A method for high-density cultivation of endophytes

Abstract

The present invention discloses a method for high-density cultivation of endophytes, which relates to the technical field of endophyte cultivation. The method comprises the following steps: soaking astragalus powder with an extract containing phosphatidylcholine under ultrasonic conditions to obtain an extract containing astragalus active ingredients; inoculating astragalus endophytes on a solid medium containing the extract for screening cultivation and activation; inoculating the activated astragalus endophytes into a liquid medium for cultivation to obtain an endophyte seed solution; fermenting and culturing the endophyte seed solution in a culture solution to obtain high-density astragalus endophytes; wherein the culture solution comprises: 10-15 parts of the extract, 4-6 parts of peptone, 1.5-3 parts of yeast powder, 4-6 parts of glucose, 1.5-2.5 parts of trace salt solution, 0.2-0.3 parts of copper citrate, 0.1-0.20 parts of diammonium citrate, 1.5-2.5 parts of C6 volatile matter, 1-2 parts of hydroxyoctadecadienoic acid, and 1000 parts of LB medium; the present invention can achieve the improvement of the cell yield and the reduction of the use of manpower and material resources.

Description

Technical Field

[0001] The present invention relates to the field of endophyte cultivation, and in particular to a method for high-density endophyte cultivation. Background Art

[0002] Astragalus is the dried root of Astragalus mongolica or Astragalus membranaceus, a plant of the Leguminosae family. It is a commonly used tonic Chinese medicine and is currently produced in Heilongjiang, Gansu, Inner Mongolia, Shanxi, Shaanxi and other places in my country. Astragalus from different origins contains n-hexanal, n-hexanol and E-2-hexenal, which are the main odor components of the bean aroma of authentic Astragalus.

[0003] Plant endophytes refer to a class of microorganisms (mainly fungi and bacteria) that spend all or nearly all of their life cycles in healthy plant hosts without causing any symptoms to the host. They live inside various tissues and organs of plants and are a natural part of the plant's internal ecosystem. In the long-term co-evolution process, endophytes and plants have formed a mutually beneficial relationship. Studies have shown that in the co-evolution process between endophytes and plants, endophytes can not only produce special secondary metabolites, but also induce the synthesis of secondary metabolites of host plants. Endophytes can also promote plant growth by secreting plant hormones or promoting plant hormone secretion, increase the total biomass of plants, improve plant seed vitality, promote seedling survival and tillering growth, thereby increasing the yield of medicinal plants and increasing secondary metabolites.

[0004] At present, the main method for promoting the growth of endophytes in Astragalus is to add E-2-hexenal and n-hexanol to the culture to promote the growth of nitrogen-fixing endophytic fungi; or to add vitamins to the culture and control the phosphorus element to promote the growth of bacteria. However, the growth-promoting effect is mainly to enhance the growth of endophytes in Astragalus under stress conditions, and cannot be applied to other situations and high-density endophyte culture methods. Moreover, constantly adding materials according to actual conditions increases the manpower and material resources, and cannot achieve better results. Summary of the invention

[0005] The embodiment of the present invention provides a method for high-density culturing of endophytes, thereby solving the problem that the prior art cannot be used for high-density culturing and wastes manpower and material resources, and realizes high-density rapid culturing, saving manpower and material resources.

[0006] The embodiment of the present invention provides a method for high-density culturing of endophytes, comprising the following steps:

[0007] Using an extract containing phosphatidylcholine to soak the astragalus powder under ultrasonic conditions to obtain an extract containing the active ingredient of the astragalus;

[0008] Inoculating the endophytic bacteria of Astragalus membranaceus on a solid culture medium containing the extract to perform screening culture and activation;

[0009] inoculating the activated Astragalus endophytes into a liquid culture medium to obtain an endophyte seed solution;

[0010] Placing the endophytic bacteria seed solution in a culture solution for fermentation and culture to obtain high-density endophytic bacteria of Astragalus membranaceus;

[0011] The culture solution comprises: 10-15 parts of leaching solution, 4-6 parts of peptone, 1.5-3 parts of yeast powder, 4-6 parts of glucose, 1.5-2.5 parts of trace salt solution, 0.2-0.3 parts of copper citrate, 0.1-0.20 parts of diammonium citrate, 1.5-2.5 parts of C6 volatiles, 1-2 parts of hydroxyoctadecadienoic acid, and 1000 parts of LB culture medium.

