CN110684708A - A kind of method for enlarged cultivation of Luo Han Guo suspension cells - Google Patents

Abstract

The present invention provides a method for enlarged cultivation of Luo Han Guo suspended cells. The method adds two or more of phenylalanine, methyl jasmonate and betaine during the process of enlarged cultivation of Luo Han Guo suspended cells. The method overcomes the shortcomings of the existing Luo Han Guo suspension culture at the level of shaking flasks, low cell yield, and inability to carry out large-scale culture, and obtains the optimized culture medium and adding process for promoting product synthesis through screening, which greatly improves the The synthesis rate of Mogroside V was studied, which provided a certain technical method and basis for the larger-scale suspension culture of Luohanguo cells.

Description

A kind of method for enlarged cultivation of Luo Han Guo suspension cells

technical field

The invention relates to the field of biotechnology, in particular to a method for amplifying the suspension cells of Luo Han Guo.

Background technique

Siraitia grosvenorii, originating from the cucurbit family Momordica, is a precious medicinal and edible plant endemic to Guilin, Guangxi. Mogroside (mogroside) is the main active ingredient in Luo Han Guo, which has the functions of clearing away heat and moistening the lungs, soothing the throat, and relaxing the bowels. Recent studies have shown that Luo Han Guo also has antioxidant, anti-diabetic and anti-cancer properties. Monk fruit is loved by people because of its novel taste, pure taste and unique health care function. It is the best sweetener and health care product for patients with obesity, high blood pressure and diabetes, and has a wide range of application value. The existing method for obtaining Mogroside V is mainly through the method of extracting from Luo Han Guo, but the extraction yield from Luo Han Guo fruit is only 1%, and the extraction from fruit is affected by region and season. The second is to obtain mogroside V by suspension culture of Luo Han Guo cells.

The production of target secondary metabolites by plant cell culture technology is one of the effective means to increase the content of target components in natural plants. Cell suspension culture has the characteristics of fast propagation speed, large culture scale and providing a large number of uniform and uniform plant cell cultures. Therefore, the cultivation and production of mogroside V through plant cell culture technology can effectively increase the content of mogroside V and reduce the production cost, which is beneficial to the further marketization of mogroside V. For example, Chinese patent CN102174463B discloses "a method for culturing Luo Han Guo callus cell suspension system", and cultured Luo Han Guo suspension cells with total glycosides and Mogroside V accounting for 8.11% and 5.77% of the cell weight respectively. However, this method only stays at the level of laboratory shake flask fermentation and cannot be applied to large-scale industrial production.

The article "The Growth of Monk Fruit Suspension Culture Cells and the Kinetics of Mogroside Accumulation" published in "Chinese Journal of Experimental Prescriptions", 2011 No. 15, this study investigated the fresh weight, dry weight and dry weight of Monk fruit suspension cells in one culture cycle. The quality and mogroside content, the growth curve and the growth rate curve were drawn, and the dynamic change rule of product accumulation was clarified, but the research stayed at the laboratory level, and the scale-up culture of Luo Han Guo suspension cells could not be realized.

On the other hand, in the process of plant secondary metabolism, inducer, as a special biochemical molecule, can rapidly, specifically and selectively induce the expression of some specific genes, thereby increasing the activity of related enzymes in the metabolic pathway, thereby promoting the Or inhibit the production of secondary products regulated by the enzyme. Methyl jasmonate (MeJA) is an abiotic inducer that stimulates the accumulation of secondary metabolites. Adding methyl jasmonate to different plant suspension culture systems can induce an increase in the accumulation of secondary metabolites in cells. Maackia amurensis Rupr.etMaxim was treated with 200 μmol/L methyl jasmonate for 9 days, and the isoflavone content increased to 417.18% of the control at the same time; chinensis), the paclitaxel content increased 10-fold. However, the addition of methyl jasmonate to some plant suspension culture systems also easily induces allergic reactions in suspension cells or promotes the release of phenolic compounds, resulting in browning of suspension cells. When the kudzu suspension cells were treated with 1.0 mg/L methyl jasmonate, the content of puerarin in the cell culture was increased, but the cells were obviously browned. There is no report on the treatment of Luo Han Guo suspension cell culture system with phenylalanine, methyl jasmonate and betaine.

