CA3138136A1 - Method for producing maackiain by using plant cell fermentation - Google Patents

Abstract

The present disclosure relates to the field of biotechnology, specifically to a method for inducing the production of maackiain by using suspension cells from Millettia specisoa Champ. The method comprises adding two or more selected from methyl jasmonate, phenylalanine and chitosan glutamate during the process of scale-up culture of the suspension cells from Millettia specisoa Champ. The present disclosure overcomes the shortcomings of the existing suspension culture of Millettia specisoa Champ at the shake flask level, having low cell yield, and incapable of carrying out large-scale culture. In addition, the present disclosure screens and obtains the optimal optimized medium and addition process for promoting the synthesis of the product, which greatly improves the synthesis rate of Millettia specisoa Champ maackiain, and provides a new method for the subsequent larger-scale culture of suspension cells from Millettia specisoa Champ.

Description

METHOD FOR PRODUCING MAACKIAIN BY USING PLANT CELL
FERMENTATION
FIELD
[0001] The present disclosure relates to the field of biotechnology, specifically to a method for inducing the production of maackiain by using suspension cells from Millettia specisoa Champ.
BACKGROUND

[0002] Millettia specisoa Champ (royal king beautiful millettia root) is the dry rhizome of Milttia specisoa Champ, which is a plant belonging to leguminosae, papillionoideae, Millettia Wight et Am. Millettia specisoa Champ has a long history of medicinal use, and the roots are used as a traditional Chinese medicine. It has been recorded in "Drug property of Herbage"
("Shengcao Yaoxing Beiyao"): "Strengthen muscles and activating collaterals, reinforcing deficiency and moistening lungs. Treating pain in lower extremities and waist, wind-damp arthralgia, chronic hepatitis, tuberculosis". "Lu Chuan Materia Medica" ("Lu Chuan Bencao") also records it: "clearing lungs and relieving cough, clearing heat and removing toxicity. Treating hemoptysis, dysentery, fever and thirst caused by warm disease, and dizziness." Besides, it has been clinically proven to have therapeutic effects on many chronic diseases, for treating low back pain, nephrasthenia leukorrhagia, rheumatoid arthritis, lumbar muscle strain, chronic hepatitis, and deficiency after illness.

[0003] Maackiain is naturally present in plants such as Millettia specisoa Champ, Maackia amurensis, and Trifolium pratense L.. Maackiain has strong antifungal effects and anti-tumor activity. It has a significant inhibitory effect on aromatic hydroxylase and can be used in the treatment of high-incidence lung cancer. It has a promoting effect on inducing leukemia cell apoptosis and has a certain adjuvant effect on the treatment of leukemia. It has no obvious activity on estrogen receptors, and has an inhibitory effect on the production of nitric oxide induced by lipid metabolism and a potent bactericidal effect.

[0004] The current methods of obtaining Millettia specisoa Champ maackiain is mainly through extracting it from Millettia specisoa Champ, but the extraction yield from the rhizome of Millettia specisoa Champ is only 0.01%, and affected by regions and seasons.
The production of target secondary metabolites through plant cell culture techniques is one of the effective means to increase the content of target components in natural plants. Cell suspension culture has the characteristics of fast reproduction speed, large scale of culture and providing a large number of uniform plant cell cultures. Therefore, culturing and producing Millettia specisoa Champ maackiain through plant cell culture can effectively increase the yield of Millettia specisoa Champ maackiain and reduce production costs, which is beneficial to the further marketization of Millettia specisoa Champ maackiain.

[0005] On the other hand, during the process of plant secondary metabolism, inducers, as a special biochemical molecule, can quickly, specifically and selectively induce the expression of certain specific genes, thereby increasing the activity of related enzymes in the metabolic pathway, and then promoting or inhibiting the production of secondary products regulated by the enzymes. Methyl Jasmonate (MeJA) is a non-biological inducing factor that stimulates the accumulation of secondary metabolites. The addition of methyl jasmonate to different plant suspension culture systems can induce an increase in the accumulation of secondary metabolites in cells. At present, there is no report on the treatment of the culture system of suspension cells from Millettia specisoa Champ with methyl jasmonate, phenylalanine and chitosan glutamate.

