CN105861589A - Method for producing dendrobium huoshanense polysaccharide through suspension culture cells - Google Patents
Method for producing dendrobium huoshanense polysaccharide through suspension culture cells Download PDFInfo
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- CN105861589A CN105861589A CN201610353014.0A CN201610353014A CN105861589A CN 105861589 A CN105861589 A CN 105861589A CN 201610353014 A CN201610353014 A CN 201610353014A CN 105861589 A CN105861589 A CN 105861589A
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Abstract
The invention discloses a method for producing dendrobium huoshanense polysaccharide through suspension culture cells. According to the method, dendrobium huoshanense polysaccharide is produced by conducting suspension culture on the dendrobium huoshanense cells through a cell culture reactor. The method comprises the following steps of 1, single-cell preparation; 2, culture of a single-cell system; 3, amplification culture of a cell population; 4, extraction of dendrobium huoshanense polysaccharide, wherein when the cell density reaches 60000-80000 cells/ml, the dendrobium huoshanense cells are collected, and dendrobium huoshanense polysaccharide is extracted. By means of the method, the cell culture cycle can be remarkably shorted, the yield of dendrobium huoshanense polysaccharide products is raised, the process is simple and stable, and the method is suitable for industrialized production.
Description
Technical field
The present invention relates to Herba Dendrobii technical field, particularly relate to a kind of suspended culture cell production Herba Dendrobii many
The method of sugar.
Background technology
Herba Dendrobii polysaccharide is one of main component of Herba Dendrobii, the pharmacological action master of Herba Dendrobii polysaccharide
Show the effects such as immune, strong, defying age, antitumor.Along with social development, people are to health
Paying close attention to more and more higher, its demand also can be increasing, but the extracting method of tradition Herba Dendrobii polysaccharide is to use
The plants such as the root of Herba Dendrobii platymiscium, stem, leaf extract gained, and the product of this technique gained is the most not
The future market demand to Herba Dendrobii intensive processing can be met.
Along with the development of plant cell engineering technology, carry out artificial culture amplification with cell and extract the metabolism of plant
Product, can not be suitable to industrialization by limitations affect such as land area, climatic environment, region, pest and disease damages
Produce.Utilize cell engineering to produce the technology of Herba Dendrobii polysaccharide, at home and abroad early have research, also take
Obtained many achievements, but there is a lot of difficult problem problem in process of production, such as complex process, Biomass speedup
Low, target product yield is low, production cycle length etc..About utilizing airlift agitation tank suspension culture Herba Dendrobii
Cell produce Herba Dendrobii polysaccharide research it is not yet reported that, therefore a kind of efficiently to produce Herba Dendrobii many in exploitation
The method of saccharide natural product is the most significant.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that a kind of technique is simple, stable, is suitable for industrialized production
Suspended culture cell produces the method for Herba Dendrobii polysaccharide.
The present invention goes to solve above-mentioned technical problem by techniques below means: a kind of suspended culture cell produces
The method of Herba Dendrobii polysaccharide, comprises the following steps:
(1) unicellular preparation: carry out aseptic seeding with solid medium after taking the sterilization of Herba Dendrobii capsule,
Take out after sprouting into protocorm, use enzymatic isolation method separation to prepare unicellular, and centrifugal filtration remove impurity, collect
Unicellular；
(2) monoclonal is cultivated: unicellular be inoculated in a MS fluid medium training by collect
Support；
(3) cell mass amplification culture: cultured monoclonal is inoculated in the 2nd MS fluid medium
Middle cultivation；In described 2nd MS fluid medium 2-3 times that P element content is MS fluid medium,
Described 2nd MS fluid medium is also added with the Ge element of 1-2mg/L, 0.02-0.1mg/L selenium element and
The acid hydrolyzed casein of 0.1-0.2g/L, pH value is 5.8 ± 0.4.
Use the 2nd MS fluid medium of the present invention, can extend relative to MS fluid medium and train in batches
Support cell growth time more than 2 times, improve cell density more than 2 times.Ge element, selenium element, acid hydrolysis
Casein can dramatically increase the fresh weight of Herba Dendrobii suspension cell, improve protein and chlorophyllous content, makes
Cellular anti-oxidant activity is significantly raised.
