CN109362569A - A kind of production method for the beautiful millettia root polysaccharide that culture medium recycles - Google Patents
A kind of production method for the beautiful millettia root polysaccharide that culture medium recycles Download PDFInfo
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- CN109362569A CN109362569A CN201811513390.7A CN201811513390A CN109362569A CN 109362569 A CN109362569 A CN 109362569A CN 201811513390 A CN201811513390 A CN 201811513390A CN 109362569 A CN109362569 A CN 109362569A
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention discloses a kind of production methods for the beautiful millettia root polysaccharide that culture medium recycles, comprising: step 1) chooses Caulis Spatholobi i.e. for mature seed, and disinfection carries out sprouting culture；Step 2) takes tender leaf to carry out Fiber differentiation as explant and obtains callus；Step 3) is at least secondary by callus progress squamous subculture, is grown to serve as cell mass；Cell mass is carried out suspension Primary culture by step 4), obtains suspension cell line；Suspension cell line is carried out Multiplying culture by step 5), obtains Caulis Spatholobi suspension cell line.Step 6) collects cell, extracts, centrifugation, obtains beautiful millettia root polysaccharide after precipitating is dry.Wherein, sprouting fluid nutrient medium includes: 1/4~1/2MS, sucrose and 6-BA, initial pH value is 5.8, or acquisition methods are as follows: the proliferation fluid nutrient medium after the start liquid culture medium and/or utilization after taking primary utilize, filtering, pH is adjusted to 5.8, and 6-BA is added in sterilizing, as sprouting fluid nutrient medium.
Description
Technical field
The present invention relates to a kind of lifes for the beautiful millettia root polysaccharide that technical field of Chinese herbal medicine cultivation more particularly to culture medium recycle
Production method.
Background technique
Caulis Spatholobi, Chinese medicine name.For the drying rattan of leguminous plant spatholobus suberectus Spatholobus suberectus Dunn.
Autumn, the harvesting of two season of winter, branches and leaves are removed, slice dries.It is distributed in the ground such as Guangdong, Guangxi, Yunnan.With blood circulation promoting and enriching, menstruation regulating stops
Bitterly, the effect of relaxing tendons and activating collaterals.For irregular menstruation, dysmenorrhea, menostasis, rheumatic arthralgia, paralysis and numbness, blood deficiency chlorosis.With research
Go deep into, scientist's discovery, the root of Caulis Spatholobi, beautiful millettia root has apparent antitumor action rich in many kinds of substance such as polysaccharide,
Demand medically is increasing.The yield of wild Caulis Spatholobi plant is no longer satisfied the market demand.To meet to this
The demand of Chinese medicine gradually adopts the mode of artificial cultivation and tissue cultures to cultivate Caulis Spatholobi in recent years.Tissue cultures
Due to having many advantages, such as that incubation time is short, being not take up arable land, gradually fast development is got up.However, in tissue cultures, due to training
The feeding time is shorter, thus in Caulis Spatholobi suspension cell the substances such as polysaccharide content it is few compared with wild type.How in a relatively short period of time
So that the polysaccharide containing more amount in the Caulis Spatholobi suspension cell of culture, is the technical issues of currently actively probing into.
Summary of the invention
It is an object of the invention to solve at least the above problems, and provide the advantages of at least will be described later.
It is a still further object of the present invention to provide a kind of production methods for the beautiful millettia root polysaccharide that culture medium recycles, comprising:
A kind of production method for the beautiful millettia root polysaccharide that culture medium recycles, comprising:
Step 1) chooses Caulis Spatholobi i.e. by mature seed, then to mature seed carries out disinfection, then at temperature 0 first
24~48h is handled at~2 DEG C, is finally placed on sprout cultivating to it in sprouting fluid nutrient medium growing 1~10mm
The tender leaf of length, i.e., the seed of the Caulis Spatholobi plant for being Post flowering 90~130 days by mature seed, the disinfection include:
First using the solution cleaning containing mass-volume concentration 1~5g/mL calcium hypochlorite it is described i.e. by mature seed several times, then
Seed after cleaning is handled 20~30 seconds at 70~80 DEG C of temperature；
Step 2), which is chosen, cultivates the tender leaf grown as explant by sprouting, and the explant is placed in induction solid training
It supports and carries out Fiber differentiation on base, obtain callus, the induction solid medium includes: 0.2~0.5mg/L of MS, 6-BA,
0.02~0.1mg/L of NAA, 10~15g/L of sucrose, 5~10g/L of propolis, 2~5g/L of yeast extract, mass fraction 2~
10% ginkgo leaf juice and agar 8g/L；
It is at least secondary that the callus is carried out squamous subculture by step 3) in subculture solid medium, until it grows into
For absinthe-green granular loose cell mass, the subculture solid medium includes: 0.5~1mg/L of MS, 6-BA,
NAA0.02~0.1mg/L, 5~10g/L of sucrose, 15~20g/L of propolis, 5~10g/L of yeast extract, mass fraction 5~
15% ginkgo leaf juice and agar 8g/L；
The cell mass is transferred in start liquid culture medium and carries out suspension Primary culture by step 4), obtains suspension cell
System, the start liquid culture medium includes: 1/4~1/2MS, 0.02~0.1mg/L of 6-BA 0.2~0.6mg/L, NAA, sucrose
The ginkgo leaf juice of 2~5g/L, 10~15g/L of propolis, 2~5g/L of yeast extract and mass fraction 2~10%；
The suspension cell line is seeded in proliferation fluid nutrient medium and carries out Multiplying culture by step 5), and it is outstanding to obtain Caulis Spatholobi
Floating cell line, the proliferation fluid nutrient medium includes: 0.02~0.1mg/L of 1/2MS, 6-BA 0.5~1mg/L, NAA, sucrose 2
The ginkgo leaf juice of~5g/L, 10~15g/L of propolis, 2~5g/L of yeast extract and mass fraction 2~10%.