[0006] At present, there are few research reports on the suspension culture of Millettia specisoa Champ cells, and many current research only stay at the shake flask level. There has been no relevant report on the suspension culture of Millettia specisoa Champ cells at the reactor level, and how to promote the rapid synthesis of products to a greater extent has always been a problem that needs to be solved in suspension cell culture.
SUMMARY

[0007] In view of this, the present disclosure provides a method for scale-up culture ofsuspension cells from Millettia specisoa Champ, comprising adding two or more selected from methyl jasmonate, phenylalanine and chitosan glutamate during the process of the suspension culture of Millettia specisoa Champ cells, which greatly promotes the synthesis and accumulation of Millettia specisoa Champ maackiain, and can be applied to large-scale industrial production processes. The present disclosure overcomes the shortcomings of the existing suspension culture of Millettia specisoa Champ staying at the shake flask level, having low cell yield, and incapable of carrying out large-scale culture.

[0008] In order to achieve the above-mentioned purpose of the invention, the present disclosure provides the following technical solutions.

[0009] The present disclosure provides use of methyl jasmonate, phenylalanine and/or chitosan glutamate in the induction of the production of maackiain by using suspension cells from Millettia specisoa Champ. In some specific embodiments of the present disclosure, the concentration of the phenylalanine is 250-1000 mg/L, the concentration of the methyl jasmonate is 5-50 mM, and the concentration of the chitosan glutamate is 0.5-10 g/L. In some specific embodiments of the present disclosure, the concentration of the phenylalanine is 450-550 mg/L, the concentration of the methyl jasmonate is 15-25 mM, and the concentration of the chitosan glutamate is 1-5 g/L.

[0010] The present disclosure also provides an inducer for inducing the production of maackiain by using suspension cells from Millettia specisoa Champ, comprising a combination of two or more selected from methyl jasmonate, phenylalanine and chitosan glutamate. In some specific embodiments of the present disclosure, the concentration of the phenylalanine is 250-1000 mg/L, the concentration of the methyl jasmonate is 5-50 mM, and the concentration of the chitosan glutamate is 0.5-10 g/L. In some specific embodiments of the present disclosure, the concentration of the phenylalanine is 450-550 mg/L, the concentration of the methyl jasmonate is 15-25 mM, and the concentration of the chitosan glutamate is 1-5 g/L.

[0011] The present disclosure also provides a method for scale-up culture of suspension cells from Millettia specisoa Champ, comprising adding the inducer during the process of scale-up culture of the suspension cells from Millettia specisoa Champ.

[0012] The present disclosure also provides a method for inducing the production of maackiain by using suspension cells from Millettia specisoa Champ, comprising adding the inducer during the process of scale-up culture of the suspension cells to obtain the maackiain.

[0013] In some specific embodiments of the present disclosure, the condition for the scale-up culture of suspension cells from Millettia specisoa Champ is: 20-30 C, air flow of 100-800 L/h, tank pressure of 0.01-0.03 MPa, rotation speed of 20-50 rpm, and culture time of 10-30 days.

[0014] In some specific embodiments of the present disclosure, the time point for adding the inducer is Day 15 to Day 20 of the scale-up culture.

[0015] In some specific embodiments of the present disclosure, the condition for the scale-up culture of suspension cells from Millettia specisoa Champ is: 23-28 C, air flow of 150-600 L/h, tank pressure of 0.01-0.03 MPa, rotation speed of 25-40 rpm, and culture time of 15-25 days.

[0016] In some specific embodiments of the present disclosure, the method for preparing the suspension cells from Millettia specisoa Champ comprises the following steps:

[0017] Step 1: selecting Millettia specisoa Champ seed embryos, performing disinfection treatment, rinsing after disinfection, cutting a wound on the seed embryos and adding them to solid MS medium containing hormones, wherein the solid MS medium are MS medium with 0.2-5 mg/L 6-BA, 0.5-4 mg/L Picloram, 10-50 g/L sucrose, 5-10 g/L agar, and pH
is 5.55-5.95;
after induction of 20-30 days, selecting callus for subculture, wherein the subculture is performed 8-12 times (8-12 passages);

[0018] Step 2: inoculating the callus into a liquid medium (the liquid medium has the same components and ratio as the solid MS medium in step 1 except for the agar), and the inoculation amount is 25-100 g/L of wet weight; in 5-15 days of the initial culture, using shake flasks for the culture; then, fully dispersing the cells, using a 35-mesh sieve to sieve out large cell clusters, replenishing the filtered small cell clusters and single cell suspension into the liquid medium to establish a liquid suspension culture system, and the subculture is performed 4-6 times.

