CN105755090B - Method for obtaining secondary metabolites such as panax japonicus saponin and the like by utilizing panax japonicus cell large-scale culture and biotransformation technology - Google Patents
Method for obtaining secondary metabolites such as panax japonicus saponin and the like by utilizing panax japonicus cell large-scale culture and biotransformation technology Download PDFInfo
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Abstract
The invention relates to the field of plant cell culture, and provides a method for obtaining secondary metabolites such as panax japonicus saponin and the like by utilizing a large-scale culture and biotransformation technology of panax japonicus cells, which comprises the following steps: (1) adding auxin, cytokinin and coagulator into RW culture medium; (2) sterilizing the surface of the explant of the panax japonicus and inducing to form callus; (3) subculturing the panax japonicus callus to obtain loose cell clusters; (4) preparing a culture solution; (5) transferring the loose cell mass into a culture solution for culture; (6) culturing and screening out high-yield cell strains; (7) carrying out amplification culture on the high-yield cell strain; (8) preparing a synthetic culture medium; (9) continuously culturing the cells in a synthetic culture solution to complete biotransformation; (10) and (5) purifying after solvent extraction. The method provided by the invention can produce the secondary metabolite of panax japonicus, has low production cost and short culture period, is not limited by natural environment, and can realize annual production.
Description
Technical Field
The invention relates to a method for obtaining a secondary metabolite of panax japonicus, in particular to a method for obtaining compounds such as panax japonicus saponin and the like by utilizing a large-scale culture and biotransformation technology of panax japonicus cells.
Background
Panax japonicus (Panax japonica C.A.Mey), also known as Panax japonicus or Panax notoginseng, is a plant of the genus Panax of the family Araliaceae. Commonly known as the king of the herbal medicine. Mainly distributed under mountain shrubs in provinces such as Jian West, Hubei, Guangxi, Sichuan, Guizhou, Yunnan and Tibet of China, beside a negative wetland or a rock ditch. It has many different names, and the Chinese medicine dictionary says that: different books have different names, such as: the traditional Chinese medicine composition comprises indigowoad root, bloody ginseng (Huajing), Japanese panax root, rhizoma Panacis Japonici (herbal convenient prescription), Japanese ginseng (scientific folk medicine grass), rhizome of bamboo pseudo-ginseng, root of Arhat pseudo-ginseng (Chinese medicine plant record), rhizoma Panacis Japonici (experience identification method for Chinese medicine shape), radix Raphani, radix et rhizoma Gynurae Divaricatae (Chinese medicinal material variety discussion), rhizoma Panacis japonici (Guizhou herbal medicine), radix Changii, wild pseudo-ginseng and herba Achillea Wilsonianae (Yunnan economic plant), and various miscellaneous abnormal names, which are fully known as the history and status of the Japanese ginseng in traditional Chinese medicine. The folk medicine is used for nourishing and strengthening, eliminating blood stasis and relieving pain, stopping bleeding and the like. The effects of panax japonicus are well documented, such as: the herbal toilet prescription contains clouds: in recent years, reports prove that the panax japonicus saponin has the effects of dilating coronary arteries and reducing myocardial oxygen consumption. Meanwhile, the panaxoside is found to obviously increase the ratio of CAMP/CGMP in mouse cardiac muscle cells, and the effect is related to the enhancement of heart force and the expansion of coronary arteries; in addition, the panax japonicus saponin is also found to have good pharmaceutical activities of relieving pain, diminishing swelling, promoting granulation, resisting anoxia, resisting oxidation, resisting radiation and the like, has great potential of medicinal value and becomes a research hotspot.
The growth of the panax japonicus is slow, 1 node of the panax japonicus is grown annually, the biomass is extremely limited, and the wild panax japonicus used as the medicine generally grows in 5 to decades. According to statistics, the reserve volume of panax japonicus in China is only about 2 tons, belongs to rare and rare medicinal materials, is only conditionally used by local doctors in the mountain areas of Hubei and the mountain areas of West and Sichuan, and is not sold in hospitals and drug stores in various major cities. Although artificial cultivation is carried out in Jiangxi, Hunan, Hubei and other places, large-area artificial cultivation is difficult to realize for a long time because of limited growth amount, difficult propagation and easy trouble of plant diseases and insect pests. Therefore, the substitution product is produced in a large scale by utilizing the biotechnology, and the specific new medicine for treating the cardiovascular and cerebrovascular diseases is developed, and has important economic value and social value.
Therefore, a new technology for converting the natural production mode of the panax japonicus into the industrial production mode is developed after years of research. The application of the technology can greatly reduce the picking and dependence of people on wild resources, and simultaneously can obtain a large number of panax japonicus products required by people. Lays a material foundation for the deep research of the medical value of the panax japonicus and provides excellent cheap medicines for the majority of patients.
Disclosure of Invention
The invention aims to provide a method for obtaining secondary metabolites such as panax japonicus saponin and the like by utilizing a biological engineering technology under the condition of not damaging natural resources.
The saponin extract containing the panax japonicus saponin, the notoginsenoside and the ginsenoside extracted by the method disclosed by the invention has the effective component of the panax japonicus saponin which is 5-10 times of that of the corresponding extract of natural panax japonicus obtained by the same extraction method.