(4) extract Herba Dendrobii polysaccharide: when cell density reaches 60000-80000/ml, use centrifugal
Collect Herba Dendrobii cell；Extract Herba Dendrobii polysaccharide again.
Preferably, the culture medium that described step (1) sowing uses is the solid culture being suitable for orchid growth
Base.
Preferably, described solid medium is 1/2MS culture medium.In 1/2MS culture medium, a great number of elements halves,
It is suitable for germination of arethusa seeds.
Preferably, in described step (2), unicellular inoculum concentration is 25 ± 2g/L, and light intensity is 2000-4000lux,
Colour temperature is 4000-6000K, and concussion is cultivated, temperature 22 ± 2 DEG C, when cell density reaches 10000-20000
/ mL, gets final product amplification culture.This range of light intensities contributes to the morphogenesis of cell and vigor promotes, too high then
Waste electric energy, too low, do not reach effect.This colour temperature is warm light, and royal purple light more equalizes, beneficially metabolism
The synthesis of product.This temperature range is the temperature that Herba Dendrobii growth equalizes the most with metabolism.Under this density,
The population growth of cell is in finger logarithm period, the period that i.e. vigor is the most vigorous.
Preferably, in described step (3), monoclonal inoculum concentration is 30 ± 2g/L, and light intensity is 2000-4000lux,
Colour temperature is 4000-6000K, airlift agitation suspension culture, and regulation Ventilation Rate and stir speed (S.S.) maintain dissolved oxygen to exist
30%.
Preferably, described 2nd MS fluid medium 0.1-2mg/L NAA, 0.1-2mg/L have been also added with
6-BA, 50-100mg/L pectase, 10-50mg/L cellulase, pH value is 5.8 ± 0.4.Add plant
Growth regulator and biological enzyme formulation in culture medium, can active cell merisis, and prevent cell aggregation
Caking, it is ensured that nutrition and the supply of molten oxygen and carbon dioxide are uniformly, fully.
Preferably, the 2-3 that P element content is MS fluid medium in a described MS fluid medium
Times, pH value is 5.8 ± 0.4.
Preferably, described Ge element derives from GeO2Or organic germanium.
Preferably, described selenium element derives from Na2SeO3Or organic selenium
It is an advantage of the current invention that: first Herba Dendrobii aseptically sowing seeds is sprouted the protocorm formed by the present invention
Stem, utilize that enzymatic isolation method isolates in high vigor protocorm is unicellular, is trained monoclonal further,
After carry out cell mass amplification culture, in amplification culture, add plant growth regulator and biological enzyme formulation and arrive
In culture medium, energy active cell merisis, and prevent cell aggregation from luming, it is ensured that nutrition and dissolved oxygen and two
Carbonoxide supply is uniformly, fully.The present invention can be greatly shortened cell culture period, improves product yield,
And technique is simple, stable, it is suitable for industrialized production.It addition, in the cell mass amplification culture stage, use this
2nd MS fluid medium of invention, when can extend the growth of batch culture cell relative to MS fluid medium
Between more than 2 times, improve cell density more than 2 times.
Detailed description of the invention
The detection method of polyoses content of the present invention
1, with reference to Herba Dendrobii quality standard in 2010 editions pharmacopeia, the Herba Dendrobii polysaccharide that embodiment is extracted
Being analyzed, specific analytical method is as follows.
The preparation of reference substance solution: take 105 DEG C and be dried to the anhydrous glucose reference substance of constant weight appropriate, accurate title
Fixed, it is dissolved in water and quantitatively makes the solution containing 100 μ g in every 1ml, to obtain final product.
