CN1103372C - Production method of cell containing resveratrol - Google Patents
Production method of cell containing resveratrol Download PDFInfo
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- CN1103372C CN1103372C CN 00113914 CN00113914A CN1103372C CN 1103372 C CN1103372 C CN 1103372C CN 00113914 CN00113914 CN 00113914 CN 00113914 A CN00113914 A CN 00113914A CN 1103372 C CN1103372 C CN 1103372C
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Abstract
The present invention relates to a producing method for cells containing resveratrol. In the method, the callus induction and the single cell separation of plant body materials containing resveratrol are carried out, high yield cell strains are induced, screened and cultured in the enlarged mode, and the production culture of a large quantity of cells is realized; consequently, a cell product containing resveratrol is finally obtained. The cell product can be used for the production and the extraction of resveratrol, health care food addition and cosmetic addition. The method has the advantages of high and stable content and freedom from the limitation of raw materials and seasons; industrialized production without dependence on natural resources is realized so that the method is of great significance for the protection of natural resources.
Description
The invention belongs to the production method of raw material thing in the active ingredients of plants extraction, relate in particular to culture plant cell (clone) technical field.
Trans-resveratrol (Resveratrel) chemistry resvertrol by name is that a kind of degeneration-resistant material with vitis spp at first is found, and finds in succession in other many plants (as: giant knotweed, Stem of Smalleaf Jointfir, Semen arachidis hypogaeae, Maackia amurensis Rupr et Maxim, European spruce etc.) again afterwards.Since finding trans-resveratrol, people have done big quantity research to the feature of aspects such as its molecular structure, chemical property.After particularly finding the trophism in grape wine, big quantity research has been carried out in its pharmacological action, the result thinks that it probably uses as a kind of novel drugs.According to the literature, trans-resveratrol has antibiotic, anti peroxidation of lipid, preventing heart disease, anticancer, anti-platelet aggregation, effects such as lax blood vessel, reducing blood-fat and antimutagenic.Especially as the natural antitumor agent and the generation of inducing SOD, more and more be subject to people's attention.At present, mainly concentrate on the content in pharmacological action, extraction separation and evaluation, the grape wine and the research of trans-resveratrol synthetic enzyme about Study of Resveratrol work, and extraneous factor is to trans-resveratrol synthetic influence etc.
Trans-resveratrol exists in the cell of various plants, generally is to obtain product by extracting.Because the content in each kind of plant is very low, its production be subjected to raw material and season condition restriction, it is very big to form the difficulty that industrial scale produces, the acquisition amount is very limited, is difficult to meet the need of market.
Method screening high yield clone with cell engineering, pass through cell clone, then carry out cell cultures and obtain useful natural product,, more high yield, more stable more direct than from the fresh plant material, extracting, and be not subjected to natural resources and condition effect, significant for the production problem that solves natural secondary metabolite.Utilize plant cell culture technology can produce many secondary metabolites such as alkaloid, terpene, steroidal, phenols, quinones at present, be employed as medicine, essence, pigment, seasonings and agrochemical.Because the problem that content is low, cost is high once made cell cultures be difficult to use aborning.In recent years,, select high yielding cell sarain, greatly improved the content of secondary metabolite in the culture plant cell by changing substratum and culture condition.Also obtained important results with immobilization technology and genetic manipulation aspect the output of secondary metabolite in improving culturing cell recently, the content of the secondary metabolite that existing at present tens kind of plant cell cultures form is equal to or higher than former vegetable material.And existing the cultivation with tobacco cell produced phosphodiesterase and ubiquinone, cultivate the production Shikonin with paniculatum cell, produce ginsenoside with the genseng cell cultures, produce alkaloid or the like with the purple foxglove cell cultures, all reached industrialized industrial scale, produced anti-cancer alkaloid with the Vinca cell cultures and reached the pilot scale level.
At present, the research to the grape cell cultures mainly is the production that concentrates on the grape flower pigment both at home and abroad, and relation and extracting method between formation mechanism, cell seed selection, substratum composition, cell growth and the anthocyanidin generation of cyanidin(e) have been done a large amount of work.But yet there are no report about the work of producing trans-resveratrol with cell cultures.
The objective of the invention is to propose the method that a kind of production contains the trans-resveratrol cell, will contain the cell strain mass production of trans-resveratrol exactly with the method for cell cultures, lack and marketing problem in short supply to solve natural resources.