Step 6) is by the Caulis Spatholobi suspension cell line to be centrifuged 3~5 at 2000~3000rpm of revolving speed, 0~4 DEG C of temperature
Minute collect cell, then at 90~100 DEG C of water bath with thermostatic control, respectively with pure water extract 3 times, first time extraction it is pure
The ratio of the dry weight of water consumption and cell is 10:1, and extraction time first time is 40min, and the pure water of second of extraction flows
The ratio of amount and the dry weight of cell is 5:1, and second of extraction time is 10min, and the pure water of third time extraction is in cell
The ratio of dry weight is 2:1, and third time extraction time is 5min, merges extracting solution three times, percent by volume is added thereto later
75% ethyl alcohol, to discard waste liquid after 3~5min of centrifugation at 6000~8000rpm of revolving speed, 0~4 DEG C of temperature, after drying being precipitated
Obtain beautiful millettia root polysaccharide；
Wherein, the sprouting fluid nutrient medium includes: 1/4~1/2MS, 0.1~0.3mg/ of sucrose 7~15g/L and 6-BA
L, initial pH value 5.8, alternatively,
The acquisition methods for sprouting fluid nutrient medium are as follows: take the start liquid culture after once utilizing in step 4)
Proliferation fluid nutrient medium after utilizing in base and/or step 5), is collected by filtration filtrate with 4~10 mesh filter screen, then will be described
The pH of filtrate is adjusted to 5.8, then places it at 110~117 DEG C of temperature and carry out closing 10~20min of sterilizing, after obtaining sterilizing
Culture medium, then thereto be added 0.1~0.3mg/L of 6-BA, using as sprout fluid nutrient medium.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, the sprouting Liquid Culture
The acquisition methods of base are as follows: take the proliferation after utilizing in the start liquid culture medium and step 5) after once utilizing in step 4)
Fluid nutrient medium is collected by filtration filtrate with 6 mesh filter screen, the pH of the filtrate is then adjusted to 5.8, then place it in temperature
Closing sterilizing 15min, the culture medium after sterilize, then addition 6-BA 0.2mg/L thereto are carried out at 115 DEG C, using as sprouting
Send out fluid nutrient medium.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, the sprouting Liquid Culture
A filter paper is placed with above base, it is described that mature seed is distributed on the filter paper.Preferably, the culture medium is sharp again
In the production method of beautiful millettia root polysaccharide, in step 1), i.e., the Caulis Spatholobi plant for being Post flowering 110 days by mature seed
Seed.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, in step 1), the disinfection
Include: first using the solution cleaning containing mass-volume concentration 3g/mL calcium hypochlorite it is described i.e. by mature seed several times, so
The seed after cleaning is handled 25 seconds at 75 DEG C of temperature afterwards.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, in step 1), the sprouting
Fluid nutrient medium includes: 1/43MS, sucrose 11g/L and 6-BA 0.2mg/L.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, in step 4), the suspension
The inoculum concentration of Primary culture is 3~5g/L squamous subculture product.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, in step 5), the proliferation
The inoculum concentration ratio of culture are as follows: the culture after taking the suspension Primary culture for accounting for the proliferation fluid nutrient medium 10%~20%.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, the ginkgo leaf juice
Acquisition methods are as follows: take Folium Ginkgo that water is added according to mass ratio 10:1, crush later, and with the filtering of 200 mesh filter screen, filtered
Liquid is ginkgo leaf juice.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, in step 1), the sprouting
The condition of culture of culture are as follows: 800~1500lux of intensity of illumination, 8~12 hour/day of photoperiod, cultivation temperature are 24~28 DEG C,
Incubation time is 4~10 days.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, in step 2), the induction
The condition of culture of culture are as follows: 1500~3000lux of intensity of illumination, 10~12 hour/day of photoperiod, cultivation temperature are 24~28
DEG C, incubation time is 20~40 days.
Preferably, described every time in step 3) in the production method for the beautiful millettia root polysaccharide that the culture medium recycles
The condition of culture of squamous subculture are as follows: 2000~4000lux of intensity of illumination, 10~12 hour/day of photoperiod, cultivation temperature be 24~
28 DEG C, incubation time is 4~8 days.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, in step 4), the suspension
The condition of culture of Primary culture are as follows: dark culture, cultivation temperature are 24~28 DEG C, 100~120rpm revolving speed shake culture, when culture
Between be 2~3 days.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, in step 5), the proliferation
The condition of culture of culture are as follows: dark culture, cultivation temperature are 24~28 DEG C, 100~200rpm revolving speed shake culture, and incubation time is
7~15 days.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, in step 1), the sprouting
A filter paper is placed with above fluid nutrient medium, it is described that mature seed is distributed on the filter paper.
Preferably, in the production method for the beautiful millettia root polysaccharide that the culture medium recycles, the start liquid culture
Base includes: 1/3MS, 6-BA 0.4mg/L, NAA 0.06mg/L, sucrose 2.5g/L, propolis 12.5g/L, yeast extract 3.5g/
The ginkgo leaf juice of L and mass fraction 6%.
The present invention is include at least the following beneficial effects:
The present invention provides a kind of production method of beautiful millettia root polysaccharide that culture medium recycles, comprising:
Step 1) chooses Caulis Spatholobi i.e. by mature seed, then to mature seed carries out disinfection, then at temperature 0 first
24~48h is handled at~2 DEG C, is finally placed on sprout cultivating to it in sprouting fluid nutrient medium growing 1~10mm
The tender leaf of length, i.e., the seed of the Caulis Spatholobi plant for being Post flowering 90~130 days by mature seed, the disinfection include:
First using the solution cleaning containing mass-volume concentration 1~5g/mL calcium hypochlorite it is described i.e. by mature seed several times, then
Seed after cleaning is handled 20~30 seconds at 70~80 DEG C of temperature, the sprouting fluid nutrient medium includes: 1/4~1/2MS,
Sucrose 0.1~0.3mg/L of 7~15g/L and 6-BA, initial pH value 5.8, alternatively, the acquisition for sprouting fluid nutrient medium
Method are as follows: take the proliferation liquid after utilizing in the start liquid culture medium and/or step 5) after once utilizing in step 4)
Culture medium is collected by filtration filtrate with 4~10 mesh filter screen, the pH of the filtrate is then adjusted to 5.8, then place it in temperature
Carried out at 110~117 DEG C closing sterilizing 10~20min, the culture medium after sterilize, then thereto addition 6-BA 0.1~
0.3mg/L, using as sprout fluid nutrient medium；The present invention is chosen i.e. first by mature seed, is carried out disinfection to it, to avoid production
Endophytic bacteria pollute later period incubation, also, disinfection of the invention use calcium hypochlorite and low temperature instantaneous sterilizing, to seed into
While row disinfection, can also sprout to it is stimulated, and promotes its sprouting.Meanwhile it selecting and mature seed is subjected to sprouting training
It supports, since kind of a skin has not yet fully hardened, is easy to sprout, germination rate has reached 100%.Step 2) is chosen by sprouting culture
The explant is placed on induction solid medium as explant and carries out Fiber differentiation, obtain callus group by the tender leaf grown
It knits, the induction solid medium includes: MS, 6-BA 0.2~0.5mg/L, NAA 0.02~0.1mg/L, 10~15g/ of sucrose
L, the ginkgo leaf juice and agar 8g/L of 5~10g/L of propolis, 2~5g/L of yeast extract, mass fraction 2~10%；Firstly,
For the present invention using the tender leaf of sprouting as explant, the differentiation of explant is small, and the suspension cell of acquisition is more vibrant, and metabolism is more
Fastly, more preferable to the absorption of nutrient.The callus is carried out squamous subculture at least two by step 3) in subculture solid medium