[0012] According to an embodiment of the present invention, the step of soaking the astragalus powder in an extract containing phosphatidylcholine under ultrasonic conditions to obtain an extract containing active ingredients of astragalus comprises:

[0013] The astragalus is dried, powdered and sterilized to obtain pretreated astragalus powder;

[0014] The astragalus powder and the extract are ultrasonically extracted at 45° C.-60° C. for 0.5 h-2 h, and then the filter residue is filtered out to obtain the extract.

[0015] According to an embodiment of the present invention, the extract comprises the following components in parts by mass:

[0016] 1-3 parts of phosphatidylcholine, 3-5 parts of n-hexanol, 1-5 parts of sodium chloride, 2-4 parts of polysorbates, 0.1-0.2 parts of E-2-hexenal and 100 parts of water.

[0017] According to an embodiment of the present invention, the components of the solid culture medium include: 4-6 parts of peptone, 1.5-3 parts of yeast powder, 4-6 parts of glucose, 0.4-0.5 parts of B vitamins, 10-15 parts of leaching solution, 1.5-2.5 parts of trace salt solution, 15 parts of agar, 1.5-2.0 parts of sucrose, 0.7-1.0 parts of catechol, and 1000 parts of sterile water.

[0018] According to an embodiment of the present invention, the components of the B vitamins include, by mass: 1-2 parts of vitamin B1, 1-2 parts of vitamin B2, 1-2 parts of vitamin B3, 1-2 parts of vitamin B5, 1-2 parts of vitamin B7, 1-2 parts of vitamin B9, and 0.5-1 parts of vitamin PP.

[0019] According to an embodiment of the present invention, the initial concentration of endophytes in the culture solution is not less than 1.6×10 7 cells / L, and the culture temperature is controlled to be 25-30° C., the culture time is 2-6 days, and the rotation speed is 150-200 rpm.

[0020] According to an embodiment of the present invention, the C6 volatiles include one or more of n-hexanal, Z-3-hexenal, n-hexanol, and esters formed by n-hexanal, esters formed by Z-3-hexenal, and esters formed by n-hexanol.

[0021] According to an embodiment of the present invention, the initial pH value of the culture medium is 7.0 - 8.0, the continuously increasing pH is always controlled not to be lower than 7, the dissolved oxygen content is 50 - 60%, the culture temperature is 28 °C, and the rotation speed is 100 rpm;

[0022] The culture medium is added to the fermentation device in a continuously decreasing manner to control the copper element in the early stage in the fermentation device to be 0.04 - 0.06 g / L, and the copper element in the later stage to be less than 0.01 g / L.

[0023] According to an embodiment of the present invention, the early stage refers to within 12 h after adding the endophyte seed liquid for culture;

[0024] The later stage refers to after 4 days of adding the endophyte seed liquid for culture;

[0025] The continuously increasing amount means that the amount of diammonium citrate gradually increases after 1 day.

[0026] According to an embodiment of the present invention, the composition components of the trace salt solution include, by mass: 0.5 - 1 part of FeSO4·7H2O, 0.25 - 0.5 part of MnCl2·4H2O, 0.5 - 1 part of MgSO4·7H2O, 0.5 - 1 part of NaCl, 0.5 - 1 part of CuSO4·5H2O, and 100 parts of sterile water.

[0027] One or more technical solutions provided in the embodiments of the present invention have at least the following technical effects or advantages:

[0028] The astragalus powder is extracted in the leaching solution to leach out polysaccharides, flavonoids, polyphenols and other substances in astragalus, and in the subsequent co-culture, it can better provide a growth environment similar to that in the astragalus plant for the endophyte, which is beneficial to the growth of the endophyte;

[0029] In high-density culture, since various enzymes produced during the growth of the bacterial cells and their own growth require copper elements, a large amount of endophytes grow in the early stage, so more copper elements are needed. In the later stage, the growth of the endophytes is slow, the enzymes are saturated, and too high copper concentration is likely to cause damage to the bacterial cells, so less copper elements are needed; the cooperation between copper and iron promotes the absorption and utilization of copper and iron more; vitamin B group not only benefits the growth of the bacterial cells, but can also combine with copper to increase the water solubility of copper and increase its utilization degree.

