WO2019136924A1

WO2019136924A1 - Method for promoting suspension cell growth of momordica grosvenori and improving content of mogroside v by means of salt stress

Info

Publication number: WO2019136924A1
Application number: PCT/CN2018/091294
Authority: WO
Inventors: 郭美锦; 宋云飞; 陈雨霞; 王泽建; 李�杰; 杨文国; 庄英萍; 储炬; 肖慈英
Original assignee: 华东理工大学; 桂林莱茵生物科技股份有限公司
Priority date: 2018-01-15
Filing date: 2018-06-14
Publication date: 2019-07-18
Also published as: CN108251349A; CN108251349B

Abstract

A method for promoting the suspension cell growth of momordica grosvenori and improving the content of mogroside V by means of salt stress, comprising: controlling the concentration of potassium ions to be 100-2,000 mg/kg in a process of culturing ripe momordica grosvenori suspension cells by means of a primary momordica grosvenori suspension cell system, the concentration ratio of potassium and sodium ions being (5-20):1. According to the method, the growth amount and the specific growth rate of momordica grosvenori and the yield and synthesis rate of the mogroside V are improved, and momordica grosvenori suspension cell growth and large-scale market production of the mogroside V are facilitated. In addition, the production and culture cycle of cells is effectively shortened, and the biomass of the momordica grosvenori suspension cells and the production process of the mogroside V are regulated manually, so that quality control and management of the product can be facilitated.

Description

Method for promoting growth of suspension cells of mangosteen by salt stress and enhancing content of sweet glycoside V

Technical field

The invention relates to the field of biotechnology, in particular to a method for promoting the growth of suspension cells of mangosteen by salt stress and increasing the content of sweet and glucoside V.

Background technique

Mangosteen sweet 甙V, also known as mogroside, is the main sweet ingredient of grosvenor grosvenor, and its sweetness is 350 times that of sucrose. Seven kinds of monomer components have been identified, among which Luo Han Guo sweet 甙V and match The glucoside I has the strongest sweetness. Mangosteen sweet 甙V has the medicinal effect of clearing away heat and moistening the lungs, opening the sound of pharynx, and relaxing the bowels. At the same time, the sweet-scented scent of Luo Han Guo also has the characteristics of novel taste and pure taste, which makes it deeply loved by people and is obese. The most suitable sweeteners and health supplements for special populations such as patients with hypertension, diabetes, etc., have a wide range of applications. At present, the method for obtaining the Luo Han Guo sweet 甙V is mainly prepared by using natural Luo Han Guo as a raw material and extracting by heating or the like. However, the intrinsic Luo Han Guo sweet 甙V component in natural Luo Han Guo is extremely low. The utilization rate of Luo Han Guo and the extraction rate of Luo Han Guo sweet 甙 V are not high in the prior art, which causes great waste of raw materials and cannot meet the growing Market demand.

Plant cell suspension culture refers to the formation of a callus or other easily dispersible tissue by a hormone-induced control of a part of the plant tissue in vitro, and then inoculated into a liquid medium to form a uniform liquid suspension culture system. a technique. Because of the pluripotency of the cells, in principle, any part of the plant explants can be used to induce callus formation on a suitable medium to form a suspension culture system, which is also the most important way to establish a plant suspension culture system.

At present, the method for obtaining Luo Han Guo sweet 甙V is mainly based on natural Luo Han Guo, and is prepared by heating and the like. Most of these natural products belong to the secondary metabolites of plants, and the content in plants is generally low, directly from Plant extraction not only occupies large areas of cultivated land, but may also cause the extinction of some rare and rare plant species. The production of secondary metabolites by plant cell culture technology is one of the effective means to increase the content of target components in natural plants. In recent years, it has received extensive attention, and cell culture is gradually replacing the traditional agricultural planting methods of these plants, alleviating the production pressure of secondary metabolites in the industry. Cell suspension culture is characterized by rapid propagation, large scale of culture, and the provision of a large number of uniform plant cell cultures. Salt stress usually causes ionic imbalance, oxidative stress and osmotic stress on plants, and salt concentration may lead to plant death. In the process of adapting to salt stress, plants gradually evolved a series of metabolic mechanisms and network regulation systems such as ion selective absorption/exhaustion, Na ⁺ compartmentalization, detoxification by antioxidant system, and accumulation of osmotic protective substances. Plant suspension cells should respond to salt stress, and selective absorption/excretion and compartmentalization of Na ⁺ play an important role in plant response to salt stress. The most obvious physiological response of Suspension cells in stress conditions is biomass and mogroside V, which are the final result of a series of physiological and biochemical responses of cells, which are caused by changes in the metabolic network. There are many factors that cause changes in the metabolic network, which may be due to the blockade of metabolic competition pathways, changes in key enzyme activities of metabolic pathways, regulation of metabolic regulators, changes in intracellular oxidative stress states, or changes in key gene expression. of.