At present, there are few reports on the suspension culture of Luo Han Guo cells, and many of the current research results are only at the level of shake flasks. There has been no relevant reports on the suspension culture of Luo Han Guo cells at the reactor level, and how to maximize the rapid synthesis of products It has always been a difficult problem to be solved in suspension cell culture.

SUMMARY OF THE INVENTION

The invention provides a method for amplifying the cultivation of Luo Han Guo suspended cells. The synthesis and accumulation of the product mogroside V is promoted, which can be applied to a large-scale industrial production process. It overcomes the problems that the existing Luo Han Guo suspension culture stays at the level of shake flask, the cell yield is low, and large-scale culture cannot be carried out.

The object of the present invention can be realized through the following technical solutions:

A method for amplifying and culturing Luo Han Guo suspended cells, characterized in that, in the process of amplifying and culturing Luo Han Guo suspended cells, two or more of phenylalanine, methyl jasmonate and betaine are added;

Preferably, 250-1000 mg/L of phenylalanine, 5-50 mM methyl jasmonate, and 250-1000 mg/L of betaine are added in the process of the enlarged cultivation of the Luo Han Guo suspended cells;

Preferably, 450-550 mg/L of phenylalanine, 15-25 Mm methyl jasmonate, and 450-550 mg/L of betaine are added in the process of the enlarged cultivation of the Luo Han Guo suspended cells;

Preferably, the conditions for the enlarged cultivation of the Luo Han Guo suspension cells are: 20-30° C., aeration volume of 50-150 L/h, tank pressure of 0.01-0.03 MPa, rotation speed of 50-150 rpm, and culture time of 20-40 days;

Preferably, the conditions for the enlarged cultivation of the Luo Han Guo suspension cells are: 24-28° C., aeration volume of 80-100 L/h, tank pressure of 0.01-0.03 MPa, rotational speed of 100-120 rpm, and culture time of 25-35 days;

Preferably, the time for adding two or more of the phenylalanine, methyl jasmonate and betaine is the 13th to 16th day of the amplified culture;

Preferably, the liquid suspension culture system in the early stage of the amplification culture of the Luo Han Guo suspension cells is obtained through the following steps:

Step 1: Select the seed embryos of Luo Han Guo for disinfection treatment, rinse them after disinfection, cut the seed embryos out of the wound and add them to the solid B5 medium containing hormones. The solid medium is composed of B5 medium supplemented with 0.5～1.5mg/L 6～BA, 0.5～1.5mg/LNAA, 10～50g/L sucrose, 50～150mg/L inositol, 3～6g/L agar, induction 20～ After 30 days, select callus for subculture;

Step 2: The embryogenic callus is inserted into the liquid medium, and the inoculation amount is 25-100 g/L of wet weight. Shake flasks are used within 5 to 15 days of the initial culture. After the cells are fully dispersed, a mesh screen is used to screen out large cell clusters. The filtered small cell clusters and single cell suspension are added to the liquid medium to form a liquid suspension culture system. subculture;

Preferably, the sub-generation times of the step 1 are 8 to 12 times;

Preferably, the number of sub-generations in the second step is 4 to 6 times.

Advantages of the present invention:

1. The present invention overcomes the shortcoming that the existing Luo Han Guo suspension culture stays at the shake flask level, the cell yield is lower, and cannot carry out large-scale culture, and provides certain technical methods and foundations for larger-scale Luo Han Guo cell suspension culture later.

2. The growth rate of the suspended cells of the present invention is faster than that of the shake flask, and the average specific growth rate μ reaches 0.439d ^-1 . At the same time, the cells enter the rapid growth and proliferation stage soon after inoculation. The growth rate has always remained in a relatively stable state.

3. The present invention solves the inhibition problem of phenylalanine and methyl jasmonate on the growth of Luo Han Guo cells in the amplifying culture of Luo Han Guo suspended cells by controlling the addition of phenylalanine, methyl jasmonate and betaine, Make full use of phenylalanine and methyl jasmonate to promote the synthesis of mogroside V.

Description of drawings

Fig. 1 Effects of phenylalanine Phe on growth, viability and induction of glucoside V in suspension cells.

Fig. 2 Effects of methyl jasmonate MeJA on the growth, viability and induction of glucoside V in suspension cells.

Figure 3. Effects of betaine on growth, viability and induction of glycoside V in suspension cells.

