CN109588319A - A method of Radix Astragali active constituent is produced using immobilization Radix Astragali cell - Google Patents
A method of Radix Astragali active constituent is produced using immobilization Radix Astragali cell Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The present invention provides a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent, this method is realized by the disinfection of Radix Astragali explant, the induction of callus and proliferation, the suspension of Radix Astragali cell and dispersion, the immobilization of Radix Astragali cell, the magnetic field processing of immobilized cell and the culture of immobilized cell this techniqueflow.The present invention immobilizes it using embedded material on the basis of Radix Astragali cell suspension cultures, bioreactor is recycled to be cultivated under given conditions by magnetized immobilized cell to obtain the culture solution containing Radix Astragali active constituent, the content of astragalus polyose is up to 68.7mg/g in culture solution, the content of astragaloside is up to 42.9mg/g, it is significantly higher than conventional Radix Astragali cell suspension cultures, and immobilized cell can also reuse.The present invention provides new way for the acquisition of Radix Astragali active constituent, and also the large-scale industrial production for Radix Astragali active constituent provides theoretical and technical foundation.
Description
Technical field
The present invention relates to Medicinal Plant Cell engineering fields, it is more particularly related to a kind of yellow using immobilization
Stilbene cell produces Radix Astragali active constituent-astragalus polyose and astragaloside method.
Background technique
Radix Astragali also known as continuous stilbene, astragali, first stilbene or astragalus, are pulse family Astragalus herbaceos perennials, in China distribution compared with
Extensively, the ground such as the Inner Mongol, Shanxi, Gansu, Heilungkiang, Jilin, Liaoning, Shaanxi, Hebei, Shandong, Ningxia are mainly grown on, it is furthermore green
Sea, Sichuan, Xinjiang etc. area and Korea, Mongolia, Russia be also distributed.It is included according to the Pharmacopoeia of the People's Republic of China,
Chinese herbal medicine astragalus is the dry root of Astragalus membranacus and astragalus mongolicus, is China's tradition tonic Chinese herbal medicine material, has more than 400 years medicines so far
With history, one of most common drug still is treated for tcm clinical practice at present.Radix Astragali has early in just on the books in Shennong's Herbal
The effect of help is strong.Traditional Chinese medicine thinks, Radix Astragali have invigorating qi for strengthening superficies, diuresis toxin expelling, expelling pus and promoting granulation, tonifying middle-Jiao and Qi work
With.With the rapid development of science and technology, era characteristics are had become using modern science and technology means research traditional Chinese medicine, passed through
Research confirms that Radix Astragali contains a variety of active ingredients, wherein most important have polysaccharide, saponins, flavone compound, also contains
A variety of amino acid, microelement and other compounds.These active constituents make Radix Astragali have following pharmacological action: 1, enhancing machine
Body immunity function participates in Organism immunoregulation, promotes the generation of immune factor；2, blood pressure lowering, hypoglycemic, the cardiovascular disease for the treatment of
Disease；3, antitumor, antiviral；4, interior free yl, anti-oxidant, anti-aging are removed；5, diuresis, removing toxic substances.
With the enhancing of people's health care consciousness, Chinese medicine and various derivative health care products with health-care efficacy are increasingly smooth
Pin, therefore the demand of Radix Astragali also continues to increase.But due to long-term a large amount of excavation, the quantity of wild Radix Astragali was sharply in recent years
It reduces, has the danger for tending to die out, therefore wild Radix Astragali has been listed in state three level protecting plant.Nowadays Radix Astragali Chinese medicine
Source is mainly artificial growth, but artificial growth is there is also many problems, if Radix Astragali grow it is more slow, generally from being seeded into
Harvesting was at least needed by 3 years；Influence during artificial growth vulnerable to seasonal variations and pest and disease damage；There are pesticides for the Radix Astragali of plantation
And the problem of heavy-metal residual；The wilder Radix Astragali of active component content is low etc. in the Radix Astragali of plantation.Studies have shown that medicinal plant
Active constituent is mainly its secondary metabolite, is to overcome by producing secondary metabolite using Medicinal Plant Cell culture
One of wild resource shortage, the effective way for solving artificial growth problems, and the essence of production of crude drugs is reformed.