Secondary, until it is grown to serve as absinthe-green granular loose cell mass, the subculture solid medium includes: MS, 6-BA
0.02~0.1mg/L of 0.5~1mg/L, NAA, 5~10g/L of sucrose, 15~20g/L of propolis, 5~10g/L of yeast extract, matter
Measure the ginkgo leaf juice and agar 8g/L of score 5~15%；The cell mass is transferred in start liquid culture medium by step 4)
Suspension Primary culture is carried out, obtains suspension cell line, the start liquid culture medium includes: 1/4~1/2MS, 6-BA 0.2~
0.02~0.1mg/L of 0.6mg/L, NAA, 2~5g/L of sucrose, 10~15g/L of propolis, 2~5g/L of yeast extract and quality point
The ginkgo leaf juice of number 2~10%；The suspension cell line is seeded in proliferation fluid nutrient medium and carries out proliferation training by step 5)
It supports, obtains Caulis Spatholobi suspension cell line, the proliferation fluid nutrient medium includes: 1/2MS, 6-BA 0.5~1mg/L, NAA 0.02
The ginkgo of~0.1mg/L, 2~5g/L of sucrose, 10~15g/L of propolis, 2~5g/L of yeast extract and mass fraction 2~10%
Leaf sap.Step 6) is by the Caulis Spatholobi suspension cell line to be centrifuged 3~5 points at 2000~3000rpm of revolving speed, 0~4 DEG C of temperature
Clock collects cell, then at 90~100 DEG C of water bath with thermostatic control, is extracted 3 times with pure water respectively, the pure water of first time extraction
The ratio of dosage and the dry weight of cell is 10:1, and extraction time first time is 40min, the pure water flow of second of extraction
Ratio with the dry weight of cell is 5:1, and second of extraction time is 10min, and the pure water of third time extraction is in the dry of cell
The ratio of weight is 2:1, and third time extraction time is 5min, merges extracting solution three times, percent by volume is added thereto later
75% ethyl alcohol discards waste liquid be centrifuged after 3~5 at 6000~8000rpm of revolving speed, 0~4 DEG C of temperature, will obtain after precipitating drying
To beautiful millettia root polysaccharide.In step of the invention, the polysaccharide in 3 Caulis Spatholobi suspension cells is extracted with pure water respectively, it can be most
That changes greatly extracts the polysaccharide in Caulis Spatholobi suspension cell, and the extracted amount of polysaccharide improves 10%~15%.The present invention
Subculture solid medium, start liquid culture medium and proliferation fluid nutrient medium in contain propolis, yeast extract and ginkgo
Leaf sap, in this way, cell can absorb and utilize more flavone compound, protein-based compound and other nutrients
Matter excites its own synthesis to flavone compound, so that its be promoted to generate more flavone compounds.Meanwhile this hair
The bright different upgrowth situations according to material different phase, the content of each component of the culture medium of reasonable disposition various concentration, from
And promote the fast-growth of more a stage different materials and the absorption to nutritional ingredient.Culture is obtained using method of the invention
The Caulis Spatholobi suspension cell period it is short, and can extract in every liter of Caulis Spatholobi suspension cell to 1.90~2.76g polysaccharide, realize
The purpose of a large amount of effective component is obtained in short period.The present invention is to start liquid culture medium and/or increment fluid nutrient medium
It is reused, it is energy saving, simultaneously as there is the residual of a small amount of more nutriment abundant, accelerate the effect of sprouting
Rate, and the consistency sprouted is also very high, whole emergence rate and consistency reach 98%, whole production level is improved, and
Raising is also obtained in the output of beautiful millettia root polysaccharide.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Specific embodiment
The present invention is described in further detail below, to enable those skilled in the art's refer to the instruction text being capable of evidence
To implement.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
The present invention provides a kind of production method of beautiful millettia root polysaccharide that culture medium recycles, comprising:
Step 1) chooses Caulis Spatholobi i.e. by mature seed, then to mature seed carries out disinfection, then at temperature 0 first
24~48h is handled at~2 DEG C, is finally placed on sprout cultivating to it in sprouting fluid nutrient medium growing 1~10mm
The tender leaf of length, i.e., the seed of the Caulis Spatholobi plant for being Post flowering 90~130 days by mature seed, the disinfection include:
First using the solution cleaning containing mass-volume concentration 1~5g/mL calcium hypochlorite it is described i.e. by mature seed several times, then
Seed after cleaning is handled 20~30 seconds at 70~80 DEG C of temperature, the sprouting fluid nutrient medium includes: 1/4~1/2MS,
Sucrose 0.1~0.3mg/L of 7~15g/L and 6-BA, initial pH value 5.8；The present invention is chosen first i.e. by mature seed, right
It carries out disinfection, and to avoid germ contamination later period incubation is generated, also, disinfection of the invention uses calcium hypochlorite and low temperature
Instantaneous sterilizing, while carrying out disinfection to seed, can also sprout to it is stimulated, and promotes its sprouting.Meanwhile selection will
Mature seed carries out sprouting culture, since kind of a skin has not yet fully hardened, is easy to sprout, germination rate has reached 100%.
Step 2), which is chosen, cultivates the tender leaf grown as explant by sprouting, and the explant is placed in induction solid training
It supports and carries out Fiber differentiation on base, obtain callus, the induction solid medium includes: 0.2~0.5mg/L of MS, 6-BA,
0.02~0.1mg/L of NAA, 10~15g/L of sucrose, 5~10g/L of propolis, 2~5g/L of yeast extract, mass fraction 2~
10% ginkgo leaf juice and agar 8g/L；Firstly, the present invention is using the tender leaf of sprouting as explant, the differentiation of explant is small,
The suspension cell of acquisition is more vibrant, and metabolism is faster, more preferable to the absorption of nutrient.
It is at least secondary that the callus is carried out squamous subculture by step 3) in subculture solid medium, until it grows into
For absinthe-green granular loose cell mass, the subculture solid medium includes: 0.5~1mg/L of MS, 6-BA,
NAA0.02~0.1mg/L, 5~10g/L of sucrose, 15~20g/L of propolis, 5~10g/L of yeast extract, mass fraction 5~
15% ginkgo leaf juice and agar 8g/L；
The cell mass is transferred in start liquid culture medium and carries out suspension Primary culture by step 4), obtains suspension cell
System, the start liquid culture medium includes: 1/4~1/2MS, 0.02~0.1mg/L of 6-BA 0.2~0.6mg/L, NAA, sucrose
The ginkgo leaf juice of 2~5g/L, 10~15g/L of propolis, 2~5g/L of yeast extract and mass fraction 2~10%；
The suspension cell line is seeded in proliferation fluid nutrient medium and carries out Multiplying culture by step 5), and it is outstanding to obtain Caulis Spatholobi
Floating cell line, the proliferation fluid nutrient medium includes: 1/2MS, 6-BA0.5~1mg/L, NAA0.02~0.1mg/L, sucrose 2~
The ginkgo leaf juice of 5g/L, 10~15g/L of propolis, 2~5g/L of yeast extract and mass fraction 2~10%.
Step 6) is by the Caulis Spatholobi suspension cell line to be centrifuged 3~5 at 2000~3000rpm of revolving speed, 0~4 DEG C of temperature
Minute collect cell, then at 90~100 DEG C of water bath with thermostatic control, respectively with pure water extract 3 times, first time extraction it is pure
The ratio of the dry weight of water consumption and cell is 10:1, and extraction time first time is 40min, and the pure water of second of extraction flows
The ratio of amount and the dry weight of cell is 5:1, and second of extraction time is 10min, and the pure water of third time extraction is in cell
The ratio of dry weight is 2:1, and third time extraction time is 5min, merges extracting solution three times, percent by volume is added thereto later
75% ethyl alcohol, to discard waste liquid after 3~5min of centrifugation at 6000~8000rpm of revolving speed, 0~4 DEG C of temperature, after drying being precipitated
Obtain beautiful millettia root polysaccharide；In step of the invention, the polysaccharide in 3 Caulis Spatholobi suspension cells is extracted with pure water respectively, it can
Maximumlly the polysaccharide in Caulis Spatholobi suspension cell is extracted, the extracted amount of polysaccharide improves 10%~15%.
