Method for culturing vietnamese sophora root suspension cells by using bioreactor

Abstract

The invention discloses a method for culturing subprostrate sophora suspension cells by using a bioreactor, and belongs to the technical field of plant tissue culture. The method comprises the following steps: selecting a vietnamese sophora root tissue culture seedling petiole as an explant, inoculating the petiole to a solid culture medium to induce a callus, and carrying out subculture to obtain a distraction callus; transferring into a liquid culture medium, and carrying out shaking table subculture to obtain suspension cell particles; and inoculating to a bubbling balloon bioreactor, and carrying out scale-up culture to obtain subprostrate sophora suspension cells. The method can efficiently obtain a large number of vietnamese sophora root suspension cells, is used for solving the problem of shortage of vietnamese sophora root resources, and provides a way for extraction of effective components and commercial production of vietnamese sophora root.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant tissue culture. More particularly, the present application relates to a method for culturing P. delavayi suspension cells by using a bioreactor. BACKGROUND

[0002] P. delavayi is a famous local medicinal material in Guangxi, a commonly used bulk traditional Chinese medicine, which is the dry root and rhizome of Millettia pachycarpa T. Chen var. delavayi (Dunn) Huang et al. Sophora tonkinensis P. delavayi is widely used in the medical field and has a large market demand. However, the natural growth of P. delavayi is limited. Its wild population is mainly distributed in the rock crevices of specific areas, and its growth environment is special and narrow, and its natural reproductive capacity is weak. In artificial cultivation, P. delavayi seeds are prone to fall off when mature, and the seed vigor declines rapidly after harvesting, resulting in low germination rate, insufficient seedling supply, and difficulty in large-scale planting, which cannot meet the market demand for P. delavayi resources. At the same time, over-exploitation of wild P. delavayi resources has led to a sharp decrease in resource reserves, and the resource shortage problem has become increasingly prominent.

[0003] In the prior art, there is a lack of a standardized process from inducing callus from P. delavayi explants to obtaining suspension cells by shake flask culture, and then realizing large-scale culture by using a bioreactor, which makes it difficult to stably and efficiently obtain a large amount of high-quality P. delavayi suspension cells. This makes it difficult to effectively alleviate the shortage of P. delavayi resources, and restricts the extraction and commercial production of its effective components. SUMMARY

[0004] The present application provides a method for culturing P. delavayi suspension cells by using a bioreactor, which can efficiently obtain a large amount of P. delavayi suspension cells, and solve the problem of P. delavayi resource shortage, and provide a way for the extraction and commercial production of its effective components.

[0005] In order to achieve these objects and other advantages of the present application, a method for culturing P. delavayi suspension cells by using a bioreactor is provided, which comprises the following steps: Step 1: selecting P. delavayi tissue culture seedling petioles as explants, cutting them into small pieces, and inoculating them into a solid culture medium to induce callus under dark conditions and at 25±2℃; subculturing every 18-22 days, and continuously subculturing 5-6 times until obtaining waterlogged callus with vigorous division, loose and easy to break, and loose; Step 2: transferring the callus into a liquid culture medium, and culturing it by shaking in a shaking bed under alternating light conditions of 12h / d darkness and 12h / d 2500-3000Lux, and at 25±2℃; subculturing every 14-16 days, and continuously subculturing 4-5 times in a shake flask to obtain suspension cell particles; Step 3: inoculate the suspension cell particles to a bubble-type air ball bioreactor, inject liquid medium into the bioreactor, and introduce air, and then carry out the bioreactor scale-up culture for 25-30 days under the conditions of 12h / d light cycle and 25±2℃ to obtain the suspension cells of S. mirrhiza.

[0006] Preferably, the solid medium in step 1 comprises MS basal medium, 0.4-0.6 mg / L 2,4-D, 1.8-2.2 mg / L NAA, 1.8-2.2 mg / L 6-BA, 28-32 g / L sucrose, 4.0-5.0 g / L agar, and 55-65 mg / L citric acid, and the pH is 5.7-5.9. The same liquid medium is used in step 2 and step 3, which comprises MS basal medium, 0.4-0.6 mg / L 2,4-D, 1.8-2.2 mg / L NAA, 1.8-2.2 mg / L 6-BA, and 28-32 g / L sucrose, and the pH is 5.7-5.9.

[0007] Preferably, the citric acid in step 1 is prepared into a solution, filtered through a 0.22 μm filter membrane, and then added to the warm solid medium which has not yet coagulated.