Currently, having much about the research report of Medicinal Plant Cell culture both at home and abroad, but it is applied to production
Sub-fraction is only accounted for, reason is analyzed, be primarily present following problems: the yield of secondary metabolite is too low, and amplification culture is difficult, plants
The speed of growth of object cell is excessively slow and unstable etc. during the cultivation process.Many studies have shown that by adjusting the hands such as microenvironment
Section can increase the yield of Secondary metabolites.In addition, having carried out purpose secondary metabolite using elicitor
It is widely used in culture plant cell.Elicitor is the general designation for referring to the factor that induction plant generates defense reaction, is planted
For object in the stimulation by elicitor, itself can generate a series of defense reaction, such as allergic reaction, egg related with plant pathogenic
The synthesis and accumulation etc. of white expression and all kinds of resistance compounds, therefore elicitor can be used for improving Secondary metabolites
Content.Bioreactor culture is to realize a kind of mode of plant cell amplification culture, big with working volume, is produced into
This is low, and unit volume production capacity is high, and can be medicinal plant the advantages that do not limited by when and where, can carry out whole year production
Object cell culture and the production of secondary metabolite provide the physicochemical environment of optimization, therefore have a vast market foreground.
Immobilization plant cell technology be free plant cell is limited to chemically or physically method it is a certain specific
In space, activity can be kept, carries out the technology of normal growth metabolism.Compared with plant cell suspension culture, immobilization
Cultivating has lot of advantages, such as raising anti-shear ability, holding cell viability, the synthesis of promotion secondary metabolite and secretion,
It is reusable and can improve production efficiency, be readily produced post-processing etc..Nowadays, it includes silver that domestic and international researcher, which has had been established,
The Immobilized culture system of more than 50 plant cells including the medicinal plants such as apricot, catharanthus roseus, Chinese yew, but yet there are no related
The report of Radix Astragali cell fixation culture.For this purpose, the present invention explores Radix Astragali cell to produce Radix Astragali active constituent as target
Process for fixation, and the Radix Astragali cell of immobilization is used for small-scale production, obtain success.
Summary of the invention
Immobilization Radix Astragali cell production Radix Astragali activity is utilized in view of the deficiencies of the prior art, the present invention provides a kind of
The method of ingredient-astragalus polyose and astragaloside provides theoretical and technology for the large-scale industrial production of Radix Astragali active constituent
Basis.
The purpose of the present invention is what is be achieved through the following technical solutions:
A method of Radix Astragali active constituent is produced using immobilization Radix Astragali cell, which is characterized in that the techniqueflow of this method
It is as follows:
1) disinfection of Radix Astragali explant: the tender stem section of Radix Astragali is chosen as explant, 10 ~ 20min is rinsed with tap water, in nothing
First with 75% ethanol disinfection 30s under the conditions of bacterium, then with 0.1% mercuric chloride 3min is sterilized, finally used aseptic water washing 4 ~ 5 times, filter paper is inhaled
Solid carbon dioxide point, obtains sterile Radix Astragali explant；
2) induction and proliferation of callus: the segment that Radix Astragali explant is cut into 0.5 ~ 0.7cm long is inoculated in containing Fiber differentiation
On the culture dish of base, be placed in 25 ± 1 DEG C, culture induces callus under dark condition, select well-grown callus
