CN108277197A - A method of improving mogroside Ⅴ content in Siraitia grosvenorii suspension cell - Google Patents
A method of improving mogroside Ⅴ content in Siraitia grosvenorii suspension cell Download PDFInfo
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Abstract
The present invention relates to a kind of methods of mogroside Ⅴ content in raising Siraitia grosvenorii suspension cell.The method passes through at the 5th~11 day of the Siraitia grosvenorii suspension cell culture period, the methyl jasmonate of the yeast inducer of 10~200mg/L of addition and 50~260mg/L respectively, to improve the synthesis rate and yield of mogroside Ⅴ, while reducing the secretory volume of mogrosides II.The method is conducive to marketization large-scale production and the quality control management of mogroside Ⅴ.
Description
Technical field
The present invention relates to biotechnology, mogroside Ⅴ content in especially a kind of raising Siraitia grosvenorii suspension cell
Method.
Background technology
Mogroside Ⅴ, also known as mogroside are Curcurbitaceae Siraitia grosvenorii category Siraitia grosvenorii (Siraitia
Grosvenori main sweet ingredient), sugariness are 350 times of sucrose, have identified seven kinds of monomer components at present, wherein with
The sugariness of mogroside Ⅴ and siamenoside Ⅰ is most strong.There are mogroside Ⅴ clearing heat and moistening lung, relieving sore-throat to open sound, relax bowel
Medicinal efficacy；Meanwhile mogroside Ⅴ also has the characteristics that taste novelty, pure taste, to make its depth be liked by people
Love, is the optimum sweetener of the special populations such as obesity, hypertension, diabetic and health products, has a wide range of applications
Value.Currently, obtain mogroside Ⅴ method mainly using natural Siraitia grosvenorii as raw material, and pass through heat the methods of extraction
It is prepared.But the intrinsic mogroside Ⅴ ingredient in natural Siraitia grosvenorii is extremely low, utilization of the prior art for Siraitia grosvenorii
The recovery rate of rate and mogroside Ⅴ is not high, causes great wastage of material, while can not meet growing
The market demand.
Purpose secondary metabolite is produced by plant cell culture technology, is to improve target component content in natural plants
One of effective means.Cell suspension cultures are fast with reproduction speed, culture scale is big and it is thin to provide a large amount of uniformity plants
The characteristics of born of the same parents' culture.Therefore, mogroside Ⅴ is produced by plant cell culture technology culture, Siraitia grosvenorii can be effectively improved
The content and reduction production cost of sweet tea glucoside V, are conducive to the further marketization of mogroside Ⅴ.Such as 1 publication number of documents
To disclose a kind of " culture side of Siraitia grosvenorii callus cell suspension system in the Chinese patent text of CN102174463B
Method ", culture obtain the total glycosides of Siraitia grosvenorii, mogroside Ⅴ account for respectively cell weight 8.11%, 5.77% arhat fruit suspension it is thin
Born of the same parents.But the cultivation cycle of this method is up to 21d, increases the difficulty and cost of Operation and Maintenance, and gained content far can not also
Meets the needs of mogroside Ⅴ marketization.
During Secondary Metabolism of Plant, inducer can be quick, single-minded as a kind of special biochemical molecular and has choosing
Induce certain specific genes expression with selecting to improve the activity of relevant enzyme in metabolic pathway, and then promotes or inhibits by this
The yield of the secondary metabolite of enzyme regulation and control.Fungal Elicitor is derived from a kind of chemical signal of determination of microbial molecules, can
Plant cell is stimulated, makes it that defense response reaction rapidly occur, so that height is single-minded and the selectively specific base of activated plant
The expression of cause, and then specific secondary metabolism approach is activated, promote the synthesis of secondary metabolite.Such as documents 2《Siraitia grosvenorii
The foundation of cell culture system and sweet tea glucoside v-shaped at regulation and control》The Chinese document of (Chen Langui, Guangxi Normal University, 2010) is recorded
The component (protein+polysaccharide) being added in the aspergillus niger crude extract that total sugar concentration is 300mg/L can make mogroside Ⅴ
Total amount improves 246.72% than control group；Addition 500mg/L phenylalanines can be obviously promoted cell synthesis mogroside Ⅴ,
The total amount of mogroside Ⅴ improves 260.38% than control group.Although this method improves the content of mogroside Ⅴ,
But the secretion synthesis for also increasing bitter taste ingredient simultaneously, increases and is isolated and purified to mogroside Ⅴ in follow-up preparation flow
Difficulty and cost.