[0008] Preferably, mechanical dispersion is carried out during the callus shake culture in step 2. Every 120 h, the shake flask is moved to a sterile operating table, the suspension culture is filtered through a 200-mesh stainless steel screen, the retained material is transferred to a 50 mL centrifuge tube, 10 mL of MS basal salt solution containing 0.1 mol / L mannitol is added to the centrifuge tube, a 10 mL syringe is used to extract the mixture, and the mixture is repeatedly injected at a uniform speed of 3 seconds per time at a distance of 1 cm below the liquid surface for 10-15 times. After blowing, the suspension is mixed with the filtrate under the screen and returned to the original shake flask for continuous culture.

[0009] Preferably, the bioreactor comprises an aeration pump (1), a filter head (2), a bubble disc (3), a culture container (4), and a rubber plug (5). The aeration pump (1) is connected to the filter head (2) and the bubble disc (3) in sequence through pipelines. The bubble disc (3) is arranged at the bottom of the culture container (4). The aeration pump (1) is used to drive air to pass through the filter membrane for sterilization, and then the air is dispersed into micro-bubbles by the bubble disc (3) and enters the culture solution. The opening at the upper part of the culture container (4) is sealed by the rubber plug (5). A breathable sealing film (6) is attached to the surface of the rubber plug (5). A stainless steel injection port is additionally arranged beside the rubber plug (5) on the top cover, and a high-pressure resistant silicone rubber spacer is covered on the surface of the injection port.

[0010] Preferably, metabolic induction is carried out during the bioreactor scale-up culture in step 3. From the 7th day of culture, the cell density was determined every 24 hours, and when the density reached 2.8-3.2 times of the initial inoculation amount, i.e. in the middle of the exponential growth phase, induction was started; Through the sterile injection hole with silica gel spacer pre-installed on the top cover of the bioreactor, Tween-80 aqueous solution was injected to make the final concentration of Tween-80 in the system 0.006±0.001% by volume fraction, and after 30±5 minutes, methyl jasmonate ethanol mother liquor with a concentration of 50 mmol / L was injected in two times, 60% of the total amount was injected at the first time, and the remaining 40% was injected after 2 hours, so that the final concentration of methyl jasmonate was 100±10 μmol / L; During the injection, the air flow was maintained at 1.0vvm, the injection flow rate was 8-12 mL / min, and after the last addition was completed, the temperature was reduced to 23.5±0.5℃ within 2 hours and maintained for 36-48 hours, and then restored to 25±2℃.

[0011] The present application at least includes the following beneficial effects: Firstly, the present application aims at the standardization problem of large-scale culture of Euchresta japonicus suspended cells, and a systematic standardized process is constructed: by unifying the optimization of medium formula, sterilization conditions and temperature, illumination and other core culture parameters, standardizing the operation nodes such as inoculation, subculture and sampling, the stability and controllability of the culture environment and process are realized. The bubble type balloon bioreactor provides key support for the process: the built-in bubble disc realizes uniform aeration of micro-bubbles, and cooperates with the precise regulation of the air pump and the filter head to ensure the stability of dissolved oxygen under different culture scales; the sealing design combined with the sterile injection port effectively reduces the risk of contamination, and finally realizes the standardization of the culture process, the controllability of the parameters and the repeatability of the results, providing reliable support for industrialization amplification.

[0012] Secondly, the present application can significantly reduce the cell cluster particle size (D 50 ) of Euchresta japonicus suspended cells, improve the uniformity and survival rate of the cells, enhance the stability of the culture process, and reduce the fluctuation of biomass and component accumulation. Citric acid plays the role of an anti-browning agent, and as an antioxidant, it can prevent the oxidation of phenolic compounds from the source, has good anti-browning effect, and has no adverse effects on explants. Since citric acid is easily decomposed by heat, the method of adding it is to filter the prepared citric acid solution through a 0.22 μm filter membrane and then add it to the warm solid medium that has not yet coagulated.

[0013] Thirdly, the present application implements methyl jasmonate metabolic induction in the middle of the exponential growth phase, which can directionally activate the activity of key enzymes (such as tryptophan decarboxylase) for alkaloid synthesis, significantly improves the accumulation amount of effective components such as matrine and oxymatrine under the premise of not inhibiting cell growth and maintaining stable biomass, and the induction effect is stable and controllable.

[0014] Other advantages, objects, and features of the application will be apparent from the following specification and appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The overall morphology of water spot-like callus formed in a culture dish according to one technical solution of the application; Figure 2 The surface close-up of water spot-like callus taken under a stereomicroscope according to one technical solution of the application; Figure 3 The structural schematic diagram of a bioreactor according to one technical solution of the application, in which: 1-air pump, 2-filter head, 3-bubble disc, 4-culture container, 5-rubber plug, 6-air-permeable sealing film. DETAILED DESCRIPTION

[0016] The application will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement the application according to the description and the drawings.

