CN105265312B - A kind of method that active principle in bioreactor culture elatus Nakai adventitious root is improved using elicitor - Google Patents
A kind of method that active principle in bioreactor culture elatus Nakai adventitious root is improved using elicitor Download PDFInfo
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Abstract
The present invention is a kind of method that active principle in bioreactor culture elatus Nakai adventitious root is improved using elicitor.When elatus Nakai adventitious root is in bioreactor culture to 30~40d, when biomass reaches maximum, add methyl jasmonate (200 μm of oL/L), salicylic acid (100 μm of oL/L), pulullan polysaccharide (200mg/L) post-processes 10d, as a result find that flavones content is significantly improved in adventitious root, content brings up to 45.88mg/g by 31.00mg/g, when wherein being handled using methyl jasmonate (200 μm of oL/L) and two kinds of elicitors of pulullan polysaccharide (200mg/L) adventitious root, polyoses content increases substantially in adventitious root, polyoses content has been respectively increased 53.33, 97.07mg/g, output has been respectively increased 0.348, 0.6865g/L.The present invention can effectively improve flavones and polyoses content in adventitious root; the means of conjunctive tissue culture; the elatus Nakai adventitious root of high level flavones and polysaccharide can be obtained in a short time by the culture of reactor adventitious root, a kind of convenient and effective method is provided for the utilization of resources and the protection of rare elatus Nakai.
Description
Technical field
The present invention relates to biological technical field, and bioreactor culture elatus Nakai adventitious root is improved using elicitor to be a kind of
The method of middle active principle.
Background technology
Elatus Nakai (Oplopanax elatus Nakai) also known as stichopus japonicus, Araliaceae stichopus japonicus category, perennial fallen leaves fill
Wood, it is one of distinctive plant resources in China Changbai Mountain.Elatus Nakai medical value is high, and its root, rhizome and stem are rare
Chinese medicine, rich in a variety of medicinal ingredients such as saponin(e, polysaccharide, flavones, volatile oil, anthraquinone, aliphatic acid, declined available for treatment nerve
The diseases such as weak, low blood pressure, impotence, schizophrenia, diabetes, and with antimycotic, antidepression, antipyretic, analgesia and anti-aging
Effect.Some compositions contained again in discovery leaf in recent years are close with root and stem, so leaf also has medical value.Due to its treatment
Effect is notable, and without any side effects, and " woody ginseng " is referred to as in Changbaishan area.At present, to elatus Nakai chemical composition
And the research of pharmacological action is more, but clinical application research is then less, therefore develops and develop elatus Nakai high potency drugs, right
The utilization of elatus Nakai resource is significant.Because its medical value is very high, people's excessively excavation causes its wild money
Source shortage, artificial cultivation aspect, generative propagation, setting percentage is low, and transplanting survival rate is low；There is seemingly-dead phenomenon in vegetative propagation, and extensive
Multiple growth period phase length, the shortage of propagating materials source, the influence of easy climate and chemical pesticide, yield can not protect in the course of cultivation
It is fixed to keep steady, and has greater risk, therefore finds a kind of method that can substitute Wild and home planted elatus Nakai and compeled
The eyebrows and eyelashes, and carry out natural resources of Chinese medicinal materials breeding using Plant Tissue Breeding and mass produce to turn into an effective and feasible development way
Footpath, Plant Tissue Breeding include cell culture, tissue and organ culture, but are effective one of the drawbacks of Medicinal Plant Cell culture
Component content is unstable, and this is secondary metabolite mainly due to most of active ingredients, and it is synthesized in cell stage table
It is now not strong and cause, and root culture can solve this problem.
The advantage of adventitious root culture is that it has huge biosynthesis ability possessed by primordial plant root, in root culture
The height of cell, which is broken up, causes the synthesis capability of secondary metabolite to significantly improve, at the same show obvious metabolic stability and
Vigor stability, it is combined with addition with bioreactor so that a large amount of productions of elatus Nakai adventitious root are had
The guarantee of effect, and then protection for elatus Nakai wild resource and exploitation provide an effective and feasible method.Due to life
It is particularly important in drug development to produce the high and stable elatus Nakai adventitious root of flavones and polyoses content, in recent years, using luring
Guide carries out target product regulation and control and biosynthesis has obtained the concern of increasing domestic and foreign scholars, and increases substantially
One of important method of metabolite in culture.