Wherein, the sprouting fluid nutrient medium includes: 1/4~1/2MS, sucrose 7~15g/L and 6-BA0.1~0.3mg/
L, initial pH value 5.8, alternatively,
The acquisition methods for sprouting fluid nutrient medium are as follows: take the start liquid culture after once utilizing in step 4)
Proliferation fluid nutrient medium after utilizing in base and/or step 5), is collected by filtration filtrate with 4~10 mesh filter screen, then will be described
The pH of filtrate is adjusted to 5.8, then places it at 110~117 DEG C of temperature and carry out closing 10~20min of sterilizing, after obtaining sterilizing
Culture medium, then thereto be added 6-BA0.1~0.3mg/L, using as sprout fluid nutrient medium.Subculture solid training of the invention
It supports in base, start liquid culture medium and proliferation fluid nutrient medium and contains propolis, yeast extract and ginkgo leaf juice, in this way,
Cell can absorb and using more flavone compound, protein-based compound and other nutriments, excite its own
Synthesis to flavone compound, so that it be promoted to generate more flavone compounds.Meanwhile the present invention is according to material difference
The different upgrowth situations in stage, the content of each component of the culture medium of reasonable disposition various concentration, to promote more a stage
The fast-growth of different materials and absorption to nutritional ingredient.It is suspended using method of the invention to the Caulis Spatholobi that culture obtains thin
Born of the same parents' period is short, and can extract in every liter of Caulis Spatholobi suspension cell to 1.90~2.76g polysaccharide, realizes and obtains within a short period of time
The purpose of a large amount of effective component.The present invention reuses start liquid culture medium and/or increment fluid nutrient medium, saves
The about energy accelerates the efficiency of sprouting, and sprout consistent simultaneously as there is the residual of a small amount of more nutriment abundant
Property is also very high, and whole emergence rate and consistency reach 98%, improve whole production level, and the production of beautiful millettia root polysaccharide
Raising is also obtained in amount.
The acquisition methods for sprouting fluid nutrient medium are as follows: take the start liquid culture after once utilizing in step 4)
Proliferation fluid nutrient medium after utilizing in base and step 5), is collected by filtration filtrate with 6 mesh filter screen, then by the pH of the filtrate
5.8 are adjusted to, then places it at 115 DEG C of temperature and carries out closing sterilizing 15min, the culture medium after being sterilized, then thereto
Be added 6-BA0.2mg/L, using as sprout fluid nutrient medium.
In step 1), i.e., the seed of the Caulis Spatholobi plant for being Post flowering 110 days by mature seed.
In step 1), the disinfection includes: first using the solution cleaning containing mass-volume concentration 3g/mL calcium hypochlorite
It is described i.e. by mature seed several times, then the seed after cleaning is handled 25 seconds at 75 DEG C of temperature.
In step 1), the sprouting fluid nutrient medium includes: 1/43MS, sucrose 11g/L and 6-BA 0.2mg/L.
In step 4), the inoculum concentration of the suspension Primary culture is 3~5g/L squamous subculture product.
In step 5), the inoculum concentration ratio of the Multiplying culture are as follows: take and account for the proliferation fluid nutrient medium 10%~20%
Suspension Primary culture after culture.
In step 1), the condition of culture for sprouting culture are as follows: 800~1500lux of intensity of illumination, photoperiod 8~12 are small
When/day, cultivation temperature is 24~28 DEG C, and incubation time is 4~10 days.
In step 2), the condition of culture of the Fiber differentiation are as follows: 1500~3000lux of intensity of illumination, photoperiod 10~12
Hour/day, cultivation temperature are 24~28 DEG C, and incubation time is 20~40 days.
In step 3), the condition of culture of each squamous subculture are as follows: 2000~4000lux of intensity of illumination, photoperiod 10
~12 hours/day, cultivation temperature are 24~28 DEG C, and incubation time is 4~8 days.
In step 4), the condition of culture of the suspension Primary culture are as follows: dark culture, cultivation temperature be 24~28 DEG C, 100~
120rpm revolving speed shake culture, incubation time are 2~3 days.
In step 4), the inoculum concentration of the suspension Primary culture is 3~5g/L squamous subculture product.
In step 5), the condition of culture of the Multiplying culture are as follows: dark culture, cultivation temperature be 24~28 DEG C, 100~
200rpm revolving speed shake culture, incubation time are 7~15 days.
In step 5), the inoculum concentration ratio of the Multiplying culture are as follows: take and account for the proliferation fluid nutrient medium 10%~20%
Suspension Primary culture after culture.
In step 1), a filter paper is placed with above the sprouting fluid nutrient medium, it is described that mature seed is distributed in institute
It states on filter paper.
The acquisition methods of the ginkgo leaf juice are as follows: it takes Folium Ginkgo that water is added according to mass ratio 10:1, crushes later, and
With the filtering of 200 mesh filter screen, obtaining filtrate is ginkgo leaf juice.
Embodiment 1
A kind of production method for the beautiful millettia root polysaccharide that culture medium recycles, comprising:
Step 1) chooses Caulis Spatholobi i.e. by mature seed, then to mature seed carries out disinfection, then at temperature 0 first
It handles finally to be placed on sprouting for 24 hours at DEG C and carries out sprouting the tender leaf that culture grows 1mm length to it in fluid nutrient medium,
The seed of the Caulis Spatholobi plant for being Post flowering 90 days by mature seed, the disinfection include: to use to contain quality first
The solution cleaning of volumetric concentration 1g/mL calcium hypochlorite it is described i.e. by mature seed several times, then by the seed after cleaning in temperature
It is handled 20 seconds at 70 DEG C of degree, the sprouting fluid nutrient medium includes: 1/4MS, sucrose 7g/L and 6-BA 0.1mg/L, initial pH
Value is 5.8.
Step 2), which is chosen, cultivates the tender leaf grown as explant by sprouting, and the explant is placed in induction solid training
It supports and carries out Fiber differentiation on base, obtain callus, the induction solid medium includes: MS, 6-BA 0.2mg/L, NAA
0.02mg/L, sucrose 10g/L, propolis 5g/L, yeast extract 2g/L, mass fraction 2% ginkgo leaf juice and agar 8g/L.
It is at least secondary that the callus is carried out squamous subculture by step 3) in subculture solid medium, until it grows into
For absinthe-green granular loose cell mass, the subculture solid medium includes: MS, 6-BA 0.5mg/L, NAA
0.02mg/L, sucrose 5g/L, propolis 15g/L, yeast extract 5g/L, mass fraction 5% ginkgo leaf juice and agar 8g/L；
The cell mass is transferred in start liquid culture medium and carries out suspension Primary culture by step 4), obtains suspension cell
System, the start liquid culture medium includes: 1/4MS, 6-BA 0.2mg/L, NAA 0.02mg/L, sucrose 2g/L, propolis 10g/L,
The ginkgo leaf juice of yeast extract 2g/L and mass fraction 2%；
The suspension cell line is seeded in proliferation fluid nutrient medium and carries out Multiplying culture by step 5), and it is outstanding to obtain Caulis Spatholobi
Floating cell line, the proliferation fluid nutrient medium includes: 1/2MS, 6-BA 0.5mg/L, NAA 0.02mg/L, sucrose 2g/L, propolis
The ginkgo leaf juice of 10g/L, yeast extract 2g/L and mass fraction 2%.