Detailed ways

Below in conjunction with specific embodiments, the present invention is further elaborated; the following examples use

to illustrate the present invention, but not to limit the scope of the present invention.

Example 1

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo were selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, rinsed with distilled water for 5 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium is composed of B5 medium supplemented with 0.5mg/L 6-BA, 0.5mg/LNAA, 10g/L sucrose, 50mg/L inositol, 3g/L agar, and after induction for 20 days, the embryos with loose, vigorous growth and state were selected. The more uniform callus was subcultured.

The embryogenic loose callus with stable state was subcultured for 5 times and inserted into liquid medium, and the inoculation amount was 25g/L wet weight. Within 5 days of the initial culture, a shake flask with a baffle was used. After the cells were fully dispersed, a 35-mesh mesh screen was used to screen out large cell clusters, and the filtered small cell clusters and single cell suspension were added to an appropriate amount of liquid medium. A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for inoculation and expansion in a fermenter after being subcultured for 3 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions are: 20°C, ventilation volume 50L/h, tank pressure 0.01MPa, rotational speed 50rpm, culture to 10 days, add 250mg/L phenylalanine, 5Mm methyl jasmonate, 250mg/L betaine, continue Induction culture up to 20 days.

Example 2

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo were selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, they were rinsed with distilled water for 6 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium is composed of B5 medium supplemented with 1.5mg/L 6-BA, 1.5mg/LNAA, 50g/L sucrose, 150mg/L inositol, 6g/L agar, and after 30 days of induction, the embryos with loose, vigorous growth and state were selected. The more uniform callus was subcultured.

The embryogenic loose callus with 15 passages and stable state was inserted into the liquid medium, and the inoculation amount was 100g/L of wet weight. Within 15 days of initial culture, shake flasks with baffles were used to fully disperse the cells, and then use a 35-mesh mesh screen to screen out large cell clusters, and then add the filtered small cell clusters and single cell suspension to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for fermenter inoculation and expansion after subculture for 8 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions are: 30°C, ventilation volume 150L/h, tank pressure 0.03MPa, rotational speed 150rpm, culture to 20 days, add 1000mg/L phenylalanine, 50Mm methyl jasmonate, 1000mg/L betaine, continue Induction culture up to 40 days.

Example 3

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo are selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, rinse with distilled water for 5 to 6 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium was composed of B5 medium supplemented with 1.0 mg/L 6-BA, 1.0 mg/LNAA, 30 g/L sucrose, 100 mg/L inositol, and 4.6 g/L agar.

The callus with loose embryogenicity, vigorous growth and relatively uniform state was induced for 25 days for subculture.

The embryogenic loose callus with 10 passages and stable state was inserted into the liquid medium, and the inoculation amount was 50g/L wet weight. Within 10 days of the initial culture, a shake flask with baffle was used to fully disperse the cells, and then a 35-mesh mesh screen was used to screen out large cell clusters, and the filtered small cell clusters and single cell suspension were added to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for fermenter inoculation and expansion after subculture for 5 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions are: 26°C, ventilation volume 90L/h, tank pressure 0.02MPa, rotational speed 110rpm, culture to 15 days, add 500mg/L phenylalanine, 20Mm methyl jasmonate, 500mg/L betaine, continue Induction culture up to 30 days.

Example 4

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo were selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, rinsed with distilled water for 5 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium was B5 medium supplemented with 0.7mg/L 6-BA, 0.7mg/LNAA, 20g/L sucrose, 75mg/L inositol, 4g/L agar, induced, and callus was selected for subculture after 23 days. 23 days after induction, the callus with loose embryogenicity, vigorous growth and relatively uniform state was selected for subculture.

The embryogenic loose callus with stable state was subcultured 7 times and inserted into liquid medium, and the inoculation amount was 40g/L wet weight. Within 7 days of the initial culture, a shaker with a baffle was used to fully disperse the cells, and then a 35-mesh mesh screen was used to screen out large cell clusters, and the filtered small cell clusters and single-cell suspension were added to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. After 4 passages of this culture system, it can be used as an experimental cell line for fermenter inoculation and expansion.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions are: 23°C, ventilation volume 75L/h, tank pressure 0.01MPa, rotational speed 70rpm, culture to 12 days, add 350mg/L phenylalanine, 10Mm methyl jasmonate, 400mg/L betaine, continue Induction culture up to 25 days.