The invention has the technical scheme that a method for producing panax japonicus saponin, notoginsenoside and ginsenoside by utilizing panax japonicus cells through industrial culture and biotransformation technology;
the method is characterized by comprising the following steps:
(1) adding auxin, plant cytokinin, one or two of sucrose and glucose and coagulant into RW culture medium, sterilizing at high temperature, and cooling to obtain slant induction culture medium; the high-temperature disinfection parameters are 115-120 ℃, and the disinfection lasts for 10-25 minutes under the pressure of 0.1-0.15 MPa;
(2) after surface sterilization, cutting the juvenile stems or buds of panax japonicus into 0.5-1 cm stem segments or taking the stem tips of the stem segments as explants, inoculating the stem segments or the stem tips to the slant induction culture medium in the step (1), and culturing to form callus; the surface sterilization treatment method comprises the steps of sterilizing for 30-60 seconds by using 70-75 vol% ethanol, treating for 15-25 minutes by using 5-7 mass% hypochlorite solution, and then washing for two-three times by using sterile water for later use;
(3) adding plant cytokinin, auxin, sucrose and a coagulant into a RW culture medium, and preparing a plate domestication culture medium after high-temperature disinfection; the high-temperature sterilization parameters are the same as those of the step (1);
(4) inoculating the callus of the panax japonicus induced and formed in the step (2) on an acclimatization culture medium for multiple subcultures, and screening out high-yield cell clusters with high growth speed and loose texture;
(5) adding auxin, cytokinin and cane sugar into the RW culture medium, and sterilizing at high temperature to obtain a liquid culture medium; the high-temperature disinfection parameters are the same as those in the step (1);
(6) inoculating the high-yield cell clusters screened in the step (4) into the liquid culture medium in the step (5) for suspension culture for 2-3 weeks, wherein the rotating speed of a shaking table is 100-120 r/min;
(7) adding a coagulant into the liquid culture medium same as the liquid culture medium in the step (5), sterilizing at high temperature, and cooling to prepare a plate culture medium; the high-temperature sterilization conditions are the same as the step (1);
(8) dispersedly inoculating the single cells cultured in the step (6) on the plate culture medium cultured in the step (7) by using a sterile pipette for culturing for 4-5 weeks;
(9) screening cell clusters with high growth speed and large cell clusters from the plate culture medium in the step (8) to obtain high-yield cell strains;
(10) preparing the same liquid culture medium as the step (5) and dispensing into a shake flask or a bioreactor, and inoculating the high-yield cell strain screened in the step (9) into the culture medium for suspension culture. Culturing for 3-4 weeks under a dark condition under the culture condition of 20-25 ℃ to obtain high-activity amplified cells, wherein the rotating speed of the shaking table or the bioreactor is 80-120 r/min;
(11) adding a combined inducer containing methyl jasmonate, hydrogen peroxide and salicylic acid and a combined precursor containing one or more of sodium pyruvate, acetyl coenzyme A and mevalonic acid into the culture solution obtained in the step (10) under an aseptic condition, and performing biotransformation and synthetic culture for 5-10 days;
(12) collecting culture solution and cells, and extracting glycoside compounds containing rhizoma Panacis Japonici saponin, notoginsenoside and ginsenoside from the culture solution.
In one embodiment, the slant-inducing medium of step (1) is prepared by adding 0.1-10 mg/L auxin and 0.1-10 mg/L cytokinin, 20-50 g/L sucrose or glucose, 5-8 g/L agar or 1.8-2.5 g/L Gellan Gum (Gellan Gum) as a coagulant to RW medium, sterilizing at 115-120 ℃ under 0.1-0.15 MPa for 10-25 minutes, and cooling.
In one embodiment, the panax japonicus callus obtained in the step (2) is obtained by taking young stems or buds of panax japonicus as explants, sterilizing the young stems or buds of panax japonicus as explants for 30-60 seconds by using ethanol with the volume percent of 70-75%, then transferring the young stems or buds into sodium hypochlorite or calcium hypochlorite solution with the mass percent of 5-10, preferably 5-7%, sterilizing the young stems or buds for 10-25 minutes, preferably 10-20 minutes, then washing the young stems or buds with sterile water for two-three times, cutting off the stem tips subjected to surface sterilization under the sterile condition, or cutting the young stems into 0.5-1 cm stem segments, inoculating the stem segments into the inclined plane induction culture medium obtained in the step (1), then transferring the stem segments into the inclined plane induction culture medium with the temperature of 18-25 ℃, 3000Lux under 2000 and 3000Lux for 12-16 hours, and performing induction culture to form the callus after 1-.
In one embodiment, the plate acclimatization medium in step (3) is prepared as follows: adding 0.1-5 mg/L auxin and 0.1-5 mg/L phytocytomin, 20-50 g/L sucrose, 5-8 g/L agar or 1.8-2.5 g/L gellan gum as coagulants into a RW culture medium, sterilizing at 115-120 ℃ under the pressure of 0.1-0.15 MPa for 15-18 minutes, and cooling to obtain the microbial preparation.
In one embodiment, the liquid medium of step (5) is prepared as follows: adding 20-40 g/L glucose, 0.1-3 mg/L auxin and 0.1-3 mg/L phytocytokinin into a RW culture medium, adjusting the pH value to 5.5-7.0 by using acid or alkali, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 15-18 minutes, and cooling to prepare a liquid culture medium.
In one embodiment, the scatter seeding of step (8) is characterized in that the selected cell line is a cell mass formed by multiple divisions of a single cell, and the larger the cell mass, the faster the cell division speed, the higher the cell yield, and thus the cell line is defined as a high-yield cell line.
In one embodiment, in the step (11), the combination inducer comprises 0.1-2 mg/L of methyl jasmonate, 20-80 μ L/L of hydrogen peroxide and 20-90 mg/L of salicylic acid, and the combination precursor comprises one or more of 40-80 mg/L of sodium pyruvate, 1-5 mg/L of acetyl-CoA anhydride and 20-40 mg/L of mevalonate.
