CN104711215A - Apple stem cell culture method and apple stem cells cultured by method - Google Patents

Apple stem cell culture method and apple stem cells cultured by method Download PDF

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Publication number
CN104711215A
CN104711215A CN201510166803.9A CN201510166803A CN104711215A CN 104711215 A CN104711215 A CN 104711215A CN 201510166803 A CN201510166803 A CN 201510166803A CN 104711215 A CN104711215 A CN 104711215A
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cell
biffins
apple
culture
culture medium
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陈海佳
王一飞
葛啸虎
戴国胜
程建强
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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Guangzhou Saliai StemCell Science and Technology Co Ltd
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Abstract

The invention relates to the technical field of plant stem cell culture, particularly an apple stem cell culture method and apple stem cells cultured by the method. The method comprises the following steps: by using an apple new branch as a raw material, inducing to form callus, and carrying out enrichment culture and amplification culture to obtain the apple stem cells. The method provided by the invention has the advantages of shorter period and high yield. The method can obtain abundant apple stem cells within about 48 days. The induction culture medium used in the apple stem cell culture process and the variety of the phytohormones contained in the enrichment culture medium are proper and suitable in proportion, and can ensure fast dedifferentiation and abundant reproduction of apple branch cells. The apple stem cells can be freeze-dried or extracted and used for preparing food, drugs, health products or cosmetics. The apple stem cell extraction method provided by the invention is simple and easy to implement and has higher extraction efficiency. The prepared extract contains abundant flavones and polyphenols.

Description

A kind of cultural method of biffins cell and the biffins cell of cultivation
Technical field
The present invention relates to plant stem cell culture technique field, particularly relate to a kind of cultural method of biffins cell and the biffins cell of cultivation.
Background technology
Plant stem cell (plant stem cell) is undifferentiated cell, has very strong self and lasting splitting ability.Plant stem cell culture technique, on the basis of traditional tissue culture technique, take plant stem cell as target, induction, separation and cultivation explant, set up corresponding stem cell culture system plant stem cell culture technique, not only enrich vegetable culture technique, and commercially producing for natural product, and even the development of Plant Biotechnology, provides new research direction and opportunity.
According to the difference of targeted stem cells, comparatively successfully plant stem cell cultural method is mainly divided into three kinds at present: the vascular cambium (such as: ginseng stem cell) of xylophyta vascular bundle form layers mitogenetic (such as: Ramulus et folium taxi cuspidatae stem cell), herbaceous plant storage root, quiescent center (such as: LIPIDS OF DRY RICE EMBRYO cell, Apple stem cell).
Existing Apple stem cell by the happy biochemical corp of Switzerland's rice hundred with the Switzerland apple Uttwiler of maturation develop based on fruit, and be made into liposome for cosmetics, the ability delaying senility and go to wrinkle can be had.Therefore, the cosmetics being added with Apple stem cell are in very great demand, often face the problem that supply falls short of demand.This is mainly because the output of Apple stem cell is lower.Trace it to its cause, on the one hand due to Uttwiler apple variety kind matter is too rare, and the rareness of raw material causes Apple yield of dried cell to be improved.On the other hand, Apple cell belongs to quiescent center, be in differentiation end, to be dedifferented and to obtain the difficulty of stem cell higher, not only need stronger inductive condition to make Apple cell de-differentiation, in culturing process, often need cycle of more growing and more dedifferente incubation times, this causes Apple yield of dried cell to can not get the basic reason improved.
Further, skin can be made more easily to absorb, to be made cosmetics although Apple stem cell to be made liposome.But the interpolation of liposome can cause the extraction of the reduction of active material concentration undoubtedly, and the preparation method of liposome is comparatively complicated, again extends the preparation cycle of stem cell.Further, liposome is mostly very responsive to gi tract environment, uses it for orally easily to cause the degraded of active substance or the reduction of bioavailability.
Therefore, provide a kind of can prepare biffins cell fast method so that provide a kind of active component content higher and to be applicable to oral biffins cell very necessary.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of method preparing biffins cell, method provided by the invention can be quick and a large amount of prepare biffins cell, and then provide a kind of active component content higher and be applicable to oral biffins cell extract.
The method of cultivation biffins cell provided by the invention, comprises the following steps:
Step 1: after the newborn branch sterilization of apple, removal xylem and marrow, be inoculated in inducing culture, inducing culture obtains cambial cell;
Step 2: cambial cell, through succeeding transfer culture, accesses proliferated culture medium, and it is unicellular that acquisition cultivated by shaking table;
Step 3: by the unicellular proliferated culture medium that is inoculated in through enlarged culturing, obtains biffins cell;
Inducing culture is the MS solid medium containing NAA, BA and caseinhydrolysate;
Proliferated culture medium for containing 2,4-D, the MS liquid nutrient medium of BA, caseinhydrolysate and gac.
The present invention selects apple innovation bar to carry out induction as raw material to prepare biffins cell.Apple is Rosaceae Malus, and its branch is developed by leaf bud, and profile is respectively epidermis, cortex, phloem, form layers, xylem and marrow within outer.Wherein, form layers, between phloem and xylem, is a kind of meristematic tissue, originates from and do not break up, but keeps the hyperplasia of embryo and the cell of differentiation capability.Also form layers can be formed in callus.For fruit, the cell of branch is more original, and the cell of fruit is in the end of differentiation.The newborn branch finger-type of apple becomes the branch of time within 1 year, and the young sprout of most apple variety has two secondary growths in annual cycle, is respectively the spring tip or autumn growth.The apple variety that the present invention adopts is Fuji apple (Malus pumila Mil), and it is planted extensively at home, and raw material sources are extensive.
In an embodiment of the present invention, sterilization comprises the following steps:
Step 1: after newborn for apple branch is rinsed with water, take concentration as the L-AA solution soaking of 10 ~ 1000mg/L;
Step 2: with aseptic water washing after the aqueous ethanolic solution being 60% ~ 95% with volume fraction soaks;
Step 3: with aseptic water washing after the aqueous hydrogen peroxide solution being 10% ~ 100% with volume fraction soaks.
As preferably, the length of the newborn branch of apple is 10cm.
As preferably, the concentration of L-AA is 120mg/L.
As preferably, the time of soaking described in step 1 is 0.5min ~ 10min.