Step 6) by the Caulis Spatholobi suspension cell line to be centrifuged 5 minutes collection cells at revolving speed 3000rpm, 4 DEG C of temperature,
Then it at 100 DEG C of water bath with thermostatic control, is extracted 3 times with pure water respectively, the pure water consumption of first time extraction and doing for cell
The ratio of weight is 10:1, and extraction time first time is 40min, the dry weight of the pure water flow and cell of second of extraction
Ratio is 5:1, and second of extraction time is 10min, and the pure water of third time extraction is 2:1 in the ratio of the dry weight of cell,
Third time extraction time is 5min, merges extracting solution three times, the ethyl alcohol of percent by volume 75% is added, thereto later with revolving speed
68000rpm, discard waste liquid after being centrifuged 5min at 4 DEG C of temperature, will precipitating it is dry after obtain beautiful millettia root polysaccharide.
In step 1), the condition of culture for sprouting culture are as follows: intensity of illumination 800lux, 8 hour/day of photoperiod, culture
Temperature is 24 DEG C, and incubation time is 4 days.
In step 1), a filter paper is placed with above the sprouting fluid nutrient medium, it is described that mature seed is distributed in institute
It states on filter paper.
In step 2), the condition of culture of the Fiber differentiation are as follows: intensity of illumination 1500lux, 10 hour/day of photoperiod, training
Supporting temperature is 24 DEG C, and incubation time is 20 days.
In step 3), the condition of culture of each squamous subculture are as follows: intensity of illumination 2000lux, 10 hours photoperiods/
Day, cultivation temperature is 24 DEG C, and incubation time is 4 days.
In step 4), the condition of culture of the suspension Primary culture are as follows: dark culture, cultivation temperature are 24 DEG C, and 100rpm turns
Fast shake culture, incubation time are 2 days.
In step 4), the inoculum concentration of the suspension Primary culture is 3g/L squamous subculture product.
In step 5), the condition of culture of the Multiplying culture are as follows: dark culture, cultivation temperature are 24 DEG C, the shake of 100rpm revolving speed
Culture is swung, incubation time is 7 days.
In step 5), the inoculum concentration ratio of the Multiplying culture are as follows: take the suspension for accounting for the proliferation fluid nutrient medium 10%
Culture after Primary culture.
Embodiment 2
A kind of production method for the beautiful millettia root polysaccharide that culture medium recycles, comprising:
Step 1) chooses Caulis Spatholobi i.e. by mature seed, then to mature seed carries out disinfection, then at temperature 2 first
48h is handled at DEG C, is finally placed on carrying out sprouting the tender leaf that culture grows 10mm length to it in sprouting fluid nutrient medium,
The seed of the Caulis Spatholobi plant for being Post flowering 130 days by mature seed, the disinfection include: to use to contain quality first
The solution cleaning of volumetric concentration 5g/mL calcium hypochlorite it is described i.e. by mature seed several times, then by the seed after cleaning in temperature
It is handled 30 seconds at 80 DEG C of degree, the acquisition methods for sprouting fluid nutrient medium are as follows: take after being utilized in step 5) in other embodiments
Proliferation fluid nutrient medium, filtrate is collected by filtration with 4 mesh filter screen, the pH of the filtrate is then adjusted to 5.8, then set
Closing sterilizing 10min, the culture medium after being sterilized are carried out at 110 DEG C of temperature, then 6-BA0.1mg/L is added thereto, with
As sprouting fluid nutrient medium.
Step 2), which is chosen, cultivates the tender leaf grown as explant by sprouting, and the explant is placed in induction solid training
It supports and carries out Fiber differentiation on base, obtain callus, the induction solid medium includes: MS, 6-BA0.5mg/L, NAA
0.1mg/L, sucrose 15g/L, propolis 10g/L, yeast extract 5g/L, mass fraction 10% ginkgo leaf juice and agar 8g/
L。
It is at least secondary that the callus is carried out squamous subculture by step 3) in subculture solid medium, until it grows into
For absinthe-green granular loose cell mass, the subculture solid medium includes: MS, 6-BA 1mg/L, NAA
0.1mg/L, sucrose 10g/L, propolis 20g/L, yeast extract 10g/L, mass fraction 15% ginkgo leaf juice and agar 8g/
L；
The cell mass is transferred in start liquid culture medium and carries out suspension Primary culture by step 4), obtains suspension cell
System, the start liquid culture medium includes: 1/2MS, 6-BA 2~0.6mg/L, NAA 0.1mg/L, sucrose 5g/L, propolis 15g/
L, the ginkgo leaf juice of yeast extract 5g/L and mass fraction 10%；
The suspension cell line is seeded in proliferation fluid nutrient medium and carries out Multiplying culture by step 5), and it is outstanding to obtain Caulis Spatholobi
Floating cell line, the proliferation fluid nutrient medium includes: 1/2MS, 6-BA 1mg/L, NAA 0.1mg/L, sucrose 5g/L, propolis 5g/
L, the ginkgo leaf juice of yeast extract 5g/L and mass fraction 10%.
Step 6) by the Caulis Spatholobi suspension cell line to be centrifuged 3 minutes collection cells at revolving speed 2000rpm, 0 DEG C of temperature,
Then it at 90 DEG C of water bath with thermostatic control, is extracted 3 times with pure water respectively, the pure water consumption of first time extraction and the dry weight of cell
Ratio be 10:1, extraction time first time be 40min, the ratio of the dry weight of the pure water flow and cell of second of extraction
Example is 5:1, and second extraction time is 10min, and the pure water of third time extraction is 2:1 in the ratio of the dry weight of cell, the
Extraction time is 5min three times, merges extracting solution three times, the ethyl alcohol of percent by volume 75% is added, thereto later with revolving speed
6000rpm, discard waste liquid after being centrifuged 3min at 0~4 DEG C of temperature, will precipitating it is dry after obtain beautiful millettia root polysaccharide.
In step 1), the condition of culture for sprouting culture are as follows: intensity of illumination 1500lux, 12 hour/day of photoperiod, training
Supporting temperature is 28 DEG C, and incubation time is 10 days.
In step 1), a filter paper is placed with above the sprouting fluid nutrient medium, it is described that mature seed is distributed in institute
It states on filter paper.
In step 2), the condition of culture of the Fiber differentiation are as follows: intensity of illumination 3000lux, 12 hour/day of photoperiod, training
Supporting temperature is 28 DEG C, and incubation time is 40 days.
In step 3), the condition of culture of each squamous subculture are as follows: intensity of illumination 4000lux, 12 hours photoperiods/
Day, cultivation temperature is 28 DEG C, and incubation time is 8 days.
In step 4), the condition of culture of the suspension Primary culture are as follows: dark culture, cultivation temperature are 28 DEG C, and 120rpm turns
Fast shake culture, incubation time are 3 days.
In step 4), the inoculum concentration of the suspension Primary culture is 5g/L squamous subculture product.
In step 5), the condition of culture of the Multiplying culture are as follows: dark culture, cultivation temperature are 28 DEG C, the shake of 200rpm revolving speed
Culture is swung, incubation time is 15 days.
In step 5), the inoculum concentration ratio of the Multiplying culture are as follows: take the suspension for accounting for the proliferation fluid nutrient medium 20%
Culture after Primary culture.
