Culture method for improving yield of huperzine A in huperzia serrata suspension cells

Abstract

The invention relates to a culture method for improving the yield of huperzine A of huperzia serrata suspension cells. According to the method, callus is obtained through induction of huperzia serrata diameter segments and leaves, and in the amplification culture process of huperzia serrata suspension cells, the optimal hormone ratio is determined as follows: 0.5 mg / L of 6-benzyladenine, 0.02 mg / L of alpha-naphthylacetic acid and 0.2 mg / L of 3-indolebutyric acid. According to the method, the technical limitation of suspension culture of huperzia serrata suspension cells is overcome, a rapid and efficient huperzia serrata culture system is established, the synthesis rate of huperzine A as a huperzia serrata product is greatly increased, and a certain technical method and basis are provided for establishment of a larger-scale huperzia serrata culture system.

Description

Technical Field

[0001] The invention relates to the field of fermentation, and in particular to a culture method for improving the yield of huperzine A in Huperzina serrata suspension cells. Background Art

[0002] Huperzia serrata Trev is an important traditional Chinese medicinal material in my country. The natural huperzine A extracted from Huperzia serrata Trev can be used to treat human Alzheimer's disease, myasthenia gravis, schizophrenia and other diseases. It is a highly effective, highly selective and reversible acetylcholinesterase inhibitor with a large market demand and is known as the "gold of plants".

[0003] However, the natural Huperzia serrata grows slowly and has a very low yield, which is far from meeting the market demand. At present, the research on Huperzia serrata is mainly focused on the cultivation of tissue culture seedlings and artificial cultivation in the wild. However, due to the great influence of ecological factors on the growth of Huperzia serrata, there is no practical cultivation technology, which cannot be scaled up and cannot meet the market demand. The induction and suspension culture of callus tissue of Huperzia serrata are still rarely reported. The problems that need to be solved urgently through tissue culture of Huperzia serrata are: first, the sterilization bottleneck. When sterilizing Huperzia serrata, in addition to killing surface bacteria, killing endophytic fungi is also an arduous task. Second, the establishment of a large-scale callus culture system. Third, the content of Huperzine A is low. How to induce the callus tissue of Huperzia serrata to increase the content of Huperzine A is an important task to solve the shortage of resources. The formation time of Huperzia serrata callus is relatively long, generally about 30 days. How to shorten the culture cycle, and how to disperse cells when the callus tissue is relatively compact are all issues worth studying.

[0004] Finding new, fast and efficient ways to increase the yield of Huperzine A is helpful to give full play to the medicinal value of Huperzine A and meet the needs of human disease treatment. The present invention collects fresh plants in the wild, induces callus tissue indoors, and adopts shake flask liquid suspension culture and 5L reactor fermenter small-scale liquid suspension culture to effectively increase the yield of Huperzine serrata callus cells, providing a preliminary experimental basis for large-scale industrial production of Huperzine A. Summary of the invention

[0005] The invention aims to provide a method for increasing the yield of huperzine A in suspension cells of Huperzine serrata, wherein fresh plants are collected in the wild and callus tissue is induced indoors. The addition amount of hormones: 6-benzyladenine, α-naphthylacetic acid and 3-indolebutyric acid is optimized. Liquid suspension culture in a shake flask and small-scale liquid suspension culture in a 5L reactor fermenter are adopted to effectively increase the yield of huperzine A in callus tissue cells of Huperzine serrata.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a culture method for improving the yield of huperzine A in suspension cells of Huperzine serrata. The method is characterized in that callus tissues of stems and leaves of Huperzine serrata with good growth are selected, shaken and cultured in a 250mL triangular flask, and sucrose, 6-benzyladenine, α-naphthylacetic acid and 3-indolebutyric acid are added during the process.