In one embodiment, the synthetic culture in step (11) is to culture the expanded high-activity panax japonicus cells in the culture solution of step (10) for 3-4 weeks, and continue culturing after adding the combined precursor and the combined elicitor, wherein the culture conditions are as follows: and (3) carrying out suspension culture in a shaking table or a large-scale bioreactor at the temperature of 25-30 ℃ and under the condition of keeping out of the sun for 5-10 days at 80-120 rpm.
In one embodiment, in the method for preparing the extract of saponin compounds described in step (12), the method comprises transferring the harvested cells into an extraction tank, crushing at 300-400 rpm, extracting with a solvent, concentrating under reduced pressure to obtain a crude extract, and extracting, separating and purifying to obtain the final product.
In one embodiment, the plant auxin is alpha-naphthylacetic acid (NAA), 2,4-D (2, 4-dichlorophenoxyacetic acid) or indoleacetic acid and the plant cytokinin is 6-benzylaminopurine (6-BA) or kinetin KT.
The molecular structure of the main effective component of the panax japonicus saponin in the extract obtained by the method is as follows:
molecular structure of oleana ne type panax japonicus saponin (panax japonicus saponin)
Wherein R is1GlcA (gluconic acid), Arb (arabinose) or Xyl (xylose); r2Glc (glucose)
Drawings
FIG. 1 is HPLC chromatogram of total saponins of Panax japonicus. HPLC (Shimadzu SPD-10AVP System) method used a Waters Symmetry C18 column (5 μm; 250 mm. times.4.6 mm). The column oven was set at 50 ℃ and the UV detection wavelength was set at 203 nm. The solvents were water (A) and methanol/acetonitrile (1:1, v: v) (B). Gradient elution conditions: 0-60 min, B is 0-100%; 60-70 min, B is 100% -0; 70-75 min, B is 0. The elution rate was 1.0 mL/min.
FIG. 2 is a semi-preparative chromatogram of total saponins of panax japonicus obtained by purification according to the invention. The semi-preparative chromatography (Shimadzu LC-6AD system) used Shim-Pock PREP-ODS (H) KIT (20X 250). The column oven was set at 50 ℃ and the UV detection wavelength was set at 203 nm. The solvents were water (A) and methanol/acetonitrile (1:1, v: v) (B). Gradient elution conditions: 0-60 min, B is 0-100%; 60-70 min, B is 100% -0; 70-75 min, B is 0. The elution rate was 8 mL/min. The maximum peak at the intermediate saponin was collected.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The present invention is further illustrated by the following specific examples.
Example one:
(1) adding 2mg/L of alpha-naphthylacetic acid (NAA), 2,4-D or indoleacetic acid (IAA), 4mg/L of 6-benzylaminopurine (6-BA) or Kinetin (KT) and 20g/L of cane sugar or glucose into a RW culture medium, adjusting the pH value to 5.6 by using acid or alkali, adding 2g/L of gellan gum as a coagulant, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 18 minutes, and cooling to prepare a slant induction culture medium;
(2) sterilizing young stems or young buds of Panax japonicus as explants with 70% ethanol for 50 s, sterilizing in 5% sodium hypochlorite or calcium hypochlorite solution for 20 min, washing with sterile water for three times, cutting off the stem tips after surface sterilization under aseptic condition, or cutting young stems into 0.5cm or inoculating the stem tips into the slant induction culture medium. Then transferring to 25 ℃, 2000Lux, and carrying out induction culture under 12-hour illumination conditions to form callus after 2 months;
(3) adding 1mg/L alpha-naphthylacetic acid (NAA), 2mg/L and/or Kinetin (KT), 30g/L sucrose and 1.8g/L gellan gum coagulant into a RW culture medium, sterilizing at 115-120 ℃ under the pressure of 0.1-0.15 MPa for 18 minutes, and cooling to prepare a plate domestication culture medium;
(4) inoculating the callus formed by induction into the domestication culture medium for multiple domestication subculture, and culturing and screening high-yield cell clusters with high growth speed and loose texture;
(5) adding 30g/L of sucrose, 1mg/L of alpha-naphthylacetic acid (NAA), 2mg/L of 6-benzylaminopurine (6-BA) and Kinetin (KT) on the basis of the RW culture medium, then adjusting the pH value to 5.5-7.0 by using acid or alkali, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 16 minutes, and cooling to prepare a liquid culture medium;
(6) inoculating the high-yield cell mass on a liquid culture medium, and culturing for 3 weeks at 25 ℃ under a dark condition to obtain a large amount of high-activity single cells, wherein the rotating speed of a shaking table or a bioreactor is 120 r/min during liquid culture;
(7) adding a coagulant into the liquid culture medium same as the liquid culture medium in the step (5), sterilizing at high temperature, and cooling to prepare a plate culture medium; the high-temperature sterilization conditions are the same as those in the step (1);
(8) dispersedly inoculating the single cells of the culture solution in the step (6) on the plate culture medium in the step (7) by using a sterile pipette for culturing for 4 weeks;
(9) screening out cell clusters with higher growth speed (larger cell clusters) from the plate culture medium (8) to be used as high-yield cell strains;
(10) preparing the same liquid culture medium as in (5) and dispensing into a shake flask or a bioreactor, and inoculating the high-producing cell strain selected in (9) into the culture medium for suspension culture. The culture condition is 20 ℃, and after 3 weeks of culture under the dark condition, high-activity amplified cells are obtained. The rotating speed of the shaking table or the bioreactor is 80 r/min;
(11) adding a combined precursor (including 40mg/L of sodium pyruvate, 1mg/L of acetyl-CoA anhydride or 20mg/L of mevalonic acid) and a combined inducer (20 mg/L of salicylic acid, 20. mu.l/L of hydrogen peroxide and 0.1mg/L of methyl jasmonate) into the culture solution of (10), and further culturing for 5 days;
(12) transferring the harvested cells into an extraction tank, stirring at high speed (300 r/min), crushing, extracting with solvent, concentrating under reduced pressure to obtain crude extract, directly extracting the culture solution, and separating and purifying to obtain compounds such as panax japonicus saponin with effective component content 5 times of that of the corresponding extract obtained from natural panax japonicus by the same extraction method.