[0030] The liquid culture medium is added to the fermenter in a continuously decreasing manner to achieve continuous feeding culture to control the amount of copper element in the fermenter.

[0031] By continuously increasing the control to keep the pH not lower than 7, it not only prevents the bacteria from being damaged in an acidic environment but also reduces the amount of free copper ions, which not only promotes the growth of the bacteria but also reduces the possibility of being damaged by copper ions. Description of the Drawings

[0032] Figure 1 It is a flowchart of step 9 of a method for high-density cultivation of endophytes according to the present invention. Detailed Embodiments

[0033] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings; the preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein; on the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] According to one aspect of the present invention, there is provided a method for high-density cultivation of endophytes, comprising the following steps:

[0036] S1: Soak the astragalus powder with an extract containing phosphatidylcholine under ultrasonic conditions to obtain an extract containing astragalus active ingredients;

[0037] S2: Inoculate the astragalus endophyte on a solid medium containing the extract for screening cultivation and activation;

[0038] S3: Inoculate the activated astragalus endophyte into a liquid medium for cultivation to obtain an endophyte seed solution;

[0039] S4: Ferment and culture the endophyte seed solution in a culture solution to obtain high-density astragalus endophytes;

[0040] Among them, the culture solution includes: 10 - 15 parts of the extract, 4 - 6 parts of peptone, 1.5 - 3 parts of yeast powder, 4 - 6 parts of glucose, 1.5 - 2.5 parts of trace salt solution, 0.2 - 0.3 parts of copper citrate, 0.1 - 0.20 parts of diammonium citrate, 1.5 - 2.5 parts of C6 volatile matter, 1 - 2 parts of hydroxyoctadecadienoic acid, and 1000 parts of LB medium.

[0041] Among the endophytes in Astragalus, the number of strains of Pseudomonas, Pantoea and Staphylococcus was relatively high, accounting for 80.00% of the total isolated strains. The strains with the ability to synthesize indoleacetic acid accounted for the highest proportion, followed by strains containing ACC deaminase and with nitrogen fixation activity.

[0042] In high-density culture, since the various enzymes produced during bacterial growth and the growth of the bacteria themselves require copper, the endophytes grow in large quantities in the early stage, so more copper is needed. In the later stage, the endophytes grow slowly, the enzyme production is saturated, and the excessive copper concentration can easily cause bacterial damage, so less copper is needed; and the cooperation between copper and iron further promotes the absorption and utilization of copper and iron. C6 volatiles have a promoting effect on the growth of strains containing ACC deaminase, indicating that C6 volatiles may be one of the important factors affecting the composition and function of the endophytic flora of Astragalus. C6 volatiles have been found to promote the growth of some endophytic strains. Low concentrations of n-hexanal, Z-3-hexenal and n-hexanol have been found to promote the growth of some endophytic strains containing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. Hydroxyoctadecadienoic acid, a triterpene from plant sources, exists in plants in free form or in the form of glycosides or esters combined with sugars, and has a variety of biochemical activities. They also play key roles in plant-microbe interactions, such as promoting the growth of endophytic bacteria and fungi, which in turn enhance the growth and secondary metabolite production of host plants. Terpene-promoted bacterial and fungal strains play a more active role in the microbiome.

[0043] According to an embodiment of the present invention, the extract containing phosphatidylcholine is used to soak the astragalus powder under ultrasonic conditions to obtain the extract containing the active ingredient of astragalus, which includes:

[0044] The astragalus is dried, powdered and sterilized to obtain pretreated astragalus powder;

[0045] The astragalus powder and the extract are ultrasonically extracted at 45°C-60°C for 0.5h-2h, and then the filter residue is filtered out to obtain the extract.

[0046] By utilizing the mechanical vibration and cavitation effect of ultrasound, the hydrogen bonds between molecules can be destroyed, the extraction efficiency and solubility of astragalus polysaccharides and saponins can be improved, and the solubility of flavonoids in water can be increased.

[0047] According to an embodiment of the present invention, the extract comprises the following components in parts by mass:

[0048] 1-3 parts of phosphatidylcholine, 3-5 parts of n-hexanol, 1-5 parts of sodium chloride, 2-4 parts of polysorbates, 0.1-0.2 parts of E-2-hexenal and 100 parts of water.