Elicitor includes biological elicitor and the major class of abiotic elicitor two, and it has selectivity, rapidity, concentration effect
Should, time effect and cooperative effect, reaction of the same plant to different elicitors is different, thus stimulates the metabolite of synthesis
And its content is also different, therefore, it should consider in actual applications and flexibly use, elicitor is trained in medicinal plant
Optimal facilitation is played during supporting.The present invention selects three kinds of abiotic elicitors (methyl jasmonate, salicylic acid, Propirams
Polysaccharide) treatment reactor culture elatus Nakai adventitious root, find three kinds of elicitors to flavones in elatus Nakai adventitious root
Content is improved largely, and the accumulation of methyl jasmonate and pulullan polysaccharide elicitor to polyoses content in adventitious root also has bright
Aobvious facilitation, this utilization to elatus Nakai resource have particularly significant meaning.
The present invention utilizes balloon-type airlift bioreactor culture elatus Nakai adventitious root, when indefinite root biomass reaches
Three kinds of elicitors are added to (30~40d of culture) during maximum, 10d is handled, adventitious root is given birth to after finding these three induction subprocessings
Object amount influences synthesis that is little, and promoting flavones in adventitious root, its content is significantly improved；Meanwhile utilize jasmonic first
Ester and pulullan polysaccharide can improve polyoses content and output in elatus Nakai adventitious root as elicitor.This method passes through
Can obtain the elatus Nakai flavones and polysaccharide of high level in a short time to adventitious root culture in reactor, acquisition it is efficient
The utilization of resources of the metabolite elatus Nakai adventitious root to rare elatus Nakai and exploitation Protection significance are great.
The content of the invention
Being improved it is an object of the invention to provide one kind using elicitor in bioreactor culture elatus Nakai adventitious root is had
The method for imitating material.Using the elatus Nakai adventitious root of bioreactor culture as material, with methyl jasmonate, salicylic acid and Propiram
Polysaccharide is handled adventitious root, by the screening to three kinds of elicitor concentration, is formulated and is improved in elatus Nakai flavones and more
The concrete scheme of sugared content, the protection and developmental utilization for rare elatus Nakai resource provide a kind of new method.
Present invention employs following technical scheme：
A kind of method that active principle in bioreactor culture elatus Nakai adventitious root is improved using elicitor.
1. elatus Nakai root induction and bioreactor culture
The adventitious root that elatus Nakai aseptic seedling lateral root induces is bred, the adventitious root for breeding acquisition weighs 20g
(FW) it is inoculated in the 5L balloon-type airlift bioreactors containing 4L fluid nutrient mediums, throughput 300mL/min, temperature
For light culture under the conditions of 25 DEG C.
2. induce subprocessing
When the indefinite root biomass of 30~40d of bioreactor culture reaches maximum (120~150g/L fresh materials weight), harvest reaction
Adventitious root and nutrient solution is collected in device, weigh 10g adventitious roots and be inoculated in the triangular flask containing the above-mentioned culture mediums of 100mL, together
The elicitor of tri- kinds of various concentrations of Shi Tianjia is handled, and adventitious root is harvested after 10d, then determines its biomass.
3. the measure of flavones content and output in elatus Nakai adventitious root
The drying of elatus Nakai adventitious root and the grind into powder of subprocessing will be induced, using aluminum nitrate decoration method, passed through
Ultraviolet specrophotometer (UV-2600, Shanghai Milky Way Environmental Technology AS, China) determines its flavones content and calculates its life
Yield, output (mg/L)=content × dry matter.
4. the measure of polyoses content and output in elatus Nakai adventitious root
The elatus Nakai adventitious root for inducing subprocessing is dried and pulverizes last, using Phenol sulfuric acid procedure, is passed through
Ultraviolet specrophotometer determines polyoses content and calculates its output.
Entirety of the invention has investigated different elicitors and its concentration for the treatment of to flavones and polysaccharide in elatus Nakai adventitious root
Action effect, optimize on this basis and filtered out rational elicitor concentration for the treatment of, form the increase of complete set
The approach of flavones and polyoses content and output in elatus Nakai adventitious root.It is indefinite that the present invention is remarkably improved elatus Nakai
The flavones and polysaccharide output of root.