[0017] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0018] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified; in the description of the application, it should be noted that unless otherwise explicitly specified and limited, the terms "mount", "connect", "arrange" should be understood broadly, for example, it can be fixedly connected, arranged, or detachably connected, arranged, or integrally connected, arranged. For those skilled in the art, the specific meanings of the above terms in the application can be understood according to the specific circumstances. The orientations or positional relationships indicated by the terms "transverse", "longitudinal", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the application and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the application.

[0019] <EMBODIMENT 1> The method for culturing Euchroma Urophylla suspension cells by using a bioreactor comprises the following steps: Step 1: inducing callus formation from leaf petioles of Euchroma Urophylla tissue culture seedlings: The leaf stalks of the tissue culture seedlings of Millettia pachycarpa Benth. are selected as explants, and the leaf stalks of the tissue culture seedlings of Millettia pachycarpa Benth. are cut and placed in an induction medium, the induction medium is MS basic medium + 0.5 mg / L 2,4-D + 2.0 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose + 4.5 g / L agar + 60 mg / L citric acid, and the pH of the solid medium is 5.8. The culture conditions are as follows: dark culture, culture temperature 25±2℃, culture cycle is subcultured every 20d, continuous subculture for 5 times, and a large amount of vigorous, soft and easy-to-break, and loose water-stained callus is obtained, as shown in Figures 1-2 , Figure 1 The callus is shown in the whole morphology of the water-stained callus formed in the culture dish, which is light cream to light yellow, the edge of the callus block is irregular, the surface is spongy or villous micro-protrusion, the block structure is loose, and the callus block can be broken into small blocks by light touch, and there is no obvious adhesion between the callus blocks, which is beneficial to subsequent liquid suspension culture, Figure 2 The surface of the water-stained callus is shown in the close-up taken under the stereomicroscope, which is light yellow to light cream, the surface is moist and lustrous, and the local part is accompanied by water-stained reflection, the texture is loose and porous, the tissue surface is composed of a large number of small circular or polygonal cell groups, the cell group gap is clear, the structure is loose, and it is suggested that the cell-cell connection is weak and easy to be mechanically dissociated.

[0020] Step 2: Shaking culture of Millettia pachycarpa Benth. suspension cells: The water-stained callus obtained in step 1 is inoculated in a liquid medium for shaking culture: the medium is MS basic medium + 0.5 mg / L 2,4-D + 2.0 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose, the pH of the liquid medium is 5.8, and the liquid medium is sterilized at 121℃ for 25 min. The culture conditions are as follows: alternating dark 12h / d, 2500-3000Lux 12h / d, culture temperature 25±2℃, shaking speed 120r / min, culture cycle is subcultured every 15d, continuous subculture for 4 generations, and a large amount of uniform size suspension cells are obtained.

[0021] Step 3: Scale-up culture of Millettia pachycarpa Benth. suspension cells by using a bioreactor: In a 5L bubble-type balloon bioreactor (BTBB), 4L liquid medium was injected, the medium was MS basal medium + 0.5mg / L 2,4-D + 2.0mg / L NAA + 2.0mg / L 6-BA + 30g / L sucrose, the pH of the liquid medium was 5.8, sterilized at 121℃ for 25min, and then injected into the bioreactor after cooling, and then the step 2 cultured S. mirrhiza suspension cells were inoculated into the bioreactor, the culture conditions were 12h / d light cycle, culture temperature 25±2℃, and cultured for 4w to obtain a large amount of S. mirrhiza suspension cells.

[0022] As shown in Figure 3 , the bioreactor comprises an aeration pump (1), a filter head (2), a bubble disc (3), a culture container (4) and a rubber plug (5), the aeration pump (1) is connected to the filter head (2) and the bubble disc (3) in sequence through pipelines, the bubble disc (3) is arranged at the bottom of the culture container (4), and the aeration pump (1) is used to drive air to enter the culture solution in the form of micro-bubbles after sterilization by a filter membrane and dispersion by the bubble disc (3); the opening at the upper part of the culture container (4) is sealed by the rubber plug (5), and a breathable sealing film (6) is attached to the surface of the rubber plug (5).