Example two:
(1) adding 5mg/L of alpha-naphthylacetic acid (NAA) and 5mg/L of 6-benzylaminopurine (6-BA) and 20g/L of cane sugar or glucose into a RW culture medium, adjusting the pH value to 5.5-7.0 by using acid or alkali, adding 5g/L of agar as a coagulant, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 15 minutes, and cooling to prepare an inclined plane induction culture medium;
(2) sterilizing young stems or young buds of Panax japonicus as explants with 75% ethanol for 30 s, sterilizing in 6% sodium hypochlorite or calcium hypochlorite solution for 15 min, washing with sterile water twice, cutting off the stem tips after surface sterilization under aseptic condition, or cutting young stems into 0.8cm or inoculating the stem tips into the slant induction culture medium. Then transferring to 18 ℃, 2000Lux, and carrying out induction culture under 12-hour illumination conditions to form callus after 2 months;
(3) adding 0.1mg/L indoleacetic acid (IAA), 5mg/L and 6-benzylaminopurine (6-BA), 20g/L sucrose and 5g/L agar coagulant into a RW culture medium, sterilizing at 115-120 ℃ under the pressure of 0.1-0.15 MPa for 16 minutes, and cooling to prepare a plate domestication culture medium;
(4) inoculating the callus formed by induction into the domestication culture medium for multiple domestication subculture, and culturing and screening high-yield cell clusters with high growth speed and loose texture;
(5) adding 20g/L of sucrose, 0.1mg/L of indoleacetic acid (IAA) and 2mg/L of 6-benzylaminopurine (6-BA) on the basis of the RW culture medium, then adjusting the pH value to 5.5-7.0 by using acid or alkali, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 17 minutes, and cooling to prepare a liquid culture medium;
(6) inoculating the high-yield cell mass on a liquid culture medium, and culturing for 2 weeks at 20 ℃ under a dark condition to obtain a large amount of high-activity single cells, wherein the rotating speed of a shaking table or a bioreactor is 80 r/min during liquid culture;
(7) adding a coagulant into the liquid culture medium same as the liquid culture medium in the step (5), sterilizing at high temperature, and cooling to prepare a plate culture medium; the high-temperature sterilization conditions are the same as those in the step (1);
(8) dispersedly inoculating the single cells of the culture solution in the step (6) on the plate culture medium in the step (7) by using a sterile pipette for culturing for 4 weeks;
(9) screening out cell clusters with higher growth speed (larger cell clusters) from the plate culture medium (8) to be used as high-yield cell strains;
(10) preparing the same liquid culture medium as in (5) and dispensing into a shake flask or a bioreactor, and inoculating the high-producing cell strain selected in (9) into the culture medium for suspension culture. The culture condition is 20 ℃, and after 3 weeks of culture under the dark condition, high-activity amplified cells are obtained. The rotating speed of the shaking table or the bioreactor is 80 r/min;
(11) adding a combined precursor (including 40mg/L of sodium pyruvate, 1mg/L of acetyl-CoA anhydride or 20mg/L of mevalonic acid) and a combined inducer (20 mg/L of salicylic acid, 20. mu.l/L of hydrogen peroxide and 0.1mg/L of methyl jasmonate) into the culture solution of (10), and further culturing for 5 days;
(12) transferring the harvested cells into an extraction tank, stirring at high speed (300 r/min), crushing, extracting with solvent, concentrating under reduced pressure to obtain crude extract, directly extracting the culture solution, and separating and purifying to obtain compounds such as panax japonicus saponin with effective component content 6 times of that of the corresponding extract obtained from natural panax japonicus by the same extraction method.
Example three:
(1) adding 2, 4-D0.5 mg/L, Kinetin (KT) 1mg/L and sucrose 30g/L into RW culture medium, adjusting pH to 5.5 with acid or alkali, adding 1.8g/L gellan gum as coagulant, sterilizing at 115-120 deg.C under 0.1MPa for 18 min, and cooling to obtain slant induction culture medium;
(2) sterilizing young stems or young buds of Panax japonicus as explants with 70% ethanol for 40 s, sterilizing in 6% sodium hypochlorite solution for 15 min, washing with sterile water for three times, and cutting off the stem tips after surface sterilization under aseptic condition, or cutting young stems into 0.8cm or inoculating the stem tips into the slant induction culture medium. Then transferring to 23 ℃, 2500Lux, and carrying out induction culture under the condition of 14 hours of illumination, and forming callus after 1 month;
(3) adding 2 mg/L2, 4-D, 2mg/L and 6-benzylaminopurine (6-BA), 50g/L sucrose and 6g/L agar into a RW culture medium, sterilizing at 115-120 ℃ under the pressure of 0.1-0.15 MPa for 16 minutes, and cooling to prepare a plate domestication culture medium;
(4) inoculating the callus formed by induction into the domestication culture medium for multiple domestication subculture, and culturing and screening high-yield cell clusters with high growth speed and loose texture;
(5) adding 30g/L of sucrose, 2, 4-D2 mg/L of sucrose and 2mg/L of Kinetin (KT) on the basis of a RW culture medium, then adjusting the pH value to 5.5 by using acid or alkali, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 15 minutes, and cooling to prepare a liquid culture medium;
(6) inoculating the high-yield cell mass on a liquid culture medium, and culturing for 2 weeks at 20 ℃ under a dark condition to obtain a large amount of high-activity single cells, wherein the rotating speed of a shaking table or a bioreactor is 100 r/min during liquid culture;