Example 5

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo were selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, they were rinsed with distilled water for 6 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium consists of B5 medium supplemented with 1.2mg/L 6-BA, 1.2mg/LNAA, 40g/L sucrose, 120mg/L inositol, 5g/L agar, and after 28 days of induction, select embryos with loose, vigorous growth and state The more uniform callus was subcultured.

The embryogenic loose callus with 12 passages and stable state was inserted into the liquid medium, and the inoculation amount was 75g/L wet weight. Within 12 days of initial culture, shake flasks with baffles were used to fully disperse the cells, and then use a 35-mesh mesh screen to screen out large cell clusters, and then add the filtered small cell clusters and single cell suspension to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for fermenter inoculation and expansion after subculture for 6 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions are: 28°C, ventilation volume 120L/h, tank pressure 0.03MPa, rotational speed 120rpm, culture to 18 days, add 750mg/L phenylalanine, 40Mm methyl jasmonate, 750mg/L betaine, continue Induction culture up to 35 days.

Example 6

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo were selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, they were rinsed with distilled water for 6 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium consists of B5 medium supplemented with 1 mg/L 6-BA, 1.2 mg/LNAA, 35 g/L sucrose, 85 mg/L inositol, and 5 g/L agar. After 20 days of induction, select embryos with loose embryos, vigorous growth, and relatively stable conditions. Homogeneous callus was subcultured.

The embryogenic loose callus with stable state was subcultured 8 times into liquid medium, and the inoculation amount was 58g/L wet weight. In the initial 9 days of culture, a shake flask with baffle was used to fully disperse the cells, and then a 35-mesh mesh screen was used to screen out large cell clusters, and the filtered small cell clusters and single cell suspension were added to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for fermenter inoculation and expansion after subculture for 6 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions are: 26°C, ventilation volume 76L/h, tank pressure 0.02MPa, rotational speed 60rpm, culture to 15 days, add 350mg/L phenylalanine, 30Mm methyl jasmonate, 500mg/L betaine, continue Induction culture up to 30 days.

Example 7

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo were selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, rinsed with distilled water for 5 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium was B5 medium supplemented with 0.7mg/L 6-BA, 0.7mg/LNAA, 20g/L sucrose, 75mg/L inositol, 4g/L agar, induced, and callus was selected for subculture after 23 days. 23 days after induction, the callus with loose embryogenicity, vigorous growth and relatively uniform state was selected for subculture.

The embryogenic loose callus with stable state was subcultured 7 times and inserted into liquid medium, and the inoculation amount was 40g/L wet weight. Within 7 days of the initial culture, a shaker with a baffle was used to fully disperse the cells, and then a 35-mesh mesh screen was used to screen out large cell clusters, and the filtered small cell clusters and single-cell suspension were added to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. After 4 passages of this culture system, it can be used as an experimental cell line for fermenter inoculation and expansion.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions were: 23°C, ventilation volume 75L/h, tank pressure 0.01MPa, rotational speed 70rpm, cultured for 12 days, added 350mg/L phenylalanine, 400mg/L betaine, and continued to induce culture to 25 days.

Example 8

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo were selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, they were rinsed with distilled water for 6 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium consists of B5 medium supplemented with 1.2mg/L 6-BA, 1.2mg/LNAA, 40g/L sucrose, 120mg/L inositol, 5g/L agar, and after 28 days of induction, select embryos with loose, vigorous growth and state The more uniform callus was subcultured.

The embryogenic loose callus with 12 passages and stable state was inserted into the liquid medium, and the inoculation amount was 75g/L wet weight. Within 12 days of initial culture, shake flasks with baffles were used to fully disperse the cells, and then use a 35-mesh mesh screen to screen out large cell clusters, and then add the filtered small cell clusters and single cell suspension to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for fermenter inoculation and expansion after subculture for 6 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions were: 28°C, aeration volume 120L/h, tank pressure 0.03MPa, rotation speed 120rpm, cultured for 18 days, added 40Mm methyl jasmonate, 750mg/L betaine, and continued to induce culture for 35 days.

Example 9

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo were selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, they were rinsed with distilled water for 6 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium consists of B5 medium supplemented with 1 mg/L 6-BA, 1.2 mg/LNAA, 35 g/L sucrose, 85 mg/L inositol, and 5 g/L agar. After 20 days of induction, select embryos with loose embryos, vigorous growth, and relatively stable conditions. Homogeneous callus was subcultured.