[0019] The beneficial effects of the present disclosure include but are not limited to:

[0020] 1. The present disclosure overcomes the shortcomings of the existing suspension culture of Millettia specisoa Champ at the flask level, which has low cell yield, and incapable of carrying out large-scale culture, and provides a certain methods and basis for the subsequent larger-scale suspension culture of Millettia specisoa Champ cells.

[0021] 2. By controlling the concentration of methyl jasmonate, phenylalanine and chitosan glutamate, the present disclosure solves the problem of the inhibitory effects of methyl jasmonate and phenylalanine on the growth of Millettia specisoa Champ cells in the scale-up culture of suspension cells from Millettia specisoa Champ, and makes full use of methyl jasmonate and phenylalanine to promote the synthesis of Millettia specisoa Champ maackiain.

[0022] 3. The method of the present disclosure artificially regulates the biomass of suspension cells from Millettia specisoa Champ and the production process of Millettia specisoa Champ maackiain, which is beneficial to the quality control management of the product.
DETAILED DESCRIPTION

[0023] The present disclosure discloses a method for inducing the production of maackiain by using suspension cells from Millettia specisoa Champ. Those skilled in the art can learn from the disclosure and appropriately improve the process parameters. It should be particularly indicated that, all similar replacements and changes are obvious for those skilled in the art, which are deemed to be included in the present disclosure. The method and the application of the present disclosure have been described according to the preferred embodiments, and it is obvious that the method and application described herein may be changed or appropriately modified and combined without departing from the content, spirit and scope of the present disclosure.

[0024] A method for scale-up culture of suspension cells from Millettia specisoa Champ, comprising adding two or more of methyl jasmonate, phenylalanine and chitosan glutamate during the process of scale-up culture of the suspension cells from Millettia specisoa Champ.

[0025] Preferably, during the process of scale-up culture of the suspension cells from Millettia specisoa Champ, 250-1000 mg/L of phenylalanine, 5-50 mM of methyl jasmonate, and 0.5-10 g/L of chitosan glutamate are added.

[0026] Preferably, during the process of scale-up culture of the suspension cells from Millettia specisoa Champ, 450-550 mg/L of phenylalanine, 15-25 mM of methyl jasmonate, and 1-5 g/L
of chitosan glutamate are added.

[0027] Preferably, the condition for the scale-up culture of suspension cells from Millettia specisoa Champ is: 20-30 C, air flow of 100-800 L/h, tank pressure of 0.01-0.03 MPa, rotation speed of 20-50 rpm, and culture time of 10-30 days.

[0028] Preferably, the condition for the scale-up culture of suspension cells from Millettia specisoa Champ is: 24-28 C, air flow of 150-600 L/h, tank pressure of 0.01-0.03 MPa, rotation speed of 25-40 rpm, and culture time of 15-25 days.

[0029] Preferably, the time point for adding the two or more of methyl jasmonate, phenylalanine and chitosan glutamate is Day 15 to Day 20 of the scale-up culture.

[0030] Preferably, the liquid suspension culture system in the early stage of the scale-up culture of suspension cells from Millettia specisoa Champ is obtained by the following steps:

[0031] Step 1: Selecting Millettia specisoa Champ seed embryos, performing disinfection treatment, rinsing after disinfection, cutting a wound on the seed embryos and adding them to solid MS medium containing hormones. The solid MS medium are MS medium with 0.2-5 mg/L
6-BA, 0.1-4 mg/L Picloram, 10-50 g/L sucrose, 5-10 g/L agar, and pH is 5.55-5.95; after induction of 20-30 days, selecting callus for subculture.

[0032] Step 2: Inoculating the embryogenic callus into a liquid medium, and the inoculation amount is 25-100 g/L of wet weight. In 5-15 days of the initial culture, using shake flasks. After fully dispersing the cells, using a sieve to sieve out large cell clusters, replenishing the filtered small cell clusters and single cell suspension into the liquid medium to establish a liquid suspension culture system, and subculturing the cells.

[0033] Preferably, in the step 1, the subculture is performed 8-12 times.

[0034] Preferably, in the step 2, the subculture is performed 4-6 times.