Na ⁺ is the main ion causing plant salt damage and producing salty habitats. K ⁺ is a large number of elements and important osmotic adjustment components necessary for plant growth and development. However, due to the similar radii and hydration energy of these two ions, Na ⁺ exhibits a significant competitive inhibition of K ⁺ absorption. Therefore, the degree of selective absorption of potassium and sodium ions is an important factor affecting the growth ability of plants. At home and abroad, a lot of researches have been done on the physiological, biochemical and molecular biological response mechanisms of salt in trees. However, there are no reports on salt stress and potassium-sodium ratio on suspension cells of Luo Han Guo. Therefore, it is necessary to evaluate and compare the growth of Luo Han Guo suspension cells in different proportions of potassium and sodium ratio, the synthesis of mogroside, metal ions and organic acids, in order to provide reference for the application of subsequent amplification and product synthesis. .

Summary of the invention

The object of the present invention is to find a suitable potassium to sodium concentration to promote the growth of suspension cells and the accumulation of glycosides V. The method not only effectively shortens the production and culture cycle of the cells, but also increases the biomass of the suspension cells of the Luo Han Guo, and promotes the synthesis of the mogroside V in the suspension cells of the Luo Han Guo, and significantly improves the production efficiency.

The inventors of the present invention found that in the process of culturing mature Luo Han Guo suspension cells in the suspension system of the original Luo Han Guo suspension cell, the potassium ion content is kept unchanged in the initial medium, and the suspension of the Luo Han Guo suspension cells can be improved to different degrees by controlling the ratio of potassium to sodium in the medium. The biomass, while accelerating the synthesis rate of mogroside V in suspension cells of Siraitia grosvenorii.

The content of the present invention can be implemented by the following technical solutions:

1. A method for promoting the growth of Suspension cells and increasing the content of glucosinolate V by salt stress. The potassium ion concentration in the control system is 100-2000mg/kg, potassium and sodium during the cultivation of mature Luo Han Guo suspension cells in the suspension system of the original Luo Han Guo suspension cell system. The ion concentration ratio is 5 to 20:1.

Preferably, the potassium ion concentration is 1400 mg/kg.

Preferably, the potassium to sodium ion concentration ratio is 15:1.

Preferably, the potassium to sodium ion concentration ratio is controlled on days 0-8.

Preferably, the potassium-sodium ion concentration ratio is controlled on the first day.

Preferably, the potassium sodium ion concentration ratio is controlled using sodium sulfate.

Preferably, the Luo Han Guo suspension cells are harvested on the 15th to 25th day of culture.

Preferably, the Luo Han Guo suspension cells are harvested on the 21st day of culture.

2. A method for promoting growth of suspension cells and promoting a content of aglucoside V by salt stress, wherein the primary generation of Luo Han Guo suspension cell system is obtained by the following steps:

Step 1: Take the seed of Luo Han Guo, containing sucrose 25 ~ 35g / L, 6 - benzylamino adenine 0.05 ~ 0.15mg / L, naphthalene acetic acid 2 ~ 6mg / L, inositol 50 ~ 150mg / L, agar 4 ~ Subcultured on B5 solid medium with 6g/L and pH range of 5.9-6.0, the embryogenic callus was successively subcultured 3~5 times;

Step 2: Transfer the embryogenic callus obtained in the first step to an inoculation amount of 25-75 g/L to contain 20-40 g/L of sucrose, 0.05-0.15 mg/L of 6-benzylaminoadenine, and 3-5 mg of naphthaleneacetic acid. /L, inositol 50 ~ 150mg / L, pH range of 5.9 ~ 6.0 B5 liquid medium, and the speed of 80 ~ 150r / min, culture temperature of 24 ~ 26 ° C, dark shaker, obtained The initial generation of Luo Han Guo suspension cell system.

Preferably, the number of times of the step one is four times.

The invention overcomes the following difficult technology: keeping the potassium ion content in the culture medium unchanged, adding different content of sodium sulfate to control the ratio of potassium to sodium in the medium, and increasing the dry weight of the suspended cells of the Luo Han Guo in the unit time after the addition, the specific growth rate It has been improved, and the synthesis amount of mogroside V has increased, and the synthesis rate has also increased.

Compared with the prior art, the present invention has the following advantages:

1. The invention adopts the Luo Han Guo seed embryo as the culture raw material, and the differentiation and passage ability is stronger and more stable, and is favorable for cultivating the callus of Luo Han Guo which has strong growth and stable state.

2. The cell culture system of the Luo Han Guo obtained by the method of the invention has a significantly improved biomass of the suspended cells of the Luo Han Guo, and the yield of the mogroside V is also significantly improved.

3. The suspension system of the Luo Han Guo cell obtained by the method of the invention has higher cell growth rate, shorter culture cycle time, lowers the difficulty of operation and maintenance, and can better meet the market production demand.