Yeast (Yeast) is the inducer generally used in culture plant cell, can induce or promote secondary metabolite
Formation.Studies have shown that in Ti converts Radix Salviae Miltiorrhizae (Salvia miltiorrhiza) cell culture, the addition of yeast inducer
Cryptotanshinone (Cryptotanshinone) is set to obtain twice of growth.And yeast also trains ceratostigma plumbaginoides Bunge (Plumbago roseal)
The content of plumbagin (Plumbagin) improves 3 times in supporting.But its inducing action of different systems is different.Methyl jasmonate
(Methyl Jasmonate, MeJA) is the abiotic class inducible factor for exciting secondary metabolites accumulation, different plant suspension trainings
Secondary metabolites accumulation it can increase in inducing cell after methyl jasmonate is added in the system of supporting.With 200 μm of ol/L jasmonic first
After ester processing bosom Chinese scholartree (Maackia amurensis Rupr.et Maxim) suspended culture cell 9d, isoflavone content increase is
The 417.18% of concurrent control；It is suspended with 100 μm of o l/L methyl jasmonate treatments southerm yews (Taxus chinensis)
When cell, content of taxol improves 10 times.However, methyl jasmonate is also easy after some plant suspension cultivating systems are added
Induction suspension cell occurs allergic reaction or phenolic compound is promoted to discharge, so as to cause suspension cell browning.With 1.0mg/L jasmines
When jasmine acid methyl esters handles Cell Suspension Cultures in Pueraria lobata, though the content of Puerarin is improved in cell culture, the apparent browning of cell.
It there is no the report with yeast or methyl jasmonate treatment Siraitia grosvenorii plant cell suspension cultures at present.
Invention content
In order to overcome the shortcomings of the prior art, the object of the present invention is to provide in a kind of raising Siraitia grosvenorii suspension cell
The method of mogroside Ⅴ content.The method not only shortens the productive culture period of Siraitia grosvenorii suspension cell, reduces
Operation and Maintenance cost in production process, also effectively increases the content of mogroside Ⅴ in Siraitia grosvenorii suspension cell, simultaneously
Reduce the synthesis secretion of bitter taste ingredient.
Inventor has found, ripe Siraitia grosvenorii suspension cell process is being trained with primary Siraitia grosvenorii suspension cell system
In, by the way that suitable yeast inducer and methyl jasmonate is added, Momordica-Glycosides in Siraitia grosvenorii suspension cell can be effectively improved
V content and accelerate its synthesis rate, while reducing the synthesis secretion of mogrosides II.
The purpose of the present invention can be achieved through the following technical solutions：
The present invention provides a kind of method improving mogroside Ⅴ content in Siraitia grosvenorii suspension cell, which is characterized in that
During being trained ripe Siraitia grosvenorii suspension cell with primary Siraitia grosvenorii suspension cell system, yeast inducer and jasmine is added
Sour methyl esters.
Preferably, ripe Siraitia grosvenorii suspension cell culture maturation sieve is trained with primary Siraitia grosvenorii suspension cell system described
50~100 μm of 10~50mg/L of yeast inducer, methyl jasmonate ol/L are added within the 5th~7 day in Chinese fruit suspension cell processes, the
120~260 μm of 100~200mg/L of yeast inducer, methyl jasmonate ol/L are added within 9~11 days, harvest is ripe within the 17th~25 day
Siraitia grosvenorii suspension cell.
Preferably, ripe Siraitia grosvenorii suspension cell culture maturation sieve is trained with primary Siraitia grosvenorii suspension cell system described
Yeast inducer is added within the 7th day in Chinese fruit suspension cell processes as 100 μm of 50mg/L, methyl jasmonate ol/L, is added within the 11st day
Yeast inducer is 180mg/L, methyl jasmonate 220 μm of ol/L, the ripe Siraitia grosvenorii suspension cell of the 19th day harvest.
Preferably, the primary Siraitia grosvenorii suspension cell system is obtained by following steps：
(1) Mature Embryos Among for taking Siraitia grosvenorii is containing sucrose 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate
0.5mg/L, inositol 100mg/L, agar 4.6g/L, pH ranging from 5.5~6.0 MS semisolid culturemediums on squamous subculture, choosing
It takes continuous subculture 3~5 times and growth is vigorous, quality is loose, in stable condition uniform fresh embryo callus；
(2) by fresh embryo callus that step (1) obtains with inoculum concentration 100g/L be transferred to containing sucrose 30g/L,
The MS Liquid Cultures of 6- benzyl aminoadenines 1.0mg/L, methyl α-naphthyl acetate 0.5mg/L, inositol 100mg/L, pH ranging from 5.5~6.5
In base, and in the shaking table that rotating speed is 150r/min, cultivation temperature is 25 ± 2 DEG C, light intensity 4000lux, light application time are 12h/d
Middle culture obtains primary Siraitia grosvenorii suspension cell system.
Preferably, step (1) subculture number is 4 times.