The drafting of standard curve: draw glucose control product solution (concentration is 0.1g/L) 0.1,0.2,0.3,
0.4,0.5ml is in 10mL test tube, adds distilled water respectively to 1.0ml, adds 5% phenol solution 1ml, shake
Even, then add sulphuric acid 5ml, shake up, put heating 20min in boiling water bath, take out, put into cooling 5min in ice bath,
With corresponding reagent as blank, at the wavelength of 488nm, measure absorbance, with absorbance as vertical coordinate, dense
Degree is abscissa, draws standard curve.With trap A as vertical coordinate, concentration of glucose C is abscissa,
Comparing curve, obtaining regression equation is A=0.0367C+0.0412, r=0.9998, the range of linearity 4.36～21.8
μg/mL。
The mensuration of sample: weigh the Herba Dendrobii polysaccharide sample 50mg that this experiment prepares, be diluted to solution extinction
Value after (10-100 times) dilution, is measured in standard curve range as stated above.Each sample is surveyed
Trying 10 times, the value in the range of line taking is averaged.
Embodiment 1
The present embodiment discloses a kind of method that suspended culture cell produces Herba Dendrobii polysaccharide, comprises the following steps:
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 23g/L, and light intensity is 2000lux, and colour temperature is 4000K, and concussion is cultivated, and adjusting pH value is 5.4,
Temperature 20 DEG C, when cell density reaches 10000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium is
2 times of conventional MS fluid medium)+0.1mg/LNAA (1-Naphthaleneacetic acid, naphthalene second
Acid)+0.1mg/L 6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+50mg/L neutrality pectase
The Ge element of+10mg/L neutral cellulase+1mg/L (derives from GeO2)+0.02mg/L selenium element (source
In Na2SeO3)+0.1g/L acid hydrolyzed casein fluid medium 30L gas-lifting type stirred fermentor in,
Fresh cell inoculum concentration is 28g/L, and light intensity is 2000lux, and colour temperature is 4000K, airlift agitation suspension culture,
Regulation Ventilation Rate and stir speed (S.S.) maintain molten O230%, molten CO23%, regulate feed supplement liquid stream dosage
Sugar content is kept to keep pH 5.4 at 3% and NaOH solution stream dosage；Feed supplement liquid is the second of 2 times of concentration
MS fluid medium；
(4) i.e. stop cultivating when cell density reaches 60000/ml, 15 days time, use 1000rpm
Low-speed centrifugal, 10min, collect Herba Dendrobii cell, after measured cell fresh weight 0.55g/mL in every milliliter of culture fluid,
Protein content 12.1mg/g, every gram of fresh cell Determination of Chlorophyll content 0.18mg/g in every gram of fresh cell, every gram fresh
SOD activity 302U/g in cell, completes the Herba Dendrobii cell collected latter 70 DEG C and is dried to constant weight,
And clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
(1) water carries: weighed the Herba Dendrobii powder 50g being dried of 20 mesh sieves, and added 10 times of quality
Water, boiling water extraction 2h, 4000rpm/min, centrifugal 10min, it is thus achieved that the first supernatant and the first precipitation, receives
Collect the first supernatant；
(2) precipitate with ethanol: the first above-mentioned supernatant is evaporated to 1/5 volume, adds the 95% of 3 times of volumes
Ethanol stands after being sufficiently mixed, 4000rpm/min, centrifugal 10min, it is thus achieved that the second supernatant and the second precipitation；
(3) it is dried: be deposited in 100 DEG C to second and be dried to constant weight, it is thus achieved that solubility Herba Dendrobii polysaccharide powder.
The weight weighing the dendrobium polysaccharide obtained is 7.66g, and yield is 15.32%.
The present invention concrete mode used of sterilizing is 75% alcohol disinfecting 1min, 0.1%HgCl2Sterilization 15min,
Sterilized water washs 3 times.Following example all use the method to carry out disinfection.
The enzymatic isolation method of the present invention separates and uses prior art, specifically at 20uM sucrose, and 10uM MgCl2,
In 20uM PH 7.8Tris HCL buffer, use pectase, cellulase to cell mass room temperature treatment 4-8h.
And centrifugal under the conditions of 500rpm, 10min, clean after collect unicellular.Following example all use the party
Method is entered to separate.