Embodiment 3
A kind of production method for the beautiful millettia root polysaccharide that culture medium recycles, comprising:
Step 1) chooses Caulis Spatholobi i.e. by mature seed, then to mature seed carries out disinfection, then at temperature 1 first
36h is handled at DEG C, is finally placed on carrying out sprouting the tender leaf that culture grows 5mm length to it in sprouting fluid nutrient medium,
The seed of the Caulis Spatholobi plant for being Post flowering 110 days by mature seed, the disinfection include: to use to contain quality first
The solution cleaning of volumetric concentration 3g/mL calcium hypochlorite it is described i.e. by mature seed several times, then by the seed after cleaning in temperature
It is handled 25 seconds at 75 DEG C of degree, the acquisition methods for sprouting fluid nutrient medium are as follows: take primary sharp in step 4) in other embodiments
Proliferation fluid nutrient medium after utilizing in the start liquid culture medium and step 5) after, is collected by filtration with 6 mesh filter screen
Then the pH of the filtrate is adjusted to 5.8 by filtrate, then place it at 115 DEG C of temperature and carry out closing sterilizing 15min, is obtained
Culture medium after sterilizing, then thereto be added 6-BA 0.2mg/L, using as sprout fluid nutrient medium.
Step 2), which is chosen, cultivates the tender leaf grown as explant by sprouting, and the explant is placed in induction solid training
It supports and carries out Fiber differentiation on base, obtain callus, the induction solid medium includes: MS, 6-BA 0.35mg/L, NAA
0.06mg/L, sucrose 12.5g/L, propolis 7.5g/L, yeast extract 3.5g/L, mass fraction 6% ginkgo leaf juice and fine jade
Rouge 8g/L.
It is at least secondary that the callus is carried out squamous subculture by step 3) in subculture solid medium, until it grows into
For absinthe-green granular loose cell mass, the subculture solid medium includes: MS, 6-BA 0.75mg/L, NAA
0.06mg/L, sucrose 7.5g/L, propolis 17.5g/L, yeast extract 7.5g/L, mass fraction 10% ginkgo leaf juice and fine jade
Rouge 8g/L；
The cell mass is transferred in start liquid culture medium and carries out suspension Primary culture by step 4), obtains suspension cell
System, the start liquid culture medium includes: 1/3MS, 6-BA 0.4mg/L, NAA 0.06mg/L, sucrose 3.5g/L, propolis
The ginkgo leaf juice of 12.5g/L, yeast extract 3.5g/L and mass fraction 6%；
The suspension cell line is seeded in proliferation fluid nutrient medium and carries out Multiplying culture by step 5), and it is outstanding to obtain Caulis Spatholobi
Floating cell line, the proliferation fluid nutrient medium includes: 1/2MS, 6-BA 0.75mg/L, NAA 0.06mg/L, sucrose 3.5g/L,
The ginkgo leaf juice of propolis 12.5g/L, yeast extract 3.5g/L and mass fraction 6%.
Step 6) by the Caulis Spatholobi suspension cell line to be centrifuged 4 minutes collection cells at revolving speed 2500rpm, 2 DEG C of temperature,
Then it at 95 DEG C of water bath with thermostatic control, is extracted 3 times with pure water respectively, the pure water consumption of first time extraction and the dry weight of cell
Ratio be 10:1, extraction time first time be 40min, the ratio of the dry weight of the pure water flow and cell of second of extraction
Example is 5:1, and second extraction time is 10min, and the pure water of third time extraction is 2:1 in the ratio of the dry weight of cell, the
Extraction time is 5min three times, merges extracting solution three times, the ethyl alcohol of percent by volume 75% is added, thereto later with revolving speed
7000rpm, discard waste liquid after being centrifuged 4min at 2 DEG C of temperature, will precipitating it is dry after obtain beautiful millettia root polysaccharide.
In step 1), the condition of culture for sprouting culture are as follows: intensity of illumination 1150lux, 10 hour/day of photoperiod, training
Supporting temperature is 16 DEG C, and incubation time is 7 days.
In step 1), a filter paper is placed with above the sprouting fluid nutrient medium, it is described that mature seed is distributed in institute
It states on filter paper.
In step 2), the condition of culture of the Fiber differentiation are as follows: intensity of illumination 2250lux, 11 hour/day of photoperiod, training
Supporting temperature is 26 DEG C, and incubation time is 30 days.
In step 3), the condition of culture of each squamous subculture are as follows: intensity of illumination 3000lux, 11 hours photoperiods/
Day, cultivation temperature is 26 DEG C, and incubation time is 6 days.
In step 4), the condition of culture of the suspension Primary culture are as follows: dark culture, cultivation temperature are 26 DEG C, and 110rpm turns
Fast shake culture, incubation time are 60 days.
In step 4), the inoculum concentration of the suspension Primary culture is 4g/L squamous subculture product.
In step 5), the condition of culture of the Multiplying culture are as follows: dark culture, cultivation temperature are 26 DEG C, the shake of 150rpm revolving speed
Culture is swung, incubation time is 11 days.
In step 5), the inoculum concentration ratio of the Multiplying culture are as follows: take the suspension for accounting for the proliferation fluid nutrient medium 15%
Culture after Primary culture.
Embodiment 4
A kind of production method for the beautiful millettia root polysaccharide that culture medium recycles, comprising:
Step 1) chooses Caulis Spatholobi i.e. by mature seed, then to mature seed carries out disinfection, then at temperature 1 first
30h is handled at DEG C, is finally placed on carrying out sprouting the tender leaf that culture grows 4mm length to it in sprouting fluid nutrient medium,
The seed of the Caulis Spatholobi plant for being Post flowering 100 days by mature seed, the disinfection include: to use to contain quality first
The solution cleaning of volumetric concentration 2g/mL calcium hypochlorite it is described i.e. by mature seed several times, then by the seed after cleaning in temperature
It is handled 22 seconds at 72 DEG C of degree, the acquisition methods for sprouting fluid nutrient medium are as follows: take primary sharp in step 4) in other embodiments
The start liquid culture medium after is collected by filtration filtrate with 10 mesh filter screen, is then adjusted to the pH of the filtrate
5.8, then place it at 117 DEG C of temperature and carry out closing sterilizing 20min, the culture medium after being sterilized, then 6- is added thereto
BA 0.3mg/L, using as sprout fluid nutrient medium.
Step 2), which is chosen, cultivates the tender leaf grown as explant by sprouting, and the explant is placed in induction solid training
It supports and carries out Fiber differentiation on base, obtain callus, the induction solid medium includes: MS, 6-BA 0.3mg/L, NAA
0.024mg/L, sucrose 12g/L, propolis 7g/L, yeast extract 3g/L, mass fraction 5% ginkgo leaf juice and agar 8g/
L。
It is at least secondary that the callus is carried out squamous subculture by step 3) in subculture solid medium, until it grows into
For absinthe-green granular loose cell mass, the subculture solid medium includes: MS, 6-BA 0.6mg/L, NAA
0.08mg/L, sucrose 7g/L, propolis 17g/L, yeast extract 8g/L, mass fraction 12% ginkgo leaf juice and agar 8g/
L；
The cell mass is transferred in start liquid culture medium and carries out suspension Primary culture by step 4), obtains suspension cell
System, the start liquid culture medium includes: 1/4MS, 6-BA 0.3mg/L, NAA 0.03mg/L, sucrose 3g/L, propolis 11g/L,
The ginkgo leaf juice of yeast extract 3g/L and mass fraction 4%；
The suspension cell line is seeded in proliferation fluid nutrient medium and carries out Multiplying culture by step 5), and it is outstanding to obtain Caulis Spatholobi
Floating cell line, the proliferation fluid nutrient medium includes: 1/2MS, 6-BA 0.7mg/L, NAA 0.04mg/L, sucrose 3g/L, propolis
The ginkgo leaf juice of 12g/L, yeast extract 3g/L and mass fraction 4%.