[0023] <Embodiment 2> The method for culturing S. mirrhiza suspension cells by using a bioreactor comprises the following steps: Step 1: inducing leaf petioles of S. mirrhiza tissue culture seedlings to generate callus: The leaf petioles of S. mirrhiza tissue culture seedlings were selected as explants, and the leaf petioles of S. mirrhiza tissue culture seedlings were cut and placed in an induction medium, the induction medium was MS basal medium + 0.5mg / L 2,4-D + 2.0mg / L NAA + 2.0mg / L 6-BA + 30g / L sucrose + 4.5g / L agar + 60mg / L citric acid, and the pH of the solid medium was 5.8. The culture conditions were dark culture, culture temperature 25±2℃, and subculture every 20d, and the subculture was continuously performed for 5 times, and a large amount of vigorous division, soft and easy-to-break, and loose water-stained callus was generated, as shown in Figures 1-2 , as shown in Figure 1 , the whole morphology of the vigorous division, soft and easy-to-break, and loose water-stained callus formed in the culture dish is shown, the callus block is light cream to light yellow, the block edge is irregular, the surface is spongy or villous micro-protrusion, the block structure is loose, and the callus block can be broken into small blocks by light touch, and there is no obvious adhesion between the callus blocks, which is beneficial to subsequent liquid suspension culture, Figure 2The water spot-like callus surface was photographed under a stereomicroscope. The overall color was light yellow to light cream, the surface was moist and shiny, and some parts had water spot-like reflections. The texture was loose and porous, and the tissue surface was composed of a large number of small round or polygonal cell clusters. The cell cluster gap was clear, the structure was loose, and the intercellular connection was weak, which was easy to mechanically dissociate.

[0024] Step 2: Shake culture of Millettia pulchra suspension cells The water spot-like callus obtained in step 1 was inoculated in a liquid medium for shake culture: the medium was MS basal medium + 0.5 mg / L 2,4-D + 2.0 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose, and the pH of the liquid medium was 5.8, sterilized at 121°C for 25 min. The culture conditions were alternating darkness for 12 h / d and 2500-3000 Lux for 12 h / d, the culture temperature was 25±2°C, the shaking speed was 120 r / min, every 120 h the shake flask was moved to a sterile operation table, the suspension culture was filtered through a 200 mesh stainless steel screen, the retained material was transferred to a 50 mL centrifuge tube, 10 mL of MS basal salt solution containing 0.1 mol / L mannitol was added to the centrifuge tube, a 10 mL syringe was used to extract the mixed solution, and a syringe pump was used to repeatedly inject at a speed of 3 seconds / time at 1 cm below the liquid surface for 10 times. After blowing, the suspension was mixed with the filtrate under the screen and returned to the original shake flask for continuous culture. The culture period was every 15 d for subculture, and 4 generations of continuous subculture were obtained to obtain a large amount of uniform size suspension cells.

[0025] Step 3: Scale-up culture of Millettia pulchra suspension cells using a bioreactor In a 5L bubble-type balloon bioreactor (BTBB, Bubble-Type Balloon Bioreactor), 4L of liquid medium was injected, the medium was MS basal medium + 0.5 mg / L 2,4-D + 2.0 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose, and the pH of the liquid medium was 5.8, sterilized at 121°C for 25 min, and then injected into the bioreactor after cooling. Then the Millettia pulchra suspension cells obtained in step 2 were inoculated into the bioreactor, the culture conditions were light cycle for 12 h / d, culture temperature was 25±2°C, and culture for 4w, a large amount of Millettia pulchra suspension cells were obtained.

[0026] As Figure 3As shown, the bioreactor comprises aeration pump (1), filter head (2), bubble disc (3), culture container (4) and rubber plug (5), the aeration pump (1) is connected with the filter head (2) and the bubble disc (3) in sequence through pipeline, the bubble disc (3) is arranged at the bottom of the culture container (4), the aeration pump (1) is used for driving air to enter the culture solution as micro-bubbles through the bubble disc (3) after sterilization through the filter membrane; the opening at the upper part of the culture container (4) is sealed by the rubber plug (5), the surface of the rubber plug (5) is attached with the breathable sealing film (6), a stainless steel injection port is additionally arranged beside the rubber plug (5) on the top cover, the surface is covered with high-pressure resistant silicone gasket, when the injection needle is punctured, the positive pressure air prevents external microorganisms from invading; after the needle is pulled out, the silicone gasket is self-sealed.

[0027] <Embodiment 3> The method for culturing Euchroma Urophylla suspension cells by using a bioreactor comprises the following steps: Step 1: inducing leaf petiole of Euchroma Urophylla tissue culture seedling to generate callus: The leaf petiole of Euchroma Urophylla tissue culture seedling is selected as an explant, the leaf petiole of Euchroma Urophylla tissue culture seedling is cut off, and then placed in an induction culture medium, the induction culture medium is MS basic medium + 0.5 mg / L 2,4-D + 2.0 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose + 4.5 g / L agar + 60 mg / L citric acid, and the pH of the solid culture medium is 5.8. The culture condition is dark culture, the culture temperature is 25±2℃, the culture cycle is subcultured every 20 days, and the subculture is continuously performed for 5 times, a large amount of split vigorous, soft and fragile, and loose water-stained callus appears, as shown in Figures 1-2 , Figure 1 As shown is the overall morphology of the split vigorous, soft and fragile, and loose water-stained callus formed in the culture dish, the callus block is light cream white to light yellow, the block edge is irregular, the surface is spongy or villous micro-protrusion, the block structure is loose, and can be broken into small blocks by light touch, and there is no obvious adhesion between the tissue blocks, which is beneficial to subsequent liquid suspension culture, Figure 2 As shown is the surface close-up of the water-stained callus taken under a stereomicroscope, the whole is light yellow to light cream white, the surface is wet and lustrous, and part is accompanied by water-stained reflection, the texture is loose and porous, the tissue surface is composed of a large number of small circular or polygonal cell groups, the cell group gap is clear, the structure is loose, and it is suggested that the cell-cell connection is weak and easy to be mechanically dissociated.