[0049] Astragalus powder is leached in a mixed solution of E-2-hexenal and n-hexanol. Astragalus itself contains such organic substances. At 85 °C, substances such as polysaccharides, flavonoids, and polyphenols in Astragalus are leached out, providing a growth environment similar to that in the Astragalus plant for endophytes in subsequent co-cultures, which is beneficial to the growth of endophytes. Although it is not directly soluble in water, organic solvents can be mixed with water to form a mixed solvent system to increase the solubility of flavonoid compounds. The solubility of flavonoid compounds in Astragalus can be increased by adding phosphatidylcholine, n-hexanol, polysorbates, and E-2-hexenal. Phosphatidylcholine belongs to phospholipid co-solvents, which can improve the bioavailability of drugs and have biocompatibility.

[0050] According to an embodiment of the present invention, the composition components of the solid medium include: 4-6 parts of peptone, 1.5-3 parts of yeast powder, 4-6 parts of glucose, 0.4-0.5 parts of vitamin B group, 10-15 parts of leaching solution, 1.5-2.5 parts of trace salt solution, 15 parts of agar, 1.5-2.0 parts of rhamnolic acid, 0.7-1.0 parts of catechol, and 1000 parts of sterile water.

[0051] Adding E-2-hexenal and n-hexanol during cultivation promotes the growth of nitrogen-fixing endophytic fungi; or adding vitamins during cultivation, along with controlling the phosphorus element, promotes the growth of bacteria.

[0052] According to an embodiment of the present invention, the composition components of the vitamin B group by mass include: 1-2 parts of vitamin B1, 1-2 parts of vitamin B2, 1-2 parts of vitamin B3, 1-2 parts of vitamin B5, 1-2 parts of vitamin B7, 1-2 parts of vitamin B9, and 0.5-1 part of vitamin PP.

[0053] According to an embodiment of the present invention, the initial concentration of endophytes in the culture solution is not less than 1.6×107 cells / L, and the culture temperature is controlled at 25-30 °C, the time is 2-6 d, and the rotation speed is 150-200 rpm.

[0054] According to an embodiment of the present invention, the C6 volatiles include one or more of n-hexanal, Z-3-hexenal, n-hexanol, and esters formed by n-hexanal, esters formed by Z-3-hexenal, and esters formed by n-hexanol.

[0055] According to an embodiment of the present invention, the initial pH value of the culture solution is 7.0-8.0, the pH is continuously increased and controlled to be not lower than 7, the dissolved oxygen content is 50-60%, the culture temperature is 28 °C, and the rotation speed is 100 rpm;

[0056] The culture solution is added to the fermentation device in a continuously decreasing manner to control the copper element in the fermentation device to be 0.04-0.06 g / L in the early stage and less than 0.01 g / L in the later stage.

[0057] According to an embodiment of the present invention, the early stage refers to within 12 h after inoculating the endophyte seed solution for culture;

[0058] The late stage refers to after 4 d of inoculating the endophyte seed solution for culture;

[0059] The continuous increment is that the amount of diamine citrate gradually increases after 1 d.

[0060] According to an embodiment of the present invention, the composition of the trace salt solution includes, by mass: 0.5 - 1 part of FeSO4·7H2O, 0.25 - 0.5 part of MnCl2·4H2O, 0.5 - 1 part of MgSO4·7H2O, 0.5 - 1 part of NaCl, 0.5 - 1 part of CuSO4·5H2O, and 100 parts of sterile water.

[0061] Example 1

[0062] As Figure 1 shown, culturing Sphingobacterium using a method for high-density culturing of endophytes provided by the present invention includes the following steps:

[0063] Step 1: Subject Astragalus membranaceus to drying, powdering, and sterilization treatments to obtain pretreated Astragalus membranaceus powder;

[0064] Step 2: Mix the Astragalus membranaceus powder with the extraction solution at 45°C and stir for 12 h, then filter to remove the residue to obtain the extract. The extraction solution includes the following components by mass:

[0065] 1 g of phosphatidylcholine, 5 g of n-hexanol, 1 g of sodium chloride, 4 g of polysorbate, 0.2 g of E-2-hexenal, and 100 ml of water;

[0066] Step 3: Inoculate the Astragalus membranaceus endophyte on a solid medium containing the extract, screen and culture it for two generations of activation, then streak it on the solid medium and culture at 37°C for 24 h. The composition of the solid medium includes: 6 g of peptone, 2.5 g of yeast powder, 6 g of glucose, 0.5 g of vitamin B complex, 15 ml of the extract, 2 mL of the trace salt solution, 15 g of agar, 2.0 g of rhamnose, 1.0 g of catechol, and 1 L of sterile water;

[0067] The composition of the vitamin B complex, by mass, includes: 1 part of vitamin B1, 1 part of vitamin B2, 1 part of vitamin B3, 1 part of vitamin B5, 1 part of vitamin B7, 1 part of vitamin B9, and 0.5 part of vitamin PP.