4. The method of the invention artificially regulates the biomass of Suspension Suspension cells and the production process of Mogroside V, which is beneficial to the quality control management of the product.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 Luo Han Guo suspension cell growth curve

Fig.2 Effect of different treatment conditions on dry weight and specific growth rate of suspension cells of Siraitia grosvenorii

Fig.3 Changes of organic acids during suspension culture of Luohanguo

Figure 4 MVA pathway and MEP pathway

Detailed ways

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

Step 1: Take the mature embryo of Mangosteen, B5 containing sucrose 25g/L, 6-benzylaminoadenine 0.05mg/L, naphthaleneacetic acid 2mg/L, inositol 50mg/L, agar 4g/L, pH 5.9 Subculture on solid medium, fresh embryogenic callus with continuous subculture 3 times and vigorous growth, loose texture and stable state;

Step 2: Transfer the fresh embryogenic callus obtained in the first step to an inoculation amount of 25 g/L to contain 20 g/L of sucrose, 0.05 mg/L of 6-benzylaminoadenine, 3 mg/L of naphthaleneacetic acid, 50 mg of inositol/ L. The B5 liquid medium with a pH of 5.9 was cultured in a shaker at a rotation speed of 80 r/min and a culture temperature of 24 ° C in a dark shaker to obtain a primary cell suspension system of Luo Han Guo.

Step 3: The mature Suerhansi suspension cell system is cultured in the first generation of Luo Han Guo suspension cell system obtained in the second step; the ratio of potassium to sodium in the original medium is 25:1, the potassium ion concentration is controlled to 720 mg/kg, and the sodium sulfate is added on the 0th day in the culture process. , the ratio of potassium to sodium ions in the medium is 5:1;

Step 4: On the 9th, 12th and 15th day, the suspension cells of Luo Han Guo were extracted, and the dry weight of Suspension suspension cells, the content of organic acid in the medium and the content of Mogroside IV were detected.

Step 5: Harvesting Luo Han Guo suspension cells on the 15th day of culture.

Example 2

Step 1: Take the embryo of Luo Han Guo, B5 solid containing 35g/L sucrose, 0.15mg/L 6-benzylaminoadenine, 6mg/L naphthaleneacetic acid, 150mg/L inositol, 6g/L agar, pH 6.0 Subculture on the medium, and embryogenic callus of 5 consecutive passages was selected;

Step 2: Transfer the embryogenic callus obtained in step 1 to an inoculation amount of 75 g/L to 40 g/L of sucrose, 0.15 mg/L of 6-benzylaminoadenine, 5 mg/L of naphthaleneacetic acid, and 150 mg/L of inositol. In the B5 liquid medium of pH 6.0, and cultured in a shaker at a rotation speed of 150r/min and a culture temperature of 26 ° C, a suspension cell system of the first generation of Luo Han Guo was obtained.

Step 3: The mature Suerhansi suspension cell system was cultured in the first generation of Luo Han Guo suspension cell system in step 2. The ratio of potassium to sodium in the original medium was 25:1, the potassium ion concentration was controlled at 700 mg/kg, and sodium nitrate was added on the 0th day of the culture. , the ratio of potassium to sodium ions in the medium is 10:1;

Step 5: Harvesting the suspension of Luo Han Guo on the 21st day.

Example 3

Step 1: Take the mature embryo of Mangosteen, containing 30g/L of sucrose, 0.1mg/L of 6-benzylaminoadenine, 4.0mg/L of naphthaleneacetic acid, 100mg/L of inositol, 5.0g/L of agar, pH range Hybrid subculture on 5.9 B5 solid medium, fresh embryogenic callus with continuous subculture 4 times and vigorous growth, loose texture and stable state;

Step 2: Transfer the fresh embryogenic callus obtained in step 1 to 50 g/L inoculated with sucrose 30 g/L, 6-benzylaminoadenine 0.1 mg/L, naphthaleneacetic acid 4.0 mg/L, inositol 100 mg. /L, pH range of 5.9 in B5 liquid medium, and the rotation rate of 110r / min, culture temperature of 25 ° C, dark shaker, to obtain the initial generation of Luo Han Guo suspension cell system;

Step 3: The mature Suerhansi suspension cell system was cultured in the first generation of Luo Han Guo suspension cell system obtained in the second step; the ratio of potassium to sodium in the original medium was 25:1, the potassium ion concentration was controlled to be 670 mg/kg, and the chlorination was added on the 0th day in the culture process. Sodium, the ratio of potassium to sodium ion concentration in the medium is 15:1;

Step 5: The suspension of Luo Han Guo suspension cells was harvested on the 25th day of culture.

Example 4

Step 2: Transfer the fresh embryogenic callus obtained in step 1 to 50 g/L inoculated with sucrose 30 g/L, 6-benzylaminoadenine 0.1 mg/L, naphthaleneacetic acid 4.0 mg/L, inositol 100 mg. /L, pH range of 5.9 B5 liquid medium, and the rotation rate of 110r / min, culture temperature of 26 ° C, dark shaker, to obtain the initial generation of Luo Han Guo suspension cell system;

Step 3: The mature Suerhansi suspension cell system is cultured in the first generation of Luo Han Guo suspension cell system obtained in the second step; the ratio of potassium to sodium in the original medium is 25:1, the potassium ion concentration is controlled to be 1400 mg/kg, and the sodium phosphate is added on the third day in the culture process. The ratio of potassium to sodium ions in the medium was 20:1.