Embodiment 2
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 25g/L, and light intensity is 3000lux, and colour temperature is 5000K, and concussion is cultivated, and adjusting pH value is 5.8,
Temperature 22 DEG C, when cell density reaches 15000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium is
3 times of conventional MS fluid medium)+1mg/LNAA (1-Naphthaleneacetic acid, naphthalene second
Acid)+1mg/L 6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+70mg/L neutrality pectase
The Ge element of+30mg/L neutral cellulase+1.5mg/L (derives from GeO2)+0.05mg/L selenium element (
Come from Na2SeO3) the 30L gas-lifting type stirred fermentor of fluid medium of acid hydrolyzed casein of+0.13g/L
In, fresh cell inoculum concentration is 30g/L, and light intensity is 3000lux, and colour temperature is 5000K, and airlift agitation suspends
Cultivating, regulation Ventilation Rate and stir speed (S.S.) maintain molten O230%, molten CO23%, regulate feed supplement liquid
Stream dosage keeps sugar content to keep pH 5.8 at 3% and NaOH solution stream dosage；Feed supplement liquid is 2 times of concentration
The 2nd MS fluid medium；
(4) i.e. stop cultivating when cell density reaches 70000/ml, 15 days time, use 1000rpm
Low-speed centrifugal, 10min, collect Herba Dendrobii cell, after measured cell fresh weight 0.58g/mL in every milliliter of culture fluid,
Protein content 12.0mg/g, every gram of fresh cell Determination of Chlorophyll content 0.18mg/g in every gram of fresh cell, every gram fresh
SOD activity 317U/g in cell, completes the Herba Dendrobii cell collected latter 70 DEG C and is dried to constant weight,
And clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
(1) water carries: weighed the Herba Dendrobii powder 50g being dried of 20 mesh sieves, and added 10 times of quality
Water, boiling water extraction 2h, 4000rpm/min, centrifugal 10min, it is thus achieved that the first supernatant and the first precipitation, receives
Collect the first supernatant；
(2) precipitate with ethanol: the first above-mentioned supernatant is evaporated to 1/5 volume, adds the 95% of 3 times of volumes
Ethanol stands after being sufficiently mixed, 4000rpm/min, centrifugal 10min, it is thus achieved that the second supernatant and the second precipitation；
(3) it is dried: be deposited in 100 DEG C to second and be dried to constant weight, it is thus achieved that solubility Herba Dendrobii polysaccharide powder.
The weight weighing the dendrobium polysaccharide obtained is 8.14g, and yield is 16.28%.
Embodiment 3
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 27g/L, and light intensity is 4000lux, and colour temperature is 6000K, and concussion is cultivated, and adjusting pH value is 6.2,
Temperature 24 DEG C, when cell density reaches 20000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium is
2 times of conventional MS fluid medium)+2mg/LNAA (1-Naphthaleneacetic acid, naphthalene second
Acid)+2mg/L 6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+100mg/L neutrality pectase
Ge element (deriving from the organic germanium)+0.1mg/L selenium element (source of+50mg/L neutral cellulase+2mg/L
In Na2SeO3)+0.2g/L acid hydrolyzed casein fluid medium 30L gas-lifting type stirred fermentor in,
Fresh cell inoculum concentration is 32g/L, and light intensity is 4000lux, and colour temperature is 6000K, airlift agitation suspension culture,
Regulation Ventilation Rate and stir speed (S.S.) maintain molten O230%, molten CO23%, regulate feed supplement liquid stream dosage
Sugar content is kept to keep pH 6.2 at 3% and NaOH solution stream dosage；Feed supplement liquid is the second of 2 times of concentration
MS fluid medium；
(4) i.e. stop cultivating when cell density reaches 80000/ml, 15 days time, use 1000rpm
Low-speed centrifugal, 10min, collect Herba Dendrobii cell, after measured cell fresh weight 0.59g/mL in every milliliter of culture fluid,
Protein content 11.8mg/g, every gram of fresh cell Determination of Chlorophyll content 0.20mg/g in every gram of fresh cell, every gram fresh
SOD activity 323U/g in cell, completes the Herba Dendrobii cell collected latter 70 DEG C and is dried to constant weight,
And clay into power, room temperature Seal and preservation is standby.After cell reaches this density, cannot continue to add density
, reach the agitation limit of 30L airlift fermentor.