The present invention overcomes following technical barriers：It is outstanding that the Siraitia grosvenorii in exponential phase is individually handled with methyl jasmonate
Floating cell, the synthetic quantity of mogroside Ⅴ increased in the unit interval after processing, but cell browning is apparent, total content
More normal culture group is declined.Individually addition yeast inducer, has no significant effect mogroside Ⅴ total content, but arhat
V synthesis rate of fruit sweet tea glucoside slows down.
By a series of technical optimization, find, in primary Siraitia grosvenorii suspension cell system culture the 5th~7 day, ferment to be added
50~100 μm of female 10~50mg/L of inducer, methyl jasmonate ol/L；In culture the 9th~11 day, yeast inducer 100 is added
120~260 μm of~200mg/L, methyl jasmonate ol/L；In the ripe Siraitia grosvenorii suspension cell of the 17th~25 day harvest of culture.Make
The synthesis rate and total content for obtaining mogroside Ⅴ significantly improve, and the synthesis secretion of bitter taste ingredient significantly reduces, and cell
Cultivation cycle is obviously shortened.Particularly, in culture the 7th day, addition yeast inducer is 100 μm of 50mg/L, methyl jasmonate ol/
L；In culture the 11st day, addition yeast inducer was 220 μm of 180mg/L, methyl jasmonate ol/L；Culture the 19th day harvest at
Ripe Siraitia grosvenorii suspension cell so that the synthesis rate of mogroside Ⅴ is most fast, total content highest, bitter taste ingredient Siraitia grosvenorii soap
The synthesis secretion of glucoside II is minimum.
The present invention also provides a kind of preparation methods of mogroside Ⅴ, which is characterized in that method described above prepare at
Ripe Siraitia grosvenorii suspension cell further extracts separation and prepares mogroside Ⅴ.
Compared with the prior art, the present invention has the following advantages：
1. the present invention is culture raw material with Siraitia grosvenorii embryo, differentiation passage capacity is stronger more stable, is conducive to culture and obtains
Strong and vigorous growth, in stable condition uniform Siraitia grosvenorii callus.
2. the Siraitia grosvenorii cell suspension system obtained using the method for the present invention culture, mogroside Ⅴ yield are obviously carried
Height, and the bitter taste impurity content for synthesizing secretion is less.
3. the Siraitia grosvenorii cell suspension system obtained using the method for the present invention culture, high cell growth speed, good dispersion,
The cultivation cycle time is short, reduces the difficulty of Operation and Maintenance, can preferably meet market-oriented production requirement.
4. acquiring the approach of mogroside Ⅴ by the method for the invention, a large amount of land resource is saved, favorably
In the protection of ecological environment, the economic strategy policy of China's sustainable development is not only conformed with, and to solve mogroside Ⅴ
The source problem that comes provide an effective way.
5. the method for the present invention adjusts the production process of Siraitia grosvenorii cell and mogroside Ⅴ manually, be conducive to produce
The quality control management of product.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Embodiment 1
(1) Mature Embryos Among for taking Siraitia grosvenorii is containing sucrose 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate
0.5mg/L, inositol 100mg/L, agar 4.6g/L, pH ranging from 5.5~6.0 MS semisolid culturemediums on squamous subculture, choosing
It takes continuous subculture 4 times and growth is vigorous, quality is loose, in stable condition uniform fresh embryo callus；
(2) by fresh embryo callus that step (1) obtains with inoculum concentration 100g/L be transferred to containing sucrose 30g/L,
The MS Liquid Cultures of 6- benzyl aminoadenines 1.0mg/L, methyl α-naphthyl acetate 0.5mg/L, inositol 100mg/L, pH ranging from 5.5~6.5
In base, and in the shaking table that rotating speed is 150r/min, cultivation temperature is 25 ± 2 DEG C, light intensity 4000lux, light application time are 12h/d
Middle culture obtains primary Siraitia grosvenorii suspension cell system；
(3) ripe Siraitia grosvenorii suspension cell is trained with primary Siraitia grosvenorii suspension cell system obtained by step (2)；It was cultivating
100 μm of yeast inducer 50mg/L, methyl jasmonate ol/L are added within the 7th day in journey, yeast inducer 180mg/ are added within the 11st day
L, 220 μm of ol/L of methyl jasmonate；
(4) in culture the 1st~31 day, Siraitia grosvenorii suspension cell is every other day extracted, detects mogroside Ⅴ and arhat
II content of fruit saponin.
