Pseudo-ginseng adventitious root high-density suspension culture system and method

Abstract

The invention discloses a pseudo-ginseng adventitious root high-density suspension culture system and method, the pseudo-ginseng adventitious root high-density suspension culture system comprises a ceramic blowing device provided with a blowing port and a culture tank provided with an air inlet, the blowing port of the ceramic blowing device is fixedly connected with the air inlet of the culture tank through a silicone tube, and a nano-silver ion electrostatic spinning antibacterial filter membrane is arranged in the silicone tube; the use method comprises the following steps: filling the double-chelate mineral culture medium into a culture container, inoculating pseudo-ginseng adventitious roots, and introducing gas into the culture medium through a ceramic blowing device; before entering the ceramic blowing device, the introduced gas is filtered and sterilized by a nano-silver ion electrostatic spinning antibacterial filter membrane, and after culturing for 35-40 days at 25-26 DEG C under a dark condition, the pseudo-ginseng adventitious roots are harvested, so that the aims of improving the utilization rate of nutrient elements, reducing the stress of ion concentration fluctuation on cells, improving the oxygen dissolving efficiency and improving the quality of the pseudo-ginseng adventitious roots are achieved. The airflow intensity is reduced, the root system restricts high-density culture, the suspension culture period is long, and the purpose of isolating microorganisms by a closed ventilation system is achieved.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and plant cell engineering, specifically to a high-density suspension culture system and method for Panax notoginseng adventitious roots, and particularly to an innovative system and method that integrates double-chelated mineral nutrient supply, efficient gas mass transfer using 3D-printed porous ceramics, and online antibacterial filtration technology using nano-silver ions to synergistically enhance the biomass and saponin content of Panax notoginseng adventitious roots. Background Technology

[0002] Panax notoginseng is a traditional and precious Chinese medicinal herb, and its main active ingredient is saponins. Utilizing the adventitious roots of Panax notoginseng for suspension culture is an effective way to replace wild resources and achieve sustainable saponin production.

[0003] However, the industrialization of this technology faces three major technical bottlenecks: First, in terms of nutrient supply, traditional culture media use inorganic salts such as FeSO4 and CaCl2. Among them, Fe²⁺ and Ca²⁺ are prone to form insoluble precipitates such as FePO4 and Ca3(PO4)2 with phosphate ions in the culture medium or organic acids secreted by the plant itself, resulting in low nutrient utilization and stress on cells caused by fluctuations in ion concentration.

[0004] Secondly, in terms of gas mass transfer, adventitious roots have a high oxygen requirement for growth, but their tissues are fragile and have poor shear resistance. Traditional aeration devices such as bubble heads and air stones produce large and uneven bubbles, resulting in low dissolved oxygen efficiency. Furthermore, violent airflow can easily damage the root system, which restricts high-density cultivation.

[0005] Finally, regarding process control, the long suspension culture cycle and open ventilation system make it easy for airborne microorganisms to be introduced, leading to contamination. Traditional filter membranes only have physical filtration functions, are prone to clogging, and lack active antibacterial capabilities. Once damaged, the entire batch of culture fails, resulting in high risk and high cost.

[0006] Existing technologies are mostly improvements targeting single problems, lacking integrated solutions that can systematically address the three major challenges of nutrition, mass transfer, and contamination. Summary of the Invention

[0007] The purpose of this invention is to solve the problems of low nutrient utilization rate, cell stress caused by ion concentration fluctuations, low dissolved oxygen efficiency, root damage caused by strong airflow which restricts high-density culture, long suspension culture cycle, and open ventilation system which easily introduces airborne microorganisms and causes pollution.

[0008] To solve the above problems, the present invention provides a high-density suspension culture system for adventitious roots of Panax notoginseng, including a culture tank and a ceramic aeration device. The ceramic aeration device is provided with an aeration port, and the culture tank is provided with an air inlet. The aeration port of the ceramic aeration device and the air inlet of the culture tank are fixedly connected by a silicone tube. A nano-silver ion electrospun antibacterial filter membrane is provided in the silicone tube, and the culture tank is filled with a double chelated mineral culture medium.

[0009] In a preferred embodiment, the double-chelated mineral culture medium contains an EDTA-Fe chelate as an iron source and an EDDHA-Ca chelate as a calcium source, wherein the concentration of the EDTA-Fe chelate is 27.8 mg / L and the concentration of the EDDHA-Ca chelate is 440 mg / L.

[0010] In a preferred embodiment, the surface of the ceramic blowing device is provided with a regular array of air vents with a vent diameter of 242-258 μm, through which gas is introduced into the culture medium.

[0011] In a preferred embodiment, the pore size of the nano-silver ion electrospun antibacterial filter membrane is 0.22-0.25 μm, and the loading of nano-silver ions is 0.5-1.0 wt%.