Step 4: On the 9th, 12th and 15th day, the suspension cells of Siraitia grosvenii were extracted, and the dry weight of the suspension cells, the organic acid content in the medium and the content of the mogroside V were measured.

Step 5: Harvesting the Luo Han Guo suspension cells on the 20th day of culture.

Example 5

Step 1: Take the mature embryo of Mangosteen, containing 30g/L of sucrose, 0.1mg/L of 6-benzylaminoadenine, 4.0mg/L of naphthaleneacetic acid, 100mg/L of inositol, 5.0g/L of agar, pH range Hybrid subculture on B5 solid medium of 5.9, fresh embryogenic callus with continuous subculture 2 times and vigorous growth, loose texture and stable state;

Step 2: Transfer the fresh embryogenic callus obtained in step 1 to 50 g/L inoculated with sucrose 30 g/L, 6-benzylaminoadenine 0.1 mg/L, naphthaleneacetic acid 4.0 mg/L, inositol 100 mg. /L, pH range of 5.9 B5 liquid medium, and the rotation speed of 110r / min, culture temperature of 25 ° C, dark shaker, to obtain the initial generation of Luo Han Guo suspension cell system.

Step 3: The mature Suerhansi suspension cell system is cultured in the first generation of Luo Han Guo suspension cell system obtained in the second step; the ratio of potassium to sodium in the original medium is 25:1, the potassium ion concentration is controlled to 100 mg/kg, and the chlorination is added on the first day of the culture process. Sodium, the ratio of potassium to sodium ion concentration in the medium is 15:1;

Step 5: The suspension of Luo Han Guo suspension cells was harvested on the 30th day of culture.

Example 6

Step 1: Take the mature embryo of Mangosteen, containing 30g/L of sucrose, 0.1mg/L of 6-benzylaminoadenine, 4.0mg/L of naphthaleneacetic acid, 100mg/L of inositol, 5.0g/L of agar, pH range Hybrid subculture on B5 solid medium of 6.1, fresh embryogenic callus with continuous subculture 5 times and vigorous growth, loose texture and stable state;

Step 2: Transfer the fresh embryogenic callus obtained in step 1 to 50 g/L inoculated with sucrose 30 g/L, 6-benzylaminoadenine 0.1 mg/L, naphthaleneacetic acid 4.0 mg/L, inositol 100 mg. /L, pH range of 6.1 in B5 liquid medium, and the rotation rate of 110r / min, culture temperature of 27 ° C, dark shaker, to obtain the initial generation of Luo Han Guo suspension cell system.

Step 3: The mature Suerhansi suspension cell system is cultured in the first generation of Luo Han Guo suspension cell system obtained in the second step; the ratio of potassium to sodium in the original medium is 25:1, the potassium ion concentration is 2000 mg/kg, and sodium acetate is added on the third day in the culture process. The ratio of potassium to sodium ions in the medium is 8:1;

Step 5: Harvesting the suspension of Luo Han Guo on the 16th day of culture.

Example 7

Step 1: Take the mature embryo of Mangosteen, containing 30g/L of sucrose, 0.1mg/L of 6-benzylaminoadenine, 4.0mg/L of naphthaleneacetic acid, 100mg/L of inositol, 5.0g/L of agar, pH range Hybrid subculture on 5.8 B5 solid medium, fresh embryogenic callus with continuous subculture 3 times and vigorous growth, loose texture and stable state;

Step 2: Transfer the fresh embryogenic callus obtained in step 1 to 50 g/L inoculated with sucrose 30 g/L, 6-benzylaminoadenine 0.1 mg/L, naphthaleneacetic acid 4.0 mg/L, inositol 100 mg. /L, pH range of 6.0 B5 liquid medium, and the rotation rate of 110r / min, culture temperature of 24 ° C, dark shaker, to obtain the first generation of Luo Han Guo suspension cell system.

Step 3: The mature Suerhansi suspension cell system is cultured in the first generation of Luo Han Guo suspension cell system obtained in the second step; the ratio of potassium to sodium in the original medium is 25:1, the potassium ion concentration is 1000 mg/kg, and the sodium phosphite is added on the 8th day in the culture process. , the ratio of potassium to sodium ions in the medium is 18:1;

Step 5: Harvesting the suspension of Luo Han Guo on the 19th day.

Comparative example 1

The present comparative example is used to evaluate the technical effect difference between the technical solution of the uncontrolled potassium-sodium ion concentration ratio medium, that is, the potassium ion concentration of 1400 mg/kg and the potassium-sodium ion concentration ratio of 25:1, and the technical scheme of the present invention, and the specific steps. as follows:

1) Take the mature embryo of Mangosteen, containing 30g/L of sucrose, 0.1mg/L of 6-benzylaminoadenine, 4.0mg/L of naphthaleneacetic acid, 100mg/L of inositol, 5.0g/L of agar, pH range of 5.9 Hybrid subculture on B5 solid medium of ~6.0, fresh embryogenic callus with continuous subculture 4 times and vigorous growth, loose texture and stable state;

2) Transfer the fresh embryogenic callus obtained in step 1) to 50 g/L inoculated with sucrose 30 g/L, 6-benzylaminoadenine 0.1 mg/L, naphthaleneacetic acid 4.0 mg/L, inositol 100 mg. /L, pH range of 6.0 B5 liquid medium, and the rotation speed of 110r / min, culture temperature of 26 ° C, dark shaker, to obtain the initial generation of Luo Han Guo suspension cell system.