[0028] Step 2: shock culture of Euchroma Urophylla suspension cells: The water-stained callus obtained in step 1 was inoculated in a liquid medium for shaking culture: the medium was MS basal medium + 0.5 mg / L 2,4-D + 2.0 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose, and the pH of the liquid medium was 5.8, which was sterilized at 121 ℃ for 25 min. The culture conditions were 12 h / d of alternating darkness and 2500-3000 Lux, 12 h / d, at 25±2 ℃, and the shaking speed was 120 r / min. Every 120 h, the shaking flask was moved to a sterile operating table, the suspension culture was filtered through a 200-mesh stainless steel screen, the retained material was transferred to a 50-mL centrifuge tube, 10 mL of MS basal salt solution containing 0.1 mol / L mannitol was added to the centrifuge tube, a 10-mL syringe was used to extract the mixture, and the mixture was repeatedly injected at a uniform speed of 3 seconds per time at 1 cm below the liquid surface for 10 times. After blowing, the suspension was mixed with the filtrate under the screen and returned to the original shaking flask for continuous culture. The culture period was 15 d for subculture, and the subculture was continuously performed for 4 generations to obtain a large amount of uniform-sized suspension cells.

[0029] Step 3: Scale-up culture of S. mirrhiza suspension cells using a bioreactor In a 5L bubble-type balloon bioreactor (BTBB), 4L of liquid medium was injected, the medium was MS basal medium + 0.5 mg / L 2,4-D + 2.0 mg / L NAA + 2.0 mg / L 6-BA + 30 g / L sucrose, and the pH of the liquid medium was 5.8, which was sterilized at 121 ℃ for 25 min and then injected into the bioreactor after cooling. Then, the S. mirrhiza suspension cells obtained in step 2 were inoculated into the bioreactor, and the culture conditions were 12 h / d of light cycle, 25±2 ℃, and metabolic induction was performed during the bioreactor scale-up culture: from the 7th day of culture, the cell density was determined every 24 h, and when the density reached 3 times the initial inoculation amount, it was in the middle of the exponential growth phase, and the induction was started. Through the sterile injection hole with a silicone septum pre-installed on the top cover of the bioreactor, a Tween-80 aqueous solution was injected to make the final concentration of Tween-80 0.006% by volume fraction. After 30 min, a methyl jasmonate ethanol stock solution with a concentration of 50 mmol / L was injected in two times, 60% of the total amount was injected first, and the remaining 40% was injected after 2 h, so that the final concentration of methyl jasmonate was 100 μmol / L. During this period, the air flow was maintained at 1.0vvm, the injection flow rate was 10 mL / min, and after the last addition was completed, the temperature was reduced to 23.5±0.5 ℃ within 2 h and maintained for 48 h, and then the temperature was restored to 25±2 ℃. After 4 weeks of culture, a large amount of S. mirrhiza suspension cells were obtained.

[0030] As Figure 3As shown, the bioreactor comprises aeration pump (1), filter head (2), bubble disc (3), culture container (4) and rubber plug (5), the aeration pump (1) is connected with the filter head (2) and the bubble disc (3) in sequence through pipelines, the bubble disc (3) is arranged at the bottom of the culture container (4), the aeration pump (1) is used to drive air to enter the culture solution as micro-bubbles through the bubble disc (3) after sterilization through a filter membrane; the opening at the upper part of the culture container (4) is sealed by the rubber plug (5), a breathable sealing film (6) is attached to the surface of the rubber plug (5), a stainless steel injection port is additionally arranged beside the rubber plug (5) on the top cover, a high-pressure resistant silicone gasket is covered on the surface, when the injection needle is punctured, the positive pressure air prevents external microorganisms from invading; after the needle is pulled out, the silicone gasket is self-sealed.

[0031] <Comparative Example 1> The method for culturing S. glabra suspended cells by using a bioreactor, steps 1-2 are the same as those in Example 1, and the difference is that step 3 uses a conventional 250 mL triangular flask for culture under the same culture medium and shaker conditions.