[0068] Step 4: Inoculate the activated Astragalus membranaceus endophyte into a liquid medium and culture at 37°C for 24 h to obtain an endophyte seed solution with a concentration of not less than 10 7 cells / L;

[0069] Step 5: Use a pipette to aspirate 1 mL of the above-mentioned endophyte seed solution, and inoculate it into 100 mL of the culture medium for fermentation culture at an inoculation amount of 1%, and culture it at 37°C to obtain a high-density Astragalus membranaceus endophyte. The initial concentration of the endophyte in the culture medium is not less than 1.6×10 7 cells / L, control the culture temperature at 28°C, the time at 5 d, the rotation speed at 200 rpm, the initial pH value of the culture medium at 7.5, continuously increase the amount to control the pH not lower than 7, the dissolved oxygen content at 60%, the culture temperature at 28°C, and the rotation speed at 100 rpm; the culture medium is added to the fermentation device in a continuously decreasing manner to control the copper element in the fermentation device to be 0.05 g / L in the early stage and less than 0.01 g / L in the later stage. The early stage refers to within 12 h after inoculating the endophyte seed solution; the later stage refers to 4 d after inoculating the endophyte seed solution; the continuous increase means that the amount of diamine citrate gradually increases after 1 d;

[0070] Among them, the culture medium includes: 10 mL of leaching solution, 6 g of peptone, 1.5 g of yeast powder, 6 g of glucose, 2 mL of trace salt solution, 0.3 g of copper citrate, 0.20 g of diamine citrate, 1.5 g of C6 volatile matter, 1 g of hydroxyoctadecadienoic acid, and 1 L of LB medium.

[0071] The C6 volatile matter includes Z-3-hexenal.

[0072] The composition components of the trace salt solution include by mass: 0.5 g of FeSO4·7H2O, 0.25 g of MnCl2·4H2O, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.5 g of CuSO4·5H2O, and 100 mL of sterile water.

[0073] According to this example, 2 - 3 d is the logarithmic growth period of the bacteria, and the fermentation tank is emptied during the stable logarithmic growth period. At this time, the OD value of the bacteria in the fermentation broth reaches 3.3, and the total culture time is 4 d.

[0074] The liquid culture medium is added to the fermentation tank in a continuously decreasing manner to achieve continuous feeding culture to control the amount of copper element in the fermentation tank. The number of viable bacteria in the fermentation broth obtained with the basic culture medium, i.e., MRS medium, is 4.8×10 8 cfu / mL, while the number of viable bacteria in the fermentation broth obtained by the culture method of the present invention is 1.6×10 11 cfu / mL, which is two orders of magnitude higher than the culture result in the basic culture medium.

[0075] Example 2

[0076] Using the method for high-density culture of endophytes provided by the present invention to culture Chaetomium globosum includes the following steps:

[0077] Step 1: Dry, powderize and sterilize Astragalus membranaceus to obtain pretreated Astragalus membranaceus powder;

[0078] Step 2: Mix the Astragalus membranaceus powder with the extraction solution and stir at 60 °C for 8 h, then filter to obtain the extract. The extraction solution includes the following components by mass fraction:

[0079] 3 g of phosphatidylcholine, 3 g of n-hexanol, 5 g of sodium chloride, 2 g of polysorbate, 0.1 g of E-2-hexenal, and 100 ml of water;

[0080] Step 3: Inoculate the endophytic bacteria of Astragalus membranaceus on the solid medium containing the extract, screen, culture and activate for two generations, then streak on the solid medium and culture at 37 °C for 24 h. The composition of the solid medium includes: 4 g of peptone, 3 g of yeast powder, 4 g of glucose, 0.4 g of vitamin B complex, 10 ml of extract, 2.5 ml of trace salt solution, 15 g of agar, 1.5 g of rhamnose, 0.7 g of catechol, and 1 L of sterile water;

[0081] The composition of the vitamin B complex by mass fraction includes: 1 part of vitamin B1, 1 part of vitamin B2, 1 part of vitamin B3, 1 part of vitamin B5, 1 part of vitamin B7, 1 part of vitamin B9, and 0.5 part of vitamin PP.

