CN104904491B - A kind of method that utilization ginseng pathogenic bacterium inducing improves saponin content in bioreactor culture American Ginseng adventitious root - Google Patents
A kind of method that utilization ginseng pathogenic bacterium inducing improves saponin content in bioreactor culture American Ginseng adventitious root Download PDFInfo
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Abstract
The method that the present invention improves saponin content in bioreactor culture American Ginseng adventitious root for a kind of utilization ginseng pathogenic bacterium inducing.When American Ginseng adventitious root reaches maximum in bioreactor culture to 30d, biomass, 4mg/L black spot induction subprocessing American Ginseng adventitious root is added, saponin content highest in adventitious root, is 27.4mg/g when handling 8d, corresponding saponin(e yield also highest, is 273.7mg/L.The present invention can effectively improve saponin content in adventitious root; the means of conjunctive tissue culture; the higher American Ginseng adventitious root of a large amount of content of ginsenoside can be obtained in a short time using the replacement medicinal material as American Ginseng, and this just provides a kind of convenient and effective method for the utilization of resources and protection of rare Chinese herbal medicine American Ginseng.
Description
Technical field
It is soap in a kind of son raising American Ginseng adventitious root using ginseng pathogenic bacterium inducing the present invention relates to biological technical field
The method of glycosides content.
Background technology
American Ginseng (Panax quinquefolium L.) is also known as U.S.'s ginseng, Western ginseng, American ginseng, American ginseng, Guangdong
Ginseng.Eastern United States and Canada are originated in, is the herbaceos perennial of Araliaceae Panax, is used as medicine with root.American Ginseng has
There are nourishing, cardiac stimulant, hematopoiesis, stomach invigorating, calmness, reducing blood lipid, anticancer and anti-ageing a variety of medical functions of waiting for a long time.Modern chemistry and pharmacology
Research shows that American Ginseng principle active component is saponin(e.Compared with ginseng, its property is gentleer, and total saponin content and ginseng soap
Glycosides Rb1 content is higher than ginseng, therefore is listed in rare traditional Chinese medicine.Because American Ginseng medical treatment and health purpose are very big, demand
Increase year by year, cause that supply falls short of demand, expensive situation.Similar with ginseng, American Ginseng is raw due to the physiological property of its own
It is long slow, typically need to harvest for 4~6 years and be used as medicine, and seed resource is rare.Therefore it is a large amount of using the method production of tissue cultures
American Ginseng substitute medicinal material just receive significant attention, among these including American Ginseng cell culture, the culture of hairy root and adventitious root train
In terms of supporting.And the advantage of adventitious root culture is then its huge biosynthesis ability that there is primordial plant root to have, root
The height of cell, which is broken up, in culture causes the synthesis capability of secondary metabolite to significantly improve, while it is steady to show obvious metabolism
Qualitative and vigor stability, is combined with so that a large amount of productions of American Ginseng adventitious root are obtained with bioreactor in addition
It is effective to ensure, and then provide feasible method for the protection and development of resources of American Ginseng wild resource.But American Ginseng is not
The saponin content of fixed basic body is not high, is only capable of reaching 1% or so, therefore it is indefinite to produce the American Ginseng of saponin content height and stabilization
Root is particularly important, and it is then effective approach to improve saponin content and its output using elicitor.
The fungal elicitor that elicitor is included in biological elicitor and the major class of abiotic elicitor two, biological elicitor is then deposited
Wide material sources, without artificially synthesize, just can be used directly through simple process many advantages, such as, compared with abiotic elicitor price
Cheap, applicability is wider.The present invention is most popular and endanger the ginseng disease fungus (black fleck disease of ginseng of most serious with two seed ginseng areas
Bacterium and ginseng rust rot bacteria) extract, as the American Ginseng adventitious root of elicitor treatment reactor culture, finds both fungies
Raising to saponin content in American Ginseng adventitious root is respectively provided with larger facilitation, this exploitation for American Ginseng resource
It is significant.
The present invention utilizes balloon-type airlift bioreactor culture American Ginseng adventitious root, when indefinite root biomass reaches most
Add black fleck disease of ginseng bacterium when big to continue to cultivate as elicitor with ginseng rust rot bacteria's extract, determining its saponin(e after 8d contains
Amount, it is found that both elicitors promote the synthesis of saponin(e, significantly improve its content.This method can be short by adventitious root culture
A large amount of American ginseng saponins are obtained in time, adventitious root can be as cultivation or the replacement medicinal material of wild American Ginseng, and this is for American Ginseng
The utilization of resources and exploitation protection it is significant.