The embryogenic loose callus with stable state was subcultured 8 times into liquid medium, and the inoculation amount was 58g/L wet weight. In the initial 9 days of culture, a shake flask with baffle was used to fully disperse the cells, and then a 35-mesh mesh screen was used to screen out large cell clusters, and the filtered small cell clusters and single cell suspension were added to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for fermenter inoculation and expansion after subculture for 6 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions were as follows: 26°C, ventilation volume 76L/h, tank pressure 0.02MPa, rotational speed 60rpm, cultured for 15 days, added 350mg/L phenylalanine, 30Mm methyl jasmonate, and continued to induce culture for 30 days.

Comparative Examples 1 to 3 are used to evaluate the effect of phenylalanine on the proliferation and Mogroside V production of Luo Han Guo suspension cells

Comparative Example 1: 100mg/L phenylalanine was used to replace phenylalanine, methyl jasmonate and betaine in Example 3, and other steps were the same as those in Example 3.

Comparative Example 2: 250mg/L phenylalanine was used to replace phenylalanine, methyl jasmonate and betaine in Example 3, and other steps were the same as those in Example 3.

Comparative Example 3: 500mg/L phenylalanine was used to replace phenylalanine, methyl jasmonate and betaine in Example 3, and other steps were the same as those in Example 3.

For example, the specific operations of Comparative Example 3 are as follows:

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo are selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, rinse with distilled water for 5 to 6 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium was composed of B5 medium supplemented with 1.0 mg/L 6-BA, 1.0 mg/LNAA, 30 g/L sucrose, 100 mg/L inositol, and 4.6 g/L agar.

The callus with loose embryogenicity, vigorous growth and relatively uniform state was induced for 25 days for subculture.

The embryogenic loose callus with 10 passages and stable state was inserted into the liquid medium, and the inoculation amount was 50g/L wet weight. Within 10 days of the initial culture, a shake flask with baffle was used to fully disperse the cells, and then a 35-mesh mesh screen was used to screen out large cell clusters, and the filtered small cell clusters and single cell suspension were added to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for fermenter inoculation and expansion after subculture for 5 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions were as follows: 26°C, aeration volume of 90 L/h, tank pressure of 0.02 MPa, and rotational speed of 110 rpm for 15 days, adding 500 mg/L phenylalanine, and continuing to induce culture for 30 days.

Comparative Examples 4 to 6 are used to evaluate the effect of methyl jasmonate on the proliferation and Mogroside V production of Luo Han Guo suspension cells

Comparative Example 4: 20 μmol/L methyl jasmonate was used to replace phenylalanine, methyl jasmonate and betaine in Example 3, and other steps were the same as those in Example 3.

Comparative Example 5: 50 μmol/L methyl jasmonate was used to replace phenylalanine, methyl jasmonate, and betaine in Example 3, and other steps were the same as those in Example 3.

Comparative Example 6: 100 μmol/L methyl jasmonate was used to replace phenylalanine, methyl jasmonate, and betaine in Example 3, and other steps were the same as those in Example 3.

For example, the specific operation of Comparative Example 5 is as follows:

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo are selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, rinse with distilled water for 5 to 6 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium was composed of B5 medium supplemented with 1.0 mg/L 6-BA, 1.0 mg/LNAA, 30 g/L sucrose, 100 mg/L inositol, and 4.6 g/L agar.

The callus with loose embryogenicity, vigorous growth and relatively uniform state was induced for 25 days for subculture.

The embryogenic loose callus with 10 passages and stable state was inserted into the liquid medium, and the inoculation amount was 50g/L wet weight. Within 10 days of the initial culture, a shake flask with baffle was used to fully disperse the cells, and then a 35-mesh mesh screen was used to screen out large cell clusters, and the filtered small cell clusters and single cell suspension were added to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for fermenter inoculation and expansion after subculture for 5 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions were as follows: 26°C, aeration volume of 90 L/h, tank pressure of 0.02 MPa, and rotational speed of 110 rpm, cultured for 15 days, added 50 μmol/L methyl jasmonate, and continued to induce culture for 30 days.