2, extract the stage
(1) water carries: weighed the Herba Dendrobii powder 50g being dried of 20 mesh sieves, and added 10 times of quality
Water, boiling water extraction 2h, 4000rpm/min, centrifugal 10min, it is thus achieved that the first supernatant and the first precipitation, receives
Collect the first supernatant；
(2) precipitate with ethanol: the first above-mentioned supernatant is evaporated to 1/5 volume, adds the 95% of 3 times of volumes
Ethanol stands after being sufficiently mixed, 4000rpm/min, centrifugal 10min, it is thus achieved that the second supernatant and the second precipitation；
(3) it is dried: be deposited in 100 DEG C to second and be dried to constant weight, it is thus achieved that solubility Herba Dendrobii polysaccharide powder.
The weight weighing the dendrobium polysaccharide obtained is 9.06g, and yield is 18.12%.
Embodiment 4
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 24g/L, and light intensity is 2500lux, and colour temperature is 4500K, and concussion is cultivated, and adjusting pH value is 5.6,
Temperature 21 DEG C, when cell density reaches 12000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium for routine
2 times of MS fluid medium)+0.5mg/LNAA (1-Naphthaleneacetic acid, naphthalene acetic acid)+0.7mg/L
6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+70mg/L neutrality pectase+20mg/L is neutral
Ge element (deriving from organic germanium)+0.06mg/L selenium element (deriving from organic selenium) of cellulase+1.8mg/L
In the 30L gas-lifting type stirred fermentor of the fluid medium of the acid hydrolyzed casein of+0.16g/L, fresh cell connects
Amount of planting is for 29g/L, and light intensity is 2500lux, and colour temperature is 4500K, airlift agitation suspension culture, and regulation is logical
Gas speed and stir speed (S.S.) maintain molten O230%, molten CO23%, regulation feed supplement liquid stream dosage keeps sugar
Content keeps pH 5.9 at 3% and NaOH solution stream dosage；Feed supplement liquid is the 2nd MS of 2 times of concentration
Fluid medium；
(4) i.e. stop cultivating when cell density reaches 60000/ml, 15 days time, use 1000rpm
Low-speed centrifugal, 10min, collect Herba Dendrobii cell, after measured cell fresh weight 0.54g/mL in every milliliter of culture fluid,
Protein content 12.2mg/g, every gram of fresh cell Determination of Chlorophyll content 0.19mg/g in every gram of fresh cell, every gram
SOD activity 298U/g in fresh cell, completes the Herba Dendrobii cell collected latter 70 DEG C and is dried to constant weight,
And clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
(1) water carries: weighed the Herba Dendrobii powder 50g being dried of 20 mesh sieves, and added 10 times of quality
Water, boiling water extraction 2h, 4000rpm/min, centrifugal 10min, it is thus achieved that the first supernatant and the first precipitation, receives
Collect the first supernatant；
(2) precipitate with ethanol: the first above-mentioned supernatant is evaporated to 1/5 volume, adds the 95% of 3 times of volumes
Ethanol stands after being sufficiently mixed, 4000rpm/min, centrifugal 10min, it is thus achieved that the second supernatant and the second precipitation；
(3) it is dried: be deposited in 100 DEG C to second and be dried to constant weight, it is thus achieved that solubility Herba Dendrobii polysaccharide powder.
The weight weighing the dendrobium polysaccharide obtained is 8.14g, and yield is 16.28%.