It is inoculated in the triangular flask containing proliferated culture medium, carries out shoot proliferation culture, every 21d subculture 1 time under the same conditions；
3) suspension and dispersion of Radix Astragali cell: picking out after subculture 2 ~ 3 times and grow the callus vigorous, quality is more loose,
Access shaking table shaken cultivation in the 500mL triangular flask of the suspension medium containing 100mL, condition of culture is 25 ± 1 DEG C, dark, revolving speed
100 ~ 120r/min, every 14d subculture 1 time gradually forms stable Radix Astragali cell suspension cultures system subculture 3 ~ 4 times, using enzymatic hydrolysis
Method disperses Radix Astragali cell, is then successively filtered cell suspending liquid with the sieve of 0.9mm and 0.1mm, takes filtrate
Immobilize operation；
4) immobilization of Radix Astragali cell: compound concentration be 40g/L sterile sodium alginate solution, and add 0.1%(W/V) mouse
Lee's glycolipid and 2%(W/V) Fe3O4, the filtrate of step 3) is uniformly mixed with this sodium alginate soln, with syringe by mixed liquor
The sterile CaCl that concentration is 20g/L is instilled dropwise2In solution, forming diameter under the stirring of magnetic stirring apparatus is about consolidating for 3mm
Surely change gel beads, be put into refrigerator overnight after standing 30min, take out and use brine 2 ~ 3 times；
5) the magnetic field processing of immobilized cell: immobilization gel beads are placed in sterile triangular flask, being put into magnetic field strength is 500Gs
Low high-intensity magnetic field in handle 30min；
6) culture of immobilized cell: being cultivated using airlift bioreactor, aseptically, by consolidating after magnetization
Surely to change gel beads to access in production medium with 10% inoculum concentration, liquid amount is 50 ~ 60%, and ventilatory capacity is 0.10 ~ 0.15VVM,
Temperature is 25 ± 1 DEG C, and in the bacillus suspension of the 8d access 5% of culture, the NaHS of 100 μm of ol/L continues to cultivate
Culture solution is released after 6-8d, for isolating and purifying for Radix Astragali active constituent (astragalus polyose and astragaloside).
The ingredient of induced medium is MS minimal medium+1.0g/L lactoalbumin hydrolysate+0.5mg/L in the step 2
D-VB5 calcium+1.0mg/L6-BA+0.2mg/L NAA+0.1mg/L choline chloride+30g/L sucrose+5g/L gellan gum, pH5.6 ~
6.0。
The ingredient of proliferated culture medium is MS minimal medium+2.0g/L lactoalbumin hydrolysate+0.5mg/L in the step 2
D-VB5 calcium+0.6mg/L6-BA+0.1mg/L NAA+1.0mg/L KT-30+30g/L sucrose+5g/L gellan gum, pH5.6 ~
6.0。
The inoculum concentration of callus is 40-50g/L in the step 3).
Suspension medium ingredient in the step 3) are as follows: MS+2.0 ~ 3.0g/L of minimal medium lactoalbumin hydrolysate+0.3 ~
0.5mg/L D-VB5 calcium+1.4 ~ 1.8mg/L spermidine+0.5 ~ 0.7mg/L 6-BA+0.1 ~ 0.3mg/L NAA+0.8 ~
1.2mg/L KT-30+30 ~ 40g/L sucrose, pH5.6 ~ 6.0.
The concrete operations of enzymatic isolation method in the step 3) are as follows: compound concentration is pectase liquid (the enzyme activity 40U/ of 1.0g/L
Mg), the pectase liquid of 20% volume is added into Radix Astragali cell suspending liquid, is digested on 28 DEG C, the shaking table of revolving speed 60r/min
24h。
The volume ratio of filtrate and sodium alginate soln is 1:4 in the step 4).
The ingredient of production medium in the step 5) are as follows:+9 ~ 11mg/ of MS minimal medium+16 ~ 20mg/L phenylalanine
L-Tyr+8 ~+120 ~ 130 μm of ol/L methyl jasmonates+0.5 of 10mg/L L-+6.1 ~ 6.5mg/L of cinnamic acid soybean oligopeptide ~
0.7mg/L 6-BA+0.1 ~ 0.3mg/L NAA+0.8 ~ 1.2mg/L KT-30+26 ~ 30g/L sucrose+20 ~ 24g/L sorbierite,
pH5.6~6.0。
Bacillus suspension the preparation method comprises the following steps: by bacillus subtilis and bacillus pumilus in the step 5)
The inclined-plane LB seed culture, respectively according in 4% and 2% inoculum concentration access LB liquid medium, was placed in shaking table oscillation after 24 hours
Culture, condition of culture be 35 ~ 37 DEG C of temperature, 160 ~ 180r/min of revolving speed, culture 20 ~ for 24 hours.