Step 6) by the Caulis Spatholobi suspension cell line to be centrifuged 3 minutes collection cells at revolving speed 220rpm, 1 DEG C of temperature,
Then it at 92 DEG C of water bath with thermostatic control, is extracted 3 times with pure water respectively, the pure water consumption of first time extraction and the dry weight of cell
Ratio be 10:1, extraction time first time be 40min, the ratio of the dry weight of the pure water flow and cell of second of extraction
Example is 5:1, and second extraction time is 10min, and the pure water of third time extraction is 2:1 in the ratio of the dry weight of cell, the
Extraction time is 5min three times, merges extracting solution three times, the ethyl alcohol of percent by volume 75% is added, thereto later with revolving speed
Waste liquid is discarded after being centrifuged 4min under 1 DEG C of 6400rpm, temperature min, beautiful millettia root polysaccharide will be obtained after precipitating drying.
In step 1), the condition of culture for sprouting culture are as follows: intensity of illumination 1000lux, 9 hour/day of photoperiod, culture
Temperature is 25 DEG C, and incubation time is 6 days.
In step 1), a filter paper is placed with above the sprouting fluid nutrient medium, it is described that mature seed is distributed in institute
It states on filter paper.
In step 2), the condition of culture of the Fiber differentiation are as follows: intensity of illumination 1800lux, 10 hour/day of photoperiod, training
Supporting temperature is 25 DEG C, and incubation time is 28 days.
In step 3), the condition of culture of each squamous subculture are as follows: intensity of illumination 2500lux, 10 hours photoperiods/
Day, cultivation temperature is 25 DEG C, and incubation time is 5 days.
In step 4), the condition of culture of the suspension Primary culture are as follows: dark culture, cultivation temperature be 25 DEG C, 100~
120rpm revolving speed shake culture, incubation time are 2 days.
In step 4), the inoculum concentration of the suspension Primary culture is 3g/L squamous subculture product.
In step 5), the condition of culture of the Multiplying culture are as follows: dark culture, cultivation temperature are 25 DEG C, the shake of 120rpm revolving speed
Culture is swung, incubation time is 9 days.
In step 5), the inoculum concentration ratio of the Multiplying culture are as follows: take the suspension for accounting for the proliferation fluid nutrient medium 12%
Culture after Primary culture.
Embodiment 5
A kind of production method for the beautiful millettia root polysaccharide that culture medium recycles, comprising:
Step 1) chooses Caulis Spatholobi i.e. by mature seed, then to mature seed carries out disinfection, then at temperature 2 first
40h is handled at DEG C, is finally placed on carrying out sprouting the tender leaf that culture grows 8mm length to it in sprouting fluid nutrient medium,
The seed of the Caulis Spatholobi plant for being Post flowering 120 days by mature seed, the disinfection include: to use to contain quality first
The solution cleaning of volumetric concentration 4g/mL calcium hypochlorite it is described i.e. by mature seed several times, then by the seed after cleaning in temperature
It is handled 27 seconds at 78 DEG C of degree, the acquisition methods for sprouting fluid nutrient medium are as follows: take primary sharp in step 4) in other embodiments
The start liquid culture medium after is collected by filtration filtrate with 8 mesh filter screen, the pH of the filtrate is then adjusted to 5.8,
It is placed it at 116 DEG C of temperature again and carries out closing sterilizing 18min, the culture medium after being sterilized, then 6-BA is added thereto
0.2mg/L, using as sprout fluid nutrient medium.
Step 2), which is chosen, cultivates the tender leaf grown as explant by sprouting, and the explant is placed in induction solid training
It supports and carries out Fiber differentiation on base, obtain callus, the induction solid medium includes: MS, 6-BA 0.4mg/L, NAA
0.08mg/L, sucrose 12g/L, propolis 9g/L, yeast extract 4g/L, mass fraction 9% ginkgo leaf juice and agar 8g/L.
It is at least secondary that the callus is carried out squamous subculture by step 3) in subculture solid medium, until it grows into
For absinthe-green granular loose cell mass, the subculture solid medium includes: MS, 6-BA 0.9mg/L, NAA
0.09mg/L, sucrose 9g/L, propolis 19g/L, yeast extract 9g/L, mass fraction 14% ginkgo leaf juice and agar 8g/
L；
The cell mass is transferred in start liquid culture medium and carries out suspension Primary culture by step 4), obtains suspension cell
System, the start liquid culture medium includes: 1/4MS, 6-BA 0.5mg/L, NAA 0.09mg/L, sucrose 4g/L, propolis 14g/L,
The ginkgo leaf juice of yeast extract 4g/L and mass fraction 9%；
The suspension cell line is seeded in proliferation fluid nutrient medium and carries out Multiplying culture by step 5), and it is outstanding to obtain Caulis Spatholobi
Floating cell line, the proliferation fluid nutrient medium includes: 1/2MS, 6-BA 0.9mg/L, NAA 0.09mg/L, sucrose 4g/L, propolis
The ginkgo leaf juice of 14g/L, yeast extract 4g/L and mass fraction 9%.
Step 6) by the Caulis Spatholobi suspension cell line to be centrifuged 4 minutes collection cells at revolving speed 2900rpm, 3 DEG C of temperature,
Then it at 99 DEG C of water bath with thermostatic control, is extracted 3 times with pure water respectively, the pure water consumption of first time extraction and the dry weight of cell
Ratio be 10:1, extraction time first time be 40min, the ratio of the dry weight of the pure water flow and cell of second of extraction
Example is 5:1, and second extraction time is 10min, and the pure water of third time extraction is 2:1 in the ratio of the dry weight of cell, the
Extraction time is 5min three times, merges extracting solution three times, the ethyl alcohol of percent by volume 75% is added, thereto later with revolving speed
7800rpm, discard waste liquid after being centrifuged 4min at 3 DEG C of temperature, will precipitating it is dry after obtain beautiful millettia root polysaccharide.
In step 1), the condition of culture for sprouting culture are as follows: intensity of illumination 1400lux, 10 hour/day of photoperiod, training
Supporting temperature is 27 DEG C, and incubation time is 9 days.
In step 1), a filter paper is placed with above the sprouting fluid nutrient medium, it is described that mature seed is distributed in institute
It states on filter paper.
In step 2), the condition of culture of the Fiber differentiation are as follows: intensity of illumination 2800lux, 10 hour/day of photoperiod, training
Supporting temperature is 27 DEG C, and incubation time is 35 days.
In step 3), the condition of culture of each squamous subculture are as follows: intensity of illumination 3500lux, 10 hours photoperiods/
Day, cultivation temperature is 27 DEG C, and incubation time is 7 days.
In step 4), the condition of culture of the suspension Primary culture are as follows: dark culture, cultivation temperature are 27 DEG C, and 110rpm turns
Fast shake culture, incubation time are 2 days.
In step 4), the inoculum concentration of the suspension Primary culture is 4g/L squamous subculture product.
In step 5), the condition of culture of the Multiplying culture are as follows: dark culture, cultivation temperature are 27 DEG C, the shake of 180rpm revolving speed
Culture is swung, incubation time is 14 days.