Embodiment 2
(1) Mature Embryos Among for taking Siraitia grosvenorii is containing sucrose 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate
0.5mg/L, inositol 100mg/L, agar 4.6g/L, pH ranging from 5.5~6.0 MS semisolid culturemediums on squamous subculture, choosing
It takes continuous subculture 3 times and growth is vigorous, quality is loose, in stable condition uniform, fresh embryo callus；
(2) the fresh embryo callus that step (1) obtains is transferred to inoculum concentration 100g/L (fresh weight) containing sucrose
The MS liquid of 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate 0.5mg/L, inositol 100mg/L, pH ranging from 5.5~6.5
In body culture medium, and rotating speed is 150r/min, cultivation temperature is 25 ± 2 DEG C, light intensity 4000lux, light application time 12h/d
Shaking table in cultivate, obtain primary Siraitia grosvenorii suspension cell system；
(3) ripe Siraitia grosvenorii suspension cell is trained with primary Siraitia grosvenorii suspension cell system obtained by step (2)；It was cultivating
50 μm of yeast inducer 10mg/L, methyl jasmonate ol/L are added within the 5th day in journey, be added within the 9th day yeast inducer 100mg/L,
120 μm of ol/L of methyl jasmonate；
(4) in culture the 1st~31 day, Siraitia grosvenorii suspension cell is every other day extracted, detects mogroside Ⅴ and arhat
II content of fruit saponin.
Embodiment 3
(1) Mature Embryos Among for taking Siraitia grosvenorii is containing sucrose 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate
0.5mg/L, inositol 100mg/L, agar 4.6g/L, pH ranging from 5.5~6.0 MS semisolid culturemediums on squamous subculture, choosing
It takes continuous subculture 5 times and growth is vigorous, quality is loose, in stable condition uniform fresh embryo callus；
(2) the fresh embryo callus that step (1) obtains is transferred to inoculum concentration 100g/L (fresh weight) containing sucrose
The MS liquid of 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate 0.5mg/L, inositol 100mg/L, pH ranging from 5.5~6.5
In body culture medium, and rotating speed is 150r/min, cultivation temperature is 25 ± 2 DEG C, light intensity 4000lux, light application time 12h/d
Shaking table in cultivate, obtain primary Siraitia grosvenorii suspension cell system；
(3) primary Siraitia grosvenorii suspension cell system obtained by step (2) is trained ripe Siraitia grosvenorii suspension cell；In incubation
In 75 μm of yeast inducer 25mg/L, methyl jasmonate ol/L are added within the 7th day, yeast inducer 200mg/L, jasmine is added within the 11st day
260 μm of ol/L of jasmine acid methyl esters；
(4) in culture the 1st~31 day, Siraitia grosvenorii suspension cell is every other day extracted, detects mogroside Ⅴ and arhat
II content of fruit saponin.
Embodiment 4
(1) it is inoculated with and is induced Siraitia grosvenorii callus from stem segment, is containing sucrose 2.5%, 6- benzyl aminoadenines 0.2mg/
L, methyl α-naphthyl acetate 0.04mg/L, squamous subculture on the MS culture mediums that agar 4.0mg/L, pH are 5.8, choose continuous subculture 3 times or 4 times
Or 5 fresh, soft frangible yellow-white callus；
(2) callus that step (1) obtains is transferred to inoculum concentration 300g/l containing sucrose 4.0%, 6- benzyl amino
In the sterile triangular flask of the MS fluid nutrient mediums of adenine 1.0mg/L, methyl α-naphthyl acetate 0.02mg/L, initial medium pH be 5.5~
6.0, the 25 DEG C of dark condition low suspension cultures of shaken cultivation case for being placed in 120r/min are stood afterwards for 24 hours, by upper layer culture medium together with
Unicellular and small cell cluster is transferred to sterile empty triangular flask relaying persistent oscillation culture；
(3) it is retransferred once using same method after 3d, is inoculated into suspension cell after 40 μm of screen filtrations after 7d
Fresh culture relays persistent oscillation culture, is transferred in big triangular flask after reaching certain volume, continues to add with same method
Add culture medium shaken cultivation, the cell suspending culture solution until reaching a certain number of stable homogeneous, cell suspension cultures temperature
It is 25 ± 2 DEG C, light intensity 2000lux, light application time 12h/d, obtains primary Siraitia grosvenorii suspension cell system；
(4) ripe Siraitia grosvenorii suspension cell is trained with primary Siraitia grosvenorii suspension cell system obtained by step (3)；It was cultivating
100 μm of yeast inducer 50mg/L, methyl jasmonate ol/L are added within the 7th day in journey, yeast inducer 180mg/ are added within the 11st day
L, 220 μm of ol/L of methyl jasmonate；
(5) in culture the 1st~31 day, Siraitia grosvenorii suspension cell is every other day extracted, detects mogroside Ⅴ and arhat
II content of fruit saponin.