[0032] The average inoculation amount, fresh weight, dry weight and effective ingredients of the suspended cells determined in Example 1 and Comparative Example 1 are shown in Table 1. The average inoculation amount is the fresh weight of the suspended cells initially inoculated into the bioreactor / triangular flask in step 3 (measured by an electronic balance with an accuracy of 0.01 g). Fresh weight / dry weight: total fresh weight of the cells after step 3 culture (4w) and dry weight after drying at 60°C to a constant weight. Matrine / oxymatrine content measurement steps: take 0.5 g of the cell dry sample after step 3 culture (4w) and dry to a constant weight, add 5 mL of methanol for ultrasonic extraction for 30 min (power 300 W, temperature 40°C), centrifuge to take the supernatant; repeat the extraction for 2 times, combine the supernatants and dilute to 10 mL, detect by HPLC (C18 column, 250 mm, use a container to dilute to a constant weight; mobile phase: acetonitrile-0.02 mol / L potassium dihydrogen phosphate = 10:90; detection wavelength: 220 nm; flow rate: 1.0 mL / min; column temperature: 30°C), calculate the content by standard sample comparison.

[0033] Table 1 Group Average inoculation amount (g) Fresh weight (g) Dry weight (g) Matrine (mg / g) Oxymatrine (mg / g) Example 1 13.3±3.1 245.6±50.2 30.7±7.2 2.04±1.02 1.64±0.48 Comparative Example 1 13.1±3.0 102.8±22.6 12.9±2.8 0.88±0.31 0.72±0.25 As can be seen from Table 1, the fresh weight and dry weight of the BTBB reactor cells of Example 1 indicate that the biomass is improved compared to Comparative Example 1, indicating that the aeration efficiency (micro-bubble aeration) and mixing uniformity of the BTBB reactor are significantly better than the triangular flask shaking culture, and the matrine and oxymatrine content is improved, indicating that the effective component is enriched, and the environment in the reactor is more suitable for the synthesis of secondary metabolites. This is probably because the BTBB reactor is provided with a bubble disc at the bottom for uniform aeration, on the one hand, a uniform upflow is formed at the bottom to avoid cell sedimentation and local nutrient depletion, on the other hand, micro-bubbles improve the oxygen efficiency to avoid local anoxia caused by cell death, promote cell respiration and energy metabolism, promote cell exponential proliferation, and verify the adaptability of the BTBB reactor to the scale-up culture of Sophorae Radix suspension cells.

[0034] Example 1, Example 2, determination step 2 after 4 generations of cell mass D of the suspension cells obtained 50 , survival rate, and the determination results are shown in Table 2. Cell mass D 50 defined as the particle size of 50% of the particles in the particle size cumulative distribution curve (i.e. 50% of the cell mass particle size is less than this value), 10 mL of the suspension cell suspension was taken, magnetic stirring was used to uniformly disperse the cell mass, 2 mL was taken to the laser particle size instrument, and a circulating pump was used to maintain the suspension state, and the detection was completed within 5 min. The survival rate (%) was determined by 0.4% trypan blue staining method, and the survival rate (%) = (number of viable cells / total number of cells) x 100%.

[0035] Table 2 Group Cell mass D 50 (μm) Survival rate (%) Example 1 248±82 83±7 Example 2 118±15 94±2 As can be seen from Table 2, the particle size of Example 1 (248±82 μm) is large and has significant fluctuations, reflecting the uneven size of naturally grown cell clusters. The particle size of Example 2 (118±15 μm) is reduced and the standard deviation is significantly reduced, demonstrating that mechanical dispersion effectively improves uniformity. The survival rate of Example 2 is improved compared to Example 1. This is probably because Example 2 can improve uniformity through mechanical dispersion, and 200-mesh screening can accurately retain 25-30% of large cell clusters, while small cell clusters of <74 μm pass through the screen and return to the flask, avoiding unnecessary mechanical damage to healthy small clusters, reducing the risk of cell rupture from the source. The MS base salt solution of mannitol can maintain the osmotic pressure of the system at -0.25 MPa, matching the physiological osmotic pressure of the Radix Sophorae Tonkinensis suspension cells. Without osmotic pressure protection, cells are prone to swelling and breaking due to water absorption (hypotonic) or shrinking due to water loss (hypertonic) when a syringe is blown. Mannitol balances the water potential inside and outside the cell, preventing cells from swelling and breaking due to osmotic pressure changes or shrinking due to water loss. A 10-mL syringe was used to inject 10 times at a flow rate of 3 seconds per time. This intensity just breaks the intercellular connections of large cell clusters (the main component of the intercellular layer of Radix Sophorae Tonkinensis callus is pectin, which can be damaged by shear force >0.3 Pa), but does not damage the cell membranes of single cells or small clusters (cell membranes can withstand shear force thresholds >0.5 Pa). Ultimately, the D50 of the cell clusters is reduced to 118 μm.

[0036] Example 1 and Example 2 were used to determine the fresh weight, dry weight, and active ingredients of the suspension cells. The results are shown in Table 3.