[0082] Step 4: Inoculate the activated endophytic bacteria of Astragalus membranaceus into the liquid medium and culture at 37 °C for 24 h to obtain an endophytic bacteria seed solution with a concentration of not less than 10 7 cells / L;

[0083] Step 5: Use a pipette to aspirate 1 mL of the above endophytic bacteria seed solution, that is, inoculate it into 100 mL of the culture solution for fermentation culture at an inoculation amount of 1%, and culture at 37 °C to obtain high-density endophytic bacteria of Astragalus membranaceus. The initial concentration of endophytic bacteria in the culture solution is not less than 1.6×10 7 cells / L, control the culture temperature at 25 °C, the time at 6 d, the rotation speed at 200 rpm, the initial pH value of the culture solution at 8.0, continuously increase the amount to control the pH not lower than 7, the dissolved oxygen content at 50%, the culture temperature at 28 °C, and the rotation speed at 100 rpm; the culture solution is added to the fermentation device in a continuously decreasing manner to control the copper element in the fermentation device to be 0.04 g / L in the early stage and less than 0.01 g / L in the later stage. The early stage refers to within 12 h after inoculating the endophytic bacteria seed solution; the later stage refers to 4 d after inoculating the endophytic bacteria seed solution; the continuous increase is that the amount of diammonium citrate gradually increases after 1 d;

[0084] Among them, the culture medium includes: 15 mL of leaching solution, 4 g of peptone, 3 g of yeast powder, 4 g of glucose, 2.5 mL of trace salt solution, 0.2 g of copper citrate, 0.10 g of diammonium citrate, 2.5 g of C6 volatile matter, 2 g of hydroxyoctadecadienoic acid, and 1 L of LB medium.

[0085] The C6 volatile matter includes Z-3-hexenal and n-hexanol.

[0086] The composition components of the trace salt solution by mass include: 0.5 g of FeSO4·7H2O, 0.25 g of MnCl2·4H2O, 0.5 g of MgSO4·7H2O, 1 g of NaCl, 1 g of CuSO4·5H2O, and 100 mL of sterile water.

[0087] The liquid culture medium is added to the fermenter in a continuously decreasing manner to achieve continuous feeding culture to control the amount of copper element in the fermenter. The number of viable bacteria in the fermentation broth obtained with the basic culture medium, i.e., MRS medium, is 4.5×10 8 cfu / mL, while the number of viable bacteria in the fermentation broth obtained by the culture method of the present invention is 9.4×10 10 cfu / mL.

[0088] Example 3

[0089] Using the method for high-density culture of endophytic bacteria provided by the present invention to culture Pseudomonas herbicola includes the following steps:

[0090] Step 1: The astragalus is dried, powdered, and sterilized to obtain pretreated astragalus powder;

[0091] Step 2: The astragalus powder is mixed and stirred with the leaching solution at 50 °C for 9 h, and then the filter residue is filtered off to obtain the leaching solution. The leaching solution includes the following components by mass:

[0092] 2 g of phosphatidylcholine, 4 g of n-hexanol, 3 g of sodium chloride, 3 g of polysorbate, 0.2 g of E-2-hexenal, and 100 ml of water;

[0093] Step 3: The astragalus endophytic bacteria are inoculated on the solid culture medium containing the leaching solution for screening culture and activation for two generations, then streaked on the solid culture medium and cultured at 37 °C for 24 h. The composition components of the solid culture medium include: 5 g of peptone, 1.5 g of yeast powder, 5 g of glucose, 0.4 g of vitamin B group, 12 ml of leaching solution, 1.5 ml of trace salt solution, 15 g of agar, 1.5 g of rhamnolic acid, 0.7 g of catechol, and 1 L of sterile water;

[0094] The composition components of the vitamin B group by mass include: 2 parts of vitamin B1, 2 parts of vitamin B2, 2 parts of vitamin B3, 2 parts of vitamin B5, 2 parts of vitamin B7, 2 parts of vitamin B9, and 1 part of vitamin PP.