Embodiment 5
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 25g/L, and light intensity is 3000lux, and colour temperature is 4400K, and concussion is cultivated, and adjusting pH value is 5.7,
Temperature 22 DEG C, when cell density reaches 18000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium for routine
2 times of MS fluid medium)+1mg/LNAA (1-Naphthaleneacetic acid, naphthalene acetic acid)+1.2mg/L
6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+60mg/L neutrality pectase+25mg/L is neutral
The Ge element of cellulase+1.8mg/L (derives from GeO2)+0.08mg/L selenium element (deriving from organic selenium)
In the 30L gas-lifting type stirred fermentor of the fluid medium of the acid hydrolyzed casein of+0.17g/L, fresh cell connects
Amount of planting is for 30g/L, and light intensity is 2000lux, and colour temperature is 5200K, airlift agitation suspension culture, and regulation is logical
Gas speed and stir speed (S.S.) maintain molten O230%, molten CO23%, regulation feed supplement liquid stream dosage keeps sugar
Content keeps pH 5.8 at 3% and NaOH solution stream dosage；Feed supplement liquid is the 2nd MS of 2 times of concentration
Fluid medium；
(4) i.e. stop cultivating when cell density reaches 60000/ml, 15 days time, use 1000rpm
Low-speed centrifugal, 10min, collect Herba Dendrobii cell, after measured cell fresh weight 0.58g/mL in every milliliter of culture fluid,
Protein content 11.9mg/g, every gram of fresh cell Determination of Chlorophyll content 0.18mg/g in every gram of fresh cell, every gram
SOD activity 331U/g in fresh cell, completes the Herba Dendrobii cell collected latter 70 DEG C and is dried to constant weight,
And clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
(1) water carries: weighed the Herba Dendrobii powder 50g being dried of 20 mesh sieves, and added 10 times of quality
Water, boiling water extraction 2h, 4000rpm/min, centrifugal 10min, it is thus achieved that the first supernatant and the first precipitation, receives
Collect the first supernatant；
(2) precipitate with ethanol: the first above-mentioned supernatant is evaporated to 1/5 volume, adds the 95% of 3 times of volumes
Ethanol stands after being sufficiently mixed, 4000rpm/min, centrifugal 10min, it is thus achieved that the second supernatant and the second precipitation；
(3) it is dried: be deposited in 100 DEG C to second and be dried to constant weight, it is thus achieved that solubility Herba Dendrobii polysaccharide powder.
The weight weighing the dendrobium polysaccharide obtained is 8.35g, and yield is 16.70%.
Embodiment 6
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 26g/L, and light intensity is 3000lux, and colour temperature is 5000K, and concussion is cultivated, and adjusting pH value is 6.1,
Temperature 20 DEG C, when cell density reaches 12000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium for routine
3 times of MS fluid medium)+0.3mg/LNAA (1-Naphthaleneacetic acid, naphthalene acetic acid)+1.4mg/L
6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+60mg/L neutrality pectase+30mg/L is neutral
The Ge element of cellulase+2mg/L (derives from GeO2)+0.05mg/L selenium element (deriving from organic selenium)
In the 30L gas-lifting type stirred fermentor of the fluid medium of the acid hydrolyzed casein of+0.18g/L, fresh cell connects
Amount of planting is for 30g/L, and light intensity is 3000lux, and colour temperature is 5000K, airlift agitation suspension culture, and regulation is logical
Gas speed and stir speed (S.S.) maintain molten O230%, molten CO23%, regulation feed supplement liquid stream dosage keeps sugar
Content keeps pH 5.8 at 3% and NaOH solution stream dosage；Feed supplement liquid is the 2nd MS of 2 times of concentration
Fluid medium；
(4) i.e. stop cultivating when cell density reaches 60000/ml, 15 days time, use 1000rpm
Low-speed centrifugal, 10min, collect Herba Dendrobii cell, after measured cell fresh weight 0.56g/mL in every milliliter of culture fluid,
Protein content 12.1mg/g, every gram of fresh cell Determination of Chlorophyll content 0.18mg/g in every gram of fresh cell, every gram fresh
SOD activity 317U/g in cell, completes the Herba Dendrobii cell collected latter 70 DEG C and is dried to constant weight,
And clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
(1) water carries: weighed the Herba Dendrobii powder 50g being dried of 20 mesh sieves, and added 10 times of quality
Water, boiling water extraction 2h, 4000rpm/min, centrifugal 10min, it is thus achieved that the first supernatant and the first precipitation, receives
Collect the first supernatant；
(2) precipitate with ethanol: the first above-mentioned supernatant is evaporated to 1/5 volume, adds the 95% of 3 times of volumes
Ethanol stands after being sufficiently mixed, 4000rpm/min, centrifugal 10min, it is thus achieved that the second supernatant and the second precipitation；
(3) it is dried: be deposited in 100 DEG C to second and be dried to constant weight, it is thus achieved that solubility Herba Dendrobii polysaccharide powder.
