavonoids);

[0016] Cell engineering operation unit: CRISPR / Cas9 gene editing technology is used to knock out negative regulators of metabolic pathways, or Agrobacterium-mediated transformation is used to overexpress rate-limiting enzyme genes; at the same time, single-cell cloning technology is used to screen stable and high-yielding cell lines.

[0017] 2. Intelligent large-scale cultivation module

[0018] Bioreactor unit: Airlift bioreactors or wave bioreactors are preferred (low shear force, which is conducive to the accumulation of secondary metabolites), with the volume scaled up from 5L for laboratory use to 1000L for industrial use.

[0019] Online monitoring sensor array: Real-time monitoring of temperature, pH, dissolved oxygen (DO), cell density and viability;

[0020] Process control system: Based on machine learning algorithms, the stirring rate, aeration rate and nutrient supply are dynamically adjusted to maintain optimal growth conditions (temperature 25±1℃, pH 5.8-6.0, DO≥40%).

[0021] 3. Precision Metabolic Induction Module

[0022] Elicitor addition unit: Automatically injects optimized concentrations of inducers at specific stages of cell culture (typically late logarithmic growth or early stationary phase), including:

[0023] Methyl jasmonate (50-200 μM): significantly enhances the synthesis of lotusine;

[0024] Salicylic acid (10-100μM): Promotes the accumulation of flavonoids;

[0025] Yeast extract (1-5 g / L): synergistically enhances stress response;

[0026] Precursor feeding unit: Add biosynthetic precursors of the target components (such as L-tyrosine for lotus seed alkaloid synthesis, and phenylalanine for flavonoid synthesis) at a concentration of 0.1-2 mM.

[0027] 4. Targeted Separation and Product Development Module

[0028] Harvesting unit: Cells are collected by vacuum filtration or centrifugation;

[0029] Extraction and purification unit: Ultrasonic-assisted extraction (temperature 50℃, time 10 minutes, material-to-liquid ratio 1:20g / m is preferred) or supercritical CO2 extraction (green and efficient);

[0030] Formulation Unit: After further purification of standardized extracts (such as high-speed countercurrent chromatography, preparative HPLC), they are developed into dosage forms such as capsules, oral liquids, and tablets.

[0031] (II) Method and Steps for Targeted Production of Highly Active Ingredients from Lotus Cell Suspension Factory Based on Healthy Targets

[0032] Includes the following steps:

[0033] S1: Identification of Healthy Targets and Creation of Cell Lines

[0034] Identify at least one health target and corresponding target active ingredient related to anti-epidemic (target: key enzymes of viral replication), anti-depression (target: monoamine oxidase MAO), or reproductive health (target: oxidative stress pathway);

[0035] High-yield cell lines capable of efficiently synthesizing target active ingredients can be created by screening from lotus germplasm or by using gene editing technology: for example, the LLX-1 cell line with a lotusine content of 0.8%-2.5% by dry weight was obtained by screening under methyl jasmonate stress.

[0036] S2: Intelligent Large-Scale Cultivation

[0037] High-yield cell lines were seeded into a bioreactor using an optimized culture medium (e.g., MS medium supplemented with 0.1-1 mg / L 2,4-D and 0.01-0.1 mg / L KT).

[0038] Suspension culture was carried out under the control of a process control system (inoculation density 1.0-1.5×10⁻⁶). 5 (cells / mL) to achieve rapid accumulation of cell biomass (culture cycle 14-21 days).

[0039] S3: Precise Metabolic Induction

[0040] During the 10-14 day period of culture (early stage of stabilization), add optimized inducers (e.g., 100 μM methyl jasmonate + 2 g / L yeast extract) and / or precursors (e.g., 1 mM L-tyrosine).

[0041] Continue induction culture for 4-7 days to increase the content of the target component by 2-3 times.

[0042] S4: Targeted Separation and Product Development

[0043] Harvest cells (biomass dry weight typically reaches 15-30 g / L), and use ultrasound-assisted ethanol extraction or supercritical CO2 extraction to obtain an extract rich in the target active ingredients;

[0044] After further purification, it can be integrated into functional foods, dietary supplements, or pharmaceutical products.

[0045] Compared with the prior art, the beneficial effects of the present invention are prominent and unexpected:

[0046] 1. It pioneered a new paradigm of "demand-driven production": for the first time, "health targets" were used as the source input to reverse-engineer the entire cell factory system, achieving precise alignment from ingredients and functions to products, and solving the fundamental problem of the lack of targeting in the traditional model.

[0047] 2. Order-of-magnitude increases in yield and efficiency: Through a triple strategy of creating high-yield cell lines, intelligent culture, and precise metabolic induction, the yield of target components far exceeds that of traditional cultivation. Specifically, in the preferred embodiment, the lotus seed heart cells cultured according to this invention can reach a lotus seed heart heart alkaloid content of 2.5% of the cell dry weight, which is more than 2.5 times that of traditional lotus seed heart medicinal materials (lotus seed heart heart alkaloid content is usually less than 1%). At the same time, the production cycle is shortened from one to two years in traditional cultivation to less than one month, and the efficiency is improved by more than 90%.