3) cultivating the mature Suerhansi suspension cells by the primary generation Luo Han Guo suspension cell system obtained in the step 2); no sodium salt is added as a blank control on the 0th day in the culture process;

4) On the 9th, 12th and 15th day, Suspension suspension cells were extracted to detect the dry weight of Suhales Suspension cells, the content of organic acids in the medium, the content of Mogroside V and the organic acids.

5) Harvesting of Luo Han Guo suspension cells on the 21st day of culture.

Table 1 Summary of extracellular potassium and sodium ion concentrations in different examples Unit: mg/kg

	实施例1Example 1	实施例2Example 2	实施例3Example 3	实施例4Example 4	实施例5Example 5	实施例6Example 6	实施例7Example 7	对比例1Comparative example 1
K ⁺ K ⁺	720720	700700	670670	14001400	100100	20002000	10001000	14001400
Na ⁺ Na ⁺	144144	7070	4545	4040	6.76.7	250250	5656	3232
K ⁺：Na ⁺ K ⁺ :Na ⁺	5:15:1	10：110:1	15：115:1	20：120:1	15：115:1	8：18:1	18：118:1	25：125:1

Physical and chemical indicators

1. Comparison of suspension cell growth of Luo Han Guo

1) Experimental method

Shake the culture solution, pour it into a Buchner funnel, vacuum filter, and rinse the cells three times with ultrapure water until the filter paper is no longer dripping. The filtered cells were collected, weighed, and dried at 60 ° C, and weighed to determine the cell specific growth rate.

2) Results are shown in Figure 1 and Figure 2

It can be seen from Fig. 1 that the growth cycle of Suspension suspension cell culture is about 30 days. In the first 3 days of culture, the cells grew slowly and had a significant lag period. After 3 days, the biomass increased rapidly, which was the logarithmic growth phase. The dry weight on the 21st day reached the highest value at 14.4 g/L. About 7 times the initial stage. After 21 days, due to insufficient nutrients, cell growth was limited and dry weight began to decline. The accumulation of mogroside V was approximately synchronous with cell growth, with a maximum of 0.3986 mg/g DCW. From the specific growth rate, the specific growth rate of Suspension suspension cells in the first 9 days has been increasing, but the specific growth rate has dropped sharply after the ninth day, so in order to ensure that the Suspension suspension cells have maintained a high specific growth rate, It is only necessary to consider the corresponding dry weight, specific growth rate and content of mogroside V on the 9th, 12th and 15th days. Such a method greatly shortens the cultivation time and facilitates the progress of the experiment.

Table 2 Effect of different treatment conditions on the dry weight of Suspension suspension cells: g/L

It can be seen from Table 2 that the level of biomass is an indicator reflecting the comprehensive salt tolerance of plants. The growth of Suspension suspension cells after each condition was listed in Table 2. It can be seen from Table 2 that when K ⁺ :Na ^{+ is} lower than 20:1 in the initial medium, the biomass of Suspension suspension cells is higher than that of the control, and increases with the increase of the ratio, when K ⁺ :Na ⁺ is 15: 1. The dry weight of the cells reached a maximum of 11.663 g/L on the 15th day, which was about 2 times that of the control. When the K ⁺ :Na ⁺ in the initial medium exceeded 20:1, the growth of Suspension suspension cells was inhibited, and the biomass on the 15th day was only 6.020 g/L. K ⁺ :Na ⁺ is related to the mechanism of water transport in plant cells. Some literatures have suggested the effect of salt stress on the initial direct effects of plants, that is, growth is related to water supply during salt stress. Under stress conditions, the cells consume additional sucrose in order to withstand adverse external conditions, and are used for maintenance damage repair, ion transport, and product synthesis.

Table 3 Effect of different treatment conditions on the growth rate of suspension cells of Siraitia grosvenorii

As can be seen from Table 3, the suspension growth of different K ⁺ :Na ⁺ in the initial medium was higher than that of the blank control; when the K ⁺ :Na ⁺ ratio was less than 20:1, with the initial stage The ratio of K ⁺ :Na ^{+ in the} medium increased, and the growth rate of cells under different conditions increased gradually. For the same K ⁺ :Na ⁺ ratio, the specific growth rate of 15d was higher than the specific growth rate of 12d. The blank control showed a downward trend.

2. Comparison of the content of mogroside V

1) Experimental method

The suspension cultured cells were washed 3 times with deionized water, dried at 70 ° C, crushed with roller mash, firstly leached with 1:10 methanol, placed on a 110 rpm shaker for 3 h, and then ultrasonically extracted for 40 min. The supernatant was concentrated to 1 ml, filtered through a 0.22 μm filter, and detected by liquid chromatography in a university. High performance liquid chromatography conditions: flow rate 1.0 mL/min, mobile phase: water: acetonitrile = 77:23, column temperature 30 ° C, detection wavelength 203 nm. 2) Results: The effects of different treatment conditions on mogroside V are shown in Table 4.