[0008] Preferably, during the culture of the Huperzella serrata suspension cells, the culture medium adopts a modified MS medium containing 1 / 2 mineral elements, 1 / 2 organic elements, and 1%-3% sucrose, and different concentrations of plant hormones 6-benzyladenine (6-BA), α-naphthaleneacetic acid (α-NAA) and 3-indolebutyric acid (IBA) are added.

[0009] Preferably, during the culture of the Huperzina serrata suspension cells, 0.1-1.5 mg / L 6-benzyladenine, 0-0.08 mg / L α-naphthylacetic acid, 0-1 mg / L 3-indolebutyric acid, 0.5-4% sucrose, and 5-50% liquid volume are added;

[0010] Preferably, during the culture of the Huperzina serrata suspension cells, 0.2-1.0 mg / L 6-benzyladenine, 0-0.04 mg / L α-naphthylacetic acid, 0-0.5 mg / L 3-indolebutyric acid, 1-3% sucrose, and 10-40% liquid volume are added;

[0011] Preferably, during the culture of the Huperzina serrata suspension cells, 0.5 mg / L 6-benzyladenine, 0.02 mg / L α-naphthylacetic acid and 0.2 mg / L l3-indolebutyric acid; 2% sucrose; 30% liquid volume;

[0012] The invention provides a culture method for improving the yield of huperzine A in Huperzina serrata suspension cells, which is characterized by carrying out small-scale liquid suspension culture of Huperzina serrata suspension cells in a 5L stirred bioreactor.

[0013] Preferably, the specific conditions for culturing the Huperzia serrata suspension cells are: a culture temperature of 25° C., a stirring speed of 100 rpm, and an aeration volume of 0.4 L / min.

[0014] Preferably, the culture process of the sterile explants of Huperzia serrata required for the Huperzia serrata suspension cell culture system is characterized in that the newly cut explants are rinsed with tap water and deionized water, immersed in 75% ethanol solution for 50 seconds and 4% sodium hypochlorite solution respectively, transferred to a sterilized culture dish equipped with filter paper, and cultured in the dark at 25°C.

[0015] The present invention discloses the following technical effects: when Huperzina serrata is cultured by the fermentation method of the present invention, the growth rate of cells in a 5L bioreactor is accelerated, and the content of the product Huperzine A can reach a maximum of 51.81 ug / mL within 33 days.

[0016] The present invention preliminarily discusses the carbon source, respiration intensity, hormones, liquid volume and other conditions in the culture medium of Huperzine serrata callus, and proposes a small-scale liquid suspension culture in a 5L stirred bioreactor, which effectively increases the cell mass and huperzine A yield of Huperzine serrata callus cells, and provides a preliminary experimental basis for the large-scale industrial production of huperzine A. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is the growth status of Huperzella serrata callus culture bottle;

[0019] Figure 2 To analyze the proliferation characteristics of Huperzia serrata callus;

[0020] Figure 3 The effect of the volume of liquid on the proliferation and viability of suspended cells;

[0021] Figure 4 The effect of sucrose addition on the proliferation and viability of suspended cells;

[0022] Figure 5 The changes of cell fresh weight, pH, conductivity and residual sugar in the 5L reactor;

[0023] Figure 6 is the change of carbon dioxide exchange rate and oxygen uptake rate in the 5L reactor;

[0024] Figure 7 Shows the changes in cell fresh weight and oxygen uptake rate in the 5L reactor. DETAILED DESCRIPTION

[0025] As used in the present invention, the improvement of "production efficiency (production efficiency)" includes the improvement of output and the improvement of yield rate.

[0026] The hormones and their abbreviations involved in the present invention include: 6-benzyladenine (6-BA), α-naphthylacetic acid (α-NAA) and 3-indolebutyric acid (IBA); the units and their abbreviations involved in the present invention include but are not limited to: day (d), hour (h), minute (min).

[0027] Unless otherwise stated, the calculation of time nodes in the claims and specification of the present invention is based on the start time of "fermentation" or "culture" in the bioreactor as the starting point (0 hours).