The advantages and positive effects of the present invention are:
1) present invention has found out the embedding immobilization method suitable for Radix Astragali cell, by adding surface-active in embedding medium
Agent rhamnolipid come improve Radix Astragali cell release secondary metabolites ability, to improve the content of active constituent in culture solution；
Magnetic powder Fe is added in embedding medium3O4, and magnetic field processing, the magnetic field effect that magnetic field processing generates are carried out to immobilization Radix Astragali cell
It can promote the growth metabolism of Radix Astragali cell, moreover it is possible to activate the defense reaction of Radix Astragali cell, produce to improve certain secondary metabolisms
The synthesis capability of object makes the amount of astragalus polyose and astragaloside in culture solution increase；Magnetic powder Fe3O4At externally-applied magnetic field
Reason is provided with magnetism, becomes " small magnetic field ", to continue this magnetic field effect.
2) bacillus is added in the incubation of immobilization Radix Astragali cell and is co-cultured by the present invention, and bacillus is
One kind in Radix Astragali endosymbiosis bacterium with host's coevolution and forms the symbiosis of mutual reciprocity and mutual benefit for a long time, can induce Huang
Secondary metabolite accumulates in stilbene cell, and promotes secondary metabolite to extracellular release；The NaHS of addition is H2S gas
The donor of body, H2S gas is important stress response signaling molecule, can equally induce secondary metabolite in Radix Astragali cell
Accumulation, furthermore also added other elicitors in production medium of the invention, substantially increases active constituent in culture solution
Content.
3) the preliminary method established using immobilization Radix Astragali cell production Radix Astragali active constituent of the present invention.It is outstanding with directly utilizing
Floating cell production is compared, and immobilized cell both reaches systematism and requires to again limit growth, to significantly improve secondary metabolism
The yield of product has great development prospect.
Specific embodiment
Below with reference to embodiment, the present invention is further described, following embodiments be it is illustrative, be not restrictive,
It cannot be limited the scope of protection of the present invention with following embodiments.
In following embodiments, the Radix Astragali used is the Astragalus membranacus of artificial growth, closes Chinese medicine plantation from Shandong day
Base；Consumptive material, reagent, strain for using etc. can be commercially available by commercial sources.
Embodiment 1
Following tests is carried out according to the method provided by the invention using immobilization Radix Astragali cell production Radix Astragali active constituent:
1) disinfection of Radix Astragali explant: the tender stem section of Radix Astragali is chosen as explant, 10 ~ 20min is rinsed with tap water, in nothing
First with 75% ethanol disinfection 30s under the conditions of bacterium, then with 0.1% mercuric chloride 3min is sterilized, finally used aseptic water washing 4 ~ 5 times, filter paper is inhaled
Solid carbon dioxide point, obtains sterile Radix Astragali explant.
2) induction and proliferation of callus: the segment that Radix Astragali explant is cut into 0.5 ~ 0.7cm long is inoculated in containing induction
(ingredient is MS minimal medium+1.0g/L lactoalbumin hydrolysate+0.5mg/L D-VB5 calcium+1.0mg/L6-BA+ to culture medium
0.2mg/L NAA+0.1mg/L choline chloride+30g/L sucrose+5g/L gellan gum, pH5.8) culture dish on, be placed in 25 DEG C, it is black
Culture induces callus under dark condition, and selecting well-grown callus to be inoculated in, (MS is trained substantially containing proliferated culture medium
Support base+2.0g/L lactoalbumin hydrolysate+0.5mg/L D-VB5 calcium+0.6mg/L6-BA+0.1mg/L NAA+1.0mg/L KT-30+
30g/L sucrose+5g/L gellan gum, pH5.8) triangular flask in, under the same conditions carry out shoot proliferation culture, every 21d subculture 1
It is secondary.