Table 4 Effect of different treatment conditions on mogroside V: mg/gDCW

As can be seen from Table 4, compared with Comparative Example 1, the content of mogroside V of Examples 1 to 7 was significantly increased, and the increase trend of dry weight was almost the same, which indicated that the medium was changed in the initial stage of Suspension suspension cell culture system. The potassium to sodium ratio can effectively increase the production of mogroside V. The reason may be that salt stress causes plant cells to make a series of stress responses in order to adapt to the environment. When the concentration of K ⁺ :Na ⁺ is 15:1, the production of mogroside V is the highest, indicating that some important enzyme activities may be inhibited or damaged during the process of synthesizing the glycosidic V metabolism, or salt stress may change the Luo Han Guo. The energy distribution form in suspension cells, the energy originally used for cell growth and product synthesis is mostly used for osmotic adjustment, such as the accumulation of inorganic ions in vacuoles and the synthesis of other organic osmotic adjustment substances, so the growth of Luo Han Guo cells is obviously inhibited. And product synthesis is also inhibited. Other ratios of sodium and potassium ions also significantly increased the production of mogroside.

Comparison of potassium and sodium ion absorption and transportation in suspension culture of Siraitia grosvenorii by salt stress

1) Experimental method

The fermentation broth was centrifuged to leave the supernatant for use. Ashed in the muffle furnace. The ash was dissolved in nitric acid, fixed in deionized water, placed in a 50 ml flask, and the intracellular and extracellular K ⁺ and Na ⁺ contents were determined by plasma emission spectrometry to calculate K ⁺ :Na ⁺ , and K ⁺ , Na ⁺ absorption selectivity coefficient.

S _{(K, Na absorption)} = (K _cells / Na _cells ) / (K _medium / Na _medium )

2) Results: The effects of different treatment conditions on the content of potassium, sodium and magnesium ions and the ratio of potassium to sodium in the suspension cell culture medium of Luo Han Guo are shown in Table 5.

Effects of Different Treatment Conditions on Potassium, Sodium and Magnesium Contents and Potassium-Sodium Ratio in Suspension Cell Culture Medium of Table 5 at Table 12

Unit: mg/Kg

It can be seen from Table 5 that with the increase of stress intensity, the salt stress caused the Na ⁺ concentration in the suspension cells of Luo Han Guo to increase significantly, while the K ⁺ concentration increased first and then decreased; while the extracellular Na ⁺ concentration also showed an upward trend, while the K ⁺ concentration first Increase after the decline. Salt stress also led to a decrease in extracellular K ⁺ :Na ⁺ with increasing salt concentration. High concentrations of salt stress resulted in decreased ability of cells to reject Na ⁺ , absorb K ⁺ and selectively transport K ⁺ , which increased intracellular Na ⁺ content and increased intracellular K ⁺ :Na ⁺ ratio. It is indicated that salt stress can cause ion balance imbalance. Biomass allocation strategy is one of the adaptation mechanisms of plants under salt stress. The degree of selective absorption can indirectly reflect the ability of plants to utilize K ⁺ . It can be found that when extracellular K ⁺ :Na ⁺ is 17, cell biology The highest amount is obtained, and the selective absorption coefficient S _{(K, Na absorption) is the} largest; and when K ⁺ :Na ⁺ >17, that is, when the Na ⁺ content in the medium is low, the selective absorption coefficient S _{(K, Na absorption) is} significant. Lowering, but not conducive to the growth of cells. Na ⁺ is the main ion causing plant salt damage. K ⁺ is a large number of elements and important osmotic adjustment components necessary for plant growth and development. Na ⁺ has obvious competitive inhibition on K ⁺ absorption, while cells are K ⁺ The degree of absorption can indirectly reflect the growth state of the cells. The larger the S _{(K, Na absorption)} , the stronger the ability of cells to reject Na ⁺ and absorb K ⁺ , which is more conducive to cell growth. In a salt environment, higher concentrations of Na ⁺ often inhibit K ⁺ transporters, thereby reducing K ⁺ uptake by cells, while maintaining a suitable K ⁺ :Na ⁺ ratio is one of the important ways in which cells adapt to salt stress.

Comparison of organic acid content in suspension culture of Siraitia grosvenorii by salt stress

1) Experimental method

The fermentation broth was centrifuged to retain the supernatant, and the supernatant was passed through a 0.22 um filter for use. The content of organic acid in the fermentation broth was determined by high performance liquid chromatography. The hydrogen column had a mobile phase of 5 mM sulfuric acid, a flow rate of 0.4 ml/min, a column temperature of 50 ° C, and an ultraviolet detection wavelength of 210 nm. The standard concentrations of α-ketoglutaric acid, citric acid, malic acid, oxaloacetic acid, pyruvic acid, fumaric acid, and acetic acid are 5g/L, 5g/L, 5g/L, 1g/L, 1g/L, and 1g, respectively. /L, 1g/L.