Preferably, the time of soaking described in step 1 is 1min.
As preferably, in aqueous ethanolic solution, the volume fraction of ethanol is 75%.
As preferably, the time of soaking described in step 2 is 0.1min ~ 10min.
Preferably, the time of soaking described in step 2 is 1min.
As preferably, in step 2, the volume of sterilized water is 2L.
As preferably, in aqueous hydrogen peroxide solution, the volume fraction of hydrogen peroxide is 30%.
As preferably, the time of soaking described in step 3 is 1min ~ 50min.
Preferably, the time of soaking described in step 3 is 20min.
As preferably, the time of rinsing described in step 3 is 1min ~ 50min.
Preferably, the time of rinsing described in step 3 is 10min.
Carry out sterilization with method for disinfection provided by the invention to the newborn branch of apple can kill fully except fungi entrained on the newborn branch of apple, bacterium or virus.To ensure that the newborn branch of apple is not disturbed in culturing process, thus ensure that the cell on the newborn branch of apple dedifferentes more fast.And experiment shows, method provided by the invention effectively by newborn for apple branch cell de-differentiation, through induction and enlarged culturing, can obtain a large amount of biffins cells for about 48 days.
The inducing culture that the method that the invention provides adopts is the MS solid medium containing NAA, BA and caseinhydrolysate.MS substratum is a kind of conventional plant culture, can be used for evoked callus, also can be used for the cultivation of embryo, stem section, stem apex and flower pesticide.NAA is the abbreviation of naphthylacetic acid, is a kind of common plant hormone, the division that can promote cell and expansion.BA is the abbreviation of benzyl aminoadenine, and be a kind of phytokinin, 6-BA is more common.Caseinhydrolysate (CH) propagation to cell has obvious promoter action.The present invention proves by experiment, and the kind of plant hormone, consumption and the dedifferente induction of ratio to biffins cell play vital effect.When adopting NAA, BA and caseinhydrolysate to induce apple branch, can obtain the cambial cell dedifferented fast, the cambial cell these dedifferented can obtain a large amount of biffins cells after amplification.
In embodiments of the present invention, the mass ratio of NAA, BA and caseinhydrolysate is (0.1 ~ 10): (0.1 ~ 10): (0.1 ~ 10).
In embodiments of the present invention, the mass ratio of NAA, BA and caseinhydrolysate is 0.5:2:1.
As preferably, in inducing culture, the concentration of NAA is 0.1mg/L ~ 10mg/L.
Preferably, in inducing culture, the concentration of NAA is 0.5mg/L.
As preferably, in inducing culture, the concentration of BA is 0.1mg/L ~ 10mg/L.
Preferably, in inducing culture, the concentration of BA is 2.0mg/L.
As preferably, in inducing culture, the concentration of caseinhydrolysate is 0.1mg/L ~ 10mg/L.
Preferably, in inducing culture, the concentration of caseinhydrolysate is 1.0mg/L.
As preferably, inducing culture is solid medium, and wherein the massfraction of agar is 1%.
As preferably, the pH value of inducing culture is 6.0.
In an embodiment of the present invention, the inoculation described in step 1 is, the newborn branch of apple that every g removes xylem and marrow is inoculated in 10cm 2inducing culture.
In the embodiment of the present invention, inducing culture is light culture; Temperature is 10 DEG C ~ 30 DEG C; Time is 1 day ~ 20 days.
In certain embodiments, inducing culture is light culture; Temperature is 25 DEG C; Time is 10 days.
After inducing culture, Cambium tissue is the tabular tissue of homogeneous growth, and its hetero-organization is then irregular gathering grower.
In embodiments of the invention, succeeding transfer culture is light culture; Temperature is 10 DEG C ~ 30 DEG C; Time is 1 day ~ 20 days.
In certain embodiments, succeeding transfer culture is light culture; Temperature is 25 DEG C; Time is 10 days.
In an embodiment of the present invention, succeeding transfer culture adopts and runs into substratum, and every 1g cambial cell is inoculated in 10cm 2inducing culture.
Through the succeeding transfer culture of 10 days, cambial cell formed callus, every 10cm 2inducing culture on about have callus 20.8g.
Proliferated culture medium provided by the invention be containing 2,4-D, the MS liquid nutrient medium of BA, caseinhydrolysate and gac.2,4-D and 2,4 dichlorophenoxyacetic acid are a kind of plant hormones, and for different vegetable cell, suitable concentration is different, can promote plant cell growth under suitable concentration, and concentration is improper can suppress plant cell growth.Harmful secretory product of the adsorbable plant of gac, avoids the propagation of these secretory product T suppression cell, can reduce substratum variable color, promotes cell proliferation.The present invention proves by experiment, adopts the MS liquid nutrient medium of 2,4-D, BA, caseinhydrolysate and gac can make apple callus rapid amplifying.
In an embodiment of the present invention, in proliferated culture medium, the mass ratio of 2,4-D, BA, caseinhydrolysate and gac is (0.1 ~ 10): (0.1 ~ 10): (0.1 ~ 10): (0.1 ~ 10).
In some embodiments, in proliferated culture medium, the mass ratio of 2,4-D, BA, caseinhydrolysate and gac is 2:1:1:1.
As preferably, in proliferated culture medium, the concentration of 2,4-D is 0.1mg/L ~ 10mg/L.
Preferably, in proliferated culture medium, the concentration of 2,4-D is 2.0mg/L.
As preferably, in proliferated culture medium, the concentration of BA is 0.1mg/L ~ 10mg/L.
Preferably, in proliferated culture medium, the concentration of BA is 1.0mg/L.
As preferably, in proliferated culture medium, the concentration of caseinhydrolysate is 0.1mg/L ~ 10mg/L.
Preferably, in proliferated culture medium, the concentration of caseinhydrolysate is 1.0mg/L.
As preferably, in proliferated culture medium, the concentration of gac is 0.1mg/L ~ 10mg/L.
Preferably, in proliferated culture medium, the concentration of gac is 1.0mg/L.
As preferably, the pH value of proliferated culture medium is 6.0.
In an embodiment of the present invention, every g callus access 20mL proliferated culture medium.
As preferably, when shaking table is cultivated, the diameter also added in proliferated culture medium through sterilizing is the granulated glass sphere of 0.5cm.