3) it the suspension and dispersion of Radix Astragali cell: is picked out after subculture 2 ~ 3 times and grows the callus group vigorous, quality is more loose
Knit, with the inoculum concentration of 45g/L access suspension medium containing 100mL (ingredient for MS minimal medium+2.5g/L lactoalbumin hydrolysate+
0.4mg/L D-VB5 calcium+1.6mg/L spermidine+0.6mg/L 6-BA+0.2mg/L NAA+1.0mg/L KT-30+35g/L sugarcane
Sugar, pH5.8) 500mL triangular flask in shaking table shaken cultivation, condition of culture is 25 DEG C, dark, revolving speed 110r/min, every 14d
Subculture 1 time, gradually form stable Radix Astragali cell suspension cultures system subculture 3 ~ 4 times；Compound concentration is the pectase liquid of 1.0g/L
(enzyme activity 40U/mg) pectase liquid of 20% volume is added into Radix Astragali cell suspending liquid, shakes in 28 DEG C, revolving speed 60r/min
Digesting on bed for 24 hours disperses Radix Astragali cell, is then successively filtered cell suspending liquid with the sieve of 0.9mm and 0.1mm, takes
Filtrate immobilizes operation.
4) immobilization of Radix Astragali cell: compound concentration is the sterile sodium alginate solution of 40g/L, and adds 0.1%(W/V)
Rhamnolipid and 2%(W/V) Fe3O4, the filtrate of step 3) is mixed with this sodium alginate soln with the volume ratio of 1:4
It is even, mixed liquor is instilled to the sterile CaCl that concentration is 20g/L dropwise with syringe2In solution, under the stirring of magnetic stirring apparatus
The immobilization gel beads that diameter is about 3mm are formed, refrigerator overnight is put into after standing 30min, takes out and use brine 2 ~ 3
It is secondary.
5) the magnetic field processing of immobilized cell: immobilization gel beads are placed in sterile triangular flask, being put into magnetic field strength is
30min is handled in the low high-intensity magnetic field of 500Gs.
6) culture of immobilized cell: being cultivated using 20L airlift bioreactor, aseptically, by magnetic
Immobilization gel beads after change are accessed in production medium with 10% inoculum concentration, liquid amount 55%, ventilatory capacity 0.12VVM,
Temperature is 25 DEG C, culture 8d access 5% bacillus suspension (the preparation method comprises the following steps: by bacillus subtilis and short and small
The inclined-plane the LB seed culture of bacillus, respectively according in 4% and 2% inoculum concentration access LB liquid medium, is set after 24 hours
In shaking table shaken cultivation, condition of culture is 35 DEG C of temperature, revolving speed 160r/min, cultivates 22h), the NaHS of 100 μm of ol/L,
Continue to release culture solution after cultivating 7d, for isolating and purifying for Radix Astragali active constituent；The wherein ingredient of production medium are as follows: MS base
+ 125 μ of basal culture medium+18mg/L phenylalanine+10mg/L tyrosine+9mg/L L- cinnamic acid+6.3mg/L soybean oligopeptide
Mol/L methyl jasmonate+0.6mg/L 6-BA+0.2mg/L NAA+1.0mg/L KT-30+28g/L sucrose+22g/L sorbierite,
pH5.8。
Influence of 2 rhamnolipid of embodiment to astragalus polyose in culture solution and astragaloside content
Tested according to the method that embodiment 1 provides, in step 4) the additive amount of rhamnolipid be respectively 0,0.05%, 0.1%,
0.15%, 0.2%, the assay of astragalus polyose and astragaloside is carried out after culture to the culture solution of releasing, is as a result seen below
Table 1.
As it can be seen from table 1, with the increase of rhamnolipid additive amount, Radix Astragali is more in culture solution in 0 ~ 0.1% range
Sugar and astragaloside content also gradually increase, and when rhamnolipid additive amount is 0.1%, astragalus polyose content is 68.5mg/L, yellow
Stilbene saponin content is 42.3mg/L, is maximum value, but continuing growing with rhamnolipid additive amount, astragalus polyose in culture solution
It is gradually decreased instead with astragaloside content.Rhamnolipid is a kind of surfactant of nonhazardous, and addition rhamnolipid can
Enhance Radix Astragali permeability of cell membranes, so that the ability of Radix Astragali cell release secondary metabolites is improved, but rhamnolipid adds
The integrality that cell membrane may excessively be destroyed, leads to part cell death, thus in this test 0.1% be most suitable rhamnolipid
Additive amount.