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0034] Materials and Methods

[0035] 1. Determination of fresh and dry weight of callus

[0036] Use tweezers to transfer the callus tissue to a suction filtration bottle containing an 8μm filter membrane for suction filtration. During this period, rinse the cells with ultrapure water and filter until the filter paper no longer drips water. The weight at this time is the fresh weight of the callus tissue (Fresh cell weight, FCW). The fresh weight cells after suction filtration are placed in a 60℃ oven and weighed after 24h as the dry weight of the callus tissue (Dry cell weight, DCW). Calculate the growth index (Growth index, GI), GI = (growth fresh weight / dry weight-inoculation fresh weight / dry weight) / (inoculation fresh weight / dry weight).

[0037] 2. Determination method of huperzine A content

[0038] The bacterial cells were collected by centrifugation, freeze-dried, and 0.5 g of bacterial powder was extracted with 20 mL of methanol, ultrasonicated for 20 minutes, and the clear liquid was detected by liquid chromatography. The chromatographic column was ZORBAX EXTEND-C18 (250 mm × 4.6 mm, 5 μm), the mobile phase was 0.02 mol / L potassium dihydrogen phosphate: acetonitrile solution (92: 8), the detection wavelength was 312 nm, the flow rate was 1 mL / min, the column temperature was 25 ° C, and the injection volume was 20 μL.

[0039] Implementation Example 1

[0040] Step 1: Obtaining and culturing sterile explants

[0041] After the newly cut explants were rinsed with tap water and deionized water, they were placed in a triangular flask containing 200 mL of sterile water. After washing at room temperature for 30 minutes, the surface moisture was absorbed with absorbent paper, and then immersed in 75% ethanol solution for 50 seconds and 4% sodium hypochlorite solution for 15 minutes, and then rinsed with sterile water four times, each time for 4 minutes. The explants were then transferred to a sterilized culture dish with filter paper and waited for the surface moisture to be absorbed by the filter paper. The stems and leaves were cut into 5 mm sizes and inoculated in the modified MS medium. The effects of hormones such as 0.2-1.0 mg / L 6-BA, 0-0.04 mg / L NA, and 0-0.5 mg / L IBA at the final concentration on the suspension cell culture were investigated, and the cells were cultured in the dark at a constant temperature of 25°C.

[0042] The formula for calculating callus induction rate (%) is: induction rate = number of callus produced / number of explants × 100.

[0043] Step 2: Investigate the effects of different concentrations of 6-BA, NAA and IBA on the induction of callus tissue in the stem segments of Huperzia serrata

[0044] From the results in Table 1 and Table 2, it can be seen that the healing time of the stem segment is generally 5-7 days. The callus tissue first forms at the wounds at both ends of the stem segment, and then slowly extends to both ends until the entire stem segment forms callus tissue. After 25-30 days of culture, the callus slowly expands and thickens outside. When the culture medium contains 6-BA 0.5 mg / L and NAA 0.02-0.04 mg / L, or IBA 0.2-0.5 mg / L, the induction rate of the stem segment reaches 45.7% and 40.3%, respectively.

[0045] The results showed that callus could be induced when two different types of hormones were present in the culture medium at the same time, and the callus induction rate of the two hormones combined was higher than that of a single hormone, indicating that exogenous hormones are necessary for the formation of callus in Huperzia serrata. When only one hormone was present in the culture medium, the callus induction rate of the stem segments was very low, and even no callus could be induced.