The objective of the invention is to be achieved by following technical scheme.Draw materials containing on the plant materials of trans-resveratrol, the explant material that obtains is carried out the inducing culture of callus, the callus that obtains is carried out unicellular separation, with the isolating unicellular screening of inducing of carrying out high yielding cell sarain; The high yielding cell sarain that obtains can be used for three aspects, 1. is used for the cell strain preservation, and it can be by screening once more after callus induction and unicellular the separation; 2. by after the unicellular separation, be used for cell suspension culture and obtain a large amount of clone cells; 3. be used for the cell solid culture and obtain a large amount of clone cells, the cell that solid culture obtains also can be used for cell suspension culture and obtain a large amount of clone cells after unicellular separation.
Can obtain a large amount of clone cells that contains trans-resveratrol by technical solution of the present invention production, its production that can not only be used for trans-resveratrol is extracted, and also can be directly used in the interpolation of protective foods and makeup; Have content advantage high and stable, that be not subjected to raw material and season limit, under the condition that does not rely on natural resources, realize industrial production, significance is arranged for the protection of natural resources.The present invention also has method characteristics simple, that implement easily.
Accompanying drawing contains the process flow sheet of trans-resveratrol cell for the designed production of the present invention.
Describe the enforcement requirement of technical solution of the present invention in detail below in conjunction with accompanying drawing.
1. draw materials: partly be material but can choose the tissue culture that contains trans-resveratrol, as root, stem, leaf and the grape fruit of giant knotweed, Stem of Smalleaf Jointfir, Semen arachidis hypogaeae, Maackia amurensis Rupr et Maxim, grape etc.Draw materials can be carried out inducing of callus after cleaning, sterilizing.
2. substratum: the present invention is by a large amount of experimental selection, and definite minimum medium is that MS is or/and B
5, this is at present comparatively general two kinds of substratum (work such as " Plant Biotechnology principle and method " Li Baojian, Hunan science tech publishing house published in 1992).Though these two kinds of substratum can both be suitable for, need it is improved.At first need to add stock culture juice 8-14ml/L is arranged; Secondly needing to add has NAA (naphthylacetic acid) 0.3-6mg/L and 6-BA (6 benzyl purine) 1-6mg/L, the principle that these two kinds of hormones use in cell cultivation process is, use the NAA (0.3-1mg/L) of low dosage and the 6-BA (4-6mg/L) of high dosage between a large amount of incubation periods from callus induction to cell, in cultivating, the final back that obtains cellular product then need change hormone dosage ratio in the substratum, promptly improve NAA concentration (1-6mg/L) reduce the concentration (1-4mg/L) of 6--BA, cell is broken up smoothly, help the formation of secondary metabolite.And substratum (MS and B after these two kinds of improvement
5) can make solid medium, also can make liquid nutrient medium, difference is whether be added with agar.
Produce from inducing culture in advance to the later stage the process of cultivating for same cell strain, can be with these two kinds of substratum (MS and B
5) alternately switching cultivation.Think by a large amount of experiments: generally speaking, the switching cultivation can make growth be suppressed, and it is dead to occur brownization of part then; And before its death, be transferred to again in the original substratum, can recover growth gradually; This explanation callus has dependency to its original substratum, and is relatively poor to the adaptive faculty of new environment.Yet by the cell strain of surviving in the training back of repeatedly transferring, its ability that conforms obviously improves, and the generation of trans-resveratrol also significantly improves simultaneously.
3. unicellular separation: be that callus or the cell mass that will cultivate is separated into the unicellular of dispersion.Can adopt common vegetable cell isolation technique to finish.For example, with the nutrient solution that contains polygalacturonase (1g/L) or contain the nutrient solution of N.F,USP MANNITOL (0.6mol/L) or contain the nutrient solution of these two kinds of materials, ratio in 1: 10 (W/V) adds callus or cell mass, dark left standstill 10-16 hour under 25 ℃ of conditions, stirring at low speed 5-8 minute again, can obtain the dispersive cell suspending liquid.It can be used for the propagation that liquid culture is carried out a large amount of cells, also available 100 purpose nets are overanxious obtain isolating unicellular.
4. the screening of high yielding cell sarain: be exactly to induce and filter out the high cell strain of trans-resveratrol generation, certain this screening also comprises the method for utilizing genetically engineered (importing the trans-resveratrol synthase gene) to obtain high yielding cell sarain.It is induced and normally adopt physics or/and the method for chemistry is finished in unicellular solid culture process, include illumination, laser, isotropic substance radiation, pH value, chemical inducer, switching or the like the mode of different substratum even, this all is the means that this technical field professional uses always.Behind a large amount of clones of cell, detect the generation of trans-resveratrol, filter out the cell strain of high yield, then can carry out enlarged culturing.