3 Fe of embodiment3O4The influence to astragalus polyose in culture solution and astragaloside content is handled with magnetic field
It is tested according to the method that embodiment 1 provides, Fe is not added in step 4)3O4And cancellation step 5), after culture
The assay that astragalus polyose and astragaloside are carried out to the culture solution of releasing, as a result see the table below 2.
From table 2 it can be seen that astragalus polyose content than embodiment 2 improves 16.3mg/L in embodiment 1, astragaloside contains
Amount improves 13.7mg/L than embodiment 2, shows to add magnetic powder Fe3O4And magnetic field processing is carried out to immobilization Radix Astragali cell and is generated
Beneficial effect.The magnetic field effect that magnetic field processing generates can promote the growth metabolism of Radix Astragali cell, moreover it is possible to activate Radix Astragali cell
Defense reaction make astragalus polyose and astragaloside in culture solution to improve the synthesis capability of certain secondary metabolites
Amount increases；Magnetic powder Fe3O4It is handled by externally-applied magnetic field and is provided with magnetism, become " small magnetic field ", to continue this magnetic field
Effect.
Influence of the 4 bacillus suspension of embodiment to astragalus polyose in culture solution and astragaloside content
Tested according to the method that embodiment 1 provides, in step 6) the inoculum concentration of bacillus suspension be respectively 0,2.5%,
5%, 7.5%, 10%, the assay of astragalus polyose and astragaloside is carried out after culture to the culture solution of releasing, is as a result seen below
Table 3.
From table 3 it can be seen that in 0 ~ 5% range, it is yellow in culture solution with the increase of bacillus suspension inoculum concentration
Astragalus polysaccharides and astragaloside content also gradually increase, and when bacillus suspension inoculum concentration is 5%, astragalus polyose content is
69.0 mg/L, astragaloside content are 42.7mg/L, are maximum value, but continuing growing with suspension inoculum concentration, culture solution
Middle astragalus polyose and astragaloside content gradually decrease instead.Bacillus is one kind in Radix Astragali endosymbiosis bacterium, for a long time and place
Main coevolution and the symbiosis for foring mutual reciprocity and mutual benefit can induce secondary metabolite in Radix Astragali cell to accumulate, and promote
Into secondary metabolite to extracellular release.But when bacillus suspension inoculum concentration is excessive, bacillus is in culture medium
In be largely proliferated, consume the nutriment in culture medium, be unfavorable for the growth of Radix Astragali cell, therefore 5% is most suitable bacillus
Suspension inoculum concentration.
Influence of 5 NaHS of embodiment to astragalus polyose in culture solution and astragaloside content
It is tested according to the method that embodiment 1 provides, the concentration of NaHS is respectively 0,25 μm of ol/L, 50 μ in step 6)
Mol/L, 100 μm of ol/L, 200 μm of ol/L, to the culture solution progress astragalus polyose of releasing and containing for astragaloside after culture
It is fixed to measure, and as a result see the table below 4.
From table 4, it can be seen that within the scope of 0 ~ 100 μm of ol/L, with the increase of NaHS concentration, Radix Astragali in culture solution
Polysaccharide and astragaloside content also gradually increase, and when NaHS concentration is 100 μm of ol/L, astragalus polyose content is 67.7
Mg/L, astragaloside content are 40.3mg/L, are maximum value, when NaHS concentration is 200 μm of ol/L, Radix Astragali in culture solution
Polysaccharide and astragaloside content reduce.NaHS is H2The donor of S gas, H2S gas is important stress response signal point
Son can induce the accumulation of secondary metabolite in Radix Astragali cell, but H simultaneously2S gas also has certain toxicity, if dense
Toxic action can be generated to Radix Astragali cell by spending height, therefore 100 μm of ol/L are most suitable NaHS concentration in this test.