The weight weighing the dendrobium polysaccharide obtained is 8.11g, and yield is 16.22%.
Embodiment 7
1, cultivation stage
(1) unicellular preparation
Taking Herba Dendrobii capsule, carry out aseptic seeding after requiring sterilization according to tissue culture technology, sowing culture medium is
Solid 1/2MS culture medium, takes out after sprouting into protocorm, selects very large, vigor bright in colour, individual
Vigorous protocorm, uses enzymatic isolation method separation to prepare unicellular, filters with the micropore filter of 80 micron pore size,
Filtrate separates through low speed centrifuge, collects the cell under precipitation；
(2) monoclonal is cultivated
In the unicellular flask with indentation being inoculated into containing a MS fluid medium that will collect, unicellular
Inoculum concentration is 26g/L, and light intensity is 4000lux, and colour temperature is 6000K, and concussion is cultivated, and adjusting pH value is 6.2,
Temperature 23 DEG C, when cell density reaches 13000/mL, gets final product amplification culture；
(3) cell mass amplification culture:
Cultured monoclonal is inoculated in containing the 2nd MS (P element in this MS fluid medium for routine
2 times of MS fluid medium)+1.3mg/LNAA (1-Naphthaleneacetic acid, naphthalene acetic acid)+0.8mg/L
6-BA (6-Benzylaminopurine, 6-benzyl aminopurine)+70mg/L neutrality pectase+35mg/L is neutral
The Ge element of cellulase+1.6mg/L (derives from GeO2)+0.1mg/L selenium element (deriving from organic selenium)
In the 30L gas-lifting type stirred fermentor of the fluid medium of the acid hydrolyzed casein of+0.17g/L, fresh cell connects
Amount of planting is for 30g/L, and light intensity is 3000lux, and colour temperature is 5000K, airlift agitation suspension culture, and regulation is logical
Gas speed and stir speed (S.S.) maintain molten O230%, molten CO23%, regulation feed supplement liquid stream dosage keeps sugar
Content keeps pH 5.6 at 3% and NaOH solution stream dosage；Feed supplement liquid is the 2nd MS of 2 times of concentration
Fluid medium；
(4) i.e. stop cultivating when cell density reaches 70000/ml, 15 days time, use 1000rpm
Low-speed centrifugal, 10min, collect Herba Dendrobii cell, after measured cell fresh weight 0.50g/mL in every milliliter of culture fluid,
Protein content 12.2mg/g, every gram of fresh cell Determination of Chlorophyll content 0.19mg/g in every gram of fresh cell, every gram fresh
SOD activity 314U/g in cell, completes the Herba Dendrobii cell collected latter 70 DEG C and is dried to constant weight,
And clay into power, room temperature Seal and preservation is standby.
2, extract the stage:
(1) water carries: weighed the Herba Dendrobii powder 50g being dried of 20 mesh sieves, and added 10 times of quality
Water, boiling water extraction 2h, 4000rpm/min, centrifugal 10min, it is thus achieved that the first supernatant and the first precipitation, receives
Collect the first supernatant；
(2) precipitate with ethanol: the first above-mentioned supernatant is evaporated to 1/5 volume, adds the 95% of 3 times of volumes
Ethanol stands after being sufficiently mixed, 4000rpm/min, centrifugal 10min, it is thus achieved that the second supernatant and the second precipitation；
(3) it is dried: be deposited in 100 DEG C to second and be dried to constant weight, it is thus achieved that solubility Herba Dendrobii polysaccharide powder.
The weight weighing the dendrobium polysaccharide obtained is 8.31g, and yield is 16.62%.
Comparative example 1
Choose the Seeded growth Herba Dendrobii tissue cultured seedling of 8 months, tissue cultured seedling is cleaned and completes after draining, then 70 DEG C
Being dried to constant weight, and clay into power, room temperature Seal and preservation is standby.