2) Results: The effects of different treatment conditions on the content of organic acids in the suspension cell culture medium of Luo Han Guo are shown in Table 7. The changes of organic acids during the growth of Suspension suspension cells are shown in Figure 3, and the MVA pathway and MEP pathway are shown in Figure 4.

The concentration of various organic acid standards has a good linear relationship with the peak area. From the peak of the suspension of Luo Han Guo suspension cells, the peak of the peak material is positively high at around 11.8, but it cannot match the peak time of the standard. It can be judged that this substance is not an organic acid. At the peak time of about 17.6min and about 26.3min, respectively, pyruvic acid and acetic acid, the pyruvic acid and acetic acid in the supernatant can be clearly detected.

As can be seen from Fig. 3, pyruvic acid accumulates slowly at the beginning of cell growth. When it grows to about 12d, the extracellular pyruvic acid content peaks, and then the extracellular pyruvate concentration decreases significantly. The acetic acid content is in the early stage of cell growth. The sharp decrease, reaching the lowest value around the 12th day, after which the acetic acid content began to increase, which may be because the "defense reaction" of the suspension of the Luo Han Guo suspension cells to adapt to the environment is the growth state in which the cells can reach equilibrium in the culture environment.

For Siraitia suspensa suspension cells, mogroside V is a dammarane-type tetracyclic triterpenoid which undergoes the EMP pathway, the MVA pathway and the MEP pathway. As shown in Figure 4, Suspension suspense cells are metabolized into glucose and fructose by sucrose, and pyruvate is produced by glycolytic pathway. Part of pyruvic acid is secreted extracellularly, and part of it enters mitochondria in the presence of oxygen and mitochondria, and dehydrogenated by pyruvate. The enzyme complex catalyzes oxidative decarboxylation to produce NADH, CO ₂ and acetyl-CoA. A portion of acetyl-CoA enters the MVA pathway to form methylglutaryl-CoA AHMG-CoA). The formed HMG to CoA form mevalonate MVA under the action of a reductase, and the latter forms isopentenyl pyrophosphate in the catalytic action of various enzymes. The MEP pathway, also known as the deoxyxylulose phosphate pathway, uses the glycolysis intermediate metabolite pyruvate and glyceraldehyde 3-phosphate as precursors to form DXP under the action of synthase, which is then catalyzed by reductase and isomerase. Turned into a MEP.

Table 7 Effect of Potassium and Sodium Ratio on the Content of Organic Acids in Suspension Cell Culture Medium of Different Treatment Conditions

Therefore, according to Table 7, the contents of propionic acid and formic acid under different potassium and sodium ratios of Examples 1 to 7 are generally higher than blank, while the content of malic acid and fumaric acid are generally lower than blank, indicating that in order to adapt to the environment in the medium. Change will make a certain "defense response." No pyruvate was detected in the fermentation broth, indicating that the pyruvic acid was doubled and no more secreted into the extracellular. When the ratio of potassium to sodium in the medium was 15:1, the values of malic acid and fumaric acid reached the lowest value. At this time, the decomposition of pyruvic acid into the TCA cycle decreased, and the pyruvate into the pathway of secondary metabolites increased; And the content of formic acid increased, the trend of change first decreased and then increased, at 15:1, reached the lowest value, perhaps the amount of amino acid metabolism decreased, the amount of synthesis to the product increased, so that the content of glycoside V increased, and mangosteen The results in the comparison of the content of the glycoside V were consistent.

in conclusion

First, when K ⁺ :Na ^{+ is} less than 20:1 in the initial medium, the biomass of Suspension suspension cells is higher than that of the control, and increases with the increase of the ratio. When K ⁺ :Na ⁺ is 15:1, The dry weight of the cells reached a maximum of 11.663 g/L on day 15 and was about twice as high as the control. When the K ⁺ :Na ⁺ in the initial medium exceeded 20:1, the growth of Suspension suspension cells was inhibited, and the biomass on the 15th day was only 6.020 g/L. In the initial medium, different K ⁺ :Na ⁺ Suspension suspension cells maintained growth rate compared with the blank control; when the K ⁺ :Na ⁺ ratio was less than 20:1, with the initial medium K ⁺ : The ratio of Na ⁺ increased, the growth rate of cells under different conditions increased gradually; for the same K ⁺ :Na ⁺ ratio, the specific growth rate of 15d was higher than the specific growth rate of 12d, while the blank control showed a decrease. trend.

Secondly, compared with Comparative Example 1, the content of mogroside V in Examples 1-7 was significantly increased, and the increase trend of dry weight was almost the same, which indicated that the potassium and sodium in the medium were changed in the initial stage of Suspension suspension cell culture system. The ratio can effectively increase the yield of mogroside V. The reason may be that salt stress causes plant cells to make a series of stress responses in order to adapt to the environment. When the concentration of sodium sulfate is 15:1, the production of mogroside V is the highest.