Preferably, the addition of granulated glass sphere is 100g/L proliferated culture medium.
In embodiment, cultivate and adopt triangular flask, in 1000mL triangular flask, add 200mL proliferated culture medium.
In an embodiment of the present invention, the condition that shaking table is cultivated is that the incandescent light of 20W per hour irradiates 10min.
In an embodiment of the present invention, the temperature that shaking table is cultivated is 10 DEG C ~ 30 DEG C; Rotating speed is 10 revs/min ~ 200 revs/min; Time is 8 days ~ 17 days.
In certain embodiments, the temperature that shaking table is cultivated is 25 DEG C; Rotating speed is 120 revs/min; Time is 14 days.
As preferably, after shaking table cultivates 7 days, add fresh proliferated culture medium.
As preferably, the volume adding fresh proliferated culture medium is 2 times of former proliferated culture medium.
After adding fresh proliferated culture medium, wherein the density of cell is 1 × 10 5/ ml.
Adopt method provided by the invention to carry out shaking table cultivation, callus becomes single cell suspension, and cell proliferation rate is very fast, and with 100 object stainless steel sift net filtrations, removing cell mass, granulated glass sphere and residue, obtain cell density about 1 × 10 6/ ml.
In certain embodiments, in step 3, the density of inoculation is 0.1 × 10 3/ ml ~ 1 × 10 4/ ml.
As preferably, in step 3, the density of inoculation is 1 × 10 4/ ml.
In an embodiment of the present invention, enlarged culturing is specially: in bio-reactor, cultivation adds proliferated culture medium to cell concn in 7 days is afterwards 1 × 10 5/ ml, then cultivate 1 day ~ 20 days.
In certain embodiments, enlarged culturing is specially: in the bio-reactor of 20L, add 5L proliferated culture medium, cultivate 7 days afterwards add proliferated culture medium to cell concn be 1 × 10 6/ ml, then cultivate 7 days.
Employing the invention provides method and cultivates biffins cell, and incubation time is short, and yield is high.Within about 48 days, a large amount of stem cells can be gathered in the crops.The stem cell of results be viscose shape, can after freeze-drying for or after extraction for the preparation of food, medicine, healthcare products or makeup.
The invention provides biffins cell prepared by method.
Present invention also offers the inducing culture of biffins cell, is the MS solid medium containing NAA, BA and caseinhydrolysate.
In embodiments of the present invention, the mass ratio of NAA, BA and caseinhydrolysate is (0.1 ~ 10): (0.1 ~ 10): (0.1 ~ 10).
In embodiments of the present invention, the mass ratio of NAA, BA and caseinhydrolysate is 0.5:2:1.
As preferably, in inducing culture, the concentration of NAA is 0.1mg/L ~ 10mg/L.
Preferably, in inducing culture, the concentration of NAA is 0.5mg/L.
As preferably, in inducing culture, the concentration of BA is 0.1mg/L ~ 10mg/L.
Preferably, in inducing culture, the concentration of BA is 2.0mg/L.
As preferably, in inducing culture, the concentration of caseinhydrolysate is 0.1mg/L ~ 10mg/L.
Preferably, in inducing culture, the concentration of caseinhydrolysate is 1.0mg/L.
As preferably, inducing culture is solid medium, and wherein the massfraction of agar is 1%.
As preferably, the pH value of inducing culture is 6.0.
Present invention also offers the proliferated culture medium of biffins cell, is the MS liquid nutrient medium containing 2,4-D, BA, caseinhydrolysate and gac.
In an embodiment of the present invention, in proliferated culture medium, the mass ratio of 2,4-D, BA, caseinhydrolysate and gac is (0.1 ~ 10): (0.1 ~ 10): (0.1 ~ 10): (0.1 ~ 10).
In some embodiments, in proliferated culture medium, the mass ratio of 2,4-D, BA, caseinhydrolysate and gac is 2:1:1:1.
As preferably, in proliferated culture medium, the concentration of 2,4-D is 0.1mg/L ~ 10mg/L.
Preferably, in proliferated culture medium, the concentration of 2,4-D is 2.0mg/L.
As preferably, in proliferated culture medium, the concentration of BA is 0.1mg/L ~ 10mg/L.
Preferably, in proliferated culture medium, the concentration of BA is 1.0mg/L.
As preferably, in proliferated culture medium, the concentration of caseinhydrolysate is 0.1mg/L ~ 10mg/L.
Preferably, in proliferated culture medium, the concentration of caseinhydrolysate is 1.0mg/L.
As preferably, in proliferated culture medium, the concentration of gac is 0.1mg/L ~ 10mg/L.
Preferably, in proliferated culture medium, the concentration of gac is 1.0mg/L.
As preferably, the pH value of proliferated culture medium is 6.0.
Present invention also offers a kind of preparation method of biffins cell lyophilized powder, comprising: freeze-drying after biffins cell multigelation prepared by the method for the invention provides, obtain biffins cell lyophilized powder.
Biffins cell lyophilized powder prepared by the method for the invention provides.
Biffins cell lyophilized powder provided by the invention is preparing the application in food, medicine, healthcare products or makeup.
Adopt method provided by the invention, in obtained biffins cell lyophilized powder, flavones and polyphenol content are very abundant, and experimental result shows, containing flavonoids from apple 18.9mg, polyphenol 22.9mg in every g biffins cell lyophilized powder.
Present invention also offers a kind of biffins cell extracting method, comprising: through freeze-drying, pulverizing after the biffins cell multigelation method of the invention provides prepared, is that extracting solution extraction, by force ultrasonic, concentrated, dry, pulverizing obtain biffins cell extract with aqueous ethanolic solution.
In an embodiment of the present invention, multigelation is specially: by biffins cell in the quick-frozen 1 hour ~ 10 hours in liquid nitrogen after 1 hour ~ 10 hours of-30 DEG C ~-80 DEG C pre-freezes; Then thaw 1 minute ~ 100 minutes; After 1 time repeatedly ~ 10 times, room temperature places 1min ~ 50min.
In certain embodiments, multigelation is specially: by biffins cell in the quick-frozen 2 hours in liquid nitrogen after 3 hours of-30 DEG C of pre-freezes; Then thaw 30 minutes; Room temperature places 10min after 2 times repeatedly.
Room temperature of the present invention is 15 DEG C ~ 30 DEG C.