Brief description of the drawings
Fig. 1 a：Plug b：Air pump c：Mass air flow sensor d：Filter membrane e：Jet rose (15 μm of aperture) f：Gas outlet * arrows
Represent air-flow direction
Influence of Fig. 2 methyl jasmonates concentration to flavones content and output in elatus Nakai adventitious root
Influence of Fig. 3 Determination of Salicylic Acid to flavones content and output in elatus Nakai adventitious root
Influence of Fig. 4 pulullan polysaccharides concentration to flavones content and output in elatus Nakai adventitious root
Influence of Fig. 5 methyl jasmonates concentration to polyoses content and output in elatus Nakai adventitious root
Influence of Fig. 6 Determination of Salicylic Acid to polyoses content and output in elatus Nakai adventitious root
Influence of Fig. 7 pulullan polysaccharides concentration to polyoses content and output in elatus Nakai adventitious root
Embodiment
(1) materials and methods
1. elatus Nakai root induction and bioreactor culture
The elatus Nakai seed of breaking dormancy is seeded in vermiculite, after cotyledon expansion grows new plant, by plant
Carry out disinfection, formation root in the culture dish (Φ × h=90 × 15mm) containing MS culture mediums is aseptically inoculated in, by side
Access contains 25mL culture mediums after root is cut into 1cm sizes (50g/L+ times of power of MS+IBA 3.0mg/L+ white sugar coagulates 8g/L, pH 5.8)
Culture dish in carry out adventitious root induction, the adventitious root of induction is inoculated in above-mentioned culture medium, every 25~30d subcultures train
Support once, the adventitious root that squamous subculture obtains then is weighed into 20g (FW), and be cut into 1cm sizes and be inoculated in containing the training of 4L liquid
In the 5L balloon-type airlift bioreactors for supporting base, culture medium：MS+IBA3mg/L+ white sugar 50g/L, pH 5.8, in throughput
For 300mL/min, temperature is light culture under the conditions of 25 DEG C.
2. induce subprocessing
When the indefinite root biomass of 30~40d of bioreactor culture reaches maximum (120~150g/L fresh materials weight), harvest reaction
Adventitious root and nutrient solution is collected in device, weigh 10g adventitious roots and be inoculated in the triangular flask containing the above-mentioned culture mediums of 100mL, together
The elicitor of tri- kinds of various concentrations of Shi Tianjia, methyl jasmonate concentration is set as 50,100,150,200 and 250 μm of oL/L；Water
Poplar acid concentration is set as 25,50,75,100 and 125 μm of oL/L；Pulullan polysaccharide concentration is set as 100,200,300,400 and
500mg/L.Not add elicitor as control.In oscillator (GTCS-2013B, section of Jintan City analyzer that rotating speed is 121r/min
Device Co., Ltd, China) on carry out light culture, adventitious root is taken out after 10d, with running water rinse 2~3 times, be placed in clean
Dehydration is squeezed with hand in gauze, claims fresh material weight, is then respectively put into the culture dish marked and is placed in 45 DEG C of drying boxes
Dried in (YHW1103, Tianjing Huabei Laboratory Apparatus Co., Ltd., China), claim dry matter after 2d.
3. the measure of flavones content in elatus Nakai adventitious root
The making of standard curve：Precision weighs rutin 10mg, is dissolved with 70% ethanol and is shaken with 250mL volumetric flask constant volumes
It is even.The titer that concentration is 40 μ g/L is obtained, accurate this standard liquid 0.1 of absorption, 0.2,0.4,0.6,0.8mL are placed in examination respectively
Guan Zhong, distilled water is added, using 70% ethanol as control, to be separately added into 0.3mL5% NaNO to 2mL2Solution, shake up static
6min, add 0.3mL10% Al (NO3)3Solution, static 6min is shaken up, add 4% NaOH solution 2mL, react 10min
Afterwards, its light absorption value is determined at 510nm.