Comparative Examples 7-9 were used to evaluate the effect of betaine on the proliferation and Mogroside V production of Luo Han Guo suspension cells

Comparative Example 7: 200mg/L betaine was used to replace phenylalanine, methyl jasmonate and betaine in Example 3, and other steps were the same as those in Example 3.

Comparative Example 8: 500mg/L betaine was used to replace phenylalanine, methyl jasmonate and betaine in Example 3, and other steps were the same as those in Example 3.

Comparative Example 9: 1000mg/L betaine was used to replace phenylalanine, methyl jasmonate and betaine in Example 3, and other steps were the same as those in Example 3.

For example, the specific operations of Comparative Example 8 are as follows:

Step 1: Establishment of Luo Han Guo cell liquid suspension culture system

The mature seed embryos of Luo Han Guo are selected for disinfection under aseptic conditions with 70% alcohol and sodium hypochlorite solution containing 2.5% effective chlorine. After disinfection, rinse with distilled water for 5 to 6 times to clean the disinfectant. After the seed embryo was stripped of the outer hard seed coat, the wound was cut and added to the solid B5 medium containing hormones to induce embryogenic loose callus. The solid medium was composed of B5 medium supplemented with 1.0 mg/L 6-BA, 1.0 mg/LNAA, 30 g/L sucrose, 100 mg/L inositol, and 4.6 g/L agar.

The callus with loose embryogenicity, vigorous growth and relatively uniform state was induced for 25 days for subculture.

The embryogenic loose callus with 10 passages and stable state was inserted into the liquid medium, and the inoculation amount was 50g/L wet weight. Within 10 days of the initial culture, a shake flask with baffle was used to fully disperse the cells, and then a 35-mesh mesh screen was used to screen out large cell clusters, and the filtered small cell clusters and single cell suspension were added to an appropriate amount of liquid medium A uniform liquid suspension culture system is formed. This culture system can be used as an experimental cell line for fermenter inoculation and expansion after subculture for 5 times.

Step 2: Scale-up culture of 5L stirred reactor

The characteristics of plant cells with walls make the cells themselves more sensitive to shear, so the paddle type is used as a push agitator with less shear. The tank is equipped with pH electrode and dissolved oxygen electrode respectively. The gas components were analyzed by exhaust gas mass spectrometry, and the physiological respiratory and metabolic parameters of cells were measured in real time. Before use, add an appropriate amount of water to the reactor, sterilize it at 121°C for 1 hour, and after cooling, pump out the water with a tank pressure, and connect it to a shake flask to suspend the cell seeds and medium. The culture conditions were as follows: 26°C, aeration volume of 90 L/h, tank pressure of 0.02 MPa, and rotational speed of 110 rpm, cultured for 15 days, added 500 mg/L betaine, and continued to induce culture for 30 days.

The suspended cells obtained in Examples 1 to 9 and Comparative Examples 1 to 9 were used for determination of cell dry weight, mitochondrial activity, and mogroside V content.

The specific results are as follows:

Table 1 Proliferation of Luo Han Guo suspension cells and the yield of glucoside V

Effects of phenylalanine alone

From the experimental results in Table 1 and Figure 1, it can be seen that a certain amount of phenylalanine (100, 250, 500 mg/L) is added, and with the increase of the added concentration of phenylalanine, the content of glucoside V also increases. The content of total glucosides induced by three concentrations of phenylalanine were: 30.15, 35.32, and 41.89 g/L. The experimental results showed that adding 500 mg/L of phenylalanine to the medium could maximize the The synthetic yield of Mogroside V was nearly 28.6% higher than that of Mogroside V per unit cell dry weight without the addition of inducer. However, with the increase of phenylalanine concentration, the inhibitory effect of phenylalanine on the growth of suspended cells was also greater. When the concentration of phenylalanine increased, the dry weight of cells and mitochondrial activity decreased significantly. Both the growth of the body and the rate of synthesis of the product are significantly inhibited.