[0095] Step 4: Inoculate the activated Astragalus membranaceus endophyte into a liquid medium and culture it at 37°C for 24 h to obtain an endophyte seed solution with a concentration of not less than 10 7 cells / L;

[0096] Step 5: Use a pipette to aspirate 1 mL of the above endophyte seed solution, that is, inoculate it into 100 mL of culture medium for fermentation culture at an inoculation amount of 1%, culture at 37°C to obtain a high-density Astragalus membranaceus endophyte. The initial concentration of the endophyte in the culture medium is not less than 1.6×107 cells / L, and control the culture temperature at 30°C, the time at 2 d, the rotation speed at 150 rpm, the initial pH value of the culture medium at 7.0, continuously increase the pH to keep it not lower than 7, the dissolved oxygen content at 55%, the culture temperature at 28°C, and the rotation speed at 100 rpm; the culture medium is added to the fermentation device in a continuously decreasing manner to control the copper element in the fermentation device to be 0.06 g / L in the early stage and less than 0.01 g / L in the later stage. The early stage refers to within 12 h after inoculating the endophyte seed solution; the later stage refers to 4 d after inoculating the endophyte seed solution; the continuous increase means that the amount of diamine citrate gradually increases after 1 d;

[0097] Among them, the culture medium includes: 12 mL of leaching solution, 5 g of peptone, 2.5 g of yeast powder, 5 g of glucose, 2 mL of trace salt solution, 0.3 g of copper citrate, 0.20 g of diamine citrate, 1.5 g of C6 volatiles, 1 g of hydroxyoctadecadienoic acid, and 1 L of LB medium.

[0098] The C6 volatiles include hexanal and hexanol.

[0099] The composition components of the trace salt solution include by mass: 1 g of FeSO4·7H2O, 0.5 g of MnCl2·4H2O, 0.5 g of MgSO4·7H2O, 1 g of NaCl, 0.5 g of CuSO4·5H2O, and 100 mL of sterile water.

[0100] Add the liquid medium to the fermentation tank in a continuously decreasing manner to achieve continuous feeding culture to control the amount of copper element in the fermentation tank. The number of viable bacteria in the fermentation broth obtained with the basic medium, i.e., MRS medium, is 4.2×10 8 cfu / mL, while the number of viable bacteria in the fermentation broth obtained by the culture method of the present invention is 1.2×10 11 cfu / mL.

[0101] Example 4

[0102] In the above embodiments, the continuous reduction addition of copper citrate is used to control the amount of copper element during fermentation to avoid damage to the bacterial cells, and it cooperates with vitamin B group and iron element to increase the absorption efficiency and utilization rate. To further reduce the damage to endogenous bacteria caused by copper element, the following changes are made in this embodiment compared with Embodiment 1.

[0103] The endogenous bacteria seed liquid and the liquid medium containing copper element are added to a fermenter for high-density culture to obtain high-density Astragalus membranaceus endogenous bacteria;

[0104] Control the initial pH value in the fermenter to be 7.5, the dissolved oxygen content to be 60%, the culture temperature to be 28 °C, and the rotation speed to be 100 rpm; and the liquid medium containing copper element is added to the fermenter in a continuously decreasing manner to control the copper element in the fermenter to be 0.05 g / L in the first 12 h and less than 0.01 g / L on the last day of culture. The continuous reduction is the reduction of copper element, and other components are continuously added to the fermenter. After 1 day, the amount of diammonium citrate gradually increases to control the continuously decreasing pH value.

[0105] The composition components of the liquid medium containing copper element include: 6 g of peptone, 3 g of yeast powder, 6 g of glucose, 7.5 mg of pretreated Astragalus membranaceus leaching solution, 2 mL of trace salt solution, 0.3 g of copper citrate, 0.20 g of diammonium citrate, and 1 L of sterile water;

[0106] The composition components of the trace salt solution include: 0.5 g of FeSO4·7H2O, 0.25 g of MnCl2·4H2O, 0.5 g of MgSO4·7H2O, 0.5 g of NaCl, 0.5 g of CuSO4·5H2O, and 100 mL of sterile water;

[0107] According to this embodiment, 2 - 3 d is the logarithmic growth phase of the bacterial cells, and the fermentation is terminated at the stable logarithmic growth stage. At this time, the OD value of the bacterial cells in the fermentation broth reaches 3.5, and the total culture time is 4 d.