[0048] 3. Unprecedented controllability and safety in quality: The entire process is carried out in a closed, sterile bioreactor, completely avoiding pesticide and heavy metal contamination. Through online monitoring and process control, the variability of target component content between different production batches is less than 5%, achieving ultimate uniformity and standardization of medicinal material quality, which is unattainable in traditional agriculture.

[0049] 4. Highly Sustainable and Environmentally Friendly: This invention does not rely on arable land or climate conditions and can be produced in cities or indoors, greatly protecting wild and agricultural lotus resources. Preliminary assessments show that for every kilogram of lotus seed alkaloids produced, the carbon footprint of this invention system is reduced by more than 60% compared to traditional cultivation and extraction methods, aligning with the development direction of green manufacturing.

[0050] 5. Synergistic amplification effect of technology integration: This invention is not a simple accumulation of technologies, but rather a deep integration of technologies such as multi-omics analysis, gene editing, process control, and green manufacturing, resulting in a synergistic effect where "one plus one is greater than two." For example, we found that when methyl jasmonate and yeast extract are used as a compound inducer, the induction effect on lotusine is not simply additive, but produces a significant synergistic effect, with the yield being 50% to 80% higher than when using a single inducer, which is something that those skilled in the art could not have known beforehand. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of a lotus cell suspension factory system for the targeted production of highly active ingredients based on health targets.

[0052] Figure 2 This is a schematic diagram of a process for a lotus cell suspension factory method for the targeted production of highly active ingredients based on health targets. Detailed Implementation

[0053] 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.

[0054] Example 1: Targeted production of extracts with high lotus seed alkaloid content for use in anti-epidemic functional foods

[0055] Health targets and target components: antiviral (acting on viral envelope proteins and host cell ACE2 receptors); neperine (known to inhibit NF-κB signaling pathway and CaMKII phosphorylation).

[0056] Cell line creation: Callus was induced from the hypocotyl of the medicinal lotus variety "Tianxianlian 28," which has a high lotusin content. After multiple generations of domestication and selection under methyl jasmonate (50-200 μM) stress, a high-yielding cell line, LLX-1, with a lotusin content of 0.8% by dry weight was obtained. Callus was also induced from the hypocotyl of "Tianxianlian 28" and subjected to six generations of methyl jasmonate stress domestication and selection. During the selection process, more than one hundred cell line clones were monitored, and a stable, rapidly growing, high-yielding cell line, named LLX-1, was finally obtained. This cell line can achieve a lotusin content of 0.8% of cell dry weight in basal culture without induction.

[0057] Large-scale culture: LLX-1 cell lines were seeded in a 10-liter airlift bioreactor using optimized MS medium (supplemented with 0.5 mg / L 2,4-D, 0.05 mg / L KT, and 3% sucrose), at a temperature of 25 ± 1 °C, dissolved oxygen maintained at 40%, and a stirring rate of 80 rpm. LLX-1 cell lines were also seeded in a 10-liter airlift bioreactor. On day 12, when cell growth entered the early stationary phase, methyl jasmonate at a concentration of 100 μmol and yeast extract at 2 g / L were simultaneously added to the system as a combined inducer.

[0058] Precise induction: On day 12 (early stage of stationary phase), 100 μM methyl jasmonate and 2 g / L yeast extract were added as a compound inducer and cultured for another 4 days. After another 4 days of induction, we observed a sharp increase in the content of lotusine in the cells.

[0059] Harvesting and Product Development: Cells were harvested, freeze-dried, and then extracted with ethanol using ultrasound-assisted extraction (conditions: 50℃, 10 minutes, material-to-liquid ratio 1:20). The content of basidimine in the cells after induction was found to increase to 2.5% of the dry weight. This extract was mixed with microcrystalline cellulose and magnesium stearate to prepare a 500mg / capsule dietary supplement, providing a recommended daily dose of 50mg of basidimine. Biomass was determined after freeze-drying. Ultrasound-assisted ethanol extraction was used for processing, and quantitative analysis was performed by high-performance liquid chromatography (HPLC). The results showed that the basidimine content in the induced LLX-1 cells reached 2.5% of the cell dry weight. In contrast, the basidimine content in the control group without the inducer under the same culture conditions was only 0.8%; while the contents of methyl jasmonate or yeast extract alone were 1.5% and 0.9%, respectively. This fully demonstrates the synergistic effect of the combined inducer.

[0060] Example 2: Targeted Production of Antidepressant Compound Extract

[0061] Health targets and target ingredients: antidepressant (inhibits monoamine oxidase MAO-A); lotus leaf alkaloid and luteolin.

[0062] Cell line creation: Using nuciferine and luteolin as screening indicators, cell line AD-1 (basic nuciferine content 0.5%, luteolin content 1.2%) was screened from mixed suspension cell populations using single-cell cloning technology; cell line AD-1, which can simultaneously produce high levels of both nuciferine and luteolin, was screened from mixed suspension cell populations using single-cell cloning technology.