6 immobilized cell of embodiment is compared with suspension cell culture
Radix Astragali cell suspending liquid is prepared according to 1 step 1) of embodiment, 2) method, 3), by suspension after handling into magnetic field excessively
To be cultivated in 10% inoculum concentration access production medium, cultural method is identical as 1 step 6) of embodiment, after culture
The assay that astragalus polyose and astragaloside are carried out to the culture solution of releasing, as a result see the table below 5.
As can be seen from Table 5, astragalus polyose content than embodiment 6 improves 19.3mg/L in embodiment 1, and astragaloside contains
Amount improves 20.1mg/L than embodiment 6, shows that immobilized growing cells can more promote secondary metabolism to produce than suspension cell culture
The accumulation of object improves the content of active constituent.The systematism level for cultivating cell is horizontal closer to whole plant, get over can with
The identical mode of whole plant reacts to the stimulation of environmental factor.And the more intimate vivo environment of immobilized cell and to culture
In the factor react.Second, the speed of growth of immobilized cell is generally below the speed of growth of suspended culture cell, and grows
There is correlation, immobilized cell not only reaches systematism and requires but also limit between the reduction of speed and the accumulation of secondary metabolite
Growth, therefore significantly improve the yield of secondary metabolite.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (9)
1. a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent, which is characterized in that the technical flow of this method
Journey is as follows:
1) disinfection of Radix Astragali explant: the tender stem section of Radix Astragali is chosen as explant, 10 ~ 20min is rinsed with tap water, in nothing
First with 75% ethanol disinfection 30s under the conditions of bacterium, then with 0.1% mercuric chloride 3min is sterilized, finally used aseptic water washing 4 ~ 5 times, filter paper is inhaled
Solid carbon dioxide point, obtains sterile Radix Astragali explant；
2) induction and proliferation of callus: the segment that Radix Astragali explant is cut into 0.5 ~ 0.7cm long is inoculated in containing Fiber differentiation
On the culture dish of base, be placed in 25 ± 1 DEG C, culture induces callus under dark condition, select well-grown callus
It is inoculated in the triangular flask containing proliferated culture medium, carries out shoot proliferation culture, every 21d subculture 1 time under the same conditions；
3) suspension and dispersion of Radix Astragali cell: picking out after subculture 2 ~ 3 times and grow the callus vigorous, quality is more loose,
Access shaking table shaken cultivation in the 500mL triangular flask of the suspension medium containing 100mL, condition of culture is 25 ± 1 DEG C, dark, revolving speed
100 ~ 120r/min, every 14d subculture 1 time gradually forms stable Radix Astragali cell suspension cultures system subculture 3 ~ 4 times, using enzymatic hydrolysis
Method disperses Radix Astragali cell, is then successively filtered cell suspending liquid with the sieve of 0.9mm and 0.1mm, takes filtrate
Immobilize operation；
4) immobilization of Radix Astragali cell: compound concentration be 40g/L sterile sodium alginate solution, and add 0.1%(W/V) mouse
Lee's glycolipid and 2%(W/V) Fe3O4, the filtrate of step 3) is uniformly mixed with this sodium alginate soln, with syringe by mixed liquor
The sterile CaCl that concentration is 20g/L is instilled dropwise2In solution, forming diameter under the stirring of magnetic stirring apparatus is about consolidating for 3mm
Surely change gel beads, be put into refrigerator overnight after standing 30min, take out and use brine 2 ~ 3 times；
5) the magnetic field processing of immobilized cell: immobilization gel beads are placed in sterile triangular flask, being put into magnetic field strength is 500Gs
Low high-intensity magnetic field in handle 30min；
6) culture of immobilized cell: being cultivated using airlift bioreactor, aseptically, by consolidating after magnetization
Surely to change gel beads to access in production medium with 10% inoculum concentration, liquid amount is 50 ~ 60%, and ventilatory capacity is 0.10 ~ 0.15VVM,
Temperature is 25 ± 1 DEG C, and in the bacillus suspension of the 8d access 5% of culture, the NaHS of 100 μm of ol/L continues to cultivate
Culture solution is released after 6-8d, for isolating and purifying for Radix Astragali active constituent (astragalus polyose and astragaloside).
