CN114107280A - Method for improving physiological and biochemical activities of strains through electrical stimulation and application of method - Google Patents
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Abstract
The invention discloses a method for improving physiological and biochemical activities of bacterial strains by electrical stimulation and application thereof. The method constructs an electrode system for understanding culture of the Bacillus amyloliquefaciens ZJK1 or the Bacillus belief (Bacillus velezensis) ZJK2 strain through an inert electrode, electrically stimulates the activated strain by low-voltage direct current, maintains the temperature at 30-37 ℃, cultures for 16-24 hours, can improve the biochemical activity of the functional strain by 20-50 percent, and improves the fermentation density by more than 1-2 times. The method greatly improves the biochemical activity and fermentation density of the strain, and has the advantages of simple process, simple and convenient operation and low cost.
Description
Technical Field
The invention belongs to the field of microbiology, and particularly relates to a method for improving physiological and biochemical activities of a strain by electrical stimulation and application thereof.
Background
The plant growth promoting bacteria are a general term for a group of beneficial bacteria living in the rhizosphere and having a promoting effect on plant growth. The plant growth promoting bacteria can activate soil nutrient substances through metabolic activities, promote plants to absorb nutrients, accelerate growth and the like. The application of the microbial inoculum rich in the active growth-promoting bacteria is beneficial to restoring the balance of a soil ecosystem and restoring the soil fertility, thereby arousing the wide attention of people. Researches show that the plant growth promoting bacteria can promote the growth of crops through direct and indirect ways, the direct actions include secretion of plant hormones to promote the growth, secretion of organic acids to promote conversion of insoluble phosphorus into available phosphorus, secretion of azotase and the like to increase soil fertility, and the indirect actions include induction of crop resistance, direct antagonism with pathogenic bacteria and the like. One of the key problems affecting the application of plant growth promoting bacteria at present is the screening and cultivation of excellent strains.
The low-voltage weak electric field can affect the charge distribution, arrangement and movement condition inside and outside the biological cell, thereby affecting the cell metabolism and proliferation. Based on the theory, the electric field is used as a biological stimulation factor, and a micro electric field is applied in the growth process of microorganisms, so that the method has the characteristics of low energy consumption and no generation of toxic and harmful substances, and is a green and environment-friendly technology. The electric field is used for processing the fruits and vegetables, so that the maturation period of the fruits and vegetables can be delayed, the water loss rate is slowed down, and the storage time of the fruits and vegetables is prolonged.
With the progress of research in recent years, the biological effect of the electric field has been continuously advanced from the macroscopic level to the microscopic mechanism. The data show that the electric field can affect the metabolic process of the cell, the gene expression of the cell, the cell proliferation, the enzyme activity and the cell membrane permeability, thereby affecting the free radical reaction and the biopolymer synthesis in the cell.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for improving physiological and biochemical activities of strains by electrical stimulation and application thereof.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
a method for improving physiological and biochemical activities of strains by electrical stimulation comprises constructing an electrode system for strain culture by inert electrodes, performing electrochemical induction acclimatization, and stimulating the growth of strains by low-voltage direct current to improve the biochemical activities and fermentation density to obtain culture solution of the electrically stimulated strains; the strain is bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) ZJK1 or Bacillus belgii (B.), (Bacillus velezensis）ZJK2。
As an improvement, the method for improving the physiological and biochemical activity of the strain by electric stimulation comprises the following steps:
Inoculating the strain stored at low temperature into a sterilized 500 mL triangular flask according to the volume ratio of 5-10%, activating in a sterilized beef extract peptone liquid culture medium, and shake-culturing at 28-37 deg.C for 16-24h to obtain activated strain seed liquid;
step 2, electrochemical induction
Inserting the sterilized inert electrode into a sterilized beef extract peptone liquid culture medium, inoculating the activated strain seed liquid into the liquid culture medium according to the volume ratio of 5-10%, introducing low-voltage direct current through the inert electrode, and performing shake culture at the constant temperature of 28-37 ℃ for 24h to obtain an electrochemical induction domesticated strain culture solution;
step 3, growth of the electrically stimulated Strain
Inoculating the strain culture solution subjected to electrochemical induction domestication into a sterilized beef extract peptone liquid culture medium according to the volume ratio of 5-10%, introducing low-voltage direct current to stimulate the growth of the strain, and performing constant-temperature shaking culture for 16-48h to obtain the electro-stimulation strain culture solution.