5. the detection method of trans-resveratrol: can with reference to Ji Chunru etc. compile " Chemistry for Chinese Traditional Medicine experimental technique and experiment " (P283-287) Henan science tech publishing house in 1986 publish; Work such as Wang Zhenyue " structural research of two compounds in the arteries and veins sorrel " " herbal medicine " (Tianjin Chinese materia medica institute) 1996,27 (12): 714-716; Works such as wangdan " research of polidatin content measurement in the Chinese medicine giant knotweed " " herbal medicine " 1987,18 (11): 16-18.
6. the production of a large amount of cells is cultivated: after obtaining (trans-resveratrol) high yielding cell sarain, can carry out enlarged culturing constantly by the solid culture or the method for liquid culture, to obtain a large amount of clone cells.As shown in the figure, wherein solid culture can obtain final cellular product by enlarged culturing constantly, finishes the production of a large amount of cells; Also can after solid culture obtains a certain amount of cell, change suspension culture over to, finish the production of a large amount of cells with airlift bioreactor through separation.And liquid (suspension) cultivation is to finish production to a certain amount of back by airlift bioreactor in enlarged culturing.No matter each cell cultures stage from inducing culture to the whole process of final mass production is that solid culture or its culture condition of suspension culture all adopt conventional means; Temperature is 25-30 ℃, illumination 12-24h/ days, and light intensity 1000-5000LX, a culture cycle is 3-5 week (promptly need change new substratum over to before cytodifferentiation); The screening of inducing from high yielding cell sarain simultaneously begins to last mass production, in substratum, can add in right amount chemical inducer is arranged, can select tetrahydroxyadipic acid (0-600mg/L) or/and aluminum chloride (0-2mg/L), to improve the content of trans-resveratrol in the cell.
Below do not limit the enforcement that example illustrates technical solution of the present invention with two.For for simplicity, used substratum (MS and B in the example
5) concrete prescription no longer carefully state, in detail prescription is referring to aforementioned documents.
Example one, 1. pluck that ripening degree is good, the sufficient bunch of grapes of fruit grain colouring, flushing with 5% hydrogen peroxide dipping 10 minutes, was used aseptic water washing after 4 hours in flowing water.Preparation MS (or B
5) solid medium is additional that the NAA of 10ml/L Sucus Vitis viniferae, 0.6mg/L, the 6-BA of 4.5mg/L, packing sterilization (later solid medium is all used this) arranged.
2. in the super clean bench aseptic technique, the grape material is cut into the piece of about 0.5cm size, insert in the substratum after the sterilizing and washing and cultivate.Under 25 ℃ of dark conditions, cultivated 36 hours, change over to then and cultivate evoked callus under the illumination (2000LX).After 3 weeks the callus that grows is cut apart, and be transferred to succeeding transfer culture on the new substratum.Every 2-3 week of successive propagation carries out once, should select loose, frangible callus.
3. get the tender callus of the fresh children of 10g and insert in the unicellular separation and Culture liquid of 100ml, 25 ℃ of dark left standstill 14 hours.Its nutrient solution is the mannitol solution of 0.6mol/L, PH3.5, and be added with the polygalacturonase of 0.1% (W/V).After the stirring at low speed 6 minutes, overanxious with 100 order nylon wires, after cleaning, obtain isolating unicellular.
4. unicellular transferring to induced screening and culturing on the solid medium, increasing in its substratum has inductor (aluminum chloride 1mg/L and tetrahydroxyadipic acid 300mg/L).In culturing process, it is carried out mutagenesis, filter out the high cell strain of Resveratrol content by various physics and chemical means.After separating, induce screening and culturing again, can repeat 2-5 time, finally determine high yielding cell sarain.
5. the preserving process of cell strain is actually succeeding transfer culture process constantly.
6. high yielding cell sarain is divided into polylith, is transferred to and carries out enlarged culturing on the solid medium that contains inductor, 3-5 week is an one-period, can constantly repeat to enlarge.
7. the cell mass of enlarged culturing is cut apart, be transferred to the production of carrying out a large amount of cells on the solid medium that contains inductor, the content of NAA is that the content of 4mg/L, 6-BA is 2mg/L in its substratum; Cultivate 3-5 week (before cytodifferentiation) under 30 ℃ of conditions, collecting cell group can be used for the extraction of trans-resveratrol or directly makes product for cellular product.