[0035] Advantages of the present disclosure:

[0036] 1. The present disclosure overcomes the shortcomings of the existing suspension culture of Millettia specisoa Champ at the shake flask level, which has low cell yield, and incapable of carrying out large-scale culture, and provides a certain technical method and basis for the subsequent larger-scale suspension culture of Millettia specisoa Champ cells.

[0037] 2. By controlling the concentration of methyl jasmonate, phenylalanine and chitosan glutamate, the present disclosure solves the problem of the inhibitory effects of methyl jasmonate and phenylalanine on the growth of Millettia specisoa Champ cells in the scale-up culture of suspension cells from Millettia specisoa Champ, and makes full use of methyl jasmonate and phenylalanine to promote the synthesis of Millettia specisoa Champ maackiain.

[0038] 3. The method of the present disclosure artificially regulates the biomass of suspension cells from Millettia specisoa Champ and the production process of Millettia specisoa Champ maackiain, which is beneficial to the quality control management of the product.

[0039] In the method for inducing the production of maackiain by using suspension cells from Millettia specisoa Champ provided by the present disclosure, the raw materials and reagents used can be purchased from the market.

[0040] The present disclosure will be further explained below in conjunction with examples.
Example 1

[0041] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0042] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 5 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 0.5 mg/L 6-BA, 0.5 mg/L
Picloram, 10 g/L sucrose, 6 g/L agar, and the pH value of the medium was 5.95. After 20 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0043] The loose embryogenic callus, which was subcultured 8 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 25 g/L of the wet weight of callus. In 5 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 4 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0044] Step 2: Scale-up culture in a 5L stirred tank reactor

[0045] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 24 C, air flow of 150 L/h, tank pressure of 0.01 MPa, and rotation speed of 30 rpm. At Day 15 of the culture, phenylalanine, methyl jasmonate and chitosan glutamate were added to reach a final concentration of 250 mg/L phenylalanine, 15 mM methyl jasmonate, and 0.5 g/L chitosan glutamate. The culture was induced and continued until Day 20.
Example 2

[0046] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0047] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1.5 mg/L 6-BA, 4 mg/L
Picloram, 30 g/L
sucrose, 10 g/L agar, and the pH value of the medium was 5.55. After 30 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0048] The loose embryogenic callus, which was subcultured 12 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 100 g/L of the wet weight of callus. In 15 days of the initial culture, a shake flask with a baffle was used.
After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 6 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0049] Step 2: Scale-up culture in a 5L stirred tank reactor

[0050] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 25 C, air flow of 180 L/h, tank pressure of 0.03 MPa, and rotation speed of 30 rpm. At Day 20 of the culture, phenylalanine, methyl jasmonate and chitosan glutamate were added to reach a final concentration of 1000 mg/L phenylalanine, 25 mM methyl jasmonate, and 5 mg/L chitosan glutamate. The culture was induced and continued until Day 30.
Example 3

[0051] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0052] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 5-6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1.0 mg/L 6-BA, 1.0 mg/L
Picloram, 30 g/L sucrose, 5 g/L agar, and the pH value of the medium was 5.85. After 25 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0053] The loose embryogenic callus, which was subcultured 10 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 50 g/L of the wet weight of callus. In 10 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 6 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0054] Step 2: Scale-up culture in a 5L stirred tank reactor

[0055] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 26 C, air flow of 200 L/h, tank pressure of 0.02 MPa, and rotation speed of 30 rpm. At Day 15 of the culture, phenylalanine, methyl jasmonate and chitosan glutamate were added to reach a final concentration of 500 mg/L phenylalanine, 40 mM methyl jasmonate, and 3 g/L chitosan glutamate. The culture was induced and continued until Day 30.
Example 4

[0056] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0057] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 5 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 0.7 mg/L 6-BA, 0.7 mg/L
Picloram, 25 g/L sucrose, 7.5 g/L agar, and the pH value of the medium was 5.75. After 23 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0058] The loose embryogenic callus, which was subcultured 8 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 40 g/L of the wet weight of callus. In 7 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 4 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0059] Step 2: Scale-up culture in a 5L stirred tank reactor

[0060] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. The exhaust gas mass spectrometry is used to analyze the gas components, and the physiological respiratory and metabolic parameters of the cells are measured in real time. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 26 C, air flow of 250 L/h, tank pressure of 0.01 MPa, and rotation speed of 30 rpm. At Day 15 of the culture, phenylalanine, methyl jasmonate and chitosan glutamate were added to reach a final concentration of 450 mg/L phenylalanine, 15 mM methyl jasmonate, and 4 g/L chitosan glutamate. The culture was induced and continued until Day 25.
Example 5