[0108] Generally, after culturing for 3 d, the pH drops below 6. The dissolution degree of copper ions has a high correlation with the pH of the solution. When the pH is low, the solubility of copper ions is large, and when there are more free copper ions in the solution, it will have an impact on the bacterial cells. Controlling the pH not only prevents the bacterial cells from being damaged in an acidic environment but also reduces the amount of copper ions.

[0109] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for high-density culturing of endophytes, characterized in that: The following steps are involved: The method comprises soaking the astragalus powder with an extract containing phosphatidylcholine under ultrasonic conditions to obtain an extract containing the active ingredients of the astragalus, comprising: ultrasonically extracting the astragalus powder and the extract at 45° C. to 60° C. for 0.5 h to 2 h, and then filtering out the filter residue to obtain the extract; Inoculating endophytes of Astragalus membranaceus on a solid culture medium containing the extract for screening, culture and activation, wherein the endophytes of Astragalus membranaceus are Sphingobacillus, Chaetomium globosum or Pseudomonas thaliana; inoculating the activated Astragalus endophytes into a liquid culture medium to obtain an endophyte seed solution; placing the endophytic bacteria seed solution in a culture solution for fermentation and culture to obtain high-density endophytic bacteria of Astragalus membranaceus; The components of the solid culture medium containing the leaching solution include: 4-6 parts of peptone, 1.5-3 parts of yeast powder, 4-6 parts of glucose, 0.4-0.5 parts of B vitamins, 10-15 parts of leaching solution, 1.5-2.5 parts of trace salt solution, 15 parts of agar, 1.5-2.0 parts of sucrose, 0.7-1.0 parts of catechol, and 1000 parts of sterile water; the components of the B vitamins include, by mass, 1-2 parts of vitamin B1, 1-2 parts of vitamin B2, 1-2 parts of vitamin B3, 1-2 parts of vitamin B5, 1-2 parts of vitamin B7, 1-2 parts of vitamin B9, and 0.5-1 parts of vitamin PP; The culture solution comprises: 10-15 parts of leaching solution, 4-6 parts of peptone, 1.5-3 parts of yeast powder, 4-6 parts of glucose, 1.5-2.5 parts of trace salt solution, 0.2-0.3 parts of copper citrate, 0.1-0.20 parts of diammonium citrate, 1.5-2.5 parts of C6 volatiles, 1-2 parts of hydroxyoctadecadienoic acid, and 1000 parts of LB culture medium; the C6 volatiles comprise one or more of n-hexanal, Z-3-hexenal, and n-hexanol; The initial pH value of the culture solution is 7.0-8.0, the pH is continuously increased to control it to be no less than 7, the dissolved oxygen content is 50-60%, the culture temperature is 28°C, and the rotation speed is 100 rpm; The culture solution is added to the fermentation device in a continuously decreasing manner to control the copper element in the fermentation device to be 0.04-0.06 g / L in the early stage and less than 0.01 g / L in the late stage; The early stage refers to the period within 12 hours after the endophyte seed solution is added; the late stage refers to the period after 4 days after the endophyte seed solution is added; the continuous increase refers to the gradual increase in the amount of diammonium citrate after 1 day; The extract comprises the following components in parts by mass: 1-3 parts of phosphatidylcholine, 3-5 parts of n-hexanol, 1-5 parts of sodium chloride, 2-4 parts of polysorbates, 0.1-0.2 parts of E-2-hexenal and 100 parts of water; The components of the trace salt solution include, by mass, 0.5-1 part of FeSO4·7H2O, 0.25-0.5 part of MnCl2·4H2O, 0.5-1 part of MgSO4·7H2O, 0.5-1 part of NaCl, 0.5-1 part of CuSO4·5H2O, and 100 parts of sterile water.

2. A method for high-density culturing of endophytes according to claim 1, characterized in that: The method of soaking the astragalus powder in an extract containing phosphatidylcholine under ultrasonic conditions to obtain an extract containing the active ingredients of astragalus comprises: The astragalus is dried, powdered and sterilized to obtain pretreated astragalus powder.

3. The method for high-density culturing of endophytes according to claim 1, characterized in that: The initial concentration of endophytes in the culture solution is not less than 1.6×10 7 cells / L, and control the culture temperature to 25-30℃, the time to 2-6 days, and the rotation speed to 150-200rpm.

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