[0012] A method for high-density suspension culture of Panax notoginseng adventitious roots based on the above system includes the following steps: Step 1: Fill the culture tank with the double-chelated mineral culture medium and inoculate with Panax notoginseng adventitious roots; Step 2: Introduce gas into the double-chelated mineral culture medium through the ceramic air blowing device; Step 3: Before entering the culture tank, the gas is filtered and sterilized through the nano-silver ion electrospun antibacterial filter membrane; Step 4: After cultivating at 25-26℃ in the dark for 35-40 days, harvest the adventitious roots of Panax notoginseng.

[0013] In the preferred embodiment, a constant gas flow rate of 0.25-0.5 L / min is introduced into a 50 L tank in step 2. The gas flow rate affects the dissolved oxygen efficiency, and the adventitious roots of Panax notoginseng have a high oxygen requirement for growth, so it is necessary to ensure a stable oxygen concentration in the bioreactor.

[0014] 7. The method for high-density suspension culture of Panax notoginseng adventitious roots according to claim 5, characterized in that, during the culture process, the nano-silver ion electrospun antibacterial filter membrane is replaced every 5-7 days.

[0015] The beneficial effects of this invention are: 1. Breakthrough in nutrient supply: The double chelation system fundamentally avoids the precipitation problem of Fe and Ca ions, achieves stable and slow release of ions, significantly improves nutrient utilization, and provides a stable induction environment for saponin synthesis, effectively reducing the stress on cells caused by fluctuations in ion concentration.

[0016] 2. Mass transfer efficiency innovation: The 3D printed ceramic device can generate micron-sized bubbles with uniform size and good dispersion, which greatly improves the oxygen dissolution efficiency. Compared with traditional ventilation devices, the airflow is gentler and can effectively reduce shear damage to adventitious roots, providing sufficient oxygen for high-density growth of adventitious roots.

[0017] 3. Significant pollution control: The nano silver ion filter membrane combines physical filtration and chemical antibacterial properties. It can effectively intercept microorganisms in the air and exert antibacterial effects, greatly reducing the risk of pollution during the cultivation process and ensuring the smooth progress of long-term cultivation.

[0018] 4. Significant system synergy: The three core technology modules are organically integrated to jointly construct an ideal culture microenvironment that is nutritionally balanced, gas-rich, sterile and safe, enabling the biomass growth rate of Panax notoginseng adventitious roots to reach 380% and the total saponin content to increase to 9.5%, breaking through the key technical bottleneck of industrialization. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of a scanning electron microscope (SEM) image of the double chelate mineral system of the present invention, magnified 5000×. Figure 2 This is a schematic diagram of a scanning electron microscope (SEM) image of the interior of the 3D-printed porous ceramic of the present invention, magnified 200×. Figure 3 This is a diagram showing the growth status of the adventitious roots cultivated according to the present invention; Figure 4 This is a diagram showing the growth status of adventitious roots cultivated using traditional methods. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited thereto. Example

[0021] A high-density suspension culture system and method for Panax notoginseng adventitious roots based on a double-chelated mineral system and aseptic mass transfer, comprising: system construction and culture of Panax notoginseng adventitious roots; Culture medium preparation: Prepare the double chelated mineral culture medium according to the formula shown above, and adjust the pH to 6.0 using 0.1 Mol / L NaOH or HCl solution; Figure 1 As shown, uniformly dispersed microparticles are visible, with no obvious agglomeration or large crystalline precipitates between particles, and the overall structure is loose and uniform.

[0022] Preparation of ceramic air blowing device: Hydroxyapatite powder with a particle size of 50-100 nm and calcium silicate powder are mixed at a mass ratio of 2:8, with 1% polyethylene glycol added as a dispersant. The mixture is ball-milled into a slurry, which is then printed into a ring-shaped green body using an extrusion 3D printer. The finished product is obtained after sintering at 1200℃ for 4 hours. Figure 2 The diagram shows a dendritic interconnected channel with visible multi-level branches. The main channel has a diameter of about several hundred micrometers, while the diameter of the branch channels gradually decreases to 242-258 μm. The channel walls are smooth and have good connectivity, without blockage or breakage.

[0023] Preparation of antibacterial filter membrane by electrospun nano silver ions: PVA aqueous solution and PLA chloroform solution were mixed at a mass ratio of 3:7, and 0.8 wt% of nano silver ions with a particle size of 10-20 nm were added. The mixture was electrospun (voltage 18 kV, receiving distance 15 cm) to form filter membranes with a thickness of about 60 μm, which were then stacked for use.

[0024] Culture process: Add culture medium to the culture tank and inoculate with 1.2 g (fresh weight) of adventitious roots (0.5-0.8 cm in length) induced by Panax notoginseng callus. Fix a ceramic air blowing device to the air inlet of the culture tank, and connect a nano-silver antibacterial filter membrane in series in the air inlet silicone tube. Set the air pump flow rate to 0.7 L / min, introduce sterile air, and incubate in a completely dark incubator at 25℃ for 40 days.