In step 5), the inoculum concentration ratio of the Multiplying culture are as follows: take the suspension for accounting for the proliferation fluid nutrient medium 18%
Culture after Primary culture.
Compliance test result:
Using embodiment 1 to the method for embodiment 5, polyoses content is measured using sulfuric acid-green onion ketone method, as a result such as one institute of table
Show.
Table one
| Embodiment | Polyoses content |
| Embodiment 1 | 1.94 |
| Embodiment 2 | 2.36 |
| Embodiment 3 | 2.76 |
| Embodiment 4 | 2.16 |
| Embodiment 5 | 1.90 |
| 100g beautiful millettia root | 0.88 |
As can be seen from Table I, method of the invention successfully passes the method culture of tissue cultures Caulis Spatholobi and suspends carefully
Born of the same parents, beautiful millettia root polyoses content greatly improve, and also improve 10%~15% to the extracted amount of polysaccharide, while choosing Caulis Spatholobi i.e.
Mature seed is used to sprout, germination rate has reached 100%, and whole emergence rate and consistency reach 98%.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details.
Claims (7)
1. a kind of production method for the beautiful millettia root polysaccharide that culture medium recycles characterized by comprising
Step 1) chooses Caulis Spatholobi i.e. by mature seed, then to mature seed carries out disinfection, then at temperature 0~2 first
24~48h is handled at DEG C, is finally placed on sprout cultivating to it in sprouting fluid nutrient medium growing 1~10mm length
Tender leaf, it is described i.e. by mature seed be Post flowering 90~130 days Caulis Spatholobi plant seed, the disinfection includes: first
Using the solution cleaning containing mass-volume concentration 1~5g/mL calcium hypochlorite it is described i.e. by mature seed several times, then will be clear
Seed after washing is handled 20~30 seconds at 70~80 DEG C of temperature；
Step 2) is chosen by sprouting the tender leaf cultivated and grown as explant, and the explant is placed in induction solid medium
Upper carry out Fiber differentiation obtains callus, and the induction solid medium includes: MS, 6-BA 0.2~0.5mg/L, NAA
0.02~0.1mg/L, 10~15g/L of sucrose, 5~10g/L of propolis, 2~5g/L of yeast extract, mass fraction 2~10%
Ginkgo leaf juice and agar 8g/L；
It is at least secondary that the callus is carried out squamous subculture by step 3) in subculture solid medium, until its be grown to serve as it is light
The granular loose cell mass of green, the subculture solid medium includes: MS, 6-BA 0.5~1mg/L, NAA 0.02
~0.1mg/L, the ginkgo of 5~10g/L of sucrose, 15~20g/L of propolis, 5~10g/L of yeast extract, mass fraction 5~15%
Leaf sap and agar 8g/L；
The cell mass is transferred in start liquid culture medium and carries out suspension Primary culture by step 4), obtains suspension cell line,
The start liquid culture medium includes: 1/4~1/2MS, 0.02~0.1mg/L of 6-BA 0.2~0.6mg/L, NAA, sucrose 2~
The ginkgo leaf juice of 5g/L, 10~15g/L of propolis, 2~5g/L of yeast extract and mass fraction 2~10%；
The suspension cell line is seeded in proliferation fluid nutrient medium and carries out Multiplying culture by step 5), obtains Caulis Spatholobi and suspends carefully
Born of the same parents system, the proliferation fluid nutrient medium includes: 0.02~0.1mg/L of 1/2MS, 6-BA 0.5~1mg/L, NAA, sucrose 2~
The ginkgo leaf juice of 5g/L, 10~15g/L of propolis, 2~5g/L of yeast extract and mass fraction 2~10%；
Step 6) is by the Caulis Spatholobi suspension cell line to be centrifuged 3~5 minutes at 2000~3000rpm of revolving speed, 0~4 DEG C of temperature
Cell is collected, then at 90~100 DEG C of water bath with thermostatic control, is extracted 3 times with pure water respectively, the pure water of first time extraction is used
The ratio of amount and the dry weight of cell is 10:1, and extraction time first time is 40min, the pure water flow of second of extraction with
The ratio of the dry weight of cell is 5:1, and second of extraction time is 10min, and the pure water of third time extraction is in the dry weight of cell
Ratio be 2:1, third time extraction time is 5min, merges extracting solution three times, percent by volume 75% is added thereto later
Ethyl alcohol, to discard waste liquid after being centrifuged 3~5min at 6000~8000rpm of revolving speed, 0~4 DEG C of temperature, will precipitating it is dry after obtain
Beautiful millettia root polysaccharide；
Wherein, the sprouting fluid nutrient medium includes: 1/4~1/2MS, sucrose 0.1~0.3mg/L of 7~15g/L and 6-BA,
Initial pH value is 5.8, alternatively,
The acquisition methods for sprouting fluid nutrient medium are as follows: take the start liquid culture medium after once utilizing in step 4)
And/or the proliferation fluid nutrient medium after being utilized in step 5), filtrate is collected by filtration with 4~10 mesh filter screen, then by the filter
The pH of liquid is adjusted to 5.8, then places it at 110~117 DEG C of temperature and carry out closing 10~20min of sterilizing, after being sterilized
Culture medium, then thereto be added 0.1~0.3mg/L of 6-BA, using as sprout fluid nutrient medium.
2. the production method for the beautiful millettia root polysaccharide that culture medium according to claim 1 recycles, which is characterized in that described to sprout
Send out the acquisition methods of fluid nutrient medium are as follows: take benefit in the start liquid culture medium and step 5) after once utilizing in step 4)
Filtrate is collected by filtration with 6 mesh filter screen in proliferation fluid nutrient medium after, the pH of the filtrate is then adjusted to 5.8, then will
It, which is placed at 115 DEG C of temperature, carries out closing sterilizing 15min, the culture medium after being sterilized, then 6-BA 0.2mg/ is added thereto
L, using as sprout fluid nutrient medium.
3. the production method for the beautiful millettia root polysaccharide that culture medium according to claim 1 recycles, which is characterized in that described to sprout
A filter paper is placed with above hair fluid nutrient medium, it is described that mature seed is distributed on the filter paper.
4. the production method for the beautiful millettia root polysaccharide that culture medium according to claim 1 recycles, which is characterized in that step 1)
In, the disinfection includes: described i.e. by mature kind using the solution cleaning containing mass-volume concentration 3g/mL calcium hypochlorite first
Son several times, is then handled the seed after cleaning 25 seconds at 75 DEG C of temperature.
5. the method for production beautiful millettia root polysaccharide according to claim 1, which is characterized in that in step 4), the suspension is opened
The inoculum concentration of dynamic culture is 3~5g/L squamous subculture product.
6. the method for production beautiful millettia root polysaccharide according to claim 1, which is characterized in that in step 5), the proliferation training
Feeding inoculum concentration ratio are as follows: the culture after taking the suspension Primary culture for accounting for the proliferation fluid nutrient medium 10%~20%.
7. the method for production beautiful millettia root polysaccharide according to claim 1, which is characterized in that the start liquid culture medium packet
Contain: 1/3MS, 6-BA 0.4mg/L, NAA 0.06mg/L, sucrose 2.5g/L, propolis 12.5g/L, yeast extract 3.5g/L and
The ginkgo leaf juice of mass fraction 6%.
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