[0046] Table 1 Effects of hormones 6-BA and NAA on induction of stem callus

[0047]

[0048]

[0049] Table 2 Effects of hormones 6-BA and IBA on induction of stem callus

[0050]

[0051] Implementation Example 2

[0052] Step 1: Obtaining and culturing sterile explants

[0053] After the newly cut explants were rinsed with tap water and deionized water, they were placed in a triangular flask containing 200 mL of sterile water. After washing at room temperature for 30 minutes, the surface moisture was absorbed with absorbent paper, and then immersed in 75% ethanol solution for 50 seconds and 4% sodium hypochlorite solution for 15 minutes, and then rinsed with sterile water four times, each time for 4 minutes. The explants were then transferred to a sterilized culture dish with filter paper and waited for the surface moisture to be absorbed by the filter paper. The stems and leaves were cut into 5 mm sizes and inoculated in the modified MS medium. The effects of hormones such as 0.2-1.0 mg / L 6-BA, 0-0.04 mg / L NA, and 0-0.5 mg / L IBA at the final concentration on the suspension cell culture were investigated, and the cells were cultured in the dark at a constant temperature of 25°C.

[0054] Step 2: Investigate the effects of different concentrations of 6-BA, NAA and IBA on the induction of Huperzia serrata leaf callus

[0055] As can be seen from Table 3, the healing time of leaves is longer than that of stem segments. A small amount of callus will slowly appear around the wound of the leaves in about 12-14 days. Moreover, the callus of the leaves only appears and grows at the edge of the leaves, and the entire explant cannot be induced to form callus. When the culture medium contains only NAA, the leaves can hardly be induced to produce callus. When the NAA concentration reaches 0.2 mg / L, only a small amount of leaves can produce callus. The same conclusion was reached when only 6-BA was contained. When the culture medium contains both hormones, the induction rate can reach 33.5%, indicating that auxin and mitogen play a very important role in the formation of plant callus. The callus induction rate of leaves as explants is low, indicating that leaves are not relatively good explants for inducing callus.

[0056] Table 3 Effects of hormones 6-BA and NAA on induction of leaf callus

[0057]

[0058] Implementation Example 3

[0059] Step 1: Obtaining and culturing sterile explants

[0060] After the newly cut explants were rinsed with tap water and deionized water, they were placed in a conical flask containing 200 mL of sterile water. After washing at room temperature for 30 minutes, the surface moisture was absorbed with absorbent paper, and then immersed in 75% ethanol solution for 50 seconds and 4% sodium hypochlorite solution for 15 minutes, and then rinsed with sterile water four times, each time for 4 minutes. The explants were then transferred to a sterilized culture dish with filter paper and the surface moisture was absorbed by the filter paper. The stems and leaves were cut into 5 mm sizes, inoculated in the modified MS culture medium, and cultured in the dark at a constant temperature of 25°C.

[0061] Step 2: Huperzia serrata suspension cell culture

[0062] Select callus tissues of stems and leaves of Huperzina serrata with good growth, inoculate them into 250mL Erlenmeyer flasks with 30% liquid volume, place them at 25℃ and shake at 150rpm for culture. Take samples every 5 days to measure the fresh weight and dry weight of suspended cells to detect the callus growth cycle.

[0063] Step 3: Analysis of proliferation characteristics of Huperzia serrata liquid suspension culture cells

[0064] Will Figure 1 About 2 g of callus tissue was inoculated into a 250 mL shake flask with a 75 mL liquid volume, and the fresh weight and cell viability were measured every 5 days. Figure 2The growth trend of callus tissue shows that the growth curve of Huperzia serrata callus is not like the obvious "S" curve of microorganisms or animal cells, but shows a more gentle growth trend. The callus tissue began to grow rapidly from the 10th day, indicating that the callus tissue gradually entered the logarithmic phase. Cell viability gradually increased after inoculation, and around 30 days, the cell viability showed a significant decline. At this time, the callus tissue was still growing, but the growth rate was significantly reduced. This shows that the growth of the callus tissue has entered a stable period and the stable period lasts for a short time, and then it immediately enters the decline period. The above experimental results show that the best subculture time for solid culture is 25-30 days.

