CN114107280B - Method for improving physiological and biochemical activity of strain by electric stimulation and application thereof - Google Patents
Method for improving physiological and biochemical activity of strain by electric stimulation and application thereof Download PDFInfo
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Abstract
The invention discloses a method for improving the physiological and biochemical activity of a strain by electric stimulation and application thereof. According to the method, an electrode system for culturing the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1 or bacillus bailii (Bacillus velezensis) ZJK2 strain is constructed through an inert electrode, the activated strain is stimulated by low-voltage direct current, the temperature is maintained at 30-37 ℃, and the strain is cultured for 16-24 hours, so that the biochemical activity of the functional strain can be improved by 20-50%, and the fermentation density is improved by more than 1-2 times. The method greatly improves the biochemical activity and fermentation density of the strain, and has 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 bacterial strains by electric stimulation and application thereof.
Background
Plant growth promoting bacteria are a collective term for a group of beneficial bacteria living in the rhizosphere range and having a promoting effect on plant growth. The plant growth promoting bacteria can activate soil nutrients through metabolic activity, promote plant to absorb nutrients, accelerate growth and the like. The application of the microbial inoculum rich in active growth promoting bacteria is beneficial to restoring the balance of the soil ecological system and restoring the soil fertility, and brings great attention to people. Research shows that the plant growth promoting bacteria can promote crop growth through direct and indirect two ways, and the direct action includes secretion of plant hormone to promote growth, secretion of organic acid to promote the conversion of insoluble phosphorus into available phosphorus, secretion of nitrogen fixing enzyme to raise soil fertility, and the indirect action includes inducing crop to produce resistance, antagonizing pathogenic bacteria, etc. One of the key problems affecting the application of plant growth promoting bacteria at present is the screening and cultivation of excellent bacteria.
The low voltage weak electric field can influence the charge distribution, arrangement and movement conditions inside and outside the cells of the organism, thereby influencing the metabolism and proliferation of the cells. Based on the theory, the electric field is used as a biological stimulus factor, and a tiny electric field is applied in the microorganism growth process, 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 treating the fruits and vegetables, so that the maturity of the fruits and vegetables can be delayed, the water loss rate can be slowed down, and the storage time of the fruits and vegetables can be prolonged.
With the recent development, the biological effect of the electric field has been advanced from the macroscopic level to the microscopic mechanism. The data show that the electric field can influence the metabolic process of cells, the gene expression of cells, the proliferation of cells, the activity of enzymes and the permeability of cell membranes, thereby influencing the free radical reaction and biopolymer synthesis in cells.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for improving the physiological and biochemical activity of a bacterial strain by electric stimulation and application thereof, and the method is based on weak current stimulation to the used bacterial strain to improve the biochemical activity and fermentation density of the bacterial strain, and the obtained electric stimulation bacterial strain culture solution can be used for assisting plant growth and preventing and treating gray mold and anthracnose.
In order to solve the problems in the prior art, the invention adopts the following technical scheme:
An electrode system for strain culture is constructed through an inert electrode, electrochemical induction and domestication are carried out first, then low-voltage direct current is utilized to stimulate the growth of the strain, and the biochemical activity and fermentation density of the strain are improved, so that an electric stimulation strain culture solution is obtained; the strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1 or bacillus bailii (Bacillus velezensis) ZJK2.
As an improvement, the method for improving the physiological and biochemical activity of the strain by the electrical stimulation comprises the following steps:
Step1, activation of the Strain
Taking the strain stored at low temperature, inoculating into sterilized 500mL triangular flask according to the volume ratio of 5-10%, activating in sterilized beef extract peptone liquid culture medium, and shake culturing at constant temperature of 28-37deg.C for 16-24 hr 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 shaking culture at constant temperature of 28-37 ℃ for 24 hours to obtain an electrochemically induced domesticated strain culture solution;
Step 3, growth of the electric stimulation strain
And (3) inoculating the electrochemically induced and domesticated strain culture solution 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 shaking culture at constant temperature for 16-48 hours to obtain the electric stimulation strain culture solution.
As an improvement, when the strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1, the preservation date of the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1 is 2020, 6 and 8 days, the preservation address is North Star Xiyu 1,3 in the Qingyang area of Beijing, and the preservation number is the China general microbiological culture Collection center: CGMCC No. 20036.
As an improvement, the strain is bacillus belicus (Bacillus velezensis) ZJK2 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 20037 in the year 6 and 8 of 2020.
As an improvement, the current intensity of the low-voltage direct current in the step 2 is 5-10mA.
As an improvement, the current intensity of the low-voltage direct current stimulation in the step 3 is 5-30 mA.
The obtained electric stimulation strain culture solution is applied to promoting plant growth and preventing and controlling gray mold and anthracnose.
As an improvement, when the strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1, the application is to promote the growth of strawberries or mulberries; when the strain is bacillus belicus (Bacillus velezensis) ZJK2, the application is to control gray mold or anthracnose of mulberry, strawberry or grape.
A plant growth additive comprises any of the above electro-stimulation strain culture solutions.