The content of the invention
It is indefinite using ginseng pathogenic bacterium inducing raising bioreactor culture American Ginseng it is an object of the invention to provide one kind
The method of saponin content in root.Using American Ginseng adventitious root as material, with ginseng alternaria and rust rot fungus extract to reaction
The adventitious root of device culture is handled, by the regulation and control to processing time and addition, is formulated and is improved saponin content in American Ginseng
Concrete scheme, the protection and developmental utilization for rare Chinese herbal medicine American Ginseng resource provides a kind of new method.
Present invention employs following technical scheme：
A kind of method that utilization ginseng pathogenic bacterium inducing improves saponin content in bioreactor culture American Ginseng adventitious root.
1. the preparation of fungal culture and fungal elicitor
Ginseng disease fungus selected by the present invention is that ginseng alternaria (Alternaria panax) and ginseng rust are rotten
Germ (Cylindrocarpon destructans).
Black fleck disease of ginseng bacterium and ginseng rust rot bacteria are seeded in containing potato dextrose agar (PDA) culture medium respectively
In the culture dish of (potato 200g/L+ glucose 50g/L+ agar 20g/L), cultivated at 25 DEG C.Mycelia is scraped after 20~25d simultaneously
It is inoculated into the PDA culture medium without agar and carries out agitated submerged culture.Fungal mycelium is taken out and cleaned after 15~20d,
Then dry, grind, extract fungi polysaccharide using ultrasonic extractor, 4 DEG C will be stored in after fungi polysaccharide extract solution autoclaving
It is lower standby as elicitor.
2. the culture of American Ginseng adventitious root and fungal induction subprocessing
Fresh weight is inoculated in progress propagation training in 5L balloon-type airlift bioreactors for 20g American Ginseng adventitious root
Support, the light culture under the conditions of temperature is 25 DEG C.When bioreactor culture to 30d, adventitious root increment adds two kinds when reaching maximum
Fungal elicitor.
3. saponin content is determined in American Ginseng adventitious root
The American Ginseng adventitious root for inducing subprocessing is taken out from reactor after drying and claying into power, use high-efficient liquid phase color
Spectrum (HPLC) determines saponin content and calculates saponin(e output.
The overall place for having investigated ginseng Bacteria culturing, elicitor preparation and elicitor to American Ginseng adventitious root of the invention
Reason mode, optimizes and has filtered out rational elicitor processing time and concentration for the treatment of, form complete set on this basis
And saponin content and the method and approach of saponin(e yield in perfect increase American Ginseng adventitious root.It is indefinite that the present invention can improve American Ginseng
The saponin(e output of root, is detected more accurate accurate, applicability is wider using HPLC.
Brief description of the drawings
9 kinds of saponin contents in American Ginseng adventitious root after Fig. 1 alternarias induction subprocessing
9 kinds of saponin contents in American Ginseng adventitious root after Fig. 2 rust rots bacterium induction subprocessing
Total saponin content in American Ginseng adventitious root after Fig. 3 fungal induction subprocessings
Total saposins output in American Ginseng adventitious root after Fig. 4 fungal induction subprocessings
Embodiment
(1) materials and methods
1. fungal culture
Black fleck disease of ginseng bacterium and ginseng rust rot bacteria are inoculated with the culture dish containing PDA culture medium respectively, cultivated at 25 DEG C.
Mycelia is scraped after 20~25d and is seeded in PDA liquid medium and carries out agitated submerged culture, cultivation cycle is 15~20d.
2. the measure of the preparation of fungal elicitor and polysaccharide concentration
By above-mentioned black fleck disease of ginseng bacterium and ginseng rust rot bacteria's mycelium after 15~20d of agitated submerged culture from blake bottle
After middle taking-up is cleaned with distilled water, dried at 40 DEG C.Dry mycelium is ground, the distilled water of 10 times of volumes, 45 is added
Ultrasonic extraction 30min at DEG C, in kind extracts filter residue after filtering, three times, merging filtrate totally again.Again by the two of filtering
Plant Extract of Fungi and 15min is centrifuged under conditions of 5000r/min, take supernatant to be settled to 500mL, and respectively by two kinds of extractions
Liquid is 1.3MPa in pressure, and temperature is autoclaving 15min under the conditions of 121 DEG C.Precision measures two kinds of fungies after sterilizing and extracted
Liquid 50mL, to add appropriate 95% ethanol after sevage reagent removing proteins and stand 12h with precipitate polysaccharides, after 12h suction filtration and point
Filter residue is not cleaned three times with absolute ethyl alcohol, acetone, ether.Filter residue is collected in drying fungi polysaccharide dry product at 40 DEG C.Will
1mg fungi polysaccharide dry products are dissolved in 10mL distilled water, are then calculated with phend-sulphuric acid by the tester of glucose content
The extension rate of concentration of glucose (mg/L) × polysaccharide of weight (mg)/polysaccharide liquid of conversion factor f, f=polysaccharide.As a result ginseng
Alternaria f is 1.05, and ginseng rust rot bacteria f is 2.96.Accurate two kinds of Extract of Fungi of 1mL of drawing are surveyed at 490nm respectively
Determine trap, while accurate draw 1mL glucose standards solutions (containing glucose 0.05mg), equally measured with phend-sulphuric acid
Light absorption value, is calculated as follows polysaccharide concentration in sample：Polysaccharide concentration (mg/L)=Cf × 1000.C in formula：Portugal in Extract of Fungi
Grape sugar concentration (mg/mL), f：Conversion factor.