(7) adding a coagulant into the liquid culture medium same as the liquid culture medium in the step (5), sterilizing at high temperature, and cooling to prepare a plate culture medium; the high-temperature sterilization conditions are the same as those in the step (1);
(8) dispersedly inoculating the single cells of the culture solution in the step (6) on the plate culture medium in the step (7) by using a sterile pipette for culturing for 5 weeks;
(9) screening out cell clusters with higher growth speed (larger cell clusters) from the plate culture medium (8) to be used as high-yield cell strains;
(10) preparing the same liquid culture medium as in (5) and dispensing into a shake flask or a bioreactor, and inoculating the high-producing cell strain selected in (9) into the culture medium for suspension culture. The culture condition is 22 ℃, and after culturing for 4 weeks under the dark condition, high-activity amplified cells are obtained. The rotating speed of the shaking table or the bioreactor is 110 r/min;
(11) adding a combined precursor (comprising 50mg/L of sodium pyruvate, 3mg/L of acetyl-CoA anhydride or 30mg/L of mevalonic acid) and a combined inducer (40 mg/L of salicylic acid, 40. mu.l/L of hydrogen peroxide or 1mg/L of methyl jasmonate) into the culture solution of (10), and further culturing for 6 days;
(12) transferring the harvested cells into an extraction tank, stirring at high speed (350 rpm) for crushing, extracting with a solvent, concentrating under reduced pressure to obtain a crude extract, directly extracting the culture solution, and separating and purifying to obtain compounds such as panax japonicus saponin, wherein the content of the effective component of panax japonicus saponin is 10 times of that of the corresponding extract obtained from natural panax japonicus by the same extraction method.
Example four:
(1) adding 10mg/L of indoleacetic acid (IAA) and 10mg/L of 6-benzylaminopurine (6-BA) and 30g/L of sucrose or glucose into a RW culture medium, adjusting the pH value to 5.8 by using acid or alkali, adding 8g/L of agar as a coagulant, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 18 minutes, and cooling to prepare an inclined plane induction culture medium;
(2) sterilizing young stems or young buds of Panax japonicus as explants with 75% ethanol for 50 s, sterilizing in 7% sodium hypochlorite or calcium hypochlorite solution for 20 min, washing with sterile water for three times, cutting off the stem tips after surface sterilization under aseptic condition, or cutting young stems into 1cm or inoculating the stem tips into the slant induction culture medium. Then transferring to 25 ℃, 3000Lux, and carrying out induction culture under 16-hour illumination conditions to form callus after 1 month;
(3) adding 5mg/L indoleacetic acid (IAA), 5 mg/L6-benzylaminopurine (6-BA), 40g/L sucrose and 8g/L agar into a RW culture medium, sterilizing at 115-120 ℃ under the pressure of 0.1-0.15 MPa for 15 minutes, and cooling to prepare a plate domestication culture medium;
(4) inoculating the callus formed by induction into the domestication culture medium for multiple domestication subculture, and culturing and screening high-yield cell clusters with high growth speed and loose texture;
(5) adding 40g/L of sucrose, 2mg/L of indoleacetic acid (IAA) and 2mg/L of 6-benzylaminopurine (6-BA) on the basis of a RW culture medium, then adjusting the pH value to 5.6 by using acid or alkali, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 15 minutes, and cooling to prepare a liquid culture medium;
(6) inoculating the high-yield cell mass on a liquid culture medium, and culturing for 3 weeks at 25 ℃ under a dark condition to obtain a large amount of high-activity single cells, wherein the rotating speed of a shaking table or a bioreactor is 120 r/min during liquid culture;
(7) adding a coagulant into the liquid culture medium same as the liquid culture medium in the step (5), sterilizing at high temperature, and cooling to prepare a plate culture medium; the high-temperature sterilization conditions are the same as those in the step (1);
(8) dispersedly inoculating the single cells of the culture solution in the step (6) on the plate culture medium in the step (7) by using a sterile pipette for culturing for 5 weeks;
(9) screening out cell clusters with higher growth speed (larger cell clusters) from the plate culture medium (8) to be used as high-yield cell strains;
(10) preparing the same liquid culture medium as in (5) and dispensing into a shake flask or a bioreactor, and inoculating the high-producing cell strain selected in (9) into the culture medium for suspension culture. The culture condition is 25 ℃, and after culturing for 4 weeks under the dark condition, high-activity amplified cells are obtained. The rotating speed of the shaking table or the bioreactor is 120 r/min;
(11) adding a combined precursor (comprising 80mg/L of sodium pyruvate, 5mg/L of acetyl-CoA anhydride or 40mg/L of mevalonic acid) and a combined inducer (90 mg/L of salicylic acid, 80. mu.l/L of hydrogen peroxide or 2mg/L of methyl jasmonate) into the culture solution of (10), and further culturing for 10 days;
(12) transferring the harvested cells into an extraction tank, stirring at high speed (400 rpm), crushing, extracting with solvent, concentrating under reduced pressure to obtain crude extract, directly extracting the culture solution, and separating and purifying to obtain compounds such as panax japonicus saponin with an effective component content 9 times that of the corresponding extract obtained from natural panax japonicus by the same extraction method.