[0037] Table 3 Group Fresh weight (g) Dry weight (g) Matrine (mg / g) Oxymatrine (mg / g) Example 1 289.2±35.6 36.5±4.8 3.66±0.67 2.32±0.42 Example 2 310.5±12.3 39.8±1.5 3.85±0.18 2.45±0.12 As can be seen from Table 3, the fresh weight and dry weight of Example 2 are improved compared to Example 1, and the standard deviation of the two indicators is significantly reduced, indicating that biomass accumulation is more efficient and stable. The content of matrine and oxymatrine is increased, indicating that uniform cell size promotes overall biomass growth. This is probably because the specific surface area of small and uniform cell clusters is increased, making them more fully contact with the culture medium, allowing for efficient use of nutrients, dissolved oxygen, and other resources. Dissolved oxygen can be evenly distributed to the core of the cell cluster, and all cells are in the "proliferation active phase", avoiding differences between cells that synthesize and cells that do not synthesize. Ultimately, net growth of biomass and active ingredient accumulation is achieved.

[0038] The cell density (g / L) and tryptophan decarboxylase activity (U / mg protein) of the same period of suspension cells after metabolic induction treatment in step 3 were determined, wherein the cells in Example 3 were in the stable phase after metabolic induction, and the cells in Example 2 were not induced at the same culture stage, and the determination results are shown in Table 4. Cell density: take 10 mL of the suspension, centrifuge, weigh after discarding the supernatant, and calculate the fresh weight of cells per liter of culture solution (g / L). Tryptophan decarboxylase (TDC) activity: homogenize the cells to extract total protein, the reaction system contains 0.1 mmol / L tryptophan, enzyme solution, 37°C reaction for 30 min, and the generation amount of product 5-hydroxytryptamine is measured by fluorescence method (excitation light 280 nm, emission light 340 nm), and 1 nmol of product generated per minute is defined as 1 U.

[0039] Table 4 Group Cell density (g / L) Tryptophan decarboxylase activity (U / mg protein) Example 2 77.6±3.1 12.6±1.5 Example 3 76.8±2.8 28.9±2.1 As can be seen from Table 4, the cell density of Example 3 has no significant difference from that of Example 2, indicating that the cell density is basically stable. The TDC of Example 3 is improved compared with Example 2, indicating that methyl jasmonate can up-regulate the gene expression of tryptophan decarboxylase in the alkaloid synthesis pathway and accelerate the conversion of precursors to matrine and oxymatrine. This is probably because Example 3 selects the middle of the exponential growth phase when the cell density reaches 3 times the initial inoculation amount to start induction, at which time the cells have accumulated sufficient biomass, and the nutrient supply is still in balance. Methyl jasmonate as a secondary metabolic signal molecule can directionally activate the expression of TDC related genes, Tween-80 can enhance the cell membrane permeability and the dispersibility of lipid-soluble inducers, and a concentration of 0.006% can reduce the lipid solubility aggregation of methyl jasmonate and enhance its affinity with the cell membrane, thereby improving the cell absorption efficiency of methyl jasmonate, and the two synergistically amplify the induction effect. Injecting twice avoids the toxic shock of single high concentration methyl jasmonate to the cells, so that the cells gradually adapt to the induction signal, maintain survival while ensuring the continuous effect of the inducer, and reduce metabolic disorders caused by stress. Cooperate with temperature regulation, reduce the temperature from 25°C to 23.5°C and maintain for 48 h, which can inhibit the cell division related metabolism by a moderate degree, so that more energy and carbon source can be allocated to secondary metabolism, and restore the temperature to 25°C to avoid the influence of long-term low temperature on cell activity.

[0040] The fresh weight, dry weight and active ingredients of the suspension cells in Example 2 and Example 3 were determined, and the determination results are shown in Table 5.

[0041] Table 5 Group Fresh weight (g) Dry weight (g) Matrine (mg / g) Oxymatrine (mg / g) Example 2 310.5±12.3 39.8±1.5 3.85±0.18 2.45±0.12 Example 3 312.6±15.4 40.2±1.8 5.28±0.25 3.32±0.15 As can be seen from Table 5, the fresh weight and dry weight of Example 3 have no significant difference compared with Example 2, indicating that the metabolic induction does not inhibit cell growth. The matrine and oxymatrine content is increased, and the standard deviation is in a controllable range, reflecting the stability of the induction effect and ensuring the continuity of biomass accumulation. This is probably because methyl jasmonate as a signal molecule activates the secondary metabolic pathway directionally, and Twain-80 enhances the absorption efficiency of the inducer, so that the key enzyme activity of alkaloid synthesis is up-regulated synchronously, and finally the stable accumulation of effective components is realized on the basis of ensuring the normal growth of cells.