In an embodiment of the present invention, pulverize as high speed pulverization, be crushed to 1 ~ 100 order.
In certain embodiments, 60 orders are crushed to.
In an embodiment of the present invention, in extracting solution, the volume fraction of ethanol is 50% ~ 95%.
In certain embodiments, in extracting solution, the volume fraction of ethanol is 75%.
In an embodiment of the present invention, the mass volume ratio of biffins cell and extracting solution is 1:5.
In an embodiment of the present invention, the temperature of extraction is 10 DEG C ~ 30 DEG C, stirs and extracts 10h ~ 100h.
In an embodiment of the present invention, concentrated employing rotary evaporation.
In an embodiment of the present invention, dry employing frozen drying.
In an embodiment of the present invention, 10 order ~ 200 orders are crushed to.
In certain embodiments, 100 orders are crushed to.
Biffins cell extract prepared by the method for the invention provides.
Biffins cell extract provided by the invention is preparing the application in food, medicine, healthcare products or makeup.
Adopt method provided by the invention, in obtained biffins cell extract, flavones and polyphenol content are very abundant, experimental result shows, every g biffins cell extract is dissolved in 1L distilled water, and the concentration of flavonoids from apple is 18.2 μ g/ml, apple polyphenol 22.1 μ g/ml.
The invention provides a kind of cultural method of biffins cell, the method with the newborn branch of apple for raw material, multiplication culture after induced synthesis callus enlarged culturing obtains biffins cell.Method provided by the invention prepares biffins cell, and the cycle is shorter, and productive rate is high.A large amount of biffins cells can be obtained within the time of about 48 days.The plant hormone kind comprised in the inducing culture and proliferated culture medium used in biffins cell processes is appropriate, ratio is suitable for cultivating in the present invention, can ensure that apple branch cell dedifferentes fast, and a large amount of breedings.This biffins cell can for the preparation of food, medicine, healthcare products or makeup after freeze-drying or after extracting.Biffins cell extraction method provided by the invention, freeze drying process are simple, and extraction efficiency is higher, enrich through extracting flavones and polyphenol content in obtained extract.
Embodiment
The invention provides a kind of cultural method of biffins cell and the biffins cell of cultivation, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications herein or suitably changes and combination not departing from content of the present invention, spirit and scope, realizes and applies the technology of the present invention.
The instrument that the present invention adopts is all common commercially available product, all can buy in market.
Below in conjunction with embodiment, set forth the present invention further:
The substratum adopted in the embodiment of the present invention is respectively:
Inducing culture A:MS solid medium+NAA 0.1mg/L+BA 0.1mg/L+CH 0.1mg/L, pH value is 6.0;
Inducing culture B:MS solid medium+NAA 10mg/L+BA 10mg/L+CH 10mg/L, pH value is 6.0;
Inducing culture C:MS solid medium+NAA0.5mg/L+BA 2mg/L+CH 1mg/L, pH value is 6.0;
Inducing culture D:CaCl 2332mg/L, KH 2pO 4170mg/L, KNO 31900mg/L, MgSO 4180.54mg/L, NH 4nO 31650mg/L, CoCl 26H 2o 0.025mg/L, CuSO 45H 2o 0.025mg/L, NaFeEDTA sodium 36.7mg/L, boric acid 6.2mg/L, KI 83mg/L, MnSO 4h 2o 16.9mg/L, Na 2moO 42H 2o 0.25mg/L, ZnSO 47SO48.6mg/L, inositol 100mg/L, nicotinic acid 5mg/L, glycine 2mg/L, vitamin B6 0.5mg/L, VITMAIN B1 0.5mg/L, folic acid 0.5mg/L, vitamin H 0.05mg/L, vitamins C 50mg/L, thiocarbamide 25mg/L, altheine 180mg/L, sucrose 30g/L, agar 0.8%, pH5.6.
Proliferated culture medium A:MS liquid nutrient medium+2,4-D 0.1mg/L+BA 0.1mg/L+CH0.1mg/L+AC 0.1mg/L, pH value is 6.0;
Proliferated culture medium B:MS liquid nutrient medium+2,4-D 10mg/L+BA 10mg/L+CH 10mg/L+AC 10mg/L, pH value is 6.0;
Proliferated culture medium C:MS liquid nutrient medium+2,4-D 2.0mg/L+BA 1.0mg/L+CH1.0mg/L+AC 1.0mg/L, pH value is 6.0;
Proliferated culture medium D:CaCl 2332mg/L, KH 2pO 4170mg/L, KNO 31900mg/L, MgSO 4180.54mg/L, NH 4nO 31650mg/L, CoCl 26H 2o 0.025mg/L, CuSO 45H 2o 0.025mg/L, NaFeEDTA sodium 36.7mg/L, boric acid 6.2mg/L, KI 83mg/L, MnSO 4h 2o 16.9mg/L, Na 2moO 42H 2o 0.25mg/L, ZnSO 47SO48.6mg/L, inositol 100mg/L, nicotinic acid 5mg/L, glycine 2mg/L, vitamin B6 0.5mg/L, VITMAIN B1 0.5mg/L, folic acid 0.5mg/L, vitamin H 0.05mg/L, vitamins C 50mg/L, thiocarbamide 25mg/L, altheine 180mg/L, sucrose 30g/L, pH5.6.
The sterilization of embodiment 1 apple branch
Select the apple tree without fertilizer and pesticide pollution, green planting, select the long newborn branches of 10 10cm according to certain judgment criteria; Newborn branch surface attachments is washed with sterilized water 2L; Newborn branch is put in 120mg/L L-AA and soak 1 minute; Newborn branch is put into the ethanolic soln sterilization 1 minute of 75%, clean with 2L aseptic water washing; Put into the superoxol sterilization 20 minutes of 30% again, and with aseptic water washing 10 minutes.
The induction of embodiment 2 Cambium tissue
Newborn for the apple of embodiment 1 sterilizing branch vaccinating lancet is peeled off the tissues such as form layers, phloem, cortex and epidermis, abandons xylem and marrow; The tissue of acquisition is seeded in fresh solid medium, with every 1g tissue inoculation 10cm 2the density of solid medium is inoculated, and at controlled dark indoor cultivation, after cultivation, Cambium tissue is the tabular tissue of homogeneous growth, and its hetero-organization is then irregular gathering grower, weighs to the Cambium tissue obtained.