[0061] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0062] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1.2 mg/L 6-BA, 1.2 mg/L
Picloram, 40 g/L sucrose, 6 g/L agar, and the pH value of the medium was 5.75. After 28 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0063] The loose embryogenic callus, which was subcultured 12 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 75 g/L of the wet weight of callus. In 12 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 6 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0064] Step 2: Scale-up culture in a 5L stirred tank reactor

[0065] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 27 C, air flow of 250 L/h, tank pressure of 0.03 MPa, and rotation speed of 30 rpm. At Day 18 of the culture, phenylalanine, methyl jasmonate and chitosan glutamate were added to reach a final concentration of 750 mg/L phenylalanine, 25 mM methyl jasmonate, and 7 g/L chitosan glutamate. The culture was induced and continued until Day 30.
Example 6

[0066] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0067] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1 mg/L 6-BA, 1.2 mg/L
Picloram, 35 g/L
sucrose, 5 g/L agar, and the pH value of the medium was 5.65. After 20 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0068] The loose embryogenic callus, which was subcultured 8 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 58 g/L of the wet weight of callus. In 9 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 6 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0069] Step 2: Scale-up culture in a 5L stirred tank reactor

[0070] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 26 C, air flow of 300 L/h, tank pressure of 0.02 MPa, and rotation speed of 30 rpm. At Day 20 of the culture, phenylalanine, methyl jasmonate and chitosan glutamate were added to reach a final concentration of 350 mg/L phenylalanine, 25 mM methyl jasmonate, and 5 g/L chitosan glutamate. The culture was induced and continued until Day 30.
Example 7

[0071] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0072] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 5 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 0.7 mg/L 6-BA, 0.7 mg/L
Picloram, 20 g/L sucrose, 8 g/L agar, and the pH value of the medium was 5.75. After 23 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0073] The loose embryogenic callus, which was subcultured 9 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 40 g/L of the wet weight of callus. In 7 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 4 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0074] Step 2: Scale-up culture in a 5L stirred tank reactor

[0075] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 27 C, air flow of 300 L/h, tank pressure of 0.01 MPa, and rotation speed of 25 rpm. At Day 18 of the culture, phenylalanine and chitosan glutamate were added to reach a final concentration of 350 mg/L phenylalanine and 4 g/L chitosan glutamate. The culture was induced and continued until Day 25.
Example 8

[0076] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0077] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1.2 mg/L 6-BA, 2.25 mg/L
Picloram, 40 g/L sucrose, 7.5 g/L agar, and the pH value of the medium was 5.85. After 28 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0078] The loose embryogenic callus, which was subcultured 12 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 75 g/L of the wet weight of callus. In 12 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 6 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0079] Step 2: Scale-up culture in a 5L stirred tank reactor

[0080] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 27 C, air flow of 350 L/h, tank pressure of 0.03 MPa, and rotation speed of 25 rpm. At Day 20 of the culture, methyl jasmonate and chitosan glutamate were added to reach a final concentration of 50 mM methyl jasmonate and 7 g/L chitosan glutamate.
The culture was induced and continued until Day 30.
.. Example 9

[0081] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0082] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1 mg/L 6-BA, 1.2 mg/L
Picloram, 25 g/L
sucrose, 7 g/L agar, and the pH value of the medium was 5.55. After 20 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0083] The loose embryogenic callus, which was subcultured 12 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 58 g/L of the wet weight of callus. In 9 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 6 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0084] Step 2: Scale-up culture in a 5L stirred tank reactor

[0085] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 26 C, air flow of 400 L/h, tank pressure of 0.02 MPa, and rotation speed of 20 rpm. At Day 15 of the culture, phenylalanine and methyl jasmonate were added to reach a final concentration of 350 mg/L phenylalanine and 40 mM methyl jasmonate. The culture was induced and continued until Day 30.
Comparative examples 1-4 Evaluate the effect of phenylalanine on the proliferation of suspension cells from Millettia specisoa Champ and the production of maackiain

[0086] Comparative Example 1: using 100 mg/L of phenylalanine instead of phenylalanine, methyl jasmonate and chitosan glutamate in Example 3. The other steps were the same as in Example 3.