Third, with the increase of stress intensity, salt stress caused a significant increase in Na ⁺ concentration in suspension cells of Luo Han Guo, while K ⁺ concentration increased first and then decreased; while extracellular Na ⁺ concentration also showed an upward trend, while K ⁺ concentration decreased first. increase. Salt stress also led to a decrease in extracellular K ⁺ :Na ⁺ with increasing salt concentration. When the extracellular K ⁺ :Na ⁺ was 17, the cell biomass reached the highest and the selective absorption coefficient S _{(K, Na absorption) was the} largest.

Fourth, the contents of propionic acid and formic acid under different potassium and sodium ratios of Examples 1 to 7 are generally higher than blank, while the content of malic acid and fumaric acid are generally lower than blank, indicating that in order to adapt to environmental changes in the medium, Make a certain "defense response." No pyruvate was detected in the fermentation broth, indicating that the pyruvic acid was doubled and no more secreted into the extracellular. When the ratio of potassium to sodium in the medium was 15:1, the values of malic acid and fumaric acid reached the lowest value. At this time, the decomposition of pyruvic acid into the TCA cycle decreased, and the pyruvate into the pathway of secondary metabolites increased; And the content of formic acid increased, the trend of change first decreased and then increased, at 15:1, reached the lowest value, perhaps the amount of amino acid metabolism decreased, the amount of synthesis to the product increased, so that the content of glycoside V increased, and Luo Han Guo The results in the comparison of the content of the glycoside V were consistent.

The invention explores a problem about the effect of salt stress on the growth of suspension cells and the accumulation of glycosides V, and provides a potassium-sodium ion concentration ratio which can increase the concentration and growth of mogroside V in suspension cells of Siraitia grosvenorii. The method of the invention artificially regulates the production process of the mogroside cells and the mogroside V, which is beneficial to the growth of the mogroside cells and the mass production and quality control management of the mogroside V, which has good economic value and broad application. prospect.

Although the present invention has been described in detail above with the aid of the general description, the specific embodiments and the examples of the invention, it may be obvious to those skilled in the art . Therefore, such modifications or improvements made without departing from the spirit of the invention are intended to be within the scope of the invention.

Claims

The invention relates to a method for promoting the growth of suspension cells of mangosteen by salt stress and enhancing the content of the sweet glycoside V, wherein the concentration of potassium ions in the control system is 100-2000 mg/kg during the cultivation of the mature suspension cells of the Luo Han Guo suspension cell system. The potassium to sodium ion concentration ratio is 5 to 20:1.
The method according to claim 1, wherein the potassium ion concentration is 1400 mg/kg, and the potassium ion concentration is 1400 mg/kg.
The method according to claim 1, wherein the potassium sulphate ion concentration ratio is 15:1.
The method according to claim 1, wherein the growth of the suspension cells of the mogroside is promoted by salt stress and the content of the sweet and sour V is promoted, wherein the potassium to sodium ion concentration ratio is controlled on the 0th to 8th days.
The method according to claim 1, wherein the growth of the suspension cells of the mogroside is promoted by salt stress and the content of the sweet and sour V is promoted, wherein the potassium-sodium ion concentration ratio is controlled on the first day.
The method according to claim 1, wherein the growth of the suspension cells of the mogroside is promoted by salt stress and the content of the sweet and sour V is promoted, wherein the potassium-sodium ion concentration ratio is controlled using sodium sulfate.
The method according to claim 1, wherein the growth of the suspension cells of the mogroside is promoted by salt stress and the content of the sweet glucoside V is increased, wherein the suspension of the Sussex fruit is harvested on the 15th to 25th day of the culture.
The method according to claim 1, wherein the growth of the suspension cells of the mogroside is promoted by salt stress and the content of the sweetover V is promoted, wherein the suspension of the Sussex fruit is harvested on the 21st day of culture.
The method according to any one of claims 1 to 8, wherein the growth of the suspension cells of the Luo Han Guo fruit by salt stress and the content of the sweet and sour V are promoted, wherein the primary generation of the Luo Han Guo suspension cell system is obtained by the following steps:

Step 1: Take the seed of Luo Han Guo, containing sucrose 25 ~ 35g / L, 6 - benzylamino adenine 0.05 ~ 0.15mg / L, naphthalene acetic acid 2 ~ 6mg / L, inositol 50 ~ 150mg / L, agar 4 ~ Subcultured on B5 solid medium with 6g/L and pH range of 5.9-6.0, the embryogenic callus was successively subcultured 3~5 times;

Step 2: Transfer the embryogenic callus obtained in the first step to an inoculation amount of 25-75 g/L to contain 20-40 g/L of sucrose, 0.05-0.15 mg/L of 6-benzylaminoadenine, and 3-5 mg of naphthaleneacetic acid. /L, inositol 50 ~ 150mg / L, pH range of 5.9 ~ 6.0 B5 liquid medium, and the speed of 80 ~ 150r / min, culture temperature of 24 ~ 26 ° C, dark shaker, obtained The initial generation of Luo Han Guo suspension cell system.
The method according to claim 9, wherein the step of promoting the growth of the suspension cells of the mogroside by the salt stress and increasing the content of the sweet and glucoside V is characterized in that the number of substeps is four times.