Example five:
(1) adding 2,4-D5 mg/L, Kinetin (KT)6 mg/L and sucrose 30g/L into RW culture medium, adjusting pH to 5.8 with acid or alkali, adding 2.5g/L gellan gum as coagulant, sterilizing at 115-120 deg.C under 0.1MPa for 17 min, and cooling to obtain slant induction culture medium;
(2) sterilizing young stems or young buds of Panax japonicus as explants with 75% ethanol for 50 s, sterilizing in 7% sodium hypochlorite or calcium hypochlorite solution for 20 min, washing with sterile water twice, cutting off the stem tips after surface sterilization under aseptic condition, or cutting young stems into 1cm or inoculating the stem tips into the slant induction culture medium. Then transferring to 22 ℃, 3000Lux, and carrying out induction culture under 16-hour illumination conditions to form callus after 1 month;
(3) adding 4mg/L alpha-naphthylacetic acid (NAA), 3mg/L Kinetin (KT), 20g/L cane sugar and 2.5g/L gellan gum coagulant into a RW culture medium, sterilizing at 115-120 ℃ under the pressure of 0.1-0.15 MPa for 15 minutes, and cooling to prepare a plate domestication culture medium;
(4) inoculating the callus formed by induction into the domestication culture medium for multiple domestication subculture, and culturing and screening high-yield cell clusters with high growth speed and loose texture;
(5) adding 40g/L of sucrose, 2mg/L of alpha-naphthylacetic acid (NAA) and 2mg/L of Kinetin (KT) on the basis of a RW culture medium, then adjusting the pH value to 6.0 by using acid or alkali, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 16 minutes, and cooling to prepare a liquid culture medium;
(6) inoculating the high-yield cell mass on a liquid culture medium, and culturing for 3 weeks at 25 ℃ under a dark condition to obtain a large amount of high-activity single cells, wherein the rotating speed of a shaking table or a bioreactor is 120 r/min during liquid culture;
(7) adding a coagulant into the liquid culture medium same as the liquid culture medium in the step (5), sterilizing at high temperature, and cooling to prepare a plate culture medium; the high-temperature sterilization conditions are the same as those in the step (1);
(8) dispersedly inoculating the single cells of the culture solution in the step (6) on the plate culture medium in the step (7) by using a sterile pipette for culturing for 5 weeks;
(9) screening out cell clusters with higher growth speed (larger cell clusters) from the plate culture medium (8) to be used as high-yield cell strains;
(10) preparing the same liquid culture medium as in (5) and dispensing into a shake flask or a bioreactor, and inoculating the high-producing cell strain selected in (9) into the culture medium for suspension culture. The culture condition is 25 ℃, and after 3 weeks of culture under the dark condition, high-activity amplified cells are obtained. The rotating speed of the shaking table or the bioreactor is 120 r/min;
(11) adding a combined precursor (comprising 70mg/L of sodium pyruvate, 3mg/L of acetyl-CoA anhydride or 40mg/L of mevalonic acid) and a combined inducer (60 mg/L of salicylic acid, 60. mu.l/L of hydrogen peroxide and 1.5mg/L of methyl jasmonate) into the culture solution of (10), and further culturing for 7 days;
(12) transferring the harvested cells into an extraction tank, stirring at high speed (400 rpm), crushing, extracting with solvent, concentrating under reduced pressure to obtain crude extract, directly extracting the culture solution, and separating and purifying to obtain compounds such as panax japonicus saponin with effective component content 7 times of that of corresponding extract obtained from natural panax japonicus by the same extraction method.
Example six:
(1) adding 6 mg/L indoleacetic acid (IAA) and 6 mg/L6-benzylaminopurine (6-BA) and 40g/L sucrose or glucose into a RW culture medium, adjusting the pH value to 6.0 by using acid or alkali, adding 7g/L agar as a coagulant, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 18 minutes, and cooling to prepare an inclined plane induction culture medium;
(2) sterilizing young stems or young buds of Panax japonicus as explants with 75% ethanol for 50 s, sterilizing in 5% calcium hypochlorite solution for 20 min, washing with sterile water for three times, and cutting off the stem tips sterilized under aseptic condition, or cutting young stems into 0.5cm or inoculating the stem tips into the slant induction culture medium. Then transferring to 25 ℃, 3000Lux, and carrying out induction culture under 12-hour illumination conditions to form callus after 1 month;
(3) adding 2mg/L alpha-naphthylacetic acid (NAA), 3 mg/L6-benzylaminopurine (6-BA), 20g/L sucrose and 6g/L agar into a RW culture medium, sterilizing at 115-120 ℃ under the pressure of 0.1-0.15 MPa for 16 minutes, and cooling to prepare a plate domestication culture medium;
(4) inoculating the callus formed by induction into the domestication culture medium for multiple domestication subculture, and culturing and screening high-yield cell clusters with high growth speed and loose texture;
(5) adding 20g/L of sucrose, 2mg/L of alpha-naphthylacetic acid (NAA), 2mg/L of 6-benzylaminopurine (6-BA) on the basis of the RW culture medium, then adjusting the pH value to 5.5 by using acid or alkali, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 15 minutes, and cooling to prepare a liquid culture medium;
(6) inoculating the high-yield cell mass on a liquid culture medium, and culturing for 3 weeks at 20 ℃ under a dark condition to obtain a large amount of high-activity single cells, wherein the rotating speed of a shaking table or a bioreactor is 90 r/min during liquid culture;
(7) adding a coagulant into the liquid culture medium same as the liquid culture medium in the step (5), sterilizing at high temperature, and cooling to prepare a plate culture medium; the high-temperature sterilization conditions are the same as those in the step (1);
(8) dispersedly inoculating the single cells of the culture solution in the step (6) on the plate culture medium in the step (7) by using a sterile pipette for culturing for 5 weeks;
(9) screening out cell clusters with higher growth speed (larger cell clusters) from the plate culture medium (8) to be used as high-yield cell strains;
(10) preparing the same liquid culture medium as in (5) and dispensing into a shake flask or a bioreactor, and inoculating the high-producing cell strain selected in (9) into the culture medium for suspension culture. The culture condition is 25 ℃, and after culturing for 4 weeks under the dark condition, high-activity amplified cells are obtained. The rotating speed of the shaking table or the bioreactor is 100 r/min;
(11) adding a combined precursor (comprising 70mg/L of sodium pyruvate, 2mg/L of acetyl coenzyme A or 20mg/L of mevalonate) and a combined inducer (80 mg/L of salicylic acid, 70 mu L/L of hydrogen peroxide and 1.5mg/L of methyl jasmonate) into the culture solution of (10), and continuously culturing for 8 days;
(12) transferring the harvested cells into an extraction tank, stirring at high speed (300 r/min), crushing, extracting with solvent, concentrating under reduced pressure to obtain crude extract, directly extracting the culture solution, and separating and purifying to obtain compounds such as panax japonicus saponin with effective component content 8 times of that of corresponding extract obtained from natural panax japonicus by the same extraction method.