[0065] Implementation Example 4

[0066] Step 1: Obtaining and culturing sterile explants

[0067] After the newly cut explants were rinsed with tap water and deionized water, they were placed in a conical flask containing 200 mL of sterile water. After washing at room temperature for 30 minutes, the surface moisture was absorbed with absorbent paper, and then immersed in 75% ethanol solution for 50 seconds and 4% sodium hypochlorite solution for 15 minutes, and then rinsed with sterile water four times, each time for 4 minutes. The explants were then transferred to a sterilized culture dish with filter paper and the surface moisture was absorbed by the filter paper. The stems and leaves were cut into 5 mm sizes, inoculated in the modified MS culture medium, and cultured in the dark at a constant temperature of 25°C.

[0068] Step 2: Huperzine serrata suspension cell culture

[0069] Callus tissues of stems and leaves of Huperzella serrata with good growth were selected and inoculated into 250mL Erlenmeyer flasks with 30% liquid filling volume. The flasks were placed at a constant temperature of 25°C and shaken at 150 rpm. The effects of factors such as 10, 20, 30, 40, and 50% liquid filling volume on the suspension cell culture were investigated. The fresh weight (g) and dry weight (g) of the suspension cells were measured by sampling every 5 days to detect the callus growth cycle, and the culture process of the suspension cells was optimized and obtained.

[0070] Step 3: Effects of liquid volume on proliferation and viability of Huperzia serrata suspension cells

[0071] 25 (10%), 50 (20%), 75 (30%), 100 (40%), and 125 mL (50%) of culture medium were added to a 250 mL shake flask for 18 days, and then the proliferation coefficient, viability, residual sugar, conductivity and other parameters of the cells in the culture system were measured. Figure 3 The results show that with the increase of liquid volume, the proliferation coefficient first increases and then decreases. When the liquid volume is 75mL (30%), the growth coefficient of the suspended cells is the largest, and the cell activity is also the largest under this condition. Figure 3The conductivity in C represents the amount of inorganic salts in the culture system. The smaller the conductivity, the more inorganic salts are consumed in the system. Figure 3 The experimental results of C also show that when the liquid volume is 75mL, the inorganic salt consumption in the system is the largest. The experimental results show that when the liquid volume is 75mL, it is most suitable for cell growth and proliferation. In addition to affecting dissolved oxygen, the liquid volume also affects the mixing of nutrients and the amount of shear. Too low a liquid volume will cause greater shear between cell particles, and too much liquid volume will affect the mass transfer of nutrients and oxygen in the system. The results show that 75mL (30%) liquid volume is more suitable.

[0072] Implementation Example 5

[0073] Step 1: Obtaining and culturing sterile explants

[0074] After the newly cut explants were rinsed with tap water and deionized water, they were placed in a conical flask containing 200 mL of sterile water. After washing at room temperature for 30 minutes, the surface moisture was absorbed with absorbent paper, and then immersed in 75% ethanol solution for 50 seconds and 4% sodium hypochlorite solution for 15 minutes, and then rinsed with sterile water four times, each time for 4 minutes. The explants were then transferred to a sterilized culture dish with filter paper and the surface moisture was absorbed by the filter paper. The stems and leaves were cut into 5 mm sizes, inoculated in the modified MS culture medium, and cultured in the dark at a constant temperature of 25°C.

[0075] Step 2: Huperzine serrata suspension cell culture

[0076] Callus tissues of stems and leaves of Huperzella serrata with good growth were selected and inoculated into 250mL Erlenmeyer flasks with 30% liquid volume, and cultured at a constant temperature of 25℃ and 150 rpm for shaking. The effects of adding sucrose at final concentrations of 10, 15, 20, 25, and 30% on suspension cell culture were explored. The fresh weight (g) and dry weight (g) of the suspension cells were measured by sampling every 5 days to detect the growth cycle of the callus tissue, and the culture process of the suspension cells was optimized and obtained.