Preferably, when the strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1, the plant growth additive is a plant growth promoting microbial agent; when the strain is bacillus belicus (Bacillus velezensis) ZJK2, the plant growth additive is a microbial fungicide.
The beneficial effects are that:
Compared with the prior art, the method for improving the physiological and biochemical activity of the strain by the electrical stimulation and the application thereof can improve 30-50% of the growth promoting capacity of bacillus amyloliquefaciens ZJK1 and 20-30% of the biocontrol capacity of bacillus bailii ZJK2 and can also improve the fermentation density of thalli by 1-2 times. The method has the advantages of low cost, improved capability of functional strains, easy realization of 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 is a graph showing the effect of applied current on the growth of Bacillus bailii (Bacillus velezensis) ZJK2 according to the method of the present invention.
Detailed Description
Unless otherwise defined, 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 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
When the strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1, the preservation date of the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1 is 2020, 6 and 8 days, the preservation address is North Star Xiyu No. 1, 3 of the Korean region of Beijing, and the preservation number is China general microbiological culture Collection center: CGMCC No. 20036
A method for promoting fermentation density of bacillus amyloliquefaciens ZJK1 by externally applying a direct current electric field comprises the following steps:
Firstly, sterilizing an electrified culture flask, an electrode material, a beef extract peptone liquid culture medium and the like by high-pressure steam (sterilizing 20 min at 121 ℃); then, taking 15m L of uniform bacterial suspension in an oscillation mode from bacillus amyloliquefaciens ZJK1 seed liquid subjected to electrochemical induction domestication, and respectively inoculating the bacterial suspension into 4 500m L triangular flasks filled with 300m L of culture medium subjected to high-pressure steam sterilization and cooling, wherein 1 flask is not electrified, and the culture medium is used as a blank control. The other 3 bottles were stimulated with 10mA DC power using stainless steel electrodes (apparent area 2 cm. Times.3 cm, electrode spacing 2 cm). 4 bottles of bacterial liquid 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 power.
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 non-electrified culture solution, and the bacillus amyloliquefaciens can be effectively promoted to grow and reproduce by the action of 10 mA direct current.
Example 2
Influence of electric stimulation of secretion of auxin IAA by Bacillus amyloliquefaciens culture solution
0.5 ML electric stimulation and unpowered seed liquid are inoculated into 50 mL LB liquid culture medium containing 100 mg/L L-tryptophan respectively, 1mL of each sample is taken when the culture is carried out at 30 ℃ under shaking culture of 120 r/min until the culture is 6, 18, 30 and 48 h, 10 min is centrifuged at 12000 r/min, 100 mu L of supernatant is taken and added into equal volume Salkowski colorimetric liquid, after uniform mixing, the mixture is kept away from light for 30min, OD 530 value is measured, and LB culture medium without L-tryptophan is taken as blank control. And calculating the content of the IAA in the unit volume of the bacterial liquid according to a standard curve. As a result, it was found that IAA concentration in the culture medium at 10mA was 40% or more higher than that in the control.
Example 3
Potted plant experiment for promoting growth of mulberry seedlings by using electric stimulation bacillus amyloliquefaciens culture solution
Selecting full and consistent mulberry seeds, soaking the mulberry seeds in warm boiled water at 50 ℃, and soaking the mulberry seeds in water for 12 h after cooling. After germination, seedling raising and permanent planting are carried out with two leaves which are equal in length and one center. Healthy mulberry seedlings with consistent growth vigor are selected to be transplanted into a plastic basin (containing 200g of air-dried soil), and after the mulberry seedlings are planted for one week, a root irrigation method is adopted to inoculate and culture the growth promoting bacterial liquid for 48 hours.
The treatment groups were sterilized beef extract peptone culture medium Control (CK), 200 times diluted unpowered bacterial liquid and 200 times electrified 10mA electro-stimulated Bacillus amyloliquefaciens culture medium, and each pot was irrigated with 50mL of root per 5 replicates per treatment.
When the mulberry seedlings grow into four leaves and one core, the plant heights and fresh weights of the mulberry in the experimental group and the control group are measured, then carefully peeled off from soil and dried to constant weight at 105 ℃, and the dry weight of the overground part and the dry weight of the root system are weighed, so that the following results are obtained:
As shown in the table, the electric stimulation bacillus amyloliquefaciens culture solution has significantly increased plant height, whole fresh weight, overground weight and root dry weight compared with the unpowered culture solution.
Example 4
Bacillus belicus (Bacillus velezensis) ZJK2 used in the invention is preserved in China general microbiological culture Collection center (China Committee for culture Collection), the preservation address is North Star Xili No. 1, 3 of the Korean area of Beijing, the institute of microbiology, china academy of sciences, the preservation number is CGMCC No. 20037, and the preservation date is 2020, 6, 8 days.
A method for promoting bacillus beijerinus ZJK2 high-density fermentation by externally adding a direct current electric field.