Comparative example 1
This comparative example is used to evaluate technical solution and the technology of the present invention side of addition aspergillus niger inducer and methyl jasmonate
The technique effect difference that case obtains, is as follows：
(1) Mature Embryos Among for taking Siraitia grosvenorii is containing sucrose 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate
0.5mg/L, inositol 100mg/L, agar 4.6g/L, pH ranging from 5.5~6.0 MS semisolid culturemediums on squamous subculture, choosing
It takes continuous subculture 4 times and growth is vigorous, quality is loose, in stable condition uniform fresh embryo callus；
(2) by fresh embryo callus that step (1) obtains with inoculum concentration 100g/L be transferred to containing sucrose 30g/L,
The MS Liquid Cultures of 6- benzyl aminoadenines 1.0mg/L, methyl α-naphthyl acetate 0.5mg/L, inositol 100mg/L, pH ranging from 5.5~6.5
In base, and in the shaking table that rotating speed is 150r/min, cultivation temperature is 25 ± 2 DEG C, light intensity 4000lux, light application time are 12h/d
Middle culture obtains primary Siraitia grosvenorii suspension cell system；
(3) ripe Siraitia grosvenorii suspension cell is trained with primary Siraitia grosvenorii suspension cell system obtained by step (2)；It was cultivating
100 μm of aspergillus niger inducer 50mg/L, methyl jasmonate ol/L are added within the 7th day in journey, aspergillus niger inducer are added within the 11st day
220 μm of 180mg/L, methyl jasmonate ol/L；
(4) in culture the 1st~31 day, Siraitia grosvenorii suspension cell is every other day extracted, detects mogroside Ⅴ and arhat
II content of fruit saponin.
Comparative example 2
This comparative example is used to evaluate addition phenylalanine and the technical solution of yeast inducer takes with technical solution of the present invention
The technique effect difference obtained, is as follows：
(1) Mature Embryos Among for taking Siraitia grosvenorii is containing sucrose 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate
0.5mg/L, inositol 100mg/L, agar 4.6g/L, pH ranging from 5.5~6.0 MS semisolid culturemediums on squamous subculture, choosing
It takes continuous subculture 4 times and growth is vigorous, quality is loose, in stable condition uniform fresh embryo callus；
(2) by fresh embryo callus that step (1) obtains with inoculum concentration 100g/L be transferred to containing sucrose 30g/L,
The MS Liquid Cultures of 6- benzyl aminoadenines 1.0mg/L, methyl α-naphthyl acetate 0.5mg/L, inositol 100mg/L, pH ranging from 5.5~6.5
In base, and in the shaking table that rotating speed is 150r/min, cultivation temperature is 25 ± 2 DEG C, light intensity 4000lux, light application time are 12h/d
Middle culture obtains primary Siraitia grosvenorii suspension cell system；
(3) ripe Siraitia grosvenorii suspension cell is trained with primary Siraitia grosvenorii suspension cell system obtained by step (2)；It was cultivating
Phenylalanine 50mg/L, yeast inducer 50mg/L is added within the 7th day in journey, culture be added within the 11st day phenylalanine 180mg/L,
Yeast inducer 180mg/L；
(4) in culture the 1st~31 day, Siraitia grosvenorii suspension cell is every other day extracted, detects mogroside Ⅴ and arhat
II content of fruit saponin.
Comparative example 3
This comparative example is used to evaluate the skill that the technical solution of individually addition yeast inducer is obtained with technical solution of the present invention
Art difference on effect, is as follows：
(1) Mature Embryos Among for taking Siraitia grosvenorii is containing sucrose 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate
0.5mg/L, inositol 100mg/L, agar 4.6g/L, pH ranging from 5.5~6.0 MS semisolid culturemediums on squamous subculture, choosing
It takes continuous subculture 4 times and growth is vigorous, quality is loose, in stable condition uniform fresh embryo callus；
(2) by fresh embryo callus that step (1) obtains with inoculum concentration 100g/L be transferred to containing sucrose 30g/L,
The MS Liquid Cultures of 6- benzyl aminoadenines 1.0mg/L, methyl α-naphthyl acetate 0.5mg/L, inositol 100mg/L, pH ranging from 5.5~6.5
In base, and in the shaking table that rotating speed is 150r/min, cultivation temperature is 25 ± 2 DEG C, light intensity 4000lux, light application time are 12h/d
Middle culture obtains primary Siraitia grosvenorii suspension cell system；
(3) ripe Siraitia grosvenorii suspension cell is trained with primary Siraitia grosvenorii suspension cell system obtained by step (2)；It was cultivating
Yeast inducer 50mg/L is added within the 7th day in journey, yeast inducer 180mg/L is added within the 11st day；
(4) in culture the 1st~31 day, Siraitia grosvenorii suspension cell is every other day extracted, detects mogroside Ⅴ and arhat
II content of fruit saponin.