Table 1 culture condition
Substratum Temperature Time Cambium tissue weight
Experimental group 1 Inducing culture A 10℃ 20 days 3.1±0.3
Experimental group 2 Inducing culture B 30℃ 1 day 1.3±0.1
Experimental group 3 Inducing culture C 25℃ 10 days 20.8±1.2
Control group Inducing culture D 25℃ 21 days 17.5±1.3
Embodiment 3 succeeding transfer culture Cambium tissue
The Cambium tissue of regeneration and other separate tissue are opened; With every 1g tissue inoculation 10cm 2the density of solid medium, is seeded to the Cambium tissue of regeneration in fresh solid medium, and darkroom is cultivated, and is separated callus, weighs.
Table 2 culture condition
Substratum Temperature Time Callus weight
Experimental group 1 Inducing culture A 10℃ 20 days 4.2±0.3
Experimental group 2 Inducing culture B 30℃ 1 day 1.4±0.2
Experimental group 3 Inducing culture C 25℃ 10 days 25.6±1.8
Control group Inducing culture D 25℃ 21 days 22.7±1.5
Embodiment 4 single cell culture
200ml liquid nutrient medium, the 20g of sterilising treatment, the granulated glass sphere of diameter 0.5cm is added in 1000ml triangular flask;
The embodiment 2 callus 10g that respectively group obtains is transferred in triangular flask respectively;
Triangular flask is placed on shaking table and cultivates, wherein:
The incandescent light of experimental group 1 ~ 3 20W per hour irradiates 10 minutes; 7th day, to add the fresh liquid nutrient medium of 400ml to cell density be the density of wherein cell was 1 × 10 5/ ml, then continues to cultivate; Concrete as table 3; With 100 object stainless steel sift net filtrations after cultivating, removing cell mass, granulated glass sphere and residue, obtain single cell suspension, calculates cell density.
Table 3 culture condition
Substratum Temperature Continue incubation time Cell density
Experimental group 1 Proliferated culture medium A 10℃ 10 days 2.5±0.2×10 5/ml
Experimental group 2 Proliferated culture medium B 30℃ 1 day 1.1±0.1×10 5/ml
Experimental group 3 Proliferated culture medium C 25℃ 7 days 9.5±0.8×10 5/ml
Control group is in 25 DEG C, 100rpm shaking table, and dark culturing is after 14 days, and with 100 object stainless steel sift net filtrations, removing cell mass, granulated glass sphere and residue, obtain single cell suspension, and calculating cell density is 2.8 × 10 5/ ml.
Embodiment 5 amplification culture
Carrying out large scale culturing according to being transferred in 20L bio-reactor by single cell suspension obtained for embodiment 4, in bio-reactor, holding the proliferated culture medium of 5L.When experimental group 1 ~ 3 cultivates the 7th day in bio-reactor, add the fresh liquid substratum of 10L, continue to cultivate rear harvested cell.Control group is cultivated and continue cultivation 20 days in bio-reactor, and substratum is not added in centre.Actual conditions is as table 4.Harvested cell after cultivating, weighs.
Table 4 culture condition
Substratum Inoculum density Temperature Aeration condition Continue incubation time Cell weight
Experimental group 1 Proliferated culture medium A 1×10 3/ml 10℃ 0.1vvm 20 days 0.1g
Experimental group 2 Proliferated culture medium B 0.1×10 3/ml 30℃ 0.1vvm 1 day 0.01g
Experimental group 3 Proliferated culture medium C 1×10 4/ml 25℃ 0.1vvm 7 days 1.1g
Control group Proliferated culture medium D 1×10 4/ml 25℃ 0.1vvm 21 days 0.8g
The preparation of embodiment 6 biffins cell lyophilized powder
Collect the biffins cell suspension 1um metre filter that embodiment 5 experimental group 3 is obtained, remove substratum, gather in the crops the viscose shape cell retained and take out;
Cell is put into the refrigerator pre-freeze 3 hours of-30 DEG C;
Put into liquid nitrogen container super low temperature quick frozen 2 hours;
After taking-up is thawed 30 minutes, then put into-30 DEG C of refrigerator pre-freezes and liquid nitrogen container quick-frozen, 2 times successively;
Taken out by cell, maintain 10 minutes under room temperature, enter in cryogenic freezing Vacuumdrier, after vacuum lyophilization, high speed pulverization becomes 60 order lyophilized powders.
The preparation of embodiment 7 biffins cell lyophilized powder
Collect the biffins cell suspension 1um metre filter that embodiment 5 experimental group 2 is obtained, remove substratum, gather in the crops the viscose shape cell retained and take out;
Cell is put into the refrigerator pre-freeze 10 hours of-50 DEG C;
Put into liquid nitrogen container super low temperature quick frozen 10 hours;
After taking-up is thawed 1 minute, then put into-50 DEG C of refrigerator pre-freezes and liquid nitrogen container quick-frozen, 10 times successively;
Taken out by cell, maintain 50 minutes under room temperature, enter in cryogenic freezing Vacuumdrier, after vacuum lyophilization, high speed pulverization becomes 10 order lyophilized powders.
The extracting method of embodiment 8 biffins cell
Collect the biffins cell suspension 1um metre filter that embodiment 5 experimental group 1 is obtained, remove substratum, gather in the crops the viscose shape cell retained and take out;
Cell is put into the refrigerator pre-freeze 1 hour of-80 DEG C;
Put into liquid nitrogen container super low temperature quick frozen 2 hours;
After taking-up is thawed 100 minutes, then put into-80 DEG C of refrigerator pre-freezes and liquid nitrogen container quick-frozen, 1 time successively;
Taken out by cell, maintain 1 minute under room temperature, enter in cryogenic freezing Vacuumdrier, after vacuum lyophilization, high speed pulverization becomes 100 order lyophilized powders;
The preparation of embodiment 9 biffins cell lyophilized powder
Collect the biffins cell suspension 1um metre filter that embodiment 5 control group is obtained, remove substratum, gather in the crops the viscose shape cell retained and take out;
Cell is put into the refrigerator pre-freeze 3 hours of-30 DEG C;
Put into liquid nitrogen container super low temperature quick frozen 2 hours;
After taking-up is thawed 30 minutes, then put into-30 DEG C of refrigerator pre-freezes and liquid nitrogen container quick-frozen, 2 times successively;
Taken out by cell, maintain 10 minutes under room temperature, enter in cryogenic freezing Vacuumdrier, after vacuum lyophilization, high speed pulverization becomes 60 order lyophilized powders.