3. the culture of American Ginseng adventitious root
Fresh weight is inoculated in 5L balloon-type airlift bioreactors for 20g American Ginseng adventitious root, culture medium is 3/
4MS+ white sugar 50g/L+IBA5mg/L, reactor throughput is 400mL/min.The light culture under the conditions of temperature is 25 DEG C.
4. the screening of fungal induction subprocessing number of days
When bioreactor culture is to 30d, adventitious root increment reaches maximum (350~400g fresh weights), now each reactor
Middle addition 5mL fungal elicitors, adventitious root is taken out, rushed with running water from reactor respectively when handling the 2nd, 4,6,8 and 10d
Wash 2~3 times, then the adventitious root of each processing be respectively charged into the gauze bag marked, using dewaterer (700r/min,
HQB52-8521, Hangzhou Matsushita Home Electrical Appliances Co., Ltd, China) its surface moisture is dried, claim fresh material weight.40 DEG C are put into afterwards
Dried in drying box (YHW1103, Tianjing Huabei Laboratory Apparatus Co., Ltd., China), claim dry matter after 2d, while determining soap
Glycosides content.
5. the screening of fungal elicitor concentration
According to result 4., fungal elicitor processing time is defined as 8d.The concentration of black fleck disease of ginseng elicitor is set
For 3,4,5 and 6mg/L, the concentration of ginseng rust rot elicitor is set as 15,20,25 and 30mg/L (being calculated with polysaccharide concentration),
Not add elicitor as control.Adventitious root culture, will after 8 days to the elicitor that various concentrations are added during 30d in reactor
Adventitious root takes out from reactor, surveys fresh material weight, dry matter and saponin content, method is with 4..
6. saponin content assay method
Precision weighs American Ginseng adventitious root dried powder (crossing 100 molybdenums sieve) 1g, adds 25mL 80% methanol at 80 DEG C
Extraction 2 times, each 2h.Filter latter incorporated filtrate and 5mL or so pastes are concentrated under reduced pressure at 50 DEG C, added in paste
Simultaneously constant volume is 25mL to dual distilled water.25mL ether defattings are added in sample after constant volume twice, and obtained lower floor's solution is utilized
25mL water-saturated n-butanols are extracted three times and merge the n-butanol extracting liquid after extraction, and extract solution is depressurized at 60 DEG C and is spin-dried for, obtains
Then the dry methanol constant volume arrived has engine filter to be filtered, testing sample is produced after filtering to 5mL with 0.45 μm.
Testing sample utilizes the high-efficient liquid phase color equipped with C18 chromatographic columns (250 × 4.6mm, 5 μm, Theromo scientific, the U.S.)
Spectrum (LC-15C, Shimadzu Corporation, Japan), enters at UV-vis detector (SPD-15C, Shimadzu Corporation, Japan) 203nm wavelength
Row detection.Sample size is 20 μ L, and mobile phase is acetonitrile and water, and flow velocity is 1.0mL/min, and column temperature is 30 DEG C.Ginsenoside standard
Product Rb1, Rb2, Re, Rd, Re, Rf, Rg1, Rg3 and Rh2 are bought by Chengdu Man Site bio tech ltd.Total saposins contain
Measure as monomer saponin sum, saponin(e output (mg/L)=indefinite root dry weight (g/L) × saponin content (mg/gDW).