Comparative example 4
This comparative example is used to evaluate the skill that the technical solution of individually addition methyl jasmonate is obtained with technical solution of the present invention
Art difference on effect, is as follows：
(1) Mature Embryos Among for taking Siraitia grosvenorii is containing sucrose 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate
0.5mg/L, inositol 100mg/L, agar 4.6g/L, pH ranging from 5.5~6.0 MS semisolid culturemediums on squamous subculture, choosing
It takes continuous subculture 4 times and growth is vigorous, quality is loose, in stable condition uniform fresh embryo callus；
(2) by fresh embryo callus that step (1) obtains with inoculum concentration 100g/L be transferred to containing sucrose 30g/L,
The MS Liquid Cultures of 6- benzyl aminoadenines 1.0mg/L, methyl α-naphthyl acetate 0.5mg/L, inositol 100mg/L, pH ranging from 5.5~6.5
In base, and in the shaking table that rotating speed is 150r/min, cultivation temperature is 25 ± 2 DEG C, light intensity 4000lux, light application time are 12h/d
Middle culture obtains primary Siraitia grosvenorii suspension cell system；
(3) ripe Siraitia grosvenorii suspension cell is trained with primary Siraitia grosvenorii suspension cell system obtained by step (2)；It was cultivating
100 μm of ol/L of methyl jasmonate are added within the 7th day in journey, 220 μm of ol/L of methyl jasmonate are added within the 11st day；
(4) in culture the 1st~31 day, Siraitia grosvenorii suspension cell is every other day extracted, detects mogroside Ⅴ and arhat
II content of fruit saponin.
Comparative example 5
This comparative example is used to evaluate the technique effect that technical solution disclosed in documents 1 is obtained with technical solution of the present invention
Difference is as follows：
(1) it is inoculated with and is induced Siraitia grosvenorii callus from stem segment, is containing sucrose 2.5%, 6- benzyl aminoadenines 0.2mg/
L, methyl α-naphthyl acetate 0.04mg/L, squamous subculture on the MS culture mediums that agar 4.0mg/L, pH are 5.8, choose continuous subculture 3 times or 4 times
Or 5 fresh, soft frangible yellow-white callus；
(2) callus that step (1) obtains is transferred to inoculum concentration 300g/l containing sucrose 4.0%, 6- benzyl amino
In the sterile triangular flask of the MS fluid nutrient mediums of adenine 1.0mg/L, methyl α-naphthyl acetate 0.02mg/L, initial medium pH be 5.5~
6.0, the 25 DEG C of dark condition low suspension cultures of shaken cultivation case for being placed in 120r/min are stood afterwards for 24 hours, by upper layer culture medium together with
Unicellular and small cell cluster is transferred to sterile empty triangular flask relaying persistent oscillation culture；
(3) it is retransferred once using same method after 3d, is inoculated into suspension cell after 40 μm of screen filtrations after 7d
Fresh culture relays persistent oscillation culture, is transferred in big triangular flask after reaching certain volume, continues to add with same method
Add culture medium shaken cultivation, the cell suspending culture solution until reaching a certain number of stable homogeneous, cell suspension cultures temperature
It is 25 ± 2 DEG C, light intensity 2000lux, light application time 12h/d, obtains primary Siraitia grosvenorii suspension cell system；
(4) in culture the 1st~31 day, Siraitia grosvenorii suspension cell is every other day extracted, detects mogroside Ⅴ and arhat
II content of fruit saponin.
Mogroside Ⅴ comparision contents
1. experimental method
1.5g Siraitia grosvenorii cellular dry weights are weighed, with 15:After 60% EtOH Sonicate extraction 40min is added in 1 solid-liquid ratio,
2h is extracted under 90 DEG C of slight boiling condition, merge extracting solution after extracting 3 times repeatedly is concentrated into Rotary Evaporators
5mL.The membrane filtration that concentrate is used to 0.22 μm, qualitative and quantitative analysis is carried out with high performance liquid chromatograph.
Chromatographic condition：Chromatographic column is reversed C18 columns, 4.6mm × 250mm；Mobile phase is water-acetonitrile=78:22, flow velocity
1.0mL/min；Detection wavelength 203nm.
2. result：Influence of the different disposal condition to mogroside Ⅴ yield in the unit interval the results are shown in Table 1, not exist together
Influence of the manage bar part to mogroside Ⅴ yield peak value (maximum value) the results are shown in Table 2.