Effects of methyl jasmonate alone

It can be seen from Table 1 and Figure 2 that when the concentration of methyl jasmonate is 20 and 50 μmol/L, the synthesis of the product mogroside V can be promoted, and the synthesis amount of the product is 45.32 and 51.89 g/L, respectively. , when the concentration increased to 100 μmol/L, the synthesis of mogroside V and the growth rate of cells were significantly inhibited. Under the condition of 50 μmol/L concentration, the synthesis rate of unit cells increased by more than 21.6% compared with the control. However, with the increase of the concentration of methyl jasmonate, the inhibitory effect of methyl jasmonate on the growth of suspended cells is also greater. When the concentration exceeds 50 μmol/L, the mitochondrial activity of Luo Han Guo suspended cells is less than or equal to 0.1, resulting in significantly inhibited cell growth and product synthesis rate.

Effects of betaine alone

As can be seen from Table 1 and Fig. 3, the test results of betaine (200, 500, 1000 mg/L) show that betaine can significantly promote the synthesis of mogroside V, and in the case of the addition amount of 500 mg/L, the unit bacteria Compared with the control, the synthesis amount of glucoside V was increased by more than 34%, and at the same time, it also had a good promoting effect on the growth of the bacteria. However, the indicators of dry cell weight, mitochondrial activity, and mogroside V content were all inferior to those of Example 3 of the present invention. The growth rate of the cells is lower than that of the present invention, and the cells enter the rapid growth and proliferation stage relatively slowly after seeding.

By controlling the addition amounts of phenylalanine, methyl jasmonate and betaine, the invention solves the problem of inhibiting the growth of Luo Han Guo cells by phenylalanine and methyl jasmonate in the enlarged culture of Luo Han Guo suspended cells. Fully phenylalanine and methyl jasmonate promote the synthesis of mogroside V, and at the same time add betaine to further promote the synthesis of mogroside V and the growth of suspension cells. The data of dry cell weight, mitochondrial activity, and mogroside V content of Monk fruit suspension cells in implementations 1 to 9 of the present invention are all better than those of comparative examples 1 to 9, the growth of suspension cells is not inhibited, and the synthesis of mogroside V is significantly improved .

Scale-up culture vs. laboratory shake flask level

In order to further grasp the proliferation characteristics of the amplifying cultivation of Luo Han Guo suspended cells in the present invention, the suspended cells in the stirred reactor were detected, the fresh weight, dry weight and proliferation coefficient of the cells were measured, the residual sugar in the medium was measured, and the capacitance and conductance in the medium were measured. The specific detection method is as follows:

1. Determination of cell fresh weight, dry weight and proliferation coefficient

The suspended cells were shaken and poured into a suction filter bottle for suction filtration, during which the cells were rinsed with ultrapure water, and suction filtered until the filter paper no longer dripped. The weight at this time was fresh weight (FCW), and the cells after suction filtration were placed in an oven at 60°C, and weighed as dry weight (DCW) after 24 h.

GI=(M _t ~ M ₀ )/M ₀

M _t is the fresh weight after harvest, M ₀ is the fresh weight at the time of inoculation

2. Determination of Residual Sugar in Medium

The determination of residual sugar adopts the DNS method. This method is to use DNS to react with reducing sugar under certain conditions to generate a chemical substance. The depth is proportional to the amount of reducing sugar. Since the carbon source used in the suspension culture of Luo Han Guo cells is sucrose, it should be hydrolyzed first, and after hydrolysis into reducing sugar, the residual sugar in the fermentation broth can be measured by DNS method.

3. Determination of capacitance and conductance in culture medium

The electrodes of the live cell sensor were immersed in the Luo Han Guo cell suspension, and a full-frequency scan was performed. Finally, the detection frequency of the live cell sensor was determined to be 473 kHz. At this frequency, the cell capacitance and conductance were measured. Capacitance can be used to characterize the concentration of biomass in the culture system, and conductance can be used to characterize the concentration of inorganic salt ions in the medium.

The specific results are as follows:

Table 2 Analysis of proliferation characteristics of Luo Han Guo suspended cells in 5L stirred reactor

From the point of view of the whole scale-up culture process, under this condition, Luo Han Guo cells can be grown in a 5L stirred bioreactor. The culture results showed that the growth rate of cells was faster than that of the shake flask, and the average specific growth rate μ was 0.439d ^-1 . The cells entered the stage of rapid growth and proliferation soon after inoculation, and the growth rate remained in a relatively stable state before the stationary phase and the dying phase. When inoculated with 50 g/L fresh weight of cells, the bacterial yield finally reached 9.5 g/L, the content of mogroside V was 8.9 mg/g DCW after detection, and the final yield reached 85.1 mg/L.

in conclusion

1. The present invention overcomes the shortcoming that the existing suspension culture of Luo Han Guo stays at the shake flask level, the cell yield is low, and the large-scale culture cannot be carried out, and the optimized culture medium and the addition process for promoting the synthesis of the product are obtained through screening, and the large The rate of synthesis of Mogroside V has been greatly improved, providing a certain technical method and basis for larger-scale suspension culture of Monk fruit cells in the future.