The preparation of sample solution：By the elatus Nakai adventitious root bole abrasive lapping of above-mentioned processing, precision weighs sample
0.1g, it is placed in centrifuge tube, 2~3h in 60 DEG C of water-bath is placed in after adding 10mL70% ethanol, after cooling, is in temperature
4 DEG C, rotating speed be 5000r/min under the conditions of centrifuge 15min, take supernatant to be settled to 10mL, be taken out 1mL and be diluted to 25mL.
Precision is drawn 1mL and operated by standard curve item, and calculates flavones content.
4. measurement of the polysaccharide content in elatus Nakai adventitious root
The making of standard curve：Precision weighs the appearance dried and to the glucose 100mg of constant weight, be dissolved in 100mL at room temperature
In measuring bottle, the glucose standard that concentration is 1mg/mL is obtained.Recycle this titer compound concentration for 0.01,0.025,
0.05th, 0.075,0.1,0.15mg/mL glucose control solution, respectively takes glucose control liquid 1mL to be placed in test tube, with distillation
Then water determines its light absorption value with Phenol sulfuric acid procedure as blank control at 490nm.
The extraction of polysaccharide：Precision weighs elatus Nakai adventitious root powder 10g, is soaked 2 times, each 6h with 90% ethanol,
Addition 100mL distilled water water-bath 1h, filter paper at 45 DEG C filter and collect extract solution after ethanol volatilizes, and filter residue are added equal
The distilled water of volume again extract by water-bath, is repeated 3 times, and 3 times is extracted obtained mixed liquor and is centrifuged under the conditions of 5000r/min
15min, obtained supernatant Sevage reagents removing protein 3 times.The 95% static 12h of ethanol is added, for precipitate polysaccharides,
Filtered afterwards, filter residue is cleaned 3 times with absolute ethyl alcohol, acetone, ether respectively, is placed in after filter residue cleaning in 45 DEG C of drying baker
Drying, both obtain elatus Nakai polysaccharide.The polysaccharide that precision weighs 10mg adds distilled water constant volume to put in 100mL volumetric flask, both
0.1mg/mL polysaccharide solution.Conversion factor：The grape of the weight (mg) of f=polysaccharide/polysaccharide solution volume (mL) × polysaccharide liquid
Sugared concentration (mg/L).
The preparation of sample solution：Precision weighs 0.1g elatus Nakai adventitious root powder, and 90% ethanol soaks 2 times, every time
6h, addition 50mL distilled water water-bath 3h at 45 DEG C, 100mL is put by filtrate constant volume after ethanol volatilizes, and both obtains sample solution, together
On using Phenol sulfuric acid procedure determination sample light absorption value.By following equation calculate polyoses content, polyoses content (mg/g)=Cf ×
1000
(2) result and analysis
By the measure to adventitious root fresh material weight and dry matter, and utilize ultraviolet specrophotometer indefinite to elatus Nakai
Flavones and polyoses content in root are analyzed, and obtain following result：
1. influence of the elicitor to flavones content in adventitious root and output
Methyl jasmonate concentration is set as 50,100,150,200,250 μm of oL/L；Determination of Salicylic Acid is set as 25,50,
75th, 100 and 125 μm of oL/L；Pulullan polysaccharide concentration is set as 100,200,300,400 and 500mg/L.Adventitious root reactor is trained
When supporting 30~40d, the elicitor of various concentrations is separately added into, adventitious root is taken out after processing 10d and claims fresh material weight and dry matter, and
Flavones content is determined with ultraviolet specrophotometer and calculates its output, is as a result found：Adventitious root after three kinds of induction subprocessings
Fresh material weight and dry matter are handled adventitious root without significant change, 200 μm of oL/L methyl jasmonates, and obtained flavones content is most
High (42.73mg/g), and do not carry out inducing in the adventitious root of subprocessing flavones content and output be only 28.33mg/g and
219.84mg/L.When salicylic acid concentration for the treatment of is 100 μm of oL/L, flavones content is carried by untreated 31.00mg/g in adventitious root
Height arrives 45.88mg/g, and when the concentration of pulullan polysaccharide is 200mg/L, the content of flavones is improved by 33.42mg/g in adventitious root
To 43.51mg/g.As can be seen here, three kinds of elicitors of debita spissitudo increase to flavones content in adventitious root.