[0042] The number of devices and the scale of processing described herein are intended to be illustrative of the application. Applications, modifications and variations of the application will be apparent to those skilled in the art.

[0043] Although embodiments of the application have been disclosed in connection with the specified embodiments, it should be understood that they are not intended to limit the scope of the application to the particular configurations and arrangements described. Accordingly, there are many other variations of the application, which are covered by the application and are within the scope of the claims.

Claims

1. A method for culturing Euchistia japonica suspended cells using a bioreactor, characterized by, The method comprises the following steps: Step 1: selecting leaf petiole of tissue culture seedling of S. soongorica as explant, cutting into small pieces and inoculating into solid culture medium, and inducing callus under dark condition and at 25±2 DEG C; Subculturing once every 18-22 days, and continuously subculturing for 5-6 times until obtaining water-logged callus with vigorous division, loose and easy to break, and having loose nature; Step 2: transferring the callus into liquid culture medium, and culturing under the condition of 12h / d dark, 2500-3000 Lux 12h / d alternate light, and 25±2 DEG C; Subculturing once every 14-16 days, and continuously subculturing for 4-5 times to obtain cell suspension particles; Step 3: inoculating the cell suspension particles into a bubble type balloon bioreactor, injecting liquid culture medium into the bioreactor, and introducing air, and culturing in the bioreactor under the condition of 12h / d light cycle and 25±2 DEG C for 25-30 days to obtain S. soongorica cell suspension.

2. The method of claim 1, wherein, The solid culture medium in step 1 comprises MS basic medium, 0.4-0.6 mg / L 2,4-D, 1.8-2.2 mg / L NAA, 1.8-2.2 mg / L 6-BA, 28-32 g / L sucrose and 4.0-5.0 g / L agar, and further comprises 55-65 mg / L citric acid, and the pH is 5.7-5.9; The liquid culture medium used in steps 2 and 3 comprises MS basic medium, 0.4-0.6 mg / L 2,4-D, 1.8-2.2 mg / L NAA, 1.8-2.2 mg / L 6-BA and 28-32 g / L sucrose, and the pH is 5.7-5.

9.

3. The method of claim 2, wherein, In step 1, the citric acid is prepared into a solution, filtered through a 0.22 mu m filter membrane, and then added into the warm solid culture medium which has not been condensed.

4. The method of claim 1, wherein, In step 2, mechanical dispersion is implemented during the callus shake culture process: Every 120 hours, the shake flask is moved to a sterile operation table, the suspension culture is filtered through a 200-mesh stainless steel screen, the retained material is transferred to a 50 mL centrifuge tube, 10 mL of MS basic salt solution containing 0.1 mol / L mannitol is added to the centrifuge tube, a 10 mL syringe is used to extract the mixed solution, and the solution is repeatedly injected at a uniform speed of 3 seconds per time at a position 1 cm below the liquid surface for 10-15 times, after blowing, the suspension is mixed with the filtrate under the screen, and then returned to the original shake flask for continuous culture.

5. The method of claim 2, wherein, The bioreactor comprises an air pump (1), a filter head (2), a bubble disc (3), a culture container (4) and a rubber plug (5), the air pump (1) is connected with the filter head (2) and the bubble disc (3) through pipelines in sequence, the bubble disc (3) is arranged at the bottom of the culture container (4), the air pump (1) is used to drive air to enter the culture liquid in the form of micro-bubbles through the bubble disc (3) after being sterilized through a filter membrane, the upper opening of the culture container (4) is sealed through the rubber plug (5), a breathable sealing film (6) is attached to the surface of the rubber plug (5), a stainless steel injection port is additionally arranged beside the rubber plug (5) on the surface, and high-pressure resistant silicone rubber is coated on the surface of the rubber plug (5) as a spacer.

6. The method of claim 5, wherein, In step 3, metabolic induction is implemented during the bioreactor scale-up culture process: From the 7th day of culture, cell density was determined every 24 h, and when the density reached 2.8-3.2 times the initial inoculation amount, i.e. in the middle of the exponential growth phase, induction was started; Through a sterile injection hole with a silica gel spacer pre-installed on the top cover of the bioreactor, inject a Tween-80 aqueous solution to make the final concentration of Tween-80 in the system 0.006±0.001% by volume fraction, after an interval of 30±5 min, inject a methyl jasmonate ethanol mother liquor with a concentration of 50 mmol / L, in two injections, 60% of the total amount of the first injection, and the remaining 40% is injected after an interval of 2 h, to make the final concentration of methyl jasmonate 100±10 μmol / L; During the injection, maintain the air flow rate at 1.0vvm, and the injection flow rate at 8-12 mL / min, after the completion of the last addition, reduce the temperature to 23.5±0.5℃ within 2 h and maintain for 36-48 h, and then restore to 25±2℃.

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