2. The present invention solves the inhibition of the growth of Luo Han Guo cells by phenylalanine, methyl jasmonate and betaine by controlling the three additions of phenylalanine, methyl jasmonate and betaine in the suspension cell amplification culture of Luo Han Guo. The problem is to fully promote the synthesis of mogroside V by phenylalanine and methyl jasmonate, and at the same time, by adding betaine, the synthesis of mogroside V and the growth of suspension cells are further promoted.

3. The growth rate of the suspended cells of the present invention is faster than that of the shake flask, and the average specific growth rate μ reaches 0.439d ^-1 . At the same time, the cells enter the rapid growth and proliferation stage soon after inoculation. The growth rate has always remained in a relatively stable state.

Although the present invention has been described in detail above with general description, specific embodiments and tests, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art . Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (9)

1. a method for amplifying cultivation of Luo Han Guo suspended cells, is characterized in that, in the process of Luo Han Guo suspended cells amplification cultivation, two or more of phenylalanine, methyl jasmonate and betaine are added. 2. the method for a kind of Luo Han Guo suspension cell amplification culture as claimed in claim 1, is characterized in that, in the process of described Luo Han Guo suspension cell amplification culture, adds the phenylalanine of 250～1000mg/L, 5～50Mm Methyl jasmonate, betaine at 250-1000 mg/L. 3. the method for enlarged cultivation of a kind of Luo Han Guo suspended cell as claimed in claim 1 is characterized in that, in the process of described Luo Han Guo suspension cell enlarged culture, add the phenylalanine of 450～550mg/L, 15～25Mm Methyl jasmonate, betaine at 450-550 mg/L. 4. the method for a kind of Luo Han Guo suspension cell amplification culture as claimed in claim 1, is characterized in that, the condition of described Luo Han Guo suspension cell amplification culture is: 20～30 ℃, ventilation volume 50～150L/h, tank pressure 0.01 ～0.03MPa, rotating speed 50～150rpm, culture time 20～40 days. 5. The method for amplifying the cultivation of suspension cells of Luo Han Guo as claimed in claim 1, wherein the conditions for the amplification of suspension cells of Luo Han Guo are: 24-28° C., ventilation volume 80-100 L/h, tank pressure 0.01-0.03 MPa, The rotation speed was 100-120 rpm, and the incubation time was 25-35 days. 6. the method for a kind of Luo Han Guo suspended cell amplification culture as claimed in claim 1, is characterized in that, adding two or more time in described phenylalanine, methyl jasmonate and betaine is Days 13-16 of the scale-up culture. 7. the method for a kind of Luo Han Guo suspension cell amplification culture as claimed in claim 1, is characterized in that, the liquid suspension culture system in the early stage of described Luo Han Guo suspension cell amplification culture is obtained by following steps: Step 1: Select the seed embryos of Luo Han Guo for disinfection treatment, rinse them after disinfection, cut the seed embryos out of the wound and add them to the solid B5 medium containing hormones. The solid medium is composed of B5 medium supplemented with 0.5～1.5mg/L 6～BA, 0.5～1.5mg/LNAA, 10～50g/L sucrose, 50～150mg/L inositol, 3～6g/L agar, induction 20～ After 30 days, select callus for subculture; Step 2: The embryogenic callus is inserted into the liquid medium, and the inoculation amount is 25-100 g/L of wet weight. Shake flasks are used within 5 to 15 days of the initial culture. After the cells are fully dispersed, a mesh screen is used to screen out large cell clusters. The filtered small cell clusters and single cell suspension are added to the liquid medium to form a liquid suspension culture system. Subculture. 8 . The method for amplifying and culturing Luo Han Guo suspension cells according to claim 7 , wherein the number of passages in the first step is 8 to 12 times. 9 . 9 . The method for amplifying and culturing Luo Han Guo suspension cells according to claim 7 , wherein the number of passages in the second step is 4 to 6 times. 10 .

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