Embodiment 10 biffins cell lyophilized powder quality evalution
Apple polyphenol is the common name of contained polyatomic phenol material in apple, apple polyphenol has very strong oxidation-resistance, removes the plurality of health care functions such as interior free yl, preventing hypertension, antitumor, anti-ageing, anti-mutation, radioprotective, antianaphylaxis, prevention of dental caries, muscle strength reinforcing, is usually used in food and healthcare products.The Flavonoid substances contained in apple is a kind of high-efficiency antioxidant agent, the blood vessel cleaning agent that it is still not best, and is the jinx of cancer.Therefore, polyphenol in biffins cell and flavones content is detected thus the quality of qualification biffins cell.
By biffins cell lyophilized powder prepared by embodiment 6 ~ 9, with 75% extraction using alcohol 3 times, united extraction liquid after rotary evaporation, lyophilize, after dissolving with distilled water, measure polyphenol content with Folin-ciocalteu method, do standard curve determination flavones content with rutin.
Concrete: the mensuration of apple polyphenol content adopts Folin-ciocalteu method, take gallic acid as standard substance, accurately draws 0.1mg/ml gallic acid standard solution 0.4,0.6,0.8,1.0,1.2,1.4,1.6ml, is placed in 100ml volumetric flask respectively, adds 50ml distilled water, add 5ml Folin-ciocalteu reagent again, after shaking up, add 15ml 1mol/L Na again 2cO 3solution, in room temperature reaction 2h.Prepare blank solution: in 100ml volumetric flask, add 5ml Folin-ciocalteu reagent, then add 15ml 1mol/LNa simultaneously 2cO 3solution, constant volume, room temperature reaction 2h.Measure the absorbancy of reference substance solution at 760nm wavelength place, the typical curve regression equation of mensuration is: A=0.00049x-0.0131, R 2=0.9932
X in formula---reference substance content, ug
A---absorbance.
After 1g biffins cell powder is dissolved with 100ml distilled water, get 2,4,6ml respectively and replace gallic acid reference substance, by the polyphenol content in standard curve making method working sample.
Flavonoid compound and aluminum nitrate effect, generate the salt complex ion of flavones, and in yellow, the depth of color becomes certain proportionlity with the content of flavones.According to Moore's Law, utilize spectrophotometer to carry out colorimetric assay mensuration in 510nm light wave place, make typical curve with rutin, thus flavones content in quantitative assay sample.
After 1g biffins cell powder is dissolved with 100ml distilled water, get 1ml in 50ml colorimetric cylinder, add 5% sodium nitrite solution 0.75ml and shake up and put 5min, accurately add 10% aluminum nitrate solution 0.75ml and place 5min, add 10ml 1mol/L NaOH again, use water constant volume.Survey absorbancy with spectrophotometer at 510nm place, every sample is parallel to be done 2 times and averages.The absorbance recorded is updated in rutin standard curve equation, just can tries to achieve wherein flavones content.
Detected result is as shown in table 5:
Table 5 detected result
Flavones content (mg/g) Polyphenol content (mg/g)
Embodiment 6 18.6 21.7
Embodiment 7 17.5 21.1
Embodiment 8 18.9 22.9
Embodiment 9 7.5 10.8
Result shows, in biffins cell prepared by the present invention, the content of flavones and polyphenol is all higher.
The extracting method of embodiment 11 biffins cell
Collect the biffins cell suspension 1um metre filter that embodiment 5 experimental group 3 is obtained, remove substratum, gather in the crops the viscose shape cell retained and take out;
Cell is put into the refrigerator pre-freeze 3 hours of-30 DEG C;
Put into liquid nitrogen container super low temperature quick frozen 2 hours;
After taking-up is thawed 30 minutes, then put into-30 DEG C of refrigerator pre-freezes and liquid nitrogen container quick-frozen, 2 times successively;
Taken out by cell, maintain 10 minutes under room temperature, enter in cryogenic freezing Vacuumdrier, after vacuum lyophilization, high speed pulverization becomes 60 order lyophilized powders;
Put into 3000ml 75% ethanolic soln, indirectly heat 26 DEG C, and stir 48 hours;
Put into ultrasonic wave separation and Extraction machine and carry out strong ultrasonication tenuigenin, then ultrasound filtration, collect filtrate;
Concentrate with rotating vacuum evaporator 26 DEG C;
Enriched material is put into cryogenic freezing Vacuumdrier, vacuum lyophilization;
High speed pulverization 100 order, vacuum packing is preserved, and makes Fructus Mali pumilae extract.
The extracting method of embodiment 12 biffins cell
Collect the biffins cell suspension 1um metre filter that embodiment 5 experimental group 2 is obtained, remove substratum, gather in the crops the viscose shape cell retained and take out;
Cell is put into the refrigerator pre-freeze 1 hour of-80 DEG C;
Put into liquid nitrogen container super low temperature quick frozen 2 hours;
After taking-up is thawed 100 minutes, then put into-80 DEG C of refrigerator pre-freezes and liquid nitrogen container quick-frozen, 1 time successively;
Taken out by cell, maintain 1 minute under room temperature, enter in cryogenic freezing Vacuumdrier, after vacuum lyophilization, high speed pulverization becomes 10 order lyophilized powders;
Put into 100ml 50% ethanolic soln, indirectly heat 10 DEG C, and stir 10 hours;
Put into ultrasonic wave separation and Extraction machine and carry out strong ultrasonication tenuigenin, then ultrasound filtration, collect filtrate;
Concentrate with rotating vacuum evaporator 26 DEG C;
Enriched material is put into cryogenic freezing Vacuumdrier, vacuum lyophilization;
High speed pulverization 10 order, vacuum packing is preserved, and makes Fructus Mali pumilae extract.