[0087] Comparative Example 2: using 250 mg/L of phenylalanine instead of phenylalanine, methyl jasmonate and chitosan glutamate in Example 3. The other steps were the same as in Example 3.

[0088] Comparative Example 3: using 500 mg/L of phenylalanine instead of phenylalanine, methyl jasmonate and chitosan glutamate in Example 3. The other steps were the same as in Example 3.

[0089] Comparative Example 4: using 1000 mg/L of phenylalanine instead of phenylalanine, methyl jasmonate and chitosan glutamate in Example 3. The other steps were the same as in Example 3.

[0090] For example, the specific operation of Comparative Example 3 is as follows.

[0091] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells

[0092] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 5-6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1.0 mg/L 6-BA, 1.0 mg/L
Picloram, 30 g/L sucrose, 5 g/L agar, and the pH value of the medium was 5.85. After 25 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.

[0093] The loose embryogenic callus, which was subcultured 8 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 50 g/L of the wet weight of callus. In 10 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 5 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.

[0094] Step 2: Scale-up culture in a 5L stirred tank reactor

[0095] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 26 C, air flow of 400 L/h, tank pressure of 0.02 MPa, and rotation speed of 20 rpm. At Day 15 of the culture, phenylalanine was added at a final concentration of 500 mg/L.
The culture was induced and continued until Day 30.

Comparative examples 5-8 Evaluate the effect of methyl jasmonate on the proliferation of suspension cells from Millettia specisoa Champ and the production of maackiain

[0096] Comparative Example 5: using 1 umol/L of methyl jasmonate instead of phenylalanine, methyl jasmonate, and chitosan glutamate in Example 3. The other steps were the same as in Example 3.

[0097] Comparative Example 6: using 20 umol/L of methyl jasmonate instead of phenylalanine, methyl jasmonate, and chitosan glutamate in Example 3. The other steps were the same as in Example 3.

[0098] Comparative Example 7: using 50 umol/L of methyl jasmonate instead of phenylalanine, methyl jasmonate, and chitosan glutamate in Example 3. The other steps were the same as in Example 3.

[0099] Comparative Example 8: using 100 umol/L of methyl jasmonate instead of phenylalanine, methyl jasmonate, and chitosan glutamate in Example 3. The other steps were the same as in Example 3.
.. [00100] For example, the specific operation of Comparative Example 5 is as follows.
[00101] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells [00102] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed .. with distilled water 5-6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1.0 mg/L 6-BA, 1.0 mg/L
Picloram, 30 g/L sucrose, 5 g/L agar, and the pH value of the medium was 5.85. After 25 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.
[00103] The loose embryogenic callus, which was subcultured 8 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 50 g/L of the wet weight of callus. In 10 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 5 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.
[00104] Step 2: Scale-up culture in a 5L stirred tank reactor [00105] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 26 C, air flow of 400 L/h, tank pressure of 0.02 MPa, and rotation speed of 20 rpm. At Day 15 of the culture, methyl jasmonate was added at a final concentration of 1 [tmol/L.
The culture was induced and continued until Day 30.
Comparative examples 942 Evaluate the effect of chitosan glutamate on the proliferation of suspension cells from Millettia specisoa Champ and the production of maackiain [00106] Comparative Example 9: using 0.1 g/L of chitosan glutamate instead of phenylalanine, methyl jasmonate and chitosan glutamate in Example 3. The other steps were the same as in Example 3.
[00107] Comparative Example 10: using 2 g/L of chitosan glutamate instead of phenylalanine, methyl jasmonate and chitosan glutamate in Example 3. The other steps were the same as in Example 3.
[00108] Comparative Example 11: using 5 g/L of chitosan glutamate instead of phenylalanine, methyl jasmonate and chitosan glutamate in Example 3. The other steps were the same as in Example 3.
[00109] Comparative Example 12: using 20 g/L of chitosan glutamate instead of phenylalanine, methyl jasmonate and chitosan glutamate in Example 3. The other steps were the same as in Example 3.
[00110] For example, the specific operation of Comparative Example 11 is as follows.