2. influence of the elicitor to polyoses content in adventitious root and output
The elicitor of various concentrations is separately added into during elatus Nakai adventitious root 30~40d of bioreactor culture, by jasmonic
Methyl acetate concentrations are set as 50,100,150,200,250 μm of oL/L；Determination of Salicylic Acid is set as 25,50,75,100 and 125 μm of oL/
L；Pulullan polysaccharide concentration is set as 100,200,300,400 and 500mg/L, takes out adventitious root after handling 10d, determines its life
Object amount and polyoses content and output.Result of study find three kinds of elicitors on the biomass accumulation of adventitious root without influence, still
Only certain density methyl jasmonate and pulullan polysaccharide have facilitation to polyoses content in adventitious root.Work as methyl jasmonate
Concentration is advantageous to the accumulation of polyoses content when being 150~200 μm of oL/L, and polyoses content brings up to 186.93 by 153.87mg/g~
207.20mg/g, 200 μm of oL/L methyl jasmonate treatments adventitious root its polysaccharide outputs are significantly higher than other processing.Propiram is more
When sugared concentration is 200mg/L, polyoses content brings up to 205.44mg/g by 108.37mg/g in adventitious root, it can be seen that, add
200mg/L pulullan polysaccharide is significantly improved effect to the polyoses content of adventitious root in reactor.
Pass through the selection result to three kinds of elicitor concentration, it is known that 30~40d of bioreactor culture adventitious root lures through three kinds
After guide processing, the fresh material weight and dry matter of adventitious root are influenceed and little, meanwhile, find three kinds of elicitors to yellow in adventitious root
The accumulation of ketone increases, methyl jasmonate (200 μm of oL/L), salicylic acid (100 μm of oL/L), pulullan polysaccharide (200mg/
L) handle flavones output after adventitious root be respectively increased 98.07,104.94,64.7mg/L；Wherein, methyl jasmonate and Pu Lu
Blue two kinds of elicitors of polysaccharide are also beneficial to the accumulation of polyoses content in adventitious root.
Therefore, in the production of elatus Nakai adventitious root, jasmonic first can be added when indefinite root biomass reaches maximum
Ester (200 μm of oL/L) or pulullan polysaccharide (200mg/L) are handled to reach the purpose of raising flavones and polysaccharide yield, if
Target product can also be used salicylic acid (100 μm of oL/L) for flavones and adventitious root handled.
Claims (1)
1. one kind is indefinite using pulullan polysaccharide raising bioreactor culture elatus Nakai (Oplopanax elatus Nakai)
The method of polyoses content in root：When elatus Nakai adventitious root cultivates 30~40d in bioreactor, adding concentration is
200mg/L pulullan polysaccharide is handled.
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| CN104920209A (en) * | 2015-05-18 | 2015-09-23 | 天津大学 | Method for taking two inducers to promote ginseng adventitious saponin accumulation |
| CN111134008A (en) * | 2018-11-02 | 2020-05-12 | 延边大学 | Method for increasing polysaccharide content in adventitious roots of northeast stichopus japonicus |
| CN111134007B (en) * | 2018-11-02 | 2023-08-29 | 华南理工大学 | Method for improving flavone content in adventitious roots of northeast thorn ginseng |
| CN116138167A (en) * | 2023-01-09 | 2023-05-23 | 延边大学 | Method for improving flavone and phenol content and antioxidant activity of adventitious roots of North-east radix ginseng |
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| WO2008100016A1 (en) * | 2007-02-14 | 2008-08-21 | Cbnbiotech Co., Ltd | Method of bioreactor culture of echinacea purpurea adventitious roots |
| CN103141394A (en) * | 2013-03-28 | 2013-06-12 | 延边大学 | Method for culturing adventitious roots of oplopanax elatus Nakai by utilizing bioreactor |
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| WO2008100016A1 (en) * | 2007-02-14 | 2008-08-21 | Cbnbiotech Co., Ltd | Method of bioreactor culture of echinacea purpurea adventitious roots |
| CN103141394A (en) * | 2013-03-28 | 2013-06-12 | 延边大学 | Method for culturing adventitious roots of oplopanax elatus Nakai by utilizing bioreactor |
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