Determine the mobile phase elution requirement such as following table of content of ginsenoside：
Gradient elution program
(2) result and analysis
By the measure to adventitious root fresh material weight and dry matter, and using HPLC to 9 kinds of monomers in American Ginseng adventitious root
The content of saponin(e (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3 and Rh2) is analyzed, and obtains following result：
1. the screening of fungal induction subprocessing number of days
Using black fleck disease of ginseng bacterium and the American Ginseng adventitious root of rust rot bacterium elicitor treatment reactor culture, processing 2,
4th, 6,8, adventitious root is taken out from reactor after 10d and claims fresh material weight and dry matter, and saponin content is determined with HPLC, is as a result sent out
It is existing：It is little with adventitious root fresh material weight and dry matter change in 2~10d of black spot induction subprocessing, respectively by untreated
384.5g and 146.7g are reduced to 335.6g and 41.6g；And during utilizing rust rot induction subprocessing, the fresh material of adventitious root
Weight and dry matter are changed greatly, and 243.1g and 23.2g are reduced to respectively.But saponin content is at two kinds of fungal elicitors
Reach maximum, respectively alternaria within the 8th day after reason：21.7mg/g DW, rust rot bacterium：21.9mg/g DW, saponin(e life
Yield respectively reaches 225.7mg/L and 170.7mg/L；And saponin content and output in the adventitious root of induction subprocessing are not carried out
Only 8.6mg/gDW and 100.4mg/L.
2. the screening of elicitor concentration
The concentration of black fleck disease of ginseng elicitor is set as 3,4,5 and 6mg/L, the concentration of rust rot elicitor is set as
15th, 20,25 and 30mg/L, post-processes 8d in adventitious root bioreactor culture 30d, then takes out adventitious root from reactor, surveys
Its fresh material weight, dry matter and saponin content.As a result find after fungal induction subprocessing American Ginseng adventitious root, each monomer in adventitious root
Saponin content has increase, but increasing degree differs.After 4mg/L black spot induction subprocessing American Ginseng adventitious root, no
The content for determining Rb1 in root brings up to 8.71mg/g by untreated 2.26mg/g, and Rb2 content brings up to 4.89mg/g and (do not located
Manage as 1.71 mg/g), Rg3 and Rh2 also increase to 1.34mg/g and 1.75mg/ by untreated 0.28mg/g and 0.32mg/g
g.And 20mg/L ginseng rust rot elicitor processing American Ginseng adventitious root is utilized, monomer saponin Rf contents in adventitious root
(3.43mg/g) is untreated 10 times, and Rb2 and Rd content are also brought up to by untreated 1.71mg/g and 1.63mg/g
6.41mg/g and 7.23mg/g, Rg3 and Rh2 content increase as 1.94mg/g and 5.78mg/g respectively.
But we have found simultaneously, fresh material weight and dry of the black spot elicitor to adventitious root in both fungal elicitors
Ghost image rings and little, and rust rot elicitor then has considerable influence, and accumulation of the various concentrations elicitor to saponin(e has different journeys
The effect of degree.Saponin content highest (27.4mg/g DW) when black spot elicitor concentration for the treatment of is 4mg/L, 4L reactor training
The indefinite root dry weight of American Ginseng raised is 40.0g, and saponin(e output now is also maximum (273.7mg/L)；And rust rot is induced
When sub- concentration is 20mg/L, though saponin content is significantly higher than the processing of other concentration, 35.6mg/g DW are reached, yet with stronger
Suppression growth, after processing 8d, the American Ginseng indefinite root dry weight only 26.5g that 4L bioreactor culture goes out, saponin(e life
Yield only reaches 235.9mg/L.It can be seen that the effect of black spot elicitor is better than rust rot elicitor in two kinds of fungal elicitors.
Therefore, in the production of American Ginseng adventitious root, 4mg/L can be added when indefinite root biomass reaches maximum (with polysaccharide
Concentration calculate) black fleck disease of ginseng elicitor handled with reach raising saponin(e yield purpose.
Claims (1)
1. one kind improves bioreactor culture American Ginseng (Panax quinquefolium L.) no using ginseng pathogenic bacterium inducing
Determine the method for saponin content in root, it is characterised in that：Ginseng pathogen used is ginseng alternaria (Alternaria
) and ginseng rust rot bacteria (Cylindrocarpon destructans) panax；Induction first is made in the ginseng pathogen of culture
Son, when American Ginseng adventitious root cultivates 30d, biomass in bioreactor and reaches maximum, adds ginseng pathogenic bacterium inducing
Handled；The saponin content that this method is obtained in a large amount of American Ginseng adventitious roots, its adventitious root in a short time is relatively high and stable,
For the replacement medicinal material as American Ginseng, with higher value of exploiting and utilizing.
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