The extracting method of embodiment 13 biffins cell
Collect the biffins cell suspension 1um metre filter that embodiment 5 experimental group 1 is obtained, remove substratum, gather in the crops the viscose shape cell retained and take out;
Cell is put into the refrigerator pre-freeze 10 hours of-50 DEG C;
Put into liquid nitrogen container super low temperature quick frozen 10 hours;
After taking-up is thawed 1 minute, then put into-50 DEG C of refrigerator pre-freezes and liquid nitrogen container quick-frozen, 10 times successively;
Taken out by cell, maintain 50 minutes under room temperature, enter in cryogenic freezing Vacuumdrier, after vacuum lyophilization, high speed pulverization becomes 100 order lyophilized powders;
Put into 10000ml 95% ethanolic soln, indirectly heat 30 DEG C, and stir 100 hours;
Put into ultrasonic wave separation and Extraction machine and carry out strong ultrasonication tenuigenin, then ultrasound filtration, collect filtrate;
Concentrate with rotating vacuum evaporator 30 DEG C;
Enriched material is put into cryogenic freezing Vacuumdrier, vacuum lyophilization;
High speed pulverization 200 order, vacuum packing is preserved, and makes Fructus Mali pumilae extract.
The preparation of embodiment 14 biffins cell lyophilized powder
Collect the biffins cell suspension 1um metre filter that embodiment 5 control group is obtained, remove substratum, gather in the crops the viscose shape cell retained and take out;
Cell is put into the refrigerator pre-freeze 3 hours of-30 DEG C;
Put into liquid nitrogen container super low temperature quick frozen 2 hours;
After taking-up is thawed 30 minutes, then put into-30 DEG C of refrigerator pre-freezes and liquid nitrogen container quick-frozen, 2 times successively;
Taken out by cell, maintain 10 minutes under room temperature, enter in cryogenic freezing Vacuumdrier, after vacuum lyophilization, high speed pulverization becomes 60 order lyophilized powders;
Put into 3000ml 75% ethanolic soln, indirectly heat 26 DEG C, and stir 48 hours;
Put into ultrasonic wave separation and Extraction machine and carry out strong ultrasonication tenuigenin, then ultrasound filtration, collect filtrate;
Concentrate with rotating vacuum evaporator 26 DEG C;
Enriched material is put into cryogenic freezing Vacuumdrier, vacuum lyophilization;
High speed pulverization 100 order, vacuum packing is preserved, and makes Fructus Mali pumilae extract.
The Quality Identification of embodiment 15 biffins cell extract
The method provided with embodiment 10 detects the quality of the obtained biffins cell extract of embodiment 11 ~ 14.And it is compared with Switzerland PhytoCelltec biffins cell lyophilized powder.Switzerland PhytoCelltec biffins cell is mainly used in cosmetics and skincare product at present, it is said to have anti-wrinkle cosmetic effect.Select Switzerland's biffins cell 10% stoste elite (30ml dress) in contrast, compare itself and the product of the present invention difference at the effective active such as flavonoids from apple, apple polyphenol component content.
1g Fructus Mali pumilae extract 1000ml distilled water is dissolved (concentration is 0.1%), get 1ml, and get Switzerland biffins cell 10% stoste elite 1ml, measure the content of apple polyphenol, flavonoids from apple material respectively by the method for embodiment 6, result display (see table 6):
Table 6 qualification result
Flavones content (μ g/ml) Polyphenol content (μ g/ml)
Embodiment 11 18.2 22.1
Embodiment 12 17.9 21.7
Embodiment 13 17.5 21.1
Embodiment 14 18.6 22.9
Switzerland's biffins cell 0.9 1.2
The product of 0.1% concentration of the present invention, than the product of Switzerland's biffins cell 10% stoste, the former apple polyphenol, the content of flavonoids from apple are far above the latter.
Embodiment 16 biffins cell healthcare products
First biffins cell lyophilized powder, oligofructose are carried out pulverizing, sieving, then by pulverizing, screened material mixes in mixing machine with poly-dextrose, carry out packing with portioning machine, namely obtained have the biffins cell powder (pulvis) improving gi tract nourishing function.
Embodiment 17 biffins cell healthcare products
First biffins cell extract, oligofructose are carried out pulverizing, sieving, then by pulverizing, screened material mixes in mixing machine with poly-dextrose, carry out packing with portioning machine, namely obtained have the biffins cell powder (pulvis) improving gi tract nourishing function.
Below be only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (13)

1. cultivate a method for biffins cell, it is characterized in that, comprise the following steps:
Step 1: after the newborn branch sterilization of apple, removal xylem and marrow, be inoculated in inducing culture, inducing culture obtains cambial cell;
Step 2: described cambial cell, through succeeding transfer culture, accesses proliferated culture medium, and it is unicellular that acquisition cultivated by shaking table;
Step 3: by the described unicellular proliferated culture medium that is inoculated in through enlarged culturing, obtains biffins cell;
Described inducing culture is the MS solid medium containing NAA, BA and caseinhydrolysate;
Described proliferated culture medium be containing 2,4-D, the MS liquid nutrient medium of BA, caseinhydrolysate and gac.
2. method according to claim 1, is characterized in that, described inducing culture is light culture; Temperature is 10 DEG C ~ 30 DEG C; Time is 1 day ~ 20 days.
3. method according to claim 1, is characterized in that, described succeeding transfer culture is light culture; Temperature is 10 DEG C ~ 30 DEG C; Time is 1 day ~ 20 days.
4. method according to claim 1, is characterized in that, the temperature that described shaking table is cultivated is 10 DEG C ~ 30 DEG C; Rotating speed is 10 revs/min ~ 200 revs/min; Time is 8 days ~ 17 days; Illumination 10min per hour.
5. the biffins cell that as described in any one of Claims 1 to 4 prepared by method.
6. an inducing culture for biffins cell, is characterized in that, is the MS solid medium containing NAA, BA and caseinhydrolysate.
7. a proliferated culture medium for biffins cell, is characterized in that, is the MS liquid nutrient medium containing 2,4-D, BA, caseinhydrolysate and gac.
8. a preparation method for biffins cell lyophilized powder, is characterized in that, freeze-drying after biffins cell multigelation method described in any one of Claims 1 to 4 prepared, and obtains biffins cell lyophilized powder.
9. the biffins cell lyophilized powder prepared of method as claimed in claim 8.
10. the biffins cell lyophilized powder that as claimed in claim 8 prepared by method is preparing the application in food, medicine, healthcare products or makeup.