2. a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent according to claim 1, feature
It is, the ingredient of induced medium is that MS minimal medium+1.0g/L lactoalbumin hydrolysate+0.5mg/L D- is general in the step 2
Sour calcium+1.0mg/L6-BA+0.2mg/L NAA+0.1mg/L choline chloride+30g/L sucrose+5g/L gellan gum, pH5.6 ~ 6.0.
3. a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent according to claim 1, feature
It is, the ingredient of proliferated culture medium is that MS minimal medium+2.0g/L lactoalbumin hydrolysate+0.5mg/L D- is general in the step 2
Sour calcium+0.6mg/L6-BA+0.1mg/L NAA+1.0mg/L KT-30+30g/L sucrose+5g/L gellan gum, pH5.6 ~ 6.0.
4. a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent according to claim 1, feature
It is, the inoculum concentration of callus is 40-50g/L in the step 3).
5. a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent according to claim 1, feature
It is, suspension medium ingredient in the step 3) are as follows: MS+2.0 ~ 3.0g/L of minimal medium lactoalbumin hydrolysate+0.3 ~
0.5mg/L D-VB5 calcium+1.4 ~ 1.8mg/L spermidine+0.5 ~ 0.7mg/L 6-BA+0.1 ~ 0.3mg/L NAA+0.8 ~
1.2mg/L KT-30+30 ~ 40g/L sucrose, pH5.6 ~ 6.0.
6. a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent according to claim 1, feature
It is, the concrete operations of enzymatic isolation method in the step 3) are as follows: compound concentration is the pectase liquid (enzyme activity 40U/mg) of 1.0g/L, to
The pectase liquid of 20% volume is added in Radix Astragali cell suspending liquid, is digested for 24 hours on 28 DEG C, the shaking table of revolving speed 60r/min.
7. a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent according to claim 1, feature
It is, the volume ratio of filtrate and sodium alginate soln is 1:4 in the step 4).
8. a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent according to claim 1, feature
It is, the ingredient of production medium in the step 5) are as follows: MS minimal medium+16 ~ 20mg/L phenylalanine+9 ~ 11mg/L junket
Propylhomoserin+8 ~+120 ~ 130 μm of ol/L methyl jasmonates+0.5 of 10mg/L L-+6.1 ~ 6.5mg/L of cinnamic acid soybean oligopeptide ~
0.7mg/L 6-BA+0.1 ~ 0.3mg/L NAA+0.8 ~ 1.2mg/L KT-30+26 ~ 30g/L sucrose+20 ~ 24g/L sorbierite,
pH5.6~6.0。
9. a kind of method using immobilization Radix Astragali cell production Radix Astragali active constituent according to claim 1, feature
It is, bacillus suspension the preparation method comprises the following steps: by the LB of bacillus subtilis and bacillus pumilus in the step 5)
After inclined-plane seed culture 24 hours, respectively according in 4% and 2% inoculum concentration access LB liquid medium, it is placed in shaking table oscillation training
Support, condition of culture be 35 ~ 37 DEG C of temperature, 160 ~ 180r/min of revolving speed, culture 20 ~ for 24 hours.
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| CN111280063A (en) * | 2020-04-02 | 2020-06-16 | 安赛搏(重庆)生物技术有限公司 | Rapid callus induction and large-scale suspension culture method for desert rose |
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| CN111587785A (en) * | 2020-05-06 | 2020-08-28 | 内蒙古自治区生物技术研究院 | Method for culturing astragalus membranaceus hairy roots for promoting accumulation of flavonoids |
| CN114958684B (en) * | 2022-06-22 | 2024-06-11 | 上海龙殷生物科技有限公司 | Method for improving competent cell conversion rate |
| CN116574668A (en) * | 2023-06-13 | 2023-08-11 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN116574668B (en) * | 2023-06-13 | 2023-12-05 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
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