As an improvement, the strain is bacillus amyloliquefaciens (B.amyloliquefaciens)Bacillus amyloliquefaciens) ZJK1, Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) The preservation date of ZJK1 is 6.8 days in 2020, the preservation address is No. 3 Xilu No. 1 Beijing north Chen of the Chaoyang district, the China general microbiological culture Collection center, the preservation number is: CGMCC number 20036.
As an improvement, the strain is Bacillus belgii (B.) (Bacillus velezensis) ZJK2 was deposited in China general microbiological culture Collection center (CGMCC) on 8.6.2020 with the accession number of CGMCC 20037.
The improvement is that the current intensity of the low-voltage direct current in the step 2 is 5-10 mA.
The improvement is that the current intensity of the low-voltage direct current stimulation in the step 3 is 5-30 mA.
The obtained electro-stimulation strain culture solution is applied to promoting plant growth and preventing and treating gray mold and anthracnose.
As an improvement, when the strain is bacillus amyloliquefaciens (B.amyloliquefaciens)Bacillus amyloliquefaciens) ZJK1, the application is for promoting strawberry or mulberryGrowing; when the strain is Bacillus belgii (B)Bacillus velezensis) ZJK2, the application is to prevent and treat gray mold or anthracnose of mulberry, strawberry or grape.
A plant growth additive contains any one of the above electro-stimulating strain culture solutions.
Preferably, when the strain is Bacillus amyloliquefaciens (B.amyloliquefaciens)Bacillus amyloliquefaciens) When ZJK1, the plant growth additive is a plant growth promoting microbial inoculum; when the strain is Bacillus belgii (B)Bacillus velezensis) ZJK2, the plant growth additive is a microbial fungicide.
Has the advantages that:
compared with the prior art, the method for improving physiological and biochemical activities of the strain by electrical stimulation and the application thereof can improve the growth promoting capacity of the bacillus amyloliquefaciens ZJK1 by 30-50% and the biocontrol capacity of the bacillus belgii ZJK2 by 20-30%, and can improve the fermentation density of the strain by 1-2 times. The method has the advantages of low cost, capability of improving functional strains, easiness in realizing industrial production and good development and application prospects.
Drawings
FIG. 1 Effect of impressed current on the ability of a strain to secrete IAA;
FIG. 2 shows the method of the present invention for applying electric current to Bacillus belgii (B) ((B))Bacillus velezensis) Growth profile effect of ZJK 2.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
Example 1
The strain is bacillus amyloliquefaciens (A), (B) and (C)Bacillus amyloliquefaciens) ZJK1, Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) The preservation date of ZJK1 is 6.8 days in 2020, the preservation address is No. 3 Xilu No. 1 Beijing north Chen of the Chaoyang district, the China general microbiological culture Collection center, the preservation number is: CGMCC number 20036
A method for promoting fermentation density of Bacillus amyloliquefaciens ZJK1 by an external direct current electric field comprises the following steps:
sterilizing an electrified culture bottle, an electrode material, a beef extract peptone liquid culture medium and the like by high-pressure steam (sterilizing for 20 min at 121 ℃); then taking 15m L of bacterial suspension which is uniformly shaken from the seed solution of the electrochemically induced and domesticated bacillus amyloliquefaciens ZJK1, and respectively inoculating the bacterial suspension into 4 500m L triangular flasks containing 300m L of culture medium which is sterilized and cooled by high-pressure steam, wherein 1 flask is not electrified to be used as a blank control. The other 3 bottles adopt stainless steel electrodes (apparent area 2cm x 3cm, electrode spacing 2cm), and 10mA direct current is added for electrical stimulation. 4 bottles of the bacterial solution are simultaneously placed in a constant temperature shaking table (180 r/min) at 37 ℃ for culture.
The results show that the bacterial growth curve stimulated by the applied direct current is obviously improved compared with the control group without electrification.
The bacterial count method shows that the concentration of the thallus in the electrified culture solution is about 1.8 times of that of the unenergized culture solution, and the direct current of 10mA introduced into the bacillus amyloliquefaciens can effectively promote the growth and the propagation of the bacillus amyloliquefaciens.