Example two, 1. prepare B
5(or MS) liquid nutrient medium is additional Sucus Vitis viniferae 12ml/L, and NAAO.4mg/L, 6-BA4.5mg/L, aluminum chloride 2mg/L sterilize after the packing.(later liquid nutrient medium is all used this).
2. with in the example one 4., 5., 6. the cell mass that finally obtains of arbitrary step is transferred to one week of suspension culture in the liquid nutrient medium after separating, the access amount is 1/5 (W/V) of nutrient solution, this cultivates for adapting to.
3. after adapting to the cultivation end,, repeat 1-4 time by this and carry out enlarged culturing suspension culture 3-5 week (before the cytodifferentiation) in the liquid nutrient medium of 5 times of amounts of the whole immigration of culture suspension.
4. the cell suspending liquid after the enlarged culturing is moved in the fresh liquid nutrient medium (wherein NAA is 5mg/L, and 6-BA is 3mg/L, tetrahydroxyadipic acid 600mg/L), make cell density reach 10
5Individual/ml, insert to produce in the airlift bioreactor and cultivate 4 weeks (before cytodifferentiation), cultivate and finish the method collecting cell product of back with overanxious (or centrifugal), can be used for the extraction of trans-resveratrol or directly make product.
Claims (6)
1. production method that contains the trans-resveratrol cell, it is characterized in that, draw materials containing on the plant materials of trans-resveratrol, the explant material that obtains is carried out the inducing culture of callus, the callus that obtains is carried out unicellular separation, with the isolating unicellular screening of inducing of carrying out high yielding cell sarain; The high yielding cell sarain that obtains can be used for three aspects, 1. is used for the cell strain preservation, and it can be by screening once more after callus induction and unicellular the separation; 2. by after the unicellular separation, be used for cell suspension culture and obtain a large amount of clone cells; 3. be used for the cell solid culture and obtain a large amount of clone cells, the cell that solid culture obtains also can be used for cell suspension culture and obtain a large amount of clone cells after unicellular separation; Cultivate used minimum medium and be MS or/and B5 medium, and be added with stock culture juice 8-14ml/L, NAA0.3-6mg/L, 6-BA1-6mg/L; Affiliated NAA and the using priciple of 6-BA are: callus induction uses NAA to be 4-6mg/L as 0.3-1mg/L, 6-BA between a large amount of incubation periods, and NAA is that 1-6mg/L, 6-BA are 1-4mg/L in the back cultivation of final acquisition cellular product.
2. method according to claim 1 is characterized in that the plant bulk material of being got is a grape fruit.
3. method according to claim 2 is characterized in that, from inducing the mass production of screening to last cell, adding in substratum has chemical inducer; Can select tetrahydroxyadipic acid 0-600mg/L, aluminum chloride 0-2mg/L.
4. method according to claim 3 is characterized in that, the final production of cellular product is to be cultivated by airlift bioreactor by a large amount of cells that suspension culture obtains to finish.
5. method according to claim 3 is characterized in that, the final production of cellular product is to be cultivated by airlift bioreactor by a large amount of cells that solid culture obtains to finish.
6. method according to claim 3 is characterized in that, the final production of cellular product is to be cultivated by the solid cell of continuous expansion to finish.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100374560C (en) * | 2004-08-27 | 2008-03-12 | 富岩 | Configuration of high effect expression serum albumin yeast strain and fermentation purification technology |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2190771B2 (en) * | 2002-01-24 | 2004-03-01 | Univ Alicante | PROCEDURE FOR THE PRODUCTION OF RESVERATROL IN CELL CULTURES. |
| CN103898130B (en) * | 2014-03-04 | 2016-04-20 | 天津大学 | The clone of mulberry fruit resveratrol synthase gene and the structure of plant expression vector |
| CN104313061B (en) * | 2014-09-05 | 2018-04-27 | 潍坊学院 | A kind of culture medium for improving Resveratrol in Rhizoma Polygoni Cuspidati content and preparation method thereof |
| CN106244517B (en) * | 2016-10-12 | 2019-12-20 | 中国农业科学院特产研究所 | Somatic embryo of vitis amurensis and culture method, culture medium and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100374560C (en) * | 2004-08-27 | 2008-03-12 | 富岩 | Configuration of high effect expression serum albumin yeast strain and fermentation purification technology |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
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