Example seven:
(1) adding 2mg/L of indoleacetic acid (IAA) and 8 mg/L of 6-benzylaminopurine (6-BA) and 30g/L of sucrose or glucose into a RW culture medium, adjusting the pH value to 5.7 by using acid or alkali, adding 6g/L of agar as a coagulant, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 15 minutes, and cooling to prepare an inclined plane induction culture medium;
(2) sterilizing young stems or young buds of Panax japonicus as explants with 70% ethanol for 50 s, sterilizing in 5% sodium hypochlorite or calcium hypochlorite solution for 20 min, washing with sterile water twice, cutting off the stem tips after surface sterilization under aseptic condition, or cutting young stems into 1cm or inoculating the stem tips into the slant induction culture medium. Then transferring to 25 ℃, 2000Lux, and carrying out induction culture under 12-hour illumination conditions to form callus after 2 months;
(3) adding 3mg/L alpha-naphthylacetic acid (NAA), 4 mg/L6-benzylaminopurine (6-BA), 30g/L sucrose and 7g/L agar into a RW culture medium, sterilizing at 115-120 ℃ under the pressure of 0.1-0.15 MPa for 15 minutes, and cooling to prepare a plate domestication culture medium;
(4) inoculating the callus formed by induction into the domestication culture medium for multiple domestication subculture, and culturing and screening high-yield cell clusters with high growth speed and loose texture;
(5) adding 20g/L of sucrose, 1mg/L of alpha-naphthylacetic acid (NAA), 1mg/L of 6-benzylaminopurine (6-BA) and 1mg/L of RW culture medium, adjusting the pH value to 5.6 by using acid or alkali, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 15 minutes, and cooling to prepare a liquid culture medium;
(6) inoculating the high-yield cell mass on a liquid culture medium, and culturing for 2 weeks at 25 ℃ under a dark condition to obtain a large amount of high-activity single cells, wherein the rotating speed of a shaking table or a bioreactor is 90 r/min during liquid culture;
(7) adding a coagulant into the liquid culture medium same as the liquid culture medium in the step (5), sterilizing at high temperature, and cooling to prepare a plate culture medium; the high-temperature sterilization conditions are the same as those in the step (1);
(8) dispersedly inoculating the single cells of the culture solution in the step (6) on the plate culture medium in the step (7) by using a sterile pipette for culturing for 4 weeks;
(9) screening out cell clusters with higher growth speed (larger cell clusters) from the plate culture medium (8) to be used as high-yield cell strains;
(10) preparing the same liquid culture medium as in (5) and dispensing into a shake flask or a bioreactor, and inoculating the high-producing cell strain selected in (9) into the culture medium for suspension culture. The culture condition is 25 ℃, and after culturing for 4 weeks under the dark condition, high-activity amplified cells are obtained. The rotating speed of the shaking table or the bioreactor is 110 r/min;
(11) adding a combined precursor (comprising 50mg/L of sodium pyruvate, 4mg/L of acetyl-CoA anhydride or 35mg/L of mevalonic acid) and a combined inducer (600 mg/L of salicylic acid, 50. mu.l/L of hydrogen peroxide and 1mg/L of methyl jasmonate) into the culture solution of (10), and continuously culturing for 8 days;
(12) transferring the harvested cells into an extraction tank, stirring at high speed (400 rpm), crushing, extracting with solvent, concentrating under reduced pressure to obtain crude extract, directly extracting the culture solution, and separating and purifying to obtain compounds such as panax japonicus saponin, wherein the content of effective component panax japonicus saponin is 10 times of that of corresponding extract obtained from natural panax japonicus by the same extraction method.