Firstly, sterilizing an electrified culture flask, an electrode material, a beef extract peptone liquid culture medium and the like by high-pressure steam (sterilizing 20min at 121 ℃); then taking 3m L of uniform bacterial suspension in an oscillating way from bacillus bailii ZJK2 seed liquid subjected to electrochemical induction domestication, and respectively inoculating the bacterial suspension into 4 500m L triangular flasks filled with 300m L culture medium subjected to high-pressure steam sterilization and cooling, wherein 1 flask is not electrified, and taking the bacterial suspension as a blank control. The other 3 bottles were stimulated with 10mA DC power using stainless steel electrodes (apparent area 2 cm. Times.3 cm, electrode spacing 2 cm). 4 bottles of bacterial liquid 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 power. As a result of the bacterial count method, the cell concentration in the culture medium was about 2 times that in the culture medium which was not energized. It can be seen that the bacillus belicus ZJK2 can be effectively promoted to proliferate by introducing 10 mA direct current.
Example 5
Diluting bacterial solutions of bacillus beljalis ZJK2 which are electrified (10 mA) and not electrified for 24 hours by 200 times and 100 times respectively; taking out a round block with the same quality from a PDA solid culture medium with the gray mold of the strawberry by using a puncher with the diameter of 5mm, taking out the round block, and inoculating the round block to one side of a PDA flat plate; and then the other side of the PDA plate is punched with a puncher with the diameter of 5mm to obtain round blocks with the same quality, and the round blocks are taken out, respectively inoculated with 100 mu L of sterile water, electrified and unpowered diluted bacteria liquid, cultured for 7d at the temperature of 28 ℃ and observed. The inhibition rate of different treatment fluids to the botrytis cinerea on the flat plate is calculated as follows:
the bacteriostasis rates of the bacillus beijerinatus ZJK2 when being electrified and not electrified are 93.6 percent and 72.5 percent respectively, and the direct current of 10mA is used for improving the antibacterial capacity of the bacillus beijerinatus ZJK2 by about 30 percent.
In the foregoing, only some embodiments of the present invention are described, and the scope of the present invention is not limited thereto, but any simple changes or equivalent substitutions of the technical solutions that are obvious to those skilled in the art within the technical scope of the present invention disclosed in the present invention fall within the scope of the present invention.
Claims (2)
1. A method for improving the physiological and biochemical activity of a strain by electric stimulation is characterized in that an electrode system for strain culture is constructed through an inert electrode, electrochemical induction and domestication are carried out first, then the growth of the strain is stimulated by low-voltage direct current, and the biochemical activity and fermentation density of the strain are improved, so that an electric stimulation strain culture solution is obtained; the strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1 or bacillus bailii (Bacillus velezensis) ZJK2, when the strain is bacillus amyloliquefaciens (Bacillus amylohliquefaciens) ZJK1, the preservation date of the bacillus amyloliquefaciens (Bacillus amylohquefaciens) ZJK1 is 2020 month 8, the preservation address is No. 3 of Xiyang North Star West-1 in Beijing, and the preservation number is the China general microbiological culture Collection center: the CGMCC No.20036, the improved biological activity is to promote the growth of mulberry, when the strain is Bacillus bailii (Bacillus velezensis) ZJK2, the Bacillus bailii (Bacillus velezensis) ZJK2 is preserved in the China general microbiological culture Collection center of China general microbiological culture Collection center (CGMCC No. 20037) on the 6 th month 8 day of 2020, and the improved biological activity is the antibacterial capability of Botrytis cinerea;
the method comprises the following steps:
step1, activation of strains:
Taking the strain stored at low temperature, inoculating into a sterilized 500mL triangular flask according to the volume ratio of 5-10%, and culturing for 16-24 hours at a constant temperature of 28-37 ℃ to obtain an activated strain seed liquid, wherein the triangular flask is filled with sterilized beef extract peptone liquid culture medium;
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 shaking culture at constant temperature of 28-37 ℃ for 24 hours to obtain an electrochemically induced domesticated strain culture solution; the current intensity of the low-voltage direct current is 5-10mA;
step 3, growth of the electric stimulation strain:
And (3) inoculating the electrochemically induced and domesticated strain culture solution 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 shaking culture at constant temperature for 16-48h to obtain the electric stimulation strain culture solution, wherein the current intensity of the low-voltage direct current stimulation is 10mA.
2. Use of the electrical stimulation strain culture solution according to claim 1 for promoting plant growth and preventing gray mold, wherein when the strain is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) ZJK1, the use is to promote the growth of mulberry; when the strain is bacillus belicus (Bacillus velezensis) ZJK2, the application is to control gray mold of strawberries.
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| CN112458011A (en) * | 2020-11-23 | 2021-03-09 | 江苏科技大学 | Bacillus amyloliquefaciens ZJK1 and application thereof |
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| CN112458012B (en) * | 2020-11-24 | 2022-10-11 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | Bacillus belgii microbial agent and application thereof |
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| CN112458011A (en) * | 2020-11-23 | 2021-03-09 | 江苏科技大学 | Bacillus amyloliquefaciens ZJK1 and application thereof |
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