Example 2
Effect of electrical stimulation of Bacillus amyloliquefaciens culture solution on secretion of auxin IAA
Respectively inoculating 0.5 mL of electrically stimulated and non-energized seed solutions into 50mL of LB liquid culture medium containing 100 mg/L L-tryptophan, culturing at 30 deg.C and 120 r/min in a shaking table for 6, 18, 30, and 48h, respectively taking 1mL of sample, centrifuging at 12000 r/min for 10 min, taking 100 μ L of supernatant, adding equal volume of Salkowski colorimetric solution, mixing, standing in dark place for 30 min, measuring OD530The blank was LB medium without L-tryptophan. And calculating the content of the bacterial liquid IAA in unit volume by referring to a standard curve. ResultsThe IAA concentration in the culture medium was found to be 40% or more higher than that in the control when the electric current was applied to 10 mA.
Example 3
Pot culture experiment for promoting growth of mulberry seedlings by electrically stimulating bacillus amyloliquefaciens culture solution
Selecting mulberry seeds which are full and consistent, soaking the mulberry seeds in warm boiled water at 50 ℃, and soaking the mulberry seeds for 12 hours after the mulberry seeds are cooled in the water. And (4) seedling culture is carried out after germination acceleration, and planting and field planting are carried out when two leaves and one heart are equilong. Healthy mulberry seedlings with consistent growth vigor are selected and transplanted into a plastic basin (containing 200g of air-dried soil), and after the mulberry seedlings are planted for one week, the rooting method is adopted to inoculate and culture the growth-promoting bacteria liquid for 48 hours.
The treatment groups comprise sterilized beef extract peptone culture solution Control (CK), 200-time diluted non-electrified bacterial solution and 200-time electrified 10mA electrically stimulated bacillus amyloliquefaciens culture solution, and 50mL of roots are irrigated into each pot after 5 parallel samples are treated.
When the mulberry seedlings grow into four leaves and one core, measuring the plant height and fresh weight of the mulberry of the experimental group and the mulberry of the control group, carefully peeling off the soil, drying at 105 ℃ to constant weight, and weighing the dry weight of the overground part and the dry weight of the root system, wherein the results are as follows:
as can be seen from the table, the electrically stimulated amylolytic bacillus culture solution significantly increased the height of the mulberry seedling, the fresh weight of the whole plant, the weight of the overground part and the dry weight of the root system compared with the unenergized one.
Example 4
Bacillus belgii (B) used in the present inventionBacillus velezensis) ZJK2, deposited in China general microbiological culture Collection center, with the deposit address of No. 3 of Xilu No. 1 of Suzhong, the area facing the sun, Beijing, the institute of microbiology, China academy of sciences, with the deposit number of CGMCC No. 20037 and the date of deposit of 2020, 6 months and 8 days.
A method for promoting high-density fermentation of Bacillus beilis ZJK2 by applying a direct current electric field.
Sterilizing an electrified culture bottle, an electrode material, a beef extract peptone liquid culture medium and the like by high-pressure steam (sterilizing for 20 min at 121 ℃); then 3m L of uniform shaking bacterial suspension is taken from the seed liquid of the electro-chemically induced domesticated Bacillus belgii ZJK2 and respectively inoculated into 4 triangular flasks of 500m L containing a culture medium of 300m L which is sterilized and cooled by high-pressure steam, wherein 1 flask is not electrified to be used as a blank control. The other 3 bottles adopt stainless steel electrodes (apparent area 2cm x 3cm, electrode spacing 2cm), and 10mA direct current is added for electrical stimulation. 4 bottles of the bacterial solution are simultaneously placed in a constant temperature shaking table (180 r/min) at 37 ℃ for culture.
The results show that the bacterial growth curve stimulated by the applied direct current is obviously improved compared with the control group without electrification. The concentration of cells in the electrified culture medium was about 2 times that in the non-electrified culture medium, as determined by the bacteria counting method. Therefore, the proliferation of the bacillus beilesensis ZJK2 can be effectively promoted under the action of introducing 10mA direct current.