The extracting method of 11. 1 kinds of biffins cells, it is characterized in that, freeze-drying after biffins cell multigelation method described in any one of Claims 1 to 4 prepared, pulverizing are that extracting solution obtains biffins cell extract through extraction, by force ultrasonic, concentrated, dry, pulverizing with aqueous ethanolic solution.
The 12. biffins cell extracts prepared of method as claimed in claim 11.
The 13. biffins cells that as claimed in claim 11 prepared by method are preparing the application in food, medicine, healthcare products or makeup.
CN201510166803.9A 2015-04-09 2015-04-09 Apple stem cell culture method and apple stem cells cultured by method Pending CN104711215A (en)

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CN105852125A (en) * 2016-04-27 2016-08-17 深圳爱生再生医学科技有限公司 Aloe stem cell lyophilized powder as well as preparation method and application thereof
CN106075112A (en) * 2016-06-20 2016-11-09 陈云海 A kind of plant stem cell Chinese medicine
CN106361846A (en) * 2016-08-29 2017-02-01 天津市尖峰天然产物研究开发有限公司 Method for preparing apple extract with low pesticide residue content and high phlorizin content
CN106511530A (en) * 2016-11-04 2017-03-22 广州赛莱拉干细胞科技股份有限公司 Papaya stem cell capsules
CN106635957A (en) * 2016-12-23 2017-05-10 叶宗耀 Stem cell culture medium and preparation method thereof
CN107648169A (en) * 2017-10-31 2018-02-02 广州赛莱拉干细胞科技股份有限公司 A kind of moisturizing face nourishing skin care products of the cell extract containing banana figs and preparation method thereof
CN107616954A (en) * 2017-10-31 2018-01-23 广州赛莱拉干细胞科技股份有限公司 A kind of moist skin care item and preparation method thereof of releiving of the cell extract containing banana figs
CN107714634A (en) * 2017-10-31 2018-02-23 广州赛莱拉干细胞科技股份有限公司 A kind of whitening antiallergic skin care item of the cell extract containing banana figs and preparation method thereof
CN107714639A (en) * 2017-11-01 2018-02-23 广州赛莱拉干细胞科技股份有限公司 A kind of blackhead skin care item of dispelling containing Chinese herbaceous peony stem cell extract
CN107638376A (en) * 2017-11-01 2018-01-30 广州赛莱拉干细胞科技股份有限公司 A kind of anti-wrinkle skin care item containing Chinese herbaceous peony stem cell extract
CN107638378A (en) * 2017-11-01 2018-01-30 广州赛莱拉干细胞科技股份有限公司 A kind of Moisturizer containing Chinese herbaceous peony stem cell extract
CN107961209A (en) * 2017-12-04 2018-04-27 广州赛莱拉干细胞科技股份有限公司 A kind of skin care item of whitening anti-aging and preparation method thereof
CN108030755A (en) * 2017-12-05 2018-05-15 广州赛莱拉干细胞科技股份有限公司 It is a kind of to dispel blackhead skin care item and preparation method thereof containing Chinese wolfberry fruit dry cell extract
CN108030756A (en) * 2017-12-05 2018-05-15 广州赛莱拉干细胞科技股份有限公司 It is a kind of to dispel livid ring around eye skin care item and preparation method thereof containing Chinese wolfberry fruit dry cell extract
CN107982200A (en) * 2017-12-05 2018-05-04 广州赛莱拉干细胞科技股份有限公司 A kind of anti-wrinkle skin care item containing Chinese wolfberry fruit dry cell extract and preparation method thereof
CN107823090A (en) * 2017-12-05 2018-03-23 广州赛莱拉干细胞科技股份有限公司 A kind of active white skin care solution and preparation method containing ambergris
CN107802589A (en) * 2017-12-06 2018-03-16 广州赛莱拉干细胞科技股份有限公司 A kind of moisturizing containing pot marigold stem cell extract, anti-oxidant skin care item
CN107951824A (en) * 2017-12-06 2018-04-24 广州赛莱拉干细胞科技股份有限公司 A kind of Moisturizer containing pot marigold stem cell extract
CN107854421A (en) * 2017-12-06 2018-03-30 广州赛莱拉干细胞科技股份有限公司 A kind of anti-wrinkle skin care item of the extract of stem cell containing folium artemisiae argyi and preparation method thereof
CN107823112A (en) * 2017-12-06 2018-03-23 广州赛莱拉干细胞科技股份有限公司 A kind of anti-oxidant skin care item containing pot marigold stem cell extract
CN107951827A (en) * 2017-12-07 2018-04-24 广州赛莱拉干细胞科技股份有限公司 A kind of anti-oxidant skin care item and preparation method containing rosemary stem cell extract
CN107789316A (en) * 2017-12-07 2018-03-13 广州赛莱拉干细胞科技股份有限公司 A kind of moisturizing antioxidant composition of extract of stem cell containing pot marigold and its application
CN107823114A (en) * 2017-12-08 2018-03-23 广州赛莱拉干细胞科技股份有限公司 A kind of anti-acne skin care item containing sealwort stem cell extract
CN108165520A (en) * 2018-01-26 2018-06-15 深圳欧珈再生医学抗衰生物工程有限公司 A kind of bud stem cell and extracting method, bud stem cell Essence
CN108967200A (en) * 2018-09-30 2018-12-11 广州赛莱拉干细胞科技股份有限公司 The induced medium of plant callus and the preparation method of plant stem cell
CN113993532A (en) * 2018-12-06 2022-01-28 M·拉希米 oral treatment
CN110269820A (en) * 2019-05-31 2019-09-24 杨宇翔 A kind of preparation method of the two-in-one whole plant shampoo based on nanometer plant stem cell
CN110496095A (en) * 2019-07-15 2019-11-26 上海新高姿化妆品有限公司 Composition with collaboration anti-oxidation efficacy and its application in cosmetics
CN112852707A (en) * 2019-11-27 2021-05-28 深圳欧珈再生医学抗衰生物工程有限公司 Bud stem cell, extraction method and bud stem cell essence
CN111869564A (en) * 2020-07-07 2020-11-03 深圳市清大生物科技有限公司 Preparation method for extracting plant stem cells by ultralow temperature freeze-drying grinding
CN111920941A (en) * 2020-09-24 2020-11-13 钟发 Beauty treatment anti-aging oral liquid and preparation method thereof

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