[0077] Step 3: Effects of sucrose addition on proliferation and cell viability of Huperzia serrata cells

[0078] Figure 4 The results showed that when the amount of sucrose added was 2% (20g / L), the cell proliferation coefficient was the largest, which was beneficial to cell reproduction. However, the cell viability was higher at a concentration of 1.5% (15g / L), and it was found that at a sucrose concentration of 1.5% (15g / L), the cell morphology was better, but at concentrations of 1% (10g / L) and 3% (30g / L), the cell growth and viability were relatively poor, so the more suitable sucrose concentration range was 1.5%-2.5% (15-25g / L).

[0079] Implementation Example 6

[0080] Step 1: Obtaining and culturing sterile explants

[0081] After the newly cut explants were rinsed with tap water and deionized water, they were placed in a conical flask containing 200 mL of sterile water. After washing at room temperature for 30 minutes, the surface moisture was absorbed with absorbent paper, and then immersed in 75% ethanol solution for 50 seconds and 4% sodium hypochlorite solution for 15 minutes, and then rinsed with sterile water four times, each time for 4 minutes. The explants were then transferred to a sterilized culture dish with filter paper and the surface moisture was absorbed by the filter paper. The stems and leaves were cut into 5 mm sizes, inoculated in the modified MS culture medium, and cultured in the dark at a constant temperature of 25°C.

[0082] Step 2: Huperzine serrata suspension cell culture

[0083] Select callus tissues of stems and leaves of Huperzina serrata with good growth, inoculate them into 250mL Erlenmeyer flasks with 30% liquid volume, place them at a constant temperature of 25°C and shake at 150 revolutions to obtain seed cells.

[0084] Step 3: Suspension culture during stirred tank reactor cultivation

[0085] A 5L stirred reactor fermenter was selected to scale up the culture of suspended cells. The culture temperature was 25°C, the stirring speed was 100 rpm, and the ventilation volume was 0.4L / min. During the culture process, pH electrodes, dissolved oxygen electrodes, tail gas mass spectrometers, and living cell sensors were used to monitor the cell growth process in real time. Multi-parameter acquisition was used to analyze the culture characteristics of the suspended cells, and the scale-up culture process of the 5L stirred reactor was obtained.

[0086] Depend on Figure 5 It can be seen that Huperzia serrata cells can be grown in a 5L stirred tank bioreactor. The culture results showed that the growth rate of cells was faster than that of the shake flask level, with an average specific growth rate (μ) of 0.139d -1 , compared with the specific growth rate of the cell shake flask (0.0421d -1) is higher, and the doubling time is also shortened. Different from the cell growth trend at the shake flask level, the cell lag period in the reactor is shorter. The cells quickly enter the rapid growth and proliferation stage after inoculation, and the growth rate before the stable period and the decay period always remains in a relatively stable state. When inoculated with 15g / L cell fresh weight, the bacterial yield eventually reached 70g / L (FCW). With the continuous increase in cell growth, the consumption of conductivity and carbon source in the culture medium also remained at a relatively slow rate throughout the process. With the continuous consumption of nutrients, the cells began to enter the stable period and the decay period after 26 days. The change trend of pH value in the culture medium is the same as that of the shake flask, and it also shows a trend of first decreasing and then rising.

[0087] Figure 6 It is the change of CO2 release rate (Carbon-dioxide Escape Rate, CER) and oxygen uptake rate (Oxygen Uptake Rate, OUR) during the suspension culture of Huperzia serrata cells. From the experimental results, it can be seen that the respiration intensity of plant cells is very weak compared with that of microorganisms, which is determined by the slow growth rate of plant cells and the characteristics of the plants themselves. Figure 7 The results showed that the respiratory metabolic intensity of cells showed a gradual upward trend with the growth of cell mass. When the nutrients such as conductivity and carbon source in the culture medium were exhausted, the respiratory intensity gradually decreased, the cells stopped proliferating, and entered a stable period. The reason for the cessation of cell growth was no longer just the influence of environmental pH, but also related to the consumption of nutrients and sugars in the culture medium. The content test results of the product Huperzine A showed that its synthesis rate was faster from 21 to 25 days, and the synthesis rate slowed down after 25 days. The highest content on the 33rd day could reach 51.81ug / mL.