Influence (n=5 of the 1 different disposal condition of table to mogroside Ⅴ yield in the unit intervalg/L)
| Embodiment 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | |
| 1d | 31.1±0.8 | 31.4±0.6 | 32.6±0.5 | 31.5±0.5 | 31.1±0.8 | 30.0±1.0 |
| 3d | 32.6±1.2 | 31.9±1.4 | 33.0±1.6 | 31.8±2.1 | 32.6±0.9 | 31.6±0.6 |
| 5d | 33.6±1.1 | 32.9±1.2 | 33.2±1.4 | 32.8±1.8 | 33.2±1.1 | 32.2±0.8 |
| 7d | 34.2±1.4 | 33.2±1.1 | 34.2±1.8 | 33.0±1.6 | 33.5±1.5 | 33.6±1.2 |
| 9d | 43.5±2.1 | 33.8±1.3 | 35.6±1.5 | 33.6±1.4 | 39.5±2.0 | 39.8±1.5 |
| 11d | 52.6±1.8 | 34.2±1.6 | 36.8±1.6 | 34.0±1.2 | 46.5±1.8 | 45.5±1.7 |
| 13d | 64.8±2.0 | 34.7±1.5 | 41.5±2.0 | 34.5±2.2 | 51.6±1.8 | 50.5±2.2 |
| 15d | 73.8±2.8 | 35.2±2.2 | 47.5±2.6 | 36.8±1.6 | 53.6±1.5 | 56.4±2.4 |
| 17d | 79.9±2.6 | 36.2±2.1 | 53.4±2.2 | 43.5±1.8 | 54.5±1.6 | 60.8±2.8 |
| 19d | 85.2±2.5 | 37.8±2.4 | 60.8±2.1 | 49.5±2.0 | 55.7±1.8 | 67.5±2.5 |
| 21d | 79.5±2.6 | 39.4±2.5 | 67.2±2.3 | 55.4±2.2 | 56.8±2.0 | 70.7±3.1 |
| 23d | 78.6±2.8 | 52.6±3.0 | 72.0±2.8 | 61.8±2.6 | 58.2±2.2 | 69.2±3.2 |
| 25d | 76.5±2.5 | 64.8±2.8 | 75.6±2.6 | 68.5±2.5 | 55.2±2.4 | 65.8±3.0 |
| 27d | 74.2±2.6 | 73.8±2.5 | 77.2±2.8 | 71.8±3.0 | 51.4±2.5 | 63.6±2.8 |
| 29d | 72.1±3.1 | 75.9±2.9 | 79.0±2.5 | 72.2±2.7 | 46.5±2.4 | 59.4±3.2 |
| 31d | 68.2±2.8 | 76.2±2.6 | 78.8±2.5 | 72.0±2.8 | 41.2±2.5 | 55.6±2.8 |
Influence (n=5 of the 2 different disposal condition of table to mogroside Ⅴ yield peak value (maximum value)；g/L)
| Mogroside Ⅴ yield peak value (maximum value) | |
| Embodiment 1 | 85.2±2.5 |
| Embodiment 2 | 84.9±1.8 |
| Embodiment 3 | 86.7±2.2 |
| Embodiment 4 | 85.1±3.0 |
| Comparative example 1 | 76.2±2.6 |
| Comparative example 2 | 79.0±2.5 |
| Comparative example 3 | 72.2±2.7 |
| Comparative example 4 | 58.2±2.2 |
| Comparative example 5 | 70.7±3.1 |
II comparision contents of mogrosides
1. experimental method
1.5g Siraitia grosvenorii cellular dry weights are weighed, with 15:After 60% EtOH Sonicate extraction 40min is added in 1 solid-liquid ratio,
2h is extracted under 90 DEG C of slight boiling condition, merge extracting solution after extracting 3 times repeatedly is concentrated into Rotary Evaporators
5mL.The membrane filtration that concentrate is used to 0.22 μm, qualitative and quantitative analysis is carried out with high performance liquid chromatograph.
Chromatographic condition：Chromatographic column is ZORBAX SBC C18 columns, 4.6mm × 150mm；Mobile phase is that water-acetonitrile gradient is washed
De- (0~25min, 13.5%~35% acetonitrile), flow velocity 0.8mL/min；Detection wavelength 203nm.
2. result：The influence of II content of triterpene glucoside the results are shown in Table 3 when different disposal condition is to sweet tea glucoside v peak values.