Claims (7)
1. A method for producing saponin extract containing chikusetsusaponin, notoginsenoside and ginsenoside by using chikusetsusaponin cells via industrialized culture and biotransformation technology; the method is characterized by comprising the following steps:
(1) adding auxin, plant cytokinin, one or two of sucrose and glucose and coagulant into RW culture medium, sterilizing at high temperature, and cooling to obtain slant induction culture medium; the high-temperature disinfection parameters are 115-120 ℃, and the disinfection lasts for 10-25 minutes under the pressure of 0.1-0.15 MPa;
(2) after surface sterilization, cutting the juvenile stems or buds of panax japonicus into 0.5-1 cm stem segments or taking the stem tips of the stem segments as explants, inoculating the stem segments or the stem tips to the slant induction culture medium in the step (1), and culturing to form callus; the surface sterilization treatment method comprises the steps of sterilizing for 30-60 seconds by using 70-75 vol% ethanol, treating for 15-25 minutes by using 5-7 mass% hypochlorite solution, and then washing for two-three times by using sterile water for later use;
(3) adding plant cytokinin, auxin, sucrose and a coagulant into a RW culture medium, and preparing a plate domestication culture medium after high-temperature disinfection; the high-temperature sterilization parameters are the same as those of the step (1); wherein the preparation of the plate domestication culture medium in the step (3) is as follows: adding 0.1-5 mg/L auxin and 0.1-5 mg/L phytocytomin, 20-50 g/L sucrose, 5-8 g/L agar or 1.8-2.5 g/L gellan gum as coagulants into a RW culture medium, sterilizing at 115-120 ℃ under the pressure of 0.1-0.15 MPa for 15-18 minutes, and cooling to obtain the microbial preparation;
(4) inoculating the callus of the panax japonicus induced and formed in the step (2) on an acclimatization culture medium for multiple subcultures, and screening out high-yield cell clusters with high growth speed and loose texture;
(5) adding auxin, cytokinin and sucrose into RW culture medium, and sterilizing at high temperature to obtain liquid culture medium; the high-temperature disinfection parameters are the same as those in the step (1); wherein the liquid medium of step (5) is prepared as follows: adding 20-40 g/L of sucrose, 0.1-3 mg/L of auxin and 0.1-3 mg/L of cytokinin into a RW culture medium, then adjusting the pH value to 5.5-7.0 by using acid or alkali, sterilizing at 115-120 ℃ under the pressure of 0.1MPa for 15-18 minutes, and cooling to prepare a liquid culture medium;
(6) inoculating the high-yield cell clusters screened in the step (4) into the liquid culture medium in the step (5) for suspension culture for 2-3 weeks, wherein the rotating speed of a shaking table is 100-120 r/min;
(7) adding a coagulant into the liquid culture medium same as the liquid culture medium in the step (5), sterilizing at high temperature, and cooling to prepare a plate culture medium; the high-temperature sterilization conditions are the same as the step (1);
(8) dispersedly inoculating the single cells cultured in the step (6) on the plate culture medium cultured in the step (7) by using a sterile pipette for culturing for 4-5 weeks;
(9) screening cell clusters with high growth speed and large cell clusters from the plate culture medium in the step (8) to obtain high-yield cell strains;
(10) preparing a liquid culture medium which is the same as the liquid culture medium prepared in the step (5), injecting the liquid culture medium into a shake flask or a bioreactor, inoculating the high-yield cell strain screened in the step (9) into the culture solution for suspension culture, and culturing for 3-4 weeks under the dark condition at the culture condition of 20-25 ℃ to obtain high-activity amplified cells, wherein the rotation speed of the shaking table or the bioreactor is 80-120 r/min;
(11) adding a combined inducer containing methyl jasmonate, hydrogen peroxide and salicylic acid and a combined precursor containing one or more of sodium pyruvate, acetyl coenzyme A and mevalonic acid into the culture solution obtained in the step (10) under an aseptic condition, and performing biotransformation and synthetic culture for 5-10 days; in the step (11), the combined elicitor comprises 0.1-2 mg/L of methyl jasmonate, 20-80 mu L/L of hydrogen peroxide and 20-90 mg/L of salicylic acid, and the combined precursor comprises one or more of 40-80 mg/L of sodium pyruvate, 1-5 mg/L of acetyl coenzyme A and 20-40 mg/L of mevalonic acid;
(12) collecting culture solution and cells, and extracting saponins extract containing chikusetsusaponin, notoginsenoside and ginsenoside from the culture solution.
2. The method according to claim 1, wherein the slant-inducing medium of step (1) is prepared by adding 0.1 to 10mg/L auxin and 0.1 to 10mg/L cytokinin, 20 to 50g/L one or two selected from sucrose and glucose, 5 to 8g/L agar or 1.8 to 2.5g/L gellan gum as a coagulant to RW medium, sterilizing the medium at 115 to 120 ℃ under 0.1 to 0.15MPa for 10 to 25 minutes, and cooling the resultant product.
3. The method as claimed in claim 1, wherein the callus of panax japonicus in step (2) is obtained by sterilizing young stems or buds of panax japonicus as explants with 70-75 vol% ethanol for 30-60 s, sterilizing the young stems or buds by transferring the young stems or buds into 5-7 mass% sodium hypochlorite or calcium hypochlorite solution for 15-25 min, washing the young stems or buds with sterile water for two-three times, cutting off stem tips after surface sterilization under sterile conditions, or cutting young stems into 0.5-1 cm stem segments, inoculating the stem segments into the slant induction culture medium in step (1), transferring the stem segments into the slant induction culture medium at 18-25 ℃, 2000 + 3000Lux under 12-16 h illumination conditions, and performing induction culture for 1-2 months to form callus.
4. The method according to any one of claims 1 to 3, wherein the scatter seeding in step (8) is characterized in that the selected cell line is a cell mass formed by multiple divisions of a single cell, and the larger the cell mass is, the faster the cell division speed is, the higher the cell yield is, thus defining as a high-yielding cell line.
5. The method according to any one of claims 1 to 3, wherein the synthetic culture in step (11) is performed by culturing the expanded high-activity panax japonicus cells in the culture solution of step (10) for 3 to 4 weeks under the following culture conditions: and (3) carrying out suspension culture in a shaking table or a large-scale bioreactor at the temperature of 20-25 ℃ and under the condition of keeping out of the sun for 5-10 days at 80-120 rpm.
6. A process according to any one of claims 1 to 3, wherein in the method of preparing the saponin extract in step (12), the method comprises transferring the harvested cells to an extraction tank, disrupting at 300 to 400 rpm, extracting with a solvent and concentrating under reduced pressure to obtain a crude extract, and separating and purifying by extraction.
7. The method of any one of claims 1 to 3 wherein the plant auxin is alpha-naphthylacetic acid, 2, 4-dichlorophenoxyacetic acid or indoleacetic acid and the plant cytokinin is 6-benzylaminopurine or kinetin KT.
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