[0088] Table 4 Changes in the content of Huperzine A during the culture process

[0089]

[0090] The present invention provides a culture method for improving the production of Huperzine A in suspension cells of Huperzine serrata. The method comprises obtaining callus tissue by inducing the diameter segments and leaves of Huperzine serrata, and finding the best hormone ratio of 0.5 mg / L 6-BA, 0.02 mg / L NAA and 0.2 mg / L IBA. Through the analysis of the proliferation characteristics of cells in the suspension culture cycle of Huperzine serrata callus tissue, 9-20 days are the logarithmic growth period and the best subculture time, 30% liquid volume in a 200mL triangular flask is more suitable, and 2% sucrose is most suitable for cell growth. The present invention conducts a preliminary investigation on the carbon source concentration, respiration intensity, hormones, liquid volume and other conditions in the Huperzine serrata callus tissue culture medium, and the average specific growth rate (μ) in a 5L stirred bioreactor is 0.139d -1 When inoculated with 15 g / L fresh cell weight, the bacterial yield finally reached 70 g / L (FCW), and the maximum content of huperzine A could reach 51.81 ug / mL after 33 days.

[0091] The technical solution of the present application can increase the cell mass and huperzine A production of Huperzina serrata suspension cells.

[0092] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for increasing the production of huperzine A in suspension cells of Huperzina serrata, characterized in that: The suspension cells of Huperzella serrata were cultured in liquid suspension culture in shake flasks and in small-scale liquid suspension culture in 5L reactor fermenters.

2. The method according to claim 1, characterized in that The callus tissues of stems and leaves of Huperzella serrata with good growth were selected and cultured in 250 mL Erlenmeyer flasks with shaking, and sucrose, 6-benzyladenine, α-naphthylacetic acid and 3-indolebutyric acid were added during the process.

3. The method according to claim 2, characterized in that During the Huperzia serrata suspension cell culture process, 0.1-1.5 mg / L 6-benzyladenine, 0-0.08 mg / L α-naphthylacetic acid, 0-1 mg / L 3-indolebutyric acid, and 0.5-4% sucrose are added. The liquid volume is 5-50%.

4. The method according to claim 2, characterized in that During the Huperzia serrata suspension cell culture, 0.2-1.0 mg / L 6-benzyladenine, 0-0.04 mg / L α-naphthylacetic acid, 0-0.5 mg / L 3-indolebutyric acid, 1-3% sucrose and 10-40% liquid volume are added.

5. The method according to claim 2, characterized in that During the Huperzine serrata suspension cell culture, 0.5 mg / L 6-benzyladenine, 0.02 mg / L α-naphthylacetic acid and 0.2 mg / L 13-indolebutyric acid and 2% sucrose were added. 30% liquid volume.

6. The method according to claim 1, characterized in that The required culture process of sterile explants of Huperzia serrata was as follows: the freshly cut explants were rinsed with tap water and deionized water, immersed in 75% ethanol solution for 50 seconds and 4% sodium hypochlorite solution respectively, transferred to a sterilized culture dish equipped with filter paper, and cultured at 25°C in the dark.

7. The method according to claim 1, characterized in that Small-scale liquid suspension culture of Huperzella serrata suspension cells was carried out in a 5 L stirred tank bioreactor.

8. The method according to claim 6, characterized in that The specific conditions for the Huperzine serrata suspension cell culture are: a culture temperature of 25° C., a stirring speed of 100 rpm, and a ventilation volume of 0.4 L / min.

9. The method according to claim 1, characterized in that Cultivation of Huperzia serrata Trev producing Huperzine A.

10. The method according to claim 1, characterized in that Application in fermentation production of huperzine A.

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