[0063] Large-scale culture and induction: AD-1 cell lines were cultured in a 5-liter wave-type bioreactor (seeding density 1.2 × 10⁶ cells / year). 5 (cells / mL). In a 5-liter wave bioreactor, 50 μmol of salicylic acid was added on day 10 of culture for specific induction.

[0064] Harvesting and Product Development: Cells were harvested after five days of induction culture. Analysis showed that the content of nuciferine in the cells increased from 0.5% before induction to 1.2%; the content of luteolin increased from 1.2% to 2.1%. The extract demonstrated strong inhibitory activity against monoamine oxidase (MAO-A) in in vitro enzyme activity experiments.

[0065] Example 3: Targeted Production of Reproductive Health Protective Components

[0066] Health targets and target ingredients: reproductive health (antioxidant and hormone regulation); epigallocatechin gallate (EGCG) and specific proanthocyanidins.

[0067] Cell line creation: Using CRISPR / Cas9 technology, a gene highly homologous to TT8, a negative regulator of proanthocyanidin synthesis, was precisely knocked out in lotus cells. Molecular identification and metabolomics analysis yielded the engineered cell line RG-1. This cell line, without induction, achieved a basal proanthocyanidin content of 5.2% of dry weight, which is 2.3 times that of wild-type cells.

[0068] Large-scale induction and data: RG-1 cells were cultured in a 20-liter reactor, and on day 11, 75 μmol methyl jasmonate and 1.5 g / L yeast extract were added for enhanced induction. After induction, the proanthocyanidin content in the cells further increased to 12%, and the content of another important antioxidant, epigallocatechin gallate, also reached 1.8% of dry weight.

[0069] Extraction technology: In this case, a water-based extraction method was used to replace organic solvents. The entire production process has no organic solvent residues or emissions, making it safer and more environmentally friendly, and significantly reducing costs.

[0070] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A medicinal plant cell suspension factory system for the targeted production of high-activity ingredients based on health targets, characterized by, The application comprises the following steps: a) determining the target active ingredient corresponding to the health target point, and creating a high-yield cell line of the medicinal lotus that can efficiently synthesize the ingredient; b) performing large-scale suspension culture of the high-yield cell line in a bioreactor; c) applying induction strategies at specific stages of culture to improve the yield of the target active ingredient; d) harvesting cells, extracting and purifying the target active ingredient, and developing it into a health product.

2. The plant cell suspension factory system based on the health target for the production of high-activity ingredients according to claim 1, characterized in that, The health target point includes anti-epidemic, anti-depression, or reproductive health; the target active ingredient includes lotus heart alkaloids, lotus leaf alkaloids, quercetin, chrysanthemum glycosides, or epigallocatechin gallate.

3. A method for producing a high active ingredient based on a health target point oriented pharmaceutical plant cell suspension factory, characterized by, The application comprises the following steps: a) determining the target active ingredient corresponding to the health target point, and creating a high-yield cell line of the medicinal lotus that can efficiently synthesize the ingredient; b) performing large-scale suspension culture of the high-yield cell line in a bioreactor; c) applying induction strategies at specific stages of culture to improve the yield of the target active ingredient; d) harvesting cells, extracting and purifying the target active ingredient, and developing it into a health product.

4. The method of claim 3, wherein the method is a plant cell suspension factory process for the targeted production of high activity ingredients based on health targets. In step a), the method for creating the high-yield cell line includes: developing molecular markers based on multi-omics analysis for assisted screening, or using CRISPR / Cas9 gene editing technology to knockout negative regulators in the metabolic pathway.

5. The method of claim 3, wherein the method is a plant cell suspension factory process for the targeted production of high activity ingredients based on health targets, characterized in that, In step b), the bioreactor used is a gas-lift or wave bioreactor, and the culture process is automatically regulated by an online monitoring and feedback control system for temperature, pH, and dissolved oxygen.

6. The method of claim 3, wherein the method is a plant cell suspension factory process for the targeted production of high activity ingredients based on health targets. In step c), the induction strategy is to add one or more of methyl jasmonate, salicylic acid, or yeast extract as an inducer to the culture system at the end of the logarithmic growth phase or the early stationary phase of cell culture.

7. A method for producing high active ingredients based on health target point orientation according to claim 3 or 6, characterized in that, In step c), the induction strategy also includes adding biosynthetic precursor substances of the target active ingredient to the culture system.

8. The method of claim 1, wherein the method is a method of producing a high-activity ingredient based on a health target point in a plant cell suspension factory, characterized by, In step d), the extraction technology used is ultrasonic-assisted extraction or supercritical CO2 extraction.

9. The method of claim 1, wherein the method is a method of producing a high-activity ingredient based on a health target point in a plant cell suspension factory, characterized by, In step d), the health product is a functional food, a dietary supplement, or a pharmaceutical raw material.

10. A pharmaceutical ginseng cell extract prepared by the method of any one of claims 3 to 9, wherein the extract is characterized by, It is rich in lotus heart alkaloids, lotus leaf alkaloids, or specific flavonoid ingredients, and the content of the ingredients is not less than 1% of the dry weight of the cells.

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