(1) water carries: weighed the Herba Dendrobii powder 50g being dried of 20 mesh sieves, and added 10 times of quality
Water, boiling water extraction 2h, 4000rpm/min, centrifugal 10min, it is thus achieved that the first supernatant and the first precipitation, receives
Collect the first supernatant；
(2) precipitate with ethanol: the first above-mentioned supernatant is evaporated to 1/5 volume, adds the 95% of 3 times of volumes
Ethanol stands after being sufficiently mixed, 4000rpm/min, centrifugal 10min, it is thus achieved that the second supernatant and the second precipitation；
(3) it is dried: be deposited in 100 DEG C to second and be dried to constant weight, it is thus achieved that solubility Herba Dendrobii polysaccharide powder.
The weight weighing the Herba Dendrobii polysaccharide obtained is 8.28g, and yield is 16.57%.
By above-mentioned cultivation and extraction, the judgement cell culture method of the present invention that can will be apparent from is carefully
The Herba Dendrobii polysaccharide that born of the same parents produce after cultivating 15 days can be close to the even more than Seeded growth same period 8 on yield
The Herba Dendrobii polysaccharide that the tissue cultured seedling of individual month produces.
The method of the present invention is not intended to the production of Herba Dendrobii polysaccharide, applies also for other edible or medicinal dendrobiums
The production of platymiscium polysaccharide.The oneth MS fluid medium of the present invention is conventional MS fluid medium or P
Element is the culture medium of conventional MS fluid medium P element 2-3 times.
The foregoing is only the preferred embodiment of the invention, not in order to limit the invention,
Any amendment, equivalent and the improvement etc. made within all spirit in the invention and principle, all should
Within being included in the protection domain of the invention.
Claims (8)
1. the method that a suspended culture cell produces Herba Dendrobii polysaccharide, it is characterised in that include following step
Rapid:
(1) unicellular preparation: carry out aseptic seeding with solid medium after taking the sterilization of Herba Dendrobii capsule,
Take out after sprouting into protocorm, use enzymatic isolation method separation to prepare unicellular, and centrifugal filtration remove impurity, collect
Unicellular；
(2) monoclonal is cultivated: unicellular be inoculated in a MS fluid medium training by collect
Support；
(3) cell mass amplification culture: cultured monoclonal is inoculated in the 2nd MS fluid medium
Middle cultivation；In described 2nd MS fluid medium 2-3 times that phosphorus element content is MS fluid medium,
Described 2nd MS fluid medium is also added with the Ge element of 1-2mg/L, 0.02-0.1mg/L selenium element and
The acid hydrolyzed casein of 0.1-0.2g/L, pH value is 5.8 ± 0.4；
(4) extract Herba Dendrobii polysaccharide: when cell density reaches 60000-80000/ml, use centrifugal
Collect Herba Dendrobii cell；Extract Herba Dendrobii polysaccharide again.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii polysaccharide, its
Being characterised by, the culture medium that described step (1) sowing uses is to be suitable for the solid medium of orchid growth.
A kind of suspended culture cell the most according to claim 2 produces the method for Herba Dendrobii polysaccharide, its
Being characterised by, described solid medium is 1/2MS solid medium.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii polysaccharide, its
Being characterised by, in described step (2), unicellular inoculum concentration is 25 ± 2g/L, and light intensity is 2000-4000lux,
Colour temperature is 4000-6000K, and concussion is cultivated, temperature 22 ± 2 DEG C, when cell density reaches 10000-20000
/ mL, gets final product amplification culture.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii polysaccharide, and it is special
Levying and be, in described step (3), monoclonal inoculum concentration is 30 ± 2g/L, and light intensity is 2000-4000lux,
Colour temperature is 4000-6000K, temperature 22 ± 2 DEG C, airlift agitation suspension culture, regulation Ventilation Rate and stirring speed
Rate maintains dissolved oxygen 30%.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii polysaccharide, its
Be characterised by, described second liquid MS culture medium has been also added with 0.1-2mg/L NAA, 0.1-2mg/L 6-BA,
50-100mg/L pectase, 10-50mg/L cellulase, pH value is 5.8 ± 0.4.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii polysaccharide, its
Being characterised by, described Ge element derives from GeO2Or organic germanium.
A kind of suspended culture cell the most according to claim 1 produces the method for Herba Dendrobii polysaccharide, and it is special
Levying and be, described selenium element derives from Na2SeO3Or organic selenium.
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