[0025] Results: Adventitious roots were harvested after cultivation. The fresh weight of the adventitious roots reached 5.76 g, and the biomass growth rate was 380%. The saponin content was determined by high-performance liquid chromatography (HPLC), and the total content of R1, Rg1, and Rb1 was 9.5%. No contamination occurred during the entire cultivation period.

[0026] Comparative Example 1: The traditional method includes replacing the iron and calcium sources in the culture medium with equimolar amounts of FeSO4·7H2O and CaCl2·2H2O, replacing the aeration device with ordinary air stones, and removing the antibacterial filter membrane, with the remaining conditions being the same as in Example 1.

[0027] Results: After 18 days of culture, a large amount of white precipitate appeared at the bottom of the culture medium, the fresh weight of adventitious roots was only 2.60 g, the growth rate was 117%, the total saponin content was 7.2%, and bacterial contamination occurred on the 15th day of culture.

[0028] Comparative Example 2: The absence of the antibacterial filter membrane includes: except for the removal of the nano-silver antibacterial filter membrane in the air intake passage, all other conditions are exactly the same as in Example 1.

[0029] Results: The adventitious roots grew well, with a fresh weight of 5.10 g and a saponin content of 9.3%, but fungal contamination occurred on the 16th day of culture.

[0030] Table 1. Performance Comparison of Different Culture Systems Comparative Example 2 was terminated early due to pollution; the data are pre-pollution measurements. like Figure 3 As shown, the adventitious roots cultivated by this invention are thinner and longer, more loosely distributed, and more white new fine roots can be seen, making the whole plant appear more expansive. like Figure 4 As shown, the adventitious roots cultivated by the traditional method are relatively shorter and thicker, with a higher degree of aggregation. The yellow tissue is denser, and the number and length of fine roots are not as obvious as in the first picture.

[0031] The comparative data of the examples and comparative examples fully demonstrate that the system of the present invention performs best in four key dimensions: biomass growth, accumulation of effective components, pollution control, and culture medium stability. Compared with the traditional system, the present invention can increase the biomass growth rate by 3.2 times while increasing the saponin content by 31.9%, and there is no interference from pollutants throughout the process, providing a more reliable technical solution for the large-scale and high-quality cultivation of Panax notoginseng adventitious roots.

[0032] The above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. It should be noted that any other improvements and variations that can be directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present application should be considered to be included within the protection scope of the present invention.

Claims

1. A high-density suspension culture system for adventitious roots of Panax notoginseng, comprising a culture tank, characterized in that, A ceramic air blowing device is provided, wherein the ceramic air blowing device is provided with an air blowing port, the culture tank is provided with an air inlet, the air blowing port of the ceramic air blowing device and the air inlet of the culture tank are fixedly connected by a silicone tube, a nano silver ion electrospun antibacterial filter membrane is provided in the silicone tube, and the culture tank is filled with a double chelated mineral culture medium.

2. The system according to claim 1, characterized in that, The double-chelated mineral culture medium contains EDTA-Fe chelate as an iron source and EDDHA-Ca chelate as a calcium source. The concentration of EDTA-Fe chelate is 27.8 mg / L and the concentration of EDDHA-Ca chelate is 440 mg / L.

3. The high-density suspension culture system for adventitious roots of Panax notoginseng according to claim 1, characterized in that, The surface of the ceramic air blowing device is provided with a regular array of air vents with a pore diameter of 242-258 μm, which allows gas to be introduced into the culture medium.

4. The system according to claim 1, characterized in that, The nano-silver ion electrospun antibacterial filter membrane has a pore size of 0.22-0.25 μm and a nano-silver ion loading of 0.5-1.0 wt%.

5. A method for high-density suspension culture of Panax notoginseng adventitious roots using the system described in any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Fill the culture tank with the double-chelated mineral culture medium and inoculate with Panax notoginseng adventitious roots; Step 2: Introduce gas into the double-chelated mineral culture medium through the ceramic air blowing device; Step 3: Before entering the culture tank, the gas is filtered and sterilized through the nano-silver ion electrospun antibacterial filter membrane; Step 4: After cultivating at 25-26℃ in the dark for 35-40 days, harvest the adventitious roots of Panax notoginseng.

6. The method for high-density suspension culture of Panax notoginseng adventitious roots according to claim 5, characterized in that, In step 2, a constant gas flow rate of 0.25-0.5 L / min is introduced into a 50 L tank. The gas flow rate affects the dissolved oxygen efficiency. The adventitious roots of Panax notoginseng have a high oxygen requirement for growth, so it is important to ensure a stable oxygen concentration in the bioreactor.

7. The method for high-density suspension culture of Panax notoginseng adventitious roots according to claim 5, characterized in that, During the cultivation process, the nano-silver ion electrospun antibacterial filter membrane is replaced every 5-7 days.

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