Influence (the n=5 of II content of triterpene glucoside when 3 different disposal condition of table is to sweet tea glucoside v peak valuesg/L)
| II content of mogrosides | |
| Embodiment 1 | 17.2±2.1 |
| Embodiment 2 | 17.6±2.0 |
| Embodiment 3 | 18.5±1.4 |
| Embodiment 4 | 18.3±2.6 |
| Comparative example 1 | 42.6±4.3 |
| Comparative example 2 | 39.8±3.8 |
| Comparative example 3 | 40.2±2.2 |
| Comparative example 4 | 32.8±2.6 |
| Comparative example 5 | 30.4±1.6 |
Conclusion
Mogroside Ⅴ comparision contents result (table 1, table 2) is shown：Compared with comparative example 1~5, Examples 1 to 4
Mogroside Ⅴ content significantly improves, when peak value is 85.2 respectively, 84.9 ± 1.8,86.7 ± 2.2,85.1 ± 3.0g/L,
Illustrate, by adding yeast inducer and methyl jasmonate in Siraitia grosvenorii plant cell suspension cultures, Siraitia grosvenorii can be effectively improved
The yield of sweet tea glucoside V, technique effect are substantially better than the prior art.At the same time, compared with comparative example 1~5, embodiment 1 synthesizes
The rate of mogroside Ⅴ significantly improves, and so that mogroside Ⅴ content is higher than 70g/L in culture 15d, in cultivating 19d
Mogroside Ⅴ content is set to reach peak value.Illustrate by Siraitia grosvenorii plant cell suspension cultures add yeast inducer and
Methyl jasmonate, can effectively improve the rate of Siraitia grosvenorii suspension cell synthesis mogroside Ⅴ, and technique effect is substantially better than existing
There is technology.
II comparision contents result (table 3) of mogrosides is shown, compared with comparative example 1~5, the arhat of Examples 1 to 5
II content of fruit saponin is substantially reduced, and is illustrated by adding yeast inducer and jasmonic in Siraitia grosvenorii plant cell suspension cultures
Methyl esters, can effectively reduce the content of bitter principle mogrosides II, and technique effect is substantially better than the prior art.
Although above having used general explanation, specific implementation mode and experiment, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (7)
1. a kind of method improving mogroside Ⅴ content in Siraitia grosvenorii suspension cell, which is characterized in that with primary arhat
During fruit suspension cell system culture maturation Siraitia grosvenorii suspension cell, yeast inducer and methyl jasmonate is added.
2. according to the method described in claim 1, it is characterized in that, being trained with primary Siraitia grosvenorii suspension cell system described
Be added within the 5th~7 day during ripe Siraitia grosvenorii suspension cell culture maturation Siraitia grosvenorii suspension cell yeast inducer 10~50mg/L,
50~100 μm of ol/L of methyl jasmonate, the 9th~11 day 100~200mg/L of addition yeast inducer, methyl jasmonate 120~
260 μm of ol/L, the ripe Siraitia grosvenorii suspension cell of the 17th~25 day harvest.
3. method according to claim 1, which is characterized in that it is described be trained with primary Siraitia grosvenorii suspension cell system it is ripe
It is 50mg/L, jasmonic that the 7th day during Siraitia grosvenorii suspension cell culture maturation Siraitia grosvenorii suspension cell, which is added yeast inducer,
Methyl esters 100 μm of ol/L, be added within the 11st day yeast inducer be 180mg/L, 220 μm of ol/L of methyl jasmonate, the 19th day harvest at
Ripe Siraitia grosvenorii suspension cell.
4. according to any method of claims 1 to 3, which is characterized in that the primary Siraitia grosvenorii suspension cell system is logical
Cross following steps acquisition：
(1) Mature Embryos Among for taking Siraitia grosvenorii is containing sucrose 30g/L, 6- benzyl aminoadenine 1.0mg/L, methyl α-naphthyl acetate 0.5mg/
L, squamous subculture on the MS semisolid culturemediums of inositol 100mg/L, agar 4.6g/L, pH ranging from 5.5~6.0 is chosen continuous
Subculture 3~5 times and growth is vigorous, quality is loose, in stable condition uniform fresh embryo callus；
(2) the fresh embryo callus that step (1) obtains is transferred to inoculum concentration 100g/L containing sucrose 30g/L, 6- benzyl
Aminoadenine 1.0mg/L, methyl α-naphthyl acetate 0.5mg/L, inositol 100mg/L, pH ranging from 5.5~6.5 MS fluid nutrient mediums in,
And it is trained in the shaking table that rotating speed is 150r/min, cultivation temperature is 25 ± 2 DEG C, light intensity 4000lux, light application time are 12h/d
It supports, obtains primary Siraitia grosvenorii suspension cell system.
5. method according to claim 4, which is characterized in that step (1) subculture number is 4 times.
6. a kind of preparation method of mogroside Ⅴ, which is characterized in that with claims 1 to 3,5 either method prepare at
Ripe Siraitia grosvenorii suspension cell further extracts separation and prepares mogroside Ⅴ.
7. a kind of preparation method of mogroside Ⅴ, which is characterized in that thin with ripe arhat fruit suspension prepared by claim 4
Born of the same parents further extract separation and prepare mogroside Ⅴ.
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