Example 5
Respectively diluting bacterial liquid of bacillus beiLeisi ZJK2 cultured for 24h by electrifying (10 mA) and not electrifying by 200 and 100 times; punching a PDA solid culture medium of strawberry gray mold into round blocks with the same mass by using a puncher with the diameter of 5mm, taking out the round blocks, and inoculating the round blocks to one side of a PDA flat plate; and punching the other side of the PDA plate by using a puncher with the diameter of 5mm to obtain round blocks with the same mass, taking out the round blocks, respectively inoculating 100 mu L of sterile water, electrified and non-electrified diluted bacteria liquid, culturing at 28 ℃ for 7d, and observing. The inhibition rate of different treatment solutions on botrytis cinerea on the plates was calculated as follows:
the inhibition rates of the bacillus bleekii ZJK2 when the bacillus bleekii ZJK2 is electrified and when the bacillus bleekii ZJK2 is not electrified are respectively 93.6% and 72.5%, and the direct current of 10mA is used for improving the antibacterial capacity of the bacillus bleekii ZJK2 by about 30%.
The above description is only a partial embodiment of the present invention, and the protection scope of the present invention is not limited thereto, and any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the protection scope of the present invention.
Claims (10)
1. A method for improving physiological and biochemical activities of strains by electrical stimulation is characterized in that an electrode system for strain culture is constructed by inert electrodes, electrochemical induction domestication is firstly carried out, and then the growth of the strains is electrically stimulated by low-voltage direct current to improve the biochemical activities and fermentation density of the strains, so as to obtain culture solution of the electrically stimulated strains; the strain is bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) ZJK1 or Bacillus belgii (B.), (Bacillus velezensis）ZJK2。
2. The method for improving physiological and biochemical activities of strains through electrical stimulation according to claim 1, which comprises the following steps:
step 1, activation of the Strain
Inoculating the strain stored at low temperature into a sterilized 500 mL triangular flask according to the volume ratio of 5-10%, activating in a sterilized beef extract peptone liquid culture medium, and shake-culturing at 28-37 deg.C for 16-24h to obtain activated strain seed liquid;
step 2, electrochemical induction
Inserting the sterilized inert electrode into a sterilized beef extract peptone liquid culture medium, inoculating the activated strain seed liquid into the liquid culture medium according to the volume ratio of 5-10%, introducing low-voltage direct current through the inert electrode, and performing shake culture at the constant temperature of 28-37 ℃ for 24h to obtain an electrochemical induction domesticated strain culture solution;
step 3, growth of the electrically stimulated Strain
Inoculating the strain culture solution subjected to electrochemical induction domestication into a sterilized beef extract peptone liquid culture medium according to the volume ratio of 5-10%, introducing low-voltage direct current to stimulate the growth of the strain, and performing constant-temperature shaking culture for 16-48h to obtain the electro-stimulation strain culture solution.
3. The method for improving physiological and biochemical activities of bacterial strains through electrical stimulation according to claim 1, wherein the bacterial strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) ZJK1, Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) The preservation date of ZJK1 is 6.8 days in 2020, the preservation address is No. 3 Xilu No. 1 Beijing north Chen of the Chaoyang district, the China general microbiological culture Collection center, the preservation number is: CGMCC number 20036.
4. The method for improving physiological and biochemical activities of bacterial strains through electrical stimulation according to claim 1, wherein the bacterial strain is Bacillus belgii (Bacillus belgii) ((R))Bacillus velezensis) ZJK2 was deposited in China general microbiological culture Collection center (CGMCC) on 8.6.2020 with the accession number of CGMCC 20037.
5. The method for improving physiological and biochemical activities of bacterial strains through electrical stimulation according to claim 1, wherein the current intensity of the low-voltage direct current in the step 2 is 5-10 mA.
6. The method for improving physiological and biochemical activities of bacterial strains through electric stimulation according to claim 1, wherein the current intensity of the low-voltage direct current in the step 3 is 5-30 mA.
7. The use of the electro-stimulating strain culture solution obtained according to claim 1 or claim 2 for promoting plant growth and preventing and treating gray mold and anthracnose.
8. The use of claim 7, wherein the strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) ZJK1, the application is promoting growth of strawberry or mulberry; when the strain is Bacillus belgii (B)Bacillus velezensis) ZJK2, the application is to prevent and treat gray mold or anthracnose of mulberry, strawberry or grape.
9. A plant growth supplement comprising a culture of an electro-stimulating strain according to any of claims 1 to 8.
10. The plant growth additive according to claim 9, wherein the strain is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) When ZJK1, the plant growth additive is a plant growth promoting microbial inoculum; when the strain is Bacillus belgii (B)Bacillus velezensis) ZJK2, the plant growth additive is a microbial insecticide.
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