[00111] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells [00112] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 5-6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1.0 mg/L 6-BA, 1.0 mg/L
Picloram, 30 g/L sucrose, 5 g/L agar, and the pH value of the medium was 5.85. After 25 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.
[00113] The loose embryogenic callus, which was subcultured 8 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 50 g/L of the wet weight of callus. In 10 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 5 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.
[00114] Step 2: Scale-up culture in a 5L stirred tank reactor [00115] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 26 C, air flow of 400 L/h, tank pressure of 0.02 MPa, and rotation speed of 20 rpm. At Day 15 of the culture, chitosan glutamate was added at a final concentration of 5 g/L.
The culture was induced and continued until Day 30.

Blank Control Example [00116] Step 1: Establish a liquid suspension culture system of Millettia specisoa Champ cells [00117] The mature seed embryos of Millettia specisoa Champ were selected and subjected to disinfection treatment under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% available chlorine. After disinfection, the seed embryos were washed with distilled water 5-6 times to remove the disinfectant. The outer hard shell of the seed embryo was peeled off, and the seed embryo was cut to make a wound. The seed embryos were added to a solid MS medium containing hormones to induce the generation of a loose embryogenic callus.
The formula of the solid medium was MS medium with 1.0 mg/L 6-BA, 1.0 mg/L
Picloram, 30 g/L sucrose, 5 g/L agar, and the pH value of the medium was 5.85. After 25 days of induction, loose callus, which was embryogenic, in vigorous growth phase and relatively uniform state, was selected for subculture.
[00118] The loose embryogenic callus, which was subcultured 8 rounds and in a relatively stable state, was inoculated into the liquid medium, and the inoculation amount was 50 g/L of the wet weight of callus. In 10 days of the initial culture, a shake flask with a baffle was used. After that, the cells were fully dispersed, and a 35-mesh mesh sieve was employed to remove large cell clusters. Then the small cell clusters and single cell suspension were replenished into an appropriate amount of liquid culture medium to establish a uniform liquid suspension culture system. After 5 passages, this culture system can be used as an experimental cell line for fermenter inoculation and scale-up culture.
[00119] Step 2: Scale-up culture in a 5L stirred tank reactor [00120] The fermenter was equipped with pH electrode, dissolved oxygen electrode and temperature control system. Before culture, an appropriate amount of culture medium was added to the reactor and sterilization was performed at 121 C for 30 min. After cooling down, the seeds (suspension cells from the shake flask) were inoculated into the culture medium. The culture conditions were: 26 C, air flow of 400 L/h, tank pressure of 0.02 MPa, rotation speed of 20 rpm and culture time 30 days.

RESULTS
Determination of the cell dry weight and the content of Millettia specisoa Champ maackiain in the suspension cells obtained in Examples 1-9, Comparative Examples 1 -12, and Blank Control Example [00121] The results are shown in Table 1.
Table 1 Proliferation of the Millettia specisoa Champ cells and the production of maackiain Cell dry weight (g/L) Content of maackiain (mg/gDCW) Example 1 4.8* 4.6**
Example 2 4.7*
Example 3 5.2* 5.0**
Example 4 5.1*
Example 5 4.7* 4.6**
Example 6 4.6*
Example 7 5.3* 5.0**
Example 8 5.5* 5.1**
Example 9 4.5* 4.2**
Comparative Example 1 4.1* 2.5*
Comparative Example 2 4.0* 2.7*
Comparative Example 3 3.8 2.8*
Comparative Example 4 3.7 2.6*
Comparative Example 5 3.2 2.2*
Comparative Example 6 3.5 2.4*
Comparative Example 7 3.4 2.4*
Comparative Example 8 3.3 2.3*
Comparative Example 9 3.6 2.5*
Comparative Example 10 3.7 2.6*
Comparative Example 11 3.8 2.8*
Comparative Example 12 3.7 2.7*
Blank Control Example 2.9 1.5 [00122] Compared with Blank Control Example, *P<0.05, **13<0.01; n=3 [00123] The results of using phenylalanine alone [00124] From the experimental results in Table 1, it can be seen that when adding a certain amount of phenylalanine (100, 250, 500, 1000 mg/L), with the increase of the concentration of phenylalanine, the content of maackiain also increased, reaching the highest at 500 g/L; while with the further increase of the concentration, the production of maackiain was inhibited. The experimental results show that adding 500 mg/L of phenylalanine to the culture medium can maximize the yield of the synthesis of maackiain. However, as the concentration of phenylalanine increased, the inhibitory effect of phenylalanine on the growth of suspension cells was greater. When the added concentration of phenylalanine increased, the cell dry weight decreased significantly, and the cell growth and the synthesis rate of the product will be significantly suppressed.
[00125] The results of using methyl jasmonate alone [00126] It can be seen from Table 1 that the addition of 20 and 50 [tmol/L
methyl jasmonate can promote the synthesis of Millettia specisoa Champ maackiain. When the concentration was increased to 100 [tmol/L, the yield of the synthesis of maackiain and cell growth rate were inhibited. Under the condition of a concentration of 50 [tmol/L methyl jasmonate, the synthesis rate per cell was higher than that of the control.
[00127] The results of using chitosan glutamate alone [00128] It can be seen from Table 1 that the investigated experimental results of chitosan glutamate (0.1, 2, 5, 10 g/L) show that chitosan glutamate can significantly promote the synthesis of Millettia specisoa Champ maackiain, and besides, it also had a good promoting effect on the growth of cells.
[00129] By controlling the concentrations of methyl jasmonate, phenylalanine and chitosan glutamate, the present disclosure solves the problem of the inhibitory effects of methyl jasmonate and phenylalanine on the growth of Millettia specisoa Champ cells in the scale-up culture of suspension cells from Millettia specisoa Champ, and makes use of methyl jasmonate and phenylalanine to promote the synthesis of Millettia specisoa Champ maackiain.
In addition, by adding chitosan glutamate, the synthesis of Millettia specisoa Champ maackiain and the growth of suspension cells are further promoted. The cell dry weight of suspension cells from Millettia specisoa Champ and the content of Millettia specisoa Champ maackiain in examples 1-9 of the present disclosure were significantly better than those of Comparative Examples 1-12. The growth of the suspension cells was not inhibited, and the synthesis of Millettia specisoa Champ maackiain is significantly improved, which was significantly higher than that of Blank Control Example.
[00130] In the above, the method for producing maackiain by using plant cell fermentation techniques provided by the present disclosure has been described in detail.
The principles and .. embodiments of the present disclosure have been described with reference to specific examples, and the description of the above embodiments is only to assist in understanding the method of the present disclosure and the core idea thereof. It should be noted that, several improvements and modifications may be made by those skilled in the art to the present disclosure without departing from the principle of the present disclosure, which improvements and modifications .. also fall within the protection scope of the claims thereof.

Claims (10)

1. Use of methyl jasmonate, phenylalanine and/or chitosan glutamate in the induction of the production of maackiain by using suspension cells from Millettia specisoa Champ.

2. The use according to claim 1, wherein the concentration of the phenylalanine is 250-1000 mg/L, the concentration of the methyl jasmonate is 5-50 mM, and the concentration of the chitosan glutamate is 0.5-10 g/L.

3. The use according to claim 1, wherein the concentration of the phenylalanine is 450-550 mg/L, the concentration of the methyl jasmonate is 15-25 mM, and the concentration of the chitosan glutamate is 1-5 g/L.

4. An inducer for inducing the production of maackiain by using suspension cells from Millettia specisoa Champ, comprising a combination of two or more of methyl jasmonate, phenylalanine and chitosan glutamate.

5. The inducer according to claim 4, wherein the concentration of the phenylalanine is 250-1000 mg/L, the concentration of the methyl jasmonate is 5-50 mM, and the concentration of the chitosan glutamate is 0.5-10 g/L.

6. The inducer according to claim 4, wherein the concentration of the phenylalanine is 450-550 mg/L, the concentration of the methyl jasmonate is 15-25 mM, and the concentration of the chitosan glutamate is 1-5 g/L.

7. A method for scale-up culture of suspension cells from Millettia specisoa Champ, comprising adding the inducer according to any one of claims 4 to 6 during the process of scale-up culture of the suspension cells from Millettia specisoa Champ.

8. A method for inducing the production of maackiain by using suspension cells from Millettia specisoa Champ, comprising adding the inducer according to any one of claims 4 to 6 during the process of scale-up culture of the suspension cells and obtaining the maackiain.

9. The method according to claim 7 or 8, wherein the condition for the scale-up culture of suspension cells from Millettia specisoa Champ is: 20-30 C, air flow of 100-800 L/h, tank pressure of 0.01-0.03 MPa, rotation speed of 20-50 rpm, and culture time of 10-30 days.

10. The method according to any one of claims 7 to 9, wherein the time point for adding the inducer according to any one of claims 4 to 6 is Day 15 to Day 20 of the scale-up culture.